A type of mistletoe exosome-like nanovesicle and its application in the preparation of drugs for the treatment of temporomandibular joint osteoarthritis.

Abstract

This invention discloses a *Taxillus chinensis* exosome-like nanovesicle and its application in the preparation of a therapeutic drug for temporomandibular joint osteoarthritis. The *Taxillus chinensis* exosome-like nanovesicle is prepared by differential ultracentrifugation combined with ultrafiltration. This invention demonstrates that *Taxillus chinensis* exosome-like nanovesicle can treat temporomandibular joint osteoarthritis through the following aspects: 1) Anti-inflammatory function: effectively reducing the expression of inflammatory factors. 2) Inhibition of cartilage degeneration function: blocking the degradation pathway of cartilage matrix and slowing down cartilage tissue damage. 3) Promotion of cartilage regeneration function: activating the synthetic activity of articular chondrocytes and enhancing cartilage repair capacity. 4) Restoration of condylar bone microstructure. The *Taxillus chinensis* exosome-like nanovesicle of this invention exhibits significant protective and repairing effects in alleviating bone and cartilage damage associated with temporomandibular joint osteoarthritis, and has promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a mulberry mistletoe exosome-like nanovesicle and its application in the preparation of a drug for treating temporomandibular joint osteoarthritis. Background Technology

[0002] Temporomandibular joint osteoarthritis (TMJ OA) is a common degenerative disease characterized by degeneration of articular cartilage, bone resorption of the mandible, and joint dysfunction, severely affecting patients' chewing, swallowing, and speech functions. Current clinical treatments mainly include nonsteroidal anti-inflammatory drugs (NSAIDs), hyaluronic acid injections, physical therapy, and arthroscopic surgery. However, all of these methods suffer from unstable efficacy, significant invasiveness, or substantial side effects, making it difficult to effectively protect and repair the articular cartilage.

[0003] In recent years, extracellular vesicles (EVs), especially plant exosome-like nanovesicles (PELNs) derived from traditional Chinese medicine, have gradually attracted attention due to their low immunogenicity, good tissue penetration ability, and potential in inflammation regulation and tissue regeneration. Mistletoe (Sangjisheng) Taxillus chinensis (DC.) Danser As a traditional Chinese medicine, it has anti-inflammatory, analgesic, and bone-strengthening effects. Chinese invention patent application with publication number CN116196342A discloses the application of active compounds from mulberry mistletoe in the preparation of drugs for treating osteoarthritis. However, there are currently no reports on exosome-like nanovesicles derived from mulberry mistletoe and their specific role in TMJ OA. Summary of the Invention

[0004] The purpose of this invention is to overcome the aforementioned defects and shortcomings in the prior art and to provide a method for preparing *Taxillus chinensis* exosome-like nanovesicles. Plant exosome-like nanovesicles derived from *Taxillus chinensis* are obtained by differential ultracentrifugation combined with ultrafiltration.

[0005] The second objective of this invention is to provide the *Taxillus chinensis* exosome-like nanovesicles obtained by the above preparation.

[0006] A third objective of this invention is to provide the application of the aforementioned mulberry mistletoe exosome-like nanovesicles in the preparation of a therapeutic drug for temporomandibular joint osteoarthritis.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] A method for preparing mulberry mistletoe exosome-like nanovesicles includes the following steps: mulberry mistletoe and buffer solution are mixed and crushed to form a homogenate and filtered. The homogenate is centrifuged at 2500–3500×g for 8–12 min. The supernatant is centrifuged at 9000–11000×g for 50–70 min. The supernatant is then concentrated by ultrafiltration through a 90–110 kDa ultrafiltration tube, filtered through a 0.20–0.25 μm filter membrane, centrifuged at 140000–160000×g for 2–4 h, resuspended, and filtered through a 0.20–0.25 μm filter membrane to obtain the nanovesicles.

[0009] This invention prepares *Taxillus chinensis* exosome-like nanovesicles using differential ultracentrifugation combined with ultrafiltration. Specifically, the process includes: plant homogenization and filtration; primary impurity removal: centrifuging the extract at 2500–3500 × g to remove large particulate impurities such as fiber debris and cell remnants, obtaining a supernatant; medium-speed centrifugation: further centrifuging the supernatant at 9000–11000 × g to further remove large vesicles and residual cell components; ultrafiltration concentration: using a 90–110 kDa molecular weight cutoff ultrafiltration tube, centrifuging and collecting the supernatant to concentrate the exosome-like vesicles; re-membrane filtration and ultracentrifugation enrichment: after further removing particulate impurities using a 0.20–0.25 μm filter membrane, ultracentrifugation at 140000–160000 × g precipitates nanoscale vesicle particles; resuspension and re-filtration: resuspending the vesicle precipitate in buffer solution and filtering again through a 0.20–0.25 μm filter membrane to improve purity and homogeneity.

[0010] Preferably, the preparation method involves mixing and crushing the mulberry mistletoe and the buffer solution to form a homogenate, filtering it, centrifuging it at 3000×g for 10 min, centrifuging the supernatant at 10000×g for 60 min, concentrating the supernatant through a 100kDa ultrafiltration tube, filtering it through a 0.22μm filter membrane, centrifuging it at 150000×g for 3 h, resuspending it, and filtering it through a 0.22μm filter membrane to obtain the final product.

[0011] Furthermore, to select high-quality mulberry mistletoe medicinal materials (originating from Yongzhou, Hunan), they were sequentially washed and dried, and then mechanically pulverized into a uniform fine powder to facilitate the subsequent release of active ingredients. The mulberry mistletoe medicinal materials include fresh or dried mulberry mistletoe leaves and stem tissues.

[0012] Furthermore, the buffer solution is a PBS buffer. The pH of the PBS buffer solution is 7.0–7.5.

[0013] Preferably, the pH of the PBS buffer is 7.2.

[0014] Furthermore, the ratio of the mulberry mistletoe to the buffer solution is 1:9 to 11 g / mL.

[0015] Preferably, the ratio of the mulberry mistletoe to the buffer solution is 1:10 g / mL.

[0016] Furthermore, the fragmentation is performed using ultrasound, which further facilitates the release of the active ingredients.

[0017] The present invention also provides *Taxillus chinensis* exosome-like nanovesicles prepared by any of the above-described preparation methods. The *Taxillus chinensis* exosome-like nanovesicles have a typical cup-shaped vesicle structure and a complete cell membrane structure, with a particle size of approximately 120–140 nm and a potential of -32.97 ± 0.53 mV.

[0018] This invention demonstrates that *Taxillus chinensis* exosome-like nanovesicles can exert anti-inflammatory effects by inhibiting the expression of pro-inflammatory factors (such as TNF-α, IL-1β, and IL-6), thus reducing inflammatory responses; inhibiting cartilage degradation by regulating the expression of proteins such as MMP13 and ADAMTS5, thereby slowing down the breakdown of articular cartilage matrix; and promoting cartilage regeneration and repair by activating chondrocytes to synthesize type II collagen (Col-2), thereby enhancing the cartilage tissue repair capacity. In an animal model of temporomandibular joint osteoarthritis (TMJ) osteoarthritis, the microstructure of condylar bone in the *Taxillus chinensis* exosome-like nanovesicle group was significantly restored, indicating that *Taxillus chinensis* exosome-like nanovesicles exhibit significant protective and reparative effects in alleviating TMJ OA-related bone damage and have promising application prospects.

[0019] Therefore, the application of any of the above-mentioned mulberry mistletoe exosome-like nanovesicles in the preparation of drugs for the treatment of temporomandibular joint osteoarthritis is also provided.

[0020] Specifically, the drug achieves therapeutic effects through anti-inflammatory and analgesic effects, inhibiting condylar cartilage degeneration, promoting condylar cartilage regeneration and repair, and restoring condylar bone microstructure.

[0021] Specifically, restoring the microstructure of the condyle involves increasing the bone volume fraction, restoring the number of trabeculae, and restoring the spacing between trabeculae.

[0022] The present invention also provides a therapeutic drug containing any of the above-described mulberry mistletoe exosome-like nanovesicles.

[0023] Furthermore, the dosage form of the drug includes, but is not limited to, sprays or injections. For example, a liquid injection: a suspension with a pH suitable for the intra-articular environment; specifically, an intra-articular injection containing 0.5–2 × 10⁻⁶ exosome-like nanovesicles. 10 per mL.

[0024] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] This invention provides a *Taxillus chinensis* exosome-like nanovesicle, which is prepared by differential ultracentrifugation combined with ultrafiltration. Research in this invention shows that *Taxillus chinensis* exosome-like nanovesicles can treat temporomandibular joint osteoarthritis (TMJ) through the following aspects: 1) Anti-inflammatory function: effectively reducing the expression of inflammatory factors. 2) Inhibition of cartilage degeneration function: blocking the degradation pathway of cartilage matrix and slowing down cartilage tissue damage. 3) Promotion of cartilage regeneration function: activating the synthetic activity of articular chondrocytes and enhancing cartilage repair capacity. 4) Restoration of condylar bone microstructure. The *Taxillus chinensis* exosome-like nanovesicles of this invention exhibit significant protective and reparative effects in alleviating TMJ OA-related bone damage. Furthermore, *Taxillus chinensis* exosome-like nanovesicles are derived from natural plants, have low toxicity and side effects, and are not immune-rejected, avoiding the ethical and safety issues faced by traditional cell exosomes, and possess promising application prospects. Attached Figure Description

[0027] Figure 1 Characterization results of *Mistletoe* exosome-like nanovesicles (TENs). Among them, Figure 1 In the figure, A represents the morphology of TENs observed by transmission electron microscopy (TEM) (scale bar = 50 nm); B represents the particle size distribution of TENs detected by a nanoparticle size analyzer; and C represents the surface charge of TENs measured by Nanoview.

[0028] Figure 2 The results show the effects of TENs on condylar chondrocyte viability and inflammation. Among them, Figure 2 Image A shows the effect of different concentrations of TENs (0, 1, 2.5, 5, 12.5, 25, 50, 75, and 100 μg / mL) on the activity of rat condylar chondrocytes (rPCCs) after co-incubation for 48 and 72 hours, as determined by the CCK8 assay; Image B shows the effect of TENs on key genes ( ) in an IL-1β-induced inflammation model, as analyzed by qRT-PCR. Tnf-α , Adamts5 , Il-6 , Mmp3 and Mmp13 The regulatory effect of TENs on collagen (COL2A1) and matrix metalloproteinase (MMP) metabolic markers (MMP9, MMP3, and MMP13) was investigated. Data are expressed as mean ± standard error (n=3). Compared with the control group, *P<0.05, **P<0.01, ***P<0.001; compared with the IL-1β group, #P<0.05, ##P<0.01, ###P<0.001; ns indicates no significant difference; C represents the results of Western blot analysis of TENs on collagen (COL2A1) and matrix metalloproteinase (MMP) metabolic markers (MMP9, MMP3, and MMP13).

[0029] Figure 3The results of TENs treatment in a TMJ OA animal model are shown. Among them, Figure 3 In the image, A represents the results of Micro-CT imaging; B represents the results of bone microstructure parameter analysis. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0031] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0032] All data in the following examples are expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism software (version 10.5). Between-group comparisons were performed using one-way ANOVA, followed by Tukey's post-hoc test for multiple comparisons. For non-normally distributed data, the Kruskal-Wallis test was used, followed by the Dunn test for pairwise comparisons. A p-value less than 0.05 was considered statistically significant.

[0033] Example 1: Preparation and characterization of mistletoe exosome-like nanovesicles (TENs)

[0034] 1. Method

[0035] Fresh mistletoe (purchased from Yongzhou, Hunan) was washed with PBS buffer (pH=7.2) and juiced for 2 minutes in a high-speed blender at a ratio of 1 g mistletoe: 10 mL PBS buffer. The resulting juice was filtered through a 300-mesh sieve to remove larger particles and purified as follows: centrifugation at 3000 ×g for 10 min to remove impurities; centrifugation at 10,000 ×g for 30 min*2 times to remove large vesicles; concentration of the supernatant using a 100 kDa ultrafiltration tube (5000 ×g, 20 min); pre-filtration using a 0.22 μm filter; ultracentrifugation at 150,000 ×g for 3 h to collect nanovesicles; resuspending in PBS buffer and filtering under sterile conditions through a 0.22 μm filter membrane (Millipore, USA). The final sterile filtrate was collected as TENs and stored at -80℃.

[0036] The morphology of TENs was observed using transmission electron microscopy (TEM, Hitachi 7500, Japan): approximately 10 μL of TENs was added to a copper grid and allowed to stand for 5 minutes. After staining with 2% (w / v) phosphotungstic acid for 3 minutes, the exosome-like structure was observed using TEM. The particle size distribution of TENs was measured using a nanoparticle size analyzer. The surface charge of TENs was determined using Nanoview.

[0037] 2. Results

[0038] like Figure 1 As shown, the morphology of TENs was observed and photographed under a transmission electron microscope. The images show typical goblet vesicle structures and intact cell membrane structures. Figure 1 (A). Nanoparticle Tracking Analysis (NTA) showed that TENs had an average particle size distribution of 130.1 nm. Figure 1 (Middle B). Therefore, the average particle size of TENs determined by transmission electron microscopy and nanoparticle tracking analysis is consistent. The yield of TENs extracted from 1 kg of fresh mulberry mistletoe is approximately 20 mg. Its purity is approximately 1.4 × 10⁻⁶. 12 Particles / mg. Nanoparticle visualization showed a potential of -32.97 ± 0.53 mV ( Figure 1 (C)

[0039] Example 2: Effects of TENs on condylar chondrocyte viability and inflammation

[0040] 1. Method

[0041] (1) Extraction and culture of primary rat condylar chondrocytes (rPCCs)

[0042] Primary condylar chondrocytes (rPCCs) were isolated from 4-week-old female Sprague-Dawley (SD) rats obtained from the Guangdong Medical Laboratory Animal Center. Cells from passages 1 to 3 were used for subsequent experiments. Condylar tissue was cut into approximately 1 mm sections. 3 Fragments were digested with 0.2% type II collagenase (Sigma-Aldrich, USA) at 37°C for 4–6 hours. After digestion, cells were collected by centrifugation, resuspended in DMEM / F12 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and cultured under standard conditions. In this study, IL-1β, commonly used to mimic in vitro pathological conditions, was used to induce an in vitro model of temporomandibular joint osteoarthritis in condylar chondrocytes. Following the in vitro experimental design of previous studies, rat condylar chondrocytes were treated with 10 ng / ml IL-1β for 24 hours.

[0043] (2) Effects of TENs on cell viability

[0044] The effect of TENs on rPCC viability was assessed using a cell counting kit-8 (CCK8, Dojindo, Japan). rPCCs (5 × 10⁻⁶ cells / year) were counted. 3Cells were seeded per well into 96-well plates and treated with TENs at concentrations of (0, 1, 2.5, 5, 12.5, 25, 50, 75, and 100 μg / mL) for 48 and 72 hours. After incubation, 100 µL of 10% CCK-8 solution was added to each well, and the plates were incubated for 2 hours. Cell viability was assessed by measuring absorbance at 450 nm using a microplate reader (Thermo Fisher Scientific, USA).

[0045] (3) Grouping, drug treatment, RNA extraction and real-time quantitative PCR (RT-qPCR)

[0046] Day 0: rPCCs (3×10 5 Cells (per well) will be seeded into 6-well plates. Day 1: Cells have adhered to the plates at this time. Control group: only fresh medium was changed, no treatment was given; IL-1β group: only fresh medium was changed, no treatment was given; TENs treated with IL-1β-induced inflammation model group (TENs+IL-1β): TENs (2.5 μg / mL, 5 μg / mL) were added after fresh medium was changed and treated for 24 hours.

[0047] Day 2: Control group: No treatment; IL-1β group: IL-1β (10 ng / mL) added for 24 hours; TENs treated with IL-1β-induced inflammation model group (TENs+IL-1β): IL-1β (10 ng / mL) added for 24 hours.

[0048] Day 3: Sample Collection: rPCCs from different groups (control group, IL-1β group, and TENs+IL-1β group) were lysed in RIPA lysis buffer (Beyotime Biotechnology, China) supplemented with 1% protease and phosphatase inhibitors. Total RNA was isolated from cultured cells using an RNA extraction kit (EZ Bioscience, China) according to the manufacturer's instructions. The extracted RNA was reverse transcribed into complementary DNA (cDNA) using a one-step RT-PCR kit (Accurate Biology, China). Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using target gene-specific primers. Primer sequences are listed in Table 1. Relative mRNA expression levels were calculated using the 2^-ΔΔCt method and normalized using GAPDH as an internal reference gene.

[0049] Table 1 Primer sequences for RT-qPCR

[0050]

[0051] (4) Western blot

[0052] Protein concentrations were further quantified using the BCA Protein Assay Kit (Beyotime Biotechnology, China). Equal volumes of protein were subjected to SDS-PAGE and transferred to 0.45 µm or 0.22 µm polyvinylidene fluoride membranes (Millipore, USA). The membranes were blocked with QuickBlock™ blocking buffer (Beyotime Biotechnology, China) for 1 hour and incubated overnight at 4°C with primary antibody. The next day, the membranes were incubated with HRP-conjugated secondary antibody at 37°C for 1 hour. Protein bands were visualized using a high-sensitivity ECL chemiluminescence assay kit (Epizyme Biomedical Technology, China). Details of the primary antibodies used in Western blots are shown in Table 2.

[0053] Table 2. Detailed information on the primary antibodies used in Western blots.

[0054]

[0055] 2. Results

[0056] like Figure 2 As shown, the effects of different concentrations of TENs (0, 1, 2.5, 5, 12.5, 25, 50, 75 and 100 μg / mL) on condylar chondrocyte viability were detected by CCK-8 assay, showing that TENs had no cytotoxicity within the experimental concentration range. Figure 2 (A). The regulatory role of TENs on key genes in an IL-1β-induced inflammation model was analyzed using qRT-PCR. IL-1β stimulation significantly upregulated these genes. Tnf-α , Adamts5 , Il-6 , Mmp3 and Mmp13 Gene expression, and TEN treatment can suppress it. Tnf-α , Adamts5 , Il- 6 , Mmp3 and Mmp13 Gene expression ( Figure 2 (Figure B). Western blot further confirmed the inhibitory effect of TENs on matrix metalloproteinases (MMPs). IL-1β stimulation significantly upregulated the expression of MMP3, MMP9, and MMP13 proteins, while TEN treatment inhibited their expression (5 μg / mL group: MMP3, MMP9, and MMP13 decreased). Simultaneously, IL-1β stimulation inhibited the expression of type II collagen (COL2A1), while TEN treatment reversed this process. Figure 2(C) This indicates that TENs can reduce synovial inflammation by inhibiting the expression of pro-inflammatory factors (such as TNF-α, IL-1β, IL-6, etc.) within the joint cavity, thereby exerting an anti-inflammatory effect and relieving joint pain and tenderness. TENs can slow down the breakdown of articular cartilage matrix by regulating the expression of proteins such as MMP13 and ADAMTS5, thereby inhibiting cartilage degradation. TENs activate chondrocytes to synthesize type II collagen, further enhancing the cartilage tissue repair capacity.

[0057] Example 3: Treatment experiment of TENs on TMJ OA animal model

[0058] To evaluate the potential efficacy of TENs in the treatment of temporomandibular joint osteoarthritis (TMJ OA), this embodiment established a TMJ OA model in SD rats induced by sodium iodoacetate (MIA), and quantitatively assessed the microstructure of the condylar bone using Micro-CT scanning and three-dimensional bone parameter analysis. Details are as follows:

[0059] 1. Method

[0060] (1) Twenty-four 8-week-old female Sprague-Dawley (SD) rats were purchased from the Guangdong Medical Laboratory Animal Center. The rats were housed under specific pathogen-free conditions and provided with standard food and water. After a one-week acclimatization period, the rats were randomly divided into four groups (n=6 per group): sham-operated group (Shame), + saline (NS) group, MIA + hyaluronic acid (HA) group, and MIA + TENs group. A temporomandibular joint osteoarthritis (TMJ-OA) model was established by injecting 0.5 mg MIA into the bilateral temporomandibular joint cavity via an insulin needle. The sham-operated group (Shame) underwent only sham surgery and did not receive MIA injection. Two weeks after model induction, intra-articular injections were performed once a week for four weeks. The MIA + NS group was injected with 50 μL of saline, the MIA + HA group was treated with 50 μL of hyaluronic acid, and the MIA + TENs group was injected with 50 μL of TENs (1×10⁻⁶). 10 (Units / mL). All procedures were performed in accordance with ethical standards. Hyaluronic acid (HA), currently the first-line intra-articular injection for the treatment of TMJ OA, was used as a positive control.

[0061] (2) Micro-CT scan evaluation

[0062] Temporomandibular joint tissue samples were collected and fixed with 4% paraformaldehyde (PFA) for subsequent analysis. Coronal scans of the samples were performed using a micro-CT system (Bruker Corporation, China) at 85 kV, 200 μA, and a pixel size of 10 μm. Key bone structural parameters were quantitatively assessed, including the bone volume to total tissue volume ratio (BV / TV), trabecular separation (Tb.Sp), and trabecular number (Tb.N).

[0063] 2. Results

[0064] (1) Micro-CT imaging observation such as Figure 3 As shown in Figure A, the Sham group exhibited intact condylar structure and dense, regular trabecular arrangement; the MIA+NS group showed significant bone destruction, a rough condylar surface, reduced trabecular number, and increased gaps (indicated by red arrows); the MIA+HA group showed partial improvement in structural disorder; while the MIA+TENs group showed clear condylar contours, significantly restored trabecular structure, and an overall morphology close to normal, indicating that TENs intervention has a good structural repair effect.

[0065] (2) Results of bone microstructure parameter analysis are as follows Figure 3 Figure B shows three key bone structure parameters obtained from Micro-CT analysis: Bone volume fraction (BV / TV): significantly decreased in the MIA+NS group (**P<0.01), indicating bone loss; TENs treatment significantly improved this index (***P<0.001), superior to the HA group. Trabecular bone number (Tb.N): decreased in the MIA+NS group, and significantly recovered in the TENs group, with statistically significant differences (*P<0.05). Trabecular bone spacing (Tb.Sp): significantly increased in the OA model group, and significantly decreased in the TENs group, recovering to near-normal levels (*P<0.05). The combined morphological observation and quantitative analysis results indicate that *Taxillus chinensis* exosome-like vesicles exhibit significant protective and repairing effects in the treatment of TMJ OA-related bone destruction, and are significantly more effective than hyaluronic acid, showing promising application prospects.

Claims

1. The use of an exosome-like nanovesicle of Morus alba in the preparation of a drug for treating osteoarthritis of the temporomandibular joint, characterized in that, The preparation method of the Morinda extractive exosome-like nanovesicle comprises the following steps: mixing and crushing Morinda and a buffer to prepare a homogenate and filter, centrifuging at 2500-3500 xg for 8-12 min, centrifuging the supernatant at 9000-11000 xg for 50-70 min, concentrating the supernatant by ultrafiltration through a 90-110 kDa ultrafiltration tube, filtering through a 0.20-0.25 μm filter membrane, centrifuging at 140000-160000 xg for 2-4 h, resuspending, and filtering through a 0.20-0.25 μm filter membrane.

2. Use according to claim 1, characterized in that, The preparation method comprises the following steps: The preparation method comprises the following steps:

3. Use according to claim 1, characterized in that, The buffer is a PBS buffer.

4. The use according to claim 1, characterized in that, The ratio of Morinda to the buffer is 1:9-11 g / mL.

5. The use according to claim 1, characterized in that, The drug achieves treatment by anti-inflammatory, inhibition of condylar cartilage degeneration, promotion of condylar cartilage regeneration and repair, and recovery of condylar bone microstructure.

6. Use according to claim 5, characterized in that, The recovery of condylar bone microstructure is to improve bone volume fraction, recover bone trabecula number, and recover bone trabecula spacing.

7. The use according to claim 1, characterized in that, The dosage form of the drug is an injection.

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