Negatively charged embolization microsphere and preparation method therefor

Abstract

A negatively charged embolization microsphere and a preparation method therefor, wherein polyvinyl alcohol, which is used as a base material, cross-links with a cross-linking agent to form microspheres while a polymer material containing polyacid radical ions is introduced. The prepared modified microsphere has the same regular spherical shape, controllable size, and good suspensibility and deformation elasticity as those of polyvinyl alcohol before modification. In addition, since a negatively charged functional group is introduced, the microsphere can bind to a positively charged small molecule anti-tumor drug, which makes the microspheres have a good drug loading performance. The preparation method is a pure chemical synthesis process, the raw materials and preparation process of the microsphere are free from viral contamination, and the microsphere meets various international requirements for embolization microsphere agents, can replace various expensive embolization microsphere agent products, and can be widely used in various operations in the field of interventional therapy.

Description

Negatively charged embolic microspheres and their preparation process Technical Field

[0001] The present invention relates to an embolic microsphere that can be used for interventional surgery to treat tumor diseases and a preparation process thereof, and belongs to the field of medical devices. Background Art

[0002] Interventional therapy, a treatment method that combines diagnostic imaging with clinical treatment, entered clinical practice after rapid development in the 1970s. Based on diagnostic imaging, interventional therapy utilizes minimally invasive procedures using catheters, guidewires, and percutaneous puncture techniques to deliver medication infusions, vasodilation, or embolization to localized lesions. This approach reduces incision and pain, enhances local drug efficacy, and minimizes systemic side effects, making it simpler, more effective, and more direct than traditional surgical methods.

[0003] Embolization is a treatment method that blocks or slows the blood supply to a tissue or organ. It can be used to block blood flow from the arteries to the tumor, causing cancer cells to be deprived of nutrients and die due to ischemia. When the material used to block the blood supply also delivers chemotherapy drugs to the tumor, it is called chemoembolization. Among them, transcatheter arterial chemoembolization (TACE) is a special type of local chemoembolization that can block the hepatic artery to achieve the effect of treating unresectable or metastatic hepatocellular carcinoma (HCC). HCC is the most common type of primary liver cancer. This type of tumor grows a large number of blood vessels, and most of these blood vessels are supplied by the hepatic artery, while the rest of the liver tissue comes from the portal vein. Therefore, the blood supply to the tumor can be cut off by blocking the hepatic artery without affecting the rest of the liver.

[0004] Embolic materials available on the market are primarily categorized as liquid and solid. Liquid embolic materials are increasingly being replaced by solid embolic materials due to their fluidity, leading to unstable embolization and inability to achieve sustained-release effects when combined with chemotherapy drugs. Solid embolic materials, commonly used clinically, have undergone the following evolutionary changes: the first generation consisted of irregularly shaped gelatin sponges and polyvinyl alcohol particles. These irregular shapes led to clinically significant shortcomings such as aggregation, difficulty in injection, and incomplete vascular embolization. The second generation consisted of uniformly sized spherical blank microspheres, such as polyvinyl alcohol blank microspheres and sodium alginate microspheres. These microspheres have a regular shape and uniform particle size, can adapt to the shape of blood vessels, and provide thorough embolization. The third generation of drug-loaded microspheres not only exhibits a regular shape and uniform particle size but also possesses a certain degree of elasticity and compression deformation, enabling them to reach deeper into tumor vessels through microcatheters for more accurate embolization. At the same time, it also has the ability to carry drugs. The adsorbed drugs can be slowly released locally at the lesion, significantly increasing the local concentration of drugs in liver cells and improving the therapeutic effect of the drugs.

[0005] Among the currently marketed embolic materials capable of encapsulating chemotherapy drugs, there are DC Bead from Biocompatibles in the UK, Hepasphere from BioSphere in the US, and CalliSpheres from Hengrui Group. All three microspheres utilize polyvinyl alcohol (PVA) as the primary microsphere material. These microspheres are modified by grafting double-bonded polymer monomers onto the PVA molecular chain. These polymer monomers then polymerize and crosslink with monomers bearing ionic functional groups to form charged embolic microspheres, enabling drug loading. These embolic microspheres exhibit a moderate drug-loading capacity and compressive elasticity. However, since these microspheres are polymerized with ionic functional groups, the more ionic functional groups are present in the microspheres, resulting in a higher drug-loading capacity. However, this also increases the degree of crosslinking, resulting in poorer compressive elasticity, expansion properties, and drug-loading speed. This interaction limits the optimal drug-loading performance and compressive elasticity of the embolic microspheres. Furthermore, literature reports indicate that the high viscosity of polyvinyl alcohol (PVA) during the preparation of these ion-exchange microspheres can lead to microsphere aggregation during polymerization. Furthermore, prolonged mixing of the microspheres with the aqueous solution can sometimes lead to irregular spherical shape and rapid intravascular release of chemotherapy drugs by the drug delivery system. Therefore, new synthesis methods and microsphere structural characteristics are needed to achieve chemoembolization with more optimal performance.

[0006] Summary of the Invention

[0007] The present invention provides a simple, industrially scalable process for preparing highly elastic embolic microspheres via cross-linking reactions. These microspheres are characterized by their ability to adsorb cationic drugs or molecules both internally and externally, enhancing their ability to absorb and speed up adsorption. Furthermore, the microspheres, prepared via a reverse suspension cross-linking method, have a low density within and a high density outside, resulting in improved compressibility and enhanced internal water absorption and expansion capacity. This method requires simultaneous control of the following steps:

[0008] 1. Adjust the ratio of hydrophilic polyhydroxy polymer material and polymer material containing polyacid ions so that the microspheres contain a certain proportion of hydroxyl groups and acid radicals, which have a strong adsorption effect on positively charged drug molecules. Too much hydrophilic polyhydroxy polymer material will lead to a weakened adsorption capacity for drug molecules, and too little hydrophilic polyhydroxy polymer material will affect cross-linking into spheres.

[0009] 2. Adjust the molar ratio of the cross-linking agent to the hydrophilic polyhydroxy polymer so that the hydrophilic polyhydroxy polymer material in the microspheres maintains a certain degree of cross-linking, which can fully entangle the polyacid ion polymer material to form regular, elastic and compressible microspheres. Too much cross-linking agent will cause the microspheres to be excessively cross-linked, thereby reducing the elasticity and compressibility of the microspheres. Too little cross-linking agent will result in the inability to form microspheres or a large number of broken microspheres.

[0010] 3. Adjust the volume ratio of water in the aqueous phase and the organic solvent in the oil phase, control the uniform dispersion of the aqueous phase droplets in the oil phase, and control the appearance morphology and particle size of the microspheres.

[0011] 4. The water content of the microspheres prepared by the above process is between 90% and 97%, which makes the microspheres have good flexibility, so as to embolize the irregularly shaped blood vessel ends and obtain better embolization effect.

[0012] Therefore, in response to the above technical problems, the purpose of the present invention is to provide a method for preparing negatively charged embolic microspheres. The purpose of the present invention is also to provide negatively charged embolic microspheres with uniform particle size and strong adsorption effect on positively charged drugs.

[0013] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0014] The invention discloses an embolic microsphere with adsorption capacity, which is prepared by an inverse emulsion method (W / O). The embolic microsphere is made of a biocompatible polymer material which reacts with a cross-linking agent to form a network structure, and is entangled with each other to form regular elastic microspheres with negative charge.

[0015] A method for preparing negatively charged embolic microspheres comprises the following steps:

[0016] A) dissolving a hydrophilic polyhydroxy polymer and a polymer containing polyacid ions in water and stirring the mixture to obtain an aqueous phase;

[0017] B) dissolving the dispersant in an organic solvent and stirring uniformly to prepare an oil phase;

[0018] C) adding the aqueous phase to the oil phase for dispersion to obtain a mixed emulsion, adding a crosslinking agent to the mixed emulsion once or several times and stirring to obtain microspheres;

[0019] D) washing and filtering the microspheres obtained in step C);

[0020] After the aqueous phase is prepared in step A) or after the mixed emulsion is obtained in step C), the pH is adjusted to acidic.

[0021] Preferably, the molar ratio of the hydrophilic polyhydroxy polymer to the polymer containing polyacid ions is 0.8:1 to 12:1.

[0022] More preferably, the molar ratio of the hydrophilic polyhydroxy polymer to the polymer containing polyacid ions is 2.3:1 to 12:1.

[0023] Preferably, the total weight of the hydrophilic polyhydroxy polymer and the polymer containing polyacid ions accounts for 4% to 15% of the total weight of the aqueous phase. The calculation formula is:

[0024] The mass percentage of the total weight of the hydrophilic polyhydroxy polymer and the polymer containing polyacid ions to the total weight of the aqueous phase = (weight of the hydrophilic polyhydroxy polymer + weight of the polymer containing polyacid ions) / (weight of the hydrophilic polyhydroxy polymer + weight of the polymer containing polyacid ions + weight of water) * 100%

[0025] Preferably, the molar ratio of the crosslinking agent to the hydrophilic polyhydroxy polymer is 1:4 to 1:19.

[0026] Preferably, the volume ratio of water in the aqueous phase to the organic solvent in the oil phase is 1:1.5 to 1:20.

[0027] Preferably, the organic solvent for preparing the oil phase is one or more organic solvents such as butyl acetate, ethyl acetate, and n-heptane.

[0028] Preferably, the mass percentage of the dispersant in the oil phase is 0.8% to 5.0%, which is calculated as follows: mass percentage of the dispersant in the oil phase = mass of the dispersant / (mass of the dispersant + mass of the organic solvent) * 100%.

[0029] Preferably, the hydrophilic polyhydroxy polymer is polyvinyl alcohol (PVA) with a molecular weight of 20 to 600 kilodaltons.

[0030] Preferably, the polyanion polymer is a polymer containing multiple carboxyl functional groups and / or a polymer containing multiple sulfonic functional groups. Further preferably, the polymer containing multiple carboxyl functional groups is polyacrylic acid and / or its salt, polymethacrylic acid and / or its salt, and the polymer containing multiple sulfonic functional groups is poly(p-styrene sulfonic acid) and / or its salt, polyethylene sulfonic acid and / or its salt. Even more preferably, the polymer containing multiple carboxyl functional groups is one or more of sodium polyacrylate (PAANa), partially neutralized sodium polyacrylate, polyacrylic acid (PAA), sodium polymethacrylate and polymethacrylic acid, each of which has a molecular weight of 10 to 600 kilodaltons, wherein the mass ratio of acrylic acid to sodium acrylate in the partially neutralized sodium polyacrylate is 2:8 to 8:2; the polymer containing multiple sulfonic functional groups is one or more of sodium poly(p-styrene sulfonate), poly(p-styrene sulfonic acid), sodium polyethylene sulfonate and polyethylene sulfonic acid, each of which has a molecular weight of 20 to 2000 kilodaltons.

[0031] Preferably, the cross-linking agent is an aldehyde compound, and more preferably, the cross-linking agent is glyoxal and / or glutaraldehyde.

[0032] Preferably, the dispersant is one or a combination of Span 80, Tween 80, cellulose acetate and cellulose acetate butyrate.

[0033] Preferably, in step C), the aqueous phase is dispersed in the oil phase using shearing or stirring equipment, wherein the stirring speed is 50 rpm to 1000 rpm.

[0034] Preferably, after preparing the aqueous phase in step A) or after obtaining the mixed emulsion in step C), one or more of hydrochloric acid, sulfuric acid and acetic acid are added to adjust the pH to 3-5; the microspheres collected by filtration in step D) are washed with butyl acetate and ethyl acetate in sequence, and then washed with acetone or ethanol, and then vacuum dried to obtain dry microspheres. The dried microspheres are soaked in pure water and filtered, then soaked in sodium hydroxide solution and filtered with water, and then stirred in boiling water, filtered, sieved, and collected.

[0035] As another aspect of the present invention, there is also provided negatively charged hydroxyl cross-linked embolic microspheres prepared according to any one of the above methods.

[0036] As another aspect of the present invention, a use of the above-mentioned embolic microspheres is also provided, wherein the embolic microspheres are used to adsorb one or more of doxorubicin, epirubicin, pirarubicin, irinotecan, mitoxantrone, daunorubicin, topotecan, bleomycin, oxaliplatin, gemcitabine and 5-fluorouracil.

[0037] In a preferred embodiment of the present invention, reactive dyes may be added to the microspheres for dyeing to enhance the visibility of the microspheres so as to facilitate user differentiation. The reactive dyes include reactive blue and reactive yellow.

[0038] In a preferred embodiment according to the present invention, the wet microspheres after filtration can be sieved through stainless steel sieves of different pore sizes to remove unnecessary particle sizes and retain microspheres of the required size range, which can be sieved into various intervals such as 100-300 μm, 300-500 μm, 500-700 μm, and 700-900 μm.

[0039] The microspheres prepared according to the preferred embodiment of the present invention can be used to adsorb one or more of doxorubicin, epirubicin, pirarubicin, irinotecan, mitoxantrone, daunorubicin, topotecan, bleomycin, oxaliplatin, gemcitabine, and 5-fluorouracil.

[0040] According to the technical solution of the present invention, the following beneficial effects are achieved:

[0041] The preparation method of the present invention is relatively simple and easy to implement, can be used for industrial operation, and has universal applicability.

[0042] (1) The microspheres are highly hydrophilic and have excellent rapid drug adsorption and loading capabilities. They can adsorb more than 90% of the drug within 15 minutes after binding with the drug, and have a high drug loading capacity.

[0043] (2) It has excellent compressibility and elasticity and can quickly rebound to a spherical shape after compression.

[0044] (3) The preparation process is simple and can be completed through a single polymerization reaction, without the need for grafting modification followed by polymerization. The preparation conditions are mild and the cost is relatively low, making it suitable for large-scale batch production.

[0045] (4) It has good sustained-release ability and can release chemotherapy drugs at the tumor site for a long time.

[0046] (5) The drug-loaded embolic microspheres prepared by this preparation method can be sterilized by high-pressure steam at 121.3°C for 30 minutes after packaging, and the drug to be adsorbed can be added for mixed adsorption before use, avoiding the degradation of the drug during the preparation, sterilization and storage of the microspheres.

[0047] (6) During the preparation process, it was found that the drug-loaded embolic microspheres prepared using polymers with multiple sulfonic acid functional groups had better drug stability after drug adsorption than those prepared using polymers with multiple carboxyl functional groups. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG1 is a microscope image of microspheres prepared according to Example 1 of the present invention.

[0049] FIG2 is a microscopic image of the microspheres prepared according to Example 1 of the present invention after adsorbing doxorubicin.

[0050] FIG3 shows the in vitro dissolution curves of the microspheres prepared according to Examples 3 and 7 of the present invention and the commercially available microspheres. DETAILED DESCRIPTION

[0051] To facilitate a better understanding of the present invention, the following further describes the embolic microspheres prepared according to the present invention, combining specific examples and comparative examples with relevant performance test data and literature data from similar marketed products. These examples are intended for illustrative purposes only and are not intended to limit the scope of the present invention.

[0052] Example 1:

[0053] Add 0.9g of cellulose acetate butyrate to 100ml of butyl acetate and stir to dissolve to form an oil phase. Dissolve 3g of polyvinyl alcohol (molecular weight ~200,000 Daltons) and 1g of sodium polyacrylate (molecular weight ~150,000 Daltons) in 30g of purified water, stir and mix evenly, and add concentrated hydrochloric acid solution to adjust the pH value to form an aqueous phase. Mix the aqueous phase and the oil phase with a stirrer at a stirring speed of 350rpm to form an oil-in-water emulsion. Add 0.2ml of glutaraldehyde aqueous solution (50% wt) and react for 2 hours. Then add 0.4ml of glutaraldehyde aqueous solution (50% wt) to the reaction system. After reacting for 2 hours, add 0.6ml of glutaraldehyde aqueous solution (50% wt) again. After 2 hours, the reaction is completed. Filter the microspheres and wash them several times with butyl acetate, ethyl acetate and acetone. After vacuum drying, obtain the dry microspheres. The dried microspheres were soaked and stirred in pure water for 1 hour, filtered, and repeatedly washed with water. They were then soaked in 0.5M sodium hydroxide solution for 10 minutes and then washed with water. Reactive Blue 4 was also used to dye the microspheres to enhance their visibility. 10 ml of wet microspheres were taken, 0.1 g of Reactive Blue 4 was weighed and dissolved in 20 ml of water, and 0.08 g of sodium hydroxide was weighed and dissolved in the reactive blue aqueous solution. After complete dissolution, the solution was mixed with 10 ml of microspheres and stirred. The dyeing was continued for 1 hour, filtered, and washed with purified water. The wet microspheres were boiled in pure water for 20 minutes and filtered to obtain injectable embolic microspheres. The microspheres were wet-sieved through a stainless steel mesh to obtain embolic microspheres of different particle size distributions. Unwanted particle sizes were removed and the microspheres were sieved into the 100-300 μm range. The water content of the microspheres was measured to be 95.6%, and the elastic modulus was 87.2 Pa.

[0054] Example 2:

[0055] 3.2 g of cellulose acetate butyrate was added to 180 ml of butyl acetate and stirred to dissolve to form an oil phase. 5.5 g of polyvinyl alcohol (molecular weight ~100,000 Daltons) and 1 g of sodium polyacrylate (molecular weight ~150,000 Daltons) were dissolved in 45 g of purified water, stirred and mixed evenly, and concentrated hydrochloric acid solution was added to adjust the pH to form an aqueous phase. The aqueous phase and the oil phase were mixed by a stirrer at a stirring speed of 350 rpm to form an oil-in-water emulsion. 0.6 ml of glutaraldehyde aqueous solution (50% wt) was added to the reaction system. After reacting for 2 hours, 0.6 ml of glutaraldehyde aqueous solution (50% wt) was added to the reaction system. After reacting for 2 hours, 1.2 ml of glutaraldehyde aqueous solution (50% wt) was added again. After 2 hours, the reaction was completed. The microspheres were filtered and washed several times with butyl acetate, ethyl acetate and acetone. After vacuum drying, the dry microspheres were obtained. The dried microspheres were soaked in pure water and stirred for 1 hour, filtered and repeatedly washed with water, and then soaked in 0.5M sodium hydroxide solution for 10 minutes and then washed with water. In addition, the microspheres were dyed with Reactive Blue 4 to enhance the visibility of the microspheres. 10 ml of wet microspheres were taken, 0.1 g of Reactive Blue 4 was weighed and dissolved in 20 ml of water, and 0.08 g of sodium hydroxide was weighed and dissolved in the reactive blue aqueous solution. After complete dissolution, the solution was mixed with 10 ml of microspheres and stirred. After dyeing for 1 hour, the solution was filtered and washed with purified water. The wet microspheres were boiled in pure water for 20 minutes and then filtered to obtain injectable embolic microspheres. The microspheres were wet-sieved through a stainless steel mesh to obtain embolic microspheres with different particle size distributions. Unwanted particles were removed and sieved into the range of 100-300 μm. The water content of the microspheres was measured to be 92.2%, and the elastic modulus was 92.2 Pa.

[0056] Example 3:

[0057] 14.2g of Tween 80 was added to 520ml of butyl acetate and stirred to dissolve to form an oil phase. 1.1g of polyvinyl alcohol (molecular weight of ~250,000 Daltons) and 1.0g of sodium polyacrylate (molecular weight of ~200,000 Daltons) were dissolved in 27g of purified water, stirred and mixed evenly, and concentrated hydrochloric acid solution was added to adjust the pH value to form an aqueous phase. The aqueous phase and the oil phase were mixed by a stirrer at a stirring speed of 500rpm to form an oil-in-water emulsion. 0.6ml of glutaraldehyde aqueous solution (50% Wt) was added and the reaction was completed after stirring for 6 hours. The microspheres were filtered and washed several times with butyl acetate, ethyl acetate and acetone in succession. After vacuum drying, the dry microspheres were obtained. The dried microspheres were soaked and stirred in pure water for 1 hour, filtered and repeatedly washed with water, and then soaked in 0.5M sodium hydroxide solution for 10 minutes and washed with water. In addition, the microspheres were dyed with reactive blue 4 to enhance the visibility of the microspheres. 10 ml of wet microspheres were taken, 0.1 g of Reactive Blue 4 was weighed and dissolved in 20 ml of water, and 0.08 g of sodium hydroxide was weighed and dissolved in the reactive blue aqueous solution. After complete dissolution, the solution was mixed with 10 ml of microspheres and stirred. After dyeing was continued for 1 hour, the microspheres were filtered and rinsed with purified water. The wet microspheres were boiled in pure water for 20 minutes and then filtered to obtain injectable embolic microspheres. The microspheres were wet-sieved through a stainless steel mesh to obtain embolic microspheres with different particle size distributions. Undesirable particle sizes were removed and the microspheres were sieved to the range of 100 to 300 μm. The water content of the microspheres was measured to be 93.2%, and the elastic modulus was 75.2 Pa.

[0058] Example 4:

[0059] 4.3 g of cellulose acetate butyrate was added to 160 ml of butyl acetate and stirred to dissolve to form an oil phase. 3.0 g of polyvinyl alcohol (molecular weight of 150,000 Daltons) and 1.0 g of sodium polyacrylate (molecular weight of 150,000 Daltons) were dissolved in 35 g of purified water, stirred and mixed evenly, and concentrated hydrochloric acid solution was added to adjust the pH value to form an aqueous phase. The aqueous phase and the oil phase were mixed by a stirrer at a stirring speed of 350 rpm to form an oil-in-water emulsion. 0.7 ml of glutaraldehyde aqueous solution (50% Wt) was added and the reaction was completed after stirring for 6 hours. The microspheres were filtered and washed several times with butyl acetate, ethyl acetate and acetone in sequence. After vacuum drying, the dry microspheres were obtained. The dried microspheres were soaked and stirred in pure water for 1 hour, filtered and repeatedly washed with water, and then soaked in 0.5 M sodium hydroxide solution for 10 minutes and washed with water. In addition, the microspheres were dyed with reactive blue 4 to enhance the visibility of the microspheres. 10 ml of wet microspheres were taken, 0.1 g of Reactive Blue 4 was weighed and dissolved in 20 ml of water, and 0.08 g of sodium hydroxide was weighed and dissolved in the reactive blue aqueous solution. After complete dissolution, the solution was mixed with 10 ml of microspheres and stirred. After dyeing was continued for 1 hour, the microspheres were filtered and rinsed with purified water. The wet microspheres were boiled in pure water for 20 minutes and then filtered to obtain injectable embolic microspheres. The microspheres were wet-sieved through a stainless steel mesh to obtain embolic microspheres with different particle size distributions. Undesirable particle sizes were removed and the microspheres were sieved to the range of 100 to 300 μm. The water content of the microspheres was measured to be 97.1%, and the elastic modulus was 102.2 Pa.

[0060] Example 5:

[0061] 3.2g of cellulose acetate butyrate was added to 90ml of butyl acetate and stirred to dissolve to form an oil phase. 2.0g of polyvinyl alcohol (molecular weight of 150,000 Daltons) and 1.0g of polyacrylic acid (molecular weight of 150,000 Daltons) were dissolved in 45g of purified water, stirred and mixed evenly, and concentrated hydrochloric acid solution was added to adjust the pH value to form an aqueous phase. The aqueous phase and the oil phase were mixed by a stirrer at a stirring speed of 350rpm to form an oil-in-water emulsion. 1.2ml of glutaraldehyde aqueous solution (50% Wt) was added and the reaction was completed after stirring for 6 hours. The microspheres were filtered and washed several times with butyl acetate, ethyl acetate and acetone in sequence. After vacuum drying, the dry microspheres were obtained. The dried microspheres were soaked and stirred in pure water for 1 hour, filtered and repeatedly washed with water, and then soaked in 0.5M sodium hydroxide solution for 10 minutes and washed with water. In addition, the microspheres were dyed with reactive blue 4 to enhance the visibility of the microspheres. 10 ml of wet microspheres were taken, 0.1 g of Reactive Blue 4 was weighed and dissolved in 20 ml of water, and 0.08 g of sodium hydroxide was weighed and dissolved in the reactive blue aqueous solution. After complete dissolution, the solution was mixed with 10 ml of microspheres and stirred. After dyeing was continued for 1 hour, the microspheres were filtered and rinsed with purified water. The wet microspheres were boiled in pure water for 20 minutes and then filtered to obtain injectable embolic microspheres. The microspheres were wet-sieved through a stainless steel mesh to obtain embolic microspheres with different particle size distributions. Undesirable particle sizes were removed and the microspheres were sieved to the range of 100 to 300 μm. The water content of the microspheres was measured to be 92.6%, and the elastic modulus was 162.8 Pa.

[0062] Example 6:

[0063] 9.1g of cellulose acetate butyrate was added to 200ml of butyl acetate and stirred to dissolve to form an oil phase. 1.0g of polyvinyl alcohol (molecular weight of 150,000 daltons) and 0.6g of sodium polyvinyl sulfonate (molecular weight of 300,000 daltons) were dissolved in 40g of purified water, stirred and mixed evenly, and concentrated hydrochloric acid solution was added to adjust the pH value to form an aqueous phase. The aqueous phase and the oil phase were mixed by a stirrer at a stirring speed of 350rpm to form an oil-in-water emulsion. 0.75ml of glutaraldehyde aqueous solution (50% Wt) was added and the reaction was completed after stirring for 6 hours. The microspheres were filtered and washed several times with butyl acetate, ethyl acetate and acetone in sequence. After vacuum drying, the dry microspheres were obtained. The dried microspheres were soaked and stirred in pure water for 1 hour, filtered and repeatedly washed with water, and then soaked in 0.5M sodium hydroxide solution for 10 minutes and washed with water. In addition, the microspheres were dyed with reactive blue 4 to enhance the visibility of the microspheres. 10 ml of wet microspheres were taken, 0.1 g of Reactive Blue 4 was weighed and dissolved in 20 ml of water, and 0.08 g of sodium hydroxide was weighed and dissolved in the reactive blue aqueous solution. After complete dissolution, the solution was mixed with 10 ml of microspheres and stirred. After dyeing was continued for 1 hour, the microspheres were filtered and rinsed with purified water. The wet microspheres were boiled in pure water for 20 minutes and then filtered to obtain injectable embolic microspheres. The microspheres were wet-sieved through a stainless steel mesh to obtain embolic microspheres with different particle size distributions. Undesirable particle sizes were removed and the microspheres were sieved to a range of 100 to 300 μm. The water content was measured to be 92.5%, and the elastic modulus was 173.2 Pa.

[0064] Example 7:

[0065] 5.3 g of cellulose acetate butyrate was added to 200 ml of butyl acetate and stirred to dissolve to form an oil phase. 1.0 g of polyvinyl alcohol (molecular weight of 250,000 Daltons) and 5.0 g of sodium poly(p-styrene sulfonate) (molecular weight of 1,000,000 Daltons) were dissolved in 45 g of purified water, stirred and mixed evenly, and concentrated hydrochloric acid solution was added to adjust the pH value to form an aqueous phase. The aqueous phase and the oil phase were mixed by a stirrer at a stirring speed of 350 rpm to form an oil-in-water emulsion. 0.6 ml of 50% glutaraldehyde aqueous solution was added and the reaction was completed after stirring for 6 hours. The microspheres were filtered and washed several times with butyl acetate, ethyl acetate and acetone in sequence. After vacuum drying, the dry microspheres were obtained. The dried microspheres were soaked and stirred in pure water for 1 hour, filtered and repeatedly washed with water, and then soaked in 0.5 M sodium hydroxide solution for 10 minutes and washed with water. In addition, the microspheres were dyed with reactive blue 4 to enhance the visibility of the microspheres. 10 ml of wet microspheres were taken, 0.1 g of Reactive Blue 4 was weighed and dissolved in 20 ml of water, and 0.08 g of sodium hydroxide was weighed and dissolved in the reactive blue aqueous solution. After complete dissolution, the solution was mixed with 10 ml of microspheres and stirred. After dyeing was continued for 1 hour, the microspheres were filtered and rinsed with purified water. The wet microspheres were boiled in pure water for 20 minutes and then filtered to obtain injectable embolic microspheres. The microspheres were wet-sieved through a stainless steel mesh to obtain embolic microspheres with different particle size distributions. Undesirable particle sizes were removed and the microspheres were sieved to a range of 100 to 300 μm. The water content was measured to be 94.2%, and the elastic modulus was 154.62 Pa.

[0066] Example 8:

[0067] 2.6 g of cellulose acetate butyrate was added to 180 ml of butyl acetate and stirred to dissolve to form an oil phase. 1.0 g of polyvinyl alcohol (molecular weight of 150,000 Daltons) and 2.0 g of sodium poly(p-styrene sulfonate) (molecular weight of 1,000,000 Daltons) were dissolved in 40 g of purified water, stirred and mixed evenly, and concentrated hydrochloric acid solution was added to adjust the pH value to form an aqueous phase. The aqueous phase and the oil phase were mixed by a stirrer at a stirring speed of 350 rpm to form an oil-in-water emulsion. 0.9 ml of 50% glutaraldehyde aqueous solution was added and the reaction was completed after stirring for 6 hours. The microspheres were filtered and washed several times with butyl acetate, ethyl acetate and acetone in sequence. After vacuum drying, the dry microspheres were obtained. The dried microspheres were soaked and stirred in pure water for 1 hour, filtered and repeatedly washed with water, and then soaked in 0.5 M sodium hydroxide solution for 10 minutes and then washed with water. In addition, the microspheres were dyed with reactive blue 4 to enhance the visibility of the microspheres. 10 ml of wet microspheres were taken, 0.1 g of Reactive Blue 4 was weighed and dissolved in 20 ml of water, and 0.08 g of sodium hydroxide was weighed and dissolved in the reactive blue aqueous solution. After complete dissolution, the solution was mixed with 10 ml of microspheres and stirred. After dyeing was continued for 1 hour, the microspheres were filtered and rinsed with purified water. The wet microspheres were boiled in pure water for 20 minutes and then filtered to obtain injectable embolic microspheres. The microspheres were wet-sieved through a stainless steel mesh to obtain embolic microspheres with different particle size distributions. Undesirable particle sizes were removed and the microspheres were sieved to a range of 100 to 300 μm. The water content was measured to be 90.3%, and the elastic modulus was 85.3 Pa.

[0068] Comparative Example 1

[0069] 3.2 g of cellulose acetate butyrate was added to 180 ml of butyl acetate and stirred to dissolve to form an oil phase. 6.5 g of polyvinyl alcohol (molecular weight ~100,000 Daltons) and 1 g of sodium polyacrylate (molecular weight ~150,000 Daltons) were dissolved in 45 g of purified water and stirred to mix evenly. Concentrated hydrochloric acid solution was added to adjust the pH value to form an aqueous phase. The aqueous phase and the oil phase were mixed by a stirrer at a stirring speed of 350 rpm to form an oil-in-water emulsion. 0.6 ml of glutaraldehyde aqueous solution (50% wt) was added to the reaction system. After reacting for 2 hours, 0.6 ml of glutaraldehyde aqueous solution (50% wt) was added to the reaction system. After reacting for 2 hours, 1.2 ml of glutaraldehyde aqueous solution (50% wt) was added again. After 2 hours, the reaction was completed. The microspheres were filtered and washed several times with butyl acetate, ethyl acetate and acetone. After vacuum drying, the dry microspheres were obtained. The dried microspheres were soaked in pure water and stirred for 1 hour, filtered and repeatedly washed with water, and then soaked in 0.5M sodium hydroxide solution for 10 minutes and then washed with water. In addition, the microspheres were dyed with Reactive Blue 4 to enhance the visibility of the microspheres. 10ml of wet microspheres were taken, 0.1g of Reactive Blue 4 was weighed and dissolved in 20ml of water, and 0.08g of sodium hydroxide was weighed and dissolved in the reactive blue aqueous solution. After complete dissolution, the solution was mixed with 10ml of microspheres and stirred. After dyeing for 1 hour, the solution was filtered and washed with purified water. The wet microspheres were boiled in pure water for 20 minutes and then filtered to obtain injectable embolic microspheres. The microspheres were wet-sieved through a stainless steel mesh to obtain embolic microspheres with different particle size distributions. Unwanted particle sizes were removed and the microspheres were sieved into the range of 100-300μm. The water content was measured to be 82.6% and the elastic modulus was 386.1Pa.

[0070] In this embodiment, the molar ratio of the hydrophilic polyhydroxy polymer material to the polyacid ion-containing polymer material is 14:1. The high content of PVA in the microspheres increases the rigidity of the microspheres, reduces the water content, and weakens the elasticity, which is not conducive to embolization.

[0071] Comparative Example 2

[0072] 2.6g of Tween 80 was added to 180ml of butyl acetate and stirred to dissolve to form an oil phase. 1.1g of polyvinyl alcohol (molecular weight of ~250,000 Daltons) and 1.0g of sodium polyacrylate (molecular weight of ~200,000 Daltons) were dissolved in 27g of purified water, stirred and mixed evenly, and concentrated hydrochloric acid solution was added to adjust the pH value to form an aqueous phase. The aqueous phase and the oil phase were mixed by a stirrer at a stirring speed of 500rpm to form an oil-in-water emulsion. 1.4ml of glutaraldehyde aqueous solution (50% Wt) was added and the reaction was completed after stirring for 6 hours. The microspheres were filtered and washed several times with butyl acetate, ethyl acetate and acetone in sequence. After vacuum drying, the dry microspheres were obtained. The dried microspheres were soaked and stirred in pure water for 1 hour, filtered and repeatedly washed with water, and then soaked in 0.5M sodium hydroxide solution for 10 minutes and washed with water. In addition, the microspheres were dyed with reactive blue 4 to enhance the visibility of the microspheres. 10 ml of wet microspheres were taken, 0.1 g of Reactive Blue 4 was weighed and dissolved in 20 ml of water, and 0.08 g of sodium hydroxide was weighed and dissolved in the reactive blue aqueous solution. After complete dissolution, the solution was mixed with 10 ml of microspheres and stirred. After dyeing was continued for 1 hour, the microspheres were filtered and rinsed with purified water. The wet microspheres were boiled in pure water for 20 minutes and then filtered to obtain injectable embolic microspheres. The microspheres were wet-sieved through a stainless steel mesh to obtain embolic microspheres with different particle size distributions. Undesirable particle sizes were removed and the microspheres were sieved to a range of 100 to 300 μm. The water content was measured to be 78.3%, and the elastic modulus was 428.3 Pa.

[0073] In this embodiment, the molar ratio of the crosslinker to the hydrophilic polyhydroxy polymer is 1:3.36. The rigidity of the microspheres prepared by using too much crosslinker is significantly increased, while the water content is affected, resulting in weakened elasticity. The poor elastic deformation ability of the microspheres is not conducive to embolization.

[0074] Commercially available products:

[0075] Commercially available products: DC Bead, 100-300 μm, Biocompatibiles UK Limited.

[0076] Microsphere drug loading test

[0077] 10mg of doxorubicin hydrochloride was accurately weighed and dissolved in a 4mL bottle of pure water (at a concentration of 2.5mg / mL), and pirarubicin hydrochloride solution (at a concentration of 2.5mg / mL) and epirubicin hydrochloride solution (at a concentration of 2.5mg / mL) were prepared in the same manner. 200mg of wet microspheres with a particle size range of 100-300μm prepared in Example 1-8 were weighed, and the clear water was removed and added to the doxorubicin solution. The microspheres were gently shaken to soak evenly in the solution. 10uL of supernatant was sampled at regular intervals of 10, 15, 20, and 30min after the microspheres were added and diluted in 10mL of pure water. Doxorubicin concentration was determined by reverse-phase high performance liquid chromatography (C18, 150x4.6 mm) using a UV detector at a wavelength of 480 nm and an aqueous solution containing 30% (v / v) acetonitrile and 0.1% (v / v) trifluoroacetic acid as the mobile phase. The results are shown in Table 1. The drug loading was calculated using the formula:

[0078] Table 1 Results of drug loading of microspheres

[0079] In vitro dissolution test of microsphere drugs

[0080] Take the above-mentioned Example 3 and Example 7 microspheres loaded with doxorubicin and place them in 500mL of release medium. The release medium is a 0.9% sodium chloride buffer solution containing 10mM morpholineethanesulfonic acid and a pH of 6.0. The microspheres are stirred in the dark at 37°C in the release medium (150rpm), and 0.5mL of the supernatant is sampled at 1hr, 2hr, 3hr, 4hr, 18hr, 24hr, and 48hr time points, and an equal amount of release medium is immediately supplemented. The concentration of doxorubicin in the release medium is analyzed by the above-mentioned microsphere drug loading experiment HPLC method (see figure). The calculation formula for the amount of drug released in vitro is:

[0081] According to the release results, the microspheres prepared in Example 3 and Example 7 released more than 80% of the drug at 48 hours after being loaded with doxorubicin.

[0082] Stability evaluation test after drug loading into microspheres

[0083] Immediately after drug loading, the microspheres were frozen and stored in the dark at 2-8°C for 14 days. Samples were taken on the 7th and 14th days after drug loading and were analyzed by HPLC for changes in the main peak and impurity peaks of doxorubicin. The doxorubicin-loaded microspheres were placed in 500 mL of eluent (250 mL of a mixture of 20% w / v potassium chloride and 250 mL of anhydrous ethanol) and stirred in the dark for 7 hours. The supernatant was then analyzed by HPLC. The results are shown in Table 2.

[0084] Table 2 HPLC detection of the main peak and impurity peaks of doxorubicin

[0085] According to the test results, the microspheres prepared with polyanion polymers having sulfonic acid groups have better stability after adsorbing doxorubicin than polyanion polymers having carboxyl groups, and the degradation of doxorubicin in the microspheres is less than 1% within 14 days.

[0086] Compressibility determination of microspheres

[0087] A physical property analyzer (SMS TA.XT PlusC) equipped with a 5Kg sensor element, and a high-magnification camera and video synchronization device (VC / CSS) are used to confirm the placement of the microspheres. The rigidity of the microspheres is evaluated by measuring the elastic modulus of the microspheres. The elastic modulus can be regarded as an indicator of the ease with which a material can be compressed and deformed. The larger the value of the elastic modulus, the greater the stress required for a certain elastic deformation of the material. Therefore, the greater the rigidity, the smaller the elastic deformation. A flat single layer of microspheres is placed on the test bench, using a 5mm diameter circular probe, a speed of 1mm / s, a sensing force of 5g, and the gradient when the sample is compressed by 20% is the elastic modulus of the sample.

[0088] According to the test results, the elastic modulus values ​​of the microspheres prepared by the methods of Examples 1-8 are similar to those of commercially available products, and have good elasticity, ensuring that the microspheres can adapt to the morphology of blood vessels to the greatest extent and can fully embolize and adhere to blood vessels.

[0089] Determination of water content of microspheres

[0090] Measure the moisture content in the microspheres by measuring the difference between the weight of the hydrated microspheres and the weight of the dehydrated microspheres by gravimetric analysis. Take the microspheres prepared in the above example, remove the moisture on the surface of the microspheres and weigh them to obtain the weight of the hydrated microspheres. Dehydrate them in an 80°C oven for 24 hours, and then reweigh them every 8 hours until the mass of the microspheres is no longer reduced. The formula for calculating the moisture content of the microspheres is:

Claims

1. A method for preparing negatively charged embolic microspheres, characterized in that: The following steps are involved: A) dissolving a hydrophilic polyhydroxy polymer and a polymer containing polyacid ions in water, stirring uniformly, to obtain an aqueous phase; B) dissolving the dispersant in an organic solvent and stirring the mixture to obtain an oil phase; C) adding the aqueous phase to the oil phase for dispersion to obtain a mixed emulsion, adding a crosslinking agent to the mixed emulsion once or several times and stirring to obtain microspheres; D) washing and filtering the microspheres obtained in step C), After the aqueous phase is prepared in step A) or after the mixed emulsion is obtained in step C), the pH is adjusted to be acidic.

2. The method according to claim 1, characterized in that The molar ratio of the hydrophilic polyhydroxy polymer to the polymer containing polyacid ions is 0.8:1 to 12:

1.

3. The method according to claim 2, characterized in that The molar ratio of the hydrophilic polyhydroxy polymer to the polymer containing polyacid ions is 2.3:1 to 12:

1.

4. The method according to claim 1, characterized in that: The total weight of the hydrophilic polyhydroxy polymer and the polymer containing polyacid ions accounts for 4% to 15% of the total weight of the water phase.

5. The method according to claim 1, characterized in that The molar ratio of the crosslinking agent to the hydrophilic polyhydroxy polymer is 1:4 to 1:

19.

6. The method according to claim 1, characterized in that The volume ratio of water in the aqueous phase to the organic solvent in the oil phase is 1:1.5 to 1:

20.

7. The method according to claim 1, characterized in that The organic solvent for preparing the oil phase is one or a mixture of butyl acetate, ethyl acetate and n-heptane.

8. The method according to claim 1, characterized in that The mass percentage of the dispersant in the oil phase is 0.8% to 5.0%.

9. The method according to claim 1, characterized in that: The hydrophilic polyhydroxy polymer is polyvinyl alcohol.

10. The method according to claim 9, characterized in that The molecular weight of the polyvinyl alcohol is 20 to 600 kilodaltons.

11. The method according to claim 1, characterized in that: The polymer containing polyacid ions is a polymer containing polycarboxyl functional groups and / or a polymer containing polysulfonic functional groups.

12. The method according to claim 11, characterized in that The polymer containing multiple carboxyl functional groups is polyacrylic acid and / or its salt, polymethacrylic acid and / or its salt, and the polymer containing multiple sulfonic functional groups is poly(p-styrene sulfonic acid) and / or its salt, poly(vinyl sulfonic acid) and / or its salt.

13. The method according to claim 12, characterized in that The polymer containing multiple carboxyl functional groups is one or more of sodium polyacrylate (PAANa), partially neutralized sodium polyacrylate, polyacrylic acid (PAA), sodium polymethacrylate and polymethacrylic acid, and the molecular weight of a single type of polymer is 10 to 600 kilodaltons, wherein the mass ratio of acrylic acid to sodium acrylate in the partially neutralized sodium polyacrylate is 2:8 to 8:2; the polymer containing multiple sulfonic functional groups is one or more of sodium poly(p-styrene sulfonate), poly(p-styrene sulfonic acid), sodium poly(ethylene sulfonate) and poly(ethylene sulfonic acid), and the molecular weight of a single type of polymer is 20 to 2000 kilodaltons.

14. The method according to claim 1, characterized in that The cross-linking agent is an aldehyde compound.

15. The method according to claim 14, characterized in that The cross-linking agent is glyoxal and / or glutaraldehyde.

16. The method according to claim 1, characterized in that The dispersant is one or a combination of Span 80, Tween 80, cellulose acetate and cellulose acetate butyrate.

17. The method according to claim 1, characterized in that In step C), the aqueous phase is dispersed in the oil phase by shearing or stirring, wherein the stirring speed is 50 rpm to 1000 rpm.

18. The method according to claim 1, characterized in that After the aqueous phase is prepared in step A) or after the mixed emulsion is obtained in step C), one or more of hydrochloric acid, sulfuric acid and acetic acid are added to adjust the pH to 3-5; the microspheres collected by filtration in step D) are washed with butyl acetate and ethyl acetate in turn, and then washed with acetone or ethanol, and then vacuum dried to obtain dry microspheres, the dry microspheres are soaked in pure water and filtered, then soaked in sodium hydroxide solution and filtered with water, and then filtered, sieved and collected after stirring in boiling water.

19. Negatively charged embolic microspheres prepared according to the method of any one of the preceding claims.

20. A use of the negatively charged embolic microspheres according to claim 19, characterized in that: The embolic microspheres are used to adsorb one or more of doxorubicin, epirubicin, pirarubicin, irinotecan, mitoxantrone, daunorubicin, topotecan, bleomycin, oxaliplatin, gemcitabine and 5-fluorouracil.

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