A biodegradable hyaluronic acid gel microsphere with synergistic effects of physical embolization and macromolecular drug loading

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a biodegradable hyaluronic acid gel microsphere with synergistic effects of physical embolization and macromolecular drug loading, its preparation method, and its application in arterial chemoembolization. This embolic microsphere can load not only small molecule drugs but also ADC drugs, achieving long-term release, and is completely degradable, meeting the needs of long-term clinical application.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a biodegradable hyaluronic acid gel microsphere with synergistic effects of physical embolization and macromolecular drug loading, its preparation method, and its application in arterial chemoembolization. Background Technology

[0002] In the field of interventional treatment for solid tumors, transarterial chemoembolization (TACE) has become one of the core treatment methods for solid tumors such as mid-to-late stage liver cancer and lung cancer due to its dual advantages of "blocking the blood supply to the tumor + local drug release".

[0003] Microspheres are delivered to the tumor's blood supply artery via a catheter, first physically blocking the blood vessel and cutting off the tumor's oxygen and nutrient supply, leading to ischemic necrosis of tumor cells. Simultaneously, the microspheres act as a drug reservoir, continuously and slowly releasing high concentrations of chemotherapy drugs at the embolization site, acting on the tumor for an extended period, achieving a double blow of "starvation" and "toxicity." Currently, the drugs loaded for treatment are generally small-molecule chemotherapy drugs such as doxorubicin, pirarubicin, irinotecan, and vinorelbine, all with good drug loading capacity. However, traditional drug-loaded embolized microspheres generally suffer from the drawback of being non-degradable, severely limiting treatment efficacy and affecting patient prognosis.

[0004] Currently, ADC drugs are replacing traditional chemotherapy as the new first-line standard treatment. Clinical studies have confirmed that CalliSpheres can be loaded with large-molecule monoclonal antibody drugs (such as bevacizumab), but there are no clinical reports of loading other ADC drugs onto embolized microspheres. Summary of the Invention

[0005] The purpose of this invention is to provide a biodegradable hyaluronic acid gel microsphere with synergistic effects of physical embolization and macromolecular drug loading, its preparation method, and its application in arterial chemoembolization.

[0006] In a first aspect of the present invention, a method for preparing hyaluronic acid gel microspheres is provided, comprising the following steps: (s1) Hyaluronic acid is mixed with a functionalized modifier in the presence of an optional activating agent to obtain a functionalized hyaluronic acid derivative; (s2) Provides an aqueous phase solution and an oil phase solution; The aqueous phase solution comprises: the functionalized hyaluronic acid derivative obtained in step 1), a thermal initiator, and a functional monomer; the oil phase solution comprises: an oil phase and an emulsifier. (s3) The aqueous solution is slowly added to the oil solution to emulsify and form a stable water-in-oil emulsion. Then, a crosslinking agent is added to the system to perform thermal crosslinking to obtain the hyaluronic acid gel microspheres.

[0007] In another preferred embodiment, in step (s1), the molecular weight of the hyaluronic acid is 1 kDa to 1000 kDa, preferably 5 kDa to 80 kDa, such as 7 kDa, 10 kDa, 20 kDa, 30 kDa, or 50 kDa.

[0008] In another preferred embodiment, in step (s1), the functionalized modifier is selected from the group consisting of: methacrylic anhydride (MA), 2-aminoethyl methacrylate hydrochloride (AEMA), amino polyethylene glycol methacrylate (NH2-PEG-MA), ethyleneamine vinylformamide copolymer, maleic acid-allyl alcohol copolymer, or combinations thereof.

[0009] In another preferred embodiment, in step (s1), the activating agent is selected from two or more of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), 4-(4,6-dimethyl-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (DMTMM), or 1-hydroxybenzotriazole (HoBt).

[0010] In another preferred embodiment, in step (s1), the activating agent is a combination of EDC and NHS, or a combination of EDC and HoBt.

[0011] In another preferred embodiment, the mass ratio of EDC to NHS is (1~5):(1~5), preferably 1:1~5.

[0012] In another preferred embodiment, the mass ratio of EDC to HoBt is (1~5):(1~5), preferably 1~5:1, and more preferably (0.8-1.2):(0.8-1.2).

[0013] In another preferred embodiment, step (s1) is performed in the presence of an inert solvent (preferably a first solvent).

[0014] In another preferred embodiment, the first solvent is selected from the group consisting of: water, MES buffer, N,N'-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO).

[0015] In another preferred embodiment, the pH of the MES buffer is 5.0-6.0, for example 5.5.

[0016] In another preferred embodiment, the first solvent is a combination of MES buffer, water and DMSO, a combination of water and DMF, or DMSO.

[0017] In another preferred embodiment, the volume ratio of water to DMSO is (1~5):(1~5), preferably 1~5:1, for example 4:1.

[0018] In another preferred embodiment, the volume ratio of water to DMF is (0.5-1.5):(0.5-1.5), preferably (0.8-1.2):(0.8-1.2).

[0019] In another preferred embodiment, in step (s1), the hyaluronic acid has a mass-volume fraction of 2-30 g / L in an inert solvent, preferably 2-20 g / L, for example 8-12 g / L.

[0020] In another preferred embodiment, in step (s1), the functionalized modifier has a mass-volume fraction of 5-50 g / L in an inert solvent, preferably 5-30 g / L, for example 10-20 g / L.

[0021] In another preferred embodiment, in step (s1), the activating agent has a mass-volume fraction of 0.5-20 g / L in the inert solvent, preferably 0.5-15 g / L, for example 5-15 g / L.

[0022] In another preferred embodiment, step (s1) is performed at 10-50°C, preferably 15-45°C.

[0023] In another preferred embodiment, in step (s1), the mixing is stirring, preferably at a stirring speed of 100-500 rpm, such as 200 rpm or 300 rpm.

[0024] In another preferred embodiment, the reaction time of step (s1) is 12-72 hours, for example, 24 hours, 36 hours, or 48 hours.

[0025] In another preferred embodiment, step (s1) is optionally performed under a protective atmosphere.

[0026] In another preferred embodiment, the protective atmosphere is selected from nitrogen, helium, and neon.

[0027] In another preferred embodiment, step (s1) further includes post-processing operations such as dialysis and lyophilization.

[0028] In another preferred embodiment, in step (s1), the dialysis includes gradient dialysis.

[0029] In another preferred embodiment, in step (s1), the dialysate used for dialysis is selected from the group consisting of phosphate buffer solution, physiological saline, and purified water.

[0030] In another preferred embodiment, in step (s1), the dialysis time is 24-120h, for example 60h, 72h, or 96h.

[0031] In another preferred embodiment, in step (s1), the freeze-drying time is 24-72 hours, for example, 48 hours.

[0032] In another preferred embodiment, the gradient dialysis includes transferring the reacted solution into a pre-activated dialysis bag, initially using physiological saline as the dialysate, dialysis for approximately 24 hours, with the dialysate being replaced every 6 hours at a volume of 40-60 times its original volume; subsequently, the dialysate is replaced with purified water, and dialysis continues for approximately 48 hours, with the dialysate being replaced every 6 hours at a volume of 40-60 times its original volume.

[0033] In another preferred embodiment, step (s1) includes: mixing hyaluronic acid, functionalized modifier and inert solvent, adjusting the system to the reaction temperature, and in the mixed state, after the above materials are completely dissolved, adding active reagent in sequence, optionally turning on the protective atmosphere, and then continuing to mix for a period of time under constant temperature conditions, dialysis and freeze drying to obtain the functionalized hyaluronic acid derivative.

[0034] In another preferred embodiment, the grafting degree of the functionalized hyaluronic acid derivative is 1% to 30%, preferably 2% to 20%.

[0035] In another preferred embodiment, in the aqueous solution, the thermal initiator is selected from the group consisting of potassium persulfate (KPS), ammonium persulfate (APS), sodium persulfate, tert-butyl hydroperoxide (TBHP), 2,2'-azobisisobutylamidine dihydrochloride (AAPH), 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044), or combinations thereof.

[0036] In another preferred embodiment, in the aqueous solution, the solvent is water, preferably deionized water or purified water.

[0037] In another preferred embodiment, in an aqueous solution, the functionalized monomer is selected from the group consisting of sodium vinyl sulfonate (SVS), 2-acrylamido-2-methylpropanesulfonic acid (AMPS), sodium p-styrene sulfonate (SSS), potassium 3-sulfonate acrylate (SPA), or combinations thereof.

[0038] In another preferred embodiment, in the aqueous solution, the functionalized hyaluronic acid derivative has a mass-to-volume ratio of 0.01-1 g / mL in water, preferably 0.01-0.2 g / mL, more preferably 0.08-0.12 g / mL, for example 0.03 g / mL, 0.05 g / mL, 0.08 g / mL, or 0.15 g / mL.

[0039] In another preferred embodiment, in the aqueous solution, the thermal initiator has a mass-to-volume ratio of 1-10 g / L in water, preferably 1-8 g / L, more preferably 4-6 g / L, for example 2 g / L, 3 g / L, 5 g / L, or 7 g / L.

[0040] In another preferred embodiment, the functionalized monomer in the aqueous solution has a mass-to-volume ratio of 1-10 g / L, preferably 1-8 g / L, more preferably 4-6 g / L, for example 2 g / L, 3 g / L, 5 g / L, or 7 g / L.

[0041] In another preferred embodiment, the method for preparing the aqueous solution includes: adding a functionalized hyaluronic acid derivative to water, mixing until dissolved, then sequentially adding a thermal initiator and a functionalized monomer, and continuing to mix until all materials are completely dissolved to form a homogeneous aqueous solution.

[0042] In another preferred embodiment, the aqueous solution is prepared at room temperature, preferably 15-35°C.

[0043] In another preferred embodiment, the viscosity of the aqueous solution, as measured by a rotational viscometer, is 20–400 cps, preferably 100–300 cps, and more preferably 100–200 cps.

[0044] In another preferred embodiment, the oil phase in the oil phase solution is selected from the group consisting of petroleum ether, liquid paraffin, ethyl acetate, dichloromethane, n-heptane, or combinations thereof.

[0045] In another preferred embodiment, the emulsifier in the oil phase solution is selected from the group consisting of Span 80, Span 60, Tween 80, Tween 60, polyglycerol ricinoleate, polyisobutylene succinimide, or combinations thereof.

[0046] In another preferred embodiment, the emulsifier in the oil phase solution has a mass fraction of 1-20%, preferably 1-15%, for example 3%, 5%, 8%, or 10%.

[0047] In another preferred embodiment, the volume ratio of the oil phase solution to the aqueous phase solvent is 2-50, preferably 3-30, more preferably 5-15, and even more preferably 8-12.

[0048] In another preferred embodiment, step (s3) is performed at 20-90°C, preferably at 20-80°C, and more preferably at 40-80°C.

[0049] In another preferred embodiment, in step (s3), the crosslinking agent is selected from the group consisting of N,N-methylenebisacrylamide (MBA), tetramethylethylenediamine (TMEDA), polyethylene glycol diacrylate (PEGDA), polyethylene glycol dimethacrylate (PEGDMA), or combinations thereof.

[0050] In another preferred embodiment, in step (s3), the mass-to-volume ratio of the crosslinking agent to the solvent in the aqueous solution is 1~10 mg:1 ml, preferably 1~5 mg:1 ml.

[0051] In another preferred embodiment, the thermal crosslinking reaction time of step (s3) is 2 to 10 hours, preferably 2 to 8 hours, for example 4 hours or 6 hours.

[0052] In another preferred embodiment, step (s3) includes: slowly adding the aqueous phase solution to the oil phase solution in a mixed state for emulsification.

[0053] In another preferred embodiment, the mixing in step (s3) includes stirring.

[0054] In another preferred embodiment, in step (s3), the stirring speed is 100 to 600 rpm, preferably 100 to 500 rpm.

[0055] In another preferred embodiment, step (s3) further includes post-processing steps: filtration, washing, freeze drying, and sterilization.

[0056] In another preferred embodiment, the washing is performed in multiple washes, each wash solution comprising isopropanol, n-hexane, and phosphate buffer solution in sequence.

[0057] In another preferred embodiment, the post-processing steps include: filtering the microspheres using a sieve; then adding 6-15 times the volume of isopropanol to the microspheres, stirring and washing at 50-200 rpm for 5-20 min, followed by filtering and collecting the microspheres again, repeating this washing step 2-5 times; then adding 6-15 times the volume of n-hexane to the microspheres, stirring and washing at the same speed and time, filtering and collecting the microspheres again, repeating the washing step 2-5 times; finally adding 6-15 times the volume of phosphate buffer solution to the microspheres, stirring, filtering and washing in the same way, repeating 2-5 times; then performing freeze-drying, and after freeze-drying, sterilizing by irradiation to obtain the hyaluronic acid gel microspheres.

[0058] In another preferred embodiment, step (s3) includes: adding an oil phase solution to a reaction vessel, setting the reaction temperature, starting stirring, and under continuous stirring, slowly adding an aqueous phase solution to the oil phase solution, emulsifying to form a stable water-in-oil emulsion, then adding a crosslinking agent to the reaction system, reacting at a constant temperature for a period of time, and then post-processing to obtain the hyaluronic acid gel microspheres.

[0059] In another preferred embodiment, the method includes the following steps: (s1) Under a protective atmosphere, in the presence of an activating agent combination of EDC and NHS, 8-15 kDa hyaluronic acid was mixed with NH2-PEG-MA to obtain a functionalized hyaluronic acid derivative. (s2) Provides an aqueous phase solution and an oil phase solution; The aqueous phase solution comprises: the functionalized hyaluronic acid derivative obtained in step 1), ammonium persulfate, and 2-acrylamide-2-methylpropanesulfonic acid; the oil phase solution comprises: liquid paraffin and Tween 80. (s3) The aqueous solution is slowly added to the oil solution to emulsify and form a stable water-in-oil emulsion. Then, N,N-methylenebisacrylamide is added to the system for thermal crosslinking to obtain the hyaluronic acid gel microspheres.

[0060] In a second aspect of the invention, hyaluronic acid gel microspheres are provided, which are prepared using the method described in the first aspect of the invention.

[0061] In another preferred embodiment, the size of the microspheres is between 50-500 μm, preferably between 100-300 μm.

[0062] In another preferred embodiment, the microspheres have one or more features selected from the group consisting of: (1) When the microspheres are loaded with small molecule drugs, the drug loading within 30 minutes exceeds 50 mg / g microspheres, preferably exceeds 60 mg / g microspheres, and more preferably exceeds 70 mg / g microspheres; (2) When the microspheres are loaded with macromolecular ADC drugs, the drug loading within 30 minutes exceeds 10 mg / g microspheres, preferably exceeds 15 mg / g microspheres, and more preferably exceeds 30 mg / g microspheres; (3) After the microspheres are loaded with small molecule drugs, the in vitro release time is more than 1 month, preferably more than 1.5 months, and more preferably more than 2 months; (4) After the microspheres are loaded with macromolecular ADC drugs, the in vitro release time is more than 3 days, preferably more than 5 days, and more preferably more than 1 week; (5) The in vitro degradation time of the microspheres exceeds 2 months, preferably up to 3-6 months.

[0063] In a third aspect of the invention, a drug-loaded microsphere is provided, comprising hyaluronic acid gel microspheres as described in the second aspect of the invention, and a drug loaded thereon, wherein the drug is one or more of a small molecule drug, a DNA drug, an RNA drug, a protein, and an ADC drug.

[0064] In another preferred embodiment, the drug loading is 5-1000 mg / g microspheres, for example 10 mg / g microspheres, 20 mg / g microspheres, 50 mg / g microspheres, or 100 mg / g microspheres.

[0065] In another preferred embodiment, the drug is a chemotherapy drug.

[0066] In another preferred embodiment, the small molecule drug is selected from the group consisting of: doxorubicin, epirubicin, daunorubicin, cisplatin, carboplatin, 5-fluorouracil, gemcitabine, paclitaxel, docetaxel, irinotecan, topotecan, bleomycin, vinorelbine, or combinations thereof.

[0067] In another preferred embodiment, the ADC drug is selected from the group consisting of trastuzumab emtansine, trastuzumab dexamethasone, goxatuzumab, brentuximab vemetuximab, ozogazine, vedictitumab, somituzumab, caputuzumab, or combinations thereof.

[0068] In another preferred embodiment, complete drug release from the drug-loaded microspheres requires at least 72 hours, preferably at least 100 hours, and more preferably at least 130 hours.

[0069] In another preferred embodiment, the drug-loaded microspheres require at least 3-6 months to fully degrade in the presence of hyaluronidase.

[0070] In a fourth aspect of the invention, an embolic agent is provided, comprising hyaluronic acid gel microspheres as described in the second aspect of the invention, or drug-loaded microspheres as described in the third aspect of the invention, and a carrier acceptable in the field of embolic agents.

[0071] In a fifth aspect of the invention, the use of the hyaluronic acid gel microspheres as described in the second aspect of the invention, or the drug-loaded microspheres as described in the third aspect of the invention, or the embolic agent as described in the fourth aspect of the invention, for the preparation of a medicament for the treatment of tumors in transarterial chemoembolization (TACE).

[0072] In another preferred embodiment, the tumor is a solid tumor.

[0073] In another preferred embodiment, the tumor is a tumor suitable for arterial chemoembolization and is selected from the group consisting of: liver cancer, cholangiocarcinoma, kidney cancer, adrenal malignant tumor, hypervascular lung cancer, osteosarcoma and bone metastases, cervical cancer, and endometrial cancer.

[0074] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0075] Figure 1 Microscopic images of the microspheres in Example 1 of this application are shown.

[0076] Figure 2 The microsphere size distribution diagram of Example 1 of this application is shown.

[0077] Figure 3Microscopic images of the microspheres in Comparative Example 1 are shown. The degree of cross-linking of the microspheres is uneven, and a small number of microspheres are adhered together.

[0078] Figure 4 The image shows a microscope photograph of the microspheres in Comparative Example 1 after they were compressed by 50%, showing that some of the microspheres were damaged.

[0079] Figure 5 The drug loading efficiency of trastuzumab emtansine on microspheres in Examples 1-4 and Comparative Example 1 of this application is shown.

[0080] Figure 6 The in vitro release rates of trastuzumab emtansine loaded with microspheres in Examples 1-4 and Comparative Example 1 of this application are shown.

[0081] Figure 7 The correlation graph between absorbance and drug concentration of microsphere-loaded rubicin is shown. Detailed Implementation

[0082] Through extensive and in-depth research, the inventors have, for the first time, prepared an embolic microsphere capable of loading macromolecular ADC drugs. By controlling specific substrates, specific functionalization modifications, and specific cross-linking methods, the inventors prepared embolic microspheres that balance mechanical properties, drug loading capacity, and degradability. These embolic microspheres can load ADC drugs, achieve long-term release, and are completely degradable, meeting the needs of long-term clinical application. This invention is based on this achievement.

[0083] the term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0084] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0085] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0086] In this invention, "water" may include deionized water or purified water.

[0087] In this invention, "mixing" includes operations such as shaking, stirring, homogenizing, or vortexing. "In a mixed state" refers to the state when the shaking, stirring, homogenizing, or vortexing operation is turned on.

[0088] In this invention, "room temperature" is 5-40°C.

[0089] The gel microspheres of the present invention This invention provides a method for preparing degradable hyaluronic acid gel microspheres that combine physical embolization and macromolecular drug loading. In addition to physically blocking blood vessels and loading commonly used small molecule chemotherapy drugs (such as doxorubicin, pirarubicin, irinotecan, vinorelbine, etc.), it can also load macromolecular ADC drugs, which are released and degraded in vivo, while taking into account degradation rate, mechanical properties and drug loading capacity.

[0090] Specifically, the preparation method includes the following steps: 1) Preparation of functionalized hyaluronic acid derivatives; 2) Mix the hyaluronic acid derivative obtained in step 1) with a thermal initiator and a functional monomer; 3) Add the mixed solution obtained in step 2) to the oil phase containing the emulsifier, emulsify to obtain a water-in-oil emulsion, then add a crosslinking agent to the system and thermally crosslink the emulsion; 4) Clean and freeze-dry to obtain the finished hyaluronic acid microspheres.

[0091] In step 1), further, The hyaluronic acid has a molecular weight of 1 kDa to 1000 kDa, preferably 10 kDa to 100 kDa.

[0092] The grafting reaction between the functionalized modifier and hyaluronic acid is an amidation reaction or an esterification reaction.

[0093] The functionalized modifier may be one of the following: methacrylic anhydride (MA), 2-aminoethyl methacrylate hydrochloride (AEMA), amino polyethylene glycol methacrylate (NH2-PEG-MA), ethyleneamine vinyl formamide copolymer, and maleic acid-allyl alcohol copolymer.

[0094] The solvent used in the preparation of the functionalized hyaluronic acid derivative is purified water, MES buffer, N,N'-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), or a mixture thereof.

[0095] The activator reagent used in the preparation of the functionalized hyaluronic acid derivative is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), 4-(4,6-dimethyl-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (DMTMM), or 1-hydroxybenzotriazole (HoBt), or a mixture thereof.

[0096] In the system, the content of hyaluronic acid is 0.2-5 wt%, the content of the functionalized modifier is 0.2-5 wt%, and the content of the activating agent is 0-1 wt%.

[0097] The reaction temperature is 4–60℃, the stirring speed is 100–1000 rpm, and the reaction time is 12–48 h.

[0098] The reaction solution is purified by dialysis using a dialysis bag. Preferably, the dialysis solution is a phosphate buffer solution, physiological saline, or purified water. The dialysis time is 20–60 h.

[0099] The freeze-drying time for the reaction solution after dialysis is 20–60 h.

[0100] The grafting degree of the functionalized hyaluronic acid derivative was tested by proton nuclear magnetic resonance spectroscopy. The preferred dissolving reagent was D2O or FA-d or a mixture of both, and the grafting degree was 2% to 20%.

[0101] In step 2), further, The thermal initiator is one or two of the following: benzoyl peroxide (BPO), 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044), potassium persulfate (KPS), ammonium persulfate (APS), and tert-butyl hydroperoxide (TBHP).

[0102] The crosslinking agent is one or two of N,N-methylenebisacrylamide (MBA), tetramethylethylenediamine (TMEDA), polyethylene glycol diacrylate (PEGDA), and polyethylene glycol dimethacrylate (PEGDMA).

[0103] The functional monomer is one or two of sodium vinyl sulfonate (SVS), 2-acrylamide-2-methylpropanesulfonic acid (AMPS), sodium p-styrene sulfonate (SSS), and potassium 3-sulfonate acrylate (SPA).

[0104] In the system, the content of the aqueous phase is 0.2-5 wt%, the content of the oil phase is 0.2-5 wt%, the content of the thermal initiator is 0.2-5 wt%, the content of the crosslinking agent is 0.2-5 wt%, and the content of the functional monomer is 0.2-5 wt%.

[0105] The viscosity of the aqueous mixed solution is 20–400 cps.

[0106] The reaction temperature of the mixed solution is 20–80 degrees Celsius.

[0107] In step 3), further, The oil phase is one of petroleum ether, liquid paraffin, ethyl acetate, dichloromethane, and n-heptane.

[0108] The emulsifier is one of Span 80, Span 60, Tween 80, Tween 60, polyglycerol ricinoleate, and polyisobutylene succinimide.

[0109] The emulsifier accounts for 1% to 20% of the oil phase by mass.

[0110] The viscosity of the aqueous mixed solution is 10–200 cps.

[0111] The volume ratio of the aqueous phase mixture to the oil phase mixture is 1:5 to 1:50.

[0112] In step 4), further, The preparation system operates at a rotation speed of 100–600 rpm, a crosslinking temperature of 20–80 degrees Celsius, and a crosslinking time of 2–8 h.

[0113] The cleaning solution is an isopropanol, n-hexane, and phosphate buffer solution.

[0114] The volume ratio of the cleaning solution to the microspheres is 5:1 to 20:1, and the number of cleaning cycles is 2 to 5.

[0115] Traditional hyaluronic acid microspheres are prepared using microfluidic technology and cross-linked via photocuring. While this method produces microspheres with a narrow particle size distribution, the yield is low and material costs are high. Furthermore, for larger microspheres, photocuring may result in incomplete internal curing. The sequential flow of microspheres through the microfluidic chip outlet poses a risk of microsphere adhesion and even fusion, and varying curing times can lead to uneven cross-linking. In contrast, thermocuring allows for complete cross-linking of polymer molecular chains, resulting in microspheres with higher mechanical strength. A fixed stirring rate ensures uniform dispersion of the microspheres in the oil phase, preventing adhesion. The reactor allows for continuous batch production with high efficiency and stable capacity. The technology is mature and offers low-cost mass production, making it suitable for the large-scale adoption of traditional embolic microspheres and meeting the low-cost requirements of industrial production.

[0116] The main advantages of this invention include: (1) In addition to physically blocking blood vessels and loading commonly used small molecule chemotherapy drugs (such as doxorubicin, pirarubicin, irinotecan, vinorelbine, etc.), the gel microspheres obtained by this invention can also load large molecule ADC drugs, while taking into account mechanical properties, drug loading capacity and degradation. This completely solves the problem of permanent vascular residues, easy vascular restenosis, organ ischemia and necrosis and other long-term complications of traditional non-degradable embolization microspheres (such as PVA microspheres).

[0117] (2) The microspheres have a particle size range of 100-300 μm and a size variation coefficient (CV) of <5%. The microspheres have regular morphology and smooth surface, and can be stored for a long time (>24 months) after sterilization, meeting the needs of long-term clinical application. When loaded with epirubicin, the microspheres can achieve a drug loading of 75 mg / g within 30 minutes, with in vitro release lasting more than 2 months; when loaded with macromolecular ADC drugs, the microspheres can achieve a drug loading of 38 mg / g within 30 minutes, with in vitro release lasting more than 1 week.

[0118] (3) According to in vitro degradation tests, the microspheres can degrade over a period of 3 to 6 months, and the mild inflammatory response they cause can subside within 2 weeks, demonstrating excellent biocompatibility and bioabsorbability. The degradation products are non-toxic and harmless small molecules (such as glucose and lactic acid), which can be naturally excreted through the body's metabolic system without the need for secondary surgery, greatly reducing the patient's treatment pain and medical costs.

[0119] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0120] Example 1 1) Weigh 1 g of hyaluronic acid with a molecular weight of 100,000 and NH2-PEG-MA into a reaction flask, add 100 mL of pH 5.5 MES buffer, adjust the reaction temperature to 20 ℃, and stir at 300 rpm. After the materials are completely dissolved, add 300 mg of EDC and 1 g of NHS sequentially. Under nitrogen protection, stir at a constant temperature for 24 h. After the reaction is complete, remove the resulting mixed reaction solution and transfer it to a pre-treated dialysis bag (the dialysis bag has been pre-activated) for dialysis. Perform gradient dialysis purification on the reaction solution: First, use physiological saline as the dialysis solution and dialyze for 24 h, changing the dialysis solution every 6 h, with a dialysis solution volume of 5 L; then change the dialysis solution to purified water and continue dialysis for 48 h, changing the dialysis solution every 6 h, with a dialysis solution volume of 5 L. After dialysis, collect the purified solution in the dialysis bag and freeze-dry for 48 h to obtain the functionalized hyaluronic acid derivative.

[0121] 2) Weigh 1 g of the freeze-dried functionalized hyaluronic acid derivative and add it to 10 mL of purified water. Vortex until dissolved. Then add 50 mg APS and 50 mg AMPS in sequence and vortex until all materials are completely dissolved to form a homogeneous aqueous solution. Use a rotational viscometer to monitor the viscosity of the aqueous solution. The viscosity was measured to be 200 cps.

[0122] 3) Add 100 mL of liquid paraffin containing 5% Tween 80 to the flask, set the reaction temperature to 50 °C, and the stirring speed to 300 rpm. Under continuous stirring, slowly add the prepared aqueous solution to the oil phase. After emulsification, a stable water-in-oil emulsion is formed. Then add 60 mg of MBA to the reaction system and keep the reaction at a constant temperature for 6 h.

[0123] 4) After the reaction is complete, pour out the reaction solution and filter the microspheres through a sieve of the appropriate size to collect the obtained microsphere product. Transfer the microsphere product to a beaker, add 10 times the volume of isopropanol, and wash by stirring at 150 rpm for 10 min. Then filter through sieve 1 to collect the microspheres. Repeat this washing step 3 times. Next, add 10 times the volume of n-hexane to the microspheres, and wash by stirring at the same speed and time. Filter through sieve 2 to collect the microspheres. Repeat this washing step 3 times. Finally, add 10 times the volume of phosphate buffer solution, and stir, filter, and wash in the same way. Repeat this process 3 times to achieve complete purification of the microspheres. The finally collected hyaluronic acid microspheres are freeze-dried, and after freeze-drying, they are sterilized by irradiation to obtain the target product.

[0124] The obtained target product was observed under a microscope, and the results are as follows: Figure 1 As shown, the particle size distribution diagram is as follows: Figure 2 As shown. Furthermore, the inventors repeated the above experiment multiple times, and the coefficient of variation (CV) was <5%.

[0125] Comparative Example 1: Photocrosslinking of microspheres prepared using microfluidic technology 1) Weigh 1 g of hyaluronic acid with a molecular weight of 100,000 and 2 g of 2-aminoethyl methacrylate hydrochloride (AEMA) into a reaction flask. Add 100 mL of MES buffer at pH 5.5, adjust the reaction temperature to 20 ℃, and stir at 300 rpm. After the materials are completely dissolved, add 300 mg of EDC and 1 g of NHS sequentially. Under nitrogen protection, stir at a constant temperature for 24 h. After the reaction is complete, remove the resulting mixed reaction solution and transfer it to a pre-treated dialysis bag (the dialysis bag has been pre-activated) for dialysis. Perform gradient dialysis purification on the reaction solution: First, use physiological saline as the dialysis solution and dialyze for 24 h, changing the dialysis solution every 6 h, with a dialysis solution volume of 5 L; then change the dialysis solution to purified water and continue dialysis for 48 h, changing the dialysis solution every 6 h, with a dialysis solution volume of 5 L. After dialysis, collect the purified solution in the dialysis bag and freeze-dry for 48 h to obtain the functionalized hyaluronic acid derivative.

[0126] 2) Weigh 1 g of the freeze-dried functionalized hyaluronic acid derivative and add it to 10 mL of purified water. Vortex until dissolved. Then add 50 mg of phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide (LAP) and 50 mg of AMPS in sequence. Vortex until all materials are completely dissolved to form a homogeneous aqueous solution. The viscosity of the aqueous solution is monitored using a rotational viscometer and is measured to be 195 cps.

[0127] 3) Add 100 mL of liquid paraffin containing 5% Tween 80 to the oil phase bottle. Transfer the dissolved aqueous phase from step 2) to the aqueous phase bottle. Install the microfluidic device. The microfluidic chip channel width is 400 μm and the depth is 10 μm. Set the aqueous phase pressure to 200 mbar and the oil phase pressure to 300 mbar. Simultaneously turn on the LED light source with a wavelength of 405 nm and a power of 20 mW / cm². 2 Take a 250ml beaker from the chip outlet to receive the reaction solution.

[0128] 4) After the reaction is complete, pour out the reaction solution and filter the microspheres through a sieve of the appropriate size to collect the obtained microsphere product. Transfer the microsphere product to a beaker, add 10 times the volume of isopropanol, and wash by stirring at 150 rpm for 10 min. Then filter through sieve 1 to collect the microspheres. Repeat this washing step 3 times. Next, add 10 times the volume of n-hexane to the microspheres, and wash by stirring at the same speed and time. Filter through sieve 2 to collect the microspheres. Repeat this washing step 3 times. Finally, add 10 times the volume of phosphate buffer solution, and stir, filter, and wash in the same way. Repeat this process 3 times to achieve complete purification of the microspheres. The finally collected hyaluronic acid microspheres are freeze-dried, and after freeze-drying, they are sterilized by irradiation to obtain the target product.

[0129] The obtained product was observed under a microscope, and the results were as follows: Figure 3 As shown, the cross-linking degree of the microspheres is uneven, and a small number of microspheres are adhered together. After compressing the microspheres by 50%, the microscopic image is as follows. Figure 4 As shown, some of the microspheres have been damaged.

[0130] Example 2 1) Weigh 0.8g of hyaluronic acid with a molecular weight of 500,000 and 1g of ethyleneamine vinylformamide copolymer into a reaction flask, add 80 mL of purified water and 20 mL of DMSO, adjust the reaction temperature to 30 ℃, and stir at 300 rpm. After the materials are completely dissolved, add 500 mg of EDC and 500 mg of HOBt sequentially. Under nitrogen protection, stir at a constant temperature for 24 h. After the reaction is complete, remove the resulting mixed reaction solution and transfer it to a pre-treated dialysis bag (the dialysis bag has been pre-activated) for dialysis. Perform gradient dialysis purification on the reaction solution: first, use physiological saline as the dialysis solution and dialyze for 24 h, changing the dialysis solution every 6 h, with a dialysis solution volume of 5 L; then replace the dialysis solution with purified water and continue dialysis for 48 h, changing the dialysis solution every 6 h, with a dialysis solution volume of 5 L. After dialysis, collect the purified solution in the dialysis bag and freeze-dry for 48 h to obtain the functionalized hyaluronic acid derivative.

[0131] 2) Weigh 0.5 g of the freeze-dried functionalized hyaluronic acid derivative and add it to 10 mL of purified water. Vortex until dissolved. Then add 20 mg VA-044 and 30 mg SVS in sequence. Vortex until all materials are completely dissolved to form a homogeneous aqueous solution. The viscosity of the aqueous solution is monitored using a rotational viscometer and is measured to be 150 cps.

[0132] 3) Add 100 mL of liquid paraffin containing 3% Tween 60 to the flask, set the reaction temperature to 60 ℃, and the stirring speed to 260 rpm. Under continuous stirring, slowly add the prepared aqueous phase solution to the oil phase. After emulsification, a stable water-in-oil emulsion is formed. Then add 0.03 mL of PEGDA to the reaction system and keep the reaction at a constant temperature for 4 h.

[0133] 4) After the reaction is complete, pour out the reaction solution and filter the microspheres through a sieve of the appropriate size to collect the obtained microsphere product. Transfer the microsphere product to a beaker, add 10 times the volume of isopropanol, and wash by stirring at 150 rpm for 10 min. Then filter through sieve 1 to collect the microspheres. Repeat this washing step 3 times. Next, add 10 times the volume of n-hexane to the microspheres, and wash by stirring at the same speed and time. Filter through sieve 2 to collect the microspheres. Repeat this washing step 3 times. Finally, add 10 times the volume of phosphate buffer solution, and stir, filter, and wash in the same way. Repeat this process 3 times to achieve complete purification of the microspheres. The finally collected hyaluronic acid microspheres are freeze-dried, and after freeze-drying, they are sterilized by irradiation to obtain the target product.

[0134] Example 3 1) Weigh 0.5 g of hyaluronic acid with a molecular weight of 500,000 and 2 mL of MA into a reaction flask, add 50 mL of purified water and 50 mL of DMF, adjust the reaction temperature to 45 ℃, and stir at 300 rpm. After the materials are completely dissolved, maintain nitrogen protection and stir at a constant temperature for 36 h. After the reaction is complete, remove the resulting mixed reaction solution and transfer it to a pre-treated dialysis bag (the dialysis bag has been pre-activated) for dialysis. Perform gradient dialysis purification on the reaction solution: first, use physiological saline as the dialysis solution and dialyze for 24 h, changing the dialysis solution every 6 h, with a dialysis solution volume of 5 L; then, change the dialysis solution to purified water and continue dialysis for 48 h, changing the dialysis solution every 6 h, with a dialysis solution volume of 5 L. After dialysis, collect the purified solution in the dialysis bag and freeze-dry for 48 h to obtain the functionalized hyaluronic acid derivative.

[0135] 2) Weigh 0.5 g of the freeze-dried functionalized hyaluronic acid derivative and add it to 15 mL of purified water. Vortex until dissolved. Then add 30 mg KPS and 20 mg SSS in sequence and vortex until all materials are completely dissolved to form a homogeneous aqueous solution. The viscosity of the aqueous solution is monitored using a rotational viscometer and is measured to be 100 cps.

[0136] 3) Add 60 mL of petroleum ether containing 10% Span 80 to the flask, set the reaction temperature to 65 °C, and the stirring speed to 180 rpm. Under continuous stirring, slowly add the prepared aqueous phase solution to the oil phase. After emulsification, a stable water-in-oil emulsion is formed. Then add 0.02 mL of PEGDMA to the reaction system and keep the reaction at a constant temperature for 4 h.

[0137] 4) After the reaction is complete, pour out the reaction solution and filter the microspheres through a sieve of the appropriate size to collect the obtained microsphere product. Transfer the microsphere product to a beaker, add 10 times the volume of isopropanol, and wash by stirring at 150 rpm for 10 min. Then filter through sieve 1 to collect the microspheres. Repeat this washing step 3 times. Next, add 10 times the volume of n-hexane to the microspheres, and wash by stirring at the same speed and time. Filter through sieve 2 to collect the microspheres. Repeat this washing step 3 times. Finally, add 10 times the volume of phosphate buffer solution, and stir, filter, and wash in the same way. Repeat this process 3 times to achieve complete purification of the microspheres. The finally collected hyaluronic acid microspheres are freeze-dried, and after freeze-drying, they are sterilized by irradiation to obtain the target product.

[0138] Example 4 1) Weigh 2 g of hyaluronic acid with a molecular weight of 70,000 and 1.2 g of maleic acid-allyl alcohol copolymer into a reaction flask, add 150 mL of DMSO, adjust the reaction temperature to 38 ℃, and stir at 200 rpm. After the materials are completely dissolved, add 80 mg of EDC and 40 mg of HOBt sequentially, and stir at a constant temperature for 24 h. After the reaction is complete, take out the resulting mixed reaction solution and transfer it to a pre-treated dialysis bag (the dialysis bag has been pre-activated) for dialysis. Perform gradient dialysis purification on the reaction solution: first, use physiological saline as the dialysis solution and dialyze for 24 h, changing the dialysis solution every 6 h, with a dialysis solution volume of 5 L; then change the dialysis solution to purified water and continue dialysis for 48 h, changing the dialysis solution every 6 h, with a dialysis solution volume of 5 L. After dialysis, collect the purified solution in the dialysis bag and freeze-dry for 48 h to obtain the functionalized hyaluronic acid derivative.

[0139] 2) Weigh 1 g of the freeze-dried functionalized hyaluronic acid derivative and add it to 15 mL of purified water. Vortex until dissolved. Then add 50 mg TBHP and 100 mg SPA in sequence and vortex until all materials are completely dissolved to form a homogeneous aqueous solution. The viscosity of the aqueous solution is monitored using a rotational viscometer and is measured to be 130 cps.

[0140] 3) Add 280 mL of liquid ethyl paraffin containing 4.5% Tween 60 to the flask, set the reaction temperature to 75℃, and the stirring speed to 330 rpm. Under continuous stirring, slowly add the prepared aqueous phase solution to the oil phase. After emulsification, a stable water-in-oil emulsion is formed. Then add 0.05 mL of TMEDA to the reaction system and keep the reaction at a constant temperature for 4 h.

[0141] 4) After the reaction is complete, pour out the reaction solution and filter the microspheres through a sieve of the appropriate size to collect the obtained microsphere product. Transfer the microsphere product to a beaker, add 10 times the volume of isopropanol, and wash by stirring at 150 rpm for 10 min. Then filter through sieve 1 to collect the microspheres. Repeat this washing step 3 times. Next, add 10 times the volume of n-hexane to the microspheres, and wash by stirring at the same speed and time. Filter through sieve 2 to collect the microspheres. Repeat this washing step 3 times. Finally, add 10 times the volume of phosphate buffer solution, and stir, filter, and wash in the same way. Repeat this process 3 times to achieve complete purification of the microspheres. The finally collected hyaluronic acid microspheres are freeze-dried, and after freeze-drying, they are sterilized by irradiation to obtain the target product.

[0142] Example 5 Drug loading efficiency test method: Take one vial of trastuzumab emtansine (0.1 g) and dissolve it in 5 mL of water for injection to prepare a concentration of 20 mg / mL. Reconstitute the freeze-dried microspheres with physiological saline for 30 min to remove excess water. Weigh 200 mg (accurate to 0.001 g) of the reconstituted microsphere sample and place it in a 5 mL sterile centrifuge tube. Pipette 1.5 mL of the prepared drug solution and slowly inject it into the centrifuge tube, ensuring the microspheres are completely submerged. Collect 50 μL of the supernatant at 15 min, 30 min, and 60 min. Determine the concentration of the remaining drug in the drug-loaded supernatant using UV spectrophotometry and calculate the drug loading using the differential method.

[0143] Drug loading (mg / g) = (Drug dosage - Remaining drug mass in supernatant) / Microsphere sample mass Drug loading efficiency (%) = (Drug loading / Drug dosage) × 100% The results are as follows Figure 5 As shown, Examples 1 and 4 both exhibit relatively superior loading effects, especially Example 1. Furthermore, the drug loading capacity can reach 38 mg / g microspheres within 30 minutes.

[0144] Example 6 In vitro release test method: After loading the drug, the microspheres were filtered to remove the supernatant. The surface was washed with PBS (pH 7.4, 10 mM), and the microspheres were transferred to 15 mL centrifuge tubes. 5 mL of PBS was added to each tube, and the tubes were placed in a constant-temperature shaker at 37 ± 0.5 ℃ and 100 r / min. At 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 72 h, 120 h, 144 h, and 168 h, 1 mL of release medium was collected, and the same volume of PBS was added. The concentration of the drug in the collected solutions was determined by UV spectrophotometry, and a drug release curve was plotted.

[0145] Drug release amount at a single time point = drug concentration × drug volume (1 mL) Cumulative drug release = Sum of drug release amounts at each time point Cumulative drug release rate (%) = (Cumulative drug release amount / Total drug mass in drug-loaded microspheres) × 100% The results are as follows Figure 6 As shown, it can be seen that Examples 1 and 2 both have relatively better sustained-release effects, especially Example 1, which can release in vitro for more than 1 week.

[0146] Example 7 Take one vial of epirubicin, containing 10 mg, and dissolve it in 0.5 mL of water for injection to prepare a concentration of 20 mg / mL.

[0147] After freeze-drying, the microspheres were reconstituted with physiological saline for 30 minutes to remove excess water. 100 mg of the reconstituted microsphere sample was weighed and placed in a 5 mL sterile centrifuge tube.

[0148] To administer 80 mg / g of the drug, pipette 0.4 mL of the prepared solution and slowly inject it into a centrifuge tube, ensuring the microspheres are completely submerged. Collect 50 μL of the supernatant at 15 min, 30 min, and 60 min, respectively.

[0149] The ultraviolet absorbance of epirubicin in the drug-loaded supernatant was determined by ultraviolet spectrophotometry. The drug loading amount and drug loading efficiency were calculated according to the calculation formula in Example 5 above.

[0150] Epirubicin was prepared in purified water at concentrations of 50, 20, 10, 5, and 2 μg / mL. The integral area of ​​the elution curves for each concentration standard was measured by ultraviolet spectrophotometry. A scatter plot was plotted with concentration as the x-axis and integral area as the y-axis. Figure 7 ).

[0151] The drug loading and loading efficiency of epirubicin are shown in Table 1 below.

[0152] Table 1 Example 8 Microsphere Degradation Test Method Take 0.5 g of each of the freeze-dried and completely reconstituted microsphere samples, remove surface moisture, and add 10 mL of degradation solution. Place the samples in a constant temperature shaker at 37±0.5 ℃ and 100 r / min. The degradation solution is PBS buffer (pH 7.4, 0.01 mol / L), and the degradation enzyme is hyaluronidase. Set up one control group without enzyme and three enzyme concentration gradient groups (10 U / mL, 50 U / mL, and 100 U / mL), with three samples in each group to ensure experimental repeatability. Replace the degradation solution daily. Sampling time points: Samples were taken at degradation times of 0, 1, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 105, 119, 133, 147, 161, 175, and 180 days. The degraded microspheres were removed, surface moisture was removed, and three parallel samples were taken from each group. The average weight was calculated and used as the remaining mass of the microspheres at that time point and for that group.

[0153] The results showed that the microspheres could degrade over a period of 3 to 6 months.

[0154] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for preparing hyaluronic acid gel microspheres, characterized by, Includes the following steps: (s1) Hyaluronic acid is mixed with a functionalized modifier in the presence of an optional activating agent to obtain a functionalized hyaluronic acid derivative; (s2) Provides an aqueous phase solution and an oil phase solution; The aqueous phase solution comprises: the functionalized hyaluronic acid derivative obtained in step 1), a thermal initiator, and a functional monomer; the oil phase solution comprises: an oil phase and an emulsifier. (s3) The aqueous solution is slowly added to the oil solution to emulsify and form a stable water-in-oil emulsion. Then, a crosslinking agent is added to the system to perform thermal crosslinking to obtain the hyaluronic acid gel microspheres.

2. The production method according to claim 1, wherein In step (s1), the molecular weight of the hyaluronic acid is 1 kDa to 1000 kDa, preferably 5 kDa to 80 kDa; The functionalized modifier is selected from the group consisting of: methacrylic anhydride (MA), 2-aminoethyl methacrylate hydrochloride (AEMA), amino polyethylene glycol methacrylate (NH2-PEG-MA), ethyleneamine vinyl formamide copolymer, maleic acid-allyl alcohol copolymer, or combinations thereof; The activating agent is selected from two or more of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), 4-(4,6-dimethyl-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (DMTMM), or 1-hydroxybenzotriazole (HoBt).

3. The production method according to claim 1, wherein In an aqueous solution, the thermal initiator is selected from the group consisting of potassium persulfate (KPS), ammonium persulfate (APS), sodium persulfate, tert-butyl hydroperoxide (TBHP), 2,2'-azobisisobutylamidine dihydrochloride (AAPH), 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044), or combinations thereof; The functionalized monomer is selected from the group consisting of sodium vinyl sulfonate (SVS), 2-acrylamide-2-methylpropanesulfonic acid (AMPS), sodium p-styrene sulfonate (SSS), potassium 3-sulfonate acrylate (SPA), or combinations thereof.

4. The preparation method according to claim 1, characterized in that, In the oil phase solution, the oil phase is selected from the group consisting of: petroleum ether, liquid paraffin, ethyl acetate, dichloromethane, n-heptane, or combinations thereof; The emulsifier is selected from the group consisting of: Span 80, Span 60, Tween 80, Tween 60, polyglycerol ricinoleate, polyisobutylene succinimide, or combinations thereof.

5. The preparation method according to claim 1, characterized in that, In step (s3), the crosslinking agent is selected from the group consisting of N,N-methylenebisacrylamide (MBA), tetramethylethylenediamine (TMEDA), polyethylene glycol diacrylate (PEGDA), polyethylene glycol dimethacrylate (PEGDMA), or combinations thereof.

6. The preparation method according to claim 1, characterized in that, The method includes the following steps: (s1) Under a protective atmosphere, in the presence of an activating agent combination of EDC and NHS, 8-15 kDa hyaluronic acid was mixed with NH2-PEG-MA to obtain a functionalized hyaluronic acid derivative. (s2) Provides an aqueous phase solution and an oil phase solution; The aqueous phase solution comprises: the functionalized hyaluronic acid derivative obtained in step 1), ammonium persulfate, and 2-acrylamide-2-methylpropanesulfonic acid; the oil phase solution comprises: liquid paraffin and Tween 80. (s3) The aqueous solution is slowly added to the oil solution to emulsify and form a stable water-in-oil emulsion. Then, N,N-methylenebisacrylamide is added to the system for thermal crosslinking to obtain the hyaluronic acid gel microspheres.

7. A hyaluronic acid gel microsphere prepared using the method described in any one of claims 1-6.

8. A drug-loaded microsphere, characterized in that, It includes the hyaluronic acid gel microspheres as described in claim 7, and a drug loaded thereon, wherein the drug is one or more of a small molecule drug, a DNA drug, an RNA drug, a protein, and an ADC drug.

9. An embolic agent comprising the hyaluronic acid gel microspheres of claim 7, or the drug-loaded microspheres of claim 8, and a carrier acceptable in the field of embolic agents.

10. The use of the hyaluronic acid gel microspheres as described in claim 7, or the drug-loaded microspheres as described in claim 8, or the embolic agent as described in claim 9, characterized in that, This is used to prepare a drug for treating tumors in transarterial chemoembolization (TACE).

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