Use of bilirubin, chondroitin sulfate and rickelfylline in combination in the preparation of a medicament for treating osteoarthritis, and a sequential targeted drug delivery system for treating arthritis

Abstract

The present invention provides the use of the combined use of bilirubin, chondroitin sulfate and licofelone in the preparation of a medicament for treating osteoarthritis. The present invention also provides a sequential targeted drug delivery system for treating arthritis, which is prepared from bilirubin, chondroitin sulfate and licofelone as raw materials, and the molar ratio of the bilirubin, chondroitin sulfate and licofelone is: 1-10 parts of bilirubin, 1-20 parts of chondroitin sulfate, and 0.1-5 parts of licofelone. The present invention also provides a preparation method and use of the sequential targeted drug delivery system. The present invention discovers for the first time the synthesis of chondroitin sulfate modified with bilirubin, and encapsulates licofelone through the self-assembled micelles of the polymer, thereby realizing sequential drug delivery, efficiently regulating the abnormal sphingolipid metabolism and AA metabolism of M1 macrophages, and thus converting them into M2 macrophages. In addition, the delivery system of the present invention can prolong the residence and action time of licofelone in the joint.

Description

Technical Field

[0001] The present invention relates to the use of the combined use of bilirubin, chondroitin sulfate and licofelone in the preparation of a medicament for treating osteoarthritis, and a sequential targeted drug delivery system for treating arthritis, belonging to the field of medicaments. Background Art

[0002] Osteoarthritis (OA) is a degenerative disease mainly characterized by joint pain caused by multi-factors leading to fibrosis, cracking, ulceration and loss of articular cartilage, often involving parts such as the knee joint, hip joint, spine and hand. Its pathological features mainly include articular cartilage degeneration and destruction, subchondral bone sclerosis or cystic change, marginal osteophyte formation of the joint and synovitis, etc. OA seriously affects the joint function and quality of life of patients. Moreover, studies have found that OA, especially symptomatic knee OA, can significantly increase the all-cause mortality of patients. Unfortunately, there is currently no safe and effective treatment method at home and abroad that can delay the progression of OA.

[0003] Chondroitin sulfate is a natural component of cartilage, usually extracted from shark or bovine cartilage or synthesized artificially. It has been widely studied in the treatment of osteoarthritis and is often used in combination with glucosamine. There is evidence that the combined use of chondroitin sulfate and glucosamine can relieve joint pain, improve joint mobility, and may reduce the dosage of conventional anti-inflammatory drugs, especially when used continuously for 6 to 24 months. Chondroitin sulfate itself has no obvious benefits when used alone, but in combination with glucosamine, it may have some alleviating effects on patients with moderate to severe knee pain.

[0004] Bilirubin, the catabolic product of heme, is usually related to liver function. Clinically, some blood or liver diseases can be detected by examining the bilirubin level in the blood. Bilirubin examination includes indirect bilirubin and direct bilirubin, and the sum of the two is called total bilirubin. Changes in bilirubin levels can reflect the status of multiple organs and tissues, such as excessive destruction of red blood cells, impaired bilirubin transport in hepatocytes, defects in glucuronide conjugation, and biliary obstruction, etc.

[0005] Currently, there are literature reports on the targeted treatment of colitis with an orally convertible bilirubin self-assembly system (Advanced Healthcare Materials (lF10.0) Pub Date: 2023-06-15, DOI: 10.1002 / adhm.202300946), which discloses that chondroitin sulfate can effectively mediate the construction of BR self-assembled nano-drugs (BSNM) through the intermolecular hydrogen bond between the dense sulfate groups and carboxyl groups of chondroitin sulfate and the imino group of BR for the treatment of colitis.

[0006] There is no report on the use of bilirubin-modified chondroitin sulfate in osteoarthritis.

[0007] Licofelone is a compound with anti-inflammatory properties. As a dual inhibitor of cyclooxygenase (COX) and 5-lipoxygenase (5-LOX), it has potential applications in the treatment of osteoarthritis (OA). According to its chemical properties, the melting point of licofelone is 162 - 163 °C, the density is 1.23, and it usually exists in solid form, with a color ranging from light yellow to nearly white. Licofelone shows inhibitory concentrations (IC50) of 0.16 μM and 0.23 μM for human platelet COX and 5-LOX, respectively, in in vitro studies. As a potential drug for the treatment of osteoarthritis, licofelone exhibits an anti-inflammatory effect comparable to that of methylprednisolone in experiments and has a similar effect to celecoxib in reducing the content of the inflammatory factor prostaglandin E2 (PGE2). However, there may also be some deficiencies or limitations in its clinical application. As a non-steroidal anti-inflammatory drug, licofelone may have potential adverse reactions similar to those of other NSAIDs, especially when used long-term, it may cause adverse effects on the gastrointestinal tract, cardiovascular system, and kidneys (Guo Binsheng, et al., Research progress on drug treatment of osteoarthritis, Progress in Pharmaceutical Sciences, 2018, 42(9): 679 - 703; Zhang Hongxia, Study on the anti-inflammatory effect of licofelone, Modern Drugs & Clinical, July 2013, Vol. 28, No. 4). In addition, licofelone does not have targetability in its action on cells and cell organelles. Moreover, licofelone is easily cleared in the joint, and its joint residence time is short. Summary of the Invention

[0008] The present invention provides a new use of the combination of bilirubin, chondroitin sulfate, and licofelone, and also provides a sequential targeted drug delivery system for the treatment of arthritis.

[0009] The present invention provides the use of the combination of bilirubin, chondroitin sulfate, and licofelone in the preparation of a drug for the treatment of osteoarthritis. The drug is a drug for relieving joint pain in osteoarthritis.

[0010] Among them, the molar ratio of bilirubin, chondroitin sulfate, and licofelone is:

[0011] Bilirubin 1 - 10 parts, chondroitin sulfate 1 - 20 parts, licofelone 0.1 - 5 parts.

[0012] Further preferably, the molar ratio of bilirubin, chondroitin sulfate, and licofelone is:

[0013] Bilirubin 15 parts, chondroitin sulfate 15 parts, licofelone 27 parts.

[0014] The drug is a preparation for intra-articular administration.

[0015] The present invention also provides a sequential targeted drug delivery system for treating arthritis, which is prepared from bilirubin, chondroitin sulfate and leflunomide as raw materials. The molar ratio of bilirubin, chondroitin sulfate and leflunomide is as follows:

[0016] 1 - 10 parts of bilirubin, 1 - 20 parts of chondroitin sulfate, 0.1 - 5 parts of leflunomide.

[0017] Further preferably, the molar ratio of bilirubin, chondroitin sulfate and leflunomide is as follows:

[0018] 15 parts of bilirubin, 15 parts of chondroitin sulfate, 27 parts of leflunomide.

[0019] The sequential targeted drug delivery system of the present invention first modifies chondroitin sulfate with bilirubin, and then encapsulates leflunomide with the modified chondroitin sulfate to prepare nanoparticles containing leflunomide (LCF - CSBN), which is the sequential targeted drug delivery system of the present invention.

[0020] The present invention also provides a preparation method of the sequential targeted drug delivery system for treating arthritis, which includes the following steps:

[0021] a. Modifying chondroitin sulfate with ethylenediamine:

[0022] First, dissolve chondroitin sulfate, carbodiimide hydrochloride, 4 - dimethylaminopyridine and ethylenediamine in N,N - dimethylformamide, react under magnetic stirring at room temperature, dialyze in water to remove unreacted ethylenediamine and catalyst, and then freeze - dry to obtain ethylenediamine - modified chondroitin sulfate;

[0023] b. Modifying chondroitin sulfate with bilirubin:

[0024] Dissolve bilirubin, EDCI, carbodiimide hydrochloride and 4 - dimethylaminopyridine in dimethyl sulfoxide, react under magnetic stirring at room temperature, then dissolve the ethylenediamine - modified chondroitin sulfate prepared in step a in a mixed solution of DMSO and water and add this solution to the above reaction solution, react under magnetic stirring at 30°C, and protect the whole reaction system by introducing nitrogen; after the reaction is completed, dialyze in water to remove organic reagents, and then remove unreacted precipitates by centrifugation, and freeze - dry the supernatant to obtain bilirubin - modified chondroitin sulfate;

[0025] c. Preparation of nanoparticles loaded with leflunomide:

[0026] Dissolve leflunomide in dimethyl sulfoxide, then drop - add this solution to the aqueous solution of bilirubin - modified chondroitin sulfate prepared in step b under stirring conditions. After the dropping is completed, continue stirring, and then probe - sonicate. Remove unencapsulated leflunomide by ultrafiltration to obtain nanoparticles of leflunomide (LCF - CSBN).

[0027] The present invention also provides the use of the sequential targeted drug delivery system in the preparation of drugs for joint cavities for treating osteoarthritis.

[0028] The present invention provides a pharmaceutical composition for treating osteoarthritis, which comprises the sequential targeted drug delivery system.

[0029] Macrophages are an important part of the innate immune system. In different physiological and pathological environments, these cells can exhibit functional plasticity according to different stimuli. Macrophages play important roles in various homeostatic functions of the body, such as maintaining tissue homeostasis, tissue remodeling, metabolic regulation, and host inflammatory defense. There are two different polarization states of macrophages, namely M1 macrophages (classically activated macrophages) and M2 macrophages (alternatively activated macrophages). In OA joints, the infiltration and activation of M1 macrophages are the main characteristics. The excessive polarization of M1 macrophages plays an important role in the formation and maintenance of the inflammatory microenvironment in OA joints. Therefore, selectively enhancing the transformation of macrophage M1 to M2 phenotype may inhibit low-grade inflammation and the progression of OA. Cellular metabolism plays an important regulatory role in the activation and function of macrophages. Generally, a high concentration of reactive oxygen species (ROS) in M1 macrophages can lead to Golgi stress, thereby triggering abnormal lipid metabolism (especially sphingolipid metabolism); at the same time, arachidonic acid (AA), which is a precursor of various inflammatory mediators, also shows abnormal metabolism in M1 macrophages, and the abnormal metabolism of AA can further promote oxidative stress. Therefore, this lipid metabolism disorder in macrophages promotes the polarization of M1 macrophages through compensatory pathways of inflammatory response and oxidative stress response. Therefore, it is necessary to simultaneously regulate the abnormal sphingolipid metabolism and AA metabolism in macrophages to effectively achieve the transformation of M1 macrophages to M2 macrophages.

[0030] In the present invention, an amphiphilic polymer is obtained by coupling bilirubin and chondroitin sulfate. The polymer can self-assemble into nano-micelles, and the drug rickelfylline is loaded into the nanoparticles. This drug delivery system can bind to the CD44 receptor of M1 macrophages through chondroitin sulfate to achieve targeted drug delivery to M1 macrophages. Further, the drug delivery system can target the Golgi apparatus of M1 macrophages. After reaching the Golgi apparatus, bilirubin reduces Golgi stress by scavenging ROS, thereby regulating the disorder of Golgi-related sphingolipid metabolism. When bilirubin is oxidized, its hydrophilicity increases significantly, resulting in the depolymerization of self-assembled nano-micelles to release rickelfylline. Rickelfylline regulates the disorder of AA metabolism in M1 macrophages by binding to cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) in cells. Finally, by simultaneously correcting the disorders of sphingolipid metabolism and AA metabolism, M1 macrophages are repolarized into M2 macrophages, thereby achieving the treatment of OA. There is no report on the study of repolarizing M1 macrophages into M2 macrophages for the treatment of OA through a sequential targeted drug delivery system.

[0031] The present invention first discovers the synthesis of chondroitin sulfate modified with bilirubin, and the self-assembled micelles of this polymer are used to encapsulate rickelfylline, thereby achieving sequential drug delivery, efficiently regulating the abnormal sphingolipid metabolism and AA metabolism of M1 macrophages, and thus converting them into M2 macrophages. In addition, the delivery system of the present invention can prolong the retention and action time of rickelfylline in the joint. Brief Description of the Drawings

[0032] Figure 1 . 1H NMR spectrum of CS-BR; 1

[0033] Figure 2 . Characterization of LCF-CSBN. A) Particle size distribution diagram of LCF-CSBN; B) Stability of LCF-CSBN stored at 4 °C; c) Particle size of CS-BR at different concentrations; D) Amount of BR in LCF-CSBN in H 2 2O 2 (5 mM), 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH, 100 mM), and NaOCl (1 mM) over time; E) Particle size of LCF-CSBN after being stored in an environment containing ROS for 1 h; F) Transmission electron micrograph of LCF-PEGBN after being stored in a solution containing AAPH (100 mM) for 1 h; G) Transmission electron micrograph of LCF-CSBN after being stored in a solution containing AAPH (100 mM) for 1 h;

[0034] Figure 3. Cellular uptake assay of CSBN. A) Observation of the uptake of CSBN and PEGBN on LPS-activated RAW264.7 cells by laser confocal microscopy; B) Observation of the co-localization of CSBN, PEGBN and the Golgi apparatus of LPS-activated RAW 264.7 cells by laser confocal microscopy; C) Histogram of flow cytometry quantitative analysis of the uptake of CSBN and PEGBN by LPS-activated RAW 264.7 cells (n = 3), ****P<0.0001; D) Uptake pathway of CSBN on LPS-activated RAW 264.7 cells (n = 3), ****P<0.0001; E) Pearson correlation coefficient of the co-localization of CSBN, PEGBN and the Golgi apparatus of LPS-activated RAW 264.7 cells (n = 3), ****P<0.0001;

[0035] Figure 4 . In vitro biological activity investigation assay of LCF-CSBR. A) Intracellular ROS concentration after treatment with LCF-CSBR (DCF green staining); B) Laser confocal images of the Golgi apparatus of cells after treatment with LCF-CSBR; C) Results of green fluorescence staining of GOLPH3 after treatment with LCF-CSBR; D) Results of green fluorescence staining of GM130 after treatment with LCF-CSBR; E) Results of red fluorescence staining of iNOS and green fluorescence staining of CD206 after treatment with LCF-CSBR; F) Cytokine concentration in the cell culture supernatant after treatment with LCF-CSBR, ****P<0.0001;

[0036] Figure 5 . Retention and distribution assay of CSBN in the OA joint. A) Small animal in vivo fluorescence imaging of CSBN in the OA rat joint; B) Semi-quantitative histogram of CSBN in the OA rat joint (n = 5); C) Area under the concentration-time curve (AUC) of CSBN in the OA rat joint (n = 5), ****P<0.0001; D) Co-localization of CSBN and M1 macrophages in the synovium of the OA joint;

[0037] Figure 6 . Number of M1 and M2 macrophages in the OA joint after treatment with LCF-CSBN (n = 5). A) Fluorescence co-staining of CD68 and iNOS (M1 type) and CD68 and CD206 (M2 type) in the synovium of the OA joint; B) Proportion of M1 macrophages in the synovium of the OA joint, ***P<0.001, ****P<0.001; C) Proportion of M2 macrophages in the synovium of the OA joint, ****P<0.001;

[0038] Figure 7. After LCF-CSBN treatment, the mechanical withdrawal threshold of OA model rats (n = 7). *P < 0.05, ***P < 0.001, ****P < 0.0001, compared with the Saline group; & P < 0.05, &&&& P < 0.0001, compared with the LCF group; $$$$P < 0.0001, compared with the CSBN group. Detailed implementation method

[0039] Example 1 Preparation method of the sequential targeted drug delivery system for treating arthritis of the present invention

[0040] 1. Synthesis of bilirubin-modified chondroitin sulfate (BR-CS)

[0041] Dissolve chondroitin sulfate (CS, 0.05 mmol; Sigma-Aldrich, St. Louis, USA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 0.06 mmol; Aladdin, Shanghai, China), 4-dimethylaminopyridine (DMAP, 0.06 mmol; Aladdin, Shanghai, China) and ethylenediamine (EDA, 0.05 mmol; Aladdin, Shanghai, China) in N,N-dimethylformamide, stir magnetically at room temperature for 24 h, then dialyze in water to remove unreacted ethylenediamine and catalyst, and then obtain ethylenediamine-modified chondroitin sulfate (CS-EDA) after lyophilization.

[0042] Dissolve bilirubin (BR, 0.04 mmol; Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), EDCI (0.06 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 0.06 mmol; Aladdin, Shanghai, China) and 4-dimethylaminopyridine (DMAP, 0.06 mmol; Aladdin, Shanghai, China) in dimethyl sulfoxide (DMSO), stir magnetically at room temperature for 30 min, then dissolve the obtained CS-EDA in a mixed solution of DMSO and water (V / V = 3:1) and add this solution to the above reaction solution, stir magnetically at 30 °C for 48 h (the whole reaction system is protected by introducing nitrogen). After the reaction is completed, dialyze in water to remove organic reagents, and then remove the unreacted BR precipitate by centrifugation (centrifuge at 10,000×g for 20 min), and the supernatant is lyophilized to obtain BR-modified CS (BR-CS).

[0043] Through nuclear magnetic resonance hydrogen spectrum ( 1The BR-CS was verified by ¹H NMR. From Figure 1 it can be seen that the characteristic peaks of both BR and CS are included in BR-CS, indicating that BR-CS was successfully obtained.

[0044] 2. Preparation of licofelone nanoparticles

[0045] Licofelone (27 μmol) was dissolved in dimethyl sulfoxide, and then this solution was added dropwise to an aqueous solution of BR-CS (15 μmol) under stirring conditions. After the addition was completed, stirring was continued for 10 min, and then probe sonication was performed for 5 min. The unencapsulated licofelone was removed by ultrafiltration to obtain licofelone nanoparticles (LCF-CSBN).

[0046] Example 2 Structural characterization of licofelone nanoparticles (LCF-CSBN) prepared by the present invention

[0047] The above-prepared preparation LCF-CSBN was diluted with pure water until it had a faint blue opalescence, and its particle size was measured by a laser particle size analyzer; the zeta potential of the obtained preparation was measured directly without dilution; its morphology was observed by transmission electron microscopy. At the same time, in order to investigate the in vitro response of LCF-CSBN to ROS, we also investigated the changes in the drug release behavior and particle size of LCF-CSBN in the presence of an oxide.

[0048] From Figure 2 it can be seen that LCF-CSBN has a particle size of about 160 nm, and the polydispersity index (PDI) of the particle size is less than 0.3 ( Figure 2 A-B). The critical micelle concentration of BR-CS is about 25 μg / mL ( Figure 2 C). LCF-CSBN can be stable for at least 1 week when stored at 4 °C ( Figure 2 D). In the presence of ROS, BR in LCF-CSBN can be oxidized within a short time, and its particle size and morphology also change significantly, indicating that LCF-CSBN has obvious ROS-responsive depolymerization ( Figure 2 E-G).

[0049] Comparative example:

[0050] Amino-terminated PEG was used instead of CS-EDA, and PEG-BR was synthesized according to the method of Example 1. The remaining steps and the amounts of substances used remained unchanged.

[0051] PEG-BR was used instead of CS-BR, and nanoparticles LCF-PEGBN were prepared using the method of Example 1. The dosage ratio of LCF to PEG-BR was (molar ratio 27:390), and the remaining steps remained unchanged.

[0052] The beneficial effects of the present invention were demonstrated by the following pharmacodynamic experiments.

[0053] Experimental Example 1 In vitro uptake experiment of the inventive zilucoplan nanoparticles

[0054] LPS-activated RAW 264.7 cells were seeded on a 12-well cell culture plate and cultured for 24 h. After they adhered to the wall, the culture medium was replaced with serum-free medium containing DiD-labeled CSBN and PEGBN (DiD concentration was 1 μ / ml). After incubation for 4 h, the fluorescence intensity of the cells was detected by flow cytometry, and the fluorescence distribution of the cells was observed and photographed using a laser confocal microscope. To verify the endocytic pathway of the nanoparticles, RAW 264.7 cells were pretreated with different inhibitors (0.5 mM CS, 30 μM chlorpromazine, 60 μM amiloride, 5 mM M-β-CD or CD44 antibody) for 1 h, then DiD-labeled CSBN was added, and incubation was continued for 4 h. Fibroblasts without inhibitor treatment were used as the control group, and the fluorescence intensity of the cells was detected by flow cytometry.

[0055] From Figure 3 it can be seen that the uptake of CSBN by LPS-activated RAW 264.7 cells was significantly higher than that of PEGBN ( Figure 3 A, C); furthermore, the co-localization ability of CSBN with the Golgi apparatus of LPS-activated RAW 264.7 cells was significantly higher than that of PEGBN ( Figure 3 B, E). In addition, after pretreatment with CS and CD44 antibody, the uptake of CSBN by LPS-activated RAW 264.7 cells was significantly reduced ( Figure 3 D). The above results indicate that CSBN can be efficiently taken up by LPS-activated RAW 264.7 cells through CD44 receptor-mediated endocytosis and can be targeted to distribute on the Golgi apparatus.

[0056] Experimental Example 2 Investigation of in vitro biological activity

[0057] LPS-activated RAW 264.7 cells were seeded on a 12-well cell culture plate and cultured for 24 h. After they adhered to the plate, LCF solution, CSBN, LCF-CSBN, and LCF-PEGBN (the concentration of LCF was 2.7 μM, and the medium without drugs was used as a control) were added to the medium respectively. After incubation for 3 h, the supernatant was taken, and the concentrations of TNF-α, IL-1β, IL-6, iNOS, and IL-10 in it were measured by ELISA; the morphology of the Golgi apparatus of cells in each group was observed by transmission electron microscopy; the Golgi apparatus of cells was labeled with a GM130 antibody with green fluorescence, and the fluorescence intensity of the cells was observed by a laser confocal microscope and measured by a flow cytometer; the reactive oxygen species concentration of cells after treatment with LCF-PEGBN was examined by labeling with 5-(6)-chloromethyl-2,7-dichlorodihydrofluorescein diacetate (DCF); the Golgi stress intensity of cells after treatment with LCF-PEGBN was examined by labeling with GOLPH3 and GM130; the M1 and M2 macrophage phenotypes of cells after treatment with LCF-PEGBN were examined by labeling with iNOS and CD206 respectively.

[0058] It can be seen from Figure 4 that, compared with other treatment methods, LCF-CSBN treatment can more effectively reduce the reactive oxygen species concentration ( Figure 4 A-B) and the Golgi stress state ( Figure 4 C-D) of LPS-activated RAW264.7 cells. At the same time, LCF-CSBN treatment can efficiently repolarize M1 macrophages into M2 macrophages ( Figure 4 E). Further, LCF-CSBN treatment can significantly reduce the secretion of pro-inflammatory cytokines and increase the secretion of anti-inflammatory cytokines of LPS-activated RAW 264.7 cells ( Figure 4 F).

[0059] Experimental Example 3 Animal experiment of the sequential targeted drug delivery system of the present invention

[0060] 1) OA rat model

[0061] The rats were anesthetized with a small animal gas anesthesia machine, the hair on their right knee joints was shaved, and they were fixed in the supine position. 50 μL of monosodium iodoacetate (MIA) with a concentration of 40 mg / mL was injected into the right knee joint cavity. When the knee joints of the rats showed swelling and the pain threshold was significantly reduced, the OA rat model was obtained.

[0062] 2) In vivo distribution and enrichment experiment of M1 macrophages

[0063] The DiD solution, DiD-labeled CSBN, and PEGBN were respectively injected into the right knee joint cavity of OA rats through the joint cavity. On the day of injection and 1 day, 2 days, 3 days, 7 days, 14 days, 21 days, and 28 days after injection, the fluorescence intensity at the inflamed joints of the rats was observed and photographed using a small animal in vivo imager. After the observation with the small animal in vivo imager was completed, the rats were sacrificed, and the synovium of the inflamed joints of each group of rats was isolated, cryosectioned, and then the cell nuclei and M1 macrophages in the sections were stained with DAPI and iNOS antibody labeled with green fluorescence respectively, and observed and photographed using a laser confocal microscope. As Figure 5 can be seen, compared with PEGNP, CSBN can remain in the joint for a long time and can be effectively enriched at the position of M1 macrophages.

[0064] 3) Pharmacodynamic experiment

[0065] Through the method of joint cavity injection, normal saline, LCF solution, blank CSBN, and LCF-CSBN were respectively used to treat the right knee joint of OA rats. The administration dose of LCF was 40 μg, and it was administered once. From the first day of administration, the mechanical withdrawal threshold of the right hind legs of each group of rats was measured once a week.

[0066] As Figure 6 can be seen, after treatment with LCF-CSBN, the number of M1 macrophages in the OA joint decreased significantly, while the number of M2 macrophages increased significantly. This result further shows that LCF-CSBN can effectively repolarize M1 macrophages into M2 macrophages. As Figure 7 can be seen, compared with other methods, LCF-CSBN treatment can more effectively relieve the joint pain of OA rats. These results illustrate the effectiveness of the sequential drug delivery system of the present invention for the treatment of OA.

Claims

1. Use of bilirubin, chondroitin sulfate and levofloxacin in combination for preparing a drug for treating osteoarthritis; the molar ratio of bilirubin, chondroitin sulfate and levofloxacin is: 1-10 parts of bilirubin, 1-20 parts of chondroitin sulfate, 0.1-5 parts of lexapro; The medicine is a preparation for intra-articular administration; The drug is prepared by first modifying chondroitin sulfate with bilirubin, and then encapsulating lexapro in the modified chondroitin sulfate to form nanoparticles LCF-CSBN containing lexapro.

2. The use according to claim 1, characterized in that: The medicine is a medicine for relieving osteoarthritis joint pain.

3. The use according to claim 1, characterized in that: The molar ratio of bilirubin, chondroitin sulfate and lexapro is: 15 parts of bilirubin, 15 parts of chondroitin sulfate, and 27 parts of lexapro.

4. A sequential targeted drug delivery system for treating arthritis, characterized in that: The drug delivery system is prepared from bilirubin, chondroitin sulfate and levofloxacin as raw materials, and the molar ratio of bilirubin, chondroitin sulfate and levofloxacin is: 1-10 parts of bilirubin, 1-20 parts of chondroitin sulfate, 0.1-5 parts of lexapro; The drug delivery system is to first modify chondroitin sulfate with bilirubin, and then encapsulate lexapro in the modified chondroitin sulfate to prepare nanoparticles LCF-CSBN containing lexapro; The preparation method of the drug delivery system comprises the following steps: a. Ethylenediamine-modified chondroitin sulfate First, chondroitin sulfate, carbodiimide hydrochloride, 4-dimethylaminopyridine and ethylenediamine are dissolved in N,N-dimethylformamide, reacted under magnetic stirring at room temperature, dialyzed in water to remove unreacted ethylenediamine and catalyst, and lyophilized to obtain ethylenediamine-modified chondroitin sulfate; b. Bilirubin-modified chondroitin sulfate: Dissolve bilirubin, EDCI, carbodiimide hydrochloride, and 4-dimethylaminopyridine in dimethyl sulfoxide, and react under magnetic stirring at room temperature. Then, dissolve the chondroitin sulfate modified with ethylenediamine in step a in a mixed solution of DMSO and water and add the solution to the above reaction solution. React under magnetic stirring at 30° C., and pass nitrogen gas through the entire reaction system for protection. After the reaction is completed, dialyze in water to remove the organic reagent, and then remove the unreacted precipitate by centrifugation. After the supernatant is freeze-dried, the bilirubin-modified chondroitin sulfate is obtained. c. Preparation of Liquefied Nanoparticles: Dissolve levofloxacin in dimethyl sulfoxide, and then add the solution dropwise to the bilirubin-modified chondroitin sulfate aqueous solution prepared in step b under stirring. After the addition is completed, continue stirring, then use probe ultrasound to remove unencapsulated levofloxacin, i.e., levofloxacin nanoparticles (LCF-CSBN), by ultrafiltration.

5. The sequential targeted drug delivery system for treating arthritis according to claim 4, characterized in that: The molar ratio of bilirubin, chondroitin sulfate and lexapro is: 15 parts of bilirubin, 15 parts of chondroitin sulfate, and 27 parts of lexapro.

6. A method for preparing a sequential targeted drug delivery system for treating arthritis according to claim 4 or 5, comprising the following steps: a. Ethylenediamine-modified chondroitin sulfate First, chondroitin sulfate, carbodiimide hydrochloride, 4-dimethylaminopyridine and ethylenediamine are dissolved in N,N-dimethylformamide, reacted under magnetic stirring at room temperature, dialyzed in water to remove unreacted ethylenediamine and catalyst, and lyophilized to obtain ethylenediamine-modified chondroitin sulfate; b. Bilirubin-modified chondroitin sulfate: Dissolve bilirubin, EDCI, carbodiimide hydrochloride, and 4-dimethylaminopyridine in dimethyl sulfoxide, and react under magnetic stirring at room temperature to obtain a reaction solution; dissolve the ethylenediamine-modified chondroitin sulfate in step a in a mixed solution of DMSO and water and add the solution to the reaction solution, and react under magnetic stirring at 30° C. The entire reaction system is protected by nitrogen; after the reaction is completed, dialyze in water to remove the organic reagent, and then remove the unreacted precipitate by centrifugation, and freeze-dry the supernatant to obtain bilirubin-modified chondroitin sulfate; c. Preparation of Liquefied Nanoparticles: Dissolve levofloxacin in dimethyl sulfoxide, and then add it dropwise to the bilirubin-modified chondroitin sulfate aqueous solution prepared in step b under stirring. After the addition is completed, continue stirring, then use probe ultrasound to remove unencapsulated levofloxacin, i.e., levofloxacin nanoparticles (LCF-CSBN), by ultrafiltration.

7. Use of the sequential targeted drug delivery system according to claim 4 or 5 in the preparation of a bone-joint cavity drug for treating osteoarthritis.

8. The use according to claim 7, characterized in that: The medicine is a medicine for relieving osteoarthritis joint pain.

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