Use of exosome-like nanoparticles from fructus aurantii immaturus in preparation of drugs for treating osteoarthritis

Abstract

The application discloses application of fructus aurantii exosome-like nanoparticles in preparation of drugs for treating osteoarthritis. The fructus aurantii exosome-like nanoparticles are prepared by differential centrifugation and ultracentrifugation, and toxicological evaluation shows that the fructus aurantii exosome-like nanoparticles have good biological safety. In vivo experiments show that the osteoarthritis model mice treated by the fructus aurantii exosome-like nanoparticles. In vitro experiments verify that the fructus aurantii exosome-like nanoparticles can play a good anti-inflammatory and cartilage protection role by significantly reducing the expression level of local inflammatory factors and matrix degradation enzymes of osteoarthritis mice.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of citrus osmotic nanoparticles in the preparation of drugs for treating osteoarthritis. Background Technology

[0002] Osteoarthritis (OA) is a common degenerative joint disease characterized by cartilage degeneration and inflammation. Clinically, it is mainly characterized by the gradual degeneration of joint tissues, often presenting with joint pain, stiffness, and limited mobility in the early stages, and deformities in later stages. The pathological changes in osteoarthritis are mainly caused by the interaction of multiple factors, including degeneration of articular cartilage matrix, chronic inflammation of joint tissue structures, extracellular matrix (ECM) degradation, abnormal subchondral bone metabolism, and osteophyte formation. This complex pathophysiological process ultimately leads to joint dysfunction and progressive disability, seriously affecting patients' physical health and quality of life, and causing a huge socioeconomic burden. Currently, the etiology of osteoarthritis is complex, including multiple factors such as aging, biomechanics, increased joint friction, and genetic susceptibility. The incidence of osteoarthritis is closely related to age. In clinical practice, the treatment of osteoarthritis mainly aims to relieve symptoms, often relying on nonsteroidal anti-inflammatory drugs (NSAIDs) for temporary pain relief, but these drugs have many serious side effects, such as the risk of gastrointestinal bleeding and cardiovascular events. Although endoscopic prosthetic joint replacement is considered the authoritative treatment for end-stage osteoarthritis (OA), it still faces limitations in resource-constrained settings due to surgical complications, prosthesis durability issues, and cost constraints. In summary, existing OA treatments can only provide relief and cannot promote the regeneration of damaged articular cartilage or reverse disease progression to achieve a cure. These limited traditional treatment methods underscore the necessity for developing novel strategies for the prevention and treatment of OA. Plant-derived exosome-like nanoparticles (PDENs) have become an emerging and highly anticipated research hotspot in the interdisciplinary field of biomedicine and nanotechnology in recent years. These nanoparticles are naturally derived from various plant tissues and are rich in diverse functional bioactive components, including proteins, nucleic acids, lipids, and plant secondary metabolites. Compared with synthetic carriers, plant exosome-like nanoparticles exhibit many significant advantages, such as low immunogenicity, excellent biocompatibility, low toxicity, and superior ability to penetrate biological barriers. These properties make them highly promising for the construction of drug delivery systems and the development of precision disease treatment strategies, and have become one of the important directions in the research of novel targeted therapies and biomimetic nanomedical materials.

[0003] Huajuhong ( Exocarpium Citri grandis, ECgrandis) It is a plant of the Rutaceae family, the immature or nearly mature dried outer pericarp of Citrus grandis 'Tomentosa' from Huazhou, a well-known genuine medicinal material, which has the effects of regulating qi and relieving distension, drying dampness and resolving phlegm, and is commonly used to treat cough with excessive phlegm, food accumulation and alcohol injury, etc. Modern pharmacological studies have shown that it has activities such as anti-inflammatory, antioxidant, antibacterial, etc. However, so far, there have been no relevant reports on extracting exosome-like nanoparticles from Citrus grandis 'Tomentosa' and using them for the treatment of osteoarthritis. Summary of the Invention

[0004] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide the application of exosome-like nanoparticles from Citrus grandis 'Tomentosa' in osteoarthritis, so as to fill the blank in the existing technology.

[0005] To achieve the above purpose, the present invention provides the following technical solution: the application of exosome-like nanoparticles from Citrus grandis 'Tomentosa' in the preparation of drugs for the treatment of osteoarthritis.

[0006] Preferably, the extraction method of the exosome-like nanoparticles from Citrus grandis 'Tomentosa' is obtained by homogenizing the pericarp of Citrus grandis 'Tomentosa', followed by centrifugation, filtration, resuspension, and then filtering to remove bacteria.

[0007] Preferably, the centrifugation includes first centrifuging at 500 - 700×g for 10 - 15 minutes, 2 - 3 times, then taking the supernatant, centrifuging at 1000 - 3000×g for 25 - 35 minutes, again sucking the supernatant, discarding the precipitate, and centrifuging at 120000 - 150000×g for 120 - 150 minutes.

[0008] Preferably, the particle size of the exosome-like nanoparticles from Citrus grandis 'Tomentosa' is 140 - 160 nm. <000​​​​​​​​​​​​​​​​Another aspect of the present invention provides a pharmaceutical composition comprising the above-described medicament for treating and improving osteoarthritis.

[0015] Compared with existing technologies, the beneficial effects of this invention are: This invention provides the application of *Citrus tanguticulatus* oozosome-like nanoparticles in the treatment of osteoarthritis. This invention prepares *Citrus tanguticulatus* oozosome-like nanoparticles using differential centrifugation and ultracentrifugation methods. Toxicological evaluation shows that these nanoparticles have good safety and can be used for in vitro and in vivo treatment. In vitro experiments verify that *Citrus tanguticulatus* oozosome-like nanovesicles can exert good anti-inflammatory and chondrogenic effects by significantly downregulating the expression levels of local inflammatory factors and matrix-degrading enzymes in osteoarthritis mice. Attached Figure Description

[0016] Figure 1 The flowchart and characterization diagram of the extraction of ENVs in this invention are shown below. Figure 1 In the diagram, A represents the preparation process; B is a TEM image (scale bar = 100 nm); and C is the NTA particle size distribution.

[0017] Figure 2 This is a flowchart of lipid information analysis in Embodiment 1 of the present invention.

[0018] Figure 3 ENVs proteins (30 mg / mL) were isolated using 10% SDS-PAGE gel and detected by Coomassie brilliant blue staining.

[0019] Figure 4 The results of the in vitro toxicity test of Citrus reticulata secoirs-like nanovesicles are as follows: the cytotoxicity of different concentrations of Citrus reticulata secoirs-like nanovesicles on RAW264.7 cells.

[0020] Figure 5 To investigate the therapeutic effect of citrus secoizosome-like nanovesicles on osteoarthritis in mice. Among them, Figure 4 In the table, A represents the expression level of the inflammatory cytokine IL-1β in the serum of mice with osteoarthritis; B represents the expression level of the inflammatory cytokine IL-6 in the serum of mice; and C represents the expression level of the matrix metalloproteinase MMP13 in the serum of mice. *p<0.05, **p<0.01, ***p<0.001, ****p<0.001.

[0021] Figure 6 To investigate the anti-inflammatory and protective effects of citrus oozoform vesicles on inflammatory SW3531 cells and maintain the balance between cartilage matrix degradation and synthesis. Figure 6In the table, A represents the detection of MMP13, a key biomarker of extracellular matrix catabolism, using ELISA; BE represents the detection of key genes and inflammatory factors in extracellular matrix catabolism using qPCR. *p<0.05, **p<0.01, ***p<0.001, ****p<0.001. Figure 7 This study aimed to detect key markers of extracellular matrix catabolism (MMPs) and inflammation-related proteins and anabolism in SW1353 chondrocytes using Western blotting. *p<0.05, **p<0.01, ***p<0.001, ****p<0.001. Detailed Implementation

[0022] Citrus reticulata peel is a known safe medicinal plant, and the nanoparticles derived from it are natural products with good biocompatibility and high safety. Toxicological evaluation (cytotoxicity test) shows that it did not show significant toxic side effects at effective doses, indicating excellent safety. Furthermore, the ectosome-like nanoparticles of Citrus reticulata peel can maintain their physical stability and biological activity for a long time at -80℃, which is beneficial for drug storage, transportation, and clinical application.

[0023] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0024] Statistical methods: All experimental data are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using GraphPadPrism 8.0 software. Student's t-test was used for comparisons between two groups, and one-way ANOVA, followed by Tukey's post hoc test, was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant.

[0025] Example 1: Extraction and characterization of citrus thrombosome-like nanoparticles I. Experimental Methods 1. Extraction of Exocarpium Citri grandis-derivednanoves (ENVs) (1) Raw material pretreatment: Pick fresh Huajuhong fruit, rinse it with running pure water to remove surface dust, dry the surface moisture with sterile filter paper, and then peel off the outer peel.

[0026] (2) Homogenization: Mix the above-mentioned Citrus reticulata peel with pre-cooled phosphate buffer (PBS, 0.01 M, pH 7.4) at a weight-to-volume ratio of 1:4 (g / mL), place it in a blender, and homogenize it thoroughly until a uniform slurry is formed.

[0027] (3) Low-speed centrifugation: Centrifuge the obtained slurry at 700×g for 10 min at 4℃, discard the precipitate, and repeat the operation; then centrifuge at 2000×g for 30 min, carefully aspirate the supernatant, and discard the precipitate (mainly tissue residue and unbroken cells).

[0028] (4) Medium speed centrifugation: Centrifuge the supernatant obtained in the previous step at 4°C at 10000×g for 40 minutes, and aspirate the supernatant again to discard the precipitate (mainly cell debris and larger organelles).

[0029] (5) Ultracentrifugation: Transfer the supernatant to an ultracentrifuge tube and centrifuge at 120000×g for 120 minutes at 4℃. Carefully discard the supernatant. The yellow-green precipitate obtained at the bottom of the tube is the citrus red mitochondrial nanoparticles.

[0030] (6) Resuspension: The above precipitate was gently resuspended with pre-cooled PBS and filtered through a 0.22 μm microporous membrane for sterilization.

[0031] (7) Storage: The final obtained citrus oozosome-like nanoparticle suspension was aliquoted into sterile EP tubes and could be stored at 4℃ for short-term use (within one week) or at -80℃ for long-term storage. The protein concentration was determined using a BCA protein quantification kit.

[0032] 2. Characterization and quality control of citrus osmotic nanoparticles (1) Morphological observation: 10 μL of nanoparticle suspension was dropped onto a copper grid, negatively stained (e.g., 2% phosphotungstic acid solution), and observed and photographed using a transmission electron microscope (TEM).

[0033] (2) Particle size distribution and concentration determination: The nanoparticle suspension after appropriate dilution with PBS was detected using a nanoparticle tracking analyzer (NTA) to analyze its particle size distribution range and particle concentration.

[0034] (3) Protein content determination and characterization Add 100 μL of RIPA lysis buffer containing PMSF to the ENVs sample, mix gently, and incubate at 4°C for 20 min for lysis. Collect the lysis buffer, centrifuge at 12000 rpm for 10 min at 4°C, and store the supernatant at -80°C for later use / prepare fresh for immediate use. Perform protein quantification using the BCA method: Prepare fresh BCA working solution according to the volume ratio of reagent A to reagent B of 50 based on the amount of standard and ENVs sample to be tested, and allow it to stand in the dark until ready for use. Add protein standards in gradients of 0, 2, 4, 6, 8, 12, 16, and 20 μL to a 96-well plate, and replenish with sterile PBS solution to a final volume of 20 μL. Add an equal volume of the target dilution sample to each well of the 96-well plate. Add 200 μL of freshly prepared BCA working solution to each standard well and sample well, gently shake to mix thoroughly, and incubate at room temperature or in a constant temperature incubator for 20-40 minutes. Measure the absorbance of the liquid in the well plate at a wavelength of 562 nm. Substitute the final result into the standard curve to obtain the amount of protein contained in the sample.

[0035] Detection of ENVs proteins by SDS-PAGE electrophoresis combined with Coomassie brilliant blue staining: When preparing 10% SDS-PAGE gel, the appropriate concentration of polyacrylamide gel should be prepared first, the coagulant should be added and quickly mixed, and the gel should be poured immediately to form a solid. When preparing samples, 5 × SDS loading buffer should be added to the protein stock solution and the total volume of the system should be made up with PBS. Then, the mixture should be boiled at 95°C for 10 minutes. When loading the samples, the loading amount of each protein sample is 20 μg and the loading amount of the protein molecular weight standard is 10 μL. During electrophoresis, the voltage is set to 80 V before the sample leaves the stacking gel. After the protein molecular weight standard enters the separating gel, the voltage is increased to 140 V. The power is turned off immediately when the indicator reaches the bottom of the gel to terminate the electrophoresis step. Then, Coomassie brilliant blue staining is performed. Before staining, the staining solution needs to be prepared in advance and heated to 100°C. The electrophoresed gel is placed in the staining solution and stained on a shaker for 15 to 20 minutes until the staining is uniform and thorough. For destaining, the destaining solution is first heated to 100°C. The stained gel is then transferred to the destaining solution and shaken on a shaker for 15 to 20 minutes. The old destaining solution is then discarded, and fresh destaining solution is added to continue destaining at room temperature. The destaining solution is replaced every 4 hours. After destaining is complete, the experimental results are observed and recorded on an imaging system.

[0036] (4) Lipidomics research The experiment utilized an LC-MS analysis platform to conduct relevant lipidomics studies on the samples. Samples underwent purification to remove proteins and interfering components, thereby enriching lipid metabolites. Subsequently, data were acquired in both positive and negative ion modes using a liquid chromatography-mass spectrometry (LC-MS) system to obtain mass spectrometry and tandem mass spectrometry information of the metabolites. Analytical tools were used to identify, align, and annotate chromatographic peaks, constructing a data matrix including retention times and identification results, and organizing it into a metabolite list and numerical matrix. Lipid metabolites with significant variations were screened using a T-test combined with VIP values ​​in the OPLS-DA model. Further customized analytical methods, such as correlation analysis, cluster heatmaps, and saturation assessment, were employed to delve into the biological functions and potential mechanisms of differentially expressed lipids.

[0037] (5) LC-MS plant non-target metabolomics Analysis flowchart as follows Figure 2 As shown.

[0038] II. Experimental Results Nanoparticles were extracted from the peel of 100g of fresh tangerine peel. Figure 1 The protein (A) was finally resuspended in 1 mL of PBS to obtain a yellow-green suspension, and its protein concentration was determined to be 30 mg / mL by the BCA method.

[0039] TEM observation of 10 μL of suspension showed that the extracted citrus erythropoietin nanoparticles were relatively uniform in size, spherical or saucer-shaped, and had a complete membrane, i.e., a complete lipid bilayer membrane structure. Figure 1 (B in the middle).

[0040] NTA analysis showed that the extracted citrus mitochondrial nanoparticles were mainly distributed in the range of 140-160 nm, with a peak particle size of 159.7 nm and an average particle size of approximately 153.5 nm. The particle size distribution was uniform, and the particle concentration was 6.2 × 10^10 particles / mL. Figure 1 (C in the middle).

[0041] SDS-PAGE electrophoresis and Coomassie brilliant blue staining revealed that ENVs contained a variety of protein substances with molecular weights ranging from 10 kDa to 180 kDa. Figure 3 Simultaneously, the protein concentration of ENVs was determined to be 30 mg / mL using the BCA method; furthermore, lipidomics analysis revealed that ENVs contained various lipid contents. These results indicate that ENVs contain proteins and lipids, exhibiting typical exosome-like characteristics.

[0042] The most common classification method currently is LIPID MAPS. The Lipid Metabolism Pathway Research Project (LIPID MAPS) classifies lipid metabolites into eight major categories: fatty acids (FA), glycerolipids (GL), glycerophospholipids (GP), sphingolipids (SP), sterol lipids (ST), prenol lipids (PR), glycolipids (SL), and polyketides (PK), which are further subdivided into 96 subcategories. A total of 374 lipids were detected from ENVs. The pie chart showing the lipid content distribution indicates that they mainly include five lipids: SP (36.10%), GL (29.41%), GP (27.27%), ST (6.15%), and FA, with SP having the highest content. Within each subcategory, GP had the highest number of identified lipids and the most lipid subcategory.

[0043] Example 2: In vitro safety evaluation of Citrus reticulata secois-like nanovesicles I. Experimental Methods In vitro safety evaluation: Cytotoxicity assays were performed using the mouse mononuclear macrophage cell line RAW264.7. Cells were seeded in 96-well plates and cultured for 24 h. The medium was then replaced with fresh medium containing different concentrations of citrus oozosome-like nanovesicles (0, 10, 20, 40, 80, 160 μg / mL), with four replicates for each concentration. After another 24 h of culture, CCK-8 reagent was added to each well, and after 1 hour of incubation, the absorbance at 450 nm (OD450) was measured using a microplate reader to calculate the relative cell viability. II. Experimental Results In vitro safety evaluation, such as Figure 4 As shown, compared with the blank control group, the cell survival rate of each treatment group was increased, and the difference was statistically significant. These results indicate that within this concentration range, *Citrus reticulata* oozosome-like nanovesicles have no significant toxicity to RAW264.7 cells; on the contrary, they can promote cell proliferation. Therefore, *Citrus reticulata* oozosome-like nanovesicles have good safety and can be used for in vitro and in vivo treatment.

[0044] Example 3: Therapeutic effect of Citrus reticulata oozosome-like nanoparticles on osteoarthritis model mice. I. Experimental Methods Healthy male C57BL / 6 mice aged 6-8 weeks (SPF grade) were selected as experimental animals, and an OA mouse model was constructed by induction with sodium iodoacetate (MIA).

[0045] After a week of acclimatization, the mice were randomly divided into two groups: a control group and a model group. All mice were raised under normal conditions with sufficient food and light. Solution preparation: Dissolve 32 mg of MIA in 1 mL of physiological saline to form MIA solution. Filter this solution through a 0.22-micron filter in a clean bench to ensure sterility, and store at 4°C for later use. Drug administration: Mice were anesthetized by intraperitoneal injection of 0.2 ml / 10 g tribromoethanol. After confirming the anesthesia effect, the injection site was approximately one-third of the way down the patellar ligament. This location could be probed with the needle tip, i.e., the needle was inserted vertically along the depression of the right knee joint. Each mouse was injected with 0.3 mg / 10 μL of MIA solution. A feeling of emptiness was felt when inserting the needle. Success was indicated by slowly withdrawing the needle without any blood seepage. The control group was injected with an equal volume of physiological saline. The day of drug administration was recorded as day 0 of the experiment. Modeling group grouping: OA mice that were successfully established for 1 week were treated with citrus erythropoietin-like nanovesicles for 4 weeks. The modeling group mice were further randomly divided into 3 subgroups: the model group, the low-dose treatment group (15 μg / ml), and the high-dose treatment group (30 μg / ml).

[0046] Drug administration: On days 7, 14, 21 and 28 of the experiment, mice were given intra-articular injection of citrus oozoline vesicles. The experiment was divided into four groups: blank group, model group, low-dose group and high-dose group, with 10 mice in each group. The mice in the drug administration group were injected with 10 μL of the corresponding dose of citrus oozoline vesicle nanovesicles into the right knee joint cavity, while the blank group and the model group were injected with an equal volume of physiological saline. Immediately after day 35 of treatment, osteoarthritis mice underwent tissue sampling, including blood collection from the eyeballs, and the heart, liver, spleen, lungs, and kidneys were quickly harvested. The internal organs were fixed in 4% paraformaldehyde, and the right knee joint was cut off, excess muscle was removed, and the right knee joint was fixed in 4% paraformaldehyde. Mouse serum separation: After blood was collected from mouse eyeballs and allowed to stand for 2 hours, the supernatant was centrifuged at 3000 rpm for 15 minutes at 4°C. The supernatant was collected and centrifuged again at 3000 rpm for 15 minutes at 4°C. The supernatant was then aliquoted and stored at -80°C. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum inflammatory factors: The expression levels of inflammatory factors IL-1β and IL-6 and the expression level of MMP13, a key enzyme in the degradation of extracellular matrix of chondrocytes, in mouse serum were detected according to the ELISA kit instructions. II. Experimental Results The results are as follows Figure 5As shown in Figures A, B, and C, the serum expression levels of inflammatory factors and matrix metalloproteinases in the osteoarthritis model mice were significantly elevated, indicating a significant local inflammatory response and cartilage degradation in the joints. Regarding inflammatory factors, the levels of IL-1β and IL-6 in the model group mice were significantly higher than those in the control group. After drug intervention, both low- and high-dose groups of *Citrus tangerine peel* oozosome-like nanovesicles significantly reduced the expression levels of these inflammatory factors, showing statistically significant differences compared to the model group. Furthermore, the high-dose group showed a further statistically significant reduction compared to the low-dose group. Regarding matrix metalloproteinases, the expression level of MMP13 in the model group mice was significantly higher than that in the control group, indicating active cartilage matrix degradation in osteoarthritis mice. After intervention with *Citrus tangerine peel* oozosome-like nanovesicles, MMP13 significantly decreased in the high-dose group, showing significant differences compared to the model group and significantly lower than the low-dose group.

[0047] Example 4: Protective and anti-inflammatory effects of Citrus reticulata oozosome-like nanoparticles on chondrocytes in vitro. I. Experimental Methods 1. Cell preparation This study selected the human chondrosarcoma cell line SW1353 because it provides a robust and reproducible in vitro model for studying the response of chondrocytes to OA-related stimuli. IL-1β (10 ng / mL) is widely considered a key pro-inflammatory cytokine involved in the pathogenesis of osteoarthritis; therefore, 10 ng / mL IL-1β was used to induce mouse chondrocytes to establish an in vitro osteoarthritis model. SW2353 cells were cultured in 6-well plates and co-incubated with (10 ng / mL) IL-1β and ENVs (40, 80 μg / mL) for 24 h.

[0048] 2. Extraction of RNA from chondrocytes Cells were processed according to the experimental groups, the supernatant was discarded, and the cells were washed with pre-cooled PBS; 1 mL TRIzol was added, the cells were lysed by pipetting, and the mixture was allowed to stand for 5 min; 200 μL chloroform was added, the mixture was mixed, and the cells were allowed to stand on ice; the cells were centrifuged at 12000×g at 4℃ for 15 min, and the upper aqueous phase was collected; an equal volume of isopropanol was added, the mixture was mixed, and the cells were allowed to stand on ice; the cells were centrifuged at 12000×g at 4℃ for 10 min, and the supernatant was discarded; the precipitate was washed with pre-cooled 75% ethanol, centrifuged at 7500g at 4℃ for 5 min, and the supernatant was discarded; the precipitate was dried, dissolved in enzyme-free water, and the concentration was measured before storage at -80℃.

[0049] 3. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) According to the kit instructions, total RNA was reverse transcribed into cDNA and aliquoted for storage. A 10 μL PCR reaction mixture was prepared on ice according to the instructions: 5 μL of 2x SYBR Green, 0.5 μL each of forward and reverse primers (10 μM), 1 μL of cDNA template, and 3 μL of enzyme-free water were added to an eight-tube strip, with three replicate wells per group. The reaction tubes were then placed in a QuantStudio 6Pro system, and the appropriate instrument parameters were set for amplification.

[0050] 4. Extraction of total protein from chondrocytes Discard the culture medium supernatant on ice, wash twice with pre-cooled PBS, and aspirate the liquid. Add 100 μL of RIPA lysis buffer and protease inhibitor (100:1) and lyse the cells on ice for 15 min to ensure complete cell lysis. Scrape the cell suspension with a spatula and transfer it to a centrifuge tube. Centrifuge at 4°C and 12,000 rpm for 15 min. Aspirate the supernatant to obtain the total protein. Aliquot and store at -80°C for subsequent BCA protein quantification.

[0051] 5. Western blot (WB) assay Gel preparation: Before gel preparation, clean and dry thin and thick glass plates, close the two plates to form an inner glass groove, place them in the clamp and fix them, and place them vertically on the gel preparation frame. According to the molecular weight of the sample protein, prepare a 10% lower separating gel and stacking gel according to the instructions. After pouring the gel, insert the comb and let it stand to solidify. Sample loading and electrophoresis: Boil the sample in a 95℃ metal bath for 5 minutes, add electrophoresis solution to the electrophoresis tank, remove the gel casting comb, use a pipette to draw the protein sample into the sample well, run the stacking gel at 80V constant voltage for 30 minutes, and run the separating gel at 100V constant voltage for 1 hour. Transfer: After electrophoresis, remove the gel and stack the PVDF membrane, which has been soaked in formaldehyde for 30 seconds at room temperature, in the following order from top to bottom: sponge / filter paper / PVDF membrane / gel / filter paper / sponge, removing air bubbles from each layer. Place the tank containing the transfer solution on ice and transfer the membrane for 90 minutes at a constant current of 300mA. Blocking: Immerse the PVDF membrane in 5% BSA for blocking, and place it on a slow, uniform shaker at room temperature for 2 hours. Wash three times with TBST buffer, each time for 5 minutes. Primary antibody incubation: The primary antibody was diluted with 5% BSA according to the instructions and incubated overnight on a shaker at 4°C; Secondary antibody incubation: After the primary antibody solution was recovered, the membrane was washed three times with TBST buffer on a shaker at room temperature for 10 minutes each time. Then, an appropriate amount of secondary antibody was added, and the membrane was incubated on a shaker at room temperature for 1 hour. After terminating the reaction, the membrane was washed three times. Exposure imaging: After preparing the exposure solution, the above bands were exposed in a gel imaging analysis system and the gray values ​​of the protein bands were measured in ImageJ software.

[0052] II. Experimental Results The results are as follows Figure 6 and Figure 7 , Figure 6 In the study, A represents MMP13, a key marker of extracellular matrix catabolism detected by ELISA. The results showed that it could inhibit the activity of catabolistic enzymes induced by pro-inflammatory cytokines in cartilage. qPCR and WB experiments showed that ENVs could inhibit matrix degradation, promote cartilage matrix synthesis, maintain normal extracellular matrix metabolism, inhibit inflammatory responses in articular cartilage, and protect the cartilage matrix from IL-1β-induced damage, thus playing a protective and repairing role in osteoarthritis cartilage.

[0053] In summary, citrus thrombosome-like nanovesicles can exert good anti-inflammatory and chondrogenic effects by significantly downregulating local inflammatory factors in osteoarthritis and maintaining the balance between the degradation and synthesis of chondrocyte extracellular matrix. These effects are dose-dependent, with the most significant effects observed at high doses. The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. Application of Citrus reticulata secois-like nanoparticles in the preparation of drugs for treating osteoarthritis.

2. An application according to claim 1, characterized in that: The extraction method of the ectosome-like nanoparticles of Citrus reticulata peel is to homogenize the peel of Citrus reticulata peel, centrifuge, filter, resuspend, and then filter to remove bacteria.

3. An application according to claim 2, characterized in that: The centrifugation process includes centrifuging at 500–700 × g for 10–15 minutes, repeating this process 2–3 times, collecting the supernatant, centrifuging at 1000–3000 × g for 25–35 minutes, collecting the supernatant again, discarding the precipitate, and centrifuging at 120000–150000 × g for 120–150 minutes.

4. An application according to claim 1, characterized in that: The particle size of the citrus leukoplakia mitochondrial nanoparticles is 140 ~ 160 nm.

5. An application according to claim 2, characterized in that: The filtration process uses a microporous membrane with a pore size of 0.22-0.45 μm.

6. A medicament for treating osteoarthritis, characterized in that: The main active ingredient of the drug is citrus ootheca-like nanoparticles.

7. The drug according to claim 6, characterized in that: The drug also contains pharmaceutically acceptable excipients.

8. The drug according to claim 6, characterized in that: The extraction method of the ectosome-like nanoparticles of Citrus reticulata peel is to homogenize the peel of Citrus reticulata peel, centrifuge, filter, resuspend, and then filter to remove bacteria.

9. The medicament according to claim 8, characterized in that: The centrifugation process includes centrifuging at 500–700 × g for 10–15 minutes, repeating this process 2–3 times, collecting the supernatant, centrifuging at 1000–3000 × g for 25–35 minutes, collecting the supernatant again, discarding the precipitate, and centrifuging at 120000–150000 × g for 120–150 minutes.

10. A pharmaceutical composition, characterized in that: The composition of the drug contains the drug as described in claim 6.

Images