A coagulant-loaded embolization particle and a method of making the same

Abstract

The application relates to the technical field of biological medicine, and particularly discloses embolism developing particles loaded with coagulation accelerators and a preparation method thereof. The embolism developing particles loaded with coagulation accelerators contain ortho-carboxy benzoylated gelatin, a developing agent and a coagulation accelerator; wherein the preparation method of the ortho-carboxy benzoylated gelatin is as follows: gelatin is dissolved in water to prepare a gelatin aqueous solution with a concentration of 3-5 wt%, and the pH of the gelatin aqueous solution is adjusted to 8.5-9.5; then, under the action of stirring, o-phthalic anhydride is added into the gelatin aqueous solution, and the reaction is carried out at 40-50 DEG C for 1-2 h to obtain a reaction liquid; the ortho-carboxy benzoylated gelatin is obtained through purification and freeze-drying; and the application further provides the preparation method of the above-mentioned embolism developing particles loaded with coagulation accelerators. The ortho-carboxy benzoylated gelatin can be used to prepare embolism developing microspheres with high porosity and good water absorption, and the embolism developing particles with high coagulation accelerator loading capacity can be obtained by mixing the ortho-carboxy benzoylated gelatin with the coagulation accelerator.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to an embolic imaging particle loaded with a coagulant and its preparation method. Background Technology

[0002] In the treatment of cancer or vascular bleeding, timely occlusion of the affected blood vessels is crucial. Embolization, also known as embolization therapy, involves the controlled injection of a plug into the blood vessel supplying the diseased organ through an arterial or venous catheter, causing it to close and interrupting blood supply. This aims to control bleeding, treat tumors and vascular lesions, and eliminate the function of the diseased organ.

[0003] Thrombin is a clotting factor that converts soluble fibrinogen in plasma into insoluble fibrin. In recent years, researchers have developed chemoembolic agents by loading thrombin onto embolic particles or microspheres. These agents are then injected into the blood supply to tumors, releasing thrombin at the lesion's blood vessels and creating an environment with an effective concentration of thrombin, thus achieving a dual hemostatic effect of physical embolization and drug coagulation. However, most commercially available thrombin-loaded embolic particles currently suffer from poor water absorption and low thrombin loading. Summary of the Invention

[0004] To improve the water absorption and loading capacity of thrombin-loaded embolic particles, this application provides embolic imaging particles loaded with a coagulant and a method for preparing the same.

[0005] In a first aspect, this application provides an embolic imaging particle loaded with a coagulant, employing the following technical solution: An embolic imaging particle loaded with a coagulant comprises o-carboxybenzoyl gelatin, a developer, and a coagulant.

[0006] This application uses o-carboxybenzoyl gelatin as a carrier to prepare embolic contrast particles loaded with a contrast agent and a coagulant. These embolic contrast particles have high water absorption and thrombin loading. When injected into blood vessels via a microcatheter, they solidify in the blood to form emboli. These emboli block blood vessels and cut off blood flow. Simultaneously, thrombin effectively assists in coagulation locally, creating thrombi and achieving embolization. Furthermore, these embolic contrast particles contain contrast agents, enabling clear visualization during surgery and providing early warning of ectopic embolism.

[0007] In this application, o-carboxybenzoylated gelatin is obtained by modifying gelatin with phthalic anhydride, causing the phthalic anhydride to undergo an acylation reaction with the amino groups on the gelatin molecular chain. This modification significantly increases the carboxyl content in the gelatin system. Using the aforementioned o-carboxybenzoylated gelatin to prepare embolic imaging particles significantly improves the water absorption of these particles.

[0008] Optionally, the o-carboxybenzoyl gelatin is prepared by dissolving gelatin in water to prepare a gelatin aqueous solution with a concentration of 3-5 wt%, and adjusting the pH of the gelatin aqueous solution to 8.5-9.5; then, under stirring, phthalic anhydride is added to the gelatin aqueous solution, and the reaction is carried out at 40-50℃ for 1-2 hours to obtain a reaction solution; after purification and freeze drying, o-carboxybenzoyl gelatin is obtained.

[0009] Optionally, the weight ratio of phthalic anhydride to gelatin is (3-7):100.

[0010] In some embodiments, the weight ratio of phthalic anhydride to gelatin can be (3-5):100 or (5-7):100.

[0011] In one specific implementation, the weight ratio of phthalic anhydride to gelatin can be 3:100, 5:100, or 7:100.

[0012] In this application, the weight ratio of phthalic anhydride to gelatin affects the hydrophilicity and water-locking properties of o-carboxybenzoyl gelatin. Experimental studies have shown that controlling the weight ratio of phthalic anhydride to gelatin within the above-mentioned range can yield o-carboxybenzoyl gelatin with excellent hydrophilicity and water-locking properties. Using the above-mentioned o-carboxybenzoyl gelatin to prepare embolization imaging particles, the obtained embolization imaging particles have good water absorption and coagulant loading. Their water absorption can reach 38.2-44.1 times their own weight, and their thrombin loading can reach 252-482 U / 100mg.

[0013] Optionally, the particle size of the embolic imaging particles loaded with the coagulant is 70-1300 μm.

[0014] Secondly, this application provides a method for preparing embolic imaging particles loaded with a coagulant.

[0015] A method for preparing embolic development particles loaded with a coagulant includes the following steps: preparing a gelatin developing solution, emulsifying and crosslinking, and loading a coagulant. Preparation of gelatin developing solution: Dissolve o-carboxybenzoylated gelatin in water to prepare an o-carboxybenzoylated gelatin solution with a concentration of 5-20 wt%; then add developer and stir evenly to obtain gelatin developing solution; Emulsification and crosslinking: The gelatin developing solution is mixed with an emulsifier and stirred evenly at a speed of 300-450 rpm. Then, a crosslinking agent is added to crosslink the gelatin. After washing, freeze-drying, and sieving, embolization developing microspheres are obtained. Loading coagulant: The embolization imaging microspheres are mixed with thrombin solution and then freeze-dried to obtain embolization imaging particles loaded with coagulant.

[0016] In the preparation method of the embolic imaging particles loaded with a coagulant in this application, a gelatin developing solution is used as the aqueous phase and an emulsifier as the oil phase. Mixing the aqueous and oil phases allows them to undergo reverse polymerization, forming a stable suspension. A crosslinking agent is then added to the suspension to form stable embolic imaging microspheres. Experimental studies in this application have shown that controlling the concentration of the o-carboxybenzoyl gelatin solution and the emulsification stirring rate within the aforementioned ranges yields embolic imaging microspheres with higher thrombin loading.

[0017] In some embodiments, the stirring speed can be 300-350 rpm, 300-400 rpm, 350-400 rpm, 350-450 rpm, or 400-450 rpm.

[0018] In one specific implementation, the stirring speed can also be 300 rpm, 350 rpm, 400 rpm or 450 rpm.

[0019] Optionally, the concentration of the phthalylated gelatin solution is 8-15 wt%.

[0020] In some embodiments, the concentration of the o-carboxybenzoyl gelatin solution may be 5-8 wt%, 5-10 wt%, 5-15 wt%, 8-10 wt%, 8-15 wt%, or 10-15 wt%.

[0021] In one specific embodiment, the concentration of the o-carboxybenzoyl gelatin solution may also be 5 wt%, 8 wt%, 10 wt%, or 15 wt%.

[0022] Optionally, the volume ratio of the gelatin developing solution to the emulsifier is 1:(8-15).

[0023] Optionally, the crosslinking agent is a 40-60 wt% aqueous solution of glutaraldehyde; the crosslinking reaction time is 2-10 h.

[0024] Optionally, the emulsifier may be paraffin oil or a high molecular weight organic solution; further, the high molecular weight organic acid may be a dichloromethane solution of polylactic acid.

[0025] Optionally, the developer is selected from one or more of metal particles, metal oxide particles, metal salts, and iodine-containing developers.

[0026] Optionally, the amount of developer added is 5-15 wt% of the gelatin solution.

[0027] Optionally, the concentration of the thrombin solution is 1200-1300 U / mL.

[0028] Optionally, the dosage relationship between the embolization imaging microspheres and the thrombin solution is as follows: 100g of embolization imaging microspheres are dissolved in 0.3-0.6mL of thrombin solution.

[0029] In summary, this application has the following beneficial effects: 1. This application uses o-carboxybenzoyl gelatin as a carrier to prepare an embolic imaging particle loaded with a coagulant through emulsification, cross-linking, and loading. Compared with embolic imaging particles in related technologies, the embolic imaging particle loaded with the coagulant has a larger porosity and water absorption rate and a higher thrombin loading capacity. It can be widely used for the treatment of diseases such as arteriovenous malformations, aneurysms, venous aneurysms, benign tumors, and malignant tumors by blocking or embolizing blood vessels to reduce blood flow or block blood supply.

[0030] 2. This application obtains o-carboxybenzoyl gelatin with excellent hydrophilicity and water-locking properties by controlling the weight ratio of phthalic anhydride to gelatin to be (3-7):100. Embolization imaging particles are prepared using the above-mentioned o-carboxybenzoyl gelatin. The obtained embolization imaging particles have good water absorption and coagulant loading. The water absorption can reach 38.2-44.1 times its own weight, and the thrombin loading can reach 252-482U / 100mg.

[0031] 3. In this application, by further controlling the concentration of the o-carboxybenzoyl gelatin solution within the range of 8-15wt%, and controlling the stirring speed in the emulsification and crosslinking step between 350-450rpm, the thrombin loading of the obtained embolization imaging particles can reach more than 400U / 100mg. Attached Figure Description

[0032] Figure 1 This is a microscopic image of the embolic particles loaded with coagulant obtained in Example 1. Detailed Implementation

[0033] This application provides embolic imaging particles loaded with a coagulant, the preparation method of which includes the following steps: (1) Preparation of gelatin developing solution: Dissolve o-carboxybenzoylated gelatin in purified water to prepare an o-carboxybenzoylated gelatin solution with a concentration of 5-10 wt%; then add 5-15 wt% developer to the gelatin solution and stir evenly to obtain gelatin developing solution.

[0034] The preparation method of o-carboxybenzoylated gelatin is as follows: gelatin (natural pigskin gelatin) is dissolved in water to prepare a gelatin aqueous solution with a concentration of 3-5 wt%, and the pH of the gelatin aqueous solution is adjusted to 8.5-9.5; then, phthalic anhydride is added to the gelatin aqueous solution under stirring, and the reaction is carried out at 40-50℃ for 1-2 hours to obtain a reaction solution; after purification and freeze-drying, o-carboxybenzoylated gelatin is obtained. The weight ratio of phthalic anhydride to gelatin is (3-7):100.

[0035] (2) Emulsification and crosslinking: Gelatin developing solution and paraffin oil are mixed at a volume ratio of 1:(8-13) and stirred evenly at a speed of 300-450 rpm; then, glutaraldehyde aqueous solution with a concentration of 40-60 wt% is added dropwise for crosslinking for 2-10 h; after crosslinking is completed, the embolization developing microspheres are washed with n-hexane and purified water; after freeze drying and sieving, embolization developing microspheres are obtained.

[0036] (3) Loading coagulant: First, prepare a thrombin solution with a concentration of 1200-1300 U / mL; then mix the embolization imaging microspheres obtained in step (2) with the thrombin solution, dissolving 100g of embolization imaging microspheres in 0.3-0.6mL of thrombin solution; after freeze drying, obtain embolization imaging particles loaded with coagulant.

[0037] In this application, the raw materials, reagents, solvents, etc., can all be obtained commercially.

[0038] The following detailed description of this application is provided in conjunction with preparation examples, embodiments, and performance testing experiments.

[0039] Preparation Examples 1-3 Preparation Examples 1-3 provide an o-carboxybenzoyl gelatin.

[0040] The difference in the above preparation examples is that the weight ratio of phthalic anhydride to gelatin in the preparation method of o-carboxybenzoyl gelatin is shown in Table 1 below.

[0041] The preparation method of the above-mentioned o-carboxybenzoyl gelatin is as follows: gelatin (natural pigskin gelatin, purchased from Wuhan Jucan Biotechnology Co., Ltd.) is dissolved in water to prepare a gelatin aqueous solution with a concentration of 4wt%, and the pH of the gelatin aqueous solution is adjusted to 8.5; then, under stirring, phthalic anhydride is added to the gelatin aqueous solution, and the reaction is carried out at 40℃ for 1 hour to obtain the reaction solution; after purification and freeze drying, o-carboxybenzoyl gelatin is obtained.

[0042] Comparative preparation examples 1-2 Preparation Examples 1-2 provide an o-carboxybenzoyl gelatin.

[0043] The difference in the above preparation examples is that the weight ratio of phthalic anhydride to gelatin in the preparation method of o-carboxybenzoyl gelatin is shown in Table 1 below.

[0044] Table 1 shows the amount and weight ratio of phthalic anhydride and gelatin in Preparation Examples 1-3 and Comparative Preparation Examples 1-2. Examples 1-3 Examples 1-3 provide embolic imaging particles loaded with a coagulant.

[0045] The difference between the above embodiments is that in the embolization particles loaded with coagulants in Examples 1-3, the o-carboxybenzoyl gelatin was derived from Preparation Examples 1-3, respectively.

[0046] The preparation method of the embolic imaging particles loaded with coagulant provided in Examples 1-3 is as follows: (1) Preparation of gelatin developing solution: Dissolve o-carboxybenzoylated gelatin in purified water to prepare an o-carboxybenzoylated gelatin solution with a concentration of 8wt%; then add 10wt% of iron oxide nanoparticles (purchased from Xi'an Qiyue Biotechnology Co., Ltd.) to the gelatin solution, stir evenly, and obtain gelatin developing solution.

[0047] (2) Emulsification and crosslinking: Gelatin developing solution and paraffin oil were mixed at a volume ratio of 1:10 and stirred evenly at a speed of 350 rpm; then 50 wt% glutaraldehyde aqueous solution was added dropwise for crosslinking for 5 h; after crosslinking was completed, the embolization developing microspheres were washed with n-hexane and purified water; after freeze drying and sieving, embolization developing microspheres were obtained.

[0048] (3) Loading the coagulant: First, prepare a thrombin solution with a concentration of 1250 U / mL; then take 100 mg of the embolization imaging microspheres obtained in step (2) and mix them with 0.4 mL of the thrombin solution; after freeze drying, obtain the embolization imaging particles loaded with the coagulant.

[0049] Examples 4-7 Examples 4-7 each provide an embolic imaging particle loaded with a coagulant.

[0050] The difference between the above embodiments and Embodiment 2 is that: (2) the concentration of the ortho-carboxybenzoyl gelatin solution in the emulsification and crosslinking step is shown in Table 2 below.

[0051] Table 2 Concentrations of o-carboxybenzoyl gelatin solutions in Examples 2 and 4-7 2 8 4 5 5 10 6 15 7 20 Examples 8-10 Examples 8-10 each provide an embolic imaging particle loaded with a coagulant.

[0052] The difference between the above embodiment and embodiment 2 is that: (2) the stirring speed in the emulsification and crosslinking step is shown in Table 3 below.

[0053] Table 3. Stirring speeds for the emulsification and crosslinking steps in Examples 2 and 8-10. 2 350 8 300 9 400 10 450 Comparative Examples 1-2 Comparative Examples 1 and 2 each provide an embolic imaging particle loaded with a coagulant.

[0054] The difference between the above comparative examples and Example 2 is that in the embolization particles loaded with coagulants in Comparative Examples 1-2, the o-carboxybenzoyl gelatin was derived from Comparative Preparation Examples 1-2.

[0055] Comparative Example 3 Comparative Example 3 provides an embolic imaging particle loaded with a coagulant.

[0056] The difference between the above comparative example and Example 2 is that the o-carboxybenzoyl gelatin was replaced with gelatin (purchased from Shanxi Jinyang Pharmaceutical Excipients Co., Ltd.).

[0057] Comparative Example 4 Comparative Example 4 provides an embolic imaging particle loaded with a coagulant.

[0058] The difference between the above comparative example and Example 2 is that the o-carboxybenzoyl gelatin was replaced with methacrylated gelatin (purchased from Wuhan Kanos Technology Co., Ltd.).

[0059] Comparative Example 5 Comparative Example 5 provides an embolic imaging particle loaded with a coagulant.

[0060] The difference between the above comparative example and Example 2 is that the o-carboxybenzoyl gelatin is replaced with succinic anhydride modified gelatin.

[0061] The preparation method of the above-mentioned succinic anhydride modified gelatin is as follows: 30g of succinic anhydride is dissolved in 1L of tetrahydrofuran solvent, 300g of gelatin particles are suspended in the above solution, and 0.1g of nicotinamide and 0.1g of polyethylene glycol are added. The reaction temperature is controlled at 50℃, and the mixture is ground and reacted in a colloid mill for 4h. After filtration, succinic anhydride modified gelatin is obtained.

[0062] Performance testing The morphology, thrombin loading, and in vitro coagulation time of the embolic particles loaded with procoagulants obtained in Examples 1-10 and Comparative Examples 1-4 were tested, and the results are shown in Table 4 below.

[0063] (1) Observe the morphology of the embolic particles loaded with coagulant under a microscope and obtain the particle size.

[0064] The morphology of the embolic particles loaded with coagulant in Example 1 under a microscope is as follows: Figure 1 As shown, by Figure 1 It can be seen that the embolization particles loaded with the coagulant are uniform in size and structure, with a particle size range of 70-200μm.

[0065] (2) Method for detecting porosity: The porosity of embolization microspheres (without thrombin loading) was detected by mercury porosimetry.

[0066] (3) Water absorption ratio: Accurately weigh M1 = 100 mg of embolic contrast microspheres (unloaded with thrombin), immerse them in a beaker containing water at 20±1℃, stir until the microspheres are completely wetted and all air is expelled, and after absorbing enough water; filter out the excess water in the beaker, weigh the embolic contrast microspheres again after water absorption, record it as M2, and calculate the water absorption ratio A. The calculation formula is as follows: A = (M2 - M1) / M1.

[0067] (4) Thrombin loading: The mixed solution of embolization imaging microspheres and thrombin in step (3) of each example and comparative example was allowed to stand to allow physical adsorption; then the concentration C1 of thrombin in the solution was detected 4 hours after mixing, and the thrombin loading Q was calculated using the following formula: Q = (C0 - C1) × 0.1 Table 4. Detection results of embolic particles in Examples 1-10 and Comparative Examples 1-4 with loaded coagulants. According to the test results in Table 4, the embolic contrast microspheres obtained in Examples 1-10 of this application have a porosity of over 90% and a water absorption capacity of 38.2-44.1 times their own weight, with 100 mg capable of loading 252-482 U of thrombin. In contrast, the embolic contrast microspheres obtained in Comparative Example 3 (using gelatin), Comparative Example 4 (using methacrylated gelatin), and Comparative Example 5 (using succinic anhydride-modified gelatin) have a thrombin loading of only 92-166 U / 100 mg. Therefore, this application demonstrates that embolic contrast microspheres with high porosity and water absorption can be prepared using o-carboxybenzoyl gelatin, thereby obtaining embolic contrast particles with high thrombin loading.

[0068] The test results of Examples 1-3 and Comparative Examples 1-2 show that with the increase of phthalic anhydride added in the o-carboxybenzoyl gelatin preparation method, the porosity of the embolization imaging microspheres does not change significantly, while the water absorption rate shows a clear trend of first increasing and then remaining basically unchanged, and the thrombin loading shows a trend of first increasing and then decreasing. The thrombin loading of the embolization imaging microspheres obtained in Examples 1-3 is 263-406 U / 100 mg, while the thrombin loading of the embolization imaging microspheres obtained in Comparative Examples 1-2 is only 108-150 U / 100 mg. Therefore, it is shown that by controlling the weight ratio of phthalic anhydride to gelatin within the range of (3-7):100, this application can obtain embolization imaging particles with suitable porosity, excellent hydrophilicity, and high thrombin loading.

[0069] The test results of Examples 2 and 4-7 show that as the concentration of the o-carboxybenzoyl gelatin solution increases, the porosity of the embolization-contrast microspheres gradually decreases, while the water absorption rate gradually increases, and the thrombin loading shows a trend of first increasing and then decreasing. Further comparison revealed that the thrombin loading of the embolization-contrast microspheres obtained in Examples 2 and 5-6 is >400 U / 100 mg, while the thrombin loading of the embolization-contrast microspheres obtained in Examples 4 and 7 is <400 U / 100 mg. Therefore, this application demonstrates that by further controlling the concentration of the o-carboxybenzoyl gelatin solution within the range of 8-15 wt%, embolization-contrast particles with high thrombin loading can be obtained.

[0070] The test results of Examples 2 and 8-10 show that as the stirring speed in the emulsification and crosslinking step increases, the porosity of the embolization-contrast microspheres remains basically unchanged, while the water absorption rate gradually increases, and the thrombin loading first increases and then decreases. In particular, the thrombin loading of the embolization-contrast microspheres obtained in Examples 2 and 9-10 can reach over 400 U / 100 mg. Therefore, this application demonstrates that by controlling the stirring speed in the emulsification and crosslinking step between 350-450 rpm, embolization-contrast particles with higher thrombin loading can be obtained.

[0071] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An embolic imaging particle loaded with a coagulant, characterized in that, The embolic imaging particles loaded with a coagulant contain o-carboxybenzoyl gelatin, a developer, and a coagulant. The preparation method of the o-carboxybenzoylated gelatin is as follows: gelatin is dissolved in water to prepare a gelatin aqueous solution with a concentration of 3-5 wt%, and the pH of the gelatin aqueous solution is adjusted to 8.5-9.5; then, under stirring, phthalic anhydride is added to the gelatin aqueous solution, and the reaction is carried out at 40-50℃ for 1-2 hours to obtain a reaction solution; after purification and freeze drying, o-carboxybenzoylated gelatin is obtained. The weight ratio of phthalic anhydride to gelatin is (3-7):

100. The coagulant is thrombin.

2. The embolic imaging particles loaded with a coagulant according to claim 1, characterized in that, The particle size of the embolic imaging particles loaded with the coagulant is 70-1300 μm.

3. The method for preparing embolic imaging particles loaded with a coagulant as described in any one of claims 1-2, characterized in that, Includes the following steps: Preparation of gelatin developing solution, emulsification and cross-linking, and loading of coagulant accelerator; Preparation of gelatin developing solution: Dissolve o-carboxybenzoylated gelatin in water to prepare an o-carboxybenzoylated gelatin solution with a concentration of 5-20 wt%; then add developer and stir evenly to obtain gelatin developing solution; Emulsification and crosslinking: Gelatin developing solution and emulsifier are mixed and stirred evenly at a speed of 300-450 rpm, and then crosslinking agent is added for crosslinking. After washing, freeze drying and sieving, embolization developing microspheres are obtained; the emulsifier is paraffin oil. Loading coagulant: The embolization imaging microspheres are mixed with thrombin solution and then freeze-dried to obtain embolization imaging particles loaded with coagulant.

4. The method for preparing embolic imaging particles loaded with a coagulant according to claim 3, characterized in that, The concentration of the o-carboxybenzoyl gelatin solution is 8-15 wt%.

5. The method for preparing embolic imaging particles loaded with a coagulant according to claim 3, characterized in that, The volume ratio of the gelatin developing solution to the emulsifier is 1:(8-15).

6. The method for preparing embolic imaging particles loaded with a coagulant according to claim 3, characterized in that, The crosslinking agent is a 40-60 wt% glutaraldehyde aqueous solution; the crosslinking reaction time is 2-10 h.

7. The method for preparing embolic imaging particles loaded with a coagulant according to claim 3, characterized in that, The concentration of the thrombin solution is 1200-1300 U / mL.

8. The method for preparing embolic imaging particles loaded with a coagulant according to claim 7, characterized in that, The dosage relationship between the embolization imaging microspheres and the thrombin solution is as follows: 100 mg of embolization imaging microspheres are dissolved in 0.4 mL of thrombin solution.

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