Application of combined use of hyaluronic acid-bilirubin conjugate and honokiol in preparation of medicines for treating osteoarthritis, honokiol-loaded nanoparticles and bionic nanoparticles for treating bone joints

Abstract

The invention provides a combined use of a hyaluronic acid-bilirubin conjugate and honokiol in preparation of a medicine for treating osteoarthritis. The invention provides a honokiol-loaded nanoparticle, which is characterized in that a hyaluronic acid-bilirubin conjugate and honokiol are used as raw materials to prepare a nanoparticle HKL-PNPs, and then the nanoparticle HKL-PNPs is modified by a mitochondrial targeting peptide to obtain the honokiol-loaded nanoparticle HKL-MPNPs. The invention also provides a bionic nanoparticle for treating bone joints. The bionic nanoparticle is prepared by wrapping the honokiol-loaded nanoparticle with an engineered cartilage cell membrane (ECM). According to the invention, cartilage cells with high expression of IL-1R2 are obtained through a gene editing method for the first time, and mitochondrial targeted nanoparticle cores loaded with Sirtuin-3 agonists are coated by cell membranes of the cells, so that the bionic nano preparation is obtained and is used for treating OA. The prepared bionic nano preparation not only can neutralize IL-1beta mediated inflammatory reaction, but also can recover Sirtuin-3 mediated mitochondrial function imbalance, so that the result of synergistic treatment of OA is achieved.

Description

Technical Field

[0001] The present invention relates to the use of a hyaluronic acid-bilirubin conjugate and honokiol in combination for preparing a medicament for treating osteoarthritis, a honokiol-loaded nanoparticle, and a biomimetic nanoparticle for treating osteoarthritis. Background Art

[0002] Osteoarthritis (OA) is a multifactorial disease characterized by joint inflammation and cartilage degeneration, and is also the most prevalent joint degenerative disease, with more than 600 million patients worldwide. However, to date, no drug has been marketed to stop the progression of OA. It is generally believed that the mutual promotion between the inflammatory microenvironment and functionally imbalanced chondrocytes is the main factor causing the progressive degeneration of OA joints. The pro-inflammatory factor interleukin-1β (IL-1β) in the OA inflammatory microenvironment can activate the pro-inflammatory mechanism of cells by binding to the type 1 IL-1 receptor (IL-1R1) on the surface of chondrocytes, thereby inducing changes in the chondrocyte phenotype and further causing cartilage degeneration. Usually, in order to balance the pro-inflammatory effect of IL-1β, the type 2 IL-1 receptor (IL-1R2) is also expressed on the surface of immune cells. This receptor can bind tightly to IL-1β but does not cause an inflammatory response, thereby inhibiting IL-1β-mediated inflammation. However, the expression level of IL-1R2 on chondrocytes is quite low and cannot neutralize IL-1β in the inflamed joint. In addition to the above inflammation-induced cartilage degeneration, the deletion of deacetylase Sirtuin-3 in chondrocytes can disrupt the production of adenosine triphosphate (ATP), increase the abnormal accumulation of reactive oxygen species (ROS), accelerate chondrocyte death, thereby inhibiting cartilage formation and the progression of osteoarthritis. In addition, the deletion of Sirtuin-3 can promote mitochondrial dysfunction imbalance, thereby further promoting OA joint inflammation. Therefore, simultaneously regulating IL-1β-mediated inflammation and Sirtuin-3 in chondrocytes has great potential in the treatment of OA.

[0003] Qiao Tingting, et al., Research progress on the pharmacological effects of bilirubin nanoparticles, China Pharmacy, Vol. 34, No. 5, 2023. Bilirubin has good anti-inflammatory, antioxidant, and immunomodulatory effects. However, due to its poor water solubility and low bioavailability, its clinical application is greatly limited. Researchers have developed bilirubin into various nanoparticles, and by virtue of the dosage form advantages, effectively eliminated the limitation of low bilirubin solubility, enabling it to maximize its anti-inflammatory, antioxidant, immunomodulatory and other pharmacological activities. Bilirubin nanoparticles have great application potential in a variety of gastrointestinal diseases, liver and kidney diseases, skin diseases, autoimmune diseases, islet transplantation, and targeted therapy of tumors (which can not only directly act against tumors but also serve as a drug delivery system). Currently, there is no relevant literature report on the use of bilirubin nanoparticles for the treatment of osteoarthritis.

[0004] Application No.: 202410088609.2, Invention Title: A Biomimetic Nanodrug Delivery Carrier Targeting Chondrocytes, Its Preparation Method and Application. Disclosed is a biomimetic nanodrug delivery carrier targeting chondrocytes, its preparation method and application. The biomimetic nanodrug delivery carrier targeting chondrocytes includes polymer nanoparticles and chondrocyte membranes, and the chondrocyte membranes coat the surface of the polymer nanoparticles. The present invention develops a chondrocyte membrane-modified nanocarrier, with polymer nanoparticles capable of loading drugs, etc. as the core, and chondrocyte membranes coated on its surface to obtain a delivery carrier. The obtained carrier has the ability to target and promote chondrocyte uptake, can adhere to the extracellular matrix of chondrocytes, penetrate into the matrix interior, and stay in the knee joint for a long time, and can be used as a cartilage-specific drug depot for the long-term treatment of osteoarthritis. Summary of the Invention

[0005] In order to synergistically regulate IL-1β-mediated inflammation in the OA joint and Sirtuin-3 on chondrocyte mitochondria, the inventors first made chondrocytes highly express IL-1R2 through transgenic technology, and then used genetically modified chondrocyte membranes to wrap the core of mitochondria-targeted nanoparticles loaded with Sirtuin-3 agonists, thereby preparing a genetically engineered chondrocyte biomimetic nanoformulation. After the nanoformulation enters the joint, IL-1R2 on its surface can act as a decoy to bind IL-1β, thereby blocking the inflammatory response mediated by it. Under the mediation of chondrocyte membranes, it penetrates through the cartilage matrix and is taken up by chondrocytes, releasing the core, which targets and delivers the Sirtuin-3 agonist to mitochondria, thereby restoring the balance of mitochondrial function. Finally, it effectively inhibits OA joint inflammation and cartilage degeneration.

[0006] The present invention provides the use of the combination of hyaluronic acid-bilirubin conjugate and honokiol in the preparation of a drug for the treatment of osteoarthritis.

[0007] Among them, the weight ratio of the hyaluronic acid-bilirubin conjugate to honokiol is: 10:1.

[0008] The present invention provides a honokiol-loaded nanoparticle, which is prepared from hyaluronic acid-bilirubin conjugate and honokiol as raw materials into nanoparticles HKL-PNPs, and then modified with a mitochondrial targeting peptide to obtain honokiol-loaded nanoparticles HKL-MPNPs.

[0009] Among them, the weight ratio of the hyaluronic acid-bilirubin conjugate to honokiol is: 10:1.

[0010] The present invention also provides a preparation method of the honokiol-loaded nanoparticle, which includes the following steps:

[0011] a. Prepare hyaluronic acid-bilirubin conjugate;

[0012] Dissolve bilirubin, carbodiimide hydrochloride and N-hydroxysuccinimide in dimethyl sulfoxide. After magnetic stirring reaction at 30 °C, add 31.4 mg of adipic dihydrazide (ADH) and continue the reaction for 12 h; dissolve hyaluronic acid, carbodiimide hydrochloride and N-hydroxysuccinimide in dimethyl sulfoxide and perform magnetic stirring reaction at 30 °C; mix the above two reaction solutions and perform magnetic stirring reaction at 30 °C for 12 h. The entire reaction system is protected by introducing nitrogen. After the reaction, dialyze in water to remove organic reagents, and then remove the precipitate by centrifugation (centrifuge at 10,000×g for 20 min). The supernatant is freeze-dried to obtain bilirubin-modified hyaluronic acid (HA-BR);

[0013] b. Preparation of honokiol-loaded nanoparticles HKL-PNPs:

[0014] Dissolve phospholipid E8, Kolliphor HS-15, MAL-PEG 2000 -DSPE and honokiol in dichloromethane, remove the organic reagent by a rotary evaporator to obtain a lipid film, then hydrate and remove the film with an aqueous solution containing HA-BR, and then perform probe sonication in an ice-water bath. Remove the unencapsulated honokiol by ultrafiltration to obtain honokiol-loaded nanoparticles (HKL-PNPs);

[0015] c. Mitochondrial targeting peptide modification:

[0016] React HKL-PNPs with a cysteine-modified mitochondrial targeting peptide (amino acid sequence: Cys-Fx-r-Fx-k-Fx-r-Fx-k) by stirring at room temperature to obtain mitochondrial targeting peptide-modified honokiol-loaded nanoparticles (HKL-MPNPs).

[0017] The present invention provides a biomimetic nanoparticle for treating bone and joints, which is obtained by encapsulating the honokiol-loaded nanoparticles with engineered chondrocyte membranes (ECM).

[0018] Among them, the mass ratio of the engineered chondrocyte membranes (ECM) to the honokiol-loaded nanoparticles is 1:50 - 10:1.

[0019] Among them, the preparation method of the engineered chondrocyte membranes (ECM) is as follows:

[0020] After co-transfecting recombinant adenovirus overexpressing mouse FLAG-tagged IL-1R2 plasmid and chondrocytes for 12 hours, fresh medium was added, and the engineered chondrocytes were collected after culturing for 72 hours. The engineered chondrocytes were resuspended in a lysis buffer containing 225 mM mannitol, 30 mM Tris-HCl, 75 mM sucrose, 0.5% (w / v) bovine serum albumin, 0.5 mM ethylenediaminetetraacetic acid, and a protease and phosphatase inhibitor mixture. After sonication, it was centrifuged at 10,000×g for 15 minutes at 4°C. Then, the supernatant was ultracentrifuged at 100,000×g for 150 minutes at 4°C, and the resulting precipitate was freeze-dried to obtain engineered chondrocyte membranes (ECM).

[0021] The present invention also provides a method for preparing the bionic nanoparticles for treating osteoarthrosis, which comprises the following steps:

[0022] a. Take the engineered chondrocyte membranes (ECM) and magnolol-loaded nanoparticles, and mix them;

[0023] b. Sonicate for 10 min under ice-water bath conditions to obtain bionic nanoparticles (HKL-ECM@MPNPs) encapsulated by ECM.

[0024] The present invention also provides the uses of the magnolol-loaded nanoparticles and the bionic nanoparticles in the preparation of drugs for treating osteoarthrosis, and the drugs are drugs for intra-articular injection.

[0025] For the first time, the present invention obtains chondrocytes with high expression of IL-1R2 by gene editing method, and coats the cell membrane of the chondrocytes to load the core of mitochondrial-targeted nanoparticles of Sirtuin-3 agonist to obtain a bionic nano-preparation for the treatment of OA. The prepared bionic nano-preparation can not only neutralize the inflammatory reaction mediated by IL-1β, but also restore the mitochondrial function imbalance mediated by Sirtuin-3, so as to achieve the result of synergistic treatment of OA. Description of the Drawings

[0026] Figure 1 . Synthetic route (A) and 1 1H NMR spectrum (B) of HA-BR;

[0027] Figure 2 . Characterization of HKL-ECM@MPNPs. (A) Transmission electron micrograph of HKL-CM@MPNPs, scale bar is 50 nm; (B) Particle size distribution diagram of HKL-CM@MPNPs; (C) Zeta potential of HKL-CM@MPNPs

[0028] Figure 3.Cell uptake assay of ECM@MPNPs. (A) Laser confocal microscopy was used to observe the uptake of nanoparticles by chondrocytes, scale bar: 20 μm; (B) Laser confocal microscopy was used to observe the co-localization of nanoparticles with the Golgi apparatus in chondrocytes, scale bar: 10 μm;

[0029] Figure 4 .HKL-ECM@MPNPs reprogram the phenotype of OA chondrocytes by synergistically blocking cytokines and restoring mitochondrial SIRT3. (A) Protein expression level of SIRT3 in OA chondrocytes; (B) Mitochondrial ROS level in OA chondrocytes, scale bar: 20 μm; (C) Levels of IL-1β, IL-6 and TNF-α in the culture supernatant of OA chondrocytes (n = 3); (D) Assessment of mitochondrial respiration intensity in OA chondrocytes (n = 5); (E) Representative TEM images of mitochondrial morphology in OA chondrocytes, scale bar: 1 μm; (F and G) SA-β-gal staining results of OA chondrocytes; Protein levels of anabolism-related indicators (COLII and ACAN) and catabolism-related indicator (MMP13) in OA chondrocytes. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001;

[0030] Figure 5 .In vivo joint retention and biodistribution of ECM@MPNPs. (A) Fluorescence images in the knee joints of DMM mice after single intra-articular injection of different DiD-labeled preparations. (B) Quantitative analysis of fluorescence intensity in the knee joints after intra-articular injection of DiD-labeled nanoparticles (n = 5, mean ± standard deviation). (C) Area under the curve calculated based on the fluorescence intensity curve in (B) (n = 5, mean ± standard deviation). (D) Representative cross-sectional fluorescence images of the knee joints of DMM mice on the 3rd day after intra-articular injection of DiD solution, DiD-PNPs, DiD-MPNPs, DiD-CM@MPNPs or DiD-ECM@MPNPs. Scale bar: 50 μm. *P < 0.05, ***P < 0.001;

[0031] Figure 6. Intra-articular injection of HKL-ECM@MPNPs effectively delays the progression of OA in the DMM mouse model. (A) Pain-related behavior assessment was performed by measuring the paw withdrawal threshold using a von Frey apparatus (n = 7); (B) Cartilage degradation was evaluated based on the OARSI score of the medial femoral condyle and medial tibial plateau. The score was evaluated by three professionals under blinded conditions according to Safranin O-fast green (S.O.) staining of a series of sections (n = 7); (C) Histopathological evaluation of joints in different groups was performed by S.O. staining; Immunohistochemical staining showed COLII and MMP13, and immunofluorescent staining showed the expression of IL-1R2 and SIRT3 in articular cartilage of different groups. Scale bars: 50 μm for immunohistochemistry and immunofluorescence, and 100 μm for S.O. staining; (D) Three-dimensional reconstructed micro-CT images of the right knee joints in different groups. Red arrows indicate osteophyte formation. Statistical significance was evaluated by one-way analysis of variance (ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Detailed implementation mode

[0032] Example 1: Preparation of chondrocyte membranes highly expressing IL-R2

[0033] After mixing and transfecting recombinant adenovirus of mouse FLAG-tagged IL-1R2 plasmid with chondrocytes for 12 hours, fresh medium was added, and the engineered chondrocytes were collected after culturing for 72 hours. The engineered chondrocytes were resuspended in a lysis buffer containing 225 mM mannitol, 30 mM Tris-HCl, 75 mM sucrose, 0.5% (w / v) bovine serum albumin, 0.5 mM ethylenediaminetetraacetic acid, and a protease and phosphatase inhibitor mixture. After sonication, centrifugation was performed at 10,000×g for 15 minutes at 4°C. Then, the supernatant was ultracentrifuged at 100,000×g for 150 minutes at 4°C, and the obtained precipitate was freeze-dried to obtain engineered chondrocyte membranes (ECM). Prepared using chondrocytes in the same manner as above, ordinary chondrocyte membranes (CM) were obtained.

[0034] Example 2. Preparation of hyaluronic acid-bilirubin conjugate

[0035] Dissolve bilirubin (BR, 175 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 29 mg), and N-hydroxysuccinimide (NHS, 17.5 mg) in dimethyl sulfoxide (DMSO). After magnetic stirring at 30 °C for 10 min, add 31.4 mg of ADH and continue the reaction for 12 h. Dissolve hyaluronic acid (HA, 160 mg), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 153.4 mg), and N-hydroxysuccinimide (NHS, 92 mg) in dimethyl sulfoxide (DMSO) and magnetically stir at 30 °C for 15 min. Mix the above two reaction solutions and magnetically stir at 30 °C for 12 h (the entire reaction system is protected by nitrogen). After the reaction, dialyze against water to remove the organic reagents, and then remove the precipitate by centrifugation (centrifuge at 10,000×g for 20 min). The supernatant is freeze-dried to obtain HA modified with BR (HA-BR).

[0036] Verify HA-BR by nuclear magnetic resonance hydrogen spectrum ( 1 1H NMR). As can be seen from Figure 1 it, HA-BR contains the characteristic peaks of both BR and HA, indicating that HA-BR is successfully obtained.

[0037] Example 3. Preparation of honokiol-loaded nanoparticles

[0038] Dissolve 150 mg of phospholipid E8, 75 mg of Kolliphor HS-15, 4 mg of MAL-PEG 2000 -DSPE, and 1 mg of honokiol (HKL, 27 μmol) in dichloromethane. Remove the organic reagent by rotary evaporator to obtain a lipid film, and then hydrate and remove the film with an aqueous solution containing 10 mg of HA-BA. Then, probe sonicate in an ice-water bath for 10 min, and remove the unencapsulated honokiol by ultrafiltration to obtain honokiol-loaded nanoparticles (HKL-PNPs). React HKL-PNPs with a cysteine-modified mitochondrial targeting peptide (amino acid sequence: Cys-Fx-r-Fx-k-Fx-r-Fx-k) by stirring at room temperature to obtain mitochondrial targeting peptide-modified honokiol-loaded nanoparticles (HKL-MPNPs).

[0039] Example 4. Preparation of biomimetic nanoparticles

[0040] Mix ECM with HKL-MPNPs, and then sonicate in an ice-water bath for 10 min to obtain ECM-coated biomimetic nanoparticles (HKL-ECM@MPNPs). Prepare CE-coated biomimetic nanoparticles (HKL-CM@MPNPs) in the same way.

[0041] Example 5 Characterization of HKL-ECM@MPNPs

[0042] Dilute the above-prepared preparation with pure water until it shows a faint blue opalescence, and measure its particle size using a laser particle size analyzer; measure the zeta potential of the prepared preparation directly without dilution; observe its morphology using a transmission electron microscope.

[0043] It can be seen from Figure 2 that HKL-CM@MPNPs exhibit a spherical core-shell structure, with a particle size of approximately 80 nm. The zeta potential of HKL-CM@MPNPs is close to that of ECM, further indicating that ECM is successfully coated on the surface of the nanoparticles.

[0044] The beneficial effects of the present invention are demonstrated by the following efficacy tests.

[0045] Test Example 1 In vitro uptake experiment

[0046] Take chondrocytes and seed them on a 12-well cell culture plate. After culturing for 24 h and waiting for them to adhere, replace the culture medium with a serum-free medium containing DiD-labeled PNPs, MPNPs, CM@MPNPs, and ECM@MPNPs (DiD concentration is 1 μ / ml). After incubation for 4 h, detect the fluorescence intensity of the cells using a flow cytometer, and observe and photograph the fluorescence distribution of the cells using a laser confocal microscope.

[0047] It can be seen from Figure 3 that compared with PNPs, the uptake of ECM@MPNPs by chondrocytes is significantly improved, and at the same time, the mitochondrial distribution is also significantly increased.

[0048] Test Example 2 Investigation of in vitro biological activity

[0049] Stimulate chondrocytes with 10 ng / mL murine IL-1β to induce inflammation. Then treat the cells with different nanoparticle preparations (HKL-PNPs, HKL-MPNPs, HKL-CM@MPNPs, and HKL-ECM@MPNPs) for 24 hours (HKL concentration is 5 μM). Use an ELISA kit to measure the concentrations of cytokines IL-6, IL-1β, and TNF-α in the supernatant. Use an enhanced ATP detection kit, a glycolysis detection (extracellular acidification) kit, and an oxygen consumption rate plate detection kit to measure the ATP content, glycolysis rate, and oxygen consumption respectively.

[0050] Treat chondrocytes according to the above-related method, and then stain them with 10 μM DCFH-DA (MCE, USA) (a marker for intracellular ROS) or 2 μM MitoSOX Green mitochondrial superoxide indicator.

[0051] Use Hoechst 33342 to label the cell nuclei, and then observe and photograph them under a fluorescence microscope.

[0052] Chondrocytes were treated according to the above - related method, and then the mitochondrial morphology in chondrocytes was observed using a transmission electron microscope (TEM).

[0053] Chondrocytes were treated with a combination of 10 ng / mL IL - 1β and 50 μM H2O2, and then treated with different nanoparticle formulations (HKL - PNPs, HKL - MPNPs, HKL - CM@MPNPs, and HKL - ECM@MPNPs) and normal saline for 96 hours. SA - β - gal staining was performed using a senescence β - galactosidase staining kit, and observed and photographed through a microscope.

[0054] Chondrocytes were stimulated with 10 ng / mL murine IL - 1β to induce inflammation. Then, after treatment with different nanoparticle formulations (HKL - PNPs, HKL - MPNPs, HKL - CM@MPNPs, and HKL - ECM@MPNPs), they were fixed and treated with the following antibodies: COLII antibody, aggrecan antibody, and MMP13 antibody, and observed and photographed through a fibroscope.

[0055] As Figure 4 shown, HKL - ECM@MPNPs significantly improved the function of OA chondrocytes through the following mechanisms: restoring mitochondrial function by increasing SIRT3 expression and improving mitochondrial morphology. Antioxidant effect, significantly reducing the mitochondrial ROS level. Anti - inflammatory effect, reducing the secretion of inflammatory factors IL - 1β, IL - 6, and TNF - α. Improving energy metabolism: increasing ATP production, glycolysis, and oxidative phosphorylation levels. Inhibiting cell senescence, reducing the number of SA - β - gal - positive cells. Promoting extracellular matrix synthesis, increasing COLII and ACAN expression, and simultaneously inhibiting MMP13 expression.

[0056] Experimental Example 3 Animal Experiment

[0057] 1) OA rat model

[0058] C57BL / 6J mice were anesthetized with isoflurane; the surgery was performed by cutting the medial meniscus tibial ligament after opening the right knee joint capsule. The joint cavity was rinsed with normal saline, and the surgical wound was closed with absorbable sutures. For the sham - operation group, mice received all the same steps except cutting the medial meniscus tibial ligament.

[0059] 2) In - vivo joint retention and biodistribution experiment

[0060] One week after surgery, DMM mice were randomly divided into different groups. The right joints of each mouse received intra-articular (IA) injection with an equal volume of DiD solution or DiD-loaded nanoparticles. Images were acquired using a Caliper IVIS Lumina III in vivo imaging system at the specified time points. The images and quantitative data were processed using Living Image software. The biodistribution experiment started on the third day after intra-articular injection. After sacrificing the DMM mice, the right knee joints were collected, fixed with 4% paraformaldehyde, and embedded in a cryo-embedding medium. 5-μm-thick tissue sections were prepared using a CM1950 cryostat (Leica Biosystems), washed three times to remove the embedding medium, and stained with DAPI. The fluorescence signals in the joints were observed by fluorescence microscopy. Observations and photographs were taken using a laser confocal microscope.

[0061] It can be seen from Figure 5 that ECM@MPNPs can remain in the joint for a long time and are widely distributed in cartilage, indicating its good cartilage penetrability.

[0062] 3) Pharmacodynamic study

[0063] One week after surgery, the mice were induced with isoflurane inhalation anesthesia. Subsequently, the right knee joints of each mouse received an intra-articular (IA) injection of 10 μL of normal saline, free HKL, HKL-PNPs, HKL-MPNPs, ECM, HKL + ECM, HKL-CM@MPNPs, or HKL-ECM@MPNPs (the equivalent concentration of HKL in each injection was 10 μM). Subsequent intra-articular injections were performed every two weeks. The mice were euthanized at the ninth week, and the right joints and serum samples were collected.

[0064] Behavioral assessment

[0065] Mechanical allodynia tests were performed using an electronic von Frey tactile anesthesiometer (IITC Life Science Inc.) to evaluate the pain-related behaviors of the mice.

[0066] Histological, immunohistochemical, and immunofluorescence analyses

[0067] Two weeks after the last injection, the mice were euthanized. The right knee joints of each mouse were collected, fixed, decalcified, dehydrated, and embedded in paraffin. Human cartilage explants were processed using the same procedure. Serial sections with a thickness of 5 μm were prepared and stained with hematoxylin and eosin (H&E) or S.O. respectively. Based on the maximum score of the S.O. stained sections, the cartilage degradation was evaluated using the OARSI scoring system. Immunohistochemical staining was performed using biotinylated secondary antibodies and 3,3′-diaminobenzidine (DAB); immunofluorescent staining was performed using secondary antibodies labeled with Alexa Fluor 488 or Alexa Fluor 594. The primary antibodies included: COLII antibody, MMP13 antibody, IL-1β antibody, TNFα antibody, P16INK4A antibody, P21 antibody, SIRT3 antibody, and IL-1R2 antibody.

[0068] Micro-computed tomography (micro-CT) analysis

[0069] Two weeks after the last injection, the mice were euthanized. The right knee joints of each mouse were collected. The knee joints were fixed with 4% paraformaldehyde for 3 days before scanning and scanned using a micro-computed tomography scanner. Three-dimensional structure reconstruction was performed on the scanned images under consistent parameters.

[0070] It can be seen from Figure 6 that compared with other methods, HKL-ECM@MPNPs treatment can more effectively relieve joint pain in OA rats, indicating the effectiveness of the nanoparticles of the present invention in the treatment of OA.

Claims

1. Use of the combination of hyaluronic acid-bilirubin conjugate and honokiol in the preparation of a medicament for treating osteoarthritis.

2. The use according to claim 1, characterized in that: The weight ratio of the hyaluronic acid-bilirubin conjugate to honokiol is: 10:

1.

3. A honokiol-loaded nanoparticle, characterized in that: It is prepared by using the hyaluronic acid-bilirubin conjugate and honokiol as raw materials to prepare nanoparticles HKL-PNPs, and then modifying them with a mitochondrial targeting peptide, that is, nanoparticles HKL-MPNPs loaded with honokiol are obtained.

4. The honokiol-loaded nanoparticles according to claim 3, wherein: The weight ratio of the hyaluronic acid-bilirubin conjugate to honokiol is: 10:

1.

5. A method for preparing the honokiol-loaded nanoparticles according to claim 3 or 4, characterized in that: It comprises the following steps: a. Prepare the hyaluronic acid-bilirubin conjugate; Dissolve bilirubin, carbodiimide hydrochloride and N-hydroxysuccinimide in dimethyl sulfoxide, stir and react magnetically at 30 °C, then add 31.4 mg of adipic dihydrazide (ADH) and continue to react for 12 h; dissolve hyaluronic acid, carbodiimide hydrochloride and N-hydroxysuccinimide in dimethyl sulfoxide, stir and react magnetically at 30 °C; mix the above two reaction solutions, stir and react magnetically at 30 °C for 12 h, protect the whole reaction system by introducing nitrogen, after the reaction is completed, dialyze in water to remove organic reagents, and then remove the precipitate by centrifugation (centrifuge at 10,000×g for 20 min), and the supernatant is freeze-dried to obtain bilirubin-modified hyaluronic acid (HA-BR); b. Prepare nanoparticles HKL-PNPs loaded with honokiol: Dissolve phospholipid E8, Kolliphor HS-15, MAL-PEG 2000 -DSPE and honokiol in dichloromethane, remove the organic reagent by a rotary evaporator to obtain a lipid film, then hydrate and remove the film with an aqueous solution containing HA-BR, then probe sonicate in an ice-water bath, and remove the unencapsulated honokiol by ultrafiltration to obtain honokiol-loaded nanoparticles (HKL-PNPs); c. Modification with mitochondrial targeting peptide: Stir and react HKL-PNPs with a cysteine-modified mitochondrial targeting peptide (amino acid sequence: Cys-Fx-r-Fx-k-Fx-r-Fx-k) at room temperature to obtain nanoparticles HKL-MPNPs loaded with honokiol modified with a mitochondrial targeting peptide.

6. A biomimetic nanoparticle for treating bone and joints, characterized in that: It is prepared by wrapping the nanoparticles HKL-MPNPs loaded with honokiol described in claim 3 or 4 with engineered chondrocyte membranes (ECM).

7. The biomimetic nanoparticles for treating bone and joint according to claim 6, characterized in that: The mass ratio of the engineered chondrocyte membranes (ECM) to the nanoparticles HKL-MPNPs loaded with honokiol is: 1:50 - 10:

1.

8. The biomimetic nanoparticles for treating bone and joint according to claim 6 or 7, characterized in that: The preparation method of the engineered chondrocyte membranes (ECM) is as follows: After mixing and transfecting recombinant adenovirus overexpressing mouse FLAG-tagged IL-1R2 plasmid with chondrocytes for 12 h, add fresh medium, and collect engineered chondrocytes after culturing for 72 h; resuspend the engineered chondrocytes in a separation buffer containing 225 mM mannitol, 30 mM Tris-HCl, 75 mM sucrose, 0.5% (w / v) bovine serum albumin, 0.5 mM ethylenediaminetetraacetic acid, and a protease and phosphatase inhibitor mixture; after sonication, centrifuge at 10,000×g at 4 °C for 15 min; then ultracentrifuge the supernatant at 100,000×g at 4 °C for 150 min, and lyophilize the obtained precipitate to obtain engineered chondrocyte membranes (ECM).

9. A method for preparing the bionic nanoparticles for treating bone and joint according to claim 7 or 8, characterized in that: It comprises the following steps: a. Take the engineered chondrocyte membranes (ECM) and nanoparticles HKL-MPNPs loaded with honokiol, and mix them; b. Sonicate in an ice-water bath for 10 min to obtain biomimetic nanoparticles (HKL-ECM@MPNPs) encapsulated by ECM.

10. Use of the honokiol-loaded nanoparticles according to claim 3 or 4 and the biomimetic nanoparticles according to any one of claims 6-8 in the preparation of a drug for treating bone and joints, wherein the drug is for intra-articular injection.

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