Polyvinyl alcohol embolic microsphere with core-shell structure, preparation method therefor, and use thereof

Abstract

Disclosed are a polyvinyl alcohol embolic microsphere with a core-shell structure, a preparation method therefor, and use thereof. According to the preparation method, polyvinyl alcohol is taken as a skeleton material, two modified polyvinyl alcohols with different grafting degrees are obtained by means of reacting N-(2,2-dimethoxy)-2-methylacrylamide with polyvinyl alcohol at different ratios, a water-soluble monomer with ionic functional groups and an initiator are mixed with an aqueous solution of low-grafting-degree polyvinyl alcohol to obtain a first aqueous phase, an initiator is mixed with an aqueous solution of high-grafting-degree polyvinyl alcohol to obtain a second aqueous phase, the two aqueous phases are added to an oil phase containing an oil-soluble dispersing agent to form a water-in-oil reverse-phase suspension polymerization system, and reverse-phase suspension polymerization is performed to obtain the polyvinyl alcohol embolic microsphere with a core-shell structure. The core-shell structure polyvinyl alcohol embolization microsphere exhibits an extremely fast drug loading rate and an extremely high drug loading capacity.

Description

Core-shell structured polyvinyl alcohol embolic microspheres and their preparation method and application Technical Field

[0001] The present application belongs to the technical field of embolic microspheres, and specifically relates to a core-shell structured polyvinyl alcohol embolic microsphere and its preparation method and application. Background Art

[0002] Currently available embolic microspheres mainly use polyvinyl alcohol as the skeleton material of the microspheres. By grafting a monomer with a double bond onto the molecular chain of polyvinyl alcohol, and then polymerizing and cross-linking the monomer with a monomer with an ionic functional group, charged embolic microspheres are formed, thereby achieving drug-carrying function. The microspheres prepared with this single cross-linked structure have certain mechanical properties (strength and compressive elasticity) and drug-carrying properties. The repulsive force between the internal charges of the microspheres has a certain supporting effect on the structure of the microspheres. However, when the microspheres are loaded with drugs with opposite charges through electrostatic interactions, the repulsive force inside the microspheres disappears because the charges are neutralized, which causes the volume of the microspheres to shrink sharply, thereby affecting the further diffusion of the drugs into the microspheres, and ultimately resulting in a relatively low drug loading capacity.

[0003] CN201410232150 discloses a method for preparing single cross-linked polyvinyl alcohol embolic microspheres. The method uses polyvinyl alcohol as the backbone material. The cross-linking agent acrylamidodimethoxyacetal is first grafted onto the polyvinyl alcohol to obtain modified polyvinyl alcohol. 2-Acrylamido-2-methylpropanesulfonic acid sodium monomer, potassium persulfate as an initiator, and the modified polyvinyl alcohol are then mixed to obtain an aqueous phase. The microspheres are then obtained by reverse suspension polymerization using tetramethylethylenediamine as a catalyst, butyl acetate as a solvent, and cellulose acetate butyrate as a dispersant. The compression elasticity of these microspheres can reach over 50%, but the time required for them to reach equilibrium with doxorubicin loading is 20 minutes, which means that doctors need to wait at least 20 minutes before clinical use. Furthermore, the drug loading is only about 50%, which leads to poor therapeutic effects.

[0004] CN201910504935 discloses a method for preparing gradient cross-linked polyvinyl alcohol embolic microspheres, which uses polyvinyl alcohol as a skeleton material, directly configuring polyvinyl alcohol into an aqueous solution, then adding 2-acrylamide-2-methylpropanesulfonic acid sodium monomer, cross-linking agent N, N-methylenebisacrylamide and initiator potassium persulfate to make an aqueous phase; using cellulose acetate butyrate as a catalyst, tetramethylethylenediamine as a solvent, cellulose acetate butyrate as a dispersant, and adding glutaraldehyde as a cross-linking agent in batches during the reverse suspension polymerization process to obtain gradually cross-linked microspheres. The compression elasticity of the microspheres can reach more than 70%, but its drug loading can reach 98%, but it takes 30 minutes to reach this drug loading, which also means that doctors need to wait at least 30 minutes before clinical use.

[0005] Therefore, there is a need in the art to further develop embolic microspheres with shorter drug loading equilibrium time and higher drug loading capacity.

[0006] Summary of the Invention

[0007] The present application provides a core-shell structured polyvinyl alcohol embolic microsphere and its preparation method and application.

[0008] In a first aspect, the present application provides a method for preparing core-shell structured polyvinyl alcohol embolic microspheres, the preparation method comprising the following steps:

[0009] (1) polyvinyl alcohol reacts with a water-soluble crosslinking agent in an amount of 2% to 8% (e.g., 2%, 3%, 5%, 7% or 8%) of the weight of the polyvinyl alcohol to obtain a low-grafting degree polyvinyl alcohol aqueous solution;

[0010] (2) reacting polyvinyl alcohol with a water-soluble crosslinking agent in an amount of 16% to 32% (e.g., 16%, 18%, 20%, 25%, 28%, 30% or 32%) of the weight of the polyvinyl alcohol to obtain a polyvinyl alcohol aqueous solution with a high grafting degree;

[0011] (3) mixing a water-soluble monomer with an ionic functional group, an initiator, and the low-grafting degree polyvinyl alcohol aqueous solution obtained in step (1) to obtain a first aqueous phase;

[0012] (4) mixing the initiator with the aqueous solution of polyvinyl alcohol with a high grafting degree obtained in step (2) to obtain a second aqueous phase;

[0013] (5) mixing the first aqueous phase and the second aqueous phase to obtain a mixed aqueous phase; and

[0014] (6) adding the mixed aqueous phase obtained in step (5) to the oil phase containing the oil-soluble dispersant to form an oil-in-water reverse suspension polymerization system, and performing reverse suspension polymerization to obtain the core-shell structured polyvinyl alcohol embolic microspheres.

[0015] In the present application, step (1) prepares a low-grafting degree polyvinyl alcohol aqueous solution, and step (2) prepares a high-grafting degree polyvinyl alcohol aqueous solution. Microspheres with a core-shell structure are obtained by mixing the aqueous phase and the oil phase of these two modified polyvinyl alcohols with different grafting degrees and performing reverse suspension polymerization.

[0016] The modified polyvinyl alcohol with a low degree of grafting in the first aqueous phase has a higher degree of hydrophilicity. During the process of reverse suspension polymerization to form droplets, it will be distributed inside the droplets with the water-soluble monomers with ionic functional groups to form a core; while the modified polyvinyl alcohol with a high degree of grafting in the second aqueous phase has a relatively low degree of hydrophilicity. During the process of reverse suspension polymerization to form droplets, it will tend to be distributed on the surface of the droplets to form a shell, thereby forming droplets with a core-shell structure, which are then solidified into microspheres with a core-shell structure through reaction.

[0017] The relatively high cross-linking degree of the shell reduces the degree of particle size shrinkage of the microspheres after drug loading to a certain extent. The shell can still maintain relatively unobstructed pores, which facilitates the rapid entry of drugs from the shell into the core and improves the drug loading speed.

[0018] The outer shell contains almost no water-soluble monomers with ionic functional groups, while the inner core is rich in water-soluble monomers with ionic functional groups, forming a charge difference between the inside and the outside. Negatively charged drugs can more easily enter the inner core under the positive attraction of the inner core, thereby improving the utilization rate of sodium 2-acrylamide-2-methylpropane sulfonate and further increasing the drug loading capacity.

[0019] This special core-shell structure enables the microspheres to have an extremely fast drug loading rate and extremely high drug loading capacity; compared with the conventional drug loading capacity (80 mg, 2 mL microspheres), the microspheres with this structure can load more than 95% of the drug in 1 minute, and the maximum drug loading capacity can reach 150 mg.

[0020] In certain embodiments, before the reaction in step (1), polyvinyl alcohol is added to water, heated to 90°C-100°C (eg, 90°C, 93°C, 95°C, 98°C or 100°C), and stirred to dissolve to form a uniform polyvinyl alcohol solution.

[0021] In certain embodiments, the mass percentage concentration of polyvinyl alcohol in the polyvinyl alcohol solution is 12%-22%, for example, 12%, 15%, 18%, 20% or 22%.

[0022] In certain embodiments, the water-soluble cross-linking agent in step (1) and step (2) is independently selected from at least one of N-(2,2-dimethoxy)-2-methylacrylamide, N-(1-hydroxy-2,2-dimethoxyethyl)-2-acrylamide, N-(2,2-dimethoxy)-2-acrylamide, N-(1-hydroxy-2,2-dimethoxyethyl)-2-methylacrylamide, N-(2,2-dimethoxy)-2-methylacrylate, N-(1-hydroxy-2,2-dimethoxyethyl)-2-acrylate, N-(2,2-dimethoxy)-2-acrylate or N-(1-hydroxy-2,2-dimethoxyethyl)-2-methylacrylate.

[0023] In certain embodiments, the reaction in step (1) is carried out in the presence of an acid catalyst.

[0024] In certain embodiments, the acid catalyst is at least one of hydrochloric acid, sulfuric acid, nitric acid or p-toluenesulfonic acid, preferably hydrochloric acid.

[0025] In certain embodiments, the mass percent concentration of the acid catalyst in the reaction system is 6%-12%, for example, 6%, 7%, 8%, 9%, 10%, 11% or 12%.

[0026] In certain embodiments, the reaction temperature of step (1) is 20°C-30°C (e.g., 20°C, 23°C, 25°C, 28°C, or 30°C), and the reaction time is 10h-15h (e.g., 10h, 11h, 12h, 13h, 14h, or 15h).

[0027] In certain embodiments, before the reaction in step (2), polyvinyl alcohol is added to water, heated to 90°C-100°C (eg, 90°C, 93°C, 95°C, 98°C or 100°C), and stirred to dissolve to form a uniform polyvinyl alcohol solution.

[0028] In certain embodiments, the mass percentage concentration of polyvinyl alcohol in the polyvinyl alcohol solution is 12%-22%, for example, 12%, 15%, 18%, 20% or 22%.

[0029] In certain embodiments, the reaction in step (2) is carried out in the presence of an acid catalyst.

[0030] In certain embodiments, the acid catalyst is at least one of hydrochloric acid, sulfuric acid, nitric acid or p-toluenesulfonic acid, preferably hydrochloric acid.

[0031] In certain embodiments, the reaction temperature of step (2) is 20°C-30°C (e.g., 20°C, 23°C, 25°C, 28°C, or 30°C), and the reaction time is 10h-15h (e.g., 10h, 11h, 12h, 13h, 14h, or 15h).

[0032] In certain embodiments, the water-soluble monomer with an ionic functional group in step (3) is selected from at least one of sodium 2-acrylamide-2-methylpropanesulfonate, sodium allylsulfonate or sodium methacrylic acid.

[0033] In certain embodiments, the amount of the water-soluble monomer with ionic functional groups in step (3) is 5%-20% by weight of the polyvinyl alcohol aqueous solution, for example, 5%, 8%, 10%, 12%, 15%, 18% or 20%.

[0034] In certain embodiments, the initiator in step (3) is at least one of ammonium persulfate, sodium persulfate, or potassium persulfate.

[0035] In certain embodiments, the amount of the initiator in step (3) is 0.3%-0.8% by weight of the polyvinyl alcohol aqueous solution, for example, 0.3%, 0.4%, 0.5%, 0.6%, 0.7% or 0.8%.

[0036] In certain embodiments, the initiator in step (4) is at least one of ammonium persulfate, sodium persulfate, or potassium persulfate.

[0037] In certain embodiments, the amount of the initiator in step (4) is 0.3%-0.8% by weight of the polyvinyl alcohol aqueous solution, for example 0.3%, 0.4%, 0.5%, 0.6%, 0.7% or 0.8%.

[0038] In certain embodiments, the mass of the second aqueous phase in step (5) is 5%-20% of the mass of the first aqueous phase, for example 5%, 8%, 10%, 12%, 15%, 18% or 20%.

[0039] In certain embodiments, the oil-soluble dispersant in step (6) is selected from cellulose acetate butyrate.

[0040] In certain embodiments, the oil phase in step (6) is obtained by adding an oil-soluble dispersant to an oily solvent and dissolving the mixture with stirring at 30-50°C (e.g., 30°C, 33°C, 35°C, 38°C, 40°C, 45°C, 48°C, or 50°C) to form a uniform solution.

[0041] In certain embodiments, the mass percentage concentration of the oil-soluble dispersant in the oil phase is 2%-5%, for example, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%.

[0042] In certain embodiments, the oily solvent in the oil phase is selected from at least one of butyl acetate, ethyl acetate, methyl acetate, or propyl acetate, preferably butyl acetate.

[0043] In certain embodiments, the reverse phase suspension polymerization in step (6) is to heat the reaction system to 50°C-80°C (e.g., 60°C, 63°C, 65°C, 68°C, or 70°C), add a catalyst to the system, and react for 10h-20h, e.g., 10h, 13h, 15h, 18h, or 20h.

[0044] In certain embodiments, the catalyst is selected from tetramethylethylenediamine.

[0045] In certain embodiments, the reverse suspension polymerization in step (6) is carried out under stirring, and the stirring rate is 200 rpm-800 rpm, for example, 200 rpm, 230 rpm, 250 rpm, 280 rpm, 300 rpm, 350 rpm, 400 rpm, 450 rpm, 500 rpm, 550 rpm, 600 rpm, 650 rpm, 700 rpm, 750 rpm or 800 rpm.

[0046] In certain embodiments, the core-shell structured polyvinyl alcohol embolic microspheres have a particle size of 40-1400 μm. Depending on actual use needs, the microspheres can be sieved using sieves of different mesh sizes to form microspheres of different sizes, such as 40 μm-70 μm, 70-100 μm, 100-150 μm, 150-200 μm, 200-300 μm, 350-450 μm, 480-580 μm, 650-750 μm, 825-975 μm, 1025-1175 μm, 1225-1375 μm, etc.

[0047] In a second aspect, the present application provides core-shell structured polyvinyl alcohol embolic microspheres prepared by the preparation method described in the first aspect.

[0048] In a third aspect, the present application provides a drug-loaded microsphere, which comprises the core-shell structured polyvinyl alcohol embolic microspheres as described above and loaded drugs.

[0049] Compared with the prior art, this application has the following beneficial effects:

[0050] The core-shell structured polyvinyl alcohol embolic microspheres of the present application have an extremely fast drug loading rate and an extremely high drug loading capacity. The drug loading capacity can reach more than 95% in 1 minute, and the maximum drug loading capacity can reach 150 mg. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG1 is a microscope image of the embolic microspheres prepared in Example 1 (video display system, VHX-950F, Keyence (China) Co., Ltd.).

[0052] FIG2 is a test result showing the effect of the amount of aqueous phase 2 added on the drug loading rate of the microspheres.

[0053] FIG3 is a graph showing the test results of the maximum doxorubicin loading of different microspheres.

[0054] FIG4 is a particle size distribution diagram of the microspheres of Example 1.

[0055] FIG5 is a graph showing the particle size distribution of microspheres of commercial product 1.

[0056] FIG6 is a graph showing the particle size distribution of microspheres of Commercial Product 2.

[0057] FIG7 is a graph showing the results of investigating the embolization effect of the microspheres of Example 1 in animals.

[0058] FIG8 is a graph showing the results of investigating the drug release of the microspheres of Example 1 in the liver tissue at the embolization site of an animal. DETAILED DESCRIPTION

[0059] The technical solution of the present application is further described below through specific implementation methods. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations of the present application.

[0060] Example 1

[0061] In this embodiment, a core-shell structured polyvinyl alcohol embolic microsphere was prepared. The preparation method included the following steps:

[0062] Step 1: Prepare low grafting degree polyvinyl alcohol aqueous solution

[0063] A high molecular weight polymer (polyvinyl alcohol, 100 g) was added to water (500 mL), heated to 95°C and stirred to dissolve to form a uniform solution; then a water-soluble crosslinking agent (N-(2,2-dimethoxy)-2-methylacrylamide, 4.00 g) was added, stirred evenly, and an acid catalyst (hydrochloric acid, 30 mL) was added. The mixture was stirred continuously at 25°C for 10 hours to obtain a polyvinyl alcohol aqueous solution.

[0064] Step 2: Prepare a high grafting degree polyvinyl alcohol aqueous solution

[0065] A high molecular weight polymer (polyvinyl alcohol, 100 g) was added to water (500 mL), heated to 95°C and stirred to dissolve to form a uniform solution; then a water-soluble crosslinking agent (N-(2,2-dimethoxy)-2-methylacrylamide, 16.00 g) was added, stirred evenly, and an acid catalyst (hydrochloric acid, 30 mL) was added. The mixture was stirred continuously at 25°C for 10 hours to obtain a polyvinyl alcohol aqueous solution.

[0066] Step 3: Prepare the reverse suspension polymerization oil phase

[0067] A dispersant (cellulose acetate butyrate, 20 g) was added to an oily solvent (butyl acetate, 1000 mL), and the mixture was stirred and dissolved at 35° C. to form a uniform solution to obtain an oil phase.

[0068] Step 4: Prepare the aqueous phase 1 of the inverse suspension polymerization system

[0069] A water-soluble monomer with an ionic functional group (sodium 2-acrylamide-2-methylpropanesulfonate, 20 g), an initiator (ammonium persulfate, 1 g), and a low-grafting degree polyvinyl alcohol aqueous solution (200 g) were stirred evenly to form a uniform solution to obtain aqueous phase 1.

[0070] Step 5: Prepare the aqueous phase 2 of the inverse suspension polymerization system

[0071] An initiator (ammonium persulfate, 1 g) and a high-grafting degree polyvinyl alcohol aqueous solution (200 g) were stirred until a uniform solution was obtained to obtain aqueous phase 2.

[0072] Step 6: Prepare the mixed water phase

[0073] The aqueous phase 1 (200 g) and the aqueous phase 2 (20 g) were mixed uniformly to obtain a mixed aqueous phase.

[0074] Step 7: Reverse suspension polymerization

[0075] Under stirring conditions, the mixed aqueous phase solution was slowly added dropwise to the oil phase to form an oil-in-water reverse suspension polymerization system. After the addition was completed, the reaction system was heated to 65°C and the catalyst (tetramethylethylenediamine, 5 mL) was continued to be added dropwise to the reaction system. The reverse suspension polymerization reaction began and the reaction lasted for 15 hours. After the reaction was completed, stirring and heating were stopped, the reaction system was allowed to stand to separate, the oil phase was separated, the microspheres were collected and repeatedly washed to obtain embolic microspheres with a core-shell structure (as shown in Figure 1), with a particle size between 40μm and 1400μm.

[0076] Example 2

[0077] This embodiment provides an embolic microsphere, and the preparation method thereof is different from that of Example 1 in that: in step 6, the amount of aqueous phase 2 added is 5 g.

[0078] Example 3

[0079] This embodiment provides an embolic microsphere, and the preparation method thereof is different from that of Example 1 in that: in step 6, the amount of aqueous phase 2 added is 10 g.

[0080] Example 4

[0081] This embodiment provides an embolic microsphere, and the preparation method thereof is different from that of Example 1 in that: in step 6, the amount of aqueous phase 2 added is 15 g.

[0082] Example 5

[0083] This embodiment provides an embolic microsphere, and the preparation method thereof is different from that of Example 1 in that: in step 6, the amount of aqueous phase 2 added is 25 g.

[0084] Example 6

[0085] This embodiment provides an embolic microsphere, and the preparation method thereof is different from that of Example 1 in that: in step 6, the amount of the aqueous phase 2 added is 30 g.

[0086] Example 7

[0087] This embodiment provides an embolic microsphere, and the preparation method thereof is different from that of Example 1 in that: in step 6, the amount of aqueous phase 2 added is 0 g.

[0088] Example 8

[0089] This embodiment provides an embolic microsphere, and the preparation method thereof is different from that of Example 1 in that: in step 6, the amount of aqueous phase 2 added is 40 g.

[0090] Example 9

[0091] This embodiment provides embolic microspheres, the preparation method of which is different from that of Example 1 in that: in step 1, the amount of water-soluble cross-linking agent N-(2,2-dimethoxy)-2-methylacrylamide added is 2 g; in step 2, the amount of water-soluble cross-linking agent N-(2,2-dimethoxy)-2-methylacrylamide added is 20 g.

[0092] Example 10

[0093] This embodiment provides an embolic microsphere, and the preparation method thereof is different from that of Example 1 in that: in step 1, the amount of the water-soluble crosslinking agent N-(2,2-dimethoxy)-2-methylacrylamide added is 5 g.

[0094] Example 11

[0095] This embodiment provides embolic microspheres, the preparation method of which is different from that of Example 1 in that: in step 1, the amount of water-soluble cross-linking agent N-(2,2-dimethoxy)-2-methylacrylamide added is 6 g; in step 2, the amount of water-soluble cross-linking agent N-(2,2-dimethoxy)-2-methylacrylamide added is 16 g.

[0096] Example 12

[0097] This embodiment provides an embolic microsphere, and its preparation method differs from that of Example 1 in that: in step 1, the amount of water-soluble cross-linking agent N-(2,2-dimethoxy)-2-methylacrylamide added is 8 g; in step 2, the amount of water-soluble cross-linking agent N-(2,2-dimethoxy)-2-methylacrylamide added is 32 g.

[0098] Drug loading performance test:

[0099] The drug loading rate of the embolic microspheres was tested by mixing the microspheres (100 μm, 2 mL) with 4 mL of a 20 mg / mL doxorubicin solution. Samples were taken every minute and the residual doxorubicin in the supernatant was determined by HPLC. The drug loading of the microspheres was then calculated. The test results are shown in Table 1.

[0100] Table 1 Drug loading performance of drug loaded microspheres

[0101] Note: Commercially available product 1: DC Bead, 100 μm-300 μm, Biocompatibles UK Limited;

[0102] Commercially available product 2: Equalsphere, 100 μm, Suzhou Hengrui Jialisheng Biotechnology Co., Ltd.

[0103] FIG2 is a test result showing the effect of the amount of aqueous phase 2 added on the drug loading rate of the microspheres.

[0104] Conclusions: 1) The drug loading rate significantly increased with the increase in the amount of aqueous phase 2 added. When the amount added was greater than 20 g, 95% of the drug could be loaded in about 1 minute. However, when the amount added was greater than 40 g, the drug loading rate began to decrease, as the shell was too thick, which affected the diffusion of the drug into the core. 2) When the amount of crosslinker added to aqueous phase 1 was less than 2 g, the core shrank sharply after drug loading due to the low degree of crosslinking, resulting in a slow drug loading rate and a reduced drug loading capacity. When the amount of crosslinker added to aqueous phase 1 was greater than 8 g, the core became dense due to excessive crosslinking, which affected drug diffusion, resulting in a slow drug loading rate and a reduced drug loading capacity. 3) When the amount of crosslinker added to aqueous phase 2 was less than 16 g, the shell shrank after drug loading due to the low degree of crosslinking, resulting in a slow drug loading rate and a reduced drug loading capacity. When the amount of crosslinker added to aqueous phase 2 was greater than 32 g, the shell became dense, which affected drug diffusion, resulting in a slow drug loading rate and a reduced drug loading capacity.

[0105] Shrinkage ratio test of microspheres after drug loading:

[0106] The drug-loaded particle size of the embolic microspheres was tested by flattening the microspheres onto a glass slide and placing them under a microscope using a video display system (VHX-950F). The particle size of 200 microspheres was measured and the average value was calculated. The test results are shown in Table 2.

[0107] Table 2 Shrinkage ratio of microspheres after drug loading

[0108] Conclusion: With the increase of the amount of aqueous phase 2 added, the shell of the microspheres becomes thicker and the particle size shrinkage ratio after drug loading is significantly reduced.

[0109] Maximum drug loading test:

[0110] The maximum drug loading capacity of embolic microspheres was tested by mixing microspheres (100 μm, 2 mL) with 8 mL of a 20 mg / mL doxorubicin solution. Samples were taken periodically until the drug solution showed no noticeable color change. The residual doxorubicin in the supernatant was determined by HPLC, and the drug loading capacity of the microspheres was calculated by conversion. The test results are shown in Table 3 (Figure 3).

[0111] Table 3

[0112] Conclusions: 1) With the increase in the amount of aqueous phase 2 added, the microsphere shell became significantly thicker, the degree of shrinkage after drug loading was small, and the utilization rate of AMPS in the core was high, resulting in a high drug loading capacity. 2) When the amount of crosslinker added to aqueous phase 1 was 2g, the core shrank sharply after drug loading due to the low degree of crosslinking, resulting in a decrease in drug loading capacity. When the amount of crosslinker added to aqueous phase 1 was 8g, the core was dense due to excessive crosslinking, which affected drug diffusion and led to a decrease in drug loading capacity. 3) When the amount of crosslinker added to aqueous phase 2 was 16g, the shell shrank after drug loading due to the low degree of crosslinking, resulting in a slow drug loading rate and a decrease in drug loading capacity. When the amount of crosslinker added to aqueous phase 2 was 32g, the shell was dense, which affected drug diffusion, resulting in a slow drug loading rate and a decrease in drug loading capacity.

[0113] Particle size distribution test:

[0114] The particle size distribution of the microspheres prepared in this application was compared with that of commercially available products. Embolic microspheres (particle size range 100-150 μm) were taken, spread on a glass slide, and placed under a microscope of a video display system (VHX-950F) for testing. The particle size of 200 microspheres was tested and then plotted into a histogram for comparison.

[0115] FIG4 is a particle size distribution diagram of the microspheres of Example 1, FIG5 is a particle size distribution diagram of the microspheres of Commercial Product 1, and FIG6 is a particle size distribution diagram of the microspheres of Commercial Product 2.

[0116] It can be seen that the standard deviation of the particle size of 200 microspheres prepared in the present application is smaller than that of commercially available products, and the particle size distribution is narrower than that of commercially available products.

[0117] The microspheres (100-150 μm) of Example 1 were used in combination with the common anthracycline chemotherapy drug doxorubicin hydrochloride (drug concentration 25 mg / mL) to simulate its clinical use. The left lobe of the hepatic artery of healthy pigs was embolized by TACE (transcatheter arterial chemoembolization) interventional surgery. The effectiveness of the microspheres was evaluated by investigating the intraoperative performance of the microspheres, the embolic effect immediately and within the test period, the blood drug concentration and the tissue drug concentration after surgery. The safety of the microspheres within the test period was preliminarily evaluated by investigating the recovery of the animals after surgery, combining clinical pathology, gross anatomical observation, histopathology, etc. The investigation time points were set 1 day, 7 days, and 28 days after surgery, with at least 3 animals at each time point.

[0118] All animal surgeries were completed successfully, with no adverse events such as tube blockage, demonstrating the microspheres' good operability and permeability through the microcatheter. The animals remained in good condition postoperatively, with no unexpected deaths.

[0119] Compared with pre-embolization images, the embolization effect of the target vessel (disappearance of staining) can be clearly observed in the DSA images immediately after embolization, 1 day, 7 days, and 28 days after surgery. Figure 7 shows some of the data, indicating that the embolization effect is well maintained (the arrows indicate the embolization sites).

[0120] Blood samples were collected from the animals before and after surgery at different time points to test hematology, serum biochemistry, and coagulation. Except for slight fluctuations in individual indicators within 1 week after surgery due to drug toxicity and embolization surgery, the rest of the indicators were normal.

[0121] The gross anatomical results showed that except for the obvious macroscopic lesions at the embolization site (left lateral lobe of the liver), there were no abnormalities in other lobes of the liver, and other organs (heart, spleen, lungs, kidneys, brain, and gallbladder) were also normal, indicating that the microspheres had good targeting and no non-targeted drift occurred after the operation.

[0122] Liver tissue from the embolization site of the animal was obtained and homogenized to test the drug concentration in the tissue. The tissue concentration is shown in Figure 8. It can be seen that the drug concentration in the tissue gradually decreased over time. 28 days after surgery, it was still 9970 ng / mL, indicating that the drug was slowly released from the microspheres in the body and could maintain an effective concentration for a long time, showing a good sustained-release effect.

[0123] Pathological sections showed that the vascular damage at the embolization site was more obvious, with medial necrosis or transmural necrosis of the vascular wall, accompanied by or without inflammatory cell infiltration, and necrosis of the perivascular tissue. This was mainly caused by the combined effects of embolism and the toxicity of drugs released from the microspheres, proving the effectiveness of the microspheres at the embolization site.

[0124] The above animal experiments further demonstrated the good operational performance of the microspheres, which have excellent embolization effect, sustained drug release effect and good safety in the body, and are expected to have good effects in clinical tumor treatment.

[0125] The applicant declares that while the above-mentioned embodiments are used to illustrate the process of the present application, the present application is not limited to the above-mentioned process steps, which does not mean that the present application must rely on the above-mentioned process steps in order to be implemented. Those skilled in the art should understand that any improvements to the present application, equivalent replacements for the raw materials used in the present application, addition of auxiliary components, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present application.

Claims

1. A method for preparing core-shell structured polyvinyl alcohol embolic microspheres, wherein The following steps are involved: (1) polyvinyl alcohol reacts with a water-soluble crosslinking agent accounting for 2% to 8% of the mass of the polyvinyl alcohol to obtain a polyvinyl alcohol aqueous solution with a low grafting degree; (2) polyvinyl alcohol reacts with a water-soluble crosslinking agent accounting for 16% to 32% of the mass of the polyvinyl alcohol to obtain a polyvinyl alcohol aqueous solution with a high grafting degree; (3) mixing a water-soluble monomer with an ionic functional group, an initiator and the low-grafting degree polyvinyl alcohol aqueous solution obtained in step (1) to obtain a first aqueous phase; (4) mixing an initiator with the aqueous solution of polyvinyl alcohol with a high grafting degree obtained in step (2) to obtain a second aqueous phase; (5) mixing the first aqueous phase and the second aqueous phase to obtain a mixed aqueous phase; as well as (6) adding the mixed aqueous phase obtained in step (5) to the oil phase containing the oil-soluble dispersant to form an oil-in-water reverse suspension polymerization system, and performing reverse suspension polymerization to obtain the core-shell structured polyvinyl alcohol embolic microspheres.

2. The method for preparing the core-shell structured polyvinyl alcohol embolic microspheres according to claim 1, in, Before the reaction in step (1), polyvinyl alcohol is added into water, heated to 90° C.-100° C. and stirred to dissolve to form a uniform polyvinyl alcohol solution.

3. The method for preparing the core-shell structured polyvinyl alcohol embolic microspheres according to claim 1 or 2, in, The mass percentage concentration of polyvinyl alcohol in the polyvinyl alcohol solution is 12%-22%.

4. The method for preparing the core-shell polyvinyl alcohol embolic microspheres according to any one of claims 1 to 3, in, The water-soluble crosslinking agent in step (1) and step (2) is independently selected from at least one of N-(2,2-dimethoxy)-2-methylacrylamide, N-(1-hydroxy-2,2-dimethoxyethyl)-2-acrylamide, N-(2,2-dimethoxy)-2-acrylamide, N-(1-hydroxy-2,2-dimethoxyethyl)-2-methylacrylamide, N-(2,2-dimethoxy)-2-methylacrylate, N-(1-hydroxy-2,2-dimethoxyethyl)-2-acrylate, N-(2,2-dimethoxy)-2-acrylate or N-(1-hydroxy-2,2-dimethoxyethyl)-2-methylacrylate.

5. The method for preparing the core-shell polyvinyl alcohol embolic microspheres according to any one of claims 1 to 4, in, The reaction in step (1) is carried out in the presence of an acid catalyst; Preferably, the acid catalyst is at least one of hydrochloric acid, sulfuric acid, nitric acid or p-toluenesulfonic acid, preferably hydrochloric acid; Preferably, the mass percentage concentration of the acid catalyst in the reaction system is 6%-12%; Preferably, the reaction temperature in step (1) is 20°C-30°C, and the reaction time is 10h-15h.

6. The method for preparing the core-shell polyvinyl alcohol embolic microspheres according to any one of claims 1 to 5, in, Before the reaction in step (2), polyvinyl alcohol is added into water, heated to 90° C.-100° C., and stirred to dissolve to form a uniform polyvinyl alcohol solution; Preferably, the mass percentage concentration of polyvinyl alcohol in the polyvinyl alcohol solution is 12%-22%; Preferably, the reaction in step (2) is carried out in the presence of an acid catalyst; Preferably, the acid catalyst is at least one of hydrochloric acid, sulfuric acid, nitric acid or p-toluenesulfonic acid, preferably hydrochloric acid; Preferably, the reaction temperature in step (2) is 20°C-30°C, and the reaction time is 10h-15h.

7. The method for preparing the core-shell polyvinyl alcohol embolic microspheres according to any one of claims 1 to 6, in, The water-soluble monomer with ionic functional groups in step (3) is selected from at least one of sodium 2-acrylamide-2-methylpropane sulfonate, sodium allyl sulfonate or sodium methacrylic sulfonate; Preferably, the amount of the water-soluble monomer with ionic functional groups in step (3) is 5%-20% by weight of the polyvinyl alcohol aqueous solution; Preferably, the initiator in step (3) is at least one of ammonium persulfate, sodium persulfate or potassium persulfate; Preferably, the amount of the initiator used in step (3) is 0.3%-0.8% by weight of the polyvinyl alcohol aqueous solution.

8. The method for preparing the core-shell polyvinyl alcohol embolic microspheres according to any one of claims 1 to 7, in, The initiator in step (4) is at least one of ammonium persulfate, sodium persulfate or potassium persulfate; Preferably, the amount of the initiator used in step (4) is 0.3%-0.8% by weight of the polyvinyl alcohol aqueous solution.

9. The method for preparing the core-shell structure polyvinyl alcohol embolic microspheres according to any one of claims 1 to 8, in, Step (5) the mass of the second aqueous phase is 5% to 20% of the mass of the first aqueous phase; Preferably, the oil-soluble dispersant in step (6) is selected from cellulose acetate butyrate; Preferably, the oil phase in step (6) is obtained by adding an oil-soluble dispersant to an oily solvent and stirring and dissolving at 30° C. to 50° C. to form a uniform solution; Preferably, the mass percentage concentration of the oil-soluble dispersant in the oil phase is 2%-5%; Preferably, the oily solvent in the oil phase is selected from at least one of butyl acetate, ethyl acetate, methyl acetate or propyl acetate, preferably butyl acetate; Preferably, the reverse phase suspension polymerization in step (6) is to heat the reaction system to 50°C-80°C, add a catalyst to the system, and react for 10h-20h; Preferably, the catalyst is selected from tetramethylethylenediamine; Preferably, the reverse phase suspension polymerization in step (6) is carried out under stirring, and the stirring rate is 200 rpm-800 rpm; Preferably, the particle size of the core-shell structured polyvinyl alcohol embolic microspheres is 40 μm-1400 μm.

10. The core-shell structured polyvinyl alcohol embolic microspheres prepared by the preparation method according to any one of claims 1 to 9.

11. A drug-loaded microsphere, comprising the core-shell structured polyvinyl alcohol embolic microsphere according to claim 10 and loaded drugs.

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