Preparation method of sodium hyaluronate embolism microspheres and prepared embolism microspheres

Abstract

The invention relates to the technical field of medical interventional therapy materials, and particularly discloses a preparation method of sodium hyaluronate embolism microspheres and the prepared embolism microspheres. The preparation method of the sodium hyaluronate embolization microspheres comprises the following steps: S1, adding methacrylic anhydride, N, N-dimethylformamide and sodium hydroxide into a sodium hyaluronate aqueous solution, reacting, adding sodium chloride, reacting, adding absolute ethyl alcohol, precipitating, filtering to remove filtrate, cleaning and drying; s2, preparing a mixture of methacrylated sodium hyaluronate, a monomer with negative charges, an initiator and water into a water phase; a mixture of oil soluble liquid and a dispersing agent is used as an oil phase; and under the protection of nitrogen, carrying out reverse suspension polymerization. According to the preparation method of the sodium hyaluronate embolization microspheres, the methylacryloylated sodium hyaluronate and the negatively charged monomer are copolymerized to introduce the negatively charged group, so that the microspheres can effectively load chemotherapeutic drugs, and the compression elasticity is high.

Description

Technical Field

[0001] The present application relates to the technical field of medical interventional treatment materials, and more specifically, to a method for preparing sodium hyaluronate embolic microspheres and the prepared embolic microspheres. Background Art

[0002] In recent years, with the development of medical interventional technology, microsphere embolic agents have developed rapidly and have a wide range of applications. Polyvinyl alcohol embolic microspheres, modified sodium hyaluronate microspheres, chitosan microspheres, sodium alginate microspheres, and albumin microspheres have emerged. Each microsphere product can have physical and chemical advantages such as non-degradable, degradable, moisturizing, strong elasticity or compliance, and also shows broad application prospects in the fields of tumor embolization, medical beauty industry, drug embedding, etc.

[0003] Hyaluronic acid, as a high-molecular polysaccharide, is abundant in the extracellular matrix and contributes to the hydrodynamics and homeostasis of tissues. It has negatively charged groups, high water absorption, strong moisturizing factor, and multiple physiological functions such as mediating cell proliferation, differentiation, and immune regulation.

[0004] For example, CN114106252B discloses an embolic microsphere, which uses N-(2,2-dimethoxy)-2-methylacrylamide to modify sodium hyaluronate, so that sodium acrylate is grafted with allyl groups to further undergo polymerization reaction to obtain microspheres.

[0005] However, the raw materials of N-(2,2-dimethoxy)-2-methylacrylamide are expensive, and the steric hindrance between the dimethoxy groups in N-(2,2-dimethoxy)-2-methylacrylamide is large, and the reaction with the hydroxyl group in sodium hyaluronate is unstable, and the prepared microspheres have poor compression elasticity. Summary of the invention

[0006] In order to improve the compression elasticity of microspheres, the present application provides a method for preparing sodium hyaluronate embolic microspheres and the prepared embolic microspheres.

[0007] In the first aspect, the present application provides a method for preparing sodium hyaluronate embolic microspheres, using the following technical solution: A method for preparing sodium hyaluronate embolic microspheres comprises the following steps: S1. Preparation of methacryloyl sodium hyaluronate: add methacrylic anhydride, N,N-dimethylformamide and sodium hydroxide to a sodium hyaluronate aqueous solution, react at 25-40° C., then add sodium chloride, and after the reaction is complete, add anhydrous ethanol to precipitate, filter to remove the filtrate, wash and dry to obtain; The weight ratio of the added amount of methacrylic anhydride to sodium hyaluronate is 0.5-2:1; S2. Preparation of embolic microspheres: using a mixture of methacryloyl sodium hyaluronate, negatively charged monomers, initiators, and water as the aqueous phase; using a mixture of oil-soluble liquid and dispersant as the oil phase; and obtaining the microspheres by reverse suspension polymerization under nitrogen protection; The weight ratio of methacrylated sodium hyaluronate, negatively charged monomer and initiator is 1:0.005-0.015:0.2-0.5.

[0008] By adopting the above technical solution: This application uses methacryloyl sodium hyaluronate as raw material. Compared with sodium hyaluronate, an allyl double bond is introduced into the structure of sodium hyaluronate, and self-polymerization reaction occurs under the action of an initiator. At the same time, copolymerization reaction occurs with negatively charged monomers to introduce negatively charged groups into the side chain, which can effectively load chemotherapeutic drugs, is safe and reliable, and has excellent performance.

[0009] Methacrylated sodium hyaluronate is an esterification reaction between the carboxyl group in methacrylic anhydride and the hydroxyl group in sodium hyaluronate. This reaction is stable and reliable, and methacrylic anhydride is a conventional raw material, which has the characteristics of low price and high purity. The prepared microspheres have better compression elasticity. After testing, the elastic modulus of the microspheres of the present application at 50% compression deformation is above 43.67kPa, and the hardness of 50% compression deformation is above 103.36gf; it is much better than the elastic modulus of 50% compression deformation of the microspheres made of sodium hyaluronate modified with N-(2,2-dimethoxy)-2-methacrylamide at 25.67kPa, and the hardness of 50% compression deformation is 48.47gf.

[0010] By controlling the weight ratio of methacrylic anhydride to sodium hyaluronate, as well as the concentrations of methacrylylated sodium hyaluronate, negatively charged monomers, and initiators in the aqueous phase, the resulting embolic microspheres have a certain strength and excellent adsorption and sustained-release properties for drugs, with a drug loading capacity of more than 60 mg / ml, a drug loading efficiency of more than 90.01% in 15 minutes, and can prolong the drug efficacy.

[0011] Optionally, in step S1, the concentration of the sodium hyaluronate aqueous solution is 0.02-0.4 g / mL.

[0012] By adopting the above technical solution: the particle size of the microspheres is controlled to be suitable for conventional embolization, the microspheres have intact morphology, certain elasticity and expansibility, no toxic side effects to the human body, and good biocompatibility.

[0013] Optionally, in step S1, the molecular weight of sodium hyaluronate in the sodium hyaluronate aqueous solution is 2000-100000Da.

[0014] By adopting the above technical solution, the obtained embolic microspheres have appropriate viscoelasticity.

[0015] Optionally, in step S1, the volume ratio of the added amount of N,N-dimethylformamide to the sodium hyaluronate aqueous solution is 0.5-2:1.

[0016] By adopting the above technical solution: when the amount of N,N-dimethylformamide is within the above range, microemulsion can be effectively emulsified to form microemulsion, so as to prepare microspheres.

[0017] Optionally, in step S1, the amount of sodium hydroxide added is to adjust the pH of the system to 9-11.

[0018] Optionally, in step S2, the weight ratio of the oil-soluble liquid to the dispersant is 100g:0.5-1.8.

[0019] Optionally, the step S2, The initiator is ammonium persulfate; The oil-soluble liquid is butyl acetate or liquid paraffin; The dispersant is one of Span60, Span80, cellulose acetate, and EM-90; The negatively charged monomer is one of sodium acrylate, sodium methacrylate, sodium methacrylate sulfonate, and sodium 2-acrylamide-2-methyl-1-propane sulfonate.

[0020] By adopting the above technical solution: when one of sodium acrylate, sodium methacrylate, sodium methacrylate sulfonate and sodium 2-acrylamide-2-methyl-1-propane sulfonate is used, the microspheres maintain the main structure of sodium hyaluronate, so that the microspheres have good biocompatibility.

[0021] At the same time, sodium acetate or sodium sulfonate structure is introduced into the side chain of the microspheres, and sodium hyaluronate, sodium acrylate, sodium methacrylate, sodium methacrylate sulfonate, and sodium 2-acrylamide-2-methyl-1-propanesulfonate all have good hydrophilicity, making the microspheres hydrophilic and able to absorb 10-30 times their own weight of water. Sodium acetate or sodium sulfonate is ionized in water with a negative charge and can effectively adsorb positively charged chemotherapy drugs, such as doxorubicin, with an effective load of 30-70 mg / ml.

[0022] Optionally, in step S2, the volume ratio of the water phase to the oil phase is 1:3-10.

[0023] In the second aspect, the present application provides a sodium hyaluronate embolic microsphere, which adopts the following technical solution: A sodium hyaluronate embolic microsphere is prepared by any of the preparation methods described above, and the particle size of the embolic microsphere is 60-1200 μm.

[0024] In summary, this application has the following beneficial effects: 1. The present application uses methacrylylated sodium hyaluronate to copolymerize with negatively charged monomers, which can effectively load chemotherapy drugs, and the drug loading rate in 15 minutes is as high as 90.01% or more; at the same time, methacrylylated sodium hyaluronate is an esterification reaction between the carboxyl group in methacrylic anhydride and the hydroxyl group in sodium hyaluronate, and this reaction is stable and reliable. In addition, methacrylic anhydride is a conventional raw material, which has the characteristics of low price and high purity. The prepared microspheres have better compression elasticity, the elastic modulus of 50% compression deformation is above 43.67 kPa, and the hardness of 50% compression deformation is above 103.36 gf; it is much better than the compression elasticity of microspheres prepared by sodium hyaluronate modified with N-(2,2-dimethoxy)-2-methacrylamide; 2. In this application, various parameters such as the dosage ratio of each component, the concentration range of the sodium hyaluronate aqueous solution, the molecular weight range of sodium hyaluronate, the volume ratio of the aqueous phase and the oil phase are controlled to achieve the best performance of the prepared microspheres. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a photograph of the embolic microspheres of Example 1 of the present application; Figure 2 This is a photograph of the embolic microspheres after being loaded with doxorubicin in Example 1 of the present application. DETAILED DESCRIPTION

[0026] The present application is further described in detail below in conjunction with embodiments.

[0027] The sources of the raw materials are as follows. Except for the following special instructions, the components are commercially available: Sodium hyaluronate of various molecular weights was sourced from Shaanxi Chenming Biotechnology Co., Ltd.

[0028] Example 1 A sodium hyaluronate embolic microsphere, see attached Figure 1 , which is prepared by the following steps: S1. Preparation of sodium methacrylated hyaluronate: Sodium hyaluronate (molecular weight 2000Da) was dissolved in deionized water to obtain a sodium hyaluronate aqueous solution (concentration 0.02g / mL). 0.5L N,N-dimethylformamide and 10g methacrylic anhydride were added to 1L sodium hyaluronate aqueous solution, and sodium hydroxide was added to adjust the pH of the system to 9. The mixture was stirred at 25°C for 24h, and then 4g sodium chloride was added to react for 0.5h. 1L anhydrous ethanol was added for precipitation, and the filtrate was removed by filtration. Anhydrous ethanol, 80% ethanol water, and 60% ethanol water were added in a 1:10 solid-liquid ratio for gradient washing. The gradient washing was repeated five times, and the washed product was dried under vacuum at 40°C to obtain sodium methacrylated hyaluronate. S2. Add sodium methacrylated hyaluronate, negatively charged monomer (sodium acrylate), and initiator (ammonium persulfate) into water and mix well to obtain an aqueous phase; wherein the volume-to-weight ratio of water, sodium methacrylated hyaluronate, negatively charged monomer, and initiator is 100 mL: 2 g: 0.4 g: 0.01 g.

[0029] 0.5 g of dispersant (Span 60) was added to 100 g of oil-soluble liquid (butyl acetate), and the mixture was mixed to obtain an oil phase.

[0030] After purging 300 mL of the oil phase with nitrogen, the nitrogen atmosphere was maintained, and 100 mL of water was added dropwise under mechanical stirring at a speed of 120 rpm for reverse suspension to form an oil-in-water reverse suspension polymerization system. After the addition was completed, the polymerization system was heated to 40°C and reacted for 6 hours. The filtrate was removed by suction, and clean water was added for washing once according to a solid-liquid ratio of 1:10, and sieved to obtain embolic microspheres with a particle size of 60-200 μm. Example 2 A sodium hyaluronate embolic microsphere is prepared by the following steps: S1. Preparation of sodium methacrylated hyaluronate: Sodium hyaluronate (molecular weight 60000Da) was dissolved in deionized water to obtain a sodium hyaluronate aqueous solution (concentration 0.2g / mL). 1L N,N-dimethylformamide and 200g methacrylic anhydride were added to 1L sodium hyaluronate aqueous solution, and sodium hydroxide was added to adjust the pH of the system to 10. The mixture was stirred at 25°C for 24h, and then 40g sodium chloride was added to react for 0.5h. 1L anhydrous ethanol was added for precipitation, and the filtrate was removed by filtration. Anhydrous ethanol, 80% ethanol water, and 60% ethanol water were added in a 1:10 solid-liquid ratio for gradient washing. The gradient washing was repeated five times, and the washed product was dried under vacuum at 40°C to obtain sodium methacrylated hyaluronate. S2. Add sodium methacrylate hyaluronate, negatively charged monomer (sodium methacrylate sulfonate, and initiator (ammonium persulfate) into water and mix well to obtain an aqueous phase; wherein the volume weight ratio of water, sodium methacrylate hyaluronate, negatively charged monomer, and initiator is 100 mL: 20 g: 6 g: 0.2 g.

[0031] Add 1 g of dispersant (Span80) to 100 g of oil-soluble liquid (butyl acetate), mix well to obtain an oil phase; After purging 700mL of the oil phase with nitrogen, maintaining the nitrogen atmosphere, under the condition of mechanical stirring at a speed of 250rpm, 100mL of water was added dropwise for reverse suspension to form an oil-in-water reverse suspension polymerization system. After the addition was completed, the polymerization system was heated to 50℃ and reacted for 7h. The filtrate was removed by suction and washed once with clean water according to a solid-liquid ratio of 1:10. After sieving, embolic microspheres with a particle size of 200-800μm were obtained.

[0032] Example 3 A sodium hyaluronate embolic microsphere is prepared by the following steps: S1. Preparation of sodium methacrylated hyaluronate: Sodium hyaluronate (molecular weight 100000Da) was dissolved in deionized water to obtain a sodium hyaluronate aqueous solution (concentration 0.4g / mL). 2L N,N-dimethylformamide and 800g methacrylic anhydride were added to 1L sodium hyaluronate aqueous solution, and sodium hydroxide was added to adjust the pH of the system to 11. The mixture was stirred at 25°C for 24h, and then 40g sodium chloride was added to react for 0.5h. 1L anhydrous ethanol was added for precipitation, and the filtrate was removed by filtration. Anhydrous ethanol, 80% ethanol water, and 60% ethanol water were added in a 1:10 solid-liquid ratio for gradient washing. The gradient washing was repeated five times, and the washed product was dried under vacuum at 40°C to obtain sodium methacrylated hyaluronate. S2. Add sodium methacrylated hyaluronate, negatively charged monomer (sodium methacrylate), and initiator (ammonium persulfate) into water and mix well to obtain an aqueous phase; wherein the volume-to-weight ratio of water, sodium methacrylated hyaluronate, negatively charged monomer, and initiator is 100 mL: 40 g: 20 g: 0.6 g.

[0033] 1.8 g of dispersant (EM-90) was added to 120 g of oil-soluble liquid (liquid paraffin), and the mixture was mixed to obtain an oil phase.

[0034] After purging 700mL of the oil phase with nitrogen, maintaining the nitrogen atmosphere, under the condition of mechanical stirring at a speed of 120rpm, 100mL of water was added dropwise for reverse suspension to form an oil-in-water reverse suspension polymerization system. After the addition was completed, the polymerization system was heated to 60℃ and reacted for 4h. The filtrate was removed by suction and washed once with clean water according to a solid-liquid ratio of 1:10. After sieving, embolic microspheres with a particle size of 800-1200μm were obtained.

[0035] Comparative Example 1 A sodium hyaluronate embolic microsphere is prepared by the following steps: S1. Add 18.00 g of sodium hyaluronate (molecular weight 60,000 Da) to 150 g of water, heat to 90° C. and stir to dissolve, then add 0.25 g of N-(2,2-dimethoxy)-2-methylacrylamide, stir evenly, add 12.5 g of sulfuric acid and stir for 12 h, then add sodium hydroxide to neutralize to obtain a high molecular monomer aqueous solution; S2, 7.00 g of sodium acrylate, 1.00 g of potassium persulfate and the polymer monomer aqueous solution were mixed, and the pH value of the solution was adjusted by adding hydrochloric acid aqueous solution to obtain an aqueous phase with a pH of 2.5; S3, dissolving 20.00g of cellulose acetate butyrate in 1200g of ethyl acetate, stirring and dissolving at a temperature of 35°C to obtain an oil phase; S4, under stirring, adding water phase dropwise to the oil phase to form an oil-in-water reverse suspension polymerization system. After the addition is completed, the reverse suspension polymerization system is heated to 85°C, and then 8.00g of tetramethyldiamine is added. After reacting for 15h, heating and stirring are stopped, and the mixture is allowed to stand. After stratification, the oil phase is separated, and the embolic microspheres are collected and repeatedly washed.

[0036] Performance Testing The embolic microspheres prepared in the examples and comparative examples were subjected to the following performance tests, and the test results are recorded in Table 1.

[0037] Detection Methods 1. Drug loading performance test: Use a 20mL syringe with a 20g needle to draw 1mL of embolic microspheres, dissolve 50mg of doxorubicin hydrochloride in 10mL of normal saline, install the resulting doxorubicin hydrochloride solution on a three-way valve, mix the microspheres and doxorubicin hydrochloride solution and place at 25°C, start timing, draw the supernatant at 15min, 30min, 45min, 60min, and 120min, detect the doxorubicin concentration in the supernatant by HPLC, and calculate the drug loading by the differential method. The electron microscopy photos of the embolic microspheres loaded with doxorubicin in Example 1 are attached. Figure 2 .

[0038] 2. Compression elasticity test: Place the microsphere-saline mixture in a clean surface dish and spread the microspheres evenly on the surface dish. Use a paper towel to absorb the saline and use a universal material testing machine (including a 5mm diameter stainless steel parallel probe) for testing. Test conditions: compression speed 0.2mm / s, hold for 10s when the compression deformation is 50%, return speed 0.2mm / s, automatically collect data, time point 500pps, record the elastic modulus and hardness of the microspheres.

[0039] Table 1 Performance test results In Examples 1-3, methacrylic anhydride is used to graft sodium hyaluronate as a raw material, so that the elastic modulus of the embolic microspheres at 50% compression deformation is above 43.67 kPa, and the maximum can reach 95.76 kPa, the hardness at 50% compression deformation is above 103.36 gf, and the maximum can reach 168.81 gf, and the drug loading efficiency is above 90.01% in 15 minutes, and the drug loading efficiency is above 91.2% in 120 minutes; while in Comparative Example 1, N-(2,2-dimethoxy)-2-methylacrylamide is used to modify sodium hyaluronate as a raw material, so that the elastic modulus of the embolic microspheres at 50% compression deformation is 25.67 kPa, the hardness at 50% compression deformation is 48.47 gf, the drug loading efficiency is 61.26% in 15 minutes, and the drug loading efficiency is 61.66% in 120 minutes. This shows that the embolic microspheres of the present application have higher compression elasticity and drug loading capacity.

[0040] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed. However, as long as it is within the scope of the claims of the present application, it shall be protected by the patent law.

Claims

1. A method for preparing sodium hyaluronate embolic microspheres, characterized in that: The following steps are involved: S1. Preparation of methacryloyl sodium hyaluronate: add methacrylic anhydride, N,N-dimethylformamide and sodium hydroxide to a sodium hyaluronate aqueous solution, react at 25-40° C., then add sodium chloride, and after the reaction is complete, add anhydrous ethanol to precipitate, filter to remove the filtrate, wash and dry to obtain; The weight ratio of the added amount of methacrylic anhydride to sodium hyaluronate is 0.5-2:1; S2. Preparation of embolic microspheres: using a mixture of methacryloyl sodium hyaluronate, negatively charged monomers, initiators, and water as the aqueous phase; using a mixture of oil-soluble liquid and dispersant as the oil phase; and obtaining the microspheres by reverse suspension polymerization under nitrogen protection; The weight ratio of methacrylated sodium hyaluronate, negatively charged monomer and initiator is 1:0.005-0.015:0.2-0.

5.

2. The method for preparing sodium hyaluronate embolic microspheres according to claim 1, characterized in that: In step S1, the concentration of the sodium hyaluronate aqueous solution is 0.02-0.4 g / mL.

3. The method for preparing sodium hyaluronate embolic microspheres according to claim 2, characterized in that: In the step S1, the molecular weight of sodium hyaluronate in the sodium hyaluronate aqueous solution is 2000-100000Da.

4. The method for preparing sodium hyaluronate embolic microspheres according to claim 1, characterized in that: In step S1, the volume ratio of the added amount of N,N-dimethylformamide to the sodium hyaluronate aqueous solution is 0.5-2:

1.

5. The method for preparing sodium hyaluronate embolic microspheres according to claim 1, characterized in that: In step S1, the amount of sodium hydroxide added is to adjust the pH of the system to 9-11.

6. The method for preparing sodium hyaluronate embolic microspheres according to claim 1, characterized in that: In the step S2, the weight ratio of the oil-soluble liquid to the dispersant is 100g:0.5-1.

8.

7. The method for preparing sodium hyaluronate embolic microspheres according to claim 6, characterized in that: The step S2, The initiator is ammonium persulfate; The oil-soluble liquid is butyl acetate or liquid paraffin; The dispersant is one of Span60, Span80, cellulose acetate, and EM-90; The negatively charged monomer is one of sodium acrylate, sodium methacrylate, sodium methacrylate sulfonate, and sodium 2-acrylamide-2-methyl-1-propane sulfonate.

8. The method for preparing sodium hyaluronate embolic microspheres according to claim 1, characterized in that: In step S2, the volume ratio of the water phase to the oil phase is 1:3-10.

9. A sodium hyaluronate embolic microsphere, characterized in that: The embolic microspheres are prepared by the preparation method according to any one of claims 1 to 8, and the particle size of the embolic microspheres is 60-1200 μm.

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