Pharmaceutical preparation of macrophage-derived exosome loaded aconite decoction extract, preparation method and application thereof in preparation of drugs for preventing and treating osteoarthritis

Abstract

The present application relates to the fields of medicine, modernization of traditional Chinese medicine and drug delivery, and specifically relates to a macrophage-derived exosome loaded with aconite decoction extract, a preparation method thereof and application thereof in preparation of a drug for preventing and treating osteoarthritis. The effective component of the drug preparation is macrophage-derived exosome loaded with aconite decoction extract, which includes low-loading preparation WTDL-Exos and high-loading preparation WTDH-Exos and contains pharmaceutically acceptable excipients. The macrophage-derived exosome is preferably derived from mouse macrophage RAW264.7 and is extracted through differential centrifugation and ultracentrifugation; the aconite decoction extract is prepared from prepared monkshood, astragalus, honeyed licorice, ephedra and peony, and the concentration of the aconite decoction mother liquor is 10 mg / mL; the drug-loaded exosome is obtained by co-incubation of the aconite decoction extract and the macrophage-derived exosome and purification, and the loading rate is about 20%. In vitro tests show that WTD-Exos has no obvious effect on cell survival rate and is basically non-cytotoxic. Western blot detection in animal experiments shows that the drug preparation can down-regulate the expression of MMP-13 and TNF-α in synovial tissue and up-regulate the expression of type II collagen, thereby improving joint synovial inflammation and protecting joint cartilage. The drug preparation can be used for preparing a drug for preventing and treating osteoarthritis.

Description

Technical Field

[0001] This invention relates to the fields of pharmaceutical technology, modernization of traditional Chinese medicine technology, and biopharmaceutical delivery technology, specifically to a pharmaceutical preparation of aconite decoction extract loaded on macrophage-derived exosomes, its preparation method, and its application in the preparation of drugs for the prevention and treatment of osteoarthritis. Background Technology

[0002] Osteoarthritis (OA) is one of the most common chronic bone and joint diseases. Its main pathological processes include abnormal activation of synovial macrophages, proliferation and invasion of synovial fibroblasts, and progressive degradation of cartilage. In severe cases, it can lead to joint pain, limited mobility, and a decline in quality of life.

[0003] Aconite Decoction, originating from the *Synopsis of Prescriptions of the Golden Chamber*, is a classic formula for treating cold-dampness obstruction and joint pain. Composed of prepared aconite root, astragalus root, peony root, licorice root, and ephedra, it possesses good anti-inflammatory and analgesic effects. However, the prepared aconite root contains components such as diester-type aconitine, and traditional systemic administration methods such as oral administration or gavage carry risks of a narrow therapeutic window and cardiac, hepatic, and nephrotoxic side effects. Furthermore, the low bioavailability of traditional decoctions at the joint site also limits its application to some extent.

[0004] Exosomes (Exos) are natural nanoscale membrane vesicles with good biocompatibility, low immunogenicity, and certain delivery advantages. Macrophages are widely involved in the inflammatory response of osteoarthritis, and their derived exosomes can serve as delivery carriers for active ingredients in traditional Chinese medicine, improving the efficiency of local joint administration and reducing the risk of systemic exposure. Therefore, developing a novel pharmaceutical formulation using macrophage-derived exosomes to load aconite root extract is of great significance. Summary of the Invention

[0005] I. Purpose of the Invention: The purpose of this invention is to provide a pharmaceutical preparation of aconite decoction extract loaded on macrophage-derived exosomes, its preparation method, and its application in the preparation of drugs for the prevention and treatment of osteoarthritis, so as to improve the problems of large systemic toxic side effects and low local bioavailability of aconite decoction in traditional administration methods.

[0006] II. Technical Solution:

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] S1. Extraction of macrophage exosomes: RAW264.7 macrophages were cultured until they reached 80%-100% confluence. The culture medium was then replaced with serum-free medium and cultured for another 18-36 hours. The conditioned medium was collected and centrifuged at 4°C at 2000×g (10 min) and 10000×g (30 min) to remove cell debris. The supernatant was then ultracentrifuged at 100000-120000×g for 70 min. The precipitate was resuspended in PBS and purified by ultracentrifugation again to obtain the exosomes.

[0009] S2. Preparation of Aconitum Decoction Mother Liquid: The prepared Aconitum carmichaelii (6g), Astragalus membranaceus (9g), Glycyrrhiza uralensis (9g), Ephedra sinica (9g), and Paeonia lactiflora (9g) were used as the base. The prepared Aconitum carmichaelii was soaked alone for 1 hour and decocted for 1 hour. Then, the remaining herbs were added, and 8 times the volume of distilled water was added, followed by boiling for 1.5 hours. The mixture was then filtered. The residue was boiled again with 8 times the volume of distilled water for 1 hour. The two filtrates were combined, allowed to stand, and then filtered again. The filtrate was cooled and freeze-dried into powder. The frozen powder was dissolved in distilled water to prepare a 10mg / ml Aconitum decoction mother liquid.

[0010] S3. Co-incubation and loading: The exosome suspension and aconite decoction mother liquor were mixed in an appropriate ratio and co-incubated at 37°C to achieve drug loading using passive loading. Free drug was then removed by ultrafiltration and centrifugation to obtain a purified exosome suspension loaded with aconite decoction extract (WTD-Exos), which was stored at -80°C. HPLC analysis showed that the loading rate of WTD-Exos was approximately 20%.

[0011] In another aspect, the present invention provides a pharmaceutical composition / formulation comprising WTD-Exos obtained by the above-described preparation method, and pharmaceutically acceptable excipients. The dosage form may be an injection or a lyophilized powder for injection.

[0012] When preparing the injection, the WTD-Exos suspension is diluted with physiological saline to make the concentration of the active ingredient 2.5-40 nmol / L, and then filtered through a 0.22 μm sterile filter membrane.

[0013] When preparing lyophilized powder injections, mannitol or trehalose with a mass concentration of 5%-10% is added to the suspension as a lyophilization protectant, and the mixture is pre-frozen, freeze-dried, and then packaged.

[0014] III. Beneficial Effects:

[0015] 1. Using macrophage-derived exosomes as carriers helps improve the delivery efficiency of aconite extract at the joint site and reduce the risk of systemic exposure.

[0016] 2. The formulation can exert its effects on multiple levels, including regulation of the inflammatory microenvironment, synovial protection, and cartilage protection.

[0017] 3. The formulation helps to downregulate the expression of inflammatory factors and matrix metalloproteinases, improve synovial inflammation, and reduce cartilage degeneration and bone destruction.

[0018] 4. Cytotoxicity assays showed that WTD-Exos had comparable target cell survival rates to the control group, exhibited minimal cytotoxicity, and demonstrated good safety.

[0019] 5. The preparation process of the formulation is controllable and has good repeatability, which facilitates large-scale production. Detailed Implementation

[0020] The present invention will be further illustrated below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0021] Example 1: Extraction and Identification of Macrophage-Derived Exosomes

[0022] 1. Cell Culture

[0023] RAW264.7 macrophages were seeded in 10cm culture dishes and placed in DMEM high-glucose medium containing 10% exosome-free fetal bovine serum and 1% penicillin-streptomycin. The dishes were then incubated at 37°C, 5% CO2, and saturated humidity, and passaged every 2–3 days. When cell confluence reached approximately 90%, the original medium was discarded, and the cells were gently washed three times with sterile PBS. The medium was then replaced with serum- and antibiotic-free DMEM, and the cells were cultured for another 24 hours.

[0024] 2. Exosome extraction

[0025] Collect the cell supernatant after 24 hours of culture and place it in a 50 mL centrifuge tube. Centrifuge at 2000×g for 10 min at 4°C to remove cells and large particulate impurities. Transfer the supernatant to a new centrifuge tube and centrifuge at 10000×g for 30 min at 4°C to remove cell debris. Transfer the supernatant to an ultracentrifuge tube and centrifuge at 120000×g for 70 min at 4°C. Discard the supernatant and resuspend the precipitate in 1 mL of pre-cooled PBS. Centrifuge again at 120000×g for 70 min at 4°C to purify the precipitate. Discard the supernatant and resuspend the precipitate in 200 μL of PBS. The resulting exosomes are derived from RAW264.7 macrophages. Aliquot and store at -80°C for later use.

[0026] 3. Exosome identification

[0027] (1) Transmission electron microscopy (TEM) observation: 20 μL of exosome suspension was dropped onto a 200-mesh carbon film copper grid and allowed to stand at room temperature for 5 min. After absorbing the excess liquid with filter paper, it was counterstained with 3% phosphotungstic acid (pH 7.4) for 5 min. The staining solution was then absorbed with filter paper, and the mixture was allowed to air dry at room temperature before being observed under a transmission electron microscope. The results showed that the exosomes had typical cup-shaped or saucer-shaped structures with a diameter of about 30–150 nm.

[0028] (2) Nanoparticle tracking analysis (NTA): Exosome suspension was diluted 10-fold with sterile water and injected into a Nanosight NS300 system for detection. The results showed that the exosome particle size was mainly distributed between 30 and 150 nm. (3) Western blot detection: Exosome proteins were extracted and quantified by BCA method. 10 μg of protein sample was separated by 10% SDS-PAGE electrophoresis, wet-transferred to a PVDF membrane, blocked with 5% skim milk powder at room temperature for 1 h, and then incubated overnight at 4℃ with CD9, CD63, and TSG101 primary antibodies (1:1000); after washing the membrane 3 times with TBST, it was incubated with HRP-labeled secondary antibody (1:5000) at room temperature for 1 h; ECL color development and gel imaging analysis were performed. The results showed that CD9, CD63, and TSG101 were positive, confirming that the obtained exosomes met the typical characterization characteristics.

[0029] Example 2: Preparation of Aconitum carmichaelii extract mother liquor and drug-loaded exosomes

[0030] 1. Preparation of Aconitum carmichaelii extract mother liquor

[0031] The formula consists of 6g of prepared aconite root, 9g of astragalus root, 9g of prepared licorice root, 9g of ephedra root, and 9g of peony root, with the prepared aconite root decocted first. The prepared aconite root is soaked for 1 hour and then decocted for 1 hour. The remaining herbs are then added, and the mixture is boiled for 1.5 hours with 8 times its volume of distilled water, followed by filtration. The residue is then boiled again with 8 times its volume of distilled water for 1 hour. The two filtrates are combined, allowed to stand, filtered, and freeze-dried into powder. After reconstitution, a mother liquor of aconite root extract with a concentration of 10 mg / mL is obtained.

[0032] 2. HPLC quality control

[0033] High-performance liquid chromatography (HPLC) was used to detect key components (such as benzoyl aconitine, astragaloside A, paeoniflorin, and glycyrrhizic acid) in aconite decoction extract, and fingerprint chromatograms were established to ensure batch-to-batch stability. Chromatographic conditions: C18 column (4.6 mm × 250 mm, 5 μm), mobile phase gradient elution of acetonitrile-0.1% phosphoric acid aqueous solution, flow rate 1.0 mL / min, column temperature 30℃, detection wavelength 230 nm.

[0034] 3. Preparation of drug-loaded exosomes

[0035] Take an appropriate amount of Aconitum carmichaelii extract mother liquor and the exosome suspension obtained in Example 1, mix them in a suitable ratio, and incubate at 37°C with constant shaking for 2 hours to allow the Aconitum carmichaelii extract to be passively loaded into the exosomes. After incubation, remove the free drug by ultrafiltration centrifugation (molecular weight cutoff 100kDa, 4°C, 4000×g centrifugation for 20 min) or ultracentrifugation (120000×g, 4°C, 70 min). The resulting drug-loaded exosomes are WTD-Exos.

[0036] 4. Loading rate determination

[0037] The content of unloaded free drug was determined by HPLC and calculated using the following formula:

[0038] Loading rate (%) = (Total dosage - Free dosage) / Total dosage × 100%

[0039] The results showed that the loading rate of WTD-Exos was approximately 20%.

[0040] The formulation obtained by loading at the base loading volume is designated as the low-loading group WTDL-Exos; the formulation obtained by loading at 10 times the base loading volume is designated as the high-loading group WTDH-Exos.

[0041] Example 3: Preparation of Drug Dosage Forms

[0042] 1. Preparation of injection: Dilute WTDL-Exos or WTDH-Exos suspension with sterile physiological saline or 5% glucose solution to a suitable concentration; filter sterilize through a 0.22μm sterile polyethersulfone filter membrane, and dispense under sterile conditions into 2mL ampoules and seal.

[0043] 2. Preparation of lyophilized powder for injection: Add 5%–10% mannitol, sucrose, or trehalose as a lyophilization protectant to the drug-loaded exosome suspension. After thorough mixing, dispense 1 mL into 2 mL vials. Place the vials in a freeze dryer, pre-freeze at -40°C for 2 hours, vacuum dry for 12 hours, stopper and cap to obtain the lyophilized powder for injection. Reconstitute with 1 mL of sterile water for injection before use.

[0044] Example 4: In vitro cell biological effects

[0045] 4.1 Cell grouping

[0046] Cell experiments were conducted using a blank control group, a control group, an exosome group, a low-power aconite decoction group (1x, WTDL-Exos), and a high-power aconite decoction group (10x, WTDH-Exos). The blank control group received no inducing factors or test samples; the control group received inducing factors but no test samples; the exosome group received blank exosomes; the low-power aconite decoction group received WTDL-Exos; and the high-power aconite decoction group received WTDH-Exos.

[0047] 4.2 Cytotoxicity Detection

[0048] Synovial macrophages were used at a rate of 5 × 10 3Cells were seeded per well in 96-well plates and cultured for 24 hours until adherence. Then, the cells were subjected to the different treatments described above and cultured for 12, 24, 48, and 72 hours respectively. 10 μL of LCK-8 solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance was measured at 450 nm using a microplate reader, and cell viability was calculated using the following formula:

[0049] Cell viability (%) = (Experimental group OD - Blank group OD) / (Control group OD - Blank group OD) × 100%

[0050] The results showed that the cell viability of the WTDL-Exos group and the WTDH-Exos group at each time point was comparable to that of the control group, with no statistically significant difference (P>0.05), indicating that the drug-loaded exosomes were essentially non-cytotoxic and had good biocompatibility.

[0051] 4.3 Macrophage polarization regulation

[0052] RAW264.7 macrophages were induced to polarize towards the M1 phenotype using IFN-γ (20 ng / mL) and / or LPS (100 ng / mL), and then co-cultured with various other samples for 24 h. Flow cytometry was used to detect the expression of surface markers CD86, CD80, CD206, and CD163; qRT-PCR and Western blot were used to detect the expression of IL-1β, IL-6, iNOS, IL-10, TGF-β, and Arg-1. The results showed that the drug-loaded exosome treatment group reduced the expression of M1-related markers CD86, CD80, iNOS, IL-1β, and IL-6, and increased the expression of M2-related markers CD206, CD163, Arg-1, IL-10, and TGF-β, suggesting that WTD-Exos can regulate the M1 / M2 polarization balance of macrophages.

[0053] 4.4 Evaluation of co-culture of synovial fibroblasts and chondrocytes

[0054] A Transwell co-culture system was used, with induced and activated macrophages placed in the upper chamber and synovial fibroblasts or chondrocytes placed in the lower chamber, and co-cultured for 48 h. Cell proliferation was detected by CCK-8 assay, inflammatory factors in the supernatant were detected by ELISA, and the expression of relevant genes and proteins was detected by qRT-PCR and Western blot. The results showed:

[0055] (1) The drug-loaded exosome treatment group can inhibit the proliferation of synovial fibroblasts and reduce the expression of TNF-α, IL-6, IL-8 and MMP-1, MMP-3 and MMP-13;

[0056] (2) The drug-loaded exosome treatment group can reduce chondrocyte apoptosis and downregulate the expression of related indicators such as MMP-1, MMP-10, MMP-13, TNF-α, IL-1β, and IL-6.

[0057] Example 5: In vivo therapeutic effect in an osteoarthritis model

[0058] 5.1 Animal grouping

[0059] Sixty SPF-grade male SD rats (weighing 300±10g) were selected to establish an osteoarthritis (OA) model. The animal experiments were divided into four groups: blank group, control group, exosome group, aconite decoction gavage group, low-power aconite decoction group (1x, WTDL-Exos), and high-power aconite decoction group (10x, WTDH-Exos). The blank group received no modeling and no drug intervention; the control group received an equal volume of physiological saline after modeling; the exosome group received intra-articular injection of blank exosomes; the aconite decoction gavage group received aconite decoction gavage; the low-power aconite decoction group received intra-articular injection of WTDL-Exos; and the high-power aconite decoction group received intra-articular injection of WTDH-Exos.

[0060] 5.2 Model Establishment and Drug Administration

[0061] A model of osteoarthritis was established using the Hulth method: Rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital. The skin of the right hind limb knee joint area was prepared and disinfected. A longitudinal incision of approximately 1 cm was made on the medial side of the patella, the medial collateral ligament and anterior cruciate ligament were severed, the medial meniscus was removed, and the joint capsule and skin were sutured layer by layer. Postoperatively, penicillin was administered intramuscularly for 3 days to prevent infection. After successful model establishment, rats were grouped as described above and given corresponding interventions, administered twice weekly for 4 consecutive weeks. Behavioral observations, imaging examinations, and tissue sampling were conducted at specified time points.

[0062] 5.3 Imaging and Histological Evaluation

[0063] At the experimental endpoint, micro-CT scans were performed to analyze parameters such as joint space, bone volume fraction (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp). After sampling, synovial membrane was stained with HE, cartilage with Safranin O-Fixel, TRAP, and TUNEL staining. Results showed that synovial inflammation, cartilage degeneration, and bone destruction were significantly improved in the WTDL-Exos and WTDH-Exos groups compared to the control group. Micro-CT results indicated improvements in joint space, bone volume fraction, and trabecular structure compared to the control group.

[0064] 5.4 Molecular biological evaluation

[0065] Synovial tissue of the knee joint of rats in each group was taken, and pre-cooled RIPA lysis buffer (containing 1% PMSF and phosphatase inhibitor) was added. After homogenization on ice, the tissue was centrifuged at 12000×g for 15 min at 4℃ and the supernatant was collected. The protein concentration was determined by BCA method. 30 μg of protein sample was added to 5× loading buffer and boiled in water for 5 min for denaturation. After separation by 10% SDS-PAGE electrophoresis, the membrane was wet-transferred to a PVDF membrane and blocked with 5% skim milk powder at room temperature for 1 h. It was then incubated overnight at 4°C with primary antibodies against type II collagen (Collagen II), matrix metalloproteinase 13 (MMP-13), and tumor necrosis factor-α (TNF-α) (1:1000 dilution). The membrane was washed three times with TBST for 10 min each time. HRP-labeled secondary antibody (1:5000 dilution) was added and incubated at room temperature for 1 h, followed by three washes with TBST. ECL chromogenic solution was used for development, and images were acquired using a gel imaging system. β-actin or GAPDH was used as an internal control, and grayscale analysis was performed using ImageJ software.

[0066] The results showed that, compared with the control group, the protein expression levels of MMP-13 and TNF-α in the synovial tissue of the WTDL-Exos group and the WTDH-Exos group were significantly decreased, while the protein expression level of type II collagen was significantly increased (P<0.05 or P<0.01). Moreover, the improvement effect of the WTDH-Exos group was better than that of the WTDL-Exos group and the aconite decoction gavage group, suggesting that the drug-loaded exosomes can effectively inhibit the expression of matrix metalloproteinases and inflammatory factors in synovial tissue and promote the synthesis of type II collagen, thereby playing a role in protecting articular cartilage and reducing synovial inflammation.

[0067] Example 6: System Security Evaluation

[0068] At the experimental endpoint, whole blood and major organs of rats were collected to detect ALT, AST, BUN, Cre, and myocardial enzyme levels. HE staining was also performed on heart, liver, and kidney tissues for observation. Results showed that the related biochemical indicators and histopathological changes in the drug-loaded exosome group were milder than those in the aconite decoction gavage group, suggesting that using macrophage-derived exosomes to deliver aconite decoction extract helps reduce the risk of systemic toxicity.

Claims

1. A pharmaceutical preparation, characterized in that, The active ingredient of the pharmaceutical preparation is aconite extract (WTD-Exos) carried on macrophage-derived exosomes, and contains pharmaceutically acceptable excipients.

2. The pharmaceutical preparation according to claim 1, characterized in that, The macrophage-derived exosomes were derived from the mouse macrophage cell line RAW264.

7.

3. The pharmaceutical preparation according to claim 1 or 2, characterized in that, The aconite decoction extract is prepared from the following raw materials: 6g of prepared aconite root, 9g of astragalus root, 9g of prepared licorice root, 9g of ephedra, and 9g of peony root. The concentration of the aconite decoction extract mother liquor is 10mg / mL. This mother liquor is used to prepare the macrophage-derived exosome-loaded aconite decoction extract.

4. The pharmaceutical preparation according to any one of claims 1 to 3, characterized in that, The drug formulation includes a low-load formulation and a high-load formulation, wherein the low-load formulation is denoted as WTDL-Exos and the high-load formulation is denoted as WTDH-Exos; the low-load formulation is loaded at the basic loading amount, and the high-load formulation is loaded at 10 times the basic loading amount; the loading rate of the drug-loaded exosomes is about 20%.

5. The pharmaceutical preparation according to any one of claims 1 to 4, characterized in that, The pharmaceutically acceptable excipients include one or more of diluents and lyophilization protectants; the diluent is physiological saline or a 5% glucose solution by mass; the lyophilization protectant is mannitol, sucrose, or trehalose, and its mass fraction in the formulation is 5% to 10%; the dosage form of the pharmaceutical formulation is an injection, a suspension, or a lyophilized powder for injection.

6. A method for preparing a pharmaceutical preparation according to any one of claims 1 to 5, characterized in that, Includes the following steps: S1. Extraction of macrophage-derived exosomes; S2. Prepare the mother liquor of Aconitum carmichaelii extract; S3. The aconite decoction extract stock solution is co-incubated with the macrophage-derived exosomes for drug loading; S4. Remove free drug to obtain macrophage-derived exosome-loaded aconite decoction extract.

7. The preparation method according to claim 6, characterized in that, Step S1 includes: culturing RAW264.7 macrophages to 80%–100% confluence, then replacing the medium with serum-free medium and culturing for another 18–36 h; collecting the cell supernatant and centrifuging it sequentially at 2000×g for 10 min and 10000×g for 30 min at 4°C to remove cells and debris; taking the supernatant and ultracentrifuging it at 100000–120000×g for 60–90 min, collecting the precipitate, resuspending it in pre-cooled PBS, and purifying it again by ultracentrifugation under the same conditions to obtain macrophage-derived exosomes.

8. The preparation method according to claim 6 or 7, characterized in that, Step S2 includes: soaking prepared Aconitum carmichaelii in purified water for 1 hour and decocting for 1 hour; then adding Astragalus membranaceus, prepared Glycyrrhiza uralensis, Ephedra sinica and Paeonia lactiflora, adding 8 times the volume of distilled water, boiling for 1.5 hours and filtering; adding 8 times the volume of distilled water to the residue and boiling for 1 hour; combining the two filtrates, letting stand, filtering, freeze-drying into powder, and then dissolving in distilled water to obtain Aconitum carmichaelii extract mother liquor with a concentration of 10 mg / mL.

9. The preparation method according to any one of claims 6 to 8, characterized in that, In step S3, drug loading was performed using a co-incubation method. The exosome suspension and the mother liquor of aconite decoction extract were mixed in an appropriate ratio and co-incubated at 37°C. In step S4, free drugs were removed by ultrafiltration centrifugation and / or ultracentrifugation. High performance liquid chromatography was used for quality control of aconite decoction extract and to detect the loading rate of drug-loaded exosomes. The results showed that the drug loading rate was approximately 20%.

10. The use of the pharmaceutical preparation according to any one of claims 1 to 5 in the preparation of a medicament for the prevention and treatment of osteoarthritis, characterized in that, The pharmaceutical preparation is used for local administration to the joint, preferably by intra-articular injection; the pharmaceutical preparation is used to regulate the M1 / M2 polarization balance of synovial macrophages, inhibit the activation of synovial fibroblasts, downregulate the expression of inflammatory factors and matrix metalloproteinases, thereby improving synovial inflammation and protecting articular cartilage.