A method for preparing a dual-effect drug sustained-release microsphere system with a core-shell structure suitable for intra-articular injection

Abstract

The present invention discloses a preparation method of a dual-effect drug sustained-release microsphere system with a core-shell structure suitable for intra-articular injection, and belongs to the field of tissue engineering. The preparation method of the microsphere system of the present invention comprises the following steps: methylprednisolone sodium succinate (MPSS) solution is added to an organic solvent dissolved with polylactic acid and KGN for emulsification, the obtained W1 / O system is dropped into a polyvinyl alcohol solution for emulsification, and the obtained W1 / O / W2 system is centrifuged, cleaned, frozen, and dried to obtain the microsphere system. The present invention uses polylactic acid as a carrier material, adopts a solvent volatilization method to load the anti-inflammatory drug MPSS to the core layer, and the cartilage regeneration drug KGN is loaded to the shell layer, achieving spatiotemporal gradient sustained release of the two drugs. The microsphere system of the present invention has good cell biocompatibility, is induced into cartilage activity and anti-inflammatory activity, can achieve the short-acting sustained release of the shell drug and the long-acting sustained release of the core drug, and has potential application value in the field of intra-articular injection.

Description

Technical Field

[0001] The invention belongs to the field of tissue engineering, and in particular relates to a method for preparing a dual-effect drug sustained-release microsphere system with a core-shell structure suitable for intra-articular injection. Background Art

[0002] Temporomandibular joint osteoarthritis (TMJOA) is a progressive degenerative joint disease caused by synovitis. Currently, treatment options include joint lavage, intraarticular drug injections, ultrashort wave physical therapy, oral medications, splinting, and surgery. These treatments can improve clinical symptoms but fail to effectively repair articular cartilage. Corticosteroids and sodium hyaluronate, approved by the US Food and Drug Administration, are the mainstream medications for intraarticular administration. However, due to the drainage of synovial capillaries and lymphatic vessels, drugs rapidly enter the systemic circulation, resulting in a short residence time and short duration of efficacy within the joint cavity. This necessitates multiple administrations with uncontrolled dosages, significantly increasing adverse reactions and impaired patient compliance. To address the need for multiple intraarticular injections, combining drugs with sustained-release systems is a promising therapeutic approach. Summary of the Invention

[0003] The present invention aims to address the shortcomings and deficiencies of the prior art by providing a method for preparing a dual-action drug sustained-release microsphere system with a core-shell structure suitable for intra-articular injection. The present invention utilizes a solvent evaporation method to simultaneously encapsulate kartogenin (KGN) and methylprednisolone sodium succinate (MPSS) in a biodegradable material, PLA. The microsphere sustained-release system controls the sustained release of the loaded drugs within the joint cavity, thereby prolonging the drug's residence time within the joint cavity. The sustained-release microsphere system exhibits gradient and ordered release characteristics, achieving spatiotemporal gradient delivery of the dual drugs by loading the core layer with methylprednisolone sodium succinate (MPSS), an anti-inflammatory drug, and the shell layer with KGN, a cartilage regeneration-promoting drug.

[0004] The purpose of the present invention is achieved through the following technical solutions:

[0005] In a first aspect, the present invention provides a method for preparing a dual-action drug sustained-release microsphere system having a core-shell structure suitable for intra-articular injection, comprising the following steps:

[0006] (1) An aqueous solution of methylprednisolone sodium succinate (MPSS) (W1) is added to an organic solvent containing polylactic acid and KGN to emulsify the solution to obtain a W1 / O system. The organic solvent is preferably any one of dichloromethane, chloroform, carbon disulfide, xylene, toluene, ethyl acetate, ethyl propionate, propyl acetate, acetone, or ethanol, or a combination of at least two thereof, and more preferably dichloromethane and / or acetone.

[0007] (2) The W1 / O system is dropped into a polyvinyl alcohol aqueous solution (W2) to emulsify to obtain a W1 / O / W2 system.

[0008] (3) The W1 / O / W2 system is centrifuged, washed, frozen, and dried to obtain the dual-action drug sustained-release microsphere system.

[0009] In step (1), the concentration of the methylprednisolone sodium succinate aqueous solution is preferably 5-8 mg / mL.

[0010] In step (1), the organic solvent containing polylactic acid and KGN preferably has a concentration of 30-50 mg / mL of polylactic acid and a concentration of 0.1-0.5 mg / mL of KGN, and the organic solvent is preferably dichloromethane. The dichloromethane containing polylactic acid and KGN is preferably obtained by a method comprising the steps of first dissolving KGN in DMSO, then adding the DMSO to the dichloromethane containing polylactic acid, and mixing to obtain the dichloromethane containing polylactic acid and KGN.

[0011] In step (1), the volume ratio of the methylprednisolone sodium succinate (MPSS) aqueous solution to the organic solvent containing polylactic acid and KGN is preferably 2-4:16.

[0012] In step (1), the emulsification condition is preferably ultrasonic emulsification under ice bath conditions. Further, the ultrasonic conditions are preferably 200-300W ultrasonic waves working for 2-4 minutes, working for 3-5 seconds, and stopping for 3-5 seconds.

[0013] In step (2), the concentration of the polyvinyl alcohol aqueous solution is preferably 10-30 mg / mL.

[0014] In step (2), the volume ratio of the W1 / O system to the polyvinyl alcohol aqueous solution is preferably 1-3:10.

[0015] In step (2), the emulsification condition is preferably stirring emulsification, and the stirring speed is preferably 1000-2000 r / min.

[0016] In step (3), the centrifugation conditions are preferably 2-8°C, 8000-10000 r / min, and 8-12 min.

[0017] The second aspect of the present invention provides a dual-effect drug sustained-release microsphere system with a core-shell structure suitable for intra-articular injection, which is obtained by the preparation method described in the first aspect.

[0018] The dual-action drug sustained-release microsphere system with a core-shell structure uses racemic polylactic acid as a carrier material, and uses KGN, a cartilage regeneration promoting drug, and methylprednisolone sodium succinate, an anti-inflammatory drug, as dual-loaded drugs. The shell layer is loaded with KGN and the core layer is loaded with methylprednisolone sodium succinate. It has good cell biocompatibility and possesses the dual properties of anti-inflammatory and cartilage regeneration promoting. It simultaneously realizes spatiotemporal gradient sustained-release delivery of the dual drugs, as well as short-acting sustained-release of the shell drug and long-acting sustained-release of the core drug.

[0019] The outermost layer of the dual-effect drug sustained-release microsphere system with a core-shell structure is an aqueous phase. The microspheres are not easy to aggregate, and are easier to be evenly dispersed in the joint cavity for intra-articular injection, and its porous structure is conducive to cell adhesion. The micropores on the surface of the microspheres may be due to the volatilization of the solvent dichloromethane or water, providing a channel for the release of the drug inside the microspheres to the external solution. In addition, MPSS is a water-soluble drug. During the curing process of the microspheres, the MPSS inside the microspheres will continuously escape into the external aqueous phase. The MPSS concentration difference inside and outside the microspheres will cause the internal MPSS to transfer to the microsphere surface. Such transfer will also form some pores connecting the surface and interior of the microspheres. The pore structure will accelerate the degradation rate of the polymer polylactic acid, thereby accelerating the release of the drug.

[0020] The application of the dual-effect drug sustained-release microsphere system with a core-shell structure in the preparation of drugs for treating arthritis.

[0021] A drug for treating arthritis comprises the dual-effect drug sustained-release microsphere system with a core-shell structure.

[0022] The administration method of the arthritis treatment drug is intra-articular injection.

[0023] The present invention has the following advantages and beneficial effects: The dual-effect drug sustained-release microsphere system with a core-shell structure suitable for intra-articular injection provided by the present invention can achieve the effects of stabilizing colostrum and sustained release without the need to add additional additives to the internal aqueous phase and oil phase, thereby eliminating the difficulties caused by the subsequent residual measurement, increasing biosafety, and reducing production costs. The method of the present invention is simple to operate, requires mild conditions, and is easy to scale up industrially. The present invention overcomes the problems of rapid drug clearance and low bioavailability after intra-articular drug injection, and can solve the problem of repeated intra-articular drug injections in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The present invention is a flow chart for preparing a dual-effect drug sustained-release microsphere system (KGN-MPSS-MS) with a core-shell structure.

[0025] Figure 2 Schematic diagram of drug release and microsphere degradation by KGN-MPSS-MS.

[0026] Figure 3 This is the scanning electron microscope image of KGN-MPSS-MS.

[0027] Figure 4 is the particle size distribution of KGN-MPSS-MS.

[0028] Figure 5 The scanning electron micrographs of KGN-MPSS-MS at different degradation time points are shown in Figure 1. a: 10d, b: 20d, and c: 50d.

[0029] Figure 6 This is the standard curve of Kartogenin and methylprednisolone sodium succinate.

[0030] Figure 7 are the sustained-release concentrations and cumulative release percentages of the two drugs in KGN-MPSS-MS. DETAILED DESCRIPTION

[0031] The embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation plans of the present invention.

[0032] The present invention provides a method for preparing a dual-effect drug sustained-release microsphere system (KGN-MPSS-MS) with a core-shell structure suitable for intra-articular injection, the process of which is as follows: Figure 1 As shown, the following steps may be specifically included:

[0033] (1) A DMSO solution containing kartogenin (KGN) was added to a dichloromethane solution containing polylactic acid and mixed to obtain an oil phase O. An aqueous solution of methylprednisolone sodium succinate (MPSS) (internal aqueous phase W1) was added to the oil phase O, and ultrasonic emulsification was performed under ice bath to obtain a W1 / O system.

[0034] (2) The W1 / O system is dropped into the polyvinyl alcohol aqueous solution (external aqueous phase W2) and stirred to obtain the W1 / O / W2 system.

[0035] (3) The W1 / O / W2 system was centrifuged, washed, and freeze-dried to obtain a dual-effect drug sustained-release microsphere system with a "core-shell" structure suitable for intra-articular injection.

[0036] The schematic diagram of drug release and microsphere degradation of KGN-MPSS-MS of the present invention is as follows Figure 2As shown, MPSS is located in the core layer of the microsphere, while KGN is located in the shell layer. Once the microspheres enter the human body, changes in factors such as pH in the body cause ionic bonds to break down, leading to the slow release of the drug. The main component of the microspheres, polylactic acid, degrades into lactic acid in the body, which is ultimately metabolized into carbon dioxide and water.

[0037] The embodiments of the present invention are further described below in conjunction with the embodiments of the present invention and the accompanying drawings.

[0038] Example 1 Preparation of the dual-effect drug sustained-release microsphere system (KGN-MPSS-MS) with a core-shell structure of the present invention

[0039] First, 3 g of polyvinyl alcohol (PVA) was added to a beaker containing 150 mL of deionized water, and dissolved under magnetic stirring overnight to obtain a 2% concentration solution. 100 mL of the above solution was taken for later use.

[0040] Add 0.6 g of polylactic acid to a 50 mL centrifuge tube containing 16 mL of dichloromethane and vortex for 3 minutes. After the polylactic acid is dissolved, add 5 mg of KGN dissolved in 50 μL of DMSO to the dichloromethane and vortex for 1 minute.

[0041] Dissolve 20 mg of methylprednisolone sodium succinate (MPSS) in 3 mL of deionized water for later use.

[0042] Pour the polylactic acid and KGN dissolved in dichloromethane into a small beaker. Continue adding deionized water containing MPSS to the beaker, allowing the oil and water to separate. Place the beaker in an ice-water bath and operate a 220W ultrasonic cell disruptor for 3 minutes (3 seconds on, 3 seconds off) to emulsify and form a W1 / O system.

[0043] The W1 / O system was added dropwise to 100mL of 2% polyvinyl alcohol solution (W2) using a 5mL syringe while maintaining a strong magnetic stirring speed of 1500r / min to form a W1 / O / W2 system. After strong rotation for 30min, the emulsion was stirred at 100r / min for 5 hours to accelerate the volatilization of dichloromethane. After stirring, the solution was poured into a centrifuge tube and centrifuged at 4°C and 8000r / min for 10min. The precipitate was washed three times with deionized water, placed in a -20°C refrigerator and frozen for 24h. It was then dried in a vacuum freeze dryer for 10h to obtain the dual-effect drug sustained-release microsphere system (KGN-MPSS-MS), which was stored in a sealed glass bottle at 4°C.

[0044] Example 2 Scanning electron microscope observation of microsphere morphology

[0045] Take the microsphere KGN-MPSS-MS prepared in Example 1. Place the freeze-dried powder of the microspheres to be observed into an EP tube filled with deionized water, vortex to disperse the microspheres, and use a pipette to draw an appropriate amount of the microsphere suspension onto aluminum foil and let it dry overnight.

[0046] The aluminum foil loaded with microspheres was cut into appropriate sizes and pasted on the conductive adhesive. Gold was sprayed for 60 seconds under vacuum conditions. The vacuum degree of the scanning electron microscope reached 4.00×10 -5 When the pressure was below mbar, the operating voltage was set to 5 kV, and the morphology of the microspheres was observed and images were taken.

[0047] The morphology of microspheres is Figure 3 As shown: It is a round spherical shape with a smooth surface and obvious holes. There is no agglomeration between the microspheres and the dispersion is good.

[0048] Example 3 Analysis of Microsphere Particle Size Distribution by Laser Particle Size Analyzer

[0049] The KGN-MPSS-MS microspheres prepared in Example 1 were used to measure the particle size distribution of the microspheres using a Malvern Mastersizer 2000. The microspheres were suspended in distilled water containing 0.1% Tween 80 and treated in a 60W ultrasonic bath for 30 seconds to disperse the microspheres. The particle size distribution of the microspheres was then measured.

[0050] The particle size of microspheres is Figure 4 As shown: the particle size of the microspheres ranges from 40nm to 30μm, and most of the microspheres have a particle size of less than 10μm, which is suitable for intra-articular injection. In the present invention, both nano-scale microspheres and micron-scale microspheres are acceptable, and the advantages of both can be better utilized. The smaller the particle size of the microspheres, the faster the drug is released from the microspheres, but they are also easily phagocytosed by macrophages in the synovial fluid. This phagocytic effect can delay the clearance of the drug, and also reduce the irritation caused by the contact of high-concentration drugs with cartilage tissue. It is generally believed that particles with a size in the range of 2.5nm-10μm are more easily taken up by phagocytes, and the released drugs can inhibit inflammation. Moreover, for intra-articular administration, the particle size of the microspheres must be strictly controlled to be less than 6μm to avoid irritation to the joint cavity. Studies have shown that the pore size of the cartilage reticular structure is about 60nm, and particles with a diameter of 31-38nm can enter the articular cartilage matrix, thereby releasing drugs and increasing the utilization rate of the drugs. The larger the particle size of the microspheres, the slower the drug release, the better the drug loading and encapsulation efficiency of the microspheres, and the longer the drug release. Therefore, the coexistence of nano-sized particles and micro-sized particles can better play a role.

[0051] Example 4 Scanning electron microscopy observation of in vitro degraded microsphere morphology

[0052] 20 mg of KGN-MPSS-MS prepared in Example 1 was weighed and placed in four centrifuge tubes containing 10 mL of PBS. The four centrifuge tubes were placed in a constant temperature shaker at 37°C and 100 rpm. The buffer solution was refreshed every five days to simulate the joint cavity.

[0053] Centrifuge tubes were randomly selected on the 10th, 20th, 30th, and 50th days, centrifuged at 8000 rpm for 10 minutes, the supernatant was removed, and the remainder was freeze-dried. The morphology of the microspheres was observed using a scanning electron microscope.

[0054] The morphology of microspheres at different degradation times is as follows Figure 5 As shown: When the microspheres were degraded to the 10th day, the microspheres still had a spherical shape, good dispersion, and a smooth surface ( Figure 5 a). When the microspheres were degraded to the 20th day, they were basically spherical, but they began to aggregate and their volume increased significantly. The surface of the microspheres became rough, forming many depressions and slightly collapsed ( Figure 5 b). When the microspheres degraded to the 50th day, most of the microspheres collapsed and the spherical shape could no longer be seen, but some intact microspheres were still visible. The overall structure was severely deformed, and only particle fragments were seen to be aggregated ( Figure 5 c).

[0055] Example 5 Determination of Absorption Wavelength and Standard Curve of Kartogenin and MPSS

[0056] Kartogenin solution and methylprednisolone sodium succinate solution of different concentrations were measured by UV spectrophotometer.

[0057] A certain amount of Kartogenin was dissolved in DMSO and methylprednisolone sodium succinate was dissolved in double distilled water. DMSO solution and double distilled water were used as blank control groups, respectively. After ultrasonic dispersion and dissolution, Kartogenin was diluted stepwise into five solutions with different concentrations (1, 2, 4, 8, 16 μg / mL), and methylprednisolone sodium succinate was diluted stepwise into five solutions with different concentrations (2, 4, 8, 16, 32 μg / mL). The absorbance and the wavelength of the maximum absorption peak of the drug (the wavelength of the maximum absorption peak of KGN is 280 nm, and the wavelength of the maximum absorption peak of MPSS is 248 nm) were measured. The standard curves of Kartogenin and methylprednisolone sodium succinate were drawn with absorbance Y as the horizontal axis and drug concentration X as the vertical axis. The standard curves of Kartogenin and MPSS are shown in the table. Figure 6 .

[0058] Example 6 Determination of the sustained release behavior of two drugs in KGN-MPSS-MS using UV spectrophotometer

[0059] 20 mg of KGN-MPSS-MS prepared in Example 1 was immersed in a centrifuge tube containing 30 mL of PBS buffer and sustained-released in a shaker at 37°C and 100 rpm. At different time points (4 h, 8 h, 12 h, 24 h, 3 d, 6 d, 10 d, 15 d, 20 d, 25 d, 30 d, and 40 d), the centrifuge tube was centrifuged at 8000 rpm for 10 min, and 3 mL of the supernatant was aspirated. The supernatant was filtered through a 0.1 μm microporous membrane and then the absorbance was measured. At the same time, 3 mL of fresh PBS buffer was added to the centrifuge tube.

[0060] The absorbance of the solution at 248 nm (MPSS) and 280 nm (KGN) was measured by UV spectrophotometry, and the drug concentration was calculated according to the standard curve drawn in Example 5. Finally, the cumulative release percentage of the two drugs at each time point and the drug concentration at each time point were calculated to study the drug release characteristics of the microspheres.

[0061] The in vitro sustained release curves of the two drugs in KGN-MPSS-MS are shown in Figure 7 In the first 24 hours, the cumulative release rate of KGN reached 55.44%, and the drug release rate was relatively fast. After 24 hours, it entered the sustained release stage of the drug. During this stage, due to the slow degradation of polylactic acid and the intermolecular force between the drug and polylactic acid, the release of microspheres tended to be gentle. During the drug release process of MPSS, due to the pressure difference between the inner aqueous phase (W1) and the outer aqueous phase (W2), the MPSS coated in the inner aqueous phase is easily freed from the inside due to the pressure, forming a burst release. Subsequently, the polylactic acid swells, the pores increase, and more pores are formed. The drug encapsulated in the polylactic acid is slowly released outward through the pores, and the drug release rate is approximately constant.

[0062] Initially, a burst of release occurred due to the retention of both drugs on the microsphere surface, followed by a slower release pattern that continued for approximately 40 days, demonstrating that the microspheres prepared by the double emulsion method exhibited excellent sustained drug release. The cumulative release of KGN within the first 4 hours was approximately 47.01%, with a drug concentration of 0.94 μg / mL. The cumulative release at the 24th hour was 55.44%, with a drug concentration of 1.11 μg / mL. The release rate slowed after 24 hours, reaching a total cumulative drug release of 79.63% on the 40th day, with a drug concentration of 1.59 μg / mL on the 40th day. The cumulative release percentage of MPSS within the first 4 hours was approximately 28.28%, with a drug concentration of 0.52 μg / mL. The cumulative release after 24 hours was 33.26%, with a drug concentration of 0.72 μg / mL. The release rate slowed down after 9 days, and the total cumulative drug release on the 40th day was 75.20%, and the drug concentration on the 40th day reached 0.88μg / mL. After 40 days, both drugs were completely released.

[0063] Methylprednisolone sodium succinate has good water solubility and a small molecular weight, but it is very easy to escape to the external aqueous phase, and the drug leakage phenomenon is obvious. The hydrophobic KGN also has a low molecular weight, but its water solubility is poor, and the drug leakage phenomenon is not obvious. MPSS is a hydrophilic drug coated in the inner aqueous phase (W1), and KGN is a lipophilic drug coated in the oil phase (O), constructing a drug sustained-release system in which anti-inflammatory drugs and cartilage regeneration-promoting drugs coexist. The oil phase has a certain degree of barrier effect on the release of drugs in the inner aqueous phase, which can reduce the burst release of water-soluble drugs. The additional shell structure is also conducive to improving the encapsulation rate of the drug. The release time of both drugs is extended by the encapsulation of polylactic acid, and the drug-carrying system achieves a good sustained-release effect.

[0064] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to the specific embodiments described. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for preparing a dual-action drug sustained-release microsphere system with a core-shell structure suitable for intra-articular injection, characterized in that: The following steps are involved: (1) Adding an aqueous solution of methylprednisolone sodium succinate to an organic solvent containing polylactic acid and kartogenin to emulsify and obtain a W1 / O system; (2) dropping the W1 / O system into a polyvinyl alcohol aqueous solution to emulsify it to obtain a W1 / O / W2 system; (3) The W1 / O / W2 system is centrifuged, washed, frozen, and dried to obtain the dual-action drug sustained-release microsphere system.

2. The preparation method according to claim 1, wherein: In step (1), the organic solvent is any one of dichloromethane, chloroform, carbon disulfide, xylene, toluene, ethyl acetate, ethyl propionate, propyl acetate, acetone, and ethanol, or a combination of at least two thereof.

3. The preparation method according to claim 1, wherein: In step (1), the concentration of the methylprednisolone sodium succinate aqueous solution is 5-8 mg / mL; in the organic solvent containing polylactic acid and kartogenin, the concentration of polylactic acid is 30-50 mg / mL, and the concentration of kartogenin is 0.1-0.5 mg / mL; the volume ratio of the methylprednisolone sodium succinate aqueous solution to the organic solvent containing polylactic acid and kartogenin is 2-4:

16.

4. The preparation method according to claim 1, wherein: In step (1), the emulsification condition is ultrasonic emulsification under ice bath conditions.

5. The preparation method according to claim 1, wherein: In step (2), the concentration of the polyvinyl alcohol aqueous solution is 10-30 mg / mL.

6. The preparation method according to claim 1, wherein: In step (2), the volume ratio of the W1 / O system to the polyvinyl alcohol aqueous solution is 1-3:

10.

7. The preparation method according to claim 1, wherein: In step (2), the emulsification condition is stirring emulsification.

8. A dual-action drug sustained-release microsphere system with a core-shell structure suitable for intra-articular injection, characterized by: The method is obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the dual-action drug sustained-release microsphere system with a core-shell structure according to claim 8 in the preparation of drugs for treating arthritis.

10. A drug for treating arthritis, characterized in that: The dual-effect drug sustained-release microsphere system with a core-shell structure as claimed in claim 8.

Images