A method for preparing a thermosensitive hemostatic gel, the product, and its application.

Abstract

This invention discloses a method for preparing a thermosensitive hemostatic gel, which is prepared by mixing a thermosensitive polymer solution with a solution of micron-sized polymer particles or microspheres with hemostatic function. The concentration of the solution of micron-sized polymer particles or microspheres with hemostatic function is 3-100 mg / mL; the concentration of the thermosensitive polymer solution is 200-450 mg / mL; the diameter of the micron-sized polymer particles or microspheres with hemostatic function is 300-900 micrometers; the gelation temperature of the thermosensitive hemostatic gel from a flowing liquid state to a solid state is greater than 15 degrees Celsius, and it is a flowing liquid state at temperatures below 15 degrees Celsius. Under the condition of warming up in response to the human body temperature, the flowing liquid hemostatic gel transforms into a solid hemostatic gel, thereby achieving the hemostatic function. The micron-sized polymer particles or microspheres in the thermosensitive hemostatic gel will not enter the damaged blood vessel during the hemostasis process.

Description

Technical Field

[0001] This invention belongs to the field of hemostatic materials, and relates to a method for preparing a thermosensitive hemostatic gel, as well as the product and its application. Background Technology

[0002] Uncontrolled massive blood loss leading to complications is a major cause of numerous casualties, making rapid and effective hemostasis crucial for disease control, clinical surgery, and rescue efforts. Aqueous solutions of thermosensitive polymers such as poloxamer and hydroxypropyl cellulose can be used in thermosensitive hydrogels. These hydrogels exhibit good biocompatibility and controllable phase transitions, making them a hot research topic in the medical field. However, when applied to wounds, thermosensitive hydrogels have limited hemostatic effects because they do not have procoagulant properties and only serve to cover the wound. Meanwhile, some biocompatible polymers such as chitosan sponge particles, calcium alginate microspheres, or cross-linked gelatin microspheres, while possessing some hemostatic effect, are easily washed away by flowing blood when applied to massive bleeding wounds, failing to fully exert their hemostatic properties and thus limiting their effectiveness. Based on this, a thermosensitive hemostatic gel was prepared by mixing micron-sized polymer particles with hemostatic function and thermosensitive hydrogel in an appropriate ratio. The addition of hemostatic material endows the thermosensitive hydrogel with good hemostatic function. The thermosensitive hydrogel avoids the possibility of the hemostatic material being washed away by flowing blood when applied to the wound, allowing it to fully exert its hemostatic function. The technology of preparing a thermosensitive hemostatic gel by mixing a solution of thermosensitive polymer with micron-sized polymer particles with hemostatic function has not been reported domestically. Summary of the Invention

[0003] In view of this, the present invention provides a method for preparing a thermosensitive hemostatic gel, a product thereof, and its application. Specifically, the present invention provides the following technical solution:

[0004] 1. A method for preparing a thermosensitive hemostatic gel, comprising mixing a thermosensitive polymer solution with a solution of micron-sized polymer particles or microspheres having hemostatic function, wherein the concentration of the solution of micron-sized polymer particles or microspheres having hemostatic function is 3-100 mg / mL; the concentration of the thermosensitive polymer solution is 200-450 mg / mL; the diameter of the micron-sized polymer particles or microspheres having hemostatic function is 300-900 micrometers; the gelation temperature of the thermosensitive hemostatic gel from a flowing liquid state to a solid state is greater than 15 degrees Celsius, and it remains a flowing liquid state at temperatures below 15 degrees Celsius. Under the condition of responding to the rise in body temperature, the flowing liquid hemostatic gel transforms into a solid hemostatic gel, thereby achieving the hemostatic function. The micron-sized polymer particles or microspheres in the thermosensitive hemostatic gel will not enter the damaged blood vessel during the hemostatic process.

[0005] Furthermore, the thermosensitive polymer solution is a poloxamer solution or a mixture of poloxamer solution and hydroxypropyl methylcellulose solution, or a mixture of poloxamer solution and hydroxypropyl methylcellulose solution; the micron-sized polymer particles or microspheres with hemostatic function are calcium alginate microspheres, cross-linked gelatin microspheres, polyvinyl alcohol particles, chitosan sponge particles, chitin particles, or collagen particles; the solvent of the solution is water, physiological saline, phosphate buffer, or glucose solution.

[0006] Furthermore, when the micron-sized polymer particles or microspheres with hemostatic function are chitosan sponge particles, their concentration in the thermosensitive polymer solution is 3-30 mg / mL; when the micron-sized polymer particles or microspheres with hemostatic function are calcium alginate microspheres, their concentration in the thermosensitive polymer solution is 30-100 mg / mL; when the micron-sized polymer particles or microspheres with hemostatic function are cross-linked gelatin microspheres, their concentration in the thermosensitive polymer solution is 20-100 mg / mL, wherein the cross-linked gelatin microspheres are proanthocyanidin cross-linked gelatin microspheres.

[0007] Furthermore, the preparation steps of the aforementioned micron-sized polymer particles with hemostatic function are as follows:

[0008] a) Prepare a 10-300 mg / mL polymer aqueous solution, then transfer the polymer aqueous solution to a large-sized tray with a diameter greater than 5 cm, and freeze-dry it to form a macroscopic large-sized polymer hemostatic sponge; the preparation process also includes forming a cross-linked network through chemical or physical cross-linking process during the preparation of the polymer solution or after freeze-drying to enhance the structural strength of the sponge.

[0009] b) Cut large-sized sponges into irregular particles using a shredder;

[0010] c) Particles of 300-900 micrometers are obtained by sieving; the sieve is a 20-50 mesh sieve.

[0011] Depending on the type of polymer, the aqueous solution in step a) can be water, diluted acetic acid / hydrochloric acid aqueous solution, or a mixed solution of urea and sodium hydroxide. The crosslinking process can be the addition of a chemical crosslinking agent, an ionic crosslinking agent, or the alteration of the sponge's pH. The chemical crosslinking agent can be glutaraldehyde, tannic acid, proanthocyanidins, or a mixed solution of epichlorohydrin and sodium hydroxide. The ionic crosslinking agent can be calcium chloride.

[0012] Furthermore, the preparation method of the aforementioned micron-sized polymer microspheres with hemostatic function is as follows:

[0013] 1) Emulsion method:

[0014] a) First, prepare a 10-300 mg / mL aqueous solution of the polymer to form an aqueous phase;

[0015] b) The organic solvent and surfactant are then mixed to form an oil phase; the aqueous phase is added dropwise to the oil phase at a volume ratio of 1:5 to 1:20, the stirring speed is 400-2000 rpm, and emulsification is carried out for 20-120 min to form an emulsion.

[0016] c) Microspheres are obtained by chemically or physically cross-linking the polymer solution in the emulsion through a cross-linking process, followed by demulsification, precipitation, washing, and drying.

[0017] d) Particles of 300-900 micrometers are obtained by sieving; the sieve is a 20-50 mesh sieve.

[0018] Depending on the type of polymer, the aqueous solution in step a) can be water, a diluted acetic acid / hydrochloric acid aqueous solution, or a mixed solution of urea and sodium hydroxide. The crosslinking process in step c) can be the addition of a chemical crosslinking agent, an ionic crosslinking agent, or the change of emulsion temperature or pH. The chemical crosslinking agent is glutaraldehyde, epichlorohydrin / sodium hydroxide, tannic acid, or proanthocyanidins. The ionic crosslinking agent is calcium chloride.

[0019] 2) Spray granulation method:

[0020] a) First, prepare a 10-300 mg / mL polymer aqueous solution;

[0021] b) Add the polymer aqueous solution to the polymer solution spraying device, wait for the polymer solution sprayed by the device to form a stable bead string, and then spray the bead string into the receiving liquid. The device is a microcapsule granulator or a polymer electrostatic spraying instrument.

[0022] c) The polymer solution in the receiving liquid is chemically or physically cross-linked to form cross-linked network microspheres, which are then obtained through centrifugation, washing, and drying.

[0023] Depending on the type of polymer, the aqueous solution in step a) can be water, a diluted acetic acid / hydrochloric acid aqueous solution, or a mixed solution of urea and sodium hydroxide. The crosslinking process in step c) can be the addition of a chemical crosslinking agent, an ionic crosslinking agent, or the change of the temperature or pH of the receiving liquid. The chemical crosslinking agent is glutaraldehyde, epichlorohydrin / sodium hydroxide, tannic acid, or proanthocyanidins. The ionic crosslinking agent is calcium chloride.

[0024] Depending on the different devices used for spraying polymer solutions, different steps are selected to obtain particles of 300-900 micrometers: Before spraying the polymer solution in step b), the particle size of the beads is controlled by adding or matching the nozzle with the instrument. The nozzle is selected with a diameter of 150-700 micrometers or no nozzle is used. After step c), particles of 300-900 micrometers are obtained by sieving, and the sieve is a 20-50 mesh sieve.

[0025] 2. A thermosensitive hemostatic gel prepared according to the above preparation method.

[0026] 3. The application of the above-mentioned thermosensitive hemostatic gel in the preparation of blood coagulation-promoting products.

[0027] Furthermore, the blood clotting-promoting product is applied from outside the blood vessels to the damaged blood vessels in the wound.

[0028] Furthermore, the blood clotting-promoting product is applied from inside the blood vessel to the embolized vessel at the site of the intact blood vessel.

[0029] The beneficial effects of this invention are as follows: This invention mixes a solution of a thermosensitive polymer with micron-sized polymer particles possessing hemostatic function to prepare a thermosensitive hemostatic gel. Thermosensitive hydrogels possess good biocompatibility and controllable phase transitions; they are liquid at low temperatures and coagulate at high temperatures. Utilizing this temperature-dependent property, they can be injected and applied to the surface of bleeding wounds in humans or animals (i.e., the site of ruptured blood vessels). The relatively high temperature environment caused by body temperature transforms the fluid liquid hemostatic gel into a solidified gel, thus covering the wound. However, a simple thermosensitive gel does not possess active procoagulant properties and cannot effectively promote the coagulation reaction of blood after material contact, thus failing to achieve rapid hemostasis. Micron-sized polymer particles or microspheres with hemostatic function possess good biocompatibility and also have a certain procoagulant function. However, in actual hemostasis processes, because the wound location cannot be pressed or is difficult to press, they are often washed away by the flowing blood at the bleeding wound site, failing to fully exert their hemostatic performance. Therefore, we prepared a thermosensitive hemostatic gel by mixing micron-sized polymer particles or microspheres with hemostatic function with thermosensitive hydrogel in an appropriate ratio. The addition of hemostatic material gives the thermosensitive hydrogel good hemostatic function. The thermosensitive hydrogel does not reduce the effectiveness of the hemostatic material and avoids the phenomenon of the hemostatic material being washed away by the flowing blood when applied to the wound, allowing it to fully exert its hemostatic function and thus achieve rapid hemostasis. Similar to the above-mentioned hemostatic application applied from outside the blood vessel to the damaged blood vessel, when the thermosensitive hemostatic gel developed in this invention is applied to the vascular embolism scenario (i.e., applied from inside the blood vessel to an intact blood vessel), it will also avoid (or effectively reduce) the disadvantage of micron-sized polymer particles being carried away by the blood flow in the blood vessel, and can also give full play to its advantages over the single thermosensitive polymer solution in promoting blood coagulation and accelerating vascular embolism. Attached Figure Description

[0030] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided:

[0031] Figure 1This is a microscope view of the chitosan sponge particles (CSK) of Example 1.

[0032] Figure 2 This is a microscope view of the proanthocyanidin-crosslinked gelatin microspheres (GM1) of Example 3.

[0033] Figure 3 This is a microscope view of the calcium alginate microspheres (AM2) of Example 4.

[0034] Figure 4 The rheological curve of thermosensitive gel X3 with hemostatic properties in Example 3 is shown.

[0035] Figure 5 The rheological curve of thermosensitive gel Y1 with hemostatic properties is shown in Comparative Example 1. Detailed Implementation

[0036] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0037] Example 1

[0038] 1) Weigh 5.2g of chitosan raw material and dissolve it in 200mL of acetic acid / deionized water mixed solution (volume ratio 1 / 149) to obtain a chitosan solution of 26mg / mL. Then transfer the chitosan solution to a plastic dish with a diameter of 10cm and heat it at -80℃.

[0039] Sponges are obtained by freezing at low temperatures under certain conditions and then freeze-drying them.

[0040] 2) The freeze-dried sponge was soaked in ethanol to remove some of the acetic acid, and then vacuum dried to obtain chitosan sponge;

[0041] 3) The chitosan sponge is cut into irregular particles using a shredder;

[0042] 4) Finally, the chitosan sponge particles (CSK) of approximately 600 micrometers were obtained by sieving through 25-mesh and 30-mesh sieves. Please refer to [link to relevant documentation]. Figure 1 .from Figure 1 It can be seen that the chitosan sponge particles are plate-like, similar to dragonfly wings, with irregular particle outlines and distinct veins on the surface.

[0043] 5) Poloxamer was prepared into a solution with a concentration of 200 mg / mL using physiological saline, and CSK was added to make its concentration in the solution 5 mg / mL, thus obtaining a thermosensitive gel X1 with hemostatic properties.

[0044] Example 2

[0045] 1) Dissolve 4g of sodium alginate in 200mL of deionized water to obtain a sodium alginate solution with a concentration of 20mg / mL, forming an aqueous phase;

[0046] 2) Add liquid paraffin to Span-80 at a volume ratio of 20:1 (liquid paraffin to Span-80).

[0047] Stir at rpm to form an oil phase;

[0048] 3) After mixing the aqueous phase and the oil phase (volume ratio of aqueous phase to oil phase is 1:10), emulsify in a constant temperature water bath at 45℃ and a rotation speed of 800 rpm for 60 min to form an emulsion; add 4 mL of CaCl2 solution (mass fraction of 10%) and crosslink for 60 min.

[0049] 4) After centrifugation at 4000 rpm, the microspheres were washed with anhydrous ethanol for 3 hours and then vacuum dried for 1 hour to obtain calcium alginate microspheres; finally, the microspheres were sieved through 25-mesh and 35-mesh sieves to obtain calcium alginate microspheres (AM1) with a size of about 600 micrometers.

[0050] 5) Prepare a 200 mg / mL solution of poloxamer with physiological saline, and add AM1 to make its concentration in the solution 70 mg / mL to obtain a thermosensitive gel X2 with hemostatic properties.

[0051] Example 3

[0052] 1) Dissolve 30g of gelatin in 200mL of 50℃ deionized water to obtain a gelatin solution of 150mg / mL, and filter it once using a funnel;

[0053] 2) After adding the filtered gelatin solution to the microencapsulation granulator, maintain the system between 45-50℃. Select a granulator nozzle diameter of 300 micrometers. Once the polymer solution sprayed from the device forms stable bead strings, collect the bead strings with cold n-hexane to form stable gel spheres. Use magnetic stirring to reduce gel sphere adhesion. Dry the n-hexane by freeze-drying to obtain uncrosslinked gelatin microspheres (GM).

[0054] 3) Disperse 10g of uncrosslinked gelatin microspheres in 100mL of a mixed solution of Tris buffer and anhydrous ethanol (Tris buffer: anhydrous ethanol volume ratio 9:1), add 0.3g of proanthocyanidins, crosslink at room temperature for 10min, wash three times with deionized water, and then freeze-dry to obtain crosslinked gelatin microspheres (GM1). Please refer to [link to relevant documentation]. Figure 2 ,from Figure 2 It can be seen that the cross-linked gelatin microspheres are spherical with edges and corners, and the porous structure inside can be faintly seen. The diameter is about 700 micrometers.

[0055] 4) Prepare a 200 mg / mL solution of poloxamer with physiological saline, and add GM1 to make its concentration in the solution 50 mg / mL to obtain a thermosensitive gel X3 with hemostatic properties.

[0056] In this embodiment, the crosslinking process used involves adding a chemical crosslinking agent, namely a proanthocyanidin solution; the microsphere preparation method used is a spray method.

[0057] Example 4

[0058] 1) Dissolve 4g of sodium alginate in 200mL of deionized water to obtain a sodium alginate solution of 20mg / mL, and filter it once using a funnel.

[0059] 2) Prepare a 0.2M calcium chloride solution by dissolving 10g of anhydrous calcium chloride in 500mL of deionized water as the crosslinking solution. The nozzle diameter of the granulator is 300 μm. Wash the microspheres with 50%, 70%, and 100% ethanol solutions, respectively. After washing for 9 hours, dry in a vacuum drying oven for 1 hour to obtain calcium alginate microspheres (AM2). Please refer to [link to relevant documentation]. Figure 3 ;from Figure 3 It can be seen that the calcium alginate microspheres are pebble-shaped or spherical, with smooth surfaces and no sharp edges. A porous structure is visible in the center, and the diameter is about 500 micrometers.

[0060] 3) Poloxamer was prepared into a solution with a concentration of 200 mg / mL using physiological saline. AM2 was added to make its concentration in the solution 70 mg / mL, thus obtaining a thermosensitive gel X4 with hemostatic properties.

[0061] In this embodiment, the crosslinking process used involves adding an ionic crosslinking agent, namely calcium chloride solution; the microsphere preparation method used is the spray method.

[0062] Example 5

[0063] Poloxamer was prepared into a solution with a concentration of 300 mg / mL using physiological saline, and hydroxypropyl methylcellulose was prepared into a solution with a concentration of 25 mg / mL using physiological saline. A mixed solution of the two solutions was prepared according to the volume ratio (poloxamer solution: hydroxypropyl methylcellulose solution = 4:1). GM1 from step 3) of Example 3 was added to make its concentration in the solution 50 mg / mL, thus obtaining thermosensitive gel X5 with hemostatic properties.

[0064] Example 6

[0065] Poloxamer was prepared into a solution with a concentration of 300 mg / mL using physiological saline. GM1 from step 3) of Example 3 was added to make its concentration in the solution 50 mg / mL, resulting in a thermosensitive gel X6 with hemostatic properties.

[0066] This embodiment differs from Example 3 only in the concentration of the poloxamer solution.

[0067] Example 7

[0068] Poloxamer was prepared into a solution with a concentration of 200 mg / mL using physiological saline. CSK from step 4) of Example 1 was added to make its concentration in the solution 40 mg / mL, resulting in a thermosensitive gel X7 with hemostatic properties.

[0069] This embodiment differs from Example 1 only in the concentration of CSK particles in the gel.

[0070] Example 8

[0071] Poloxamer was prepared into a solution with a concentration of 200 mg / mL using physiological saline. AM2 from step 2) of Example 4 was added to make its concentration in the solution 20 mg / mL, resulting in a thermosensitive gel X8 with hemostatic properties.

[0072] This embodiment differs from Example 4 only in the concentration of AM2 in the gel.

[0073] Example 9

[0074] Poloxamer was prepared into a solution with a concentration of 200 mg / mL using physiological saline. GM1 from step 3) of Example 3 was added to make its concentration in the solution 15 mg / mL, resulting in a thermosensitive gel X9 with hemostatic properties.

[0075] The only difference between this embodiment and Example 3 is the concentration of GM1 particles in the gel.

[0076] Comparative Example 1

[0077] Poloxamer was prepared into a solution with a concentration of 150 mg / mL using physiological saline. GM1 from step 3) of Example 3 was added to make its concentration in the solution 50 mg / mL, resulting in a thermosensitive gel Y1 with hemostatic properties.

[0078] Compared with Example 3, the concentration of the temperature-sensitive substance (poloxam) in this comparative example is too low, resulting in poor gelation performance. For specific analytical data, please refer to the data analysis section of the test examples.

[0079] Comparative Example 2

[0080] Poloxamer was prepared into a solution with a concentration of 200 mg / mL using physiological saline. CSK from step 4) of Example 1 was added to make its concentration in the solution 1 mg / mL, resulting in a thermosensitive gel Y2 with hemostatic properties.

[0081] Compared with Example 1, the concentration of micron-sized polymer particles with hemostatic function in the gel in this comparative example is too low, resulting in poor hemostatic effect. For specific analysis data, please refer to the data analysis section of the test example.

[0082] Comparative Example 3

[0083] Poloxamer was prepared into a solution with a concentration of 200 mg / mL using physiological saline. AM2 from step 2) of Example 4 was added to make its concentration in the solution 120 mg / mL, resulting in a thermosensitive gel Y3 with hemostatic properties.

[0084] Compared with Example 4, the concentration of micron-sized polymer particles with hemostatic function in the gel was too high in this comparative example, resulting in poor hemostatic effect. For specific analysis data, please refer to the data analysis section of the test example.

[0085] Comparative Example 4

[0086] Poloxamer was prepared into a solution with a concentration of 200 mg / mL using physiological saline. GM1 from step 3) of Example 3 was added to make its concentration in the solution 110 mg / mL, resulting in a thermosensitive gel Y4 with hemostatic properties.

[0087] Compared with Example 3, the concentration of micron-sized polymer particles with hemostatic function in the gel was too high in this comparative example, resulting in poor hemostatic effect. For specific analysis data, please refer to the data analysis section of the test example.

[0088] Comparative Example 5

[0089] Poloxamer was prepared into a solution with a concentration of 200 mg / mL using physiological saline to obtain thermosensitive gel Y5.

[0090] This comparative example uses a pure adhesive system without the addition of micron-sized polymer particles that have hemostatic function, resulting in poor hemostatic effect. For detailed analysis data, please refer to the data analysis section of the test examples.

[0091] Comparative Example 6

[0092] In Example 3, step 3), GM1 refers to Y6, the gelatin microspheres cross-linked with proanthocyanidins. The particles were not compounded with poloxamer.

[0093] Comparative Example 7

[0094] In step 4) of Example 1, CSK, namely chitosan sponge particles Y7, was not compounded with poloxamer.

[0095] Test Example 1: Gel Transition Temperature Test

[0096] Testing conditions: The gel transition temperature of the materials obtained from each experimental example and comparative example was tested using a rheometer.

[0097] Detection method: The gel transition temperature (Tg) of the sample was determined using a rheometer in temperature scanning mode. The strain value was set to 1%, the heating range was 5-40℃, the heating rate was 1℃ / min, and the sample thickness was 1000 μm. The changes in the storage modulus G' and loss modulus G″ of the thermosensitive hydrogel with temperature were observed. When the storage modulus < loss modulus, the system is in a liquid or viscous state; when the storage modulus is equal to the loss modulus, the system changes from a liquid state to a semi-solid gel state, and this temperature is the gel transition temperature of the thermosensitive hydrogel; when the temperature continues to rise, the storage modulus > loss modulus, and the system has completely changed to a solid gel state. The gel transition temperature test results are shown in Table 1.

[0098] Table 1. Gel transition temperature test

[0099] sample X1 X2 X3 X4 X5 X6 X7 Y1 Transition temperature (°C) 24 25 23 26 32 15 23 none

[0100] As can be seen from Table 1, the gel transition temperature of Examples 1-7 is 15-32℃. The thermosensitive gel with this transition temperature can quickly change from a liquid gel state to a solid gel state in a human body environment of 37℃.

[0101] In Example 6, the concentration of the thermosensitive substance was 30%, and its gel transition temperature was 15°C, significantly lower than other thermosensitive gels. This thermosensitive hemostatic gel with this transition temperature is already in a solid state at room temperature. However, due to the shear-thinning principle, the solidified gel can regain its fluidity under the pressure of a syringe, allowing it to cover the wound surface. It then reverts to a solid state, achieving rapid hemostasis. Therefore, by using the preparation method of this invention, adding an appropriate amount of hemostatic micron-sized polymer particles to the thermosensitive polymer solution does not affect the thermosensitivity of the gel, resulting in a thermosensitive hemostatic gel with good rheological properties.

[0102] The reasons for the poor rheological properties of the comparative model are analyzed as follows:

[0103] Figure 4 The rheological curve of gel X3 in Example 3 is shown. Figure 5 The rheological curve of gel Y1 in Comparative Example 1 is shown.

[0104] from Figure 4 We can clearly see that the gel transition temperature of X3 is 23℃.

[0105] from Figure 5 As we can see, Y1 does not have a significant gel transition temperature and remains in a liquid or viscous state.

[0106] Comparative Example 1 reduced the concentration of the poloxamer solution in Example 3 from 200 mg / mL to 150 mg / mL. This demonstrates that the thermosensitive hemostatic gel obtained by reducing the concentration of the poloxamer solution cannot be transformed from a liquid gel state to a solid gel state because the concentration of the thermosensitive polymer is too low, reducing the gel's thermosensitive conversion ability and thus affecting its rheological properties.

[0107] Test Example 2: In Vitro Coagulation Efficacy Test

[0108] Testing conditions: The materials obtained from each experimental example and comparative example were subjected to an in vitro coagulation effect comparison experiment. The blood used for testing was fresh sodium citrate anticoagulated blood taken from the heart of a rat.

[0109] Test Method: Take 50 μL of gel. Mix 100 μL of fresh anticoagulated rat blood with 10 μL of 0.2 M CaCl2 solution, then quickly add the mixture to the material and incubate at 37°C for 50 seconds. Then slowly add 10 mL of deionized water and continue incubation for 3 minutes to fully lyse any excess blood that has not formed a clot. Finally, take 100 μL of the lysed supernatant and add it to a 96-well plate. Calculate the hemoglobin content by measuring the absorbance (Abs) at 545 nm using an ELISA reader. The blank is prepared by adding 100 μL of fresh anticoagulated blood to 10 mL of deionized water, incubating at 37°C for 3 minutes, and then measuring the absorbance (Abs) at 545 nm with 100 μL of the blank. Finally, calculate the coagulation index (BCI) using the following formula.

[0110] Coagulation index % (BCI) = (Abs sample / Abs blank) × 100% ... Equation 1

[0111] In the formula: Abs sample is the absorbance of the experimental group at 545 nm; Abs blank is the absorbance of the blank group at 545 nm. The BCI index of the examples and comparative examples is shown in Table 2.

[0112] Table 2 In vitro coagulation effect test

[0113] sample X1 X2 X3 X4 X5 X6 X7 BCI (%) 17.9 19.4 18.5 20.6 23.5 19.3 42.6 sample X8 X9 Y2 Y3 Y4 Y5 BCI (%) 44.2 40.1 57.6 70.1 67.6 68.6

[0114] BCI (Blood Clotting Index) can characterize the clotting effect of a material. Generally, the smaller the BCI value, the better the clotting effect of the material.

[0115] As shown in Table 2, the BCI indices of the thermosensitive hemostatic gels X1-X9 obtained in Examples 1-9 of this invention ranged from a minimum of 17.9% to a maximum of 47.6%, indicating that the thermosensitive hemostatic gels obtained by adding an appropriate amount of micron-sized polymer particles with hemostatic properties to the thermosensitive polymer have superior procoagulant properties. Even after adding an appropriate amount of micron-sized polymer particles with hemostatic properties to the thermosensitive polymer, its hemostatic properties can still be fully utilized, improving the adhesion and activation of platelets, thereby enhancing the hemostatic performance of the gel. Therefore, by using the preparation method of this invention, adding an appropriate amount of micron-sized polymer particles with hemostatic function to the thermosensitive polymer solution can yield a thermosensitive hemostatic gel with strong hemostatic properties.

[0116] The reasons why the hemostatic effect of the comparative ratio is not good are as follows:

[0117] Comparative Example 2 reduced the CSK addition from 5 mg / mL to 1 mg / mL in Example 1. The BCI of Example 1 was 17.9%, while that of Comparative Example 2 was 57.6%. This shows that when the CSK addition is too low, the particles embedded in the temperature-sensitive polymer are too dispersed, making it difficult to effectively aggregate blood cells and adhere to platelets, thus failing to effectively accelerate the coagulation process.

[0118] Comparative Example 3 increased the AM2 concentration from 70 mg / mL in Example 4 to 120 mg / mL. The BCI of Example 4 was 20.6%, while that of Comparative Example 3 was 70.1%. This demonstrates that a higher AM2 concentration is not necessarily better. Excessive AM2 concentration leads to the release of too many calcium ions during hemostasis. Excessive calcium ions can enhance the inhibitory effect of a protease on coagulation factors, thereby terminating the coagulation process and hindering effective and rapid hemostasis.

[0119] Comparative Example 4 increased the GM1 concentration from 50 mg / mL in Example 3 to 110 mg / mL. The BCI of Example 3 was 18.5%, while the BCI of Comparative Example 4 was 67.6%. This demonstrates that a higher GM1 concentration is not necessarily better. Excessive GM1 concentration makes it difficult to disperse effectively in the thermosensitive polymer solution, leading to particle agglomeration. This reduces the surface area of ​​the particles embedded in the thermosensitive polymer in contact with blood during hemostasis, thus failing to effectively accelerate the coagulation process.

[0120] Comparative Example 5, prepared using only poloxamer (F127) without the addition of any micron-sized polymer particles with hemostatic function, showed a BCI of 68.6%. This indicates that the hydrogel without the addition of micron-sized polymer particles with hemostatic function exhibits poor hemostatic performance.

[0121] Test Example 3: Test of Hemostatic Effect on the Femoral Artery of Rats

[0122] Test Method: For the thermosensitive hemostatic gel material: SD rats weighing 240-260g were anesthetized by intraperitoneal injection of chloral hydrate (10% deionized water, 0.5mL / 100g body weight). The epithelial tissue of the right hind limb was incised with surgical scissors to expose the femoral artery and vein. The femoral artery (along with veins and peripheral nerves that are difficult to separate from the artery) was quickly incised with a scalpel. After incising the femoral artery, free bleeding was allowed for 10 seconds. At the same time, the blood loss in the first 10 seconds was collected using pre-weighed gauze (m1) and weighed to obtain m1'. After 10 seconds of free bleeding, 400μL of the material was quickly applied to the bleeding site using a syringe. During hemostasis, any spilled blood was wiped away with pre-weighed filter paper (m2). Hemostasis was first achieved for 1.5 minutes, and bleeding was observed. If bleeding continued, it was observed every 1 minute until bleeding stopped. The bleeding time was recorded, and m2' was collected and weighed. In the experiment, rat samples with abnormal pre-bleeding (>400mg or <200mg) were excluded from the final analysis of blood loss and hemostasis time to reduce the influence of individual rat differences and procedural errors.

[0123] Calculate the initial blood loss and total blood loss using the following formula:

[0124] Pre-blood loss M1 = m1' - m1.....................Equation 2

[0125] Blood loss M2 = m2' - m2......................Equation 3

[0126] Table 3 Blood loss during femoral artery hemostasis in rats

[0127]

[0128] Table 4 Hemostasis time of femoral artery in rats

[0129]

[0130]

[0131] The rat femoral artery hemostasis test simulates and evaluates the hemostasis effect of a material in real-world application scenarios by measuring the hemostasis time and blood loss at the injury site in a rat femoral artery injury model. Generally, the better the procoagulant effect of the material, the shorter the time required for hemostasis during treatment and the less blood loss at the injury site.

[0132] Comparative Example 6 removed step 4) of Example 3, directly obtaining proanthocyanidin-crosslinked gelatin microspheres without compounding the particles with poloxamer. The blood loss of X3 was 720.6 mg, with a hemostasis time of 1 min; the blood loss of Y6 was 1415.8 mg, with a hemostasis time of 6 min. Therefore, the blood loss of Y6 was significantly greater than that of X3, and the hemostasis time was significantly longer than that of X3.

[0133] Comparative Example 7 omitted step 4) of Example 1, directly obtaining chitosan sponge particles without compounding the particles with poloxamer. The blood loss of X1 was 848.8 mg, with a hemostasis time of 1.5 min; the blood loss of Y7 was 1852.3 mg, with a hemostasis time of 8 min. Therefore, the blood loss of Y7 was significantly greater than that of X1, and the hemostasis time was significantly longer than that of X1.

[0134] As can be seen from Tables 3 and 4, the thermosensitive hemostatic gel materials obtained in Examples 1 and 3 of the present invention showed lower blood loss and hemostasis time in rat femoral artery trauma hemostasis than those in Comparative Examples 6 and 7, indicating that the thermosensitive hemostatic gel prepared by the preparation method of the present invention has better therapeutic effect in the treatment of rat femoral artery injury model.

[0135] The analysis is as follows: When using only micron-sized polymer particles with hemostatic function for hemostasis, the blood loss during the hemostasis process is greater and the time required for hemostasis is longer. This is because during the wound hemostasis process, the flow of blood at the wound site washes away the added hemostatic particles, preventing them from achieving a good hemostatic effect. However, when the hemostatic particles are compounded with a thermosensitive polymer (poloxam), a solid gel is quickly formed on the wound surface, covering it and allowing the hemostatic particles to fully exert their hemostatic function.

[0136] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method of preparing a temperature-sensitive hemostatic gel, characterized in that, The temperature-sensitive polymer solution is mixed with a solution of micrometer-sized high-molecular particles or microspheres with hemostatic function to obtain the solution, the concentration of the solution of micrometer-sized high-molecular particles or microspheres with hemostatic function is 3-100 mg / mL; the concentration of the temperature-sensitive polymer solution is 200-450 mg / mL; the diameter of the micrometer-sized high-molecular particles or microspheres with hemostatic function is 300-900 micrometers; the temperature-sensitive hemostatic gel has a gelation temperature of greater than 15 degrees Celsius for changing from a flowing liquid state to a solidified state, and is in a flowing liquid state at a temperature of less than 15 degrees Celsius; under the condition of temperature rise in response to human body temperature, the flowing liquid state of the hemostatic gel changes to the solidified state of the hemostatic gel to realize the hemostatic function, and the micrometer-sized high-molecular particles or microspheres in the temperature-sensitive hemostatic gel do not enter the damaged blood vessels in the hemostatic process; the temperature-sensitive polymer solution is a poloxamer solution or a mixed solution of a poloxamer solution and a hydroxypropyl methylcellulose solution or a mixed solution of a poloxamer solution and a hydroxypropyl cellulose solution; the micrometer-sized high-molecular particles or microspheres with hemostatic function are calcium alginate microspheres, cross-linked gelatin microspheres or chitosan sponge particles; the solvent of the solution is water, physiological saline, phosphate buffer or glucose solution; when the micrometer-sized high-molecular particles or microspheres with hemostatic function are chitosan sponge particles, the concentration of the chitosan sponge particles in the temperature-sensitive polymer solution is 3-30 mg / mL; when the micrometer-sized high-molecular particles or microspheres with hemostatic function are calcium alginate microspheres, the concentration of the calcium alginate microspheres in the temperature-sensitive polymer solution is 30-100 mg / mL; when the micrometer-sized high-molecular particles or microspheres with hemostatic function are cross-linked gelatin microspheres, the concentration of the cross-linked gelatin microspheres in the temperature-sensitive polymer solution is 20-100 mg / mL, and the cross-linked gelatin microspheres are procyanidine cross-linked gelatin microspheres.

2. A process for the preparation of a temperature sensitive hemostatic gel according to claim 1, characterized in that, The preparation steps of the micrometer-sized high-molecular particles with hemostatic function are as follows: a) preparing a high-molecular aqueous solution with a concentration of 10-300 mg / mL, and then transferring the high-molecular aqueous solution to a large-size tray with a diameter greater than 5 cm to form a macro large-size high-molecular hemostatic sponge through freeze-drying; the preparation process further includes forming a cross-linked network to enhance the structural strength of the sponge through chemical or physical cross-linking during the preparation of the high-molecular solution or after the freeze-drying; b) cutting the large-size sponge into irregular particles through a pulverizer; c) obtaining particles with a diameter of 300-900 micrometers through sieving; the sieve is a 20-50 mesh sieve; According to the type of high-molecular, the aqueous solution in step a) is water, a diluted acetic acid / hydrochloric acid aqueous solution, a mixed solution of urea and sodium hydroxide, the cross-linking process is adding a chemical cross-linking agent, an ionic cross-linking agent or changing the pH of the sponge, the chemical cross-linking agent is a mixed solution of glutaraldehyde, tannic acid, procyanidine or epichlorohydrin and sodium hydroxide, and the ionic cross-linking agent is calcium chloride.

3. A process for the preparation of a temperature sensitive hemostatic gel according to claim 1, characterized in that, The preparation method of the micrometer-sized high-molecular microspheres with hemostatic function is as follows: 1) emulsion method: a) first preparing a high-molecular aqueous solution with a concentration of 10-300 mg / mL to form an aqueous phase; b) mixing the organic solvent with the surfactant to form an oil phase; adding the water phase into the oil phase, the volume ratio of the water phase to the oil phase is 1:5-1:20, the stirring speed is 400-2000 rpm, and the emulsification is performed for 20-120 min to form an emulsion; c) forming a cross-linked network by chemically or physically cross-linking the high molecular solution in the emulsion, and obtaining the microspheres by breaking the emulsion, precipitation washing, and drying; d) obtaining the particles with a diameter of 300-900 microns by sieving, wherein the sieve is a 20-50 mesh sieve; According to different types of high molecular, the water solution in step a) is water, diluted acetic acid / hydrochloric acid aqueous solution, or a mixed solution of urea and sodium hydroxide, and the cross-linking process in step c) is adding a chemical cross-linking agent, an ionic cross-linking agent, or changing the temperature or pH of the emulsion, wherein the chemical cross-linking agent is glutaraldehyde, epichlorohydrin / sodium hydroxide, tannic acid, or procyanidin, and the ionic cross-linking agent is calcium chloride. 2) Spray granulation method: a) preparing a high molecular water solution with a concentration of 10-300 mg / mL; b) adding the high molecular water solution into a high molecular solution spraying device, and spraying the bead string into a receiving liquid after the device sprays the high molecular solution to form a stable bead string, wherein the device is a microcapsule granulator or a high molecular electrostatic spraying instrument; c) forming a cross-linked network by chemically or physically cross-linking the high molecular solution in the receiving liquid, and obtaining the microspheres by centrifugation, washing, and drying; According to different types of high molecular, the water solution in step a) is water, diluted acetic acid / hydrochloric acid aqueous solution, or a mixed solution of urea and sodium hydroxide, and the cross-linking process in step c) is adding a chemical cross-linking agent, an ionic cross-linking agent, or changing the temperature or pH of the receiving liquid, wherein the chemical cross-linking agent is glutaraldehyde, epichlorohydrin / sodium hydroxide, tannic acid, or procyanidin, and the ionic cross-linking agent is calcium chloride. According to different devices for spraying the high molecular solution, the particles with a diameter of 300-900 microns are obtained by selecting different steps: before step b), the bead size is controlled by adding a nozzle or a nozzle matched with the instrument, the nozzle has a diameter of 150-700 microns, or no nozzle is selected, and after step c), the particles with a diameter of 300-900 microns are obtained by sieving, wherein the sieve is a 20-50 mesh sieve.

4. A temperature-sensitive hemostatic gel prepared by the preparation method according to any one of claims 1-3.

5. The use of the temperature-sensitive hemostatic gel according to claim 4 in the preparation of a blood coagulation product.

6. Use of a temperature-sensitive hemostatic gel according to claim 5 for the preparation of a procoagulant product, characterized in that, The blood coagulation product is applied to a damaged blood vessel at a wound from outside the blood vessel.

7. Use of a temperature-sensitive hemostatic gel according to claim 5 for the preparation of a procoagulant product, characterized in that, The blood coagulation product is applied to an embolized blood vessel at a complete blood vessel from inside the blood vessel.

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