An environmentally responsive drug-loaded microsphere for treating arthritis, its preparation method and application

Abstract

The present invention provides an environment-responsive drug-loaded microsphere for treating arthritis, a preparation method thereof and an application thereof. By using the microfluidic technology, polyethylene glycol diacrylate molecules undergo a polymerization reaction under the action of a photoinitiator to form a first hydrogel network structure, and modified polyethylene glycol and chitosan molecules undergo a chemical reaction to generate Schiff base bonds to form a second hydrogel network structure, thereby constructing a drug-loaded microsphere with a double network structure. The preparation method of the present invention is simple, the preparation period is short, the particle size of the obtained drug-loaded microsphere is controllable, the particle size distribution is uniform, the batches are stable, and the physical and chemical properties are relatively uniform. The drug-loaded microsphere of the present invention has good application value in the field of drug carriers, has a good encapsulation effect on CST-14, the hydrogel microsphere loaded with CST-14 has a good effect on the treatment of arthritis, and has a stimulus-responsive property and can release drugs according to the change of pH.

Description

Technical Field

[0001] The present invention relates to an environmentally responsive drug-loaded microsphere for treating arthritis, a preparation method thereof, and an application thereof, and belongs to the technical fields of micro-nano materials and drug carriers. Background Art

[0002] The preparation of drug carriers is a key research focus in the field of drug controlled release systems. Hydrogel materials with responsiveness and good biocompatibility provide potential materials and applications for drug carriers. How to prepare hydrogel materials suitable for drug carriers and how to prepare hydrogel materials into micro-nano drug carriers are the key concerns of researchers. Hydrogel microspheres are a commonly used drug carrier. According to different design concepts, the preparation methods of traditional hydrogel microspheres are divided into two methods: top-down and bottom-up. The top-down method mainly refers to the mechanical fragmentation method, and the bottom-up methods include nanoprecipitation, salting out, dialysis, emulsification, spray drying, in-situ micronization, and supercritical fluid technology. However, these traditional hydrogel microsphere preparation methods have complex preparation processes, long preparation times, and problems such as different physical and chemical properties, uncontrollable particle size, and uneven particle size distribution even for hydrogel microspheres prepared in the same batch.

[0003] Osteoarthritis (OA) is a common type of arthritis worldwide. Progressive degeneration and cartilage destruction are one of the hallmarks of osteoarthritis, and this disease has become a common cause of disability for millions of adults. So far, systemic administration of anti-inflammatory drugs and intra-articular injection are the first-line treatment methods for osteoarthritis. The drug delivery of osteoarthritis is a continuous challenge, so effective drug delivery is the key to the treatment of osteoarthritis.

[0004] Microfluidic technology can rapidly produce a large number of hydrogel microspheres with controllable and uniform sizes and relatively uniform physical and chemical properties, and this method is simple, easy to operate, and has good repeatability. The preparation of hydrogel microspheres using microfluidic technology generally involves two steps: 1) Using a hydrogel prepolymer solution as the dispersed phase to form microdroplets in a microfluidic chip; 2) Using chemical and physical methods to solidify the microdroplets into hydrogel microspheres in a microchannel or a droplet collection solution.

[0005] Therefore, how to prepare drug-loaded microspheres that can respond effectively to treat arthritis with stable batches, controllable particle sizes, and uniform particle size distributions in a simple and efficient manner is worthy of further research. Summary of the Invention

[0006] In view of the deficiencies of existing drug-loaded microspheres, the present invention provides an environment-responsive drug-loaded microsphere for treating arthritis, its preparation method and application. The preparation method of the present invention is simple, the preparation period is short, the particle size of the obtained drug-loaded microspheres is controllable, the particle size distribution is uniform, the batches are stable, and the physical and chemical properties are relatively uniform. The present invention utilizes microfluidic technology, where polyethylene glycol diacrylate molecules (PEGDA) undergo a polymerization reaction under the action of a photoinitiator to form a hydrogel first network structure, and modified polyethylene glycol (DF-PEG) and chitosan (CS) molecules undergo a chemical reaction to form Schiff base bonds to form a hydrogel second network structure, thereby constructing a drug-loaded microsphere with a double network structure. The drug-loaded microspheres of the present invention have good application value in the field of drug carriers, have a good encapsulation effect on CST-14 (corticostatin 14, a cyclic peptide drug used for the treatment of osteoporosis and osteoarthritis), the hydrogel microspheres loaded with CST-14 have a good effect on the treatment of arthritis, and have a stimulus-responsive property, and can release drugs according to the change of pH.

[0007] The technical solution of the present invention is as follows:

[0008] An environment-responsive drug-loaded microsphere for treating arthritis, the drug-loaded microsphere is a double-network hydrogel microsphere composed of a hydrogel first network formed by the polymerization of polyethylene glycol diacrylate molecules (PEGDA), and a hydrogel second network formed by the reaction of benzaldehyde-terminated polyethylene glycol (DF-PEG) and chitosan (CS) molecules.

[0009] Preferably according to the present invention, the particle size range of the drug-loaded microspheres is mainly distributed from 230 to 250 μm, the average value of the particle size is 234 μm, and the coefficient of variation of the particle size is 4.70%.

[0010] The preparation method of the above-mentioned environment-responsive drug-loaded microsphere for treating arthritis includes the steps:

[0011] (1) In solvent A, under the action of a catalyst, polyethylene glycol and p-formylbenzoic acid are reacted to prepare benzaldehyde-terminated polyethylene glycol (DF-PEG);

[0012] (2) Dissolve the surfactant in solvent B to obtain a continuous phase; fully disperse polyethylene glycol diacrylate (PEGDA), a photoinitiator, chitosan, glacial acetic acid, and benzaldehyde-terminated polyethylene glycol in water to obtain a hydrogel prepolymer dispersion phase;

[0013] (3) Introduce the dispersion phase and the continuous phase into a microfluidic chip. After generating gel droplets, they are cured by ultraviolet light, collected, washed, and dried to obtain the drug-loaded microspheres.

[0014] Preferably according to the present invention, in step (1), the solvent A is one or a combination of two or more of methanol, ethanol, acetone, tetrahydrofuran or dichloromethane; preferably, the solvent A is a combination of tetrahydrofuran and dichloromethane, and the volume ratio of tetrahydrofuran to dichloromethane is 5-15:1; the mass ratio of p-formylbenzoic acid to the volume of solvent A is 0.005-0.1 g / mL.

[0015] Preferably according to the present invention, in step (1), the catalyst is a combination of dicyclohexylcarbodiimide and 4-dimethylaminopyridine, and the mass ratio of dicyclohexylcarbodiimide to 4-dimethylaminopyridine is 15-20:1; the mass of the catalyst is 1-3 times the mass of p-formylbenzoic acid. Preferably, the catalyst dicyclohexylcarbodiimide is added to the reaction system in the form of a dicyclohexylcarbodiimide solution; the solvent used for the dicyclohexylcarbodiimide solution is the same as solvent A, and the concentration of the dicyclohexylcarbodiimide solution is 0.01-0.1 g / mL.

[0016] Preferably according to the present invention, in step (1), the molar ratio of the hydroxyl group of polyethylene glycol to the carboxyl group in p-formylbenzoic acid is 1:1-1:4.

[0017] Preferably according to the present invention, in step (1), the polyethylene glycol can be linear polyethylene glycol or hyperbranched polyethylene glycol; preferably, the polyethylene glycol is linear polyethylene glycol, four-arm polyethylene glycol or eight-arm polyethylene glycol.

[0018] Preferably according to the present invention, in step (1), the number-average molecular weight of the polyethylene glycol is 200-20000; preferably, the number-average molecular weight of the polyethylene glycol is 2000-5000.

[0019] Preferably according to the present invention, in step (1), the reaction temperature is 15-50 °C. The reaction time is 10-60 hours; preferably, the reaction time is 20-30 h.

[0020] Preferably according to the present invention, in step (1), the reaction is carried out under the protection of an inert gas; the inert gas is nitrogen, argon or helium; preferably, the inert gas is nitrogen.

[0021] Preferably according to the present invention, in step (1), the post-treatment of the reaction solution obtained by the reaction of polyethylene glycol and p-formylbenzoic acid can be carried out according to the prior art. Preferably, the post-treatment steps are as follows: the reaction solution is filtered to remove the precipitate, and the solid intermediate product is obtained by rotary evaporation; the solid intermediate product is dissolved in water, filtered, and the obtained filtrate is freeze-dried to obtain benzaldehyde-terminated polyethylene glycol.

[0022] Preferably according to the present invention, in step (2), the solvent B is a liquid paraffin organic solvent; preferably, the solvent B is a normal alkane with C16 - C20; more preferably n - hexadecane.

[0023] Preferably according to the present invention, in step (2), the surfactant is a Span - type or Tween - type surfactant; preferably, the surfactant is Span80, Tween80, Tween60 or Tween20, and more preferably Span80.

[0024] Preferably according to the present invention, in step (2), the concentration of the surfactant in the continuous phase is 5wt% - 20wt%. The purpose of adding the surfactant to the solvent B is to prevent the coalescence of droplets during the droplet formation process.

[0025] Preferably according to the present invention, in step (2), the hydrogel prepolymer dispersion phase comprises the following raw materials in parts by mass: 5 - 25 parts of polyethylene glycol diacrylate, 0.1 - 1 part of chitosan, 0.1 - 5 parts of benzaldehyde - terminated polyethylene glycol, 1 - 5 parts of photoinitiator, 0.1 - 1 part of glacial acetic acid, and 63 - 94 parts of water. The size of the generated droplets is closely related to the viscosity of the dispersion phase solution. Under the condition that the flow rate and the flow rate ratio remain unchanged, when the viscosity of the dispersion phase solution is relatively low, the viscous force is low, and the shearing effect of the continuous phase is obvious, and the size of the droplets generated by the dispersion phase is relatively small; while when the viscosity of the dispersion phase solution is relatively high, the viscous force is high, and the dispersion phase is not easily broken to form droplets, and the size of the generated droplets is relatively large. Therefore, the dispersion phase with the above composition of the present invention is selected. If the composition of the hydrogel prepolymer dispersion phase of the present invention is not suitable, it will affect the structure or performance of the obtained drug - loaded microspheres; for example, an increase in the proportion of chitosan in the solution will make the solution become viscous, and the fluid velocity will decrease significantly, resulting in the production process of microspheres becoming difficult to control; if the proportion of PEGDA is too much, and the UV curing is not sufficient, the microspheres may be toxic due to the uncured PEGDA monomers.

[0026] Preferably, the hydrogel prepolymer dispersion phase comprises the following raw materials in parts by mass: 10 parts of polyethylene glycol diacrylate, 0.5 part of chitosan, 1 part of benzaldehyde - terminated polyethylene glycol, 2 parts of photoinitiator, 0.5 part of glacial acetic acid, and 86 parts of water.

[0027] Preferably according to the present invention, in step (2), the number - average molecular weight of polyethylene glycol diacrylate is 200 - 6000, preferably 200 - 1500.

[0028] Preferably according to the present invention, in step (2), the weight - average molecular weight of chitosan is 50,000 - 500,000Da, and the degree of deacetylation ≥85%; preferably, the weight - average molecular weight of chitosan is 150,000Da, and the degree of deacetylation ≥95%.

[0029] Preferably according to the present invention, in step (2), the photoinitiator is photoinitiator 2959.

[0030] Preferably according to the present invention, in step (3), the feeding rate of the continuous phase is 0.01 - 0.15 mL / min, the feeding rate of the dispersed phase is 0.001 - 0.15 mL / min, and the flow rate ratio of the continuous phase to the dispersed phase is 30 - 100:1; preferably, the feeding rate of the continuous phase is 0.15 mL / min and the feeding rate of the dispersed phase is 0.005 mL / min. The flow rates and flow rate ratio of the two phases not only determine whether droplets can be formed, but also affect the droplet size and generation rate when droplets are formed. When the flow rates of the two phases are close, the dispersed phase tends to form rod-shaped droplets or even fibers; when the flow rate ratio of the continuous phase to the dispersed phase is within a certain range, the dispersed phase can form droplets; while when the flow rate ratio of the continuous phase to the dispersed phase is too large and exceeds a certain critical value of the flow rate ratio, the dispersed phase cannot generate droplets. The critical value of the flow rate ratio has a certain relationship with the flow rates of the two phases. When droplets can be generated, the droplet size decreases with the increase of the flow rate ratio of the continuous phase to the dispersed phase, and the droplet generation rate increases with the increase of the flow rate ratio of the continuous phase to the dispersed phase.

[0031] Preferably according to the present invention, in step (3), the microfluidic chip is a flow focusing type chip or a T-type chip; preferably, the microfluidic chip is a flow focusing type chip. In the flow focusing type chip, the continuous phase applies a symmetric shear force to the dispersed phase, thereby shearing the dispersed phase into droplets. Compared with the T-type chip, the droplets generated by the flow focusing method not only have a wider particle size range distribution, but also are easier to control in size, and the droplet generation is also more stable. Therefore, the flow focusing type chip is preferably selected.

[0032] Preferably according to the present invention, in step (3), the ultraviolet curing temperature is room temperature, the ultraviolet curing time is 5 s - 30 s, and the power of the ultraviolet lamp is 10 w - 50 w.

[0033] Preferably according to the present invention, in step (3), n-hexadecane is used to collect the cured microspheres; the reagent used for washing is an aqueous solution of isopropanol, wherein the volume ratio of isopropanol to water is 1:1 - 5; preferably, the volume ratio of isopropanol to water is 1:1; the drying is freeze-drying; the obtained drug-loaded microspheres after drying are stored at (-10) - (-30) °C.

[0034] Use of the above environment-responsive drug-loaded microspheres for treating arthritis in drugs for treating arthritis.

[0035] Preferably according to the present invention, the drug-loaded microspheres are used as a carrier for loading CST-14 (corticostatin 14).

[0036] Preferably, the application method includes the steps of adding the drug-loaded microspheres to the CST-14 solution, incubating to allow the drug molecules to fully react with the gel network, and obtaining the drug-loaded microspheres.

[0037] More preferably, the concentration of the CST-14 solution is 50 - 500 μg / mL, and the solvent used is PBS buffer; preferably, the pH of the PBS buffer is 3.5 - 7.0, and more preferably, the pH of the PBS buffer is 7.0.

[0038] More preferably, each milliliter of the CST-14 solution contains 1000 - 3000 drug-loaded microspheres.

[0039] More preferably, the incubation temperature is 1 - 10°C, preferably 2°C; the incubation time is 1 - 24 h, preferably 12 h.

[0040] The technical features and beneficial effects of the present invention are as follows:

[0041] 1. The present invention selects the microfluidic technology to prepare the hydrogel microspheres. The microfluidic chip is used to prepare the hydrogel droplets with uniform size, and then the hydrogel droplets are solidified into hydrogel microspheres by ultraviolet irradiation. The preparation method of the present invention is simple, the preparation period is short, the obtained drug-loaded microspheres have controllable particle size and uniform particle size distribution, stable batches, and relatively uniform physical and chemical properties. The hydrogel microspheres prepared in the present invention, as a form of hydrogel, have a larger surface-volume ratio and excellent injectability compared with the bulk hydrogel.

[0042] 2. The present invention uses the microfluidic technology to prepare a hydrogel microsphere with a double-network structure. The polyethylene glycol diacrylate molecule (PEGDA) undergoes a polymerization reaction under the action of a photoinitiator to form the first hydrogel network structure. At the same time, the modified polyethylene glycol (DF-PEG) and chitosan (CS) molecules undergo a chemical reaction to generate Schiff base bonds, thereby forming the second hydrogel network structure, as Figure 3As shown in the figure, when chitosan and DF-PEG form a drug-anchoring network and encounter an extreme inflammatory environment (pH < 5), the bonds will break and decompose rapidly, while the PEGDA network will remain to maintain the gel form. Secondly, the presence of PEGDA improves the mechanical properties of the double network. For the simple PEGDA network structure, the obtained microspheres do not have the ability to anchor drugs; the microspheres obtained from the PEGDA and chitosan gel network are difficult to be stable in an environment with changing pH values and do not have the ability to anchor drugs; the chitosan and DF-PEG gel network structure will completely decompose in an acidic environment and do not have the gel form, which is difficult to be compatible with the in-vivo environment. When the proportion of chitosan in the solution increases, the solution becomes viscous and the fluid velocity decreases significantly, resulting in the production process of microspheres becoming difficult to control; when the proportion of PEGDA is too high, the ultraviolet curing is insufficient, and the microspheres may be toxic due to the uncured PEGDA monomers.

[0043] 3. The microspheres for drug loading of the present invention have good application value in the field of drug carriers and can be used as drug carriers to better deliver drugs. In the microspheres obtained in the present invention, the highly active aldehyde groups on the modified polyethylene glycol benzene ring can easily form Schiff base bonds with the three primary amines on the CST-14 cyclic peptide that plays a key role in the treatment of inflammation. These bonds show high stability and strong binding energy under normal conditions, but are highly sensitive to the pH value. When the pH value is low, the Schiff base bonds will break, so as to release the drug to achieve the therapeutic effect. In the present invention, the combination of the drug and the microspheres enables the drug molecules to react with the gel network and be anchored, and this interaction usually results in a longer drug molecule release time. The microspheres obtained in the present invention have a good encapsulation effect on CST-14. The hydrogel microspheres loaded with CST-14 have a good effect on the treatment of arthritis and have a stimulus-responsive performance, and can release drugs according to the change of pH; when there is inflammation in the joint, the pH in the joint will be lower, which will cause the rapid destruction of the drug-anchoring structure, thus causing the release of the drug to achieve the purpose of treating arthritis; when the disease develops slowly and the pH change is small, the drug can be stored stably. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Schematic diagrams of the flow-focusing type (left) and T-type chip (right) structures;

[0045] Figure 2 Schematic diagram of the droplet generation process in the flow-focusing type chip;

[0046] Figure 3 Schematic diagram of the double network structure of the hydrogel microspheres for drug loading of the present invention;

[0047] Figure 4 Microscopic image of the hydrogel microspheres for drug loading in Example 1;

[0048] Figure 5 It is the particle size distribution diagram of the hydrogel drug-loaded microspheres in Example 1;

[0049] Figure 6 It is the drug release diagram of the drug-loaded gel microspheres in different pH environments in the test example;

[0050] Figure 7 It is the treatment effect diagram of the drug-loaded gel microspheres for rat osteoarthritis in the test example;

[0051] Figure 8 It is the histological analysis diagram of the drug-loaded gel microspheres promoting cartilage repair in rat osteoarthritis in the test example. Detailed implementation manners

[0052] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation to the present invention. The experimental methods without specific conditions and the reagents without specified formulations in the embodiments are all according to the conventional conditions in the art.

[0053] Example 1

[0054] Preparation of hydrogel drug-loaded microspheres by microfluidic technology

[0055] Step 1: Synthesize benzaldehyde-terminated polyethylene glycol (DF-PEG). The synthesis steps are as follows: First, prepare Solution 1 by weighing 8 g of polyethylene glycol (number average molecular weight 4000), 1.8 g of p-formylbenzoic acid, and 0.18 g of 4-dimethylaminopyridine (catalyst) and dissolve them in 100 mL of tetrahydrofuran. To accelerate dissolution, add 10 mL of dichloromethane thereto and assist dissolution using an ultrasonic cleaner. Then prepare Solution 2 by weighing 3.296 g of dicyclohexylcarbodiimide (dehydrating agent) and dissolve it in 50 mL of tetrahydrofuran. Under nitrogen protection, slowly inject Solution 2 into Solution 1, and stir the mixture with a magnetic stirrer for 24 hours (27 °C). Next, perform two suction filtrations. For the first suction filtration, filter out the solid particles in the mixture, and perform rotary evaporation drying on the filtered solution to remove the organic solvents of tetrahydrofuran and dichloromethane. Then dissolve the solid obtained by rotary evaporation in deionized water. For the second suction filtration, filter out the solid particles in the above aqueous solution, and then place the filtrate in a freeze dryer for freeze drying for 24 hours to obtain the solid benzaldehyde-terminated polyethylene glycol (DF-PEG).

[0056] Step 2: Select n-hexadecane solution as the matrix solution for the continuous phase. To prevent the fusion and coalescence of droplets during the droplet generation experiment, add Span 80 surfactant to n-hexadecane. Dissolve the surfactant Span 80 fully in n-hexadecane to obtain a continuous phase with a concentration of 10 wt%.

[0057] Step 3: The dispersed phase is an aqueous solution of PEGDA&CS&DF-PEG, including the following components: 1 g of PEGDA (number average molecular weight 700), 0.2 g of photoinitiator 2959, 0.05 g of chitosan (weight average molecular weight 150,000 Da, deacetylation ≥ 95%), 0.05 g of glacial acetic acid, 0.1 g of DF-PEG, and 8.6 g of deionized water. The preparation steps of the dispersed phase are as follows: (1) First, weigh the photoinitiator 2959 with a balance and add it to deionized water, heat and stir to dissolve at a temperature of 50 °C; (2) Wait for the above solution to cool to room temperature, add glacial acetic acid, and then add chitosan, stir to dissolve; (3) Finally, add PEGDA and DF-PEG, stir to disperse evenly, remove bubbles with an ultrasonic cleaner, and store in a brown reagent bottle.

[0058] Step 4: Apply a flow-focusing microfluidic chip (the structural schematic diagram is as shown in Figure 1 the left, Figure 1 the marked dimensions are all the diameters of the channels, and the droplet generation process is as shown in Figure 2 the figure). Feed the continuous phase and the dispersed phase into the microfluidic chip simultaneously at flow rates of 0.15 mL / min and 0.005 mL / min respectively. Gel droplets are generated at the interface between the two phases under the symmetric shearing action of the continuous phase, and the generated droplets are irradiated with 12 W ultraviolet light at room temperature for 20 s for curing.

[0059] Step 5: Use n-hexadecane to collect the above-mentioned solid-liquid mixture, wash it three times with an isopropanol-aqueous solution with a volume ratio of 1:1, centrifuge to remove oil, and finally obtain drug-loaded microspheres through freeze-drying and store them at -20 °C. The schematic diagram of the double-network structure of the drug-loaded microspheres is as shown in Figure 3 the figure.

[0060] Figure 4 and 5 are respectively the microscope image and particle size distribution diagram of the hydrogel drug-loaded microspheres prepared in this example. It can be seen from the figure that the drug-loaded microspheres prepared by the present invention have good size uniformity, and the particle size range is mainly distributed from 230 to 250 μm, and the average value of the particle size is 234 μm.

[0061] The above hydrogel drug-loaded microspheres are used to prepare drug-loaded microspheres. The specific preparation method includes the steps of:

[0062] Add the hydrogel drug-loaded microspheres to the CST-14 solution (the mass concentration of the CST-14 solution is 200 micrograms per milliliter, the solvent used is PBS buffer with a pH of 7 or 3.5, and each milliliter of the CST-14 solution contains 2000 drug-loaded microspheres), incubate at 2 °C for 12 hours to allow the drug molecules to fully react with the gel network, and obtain a drug-loaded microsphere dispersion.

[0063] Example 2

[0064] The preparation method of the hydrogel-loaded drug microspheres is as described in Example 1, except that: the flow-focusing microfluidic chip is replaced by a T-shaped chip (the structural schematic diagram is as shown on the right, and the dimensions marked in it are all the diameters of the channels); other preparation steps and conditions are the same as those in Example 1. Figure 1 as shown on the right, Figure 1 and the dimensions marked in it are all the diameters of the channels); other preparation steps and conditions are the same as those in Example 1.

[0065] Example 3

[0066] The preparation method of the hydrogel-loaded drug microspheres is as described in Example 1, except that: the flow rates of the continuous phase and the dispersed phase are 0.10 mL / min and 0.001 mL / min respectively; other preparation steps and conditions are the same as those in Example 1.

[0067] Comparative Example 1

[0068] The preparation method of the hydrogel-loaded drug microspheres is as described in Example 1, except that: the dispersed phase is an aqueous solution of PEGDA, which includes the following components: 1 g of PEGDA (number average molecular weight 700), 0.2 g of photoinitiator 2959, and 8.8 g of deionized water. The specific preparation method is as follows: (1) First, weigh the photoinitiator 2959 with a balance, add it to deionized water, heat and stir to dissolve, and the temperature is 50 °C; (2) After the above solution is cooled to room temperature, add PEGDA, stir to disperse evenly, remove the bubbles with an ultrasonic cleaner, and then store it in a brown reagent bottle. Other preparation steps and conditions are the same as those in Example 1.

[0069] The method for applying the above hydrogel-loaded drug microspheres to prepare drug-loaded microspheres is the same as that in Example 1.

[0070] The microspheres prepared in this comparative example do not have the ability to anchor drugs.

[0071] Comparative Example 2

[0072] The preparation method of the hydrogel-loaded drug microspheres is as described in Example 1, except that: the dispersed phase is an aqueous solution of PEGDA&CS, which includes the following components: 1 g of PEGDA (number average molecular weight 700), 0.2 g of photoinitiator 2959, 0.05 g of chitosan (weight average molecular weight 150,000 Da, deacetylation ≥ 95%), 0.05 g of glacial acetic acid, and 8.7 g of water. The specific preparation method is as follows: (1) First, weigh the photoinitiator 2959 with a balance, add it to deionized water, heat and stir to dissolve, and the temperature is 50 °C; (2) After the above solution is cooled to room temperature, add glacial acetic acid, and then add chitosan, and stir to dissolve; (3) Finally, add PEGDA, stir to disperse evenly, remove the bubbles with an ultrasonic cleaner, and then store it in a brown reagent bottle. Other preparation steps and conditions are the same as those in Example 1.

[0073] The method for using the above hydrogel drug-loaded microspheres to prepare drug-loaded microspheres is the same as that in Example 1.

[0074] The microspheres prepared in this comparative example are difficult to be stable in an environment with pH changes and do not have the ability of drug anchoring.

[0075] Comparative Example 3

[0076] The preparation method of the hydrogel drug-loaded microspheres is as described in Example 1, except that: the dispersed phase is an aqueous solution of CS&DF-PEG, including the following components: 0.2 g of photoinitiator 2959, 0.05 g of chitosan (weight average molecular weight 150,000 Da, deacetylation ≥ 95%), 0.05 g of glacial acetic acid, 0.1 g of DF-PEG, and 9.6 g of water. The preparation steps of the dispersed phase are: (1) First, weigh the photoinitiator 2959 with a balance, add it to deionized water, heat and stir to dissolve, and the temperature is 50 °C; (2) Wait for the above solution to cool to room temperature, add glacial acetic acid, and then add chitosan, stir to dissolve; (3) Finally, add DF-PEG, stir to disperse evenly, remove bubbles with an ultrasonic cleaner, and store in a brown reagent bottle. Other preparation steps and conditions are the same as those in Example 1.

[0077] The method for using the above hydrogel drug-loaded microspheres to prepare drug-loaded microspheres is the same as that in Example 1.

[0078] The simple DF-PEG and chitosan gel network will completely decompose in an acidic environment and do not have the morphology of a gel, which is difficult to be compatible with the in-vivo environment.

[0079] Test Example:

[0080] (1) In-vitro release experiment

[0081] The in-vitro release experiment of the binding of microspheres and drugs provides a better qualitative and quantitative evaluation for the binding and separation process of drugs and the hydrogel matrix.

[0082] Add 1 mL of the drug-loaded microsphere dispersion prepared in Example 1 to 20 mL of PBS (pH 7 and 3.5), place it in a normal temperature environment, and collect the supernatant for analysis at different time intervals.

[0083] From Figure 6 It can be seen that when the pH of the system is 3.5, the concentration of CST-14 in the supernatant reaches the peak at 30 minutes, while when the pH of the system is 7, it takes twice the time. Therefore, the drug-loaded microspheres prepared by the present invention can dynamically adjust drug release according to environmental conditions, and it is easier to release drugs under acidic conditions (pH = 3.5) than in a normal environment (pH = 7).

[0084] (2) Establishment of a rat model

[0085] Anesthetize the rats with isoflurane in a rat anesthetic apparatus, and then perform arthroscopic surgery on the right joint by incising the medial meniscus ligament. The sham operation group (abbreviated as the sham group) used rats of the same age as the control, with the same incision at the right joint capsule, and the meniscus ligament was not resected. The medial meniscus ligament surgery was all performed under a microscope. According to the grouping design, on the 8th day after surgery, 100 μL of PBS (pH = 7, abbreviated as the control group), a PBS solution of the hydrogel-loaded drug microspheres prepared in Example 1 (the solvent was PBS solution with a pH of 7; each milliliter of PBS solution contained 2000 drug-loaded microspheres; abbreviated as the microspheres group), a PBS solution of CST-14 (mass concentration of 200 micrograms per milliliter, and the solvent used was PBS solution with a pH of 7, abbreviated as the CST group), and the drug-loaded microsphere dispersion prepared in Example 1 (where the pH of the PBS buffer was 7, abbreviated as the microspheres + CST group) were respectively injected into the knee joints of the rats, twice a week. The rats were sacrificed 6 weeks after surgery.

[0086] It can be seen from Figure 7 that the X-ray images showed no significant difference in the joint space width between the microspheres group (microspheres) and the control group (control), and the osteoarthritis joint space width of the microspheres + CST group was the largest. Micro-CT images showed that the joint morphology of the microspheres + CST group was better and there were fewer bone fragments, and microspheres + CST reduced the formation of osteophytes in the knee joints of mice. It shows that microspheres + CST combines the therapeutic effect of CST-14 with the drug sustained delivery and joint lubrication ability of the microspheres well. Therefore, microspheres + CST can better relieve the effect of cartilage aging in the DMM osteoarthritis model.

[0087] (3) Histological analysis of osteoarthritis

[0088] The collected knee joint specimens were fixed with 4% paraformaldehyde and embedded in paraffin. The tissue sections were mounted on glass slides with a microtome (Leica, Germany). The safranin O-fast green (S-O) staining was performed according to the standard protocol. The sections were stained with a Safranin O staining kit (G1371, Solarbio) according to the manufacturer's instructions.

[0089] Figure 8As shown in b, safranin O-fast green staining showed a decrease in proteoglycans in the control group. There were signs of damage repair in both the microspheres group and the CST group, but the CST+Microspheres group had obvious improvement. Regarding the erosion of articular cartilage, such as cracks and deformities, the erosion of articular cartilage was most obvious in the control group, followed by the microspheres group and the CST group. The CST+Microspheres group showed better morphological integrity and was most similar to the Sham group (sham operation group). At the same time, the protein expression levels of two important articular cartilage biomarkers were measured. Figure 8 As shown in c and e, there was no significant difference in the expression of Aggrecan and MMP13 proteins between the microspheres group and the control group. Compared with the control group, the expression of Aggrecan protein increased and the expression of MMP13 protein decreased in the CST group and the CST+Microspheres group. Figure 8 As shown in d and f, the changes in the expression of Aggrecan and MMP13 proteins were most obvious in the CST+Microspheres group. These results indicate that CST-loaded microspheres can improve the cartilage delivery efficiency of the therapeutic drug CST. The CST-loaded microspheres can effectively deliver the drug into cells after intra-articular injection, which can more effectively protect chondrocytes from senescence than simply injecting CST, providing an attractive method for the treatment of osteoarthritis.

Claims

1. Use of an environmentally responsive drug-loaded microsphere for treating arthritis in the preparation of a drug for treating arthritis, characterized in that, The drug-loaded microspheres are used as a carrier for loading CST-14; the application method includes the steps of adding the drug-loaded microspheres into a CST-14 solution, incubating, and allowing the drug molecules to fully react with the gel network to obtain the drug-loaded microspheres; The drug-loaded microspheres are double-network hydrogel microspheres composed of a first hydrogel network formed by the polymerization of polyethylene glycol diacrylate molecules and a second hydrogel network formed by the reaction of benzaldehyde-terminated polyethylene glycol and chitosan molecules; The preparation method of the drug-loaded microspheres includes the steps of: (1) In solvent A, under the action of a catalyst, polyethylene glycol and p-formylbenzoic acid are reacted to prepare benzaldehyde-terminated polyethylene glycol; (2) Dissolve the surfactant in solvent B to obtain a continuous phase; fully disperse polyethylene glycol diacrylate, a photoinitiator, chitosan, glacial acetic acid, and benzaldehyde-terminated polyethylene glycol in water to obtain a hydrogel prepolymer dispersion phase; (3) Introduce the dispersion phase and the continuous phase into a microfluidic chip. After generating gel droplets, cure them with ultraviolet light, and collect, wash, and dry them to obtain the drug-loaded microspheres.

2. The application according to claim 1, characterized in that, The particle size range distribution of the drug-loaded microspheres is 230-250 μm, the average value of the particle size is 234 μm, and the coefficient of variation of the particle size is 4.70%.

3. The application according to claim 1, characterized in that, In step (1) of the preparation method of the drug-loaded microspheres, one or more of the following conditions are included: i. The solvent A is one or a combination of two or more of methanol, ethanol, acetone, tetrahydrofuran, or dichloromethane; the mass ratio of p-formylbenzoic acid to the volume of solvent A is 0.005-0.1 g / mL; ii. The catalyst is a combination of dicyclohexylcarbodiimide and 4-dimethylaminopyridine, and the mass ratio of dicyclohexylcarbodiimide to 4-dimethylaminopyridine is 15-20:1; the mass of the catalyst is 1-3 times the mass of p-formylbenzoic acid; the catalyst dicyclohexylcarbodiimide is added to the reaction system in the form of a dicyclohexylcarbodiimide solution; the solvent used for the dicyclohexylcarbodiimide solution is the same as solvent A, and the concentration of the dicyclohexylcarbodiimide solution is 0.01-0.1 g / mL; iii. The molar ratio of the hydroxyl group of polyethylene glycol to the carboxyl group in p-formylbenzoic acid is 1:1-1:4; iv. The polyethylene glycol is linear polyethylene glycol or hyperbranched polyethylene glycol; v. The number-average molecular weight of the polyethylene glycol is 200-20000; vi. The reaction temperature is 15-50 °C; the reaction time is 10-60 hours; vii. The reaction is carried out under the protection of an inert gas; the inert gas is nitrogen, argon, or helium.

4. The application according to claim 3, characterized in that One or more of the following conditions are included: i. The polyethylene glycol is linear polyethylene glycol, four-arm polyethylene glycol, or eight-arm polyethylene glycol; ii. The number-average molecular weight of the polyethylene glycol is 2000-5000; iii. The reaction time is 20-30 h; iv. The solvent A is a combination of tetrahydrofuran and dichloromethane, and the volume ratio of tetrahydrofuran to dichloromethane is 5-15:

1.

5. The application according to claim 1, characterized in that In step (2) of the preparation method of the drug-loaded microspheres, one or more of the following conditions are included: i. The solvent B is a liquid paraffin organic solvent; the solvent B is a normal alkane with C16 - C20; ii. The surfactant is a Span or Tween surfactant; iii. The concentration of the surfactant in the continuous phase is 5wt% - 20wt%; iv. The number average molecular weight of polyethylene glycol diacrylate is 200 - 6000; v. The weight average molecular weight of chitosan is 50,000 - 500,000 Da, and the degree of deacetylation is ≥85%; vi. The photoinitiator is photoinitiator 2959.

6. The application according to claim 5, wherein Including one or more of the following conditions: i. The solvent B is n - hexadecane; ii. The surfactant is Span80, Tween80, Tween60 or Tween20; iii. The number average molecular weight of polyethylene glycol diacrylate is 200 - 1500; iv. The weight average molecular weight of chitosan is 150,000 Da, and the degree of deacetylation is ≥95%.

7. The application according to claim 1, characterized in that, In step (2) of the preparation method of the drug - loaded microspheres, the hydrogel prepolymer dispersion phase comprises the following raw materials in parts by mass: 5 - 25 parts of polyethylene glycol diacrylate, 0.1 - 1 part of chitosan, 0.1 - 5 parts of polyethylene glycol with benzaldehyde end - groups, 1 - 5 parts of photoinitiator, 0.1 - 1 part of glacial acetic acid, and 63 - 94 parts of water.

8. The application according to claim 7, characterized in that, The hydrogel prepolymer dispersion phase comprises the following raw materials in parts by mass: 10 parts of polyethylene glycol diacrylate, 0.5 part of chitosan, 1 part of polyethylene glycol with benzaldehyde end - groups, 2 parts of photoinitiator, 0.5 part of glacial acetic acid, and 86 parts of water.

9. The application according to claim 1, wherein In step (3) of the preparation method of the drug - loaded microspheres, including one or more of the following conditions: i. The feeding rate of the continuous phase is 0.01 - 0.15 mL / min, the feeding rate of the dispersion phase is 0.001 - 0.15 mL / min, and the flow rate ratio of the continuous phase to the dispersion phase is 30 - 100:1; ii. The microfluidic chip is a flow - focusing type chip or a T - type chip; iii. The ultraviolet light curing temperature is room temperature, the ultraviolet light curing time is 5s - 30s, and the power of the ultraviolet lamp is 10w - 50w; iv. n - hexadecane is used to collect the cured microspheres; the washing reagent is an aqueous solution of isopropanol, wherein the volume ratio of isopropanol to water is 1:1 - 5; the drying is freeze - drying; the obtained drug - loaded microspheres after drying are stored at (-10) - (-30)°C.

10. The application according to claim 9, characterized in that Including one or more of the following conditions: i. The feeding rate of the continuous phase is 0.15 mL / min, and the feeding rate of the dispersion phase is 0.005 mL / min; ii. The microfluidic chip is a flow - focusing type chip.

11. The application according to claim 1, characterized in that, The concentration of the CST - 14 solution is 50 - 500 micrograms per milliliter, and the solvent used is PBS buffer; the pH of the PBS buffer is 3.5 - 7.0; each milliliter of the CST - 14 solution contains 1000 - 3000 drug - loaded microspheres; the incubation temperature is 1 - 10°C; the incubation time is 1 - 24h.

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