WO2023236228A1

WO2023236228A1 - Drug-loaded microgel sphere, drug-loading stent and method for preparing same

Info

Publication number: WO2023236228A1
Application number: PCT/CN2022/098394
Authority: WO
Inventors: 于寅; 刘怿斯; 陈飞; 王刚
Original assignee: 深圳先进技术研究院
Priority date: 2022-06-08
Filing date: 2022-06-13
Publication date: 2023-12-14
Also published as: CN115137703B; CN115137703A

Abstract

Provided are a drug-loaded microgel sphere, a method for preparing the same, and a drug-loading stent formed by means of a photo-crosslinking reaction. The loaded drug is a disulfiram drug, and the disulfiram drug comprises disulfiram or a disulfiram derivative. The method for preparing the drug-loaded microgel sphere comprises: providing disulfiram drug nanoparticles; providing methacrylic anhydride modified gelatin; mixing the methacrylic anhydride modified gelatin and the disulfiram drug according to an equal mass ratio to obtain a water phase; mixing HFE7500 and a surfactant to obtain an oil phase; and mixing the water phase and the oil phase by means of a micro-fluidic chip technology to prepare the drug-loaded microgel sphere. The drug-loaded microgel sphere and the drug-loading stent can treat and relieve symptoms of osteoarthritis, the preparation method is simple, the drug-loading stent has a dual sustained-release effect with a better drug effect. Further provided is a new application of the disulfiram drug.

Description

Drug-loaded microgel spheres, drug-loaded scaffolds and preparation methods thereof

Technical field

The present application relates to the field of medical technology, and in particular to a drug-loaded microgel sphere, a drug-loaded stent and a preparation method thereof.

Background technique

Osteoarthritis (OA) is a chronic joint disease characterized by degeneration and destruction of articular cartilage and bone hyperplasia. It is a serious medical problem and is the fourth most disabling disease in my country and the third most disabling disease in Europe and the United States. disease.

The subchondral bone is the bony bed of the joint on which the articular cartilage lies. Traditionally, osteoarthritis (OA) has been thought of as wear and tear of articular cartilage, but recent evidence suggests that subchondral bone disorders and synovial inflammation can initiate and contribute to disease progression.

Osteoarthritis is characterized by a diverse group of diseases in which inflammation, immune, and central nervous system dysfunction play central roles in overall joint damage, injury progression, pain, and disability. Sclerosis and thickening of subchondral bone are one of the main causes of osteoarthritis. Subchondral bone is the transitional part of the osteochondral junction, between soft tissue and hard tissue. It is used to absorb stress during joint loading. When the loading is abnormal, it will lead to microfractures at the osteochondral junction and the appearance of bone. Collapse, followed by subchondral bone sclerosis. Many experiments have proven that subchondral bone regeneration or sclerosis occurs before articular cartilage degeneration. The integrity of articular cartilage depends on the biomechanical properties of the underlying bone bed. Therefore, subchondral bone sclerosis may be the initial factor in the onset of OA.

Existing technology improves the symptoms of osteoarthritis by injecting biogel into osteoarthritis areas. It uses the cell adhesion and good cell compatibility of the gel itself to play a certain role in the repair of articular cartilage. . However, the biogel itself does not contain therapeutic drugs, its joint repair ability is limited, and the therapeutic effect is poor.

Contents of the invention

The purpose of this application is to overcome the deficiencies in the prior art and provide a drug-loaded microgel sphere, a drug-loaded scaffold and a preparation method thereof, which can enhance or induce subchondral Bone rejuvenation restores the joint bone bed, thereby treating and alleviating the symptoms of osteoarthritis, making disulfiram a new application in the treatment of osteoarthritis.

In order to achieve the above purpose, the technical solutions adopted in this application are:

A drug-loaded microgel sphere, the drug contained is a disulfiram drug, the disulfiram drug includes disulfiram or a disulfiram derivative, the drug-loaded microgel sphere is used to treat and relieve bone joints symptoms of inflammation.

Further improvements to the above technical solution are:

The drug-loaded microgel spheres are used by injection into the lesion site.

The particle size range of the drug-loaded microgel spheres is 200-300 μm.

This application also provides a method for preparing drug-loaded microgel spheres, which includes the following steps:

Provide disulfiram drug nanoparticles, the disulfiram drug including disulfiram or a disulfiram derivative;

Provides methacrylic anhydride modified gelatin;

Mix the methacrylic anhydride modified gelatin and the disulfiram drug nanoparticles in an equal mass ratio to obtain an aqueous phase;

Mix HFE7500 with surfactant to obtain oil phase;

The water phase and the oil phase are mixed through microfluidic chip technology to prepare the drug-loaded microgel sphere;

The drug-loaded microgel spheres are used to treat and relieve symptoms of osteoarthritis.

Further, the disulfiram drug nanoparticles are prepared by the following method:

Weigh PLGA, PLGA-b-PEG and the drug respectively according to the mass ratio requirements, and completely dissolve them in DCM to form a first mixture with a concentration of 8%-12% w/v; the PLGA, PLGA-b- The mass ratio of PEG and drugs is 25-50:25-50:10; the drugs include disulfiram or disulfiram derivatives;

Mix and stir the first mixture and the PVA solution according to a volume ratio of 1:5-100 to obtain a second mixture;

The second mixture and the PVA solution are stirred in the dark according to a volume ratio of 1-20:10-100 to remove residual DCM to obtain a third mixture;

The third mixture is centrifuged and washed to remove residual PVA to obtain the disulfiram drug nanoparticles.

Further, the concentration of the PVA solution is 1% w/v; the weight average molecular weight of the PLGA is 35kDa.

Further, the methacrylic anhydride modified gelatin is prepared by the following method:

Completely dissolve gelatin in DPBS to form a first substance with a concentration of 8%-12% w/v;

Add MA to the first substance, stir in the dark, and then add DPBS for dilution to form a second substance; wherein the volume ratio of the first substance, MA, and DPBS is 100-150:1:100;

After the second substance is placed in a dialysis bag for dialysis, the methacrylic anhydride modified gelatin is formed.

Further, the molecular weight cutoff _MW of the dialysis bag ranges from 8000 to 14000.

Further, the microfluidic chip of the microfluidic chip technology includes PDMS and a curing agent.

The present application also provides a drug-loaded stent, which is made from drug-loaded microgel spheres prepared by the above preparation method and subjected to photo-crosslinking reaction.

According to the technical solution of the present application, it can be seen that the drug-loaded microgel balls and drug-loaded stents of the present application can slowly release the drug through the gel-loaded drug. In particular, the drug-loaded stent after photo-crosslinking can play a role in The double sustained-release effect is more conducive to drug absorption and improves bioavailability. Drug-loaded microgel balls and drug-loaded stents can directly reach the lesion site through local injection. Through the joint action of microgel and disulfiram drugs, Further improving the effect of enhancing or inducing subchondral bone regeneration, improving the efficacy while reducing systemic side effects, is of great significance in the treatment of osteoarthritis and has broad application prospects.

Description of drawings

Figure 1 is a schematic flow chart of the preparation method of the drug-loaded stent in Example 2 of the present application.

Figure 2 shows the particle size distribution of disulfiram drug nanoparticles prepared using different concentrations of PVA.

Figure 3 is a potential diagram of disulfiram drug nanoparticles in Example 1 of the present application.

Figure 4 is the drug release curve of the disulfiram drug nanoparticles in Example 1 of the present application in a 37°C shaker.

Figure 5 is the drug release curve of the drug-loaded microgel spheres in Example 1 of the present application in a 37°C shaker.

Figure 6 is a graph showing changes in fluorescence intensity in joints after fluorescent labeling of disulfiram drug nanoparticles and drug-loaded microgel spheres according to the embodiment of the present application.

Figure 7 is a fluorescence display showing that the drug-loaded microgel spheres in Example 1 of the present application successfully encapsulated drugs.

Figure 8 is a comparison chart of the measurement of important parameters of subchondral bone weight after 4 weeks of treatment with different compositions 21 days after the rat iodoacetate osteoarthritis model was established.

Figure 9 shows the comparison between the number of chondrocytes and the normal number 1 to 3 days after injection of the drug-loaded scaffold.

Figure 10 is a shear characteristic diagram of the drug-loaded stent in Example 2 of the present application.

Figure 11 is a modulus characteristic diagram of the drug-loaded stent in Example 2 of the present application.

Figure 12 is a graph showing the relationship between temperature and viscosity of the drug-loaded stent in Example 2 of the present application.

Figure 13 is a graph showing the relationship between temperature and modulus of the drug-loaded stent in Example 2 of the present application.

Figure 14 is a histological analysis of H&E staining and Safranin-O staining of the tibial plateau and femoral condyle of OA animals.

Figure 15 is a diagram showing the expression evaluation of Aggrecan and Collagen II after immunohistochemical staining of knee joint tissue sections.

Detailed ways

In order to facilitate understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are shown in the accompanying drawings. However, the present application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that a thorough understanding of the disclosure of the present application will be provided.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing specific embodiments only and is not intended to limit the application.

Example 1: This example provides a drug-loaded microgel sphere and a preparation method thereof. The drug contained is a disulfiram drug, and the disulfiram drug includes disulfiram or a disulfiram derivative. Medicinal microgel spheres are used to treat and relieve symptoms of osteoarthritis. The drug-loaded microgel spheres are used by injection into the lesion site. The particle size range of the drug-loaded microgel spheres is 200-300 μm.

As shown in Figure 1, the drug-loaded microgel spheres are prepared by the following method:

S1. Provide disulfiram drug nanoparticles. The disulfiram drug includes disulfiram or disulfiram derivatives; the specific preparation method is as follows:

S1.1. Completely dissolve PLGA (polylactic acid glycolic acid copolymer), PLGA-b-PEG, and the drug in DCM (dichloromethane) according to the mass ratio of 25-50:25-50:10 to form a concentration of 8 %-12% w/v of the first mixture; the drug includes disulfiram (DSF) or a disulfiram derivative.

S1.2. Mix and stir the first mixture and PVA (polyvinyl alcohol) solution according to a volume ratio of 1:5-100 to obtain a second mixture.

S1.3. Stir the second mixture and the PVA solution in the dark according to a volume ratio of 1-20:10-100, remove residual DCM, and obtain a third mixture.

S1.4. Centrifuge and wash the third mixture to remove residual PVA to form disulfiram drug nanoparticles.

Specifically, in this example, 45 mg of PLGA35k, 45 mg of PLGA55k-b-PEG5k, and 10 mg of the drug were weighed, and the weighed above substances were dissolved in DCM to make a solution with a concentration of 10% w/v. First mixture.

Measure 1 ml of the first mixture and slowly drop it into 10 ml of PVA solution, mix and stir to form a first emulsion. The rotation speed of the magnetic stirrer is 600 rpm, the concentration of the PVA solution is 1% w/v, and the weight average molecular weight Mw of the PVA is 25 kDa. When stirring, first stir in a high-speed vortex in a magnetic stirrer for 1 minute, and then perform ultrasonic stirring at 60% power. The number of ultrasonic stirring is 10 times, and the time of each ultrasonic stirring is 5 s. Each ultrasonic stirring is After 5 seconds, perform the next ultrasonic stirring. The entire mixing and stirring reaction process is performed at a temperature of 0°C. For example, a container containing the first mixture and the PVA solution can be placed in an ice-water mixture.

After ultrasonic stirring, the first emulsion was added dropwise to 50 ml of PVA solution, and stirred in the dark at room temperature for 6 hours to remove residual DCM and form a second emulsion. The concentration of the PVA solution is 1% w/v, and the light-proof stirring can be carried out in an opaque tin foil-wrapped container. The light-proof stirring can be performed on a centrifuge,

The second emulsion is centrifuged and washed to remove residual PVA to obtain the disulfiram drug nanoparticles. During centrifugation, the speed of the centrifuge is 10000g-14000g, the temperature during centrifugation is 1-15°C, and the centrifugation time is 5-30 minutes. Specifically, in this embodiment, the speed of the centrifuge is 14000g, the temperature during centrifugation is 4°C, and the centrifugation time is 10 minutes. The second emulsion is washed with distilled water, and after repeated washing three times, the disulfiram drug nanoparticles are stored below -80°C.

As shown in Figure 2, the particle size distribution diagram of disulfiram drug nanoparticles prepared by using different concentrations of PVA is shown. It can be seen from the figure that when the disulfiram drug nanoparticles are prepared with a concentration of 1% PVA, the The diameters are all below 500nm, and most of the particle diameters are distributed around 200-300nm, and the particle size distribution is relatively uniform. When prepared with PVA at a concentration of 0.5% and 0.2%, the particle size is relatively large, with the maximum value close to 1 μm. .

As shown in Figure 3, it is the potential diagram of disulfiram drug nanoparticles. It can be seen from the figure that dynamic light scattering (DLS) Zeta point analysis shows that the surface of the nanoparticles is negatively charged (-31.5mV). Negative materials have a certain osteogenic effect.

S2. Provide methacrylic anhydride modified gelatin. The specific preparation method is as follows:

S2.1. Completely dissolve gelatin in DPBS (phosphate buffer solution) to form the first substance with a concentration of 8%-12% w/v.

S2.2. Add MA (methacrylic anhydride) to the first substance, stir in the dark, and then add DPBS for dilution to form a second substance; wherein the volume ratio of the first substance, MA, and DPBS is 100-150:1:100.

S2.3. After placing the second substance in a dialysis bag for dialysis, the methacrylic anhydride modified gelatin is formed.

Specifically, in this example, 20 g of Gelatin (gelatin) and 200 ml of DPBS were weighed and stirred at 60° C. until completely dissolved to form the first substance with a concentration of 10% w/v.

Cool the first substance to 50°C, add it dropwise to 1.6 ml of MA while stirring, and then stir in the dark for 1-4 hours (in this embodiment, the stirring time in the dark is specifically 2 hours). ), add 100 ml of DPBS for dilution to form the second substance.

The second substance is placed in a dialysis bag for dialysis. The model of the dialysis bag is MD34. The molecular weight retained by the dialysis bag is Mw=8000-14000. The dialysis medium is ddH ₂ O (double distilled water). The dialysis time is 2-10 days. In this embodiment, the specific time of dialysis is 7 days, and ddH ₂ O is changed 2-3 times a day. The temperature range during dialysis is 22-50°C. In this embodiment, the dialysis is performed at 40°C. The method is carried out in a constant temperature shaker to form the methacrylic anhydride modified gelatin, and the methacrylic anhydride modified gelatin is freeze-dried and stored under conditions below -80°C.

S3. Microfluidic mixing forming:

S3.1. Mix the photoinitiated cross-linking agent LAP with the freeze-dried methacrylic anhydride modified gelatin, and dissolve it in a constant temperature shaker to obtain the photosensitive hydrogel; The hydrogel and the disulfiram drug nanoparticles are mixed according to an equal mass ratio to obtain an aqueous phase; the concentration range of the LAP dissolved in distilled water is 0.1%-10%, and the LAP and the methacrylic acid The mass ratio of anhydride-modified gelatin is 0.1-10:1000

Specifically, in this example, weigh 0.05-1 mg of LAP. In this example, the weight of LAP is specifically 0.1 mg, dissolve it in 10 ml of distilled water, and then add the freeze-dried solution prepared in step S2.2.3. The final methacrylic anhydride modified gelatin, the mass of the freeze-dried methacrylic anhydride modified gelatin is 1g, and the photosensitive water is obtained after dissolving in a constant temperature shaker at 37°C for 30 minutes to 1 hour. gel, and store the photosensitive hydrogel in a refrigerator at 4°C. In this embodiment, the amino substitution degree of the photosensitive hydrogel is 60%, and the specific model of the LAP is A2959.

S3.2. Mix HFE7500 (fluorinated ether) and surfactant to obtain an oil phase.

S3.3. Mix the water phase and oil phase by controlling the flow rate with air pressure, and use microfluidic technology to prepare microspherical drug-loaded microgel balls.

Specifically, in this embodiment, photosensitive hydrogel is used as the water phase.

Mix HFE7500 and 10% surfactant FluoSurf to obtain an oil phase.

The above water phase and oil phase are passed through the microfluidic chip to form 200-300 μm microspheres under a pressure of 200 Pa. After photo-cross-linking, an unloaded microgel is obtained. The microfluidic chip includes PDMS (polydimethylsiloxane) and 1-30% curing agent.

Equal masses of light-sensitive hydrogel and disulfiram drug nanoparticles are mixed in equal proportions to form the water phase; in this example, the specific concentration of the light-sensitive hydrogel is 100 mg/ml, and disulfiram drug nanoparticles are The concentration of the particles is 2mg/ml. The oil phase is mineral oil, specifically HFE3500, which contains 10% surfactant. The above water phase and oil phase are passed through the microfluidic chip to form microspheres of 200-300 μm under a pressure of 200 Pa, thereby obtaining drug-loaded microgel spheres. The microfluidic chip includes PDMS (polydimethylsiloxane) and 1-30% curing agent.

Example 2: This example provides a drug-loaded stent and a preparation method thereof. The drug-loaded stent is made of the drug-loaded microgel spheres of Example 1 through photo-crosslinking reaction.

The prepared drug-loaded microgel spheres are irradiated with ultraviolet light for about 1 minute and photo-cross-linked to obtain a stable drug-loaded stent, which has a dual sustained-release effect.

Preparation of unloaded microgel spheres: In order to facilitate comparison of the therapeutic effect of the drug-loaded stent of this embodiment, unloaded microgel spheres were specially prepared as a comparative example. The specific steps are as follows:

Use photosensitive hydrogel as the water phase.

Mix HFE7500 and 10% surfactant FluoSurf to obtain an oil phase.

The above water phase and oil phase are passed through the microfluidic chip to form 200-300 μm microspheres under a pressure of 200 Pa. After photo-cross-linking, an unloaded microgel is obtained.

In order to prove the success of drug encapsulation in this example, drug release detection was carried out. The specific detection method is as follows: High performance liquid chromatography (HPLC) was used for identification:

Take 3 ml of the disulfiram drug nanoparticles and drug-loaded stent prepared in the above example as test samples respectively, place the test samples in dialysis bags (n=5), and place the dialysis bags in test tubes containing 33 ml of deionized water. , the temperature is 37℃, at different time intervals (16h, 1, 2, 4, 6 days, 8 days, 10 days, 12 days, 14 days), 0.3mL sample solution is collected from the test tube and frozen for subsequent analysis. 0.3ml of water was replaced each time, and HPLC was used for analysis. The analysis results are shown in Figures 4 and 5 respectively. From the release detection results in Figures 4 and 5, it can be seen that the disulfiram drug nanoparticles and drug-loaded stents were successfully loaded with drugs. , the drug is slowly released over time, and the drug-loaded stent has a better sustained drug release effect.

As shown in Figure 6, it is the fluorescence intensity change curve of fluorescently labeled disulfiram drug nanoparticles and drug-loaded stent in the joint. Among them, NPs/DSF represents the disulfiram drug nanoparticle labeling group, and GelMA-NPs/DSF represents the drug-loaded stent labeling group. They are the 1st hour of the first day, the 12th hour of the first day, the 24th hour of the first day, the second day, the fourth day, the sixth day, the eighth day, the tenth day, the twelfth day, and the tenth day. Fluorescence intensity change values at four days, sixteenth days, twenty-one days and twenty-eighth days.

The success of drug encapsulation can also be visually observed from Figure 7. In Figure 7, NPs represents the fluorescence display of disulfiram nanoparticles fluorescently labeled with coumarin-6; GelMA represents the fluorescently labeled methacrylic anhydride modified with rhodamine B. The fluorescence display image of the gelatin; merge represents the fluorescence display image of the prepared drug-loaded microgel spheres. It can be seen from the picture that the disulfiram nanoparticles were successfully encapsulated.

The therapeutic effect on traumatic arthritis in rats proves that the disulfiram drug nanoparticles and drug-loaded scaffolds prepared in this example are effective in treating osteoarthritis and improving chondrocyte function. The details are as follows:

Establishment of rat iodoacetic acid osteoarthritis model: 8-month-old SD rats were anesthetized with intraperitoneal injection of pentobarbital, placed in a supine position, exposed knee joints, and 100 ul of iodoacetic acid (concentration: 4.8 mg/60 _μ L) was injected into the rat knee joint. Two weeks after the operation, the joint cysts were enlarged and the joint surface was dark. Four weeks after the operation, the articular cartilage turned yellow and small cracks appeared in the joint. Bone spurs and joint ligament adhesion could be felt at the sixth week.

Composition treatment on the rat iodoacetate osteoarthritis model: 21 days after the rat iodoacetate osteoarthritis model was established, the composition (empty microgel spheres, disulfiram drug nanoparticles or drug-loaded scaffolds) was used for treatment 4 Weekly, the key parameters of joint status such as average thickness of trabecular bone, number of trabeculae, separation of trabecular bone, bone density/bone mineral density, etc. were measured. The specific test results are shown in Figure 7.

Figure 8 shows the measurement of important parameters of subchondral bone mass 21 days after the establishment of the rat iodoacetate osteoarthritis model and 4 weeks of treatment. Figure 8A shows the measurement of Tb.Th (average thickness of trabecular bone), and Figure 8B Figure 8C is the measurement of Tb.N (number of bone trabeculae), Figure 8C is the measurement of Tb.Sp (number of trabecular separation), Figure 8D is the measurement of BMD (bone density/bone mineral density), Figure 8E is the measurement of BV/TV (relative Bone volume/bone volume fraction) determination. The groups are as follows: NC represents the normal rat group, OA represents the 21-day iodoacetic acid rat osteoarthritis model group, GelMA represents the empty microgel sphere treatment group, NPs/DSF represents the disulfiram drug nanoparticle treatment group, GelMA -NPs/DSF represents the drug-loaded stent treatment group.

As can be seen from Figure 8A, the average thickness of trabecular bone in the OA group decreased compared with the NC (normal) group, while the NPs/DSF group and GelMA-NPs/DSF group increased compared with the OA group and were close to the NC (normal) group. It can be seen from Figure 7B that the number of bone trabeculae in the OA group decreased compared with the NC (normal) group, while the NPs/DSF group and GelMA-NPs/DSF group increased compared with the OA group and were close to the NC (normal) group. It can be seen from Figure 8C that the number of trabecular bone separations in the OA group increased compared with the NC (normal) group, while the NPs/DSF group and GelMA-NPs/DSF group decreased compared with the OA group and were close to the NC (normal) group. It can be seen from Figure 8D that the bone density/bone mineral density of the OA group decreased compared with the NC (normal) group, while the NPs/DSF group and GelMA-NPs/DSF group increased compared with the OA group and were close to the NC (normal) group. . It can be seen from Figure 8E that the relative bone volume/bone volume fraction of the OA group decreased compared with the NC (normal) group, while the NPs/DSF group and GelMA-NPs/DSF group increased compared with the OA group and were close to the NC (normal) group. .

It can be seen from the above results that the disulfiram drug nanoparticles and drug-loaded scaffolds prepared in this example are effective in treating osteoarthritis and improving chondrocyte function, and because the drug-loaded scaffold has outstanding long-term sustained release effect, the drug-loaded scaffold Medicinal stents are more effective in treating and improving osteoarthritis.

Figure 9 shows the effect of the drug-loaded scaffold on the number of chondrocytes when the drug-loaded scaffold was injected for 1 to 3 days. Among them, NC-D1 represents the number of normal chondrocytes on the first day, NC-D2 represents the number of normal chondrocytes on the second day, NC-D3 represents the number of normal chondrocytes on the third day, and GelMA-D1 represents the day after injection of the drug-loaded scaffold. GelMA-D2 represents the number of chondrocytes two days after the drug-loaded scaffold was injected, and GelMA-D3 represents the number of chondrocytes three days after the drug-loaded scaffold was injected. It can be seen from the figure that the drug-loaded scaffold has no stimulating or inhibitory effect on the growth of chondrocytes.

Figure 10 is a shear characteristic diagram of the drug-loaded stent in Example 2 of the present application. The abscissa represents the shear rate, and the ordinate represents the viscosity. It can be seen from the figure that the shear thinning characteristics of the drug-loaded stent are , indicating that the drug-loaded stent can be used by injection.

Figure 11 is a modulus characteristic diagram of the drug-loaded stent in Example 2 of the present application. The abscissa represents the angular velocity, and the ordinate represents the modulus. It can be seen from the figure that the storage modulus of the drug-loaded stent is greater than the loss modulus. It is explained that The drug-loaded stent is made of elastic material.

Figure 12 is a graph showing the relationship between temperature and viscosity of the drug-loaded stent in Example 2 of the present application, in which the abscissa is the temperature value and the ordinate is the viscosity value. It can be seen from the figure that the viscosity of the drug-loaded stent is hardly affected by temperature.

Figure 13 is a graph showing the relationship between temperature and modulus of the drug-loaded stent in Example 2 of the present application, in which the abscissa represents the temperature value and the ordinate represents the modulus value. It can be seen from the figure that the modulus of the drug-loaded stent is almost not affected by temperature. It can be seen from Figures 12 and 13 that the performance of the drug-loaded stent is hardly affected by temperature.

As shown in Figure 14, it is a histological analysis of H&E staining and Safranin-O staining of the tibial plateau and femoral condyle of OA animals. Among them, OA represents the osteoarthritis group, GelMA represents the unloaded microgel sphere treatment group, DSF/NPs represents the disulfiram drug nanoparticle treatment group, and GelMA-DSF/NPs represents the drug-loaded stent treatment group. It can be seen from the color picture that according to histopathological examination, the bone is green, the cartilage is red, and the OA group proteoglycan (red) content is decreased. It can be seen that in the OA group, the surface of the articular cartilage layer is rough, the integrity is damaged, partial matrix fibrosis occurs, and visible fibrous granulation tissue filling; compared with the drug-loaded scaffold treatment group, OA-related cartilage deformation (joint joint deformation) is significantly reduced. smooth surface), cartilage destruction and proteoglycan loss are also significantly reduced.

As shown in Figure 15, it is a picture of the expression evaluation of Aggrecan and Collagen II after immunohistochemical staining of knee joint tissue sections. Aggrecan, also known as proteoglycan, is a macromolecular proteoglycan that exists in the extracellular matrix of connective tissue. It is the main structural macromolecule of cartilage; collagen II, also known as type II osteogel, is the main organic component of cartilage and joints and is rich in Contains amino acids specifically required by bone connective tissue, which can help regenerate human cartilage tissue and is widely used in the evaluation of cartilage repair. As can be seen from the figure, the positive expression protein in the treatment group increased significantly compared with the OA group, and the expression of aggrecan and Collagen II increased significantly in the drug-loaded stent treatment group. The morphology and number of chondrocytes were also increased relative to the OA group. It can be inferred from the above results that the disulfiram drug nanoparticles, drug-loaded microgel spheres, and drug-loaded scaffolds of the present application can significantly alleviate the inflammatory damage to cartilage caused by osteoarthritis, effectively treat osteoarthritis, and repair cartilage.

The above experimental data can prove that disulfiram drugs have certain effectiveness in treating osteoarthritis, opening up new application scenarios for disulfiram drugs and making disulfiram drugs more widely used.

In the embodiment of the present application, the drug-loaded stent is introduced into the joint cavity by local injection, which promotes the reshaping of subchondral bone and thereby achieves the purpose of treating osteoarthritis. Experimental tests have confirmed that the drug-loaded scaffold can induce and enhance subchondral bone remodeling, and improve key parameters of joint status such as average thickness of trabecular bone, number of trabecular bone, separation of trabecular bone, bone density/bone mineral density, etc. It was confirmed that disulfiram loaded with injectable microgel can induce and enhance subchondral bone remodeling to treat osteoarthritis. The drug-loaded stent of the present application provides a new, safe and effective treatment method for the treatment of osteoarthritis, which is of great significance in the treatment of osteoarthritis and has broad application prospects.

The technical features of the above embodiments can be combined in any way. To simplify the description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, all possible combinations should be used. It is considered to be within the scope of this manual.

The above embodiments only express preferred embodiments of the present application, and their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the patent application. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all fall within the protection scope of the present application. Therefore, the scope of protection of this patent application should be determined by the appended claims.

Claims

A drug-loaded microgel sphere, characterized in that: the drug contained is a disulfiram drug, the disulfiram drug includes disulfiram or a disulfiram derivative, and the drug-loaded microgel sphere is used for treatment and relieve symptoms of osteoarthritis.
The drug-loaded microgel sphere according to claim 1, characterized in that the drug-loaded microgel sphere is used by injecting it into the lesion site.
The drug-loaded microgel sphere according to claim 1, wherein the particle size range of the drug-loaded microgel sphere is 200-300 μm.
A method for preparing drug-loaded microgel spheres, which is characterized by comprising the following steps:

Provide disulfiram drug nanoparticles, the disulfiram drug including disulfiram or a disulfiram derivative;

Provides methacrylic anhydride modified gelatin;

Mix the methacrylic anhydride modified gelatin and the disulfiram drug nanoparticles in an equal mass ratio to obtain an aqueous phase;

Mix HFE7500 with surfactant to obtain oil phase;

The water phase and the oil phase are mixed through microfluidic chip technology to prepare the drug-loaded microgel sphere;

The drug-loaded microgel spheres are used to treat and relieve symptoms of osteoarthritis.
The preparation method of drug-loaded microgel spheres according to claim 4, characterized in that: the disulfiram drug nanoparticles are prepared by the following method:

Weigh PLGA, PLGA-b-PEG and the drug respectively according to the mass ratio requirements, and completely dissolve them in DCM to form a first mixture with a concentration of 8%-12% w/v; the PLGA, PLGA-b- The mass ratio of PEG and drugs is 25-50:25-50:10; the drugs include disulfiram or disulfiram derivatives;

Mix and stir the first mixture and the PVA solution according to a volume ratio of 1:5-100 to obtain a second mixture;

The second mixture and the PVA solution are stirred in the dark according to a volume ratio of 1-20:10-100, and the residual DCM is removed to obtain a third mixture;

The third mixture is centrifuged and washed to remove residual PVA to obtain the disulfiram drug nanoparticles.
The method for preparing drug-loaded microgel spheres according to claim 5, wherein the concentration of the PVA solution is 1% w/v; the weight average molecular weight of the PLGA is 35 kDa.
The preparation method of drug-loaded microgel spheres according to claim 4, characterized in that: the methacrylic anhydride modified gelatin is prepared by the following method:

Completely dissolve gelatin in DPBS to form a first substance with a concentration of 8%-12% w/v;

Add MA to the first substance, stir in the dark, and then add DPBS for dilution to form a second substance; wherein the volume ratio of the first substance, MA, and DPBS is 100-150:1:100;

After the second substance is placed in a dialysis bag for dialysis, the methacrylic anhydride modified gelatin is formed.
The method for preparing drug-loaded microgel spheres according to claim 7, wherein the molecular weight cutoff M W of the dialysis bag ranges from 8000 to 14000.
The method for preparing drug-loaded microgel spheres according to claim 4, characterized in that: the microfluidic chip of the microfluidic chip technology includes PDMS and a curing agent.
A drug-loaded stent, characterized in that drug-loaded microgel spheres prepared by the preparation method of any one of claims 4 to 9 are produced through photo-crosslinking reaction.