Emulsion gel microbead as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to an emulsion gel microsphere as well as a preparation method and application thereof. The preparation method specifically comprises the following steps: (1) mixing a dodecene succinic anhydride modified sodium alginate solution and fish oil to obtain a DSA emulsion; (2) mixing the DSA emulsion, a sodium alginate solution and glycocholic acid to obtain a DSA / SA emulsion; (3) mixing the DSA / SA emulsion, the surfactant solution and the co-emulsifier solution to obtain O / W / O emulsion; (4) dropwise adding a calcium chloride solution into the O / W / O emulsion to obtain emulsion gel; and (5) mixing the emulsion gel with water, stirring, standing, and removing impurities to obtain the emulsion gel microbeads. The emulsion gel microsphere prepared by the invention can be effectively combined with drug components, has good biocompatibility after being injected and used, and can achieve the drug delivery process of slow release and uniform slow release of drugs.

Description

Technical Field

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

[0002] Pickering emulsion is an emulsion obtained by using ultrafine solid particles as emulsifiers, also known as Pickering emulsion. The type of emulsion obtained depends on which phase preferentially wets the solid particles. Usually, the phase that preferentially wets the solid particles is the external phase; for example, sometimes the solid particles are more easily wetted by the oil phase, and the emulsion is a W / O (water-in-oil) type. On the contrary, 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, the solid powders used as emulsifiers or stabilizers include metal hydroxides, graphite, carbon black, nano-silica, 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, etc. Conventional oil-in-water or water-in-oil Pickering emulsions are widely used in the preparation of porous polymers; on top of this, 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 the fields of microcapsule preparation, drug encapsulation and controlled release, food nutritional additives, cosmetics, etc. due to their special "two membranes and three phases" structure.

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

[0005] At present, the clinical treatment drugs for osteoarthritis are divided into specific therapeutic drugs and non-specific therapeutic drugs. Non-specific therapeutic drugs are mainly used for analgesia and symptom control, but have no protective effect on cartilage, such as non-steroidal anti-inflammatory drugs. Specific therapeutic drugs can protect joint cartilage and delay the progression of osteoarthritis, such as glucosamine and chondroitin sulfate, but they generally take effect slowly and require several months of treatment to be effective, and 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 effect of existing drugs for treating osteoarthritis is not ideal. Therefore, how to use Pickering emulsion to further carry drugs for treating osteoarthritis to improve the therapeutic effect of the drugs is a problem that technicians in this field urgently need to solve. 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 dodecyl succinic 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) dropping a calcium chloride solution 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, 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%.

[0017] Preferably, 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; the co-emulsifier solution is glyceryl monostearate and polyethylene glycol 400, and the volume ratio is 1-3:3-1.

[0018] Preferably, 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%.

[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 the use of the latex gel microbeads in preparing drugs for promoting cartilage repair or treating osteoarthritis, wherein the latex 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 to achieve the purpose of transporting drugs and improving drug treatment effects. In the present invention, the technical effect of effectively treating osteoarthritis is achieved by carrying the osteoarthritis drug-tofacitinib.

[0026] The sodium alginate solution modified with dodecene succinic anhydride added in the preparation of the emulsion gel microbeads of the present invention is a stabilizer in the emulsion system. Compared with the emulsion stabilized with natural alginate, the modified sodium alginate can provide repulsive force between oil droplets, enhance the physical stability of the emulsion, and can also inhibit the oxidation of lipids in the emulsion, thereby increasing the stability of the emulsion.

[0027] The glycolithocholic acid added in the present invention is used as a penetration enhancer, which has the effect of promoting the release and absorption of drugs and can effectively promote the active substances of drugs to play a role. The present invention also adds a composite emulsifier, and the use of the composite emulsifier can improve the stability of the emulsion, which can achieve better drug loading and release effects, and the use of the auxiliary emulsifier also further improves the stability of the emulsion system.

[0028] Therefore, the emulsion gel microspheres 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 release of drugs, a uniform sustained-release drug administration process, ensure the concentration of the drug in the affected area, prevent the problem of uneven drug administration due to excessively high or low drug concentrations during administration, effectively reduce the toxicity and side effects of the drugs, prevent the human body from developing resistance to the drugs, and improve 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 drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0030] Figure 1 It is a 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 dodecyl succinic 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) dropping a calcium chloride solution 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 dodecene succinic 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 1:4-6, preferably 1:5.

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

[0051] The present invention also provides the use of the latex gel microbeads in preparing drugs for promoting cartilage repair or treating osteoarthritis, wherein the latex 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 in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0054] Example 1

[0055] A method for preparing emulsion gel microbeads, the steps are as follows:

[0056] (1) A 2% dodecylsuccinic anhydride modified sodium alginate solution and fish oil were mixed in 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 having 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% (with fish oil as a solvent), and a co-emulsifier solution with a volume concentration of 2% (with 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) dropping a calcium chloride solution having a mass concentration of 0.5% into the O / W / O emulsion, and continuously stirring with a magnetic force at 1000 rpm for 15 min 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 and water are mixed 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, the steps are as follows:

[0065] (1) mixing a 3% volume concentration sodium alginate solution modified with dodecyl succinic anhydride and fish oil in a volume ratio of 5:1, and homogenizing at 8000 rpm for 1 min to obtain a DSA emulsion;

[0066] (2) mixing the DSA emulsion, a sodium alginate solution having 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 surfactant solution with a volume concentration of 3% (with fish oil as solvent), and an emulsifier solution with a volume concentration of 3% (with fish oil as 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) dropping a calcium chloride solution having a mass concentration of 1.5% into the O / W / O emulsion, and continuously stirring with a magnetic force at 1000 rpm for 15 min 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 and water are mixed in a volume ratio of 1:6, 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 60 to 120 μm.

[0072] Example 3

[0073] A method for preparing emulsion gel microbeads, the steps are as follows:

[0074] (1) mixing a 2.5% volume concentration of sodium alginate solution modified with dodecyl succinic anhydride and fish oil in a volume ratio of 4:1, and homogenizing 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% (with fish oil as solvent), and an emulsifier solution with a volume concentration of 2.5% (with fish oil as 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) dropping a calcium chloride solution having a mass concentration of 1% into the O / W / O emulsion, and continuously stirring with a magnetic force at 1000 rpm for 15 min 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 and water are mixed at 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) mixing a 2.5% sodium alginate solution modified with dodecyl succinic anhydride, fish oil and tofacitinib in a volume mass ratio of 4 mL:1 mL:1 mg, and homogenizing 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% (with fish oil as solvent), and an emulsifier solution with a volume concentration of 2.5% (with fish oil as 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) dropping a calcium chloride solution having a mass concentration of 1% into the O / W / O emulsion, and continuously stirring with a magnetic force at 1000 rpm for 15 min 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 and water are mixed at 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) mixing a 2% dodecylsuccinic anhydride modified sodium alginate solution, fish oil and tofacitinib in a volume mass ratio of 4 mL:1 mL:1 mg, and homogenizing at 8500 rpm for 1 min to obtain a DSA emulsion;

[0093] (2) mixing the DSA emulsion, a sodium alginate solution having 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% (with fish oil as a solvent), and a co-emulsifier solution with a volume concentration of 2% (with 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) dropping a calcium chloride solution having a mass concentration of 0.5% into the O / W / O emulsion, and continuously stirring with a magnetic force at 1000 rpm for 15 min 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 and water are mixed 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) mixing a 3% dodecylsuccinic anhydride modified sodium alginate solution, fish oil and tofacitinib in a volume mass ratio of 5 mL:1 mL:1 mg, and homogenizing at 8000 rpm for 1 min to obtain a DSA emulsion;

[0102] (2) mixing the DSA emulsion, a sodium alginate solution having 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 surfactant solution with a volume concentration of 3% (with fish oil as solvent), and an emulsifier solution with a volume concentration of 3% (with fish oil as 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) dropping a calcium chloride solution having a mass concentration of 1.5% into the O / W / O emulsion, and continuously stirring with a magnetic force at 1000 rpm for 15 min 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 and water are mixed 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. After 1 week of adaptive feeding, the collagen-induced arthritis (CIA) model was prepared, and 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. The model was successfully established after the injection of CIA on the 21st day (day 21), and 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 an actual dosage 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 dodecene succinic 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 are the same as those of Example 6, except that only SPAN80 is added to the surfactant solution.

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

[0116] 2. Micro-CT detection

[0117] The mouse knee joint was scanned and reconstructed in three dimensions using a micro-CT instrument, with a scanning voltage of 90 kV, a current of 80 μA, and a resolution of 10 μm. The data were analyzed using analysis software (CTAn v1.9) and three-dimensional visualization software (CTVol v2.0). The area between the tibial growth plate and the tibial plateau was determined as the region of interest, and the bone volume (BV), bone volume fraction (BV / TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N) were statistically analyzed based on the tomographic data of three-dimensional morphometry.

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

[0119] The collected blood samples were centrifuged in a high-speed refrigerated centrifuge at a speed of 3500r / min, a time of 5min, and a temperature of 4℃ for a total of 2 centrifugations to collect serum. The levels of BUN and Cr in the serum of mice were detected according to the instructions of the urea nitrogen (BUN) test kit and the creatinine (Cr) test kit to evaluate the renal function of mice. The levels of AST, ALT, and AKP in the serum of mice were detected according to the instructions of the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and alkaline phosphatase (AKP) test kits using an automatic microplate reader to evaluate the liver function of mice. L-1β (EK0391, Boster) and TNF-α (FEK0527, Boster) were detected using ELISA kits according to the methods provided in the instructions.

[0120] 4. After blood collection, the mice were killed by cervical dislocation, and the liver and kidney tissues of the mice were collected for pathological examination. The mice were fixed on the operating table in supine position, and the abdominal cavity was opened from the midline of the mouse from the lower abdomen upward with surgical scissors to avoid damaging the kidneys and liver. The liver and kidneys were quickly removed, washed with saline, and immediately fixed in 10% neutral formaldehyde for liver and kidney pathological examination. The liver and kidney tissues of each group of mice were observed with the naked eye, including whether the tissues were swollen, color, morphology, gloss, texture, and whether there were 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. It was found that the liver and kidney tissues of the mice 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 mesangium of the kidney tissues of the mice in each experimental group were normal; no lesions such as tubular cell edema and tubular casts were observed.

[0123] (2) From the contents recorded in Tables 1 and 2, it can be seen that the contents of ALT, AST, AKP, Cr, and BUN in the experimental group, the control group, and the model group increased, but the difference was 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 ALT, AST, AKP level questionnaire

[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. And the therapeutic effect of the drug in the prior art group is not ideal. This may be because the drug is administered once a week in this experiment and the dosage is relatively 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 principle 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) mixing a sodium alginate solution modified with dodecyl succinic anhydride and fish oil to obtain a DSA emulsion; (2) mixing the DSA emulsion, sodium alginate solution and glycolithocholic acid to obtain a DSA / SA emulsion; (3) mixing the DSA / SA emulsion, surfactant solution, and co-emulsifier solution to obtain an O / W / O emulsion; (4) dropping a calcium chloride solution 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 by dodecene succinic anhydride in step (1) is 2-3%; the volume ratio of the sodium alginate solution modified by dodecene succinic 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: 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; the co-emulsifier solution is glyceryl monostearate and polyethylene glycol 400, and the volume ratio is 1-3:3-1.

5. 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%.

6. 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.

7. 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.

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

9. Use of the latex gel microbeads according to claim 8 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.

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

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