Coagulant-loaded embolization microsphere as well as preparation method and application thereof

Abstract

The invention discloses a coagulant-loaded embolism microsphere as well as a preparation method and application thereof, the embolism microsphere is prepared through microfluidic and free radical polymerization technologies, and the coagulant-loaded embolism microsphere has a wide application prospect in the fields of arterial embolism treatment, tumor vascular infarction and the like. The embolism microspheres have the characteristics of uniform size, efficient entrapment and continuous release of the coagulant, and can effectively promote fibrinogen to generate fibrin and accelerate coagulation of rabbit blood. Besides, the thrombin-loaded embolism microspheres can obviously induce tumor artery occlusion, and obvious blood clots are formed in far-end blood vessels, so that the growth of rabbit in-situ VX2 tumors is effectively inhibited. The embolism microsphere carrying the coagulant has the advantages of being high in safety, easy and convenient to prepare, good in embolism effect and the like, and efficient entrapment and controllable release of thrombin can be achieved. In the aspects of liver cancer transarterial embolism and tumor vascular infarction treatment, the technology has great transformation potential and is expected to promote the development of vascular embolism agents.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of embolization microspheres of procoagulant, a kind of preparation method and application of procoagulant loaded embolization microspheres, specifically to a kind of preparation method of procoagulant loaded embolization microspheres, and the application of the microsphere in the field of liver cancer intervention embolization. BACKGROUND

[0002] Transarterial embolization (TAE) and transarterial chemoembolization (TACE) as minimally invasive interventional therapy have been widely used in the treatment of advanced hepatocellular carcinoma. The principle is to guide the catheter to the tumor feeding artery in the human body from the femoral artery under the guidance of high-definition medical imaging equipment, and inject embolic agents and drugs to block the blood supply of tumor tissue, so as to achieve tumor necrosis. However, the current microsphere embolic agents (such as Embosphere, CalliSpheres and DC Bead, etc.) are difficult to enter the distal and small tumor vessels due to their large size. Although liquid embolic agents (such as iodine oil) have good fluidity and can enter small blood vessels, they are easily washed away by blood and are easily removed, and the embolic effect is not good. Therefore, it is urgent to develop an embolic agent that can block the main, distal and small vessels of the tumor at the same time to enhance the therapeutic effect of embolization therapy. Similar to TAE, tumor vascular occlusion therapy is also an attractive cancer treatment method, which blocks the blood supply of tumors by destroying tumor blood vessels or inducing blood clot formation, and is a promising cancer treatment method (Guelfi, S. Nat. Rev. Cancer 2024, 24, 655-675). Since the publication of the pioneering study in 1997, a variety of blood procoagulants based on truncated human tissue factor, thrombin, etc. have been widely used in tumor vascular embolization therapy (Nie, C. ACS Nano 2023, 17, 13211-13223). However, non-specific delivery of procoagulants often leads to poor treatment effect and serious damage to normal organs. Here, we introduce thrombin into 3Asphere to enhance the occlusion of distal and small tumor vessels. Therefore, the combination of embolization therapy and vascular occlusion therapy by loading procoagulant on microspheres has great potential to completely block the entire tumor vascular system by combining the advantages of both, thereby enhancing the therapeutic effect of cancer. SUMMARY

[0003] The purpose of the present application is to provide a preparation method of procoagulant-loaded embolization microspheres, which has good application prospects in the field of vascular embolization.

[0004] To achieve the above-mentioned purpose of the application, the technical solution adopted by the present application is: A preparation method of procoagulant-loaded embolization microspheres, comprising the following steps: mixing microspheres with procoagulant to obtain procoagulant-loaded embolization microspheres.

[0005] In the application, macromolecules are reacted with acrylic compounds in solution to obtain macromolecules with crosslinking groups; water phase material containing macromolecules with crosslinking groups and oil phase material are crosslinked and solidified by free radical polymerization through microchannel pipeline to obtain microspheres; the microchannel pipeline includes cross type, Y type, T type or flow focusing type microchannel pipeline.

[0006] In the application, macromolecules are reacted with acrylic compounds in solution to obtain macromolecules with crosslinking groups in the presence of a catalyst.

[0007] In the application, the macromolecules include hyaluronic acid, heparin, alginic acid and the like; the coagulation accelerator includes thrombin, hemocoagulase, protamine, blood coagulation factor and the like.

[0008] In the application, the water phase material includes macromolecules with crosslinking groups, buffer and initiator, and the concentration of the macromolecules with crosslinking groups is 10-100 mg / mL; the mass ratio of the macromolecules with crosslinking groups, buffer and initiator is 100:2000-5000:8-50.

[0009] In the application, the oil phase material includes surfactant and oil solvent.

[0010] In the application, the flow rate of the oil phase material is 10-50 μL / min, and the flow rate of the water phase material is 2-10 μL / min. In the application, the solidification includes ultraviolet light solidification and thermal solidification. The application discloses a coagulation accelerator carrying embolism microsphere prepared by the preparation method of the coagulation accelerator carrying embolism microsphere.

[0011] In the application, the microspheres are mixed with the coagulation accelerator in a solvent, the concentration of macromolecules of the microspheres is 5-100 mg / mL, and the concentration of the coagulation accelerator is 0.1-5 μg / mL; the solvent includes water, and the mixing is conventional oscillation mixing, which is a conventional technology.

[0012] The application discloses a medicine including the coagulation accelerator carrying embolism microsphere.

[0013] The application discloses an application of the coagulation accelerator carrying embolism microsphere in preparation of a medicine.

[0014] The application discloses an application of the coagulation accelerator carrying embolism microsphere in preparation of an antitumor medicine or a vascular embolism agent.

[0015] Compared with the prior art, the preparation method of the thromboplastin-loaded embolism microspheres has the advantages that the preparation process is simple, the material synthesis is simple, the cost is low, the thromboplastin can be well loaded and the release thereof can be controlled. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a nuclear magnetic resonance hydrogen spectrum of the hyaluronic acid methacrylate derivative (HA-AMA) in Example 1.

[0017] Figure 2 is a microscopic image of the hyaluronic acid embolism microspheres (3Asphere) in Example 1, the scale is 100 μm.

[0018] Figure 3 is a mechanical function characterization of the 3Asphere in Example 1, wherein a is a viscosity characterization, b is a modulus characterization, and c is a compression deformation curve.

[0019] Figure 4 is a loading and release characterization of the 3Asphere for thrombin in Example 2, wherein a is a thrombin loading time curve, b is a drug loading amount and drug loading efficiency, c is a change in the size and size distribution of the microspheres under different thrombin drug loading amounts, d is a morphology and size distribution of Th-3Asphere, and e is an in-vitro release of Th-3Asphere.

[0020] Figure 5 is a loading characterization of a commercial microsphere for thrombin in Example 2.

[0021] Figure 6 is a fibrin formation characterization of Th-3Asphere in Example 3, wherein a is a fibrin gel photograph, and b is an ultraviolet curve after reaction of each group.

[0022] Figure 7 is an insoluble protein fluorescence picture caused by Th-3Asphere in Example 3, the scale is 100 μm.

[0023] Figure 8 is an in-vitro rabbit blood coagulation characterization of Th-3Asphere in Example 3, wherein a is a coagulation photograph, and b is a hemoglobin absorbance of supernatant of different groups.

[0024] Figure 9 is a cell compatibility characterization of Th-3Asphere in Example 4, wherein a is L929 cells, b is Hepa1-6 cells, and c is HepG2 cells.

[0025] Figure 10 is the live and dead cell staining characterization of Th-3A sphere in Example Four, wherein a is Hepa1-6 cell; b is HepG2 cell.

[0026] Figure 11 is the embolization treatment characterization of Th-3A sphere in Example Five, wherein the image is a three-dimensional reconstruction image of CT.

[0027] Figure 12 is the tumor volume change of each group of rabbits in Example Five.

[0028] Figure 13 is the tumor blood coagulation related H&E staining image of each group of tumor sites in Example Five, scale: 100 μm.

[0029] Figure 14 is the H&E (a), TUNEL (b), CD31 (c) staining image of each group of tumor sites in Example Five.

[0030] Figure 15 is the body weight change of each group of rabbits in Example Five.

[0031] Figure 16 is the H&E staining section image of heart, liver, spleen, lung and kidney of each group in Example Five.

[0032] Figure 17 is the liver and kidney function change of each group of rabbits in Example Five.

[0033] Figure 18 is the coagulation function of each group of rabbits in Example Five.

[0034] Figure 19 is the structure schematic diagram of the microfluidic chip in Example One. DETAILED DESCRIPTION

[0035] In the prior art, commercially available Embosphere and CallisSpheres microspheres are two products currently considered to have good performance in clinical application, but their elasticity and drug loading performance, especially protein loading capacity, still need to be improved. The application discloses hyaluronic acid embolization microspheres loaded with thrombin, and a preparation method thereof, which comprises the following steps: Hyaluronic acid is added to a flask containing deionized water, and after being dissolved, 2-aminoethyl methacrylate is added, and after stirring, a catalyst 4-(4, 6-dimethoxytriazin-2-yl)-4-methyl morpholine hydrochloride is added, and the stirring reaction is continued for 20-28 hours, then the solution after reaction is dialyzed, and after freeze-drying, hyaluronic acid derivative (HA-AMA) is obtained; The HA-AMA and photoinitiator 2959 are dissolved in a phosphate buffer (PB) as the water phase material, and Span 80 is dissolved in mineral oil as the oil phase material; the water phase material and the oil phase material are injected into the microchannel of the microfluidic chip by a constant pressure pump, and the water phase material is sheared into microdroplets under the shearing force of the oil phase material; then the microspheres are prepared by free radical polymerization and cross-linking curing under ultraviolet light irradiation; after the microspheres are cured, isopropyl alcohol, n-hexane and deionized water are used for washing in sequence, and finally the hyaluronic acid embolization microspheres are obtained.

[0036] The thrombin is mixed with the microspheres in a solvent, wherein the concentration of the thrombin is 0.1-1 mg / mL, and the concentration of the microspheres is 5-15 mg / mL; then the mixture is uniformly shaken to obtain the hyaluronic acid embolization microspheres loaded with thrombin.

[0037] In the above technical solution, the mass ratio of the hyaluronic acid derivative, the phosphate buffer and the initiator in the water phase material is 100:2500-5000:10-20. The mass ratio of Span 80 and mineral oil in the oil phase material is 5-10:100; further, the mass ratio of Span 80 and mineral oil in the oil phase material is 6-10:100.

[0038] In the above technical solution, the microchannel pipeline is a flow focusing type microchannel pipeline, and the depth of the microchannel is 50-150 μm, which is a conventional technology.

[0039] In the above technical solution, the wavelength of the ultraviolet light is 365 nm, the cross-linking curing time is 10-20 min, and the intensity of the ultraviolet surface light source is 20-40 mW / cm 2 .

[0040] In the above technical solution, the flow rate of the oil phase is 10-50 μL / min, and the flow rate of the water phase is 2-10 μL / min.

[0041] In the above technical solution, the concentration of the microspheres is 10-20 mg / mL, and the concentration of the thrombin is 0.1-1 mg / mL.

[0042] The hyaluronic acid embolization microspheres prepared by the microfluidic technology in the application have regular shape and uniform size, and can realize rapid and efficient loading of thrombin and slow release in vitro. The hyaluronic acid embolization microspheres loaded with thrombin in the application can be used as or prepared into a vascular embolism agent and a vascular infarction agent.

[0043] Further, the application further discloses the application of the hyaluronic acid embolization microspheres loaded with thrombin in the preparation of a liver cancer embolization therapeutic agent.

[0044] The application discloses a preparation method of thrombin-loaded hyaluronic acid embolism microspheres and application thereof, and further describes the application by combining with drawings and examples. Raw materials used are all existing products, and specific preparation operation and performance test are all conventional technologies. Animal experiments meet relevant requirements of Suzhou University, and data statistical processing is existing technology.

[0045] Example 1: Preparation and characterization of hyaluronic acid embolism microspheres (3A sphere) Under a nitrogen atmosphere, 5 g of hyaluronic acid is dissolved in a mixture of 200 mL of deionized water and 50 mL of dimethyl sulfoxide, and stirred uniformly at 37°C; then 0.33 g, 0.66 g, 1.03 g or 1.21 g of functionalized small molecule 2-aminomethyl acrylate is added, respectively, and stirring is continued for 10 minutes, and then 0.82 g, 1.64 g, 2.57 g or 3.01 g of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride is added, respectively, and stirring is continued for 24 hours; then the reaction crude product is purified by dialysis using deionized water, and after conventional freeze-drying, a functionalized hyaluronic acid derivative (HA-AMA) is obtained, with a yield of 88.3%; the nuclear magnetic resonance characterization of HAMA (with a degree of substitution of 22.0) is shown in the following table Figure 1 , 1 H NMR (D2O / DMSO-d6): HA: δ 1.91, 3.15-4.10, 4.33-4.65; AMA: δ 1.99, 5.71, 6.14. The degree of substitution of the methacrylate group (the number of methacrylate groups in 100 sugar units) can be obtained by calculating the peak area of the signal peaks, and is 10.8, 16.5, 22 and 24.8, respectively.

[0046] 40-100 mg of hyaluronic acid derivative (HA-AMA, with a degree of substitution of 22.0) and 8-20 mg of photoinitiator I2959 are dissolved in 2 mL of PB (10 mM, pH 8.5) as an aqueous phase of microfluidics, and 5-10 wt.% of Span 80 is dissolved in mineral oil as an oil phase of microfluidics; then the aqueous phase and the oil phase are injected into a flow focusing type microfluidic chip (the chip depth is 100 microns, and the corresponding width is 210 microns) through a constant pressure pump, the water phase flow rate is adjusted to 1 μL / min, and the water / oil phase flow rate ratio is 1:3-1:5. Uniform water-in-oil droplets are formed through microfluidics, and the droplets are irradiated with ultraviolet light (30 mM / cm 2Microspheres were obtained by cross-linking and solidifying droplets under irradiation, followed by washing with isopropanol, n-hexane, and PB sequentially to finally obtain hyaluronic acid embolic microspheres (3Aspheres) in solution form. 90-micrometer 3Aspheres were successfully prepared using a polymer concentration of 40 mg / mL, an initiator concentration of 6 mg / mL, mineral oil containing 10 wt.% Span 80, and an oil-to-water ratio of 1:4. The uniformity of the 3Aspheres was observed using an inverted fluorescence microscope; the 3Aspheres exhibited a regular spherical shape and uniform size. Figure 2 Its CV is 1.93%.

[0047] Flow-focusing microfluidic chips are standard products; their structure is shown below. Figure 19 The microchannels have a rectangular structure with a depth of 70 μm and a width that is twice the depth plus 10 micrometers.

[0048] like Figure 3 As shown in Figure a, the viscosity of 3Asphere decreases significantly with increasing shear rate, exhibiting typical shear-thinning behavior. Compared to commercial microspheres Embosphere and CalliSpheres, 3Asphere exhibits lower viscosity (20-1400 mPa∙s) across different shear rate ranges, a characteristic that makes it more suitable for catheter delivery. Scanning amplitude experiments show that the maximum storage modulus of 3Asphere is approximately 90-100 Pa, while the maximum storage moduli of clinical microspheres Embosphere and CalliSpheres are approximately 1100-1200 Pa and 160-180 Pa, respectively. Figure 3 b). Furthermore, 3Asphere, Embosphere, and CalliSpheres all exhibit quasi-elastic deformation behavior. However, 3Asphere requires only about 0.8 N of compressive force to achieve 50% deformation, while Embosphere and CalliSpheres only achieve 30% and 36%, respectively. Figure 3 c). Notably, Embosphere and CalliSpheres require compressive forces of 1.8 N and 1.4 N, respectively, to compress the microspheres to 50%. These data suggest that 3Asphere, with its lower modulus and smaller compressive force, achieves large deformations and thus exhibits better elasticity.

[0049] Example 2: Preparation and in vitro release study of thrombin-loaded 3Asphere (Th-3Asphere) 0.5 mL of 3Asphere (10 mg / mL) and 0.5 mL of thrombin aqueous solutions of different concentrations (0.25, 0.53, 0.81 μg / mL, corresponding to theoretical drug loadings of 2.5, 5.0, 7.5 wt.%) were incubated in a constant temperature shaker at 25 °C and 1000 rpm to obtain Th-3Asphere. At predetermined time points (2, 5, 10, 20, 40, and 60 minutes), 20 μL of the supernatant was collected, and the thrombin concentration was quantitatively analyzed using a MicroBCA kit.

[0050] Drug loading efficiency (DLE) and drug loading capacity (DLC) are calculated using the following formula: DLC (wt.%) = (mass of loaded thrombin / mass of 3Asphere + mass of added thrombin) × 100; DLE (%) = (mass of thrombin loaded / mass of thrombin added) × 100.

[0051] In vitro thrombin release was performed using a Transwell system (8 μm pores). 0.3 mL of 3Asphere (500 μg thrombin loaded) was placed in the upper chamber, and 1.0 mL of phosphate-buffered saline (PFS) at different pH values ​​(6.5 and 7.4) was placed in the lower chamber. At predetermined time points, all release media from the lower chamber was removed and fresh media was added. The mass of the released thrombin was measured using a MicroBCA kit.

[0052] Th-3Asphere was prepared by adding thrombin solution to 3Asphere. At a theoretical drug loading of 2.5-7.5 wt.%, 3Asphere exhibited rapid and high thrombin loading, with a loading efficiency of approximately 90%. Figure 4 a, b). In this invention, unless otherwise specified, Th-3Asphere prepared by incubation for 60 minutes was used for experiments. The particle size and particle size distribution of Th-3Asphere did not differ significantly under different drug loading levels. Figure 4 c, d). Th-3Asphere continuously releases thrombin for up to 5 days under PBS release conditions. Figure 4 e).

[0053] Comparison Example Referring to Example 2, commercially available Embosphere and CalliSpheres microspheres were used instead of 3Asphere microspheres for thrombin loading experiments. The experimental results showed that when thrombin concentrations were 0.25, 0.53, and 0.81 μg / mL, the loading efficiency of both types of microspheres for thrombin was low, all less than 40%. Figure 5 ).

[0054] Example Three In vitro procoagulant characterization of Th-3Asphere Equal amount of Th (10 U / mL) and Th-3Asphere (3Asphere loaded with 5 wt.% Th) solutions were mixed with fibrinogen solution (10 mg / mL). After incubation at 37 °C for 10 min, the transmittance of each group was measured by UV spectrophotometer. In addition, fibrinogen was labeled with FITC and then added into Th and Th-3Asphere, and the formation of insoluble fibrin was observed by inverted fluorescence microscope.

[0055] Further, rabbit blood was taken from the rabbit periphery into an anticoagulant tube. 250 μL of 0.9% NaCl, Thrombin, 3Asphere and Th-3Asphere (3Asphere loaded with 5 wt.% Th) were added into 250 μL of fresh anticoagulant rabbit whole blood, and the concentration of thrombin was 10 U / mL. After incubation at 37 °C for 10 min, the blood clot photos of each group were taken. Then 5 mL of deionized water was added to each group to lyse the red blood cells, and the absorbance of the supernatant at 545 nm was measured by UV spectrophotometer to evaluate the stability of the blood clots of each group.

[0056] Similar to the fibrinogen conversion to fibrin by free thrombin, Th-3Asphere also induced fibrin gel formation, solution opacity and green insoluble fibrin by free thrombin (Fig. 6). Figure 6 In addition, after the addition of Th-3Asphere into the anticoagulant whole blood, obvious blood clots were produced, the absorbance of hemoglobin in the supernatant of the blood clots dispersed in water was low, and the stability of the blood clots was good (Fig. 7). Figure 8

[0057] Example Four Cell compatibility of Th-3Asphere Th-3Asphere (3Asphere loaded with 5 wt.% Th) prepared in Example Two was used to test the cell compatibility of microspheres and thrombin-loaded microspheres. First, fibroblasts (L929) and hepatoma cells (Hepa1-6 and HepG2) were respectively plated on 96-well cell culture plates, and the cell density was 3000 cells per well. After incubation at 37 °C, 5% carbon dioxide for 12 hours, 10 μL of different concentrations of Th and Th-3Asphere were added to each well. After incubation at 37 °C for 48 hours, 10 μL of CCK8 solution was added to each well and incubated for another 2 hours. Finally, the absorbance of each sample at 450 nm was measured by using a microplate reader. The relative survival rate of cells was obtained by comparing the absorbance at 450 nm of the control wells with only blank cells, and each experiment was repeated 5 times on average.​

[0058] Further, the cell compatibility of Th-3Asphere was evaluated by live / dead staining. Hepa1-6 and HepG2 cells were seeded into 24-well plates at a density of 20,000 cells per well and incubated at 37 °C for 12 h. Then, 200 μg / mL of 3Asphere and Th-3Asphere with 10 U / mL of thrombin were added to the culture medium and the cells were incubated at 37 °C for 48 h. Finally, the cells were washed and stained with Calcein-AM / PI cell double staining kit for live / dead cell staining.

[0059] The results showed that the survival rates of healthy cells (L929) and tumor cells (Hepa1-6 and HepG2) were all above 95% after incubation with Th-3Asphere for 48 h. Figure 9 The results of live / dead cell staining showed that almost all the cells incubated with 3Asphere or Th-3Asphere were stained with Calcein-AM (green fluorescence), which confirmed their good cell compatibility. Figure 10

[0060] Example Five In vivo Liver Cancer Treatment Study of Th-3Asphere (3Asphere loaded with 5 wt.% Th) According to the conventional method, a rabbit VX2 orthotopic liver cancer model was established by implanting VX2 (rabbit anaplastic epidermal squamous carcinoma strain) tumor tissue into the left lobe of the liver of rabbits under the guidance of CT imaging. Two weeks later, abdominal CT imaging of the rabbit was performed again to determine the location of the liver tumor and the size of the tumor volume. Then the rabbits with a tumor longest diameter of 0.7-1.5 cm were randomly divided into 3 groups, 3 rabbits in each group, and treated as follows: (1) Control; (2) 3Asphere (200 μL); (3) Th-3Asphere (200 μL, Th: 100 U / rabbit). After treatment, the tumor growth was monitored by CT imaging. The tumor volume was calculated according to the maximum direct (a) and minimum direct (b) obtained by CT imaging, as follows: Tumor volume (cm 3 ) = (a × b 2 ​) / 2. In addition, at certain time points, peripheral blood of rabbits was collected in coagulation tubes, and serum was obtained by centrifugation. Biochemical analyzer was used to detect liver and kidney function indexes of rabbits, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin (ALB), total bilirubin (TBIL), urea (UREA) and creatinine (CREA). At 14 days after operation, blood of rabbits was collected in coagulation tubes, and coagulation function indexes were determined, such as activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT) and fibrinogen (FIB). At the same time, one rabbit was euthanized after treatment, and tumors and normal organs (heart, liver, spleen, lung and kidney) were fixed in 4.0% paraformaldehyde solution, sectioned and subjected to H&E staining analysis. In addition, tumor sections were subjected to apoptosis (TUNEL) staining and immunofluorescence (Ki67 and CD31) staining.

[0061] Compared with blank 3Asphere, three-dimensional CT results showed that the inhibitory effect of Th-3Asphere on tumor growth was significantly enhanced ( Figure 11 ), and the tumor volume was significantly reduced ( Figure 12 ). Notably, the embolization effect of Th-3Asphere induced obvious blood clots in tumor arteries, while no obvious blood clots were observed in 3Asphere and control groups ( Figure 13 ). In addition, compared with 3Asphere, the degree of tumor cell necrosis (H&E) and apoptosis (TUNEL) was higher, and the tumor vascularization density (CD31) was lower in rabbits after Th-3Asphere treatment ( Figure 14 ), which confirmed that the introduction of thrombin promoted the formation of obstructive thrombus and induced obvious vascular infarction. Importantly, after Th-3Asphere treatment, the body weight change of rabbits was small, and there was no obvious damage to major organs, and the effect on liver and kidney function was negligible ( Figures 15-17 ). In addition, embolization treatment using Th-3Asphere had no significant effect on APTT, TT, PT and FIB levels ( Figure 18 ), indicating that the released thrombin was mainly located in the tumor site and had little effect on the coagulation function of the whole body.

[0062] The above examples are only used to illustrate the technical concept and characteristics of the present application, and the purpose is to enable those skilled in the art to understand and implement the technology. These examples should not be regarded as limiting the protection scope of the present application. Any equivalent changes or modifications made on the basis of the spirit of the present application should be covered within the protection scope of the present application.

Claims

1. A method for preparing a procoagulant embolizing microsphere loaded with a procoagulant agent, characterized in that, The method comprises the following steps: mixing the microspheres with the coagulation accelerator to obtain coagulation accelerator-loaded embolization microspheres.

2. The method for preparing embolic microspheres loaded with coagulant according to claim 1, characterized in that, The coagulation accelerator comprises one or more of thrombin, hemocoagulase, protamine and blood coagulation factor.

3. The method for preparing embolic microspheres loaded with coagulant according to claim 1, characterized in that, The water-phase material containing the macromolecule with cross-linking groups and the oil-phase material are cross-linked and solidified by radical polymerization through the micro-channel pipeline to obtain the microspheres.

4. The method for preparing embolic microspheres loaded with coagulant according to claim 3, characterized in that, The macromolecule is reacted with an acrylic compound in a solution to obtain the macromolecule with cross-linking groups; and the micro-channel pipeline comprises a flow focusing type micro-channel pipeline.

5. The method of claim 3, wherein the supercoagulant embolizing microspheres are prepared by the steps of: The water-phase material comprises the macromolecule with cross-linking groups, buffer and initiator, and the concentration of the macromolecule with cross-linking groups is 10-400 mg / mL; the oil-phase material comprises a surfactant and an oil solvent; the micro-channel pipeline comprises a cross type, Y type, T type or flow focusing type micro-channel pipeline; and the solidification comprises ultraviolet light solidification and thermal solidification. The mass ratio of the macromolecule with cross-linking groups, buffer and initiator is 100:2000-5000:8-50.

6. The method of claim 1, wherein the supercoagulant embolizing microspheres are prepared by the steps of: The microspheres are mixed with the coagulation accelerator in a solvent, and the concentration of the macromolecule of the microspheres is 5-100 mg / mL, and the concentration of the coagulation accelerator is 0.1-5 μg / mL.

7. The coagulation accelerator-loaded embolization microspheres prepared by the method according to any one of claims 1 to 6.

8. A medicament, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof.

7. The coagulation accelerator-loaded embolization microspheres.

9. The use of the coagulation accelerator-loaded embolization microspheres according to claim 7 in the preparation of a medicine.

10. The use of the coagulation accelerator-loaded embolization microspheres according to claim 7 in the preparation of an antitumor medicine or a vascular embolization agent.

Images