Emulsion gel microbeads and preparation method and application thereof

Abstract

The present invention relates to the field of biomedicine technology, and in particular to an emulsion gel microbead, and a preparation method and application thereof. Specifically, the process comprises the following steps: (1) mixing a sodium alginate solution modified with dodecylsuccinic anhydride and fish oil to obtain a DSA emulsion; (2) mixing the DSA emulsion, the sodium alginate solution and glycolithocholic acid to obtain a DSA / SA emulsion; (3) mixing the DSA / SA emulsion, a surfactant solution and an emulsifier solution to obtain an O / W / O emulsion; (4) dripping a calcium chloride solution into the O / W / O emulsion to obtain an emulsion gel; (5) mixing the emulsion gel with water, stirring, and standing, and removing impurities to obtain emulsion gel microbeads. The emulsion gel microbeads prepared by the present invention can effectively bind drug ingredients, have good biocompatibility after injection, and can achieve a drug release process in which the drug is slowly released and uniformly released.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to emulsion gel microbeads and a preparation method and application thereof. Background Art

[0002] Pickering emulsions are emulsions formed using ultrafine solid particles as emulsifiers, also known as Pickering emulsions. The type of emulsion obtained depends on which phase preferentially wets the solid particles. Typically, the phase that preferentially wets the solid particles is the external phase. For example, if the solid particles are more easily wetted by the oil phase, the emulsion is a W / O (water-in-oil) type. Conversely, if the solid particles are more easily wetted by the water phase, the emulsion is an O / W (oil-in-water) type. In common Pickering emulsions, solid powders used as emulsifiers or stabilizers include metal hydroxides, graphite, carbon black, nanosilica, etc.

[0003] Compared with traditional surfactant-stabilized emulsions, Pickering emulsions have many advantages, such as low cost, good stability, and environmental friendliness. Therefore, Pickering emulsions are increasingly used in cosmetics, food, medicine, and other fields. Conventional oil-in-water or water-in-oil Pickering emulsions are widely used in the preparation of porous polymers. In addition, Pickering multiple emulsions such as oil-in-water-in-oil (O / W / O) or water-in-oil-in-water (W / O / W) types have broad application prospects in microcapsule preparation, drug encapsulation and controlled release, food nutritional additives, cosmetics, and other fields due to their special "two membranes and three phases" structure.

[0004] Osteoarthritis (OA) is a common joint disease characterized by joint pain, stiffness, and cartilage damage. With the aging population, OA will become a major issue affecting people's quality of life, and the market demand for OA drugs will continue to expand.

[0005] Currently, clinical treatments for osteoarthritis are divided into specific therapeutic drugs and nonspecific therapeutic drugs. Nonspecific therapeutic drugs, such as nonsteroidal anti-inflammatory drugs, are mainly used for analgesia and symptom control, but have no protective effect on cartilage. Specific therapeutic drugs, such as glucosamine and chondroitin sulfate, can protect joint cartilage and delay the progression of osteoarthritis, but they generally have a slow onset and require several months of treatment to be effective. In addition, they have no effect on the regeneration of damaged cartilage. Therefore, developing a new osteoarthritis treatment drug with good safety and outstanding efficacy is a problem that medical workers need to solve today.

[0006] It can be seen that the therapeutic effects of existing drugs for treating osteoarthritis are not ideal. Therefore, how to use Pickering emulsion to further carry drugs for treating osteoarthritis to improve the therapeutic effects of the drugs is a problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0007] The purpose of the present invention is to provide an emulsion gel microbead and a preparation method and application thereof.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for preparing emulsion gel microbeads, comprising the following steps:

[0010] (1) mixing a sodium alginate solution modified with dodecylsuccinic anhydride and fish oil to obtain a DSA emulsion;

[0011] (2) mixing the DSA emulsion, sodium alginate solution and glycolithocholic acid to obtain a DSA / SA emulsion;

[0012] (3) mixing the DSA / SA emulsion, surfactant solution, and co-emulsifier solution to obtain an O / W / O emulsion;

[0013] (4) adding a calcium chloride solution dropwise into the O / W / O emulsion to obtain an emulsion gel;

[0014] (5) The emulsion gel is mixed with water, stirred, and allowed to stand to remove impurities, thereby obtaining emulsion gel microbeads.

[0015] Preferably, the volume concentration of the sodium alginate solution modified with dodecene succinic anhydride in step (1) is 2-3%; and the volume ratio of the sodium alginate solution modified with dodecene succinic anhydride to fish oil is 3-5:1.

[0016] Preferably, in step (2), the volume mass ratio of the DSA emulsion, sodium alginate solution and glycolithocholic acid is 2 mL: 4-6 mL: 0.2-0.4 g; and the mass concentration of the sodium alginate solution is 1.5-2.5%.

[0017] Preferably, the surfactant solution in step (3) is SPAN80, egg yolk lecithin and poloxamer 188, with a volume ratio of 10-16:5-7:2-4; the co-emulsifier solution is glyceryl monostearate and polyethylene glycol 400, with a volume ratio of 1-3:3-1.

[0018] Preferably, in step (3), the volume ratio of the DSA / SA emulsion, surfactant solution, and co-emulsifier solution is 2:6-8:2-4; the volume concentration of the surfactant solution is 2-3%; and the volume concentration of the co-emulsifier solution is 2-3%.

[0019] Preferably, the mass concentration of the calcium chloride solution in step (4) is 0.5-1.5%; and the volume ratio of the calcium chloride solution to the O / W / O emulsion is 4-6:1.

[0020] Preferably, the volume ratio of the emulsion gel to water in step (5) is 1:4-6.

[0021] The invention also provides emulsion gel microbeads prepared by the preparation method.

[0022] The present invention also provides use of the emulsion gel microbeads in preparing drugs for promoting cartilage repair or treating osteoarthritis, wherein the emulsion gel microbeads are used as carriers to carry the therapeutic drugs.

[0023] Preferably, the therapeutic drug is tofacitinib.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention prepares emulsion gel microbeads to carry therapeutic drugs, thereby achieving the purpose of transporting drugs and improving drug treatment effects. In this invention, the technical effect of effectively treating osteoarthritis is achieved by carrying the osteoarthritis drug - tofacitinib.

[0026] The sodium alginate solution modified with dodecylsuccinic anhydride added to the preparation of the emulsion gel microbeads in this invention serves as a stabilizer in the emulsion system. Compared to emulsions stabilized with natural alginates, the modified sodium alginate provides repulsive forces between oil droplets, enhancing the physical stability of the emulsion. It also inhibits the oxidation of lipids in the emulsion, increasing the stability of the emulsion.

[0027] The glycolithocholic acid used in the present invention acts as a penetration enhancer, promoting the release and absorption of drugs and effectively promoting the functioning of pharmaceutically active substances. The present invention also adds a composite emulsifier, which can improve the stability of the emulsion, thereby achieving better drug loading and release effects. The use of a co-emulsifier further improves the stability of the emulsion system.

[0028] Therefore, the emulsion gel microbeads prepared by the present invention can effectively bind to drug components, and the hydrogel microspheres formed in the later stage have a good coating and binding effect on the drugs. They have good biocompatibility after injection and can achieve a slow and uniform sustained-release drug administration process, ensuring the concentration of the drug in the affected area, preventing the problem of uneven administration due to excessively high or low drug concentrations during administration, effectively reducing the toxic and side effects of the drugs, preventing the human body from developing resistance to the drugs, and improving the effect and duration of the drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0030] Figure 1 Schematic diagram of the preparation process of the present invention.

[0031] Figure 2 This is a laser confocal image of the emulsion gel microbeads prepared in Example 3: specifically, it is a laser confocal image taken using Nile red staining, which can observe the state of the gel spheres and the oil droplets loaded with drugs inside them, where the red ones are the oil droplets wrapped in the gel. DETAILED DESCRIPTION

[0032] The present invention provides a method for preparing emulsion gel microbeads, comprising the following steps:

[0033] (1) mixing a sodium alginate solution modified with dodecylsuccinic anhydride and fish oil to obtain a DSA emulsion;

[0034] (2) mixing the DSA emulsion, sodium alginate solution and glycolithocholic acid to obtain a DSA / SA emulsion;

[0035] (3) mixing the DSA / SA emulsion, surfactant solution, and co-emulsifier solution to obtain an O / W / O emulsion;

[0036] (4) adding a calcium chloride solution dropwise into the O / W / O emulsion to obtain an emulsion gel;

[0037] (5) The emulsion gel is mixed with water, stirred, and allowed to stand to remove impurities, thereby obtaining emulsion gel microbeads.

[0038] In the present invention, the volume concentration of the sodium alginate solution modified with dodecylsuccinic anhydride in step (1) is 2-3%, preferably 2.5%.

[0039] In the present invention, the volume ratio of the dodecylsuccinic anhydride modified sodium alginate solution and the fish oil in step (1) is 3 to 5:1, preferably 4:1.

[0040] In the present invention, the volume mass ratio of the DSA emulsion, sodium alginate solution and glycolithocholic acid in step (2) is 2 mL: 4-6 mL: 0.2-0.4 g; preferably 2 mL: 5 mL: 0.2-0.4 g; further preferably 2 mL: 5 mL: 0.3 g.

[0041] In the present invention, the mass concentration of the sodium alginate solution in step (2) is 1.5-2.5%; preferably 1.7-2.3%; more preferably 1.9-2.1%; and more preferably 2%.

[0042] In the present invention, the surfactant solution in step (3) is SPAN80, egg yolk lecithin and poloxamer 188, and the volume ratio is 10-16:5-7:2-4; preferably 11-15:6:3; more preferably 12-14:6:3; more preferably 13:6:3.

[0043] In the present invention, the co-emulsifier solution in step (3) is glyceryl monostearate and polyethylene glycol 400, and the volume ratio is 1-3:3-1; preferably 2:3-1; more preferably 1:1.

[0044] In the present invention, the volume ratio of the DSA / SA emulsion, surfactant solution and co-emulsifier solution in step (3) is 2:6-8:2-4; preferably 2:7:2-4; more preferably 2:7:3.

[0045] In the present invention, the volume concentration of the surfactant solution in step (3) is 2-3%, preferably 2.5%.

[0046] In the present invention, the volume concentration of the co-emulsifier solution in step (3) is 2-3%, preferably 2.5%.

[0047] In the present invention, the mass concentration of the calcium chloride solution in step (4) is 0.5-1.5%; preferably 0.7-1.3%; more preferably 0.9-1.1%; and more preferably 1%.

[0048] In the present invention, the volume ratio of the calcium chloride solution to the O / W / O emulsion in step (4) is 4 to 6:1, preferably 5:1.

[0049] In the present invention, the volume ratio of the emulsion gel to water in step (5) is 1:4 to 6, preferably 1:5.

[0050] The invention also provides emulsion gel microbeads prepared by the preparation method.

[0051] The present invention also provides use of the emulsion gel microbeads in preparing drugs for promoting cartilage repair or treating osteoarthritis, wherein the emulsion gel microbeads are used as carriers to carry the therapeutic drugs.

[0052] In the present invention, the therapeutic drug is tofacitinib.

[0053] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0054] Example 1

[0055] A method for preparing emulsion gel microbeads, comprising the following steps:

[0056] (1) A 2% dodecylsuccinic anhydride-modified sodium alginate solution and fish oil were mixed at a volume ratio of 3:1 and homogenized at 7500 rpm for 1 min to obtain a DSA emulsion;

[0057] (2) mixing the DSA emulsion, a sodium alginate solution with a mass concentration of 1.5%, and glycolithocholic acid in a volume mass ratio of 2 mL:4 mL:0.2 g to obtain a DSA / SA emulsion;

[0058] (3) The DSA / SA emulsion, a surfactant solution with a volume concentration of 2% (using fish oil as a solvent), and a co-emulsifier solution with a volume concentration of 2% (using fish oil as a solvent) were mixed in a volume ratio of 2:6:2, and homogenized at 8500 rpm for 1 min to obtain an O / W / O emulsion;

[0059] The surfactant solution is SPAN80, egg yolk lecithin and poloxamer 188, with a volume ratio of 10:5:2;

[0060] The co-emulsifier solution is glyceryl monostearate and polyethylene glycol 400, with a volume ratio of 1:3;

[0061] (4) adding a 0.5% calcium chloride solution dropwise into the O / W / O emulsion, and continuously stirring with a magnetic stirrer at 1000 rpm for 15 minutes to obtain an emulsion gel; the volume ratio of the calcium chloride solution to the O / W / O emulsion is 4:1;

[0062] (5) The emulsion gel was mixed with water in a volume ratio of 1:4, stirred at 5000 rpm for 2 min, allowed to stand for 3 h, washed with water to remove impurities, and dried naturally to obtain emulsion gel microbeads with a particle size range of 80 to 180 μm.

[0063] Example 2

[0064] A method for preparing emulsion gel microbeads, comprising the following steps:

[0065] (1) A 3% volume concentration of sodium alginate modified with dodecylsuccinic anhydride solution and fish oil were mixed at a volume ratio of 5:1 and homogenized at 8000 rpm for 1 min to obtain a DSA emulsion;

[0066] (2) mixing the DSA emulsion, a sodium alginate solution with a mass concentration of 2.5%, and glycolithocholic acid in a volume mass ratio of 2 mL:6 mL:0.4 g to obtain a DSA / SA emulsion;

[0067] (3) The DSA / SA emulsion, a 3% surfactant solution (using fish oil as a solvent) and a 3% co-emulsifier solution (using fish oil as a solvent) were mixed in a volume ratio of 2:8:4 and homogenized at 8000 rpm for 1 min to obtain an O / W / O emulsion;

[0068] The surfactant solution is SPAN80, egg yolk lecithin and poloxamer 188, with a volume ratio of 16:7:4;

[0069] The co-emulsifier solution is glyceryl monostearate and polyethylene glycol 400, with a volume ratio of 3:1;

[0070] (4) adding a 1.5% calcium chloride solution dropwise to the O / W / O emulsion, and continuously stirring with a magnetic stirrer at 1000 rpm for 15 minutes to obtain an emulsion gel; the volume ratio of the calcium chloride solution to the O / W / O emulsion is 6:1;

[0071] (5) The emulsion gel was mixed with water in a volume ratio of 1:6, stirred at 5000 rpm for 2 minutes, allowed to stand for 3 hours, washed with water to remove impurities, and dried naturally to obtain emulsion gel microbeads with a particle size range of 60 to 120 μm.

[0072] Example 3

[0073] A method for preparing emulsion gel microbeads, comprising the following steps:

[0074] (1) A 2.5% volume concentration of sodium alginate modified with dodecylsuccinic anhydride solution and fish oil were mixed at a volume ratio of 4:1 and homogenized at 8000 rpm for 1 min to obtain a DSA emulsion;

[0075] (2) mixing the DSA emulsion, a sodium alginate solution with a mass concentration of 2%, and glycolithocholic acid in a volume mass ratio of 2 mL:5 mL:0.3 g to obtain a DSA / SA emulsion;

[0076] (3) The DSA / SA emulsion, a surfactant solution with a volume concentration of 2.5% (using fish oil as a solvent), and a co-emulsifier solution with a volume concentration of 2.5% (using fish oil as a solvent) were mixed in a volume ratio of 2:7:3, and homogenized at 8000 rpm for 1 min to obtain an O / W / O emulsion;

[0077] The surfactant solution is SPAN80, egg yolk lecithin and poloxamer 188, with a volume ratio of 13:6:3;

[0078] The co-emulsifier solution is glyceryl monostearate and polyethylene glycol 400, with a volume ratio of 1:1;

[0079] (4) adding a 1% calcium chloride solution dropwise to the O / W / O emulsion, and continuously stirring with a magnetic stirrer at 1000 rpm for 15 minutes to obtain an emulsion gel; the volume ratio of the calcium chloride solution to the O / W / O emulsion is 5:1;

[0080] (5) The emulsion gel was mixed with water in a volume ratio of 1:5, stirred at 5000 rpm for 2 minutes, allowed to stand for 3 hours, washed with water to remove impurities, and dried naturally to obtain emulsion gel microbeads with a particle size range of 50 to 150 μm.

[0081] Example 4

[0082] A method for preparing drug-loaded emulsion gel microbeads, comprising the following steps:

[0083] (1) 2.5% dodecylsuccinic anhydride-modified sodium alginate solution, fish oil, and tofacitinib were mixed at a volume mass ratio of 4 mL:1 mL:1 mg, and homogenized at 7500 rpm for 1 min to obtain a DSA emulsion;

[0084] (2) mixing the DSA emulsion, a sodium alginate solution with a mass concentration of 2%, and glycolithocholic acid in a volume mass ratio of 2 mL:5 mL:0.3 g to obtain a DSA / SA emulsion;

[0085] (3) The DSA / SA emulsion, a surfactant solution with a volume concentration of 2.5% (using fish oil as a solvent), and a co-emulsifier solution with a volume concentration of 2.5% (using fish oil as a solvent) were mixed in a volume ratio of 2:7:3, and homogenized at 8000 rpm for 1 min to obtain an O / W / O emulsion;

[0086] The surfactant solution is SPAN80, egg yolk lecithin and poloxamer 188, with a volume ratio of 13:6:3;

[0087] The co-emulsifier solution is glyceryl monostearate and polyethylene glycol 400, with a volume ratio of 1:1;

[0088] (4) adding a 1% calcium chloride solution dropwise to the O / W / O emulsion, and continuously stirring with a magnetic stirrer at 1000 rpm for 15 minutes to obtain an emulsion gel; the volume ratio of the calcium chloride solution to the O / W / O emulsion is 5:1;

[0089] (5) The emulsion gel was mixed with water in a volume ratio of 1:5, stirred at 4500 rpm for 2 minutes, allowed to stand for 3 hours, washed with water to remove impurities, and dried naturally to obtain emulsion gel microbeads with a particle size range of 90 to 200 μm.

[0090] Example 5

[0091] A method for preparing drug-loaded emulsion gel microbeads, comprising the following steps:

[0092] (1) 2% dodecylsuccinic anhydride-modified sodium alginate solution, fish oil, and tofacitinib were mixed at a volume mass ratio of 4 mL:1 mL:1 mg, and homogenized at 8500 rpm for 1 min to obtain a DSA emulsion;

[0093] (2) mixing the DSA emulsion, a sodium alginate solution with a mass concentration of 1.5%, and glycolithocholic acid in a volume mass ratio of 2 mL:4 mL:0.2 g to obtain a DSA / SA emulsion;

[0094] (3) The DSA / SA emulsion, a surfactant solution with a volume concentration of 2% (using fish oil as a solvent), and a co-emulsifier solution with a volume concentration of 2% (using fish oil as a solvent) were mixed in a volume ratio of 2:6:2, and homogenized at 8500 rpm for 1 min to obtain an O / W / O emulsion;

[0095] The surfactant solution is SPAN80, egg yolk lecithin and poloxamer 188, with a volume ratio of 10:5:2;

[0096] The co-emulsifier solution is glyceryl monostearate and polyethylene glycol 400, with a volume ratio of 1:3;

[0097] (4) adding a 0.5% calcium chloride solution dropwise into the O / W / O emulsion, and continuously stirring with a magnetic stirrer at 1000 rpm for 15 minutes to obtain an emulsion gel; the volume ratio of the calcium chloride solution to the O / W / O emulsion is 4:1;

[0098] (5) The emulsion gel was mixed with water in a volume ratio of 1:4, stirred at 5500 rpm for 2 minutes, allowed to stand for 3 hours, washed with water to remove impurities, and dried naturally to obtain emulsion gel microbeads with a particle size range of 50 to 150 μm.

[0099] Example 6

[0100] A method for preparing drug-loaded emulsion gel microbeads, comprising the following steps:

[0101] (1) 3% dodecylsuccinic anhydride-modified sodium alginate solution, fish oil, and tofacitinib were mixed at a volume mass ratio of 5 mL:1 mL:1 mg, and homogenized at 8000 rpm for 1 min to obtain a DSA emulsion;

[0102] (2) mixing the DSA emulsion, a sodium alginate solution with a mass concentration of 2.5%, and glycolithocholic acid in a volume mass ratio of 2 mL:6 mL:0.4 g to obtain a DSA / SA emulsion;

[0103] (3) The DSA / SA emulsion, a 3% surfactant solution (using fish oil as a solvent) and a 3% co-emulsifier solution (using fish oil as a solvent) were mixed in a volume ratio of 2:8:4 and homogenized at 8000 rpm for 1 min to obtain an O / W / O emulsion;

[0104] The surfactant solution is SPAN80, egg yolk lecithin and poloxamer 188, with a volume ratio of 16:7:4;

[0105] The co-emulsifier solution is glyceryl monostearate and polyethylene glycol 400, with a volume ratio of 3:1;

[0106] (4) adding a 1.5% calcium chloride solution dropwise to the O / W / O emulsion, and continuously stirring with a magnetic stirrer at 1000 rpm for 15 minutes to obtain an emulsion gel; the volume ratio of the calcium chloride solution to the O / W / O emulsion is 6:1;

[0107] (5) The emulsion gel was mixed with water in a volume ratio of 1:6, stirred at 5000 rpm for 2 minutes, allowed to stand for 3 hours, washed with water to remove impurities, and dried naturally to obtain emulsion gel microbeads with a particle size range of 50 to 150 μm.

[0108] Experimental Example 1

[0109] 1. Experimental animals, modeling, and drug administration

[0110] Before the experiment, 8-week-old male SPF mice were purchased and adapted for 1 week before the collagen-induced arthritis (CIA) model was prepared. Arthritis was induced by injecting an emulsion of Freund's adjuvant and type II collagen. In mouse CIA, mice were immunized with type II bovine collagen emulsion in complete Freund's adjuvant (CFA) and received a boost of type II bovine collagen in incomplete Freund's adjuvant (IFA) 21 days after the first injection. After the CIA injection on the 21st day (day 21), the model was successfully established. The mice were randomly divided into groups (5 mice per group), and each group was injected with the corresponding drug (intra-articular injection) once a week for 4 weeks.

[0111] The emulsion gel microbeads prepared in Example 6 were used as the experimental group. After the microbeads were diluted with water for injection, the gel microbeads were injected at a dose of 20 μl / bead / 10 g body weight for 4 weeks. The blank control group and the model group were injected with an equal amount of normal saline. The prior art group was administered 30 mg / kg tofacitinib by gavage once a week for 4 weeks.

[0112] A control group was set up: Control group 1. Other methods were the same as those in Example 6, except that the sodium alginate solution modified with dodecylsuccinic anhydride was replaced with an equal amount of sodium alginate solution.

[0113] Control group 2: other methods were the same as those in Example 6, except that glycolithocholic acid was not added.

[0114] Control group 3: other methods were the same as those in Example 6, except that only SPAN80 was added to the surfactant solution.

[0115] Control group 4: other methods were the same as those in Example 6, except that no co-emulsifier solution was added. The use of the drugs in control groups 1 to 4 was the same as that in the experimental group.

[0116] 2. Micro-CT detection

[0117] Mouse knee joints were scanned and three-dimensionally reconstructed using a micro-CT instrument with settings of 90 kV, 80 μA, and 10 μm resolution. Data were analyzed using analysis software (CTAn v1.9) and 3D visualization software (CTVol v2.0). The region of interest between the tibial growth plate and the tibial plateau was identified, and statistical analysis of bone volume (BV), bone volume fraction (BV / TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N) was performed based on the 3D morphometric cross-sectional data.

[0118] 3. Before specimen collection, mice were fasted but not watered for 12 h. After anesthesia, blood specimens were collected by removing the eyeballs of the mice, and serum was separated for liver and kidney function serum biochemical tests.

[0119] Blood samples were collected and centrifuged twice in a high-speed refrigerated centrifuge at 3500 rpm for 5 minutes at 4°C, and serum was collected. Serum urea nitrogen (BUN) and creatinine (Cr) levels were measured according to the manufacturer's instructions for the BUN and creatinine (Cr) test kits, respectively, to assess renal function. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (AKP) levels were measured using an automated microplate reader, respectively, to assess liver function. L-1β (EK0391, Boster) and TNF-α (FEK0527, Boster) were detected using ELISA kits according to the manufacturer's instructions.

[0120] 4. After blood collection, mice were sacrificed by cervical dislocation, and liver and kidney tissues were collected for pathological examination. Mice were placed in the supine position and secured on an operating table. Surgical scissors were used to open the abdominal cavity from the midline upward, avoiding damage to the kidneys and liver. The liver and kidneys were quickly removed, rinsed with saline, and immediately fixed in 10% neutral formaldehyde for liver and kidney pathological examination. Liver and kidney tissues of each group were visually inspected for swelling, color, morphology, gloss, texture, and the presence of ecchymoses and petechiae.

[0121] result:

[0122] (1) The liver and kidney tissues of the mice in each experimental group were observed with the naked eye. The results showed that the liver and kidney tissues in each experimental group were not swollen, and the color and morphology were normal. The tissue surface was smooth and there were no ecchymoses or petechiae. The kidney tissues of the mice were observed under a light microscope. The number of cells in the glomeruli, the lumen of the capillaries, and the basement membrane of the kidney tissues of the mice in each experimental group were normal. No lesions such as renal tubular cell edema and renal tubular casts were observed.

[0123] (2) As shown in Tables 1 and 2, the levels of ALT, AST, AKP, Cr, and BUN increased in the experimental group compared with the control group and the model group, but the differences were not statistically significant (P>0.05). This indicates that the gel microbeads provided by the present invention are not toxic to the liver and kidneys.

[0124] Table 1 Questionnaire for ALT, AST, and AKP levels

[0125] Group ALT (IU / L) AST (IU / L) AKP(gprot / L) Experimental group 23.57 85.14 54.63 Control group 1 22.97 84.67 53.27 Control group 2 22.34 82.45 50.37 Control group 3 20.85 79.55 52.23 Control group 4 24.36 81.36 51.89 Blank control group 21.36 84.56 52.44 Model Group 20.34 80.36 49.85

[0126] Table 2 Questionnaire for Cr and BUN levels

[0127] Group Cr (μmol / L) BUN (mmol / L) Experimental group 72.41 11.85 Control group 1 69.30 10.97 Control group 2 70.55 12.35 Control group 3 71.44 11.84 Control group 4 72.63 10.67 Blank control group 67.85 11.36 Model Group 65.32 10.68

[0128] Note: Compared with the blank control group, *P<0.05; compared with the model group, **P<0.05.

[0129] (3) As shown in Table 3, quantitative analysis of bone morphological indicators showed that compared with the model group, the gel microbeads of the present invention significantly improved the subchondral bone volume (BV), bone volume fraction (BV / TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) of the tibial side of KOA mice after treatment, and also reduced the trabecular separation (Tb.Sp) (P < 0.05).

[0130] Table 3 CT test results

[0131]

[0132] (4) As can be seen from Table 4, the gel microbeads prepared by the present invention can significantly reduce the levels of inflammatory factors IL-1β and TNF-α in the serum of KOA mice (P < .001), and can inhibit joint synovial inflammation. It can be seen from the data of the control group that only the gel microbeads prepared by the complete preparation process of the present invention can achieve the best therapeutic effect. If some of the components are discarded, the therapeutic effect of the drug will be significantly affected. In addition, from the comparison of the data of the experimental group, the control group and the prior art group, it can be seen that if gel microbeads are used to carry therapeutic drugs, the therapeutic effect of the drug will be further improved. The therapeutic effect of the drug in the prior art group is not ideal, which may be because the drug is administered once a week in this experiment and the dosage is low.

[0133] Table 4 Inflammatory factor questionnaire

[0134]

[0135]

[0136] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing emulsion gel microbeads, characterized in that: The steps include: (1) Dodecylsuccinic anhydride-modified sodium alginate solution and fish oil were mixed to obtain a DSA emulsion; (2) mixing the DSA emulsion, sodium alginate solution and glycolithocholic acid to obtain a DSA / SA emulsion; (3) The DSA / SA emulsion, surfactant solution, and co-emulsifier solution are mixed to obtain an O / W / O emulsion; the surfactant solution comprises SPAN80, egg yolk lecithin, and poloxamer 188 in a volume ratio of 10-16:5-7:2-4; the co-emulsifier solution comprises glyceryl monostearate and polyethylene glycol 400 in a volume ratio of 1-3:3-1; (4) adding a calcium chloride solution dropwise into the O / W / O emulsion to obtain an emulsion gel; (5) The emulsion gel is mixed with water, stirred, and allowed to stand to remove impurities, thereby obtaining emulsion gel microbeads.

2. The preparation method according to claim 1, characterized in that The volume concentration of the sodium alginate solution modified with dodecylsuccinic anhydride in step (1) is 2-3%; the volume ratio of the sodium alginate solution modified with dodecylsuccinic anhydride to fish oil is 3-5:

1.

3. The preparation method according to claim 1, characterized in that In step (2), the volume mass ratio of the DSA emulsion, the sodium alginate solution and the glycolithocholic acid is 2 mL: 4-6 mL: 0.2-0.4 g; and the mass concentration of the sodium alginate solution is 1.5-2.5%.

4. The preparation method according to claim 1, characterized in that In step (3), the volume ratio of the DSA / SA emulsion, the surfactant solution, and the co-emulsifier solution is 2:6~8:2~4; the volume concentration of the surfactant solution is 2~3%; and the volume concentration of the co-emulsifier solution is 2~3%.

5. The preparation method according to claim 1, characterized in that The mass concentration of the calcium chloride solution in step (4) is 0.5-1.5%; the volume ratio of the calcium chloride solution to the O / W / O emulsion is 4-6:

1.

6. The preparation method according to claim 1, characterized in that The volume ratio of the emulsion gel to water in step (5) is 1:4-6.

7. Emulsion gel microbeads obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the emulsion gel microbeads according to claim 7 in preparing a drug for promoting cartilage repair or treating osteoarthritis, characterized in that: The emulsion gel microbeads are used as carriers to carry therapeutic drugs.

9. The use according to claim 8, characterized in that The therapeutic drug is tofacitinib.

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