A porous degradable embolization microsphere and a preparation method thereof

Abstract

The application discloses a kind of porous degradable embolism microspheres and preparation method thereof, comprising the following steps: taking the aqueous solution of drug as internal water phase W1;Take modified PLGA polymer and add dichloromethane, low-temperature stirring dissolves as oil phase;Composite emulsifier is added in oil phase, W1 is added to oil phase, and stable emulsion is obtained by high shear homogenization;Stable emulsion is slowly added to PVA aqueous solution, and is fully emulsified, solvent is volatilized until microspheres solidification is complete;Microspheres are washed by rapid cooling pre-freezing and freeze-drying after water, and form porous structure degradable embolism microspheres.The microspheres prepared by this method are porous structure, particle size distribution is uniform, drug distribution is uniform, and drug can be uniformly released;Higher drug release rate is obtained by modified PLGA.

Description

Technical Field

[0001] This invention belongs to the fields of polymer materials and biomedical engineering, and specifically relates to a porous biodegradable embolic microsphere and its preparation method. Background Technology

[0002] Transarterial chemoembolization (TACE) is a commonly used non-surgical treatment for liver cancer. Depending on the embolic agent, TACE can be divided into conventional TACE (c-TACE) and drug-eluting microsphere TACE (DEB-TACE).

[0003] c-TACE refers to embolization therapy primarily using iodized oil-based chemotherapy drug emulsions, supplemented with gelatin sponge particles, blank microspheres, or PVA. DEB-TACE refers to embolization therapy primarily using drug-eluting microspheres loaded with chemotherapy drugs. Drug-loaded microspheres can be loaded with chemotherapy drugs such as doxorubicin and irinotecan. The particle sizes of these microspheres are mainly 70-150 μm, 100-300 μm, 300-500 μm, or 500-700 μm, with different sizes selected based on tumor size, blood supply, and treatment objectives. DEB-TACE can embolize the blood supply arteries of liver cancer, causing tumor ischemia and necrosis, and simultaneously serves as a carrier for chemotherapy drugs, offering the advantage of sustained and stable drug release.

[0004] Currently available DEB-TACE microspheres include: DC Bead from Biocompatibles (UK), CalliSphere drug-loadable microspheres from Hengrui Medicine (China), and HepaSphere / Quadra Sphere microspheres from BioSphere Pharmaceuticals (USA), all of which are non-degradable drug-loadable microspheres. Their main mechanism of action is to load positively charged chemotherapy drugs such as doxorubicin and irinotecan via ion exchange before TACE surgery. However, they cannot load drugs containing anionic groups or those that are difficult to dissociate. Furthermore, drug release from the microspheres after surgery is mainly concentrated in the first three days after the procedure, and cannot achieve a long-term stable release effect.

[0005] There are currently no commercially available biodegradable DEB products. Theoretically, biodegradable DEB embolizes arteries and releases chemotherapy drugs to the tumor only during treatment, alleviating post-embolization syndrome, especially long-term syndrome; the mass and mechanical strength of the microspheres decrease over time and will be gradually absorbed by surrounding tissues, allowing for recanalization of the embolized vessels; considering the cell growth cycle, multiple intermittent embolizations of the same lesion may be more beneficial, and bioabsorbable microspheres provide a route for subsequent chemotherapy administration. Summary of the Invention

[0006] The purpose of this invention is to overcome technical deficiencies and provide a porous biodegradable embolization microsphere and its preparation method. The microspheres prepared by this method have a porous structure, uniform particle size distribution, uniform drug distribution, and uniform drug release.

[0007] This invention is achieved through the following technical solution:

[0008] A method for preparing porous biodegradable embolic microspheres includes the following steps:

[0009] (1) Preparation of internal emulsion: Doxorubicin hydrochloride was completely dissolved in pure water and used as W1; modified PLGA polymer was weighed and added to dichloromethane and stirred to dissolve as oil phase; composite emulsifier was added to oil phase, W1 was added to oil phase, and high shear homogenization was performed to obtain stable emulsion.

[0010] (2) Weigh PVA, add it to water, heat and stir to dissolve it, and obtain a uniform PVA aqueous solution. After cooling, use it as the aqueous phase W.

[0011] (3) Emulsification and curing: The emulsion from step (1) is slowly added to the aqueous phase stirred at a constant speed to fully emulsify, evaporate the solvent, and completely cure the microspheres;

[0012] (4) Cleaning of microspheres: After the microspheres have solidified, stop stirring, filter, take the solid microspheres, add pure water, stir and clean, filter, and repeat the washing process.

[0013] (5) Drying of microspheres: After washing, the microspheres were pre-frozen in a refrigerator and then dried in a freeze dryer under low temperature vacuum to obtain dried PLGA microspheres.

[0014] Furthermore, the mass ratio of doxorubicin hydrochloride to pure water is 1:100; the mass ratio of modified PLGA polymer to dichloromethane is 1:20-1:5; and the mass ratio of oil phase to water phase is 1:40-1:5.

[0015] Further, the composite emulsifier in step (1) is a compound emulsifier of Tween 80 and SPAN 80, with a mass ratio of Tween 80 to SPAN 80 of 5:1.

[0016] Furthermore, the mass ratio of the composite emulsifier to the oil phase in step (1) is 1:100-1:40.

[0017] Furthermore, the shear rate in step (1) is 6000-10000 rpm, the stirring rate in step (2) is 300 rpm, and the stirring speed of the aqueous phase in step (3) is 250-600 rpm.

[0018] Furthermore, in step (5), the pre-freezing temperature is -80℃ and the low temperature is -50℃.

[0019] Further, the preparation method of the modified PLGA polymer in step (1) is as follows: dissolve the PLGA polymer in dichloromethane, add EDS and NHS, add tannin dissolved in ethanol, stir the reaction at room temperature, evaporate the organic solvent, filter and dialyze, and dry to obtain the modified PLGA polymer.

[0020] The present invention also discloses porous biodegradable embolic microspheres prepared by the above preparation method.

[0021] The microspheres have a particle size of 50-700 μm.

[0022] The beneficial effects of this invention are as follows:

[0023] This invention uses modified PLGA and grafts tannins onto it. On the one hand, this can improve the bioactivity of PLGA, and on the other hand, tannins themselves have antibacterial and other bioactivities, thus increasing the therapeutic effect.

[0024] This invention uniformly immobilizes the drug doxorubicin hydrochloride within microspheres with a particle size of 50-700 micrometers. The prepared microspheres are biodegradable, thereby achieving complete release of the loaded drug.

[0025] The microspheres prepared by this invention exhibit good biocompatibility, are biodegradable, and the degradation products are safe and non-toxic. The preparation method is simple, reproducible, operates under mild conditions, and allows for controllable particle size, showing promising application prospects in the field of tumor treatment. Attached Figure Description

[0026] Figure 1 This is a particle size distribution diagram of Example 1;

[0027] Figure 2 This is a particle size distribution diagram for Example 2;

[0028] Figure 3 This is the particle size distribution diagram for Comparative Example 1;

[0029] Figure 4 Electron micrographs of Example 1, Example 2 and Comparative Example 1;

[0030] Figure 5 This is a comparison chart of drug release rates between Example 2 and Comparative Example 1. Detailed Implementation

[0031] The present invention will be further described below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present invention, and are not intended to limit the present invention.

[0032] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The invention is further described below with reference to specific embodiments, but is not intended to limit the scope of the invention.

[0033] Main materials and reagents: PVA124 (analytical grade), PLGA polymer (50 / 50, molecular weight 30,000), doxorubicin hydrochloride (analytical grade, 98%), dichloromethane (analytical grade), tannic acid (analytical grade).

[0034] Instruments: Thermostatic magnetic stirrer, low-temperature coolant circulation pump, high-shear homogenizer, analytical balance, heat-collecting magnetic stirrer, ultra-low temperature freeze storage box, freeze dryer.

[0035] The modified PLGA polymer was prepared as follows: PLGA polymer was dissolved in dichloromethane, EDS and NHS were added, tannic acid dissolved in ethanol was added, and the mixture was stirred at room temperature for 48 hours. The organic solvent was evaporated, the mixture was filtered, and then freeze-dried to obtain the modified PLGA polymer. The molecular weight of the tannic acid was 500-3000 Da.

[0036] The mass ratio of PLGA polymer to EDS, NHS, and tannic acid is 1:1:1:1.

[0037] Example 1

[0038] (1) Dissolve 0.10g of doxorubicin hydrochloride completely in 10g of pure water to obtain W1; weigh 20g of modified PLGA polymer and add it to 100g of dichloromethane, stir and dissolve to obtain the oil phase; add 1.10g of composite emulsifier to the oil phase, which is a compound emulsifier of Tween 80 and SPAN 80, with a mass ratio of Tween 80 to SPAN 80 of 5:1. Add W1 to the oil phase and homogenize at 6000rpm to obtain a stable emulsion.

[0039] (2) Weigh 30g of PVA, add it to 2L of water, heat to 90℃, stir at 300rpm for 4h until dissolved, and obtain a uniform PVA aqueous solution. After cooling, use it as the aqueous phase W.

[0040] (3) The emulsion from step (1) was added to the aqueous phase and stirred at a constant speed of 400 rpm for emulsification. After stabilization, it was stirred at 250 rpm for 24 hours to fully emulsify, evaporate the solvent, and completely solidify the microspheres. After the microspheres solidified, stirring was stopped, and the solid microspheres were filtered. Pure water was added and stirred for 20 minutes for washing. The mixture was then filtered and washed repeatedly. After the last washing, one-fifth of the water was retained and the microspheres were pre-frozen in a -80℃ freezer. Then, they were dried in a freeze dryer at -50℃ under vacuum for 40 hours to obtain dried PLGA microspheres.

[0041] Example 2

[0042] (1) Dissolve 0.20g of doxorubicin hydrochloride completely in 20g of pure water to obtain W1; weigh 20g of modified PLGA polymer and add it to 100g of dichloromethane, stirring to dissolve it to obtain the oil phase; add 2.20g of composite emulsifier to the oil phase. The composite emulsifier is a compound emulsifier of Tween 80 and SPAN 80, with a mass ratio of Tween 80 to SPAN 80 of 5:1. Add W1 to the oil phase and homogenize at 10000rpm to obtain a stable emulsion.

[0043] (2) Weigh 30g of PVA, add it to 2L of water, heat to 90℃, stir at 300rpm for 4h until dissolved, and obtain a uniform PVA aqueous solution. After cooling, use it as the aqueous phase W.

[0044] (3) The emulsion from step (1) was added to the aqueous phase and stirred at a constant speed of 300 rpm for emulsification. After stabilization, it was stirred at 250 rpm for 24 hours to fully emulsify, evaporate the solvent, and completely solidify the microspheres. After the microspheres solidified, stirring was stopped, and the solid microspheres were filtered and washed with pure water for 20 minutes. The mixture was then filtered and washed repeatedly. After the last washing, one-fifth of the water was retained and the microspheres were pre-frozen in a -80℃ freezer. Then, they were dried in a freeze dryer at -50℃ under vacuum for 40 hours to obtain dried PLGA microspheres.

[0045] Comparative Example 1

[0046] (1) Dissolve 0.20g of doxorubicin hydrochloride completely in 20g of pure water to obtain W1; weigh 20g of unmodified PLGA polymer and add it to 100g of dichloromethane, stirring to dissolve it to obtain the oil phase; add 2.20g of composite emulsifier to the oil phase. The composite emulsifier is a compound emulsifier of Tween 80 and SPAN 80, with a mass ratio of Tween 80 to SPAN 80 of 5:1. Add W1 to the oil phase and homogenize at 10000rpm to obtain a stable emulsion.

[0047] (3) Weigh 30g of PVA, add it to 2L of water, heat to 90℃, stir at 300rpm for 4h until dissolved, and obtain a uniform PVA aqueous solution. After cooling, use it as the aqueous phase W.

[0048] (3) The emulsion from step (1) was added to the aqueous phase and stirred at a constant speed of 300 rpm for emulsification. After stabilization, it was stirred at 250 rpm for 24 hours to fully emulsify, evaporate the solvent, and completely solidify the microspheres. After the microspheres solidified, stirring was stopped, and the solid microspheres were filtered and washed with pure water for 20 minutes. The mixture was then filtered and washed repeatedly. After the last washing, one-fifth of the water was retained and the microspheres were pre-frozen in a -80℃ freezer. Then, they were dried in a freeze dryer at -50℃ under vacuum for 40 hours to obtain dried PLGA microspheres.

[0049] Effect verification

[0050] The effects of the microspheres prepared in Examples 1-2 and Comparative Example 1 were verified.

[0051] 1. Particle size measurement: The particle size and distribution of microspheres were measured using a Malvern laser particle size analyzer.

[0052] from Figures 2-4 It can be seen that in Example 1: the normal distribution is 125-243 μm, and D50 = 176 μm;

[0053] Example 2: Normal distribution 239-392μm, D50=306μm;

[0054] Comparative Example 1: Normal distribution 157-488μm, D50=298μm.

[0055] 2. Morphology test: The surface morphology of the microspheres was observed using scanning electron microscopy.

[0056] 3. Biodegradability test: Take 0.2g of sample and place it in a 25mL test tube, add 12mL of PBS solution, seal and place in a constant temperature test chamber at 37℃ to observe the degradation. After a certain time interval, freeze-dry the PLGA microspheres and examine the degradation time.

[0057] 4. Drug loading test: Accurately weigh 10 mg of PLGA porous drug-loaded microspheres and completely dissolve them in 2 mL of DCM. Add 2 mL of 0.01 M HCl to the solution, shake thoroughly to mix, centrifuge, separate the supernatant, repeat three times, combine the collected supernatants, and make up to 10 mL. Detect the drug loading of the particles by high performance liquid chromatography and calculate the drug loading of the particles.

[0058] Example Particle size distribution (D50) / μm Appearance Drug loading (mg / g) Degradation time / d 1 125-243(176) Porous microspheres 3.22 135 2 239-392(306) Porous microspheres 4.08 120 3 157-488(298) Microspheres 2.69 180

[0059] 5. In vitro drug release test: Accurately weigh 100 mg of drug-loaded microspheres and place them in a brown centrifuge tube containing 40 ml of Sorensen buffer. Seal the tube and place it in a shaking incubator with constant shaking speed (37°C, 100 r / min). At the predetermined time point, centrifuge the sample tube, take 39 ml of buffer solution, and immediately add an equal volume of Sorensen buffer solution at the same temperature and consistency. Determine the drug content in the buffer sample, calculate its release concentration and cumulative release percentage, and plot the drug release curve. Compare the drug release rates of Example 2 and the comparative example.

[0060] The test results above show that the drug-loaded microspheres prepared by modified PLGA have a narrower particle size distribution, are porous, have a higher drug loading capacity, and exhibit significantly improved degradation and drug release performance.

[0061] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of this invention should be considered within the scope of protection of this invention.

Claims

1. A method for preparing porous biodegradable embolic microspheres, characterized in that, Includes the following steps: (1) Preparation of internal emulsion: Take the aqueous solution of water-soluble drug or pure water as W1; weigh the modified PLGA polymer and add it to dichloromethane, stir and dissolve it as the oil phase; add the composite emulsifier to the oil phase, add W1 to the oil phase, and obtain a stable emulsion by high shear homogenization; the preparation method of modified PLGA polymer is as follows: dissolve PLGA polymer in dichloromethane, add EDC and NHS, add tannin dissolved in ethanol, stir and react at room temperature, evaporate the organic solvent, filter and dialyze, and dry to obtain modified PLGA polymer; (2) Weigh PVA, add it to water, heat and stir to dissolve it, and obtain a uniform PVA aqueous solution. After cooling, use it as the aqueous phase W. (3) Emulsification and curing: The emulsion from step (1) is slowly added to the stirred aqueous phase to fully emulsify, evaporate the solvent, and the microspheres are completely cured; (4) Cleaning of microspheres: After the microspheres have solidified, stop stirring, filter, take the solid microspheres, add pure water, stir and clean, filter, and repeat the washing process; (5) Drying of microspheres: After washing, the microspheres are rapidly cooled and pre-frozen, and then dried under low temperature vacuum to form pores, thus obtaining porous modified PLGA microspheres.

2. The method for preparing porous biodegradable embolic microspheres according to claim 1, characterized in that, The mass ratio of doxorubicin hydrochloride to pure water is 1:100; the mass ratio of PLGA polymer to dichloromethane is 1:20-1:5; and the mass ratio of oil phase to water phase is 1:10-1:

3.

3. The method for preparing porous biodegradable embolic microspheres according to claim 1, characterized in that, The composite emulsifier in step (1) is a compound emulsifier of Tween 80 and SPAN 80, with a mass ratio of Tween 80 to SPAN 80 of 5:

1.

4. The method for preparing porous biodegradable embolic microspheres according to claim 1, characterized in that, The mass ratio of the composite emulsifier to the oil phase in step (1) is 1:100-1:

50.

5. The method for preparing porous biodegradable embolic microspheres according to claim 1, characterized in that, The shear rate in step (1) is 6000-10000 rpm, the stirring rate in step (2) is 300 rpm, and the stirring speed of the aqueous phase in step (3) is 250-600 rpm.

6. The method for preparing porous biodegradable embolic microspheres according to claim 1, characterized in that, In step (5), the pre-freezing temperature is -80℃ and the low temperature is -50℃.

7. A porous biodegradable embolic microsphere prepared by the preparation method according to any one of claims 1-6.

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