Mitochondria-derived biomimetic vesicles, and preparation method and application thereof

Abstract

The application relates to a mitochondrion-derived biomimetic vesicle and a preparation method and application thereof. The mitochondrion-derived biomimetic vesicle is prepared by the following steps: mitochondria are separated and extracted from cells; the mitochondria are placed in a PBS solution containing drugs to obtain a PBS solution containing the mitochondria and the drugs; the PBS solution is passed through a polycarbonate membrane by using a liposome extruder to obtain a filtrate; and the filtrate is centrifuged by using an ultrafiltration tube to collect the inner tube interception liquid, so that the mitochondrion-derived biomimetic vesicle is obtained. Compared with the prior art, the mitochondrion-derived biomimetic vesicle prepared by the application has a particle size of less than 200 nm, is beneficial to cartilage penetration and cell uptake, and also retains the homologous targeting ability of mitochondria, so that the drug can be accurately delivered to a functionally impaired mitochondrion network. In addition, in-vitro experiments prove that the mitochondrion-derived biomimetic vesicle prepared by the application can significantly increase the expression of cartilage extracellular matrix core components COL2 and ACAN; and increase ATP generation and reduce ROS.

Description

Technical Field

[0001] This invention relates to the field of orthopedics, and in particular to a mitochondrial-derived biomimetic vesicle, its preparation method, and its application. Background Technology

[0002] Osteoarthritis, a chronic disabling disease characterized by progressive degeneration, loss, and even functional loss of articular cartilage, has long faced significant challenges in clinical treatment. Current interventions are mostly limited to analgesia, anti-inflammation, or late-stage joint replacement, failing to effectively halt disease progression. A deeper exploration of its pathological mechanisms reveals that the "energy crisis" in chondrocytes and mitochondrial dysfunction have become core drivers of disease development. During the progression of osteoarthritis, the dual impact of abnormal mechanical load and the chronic inflammatory microenvironment leads to significant morphological changes and functional inactivation of mitochondria within articular chondrocytes. This includes mitochondrial network fragmentation, membrane potential collapse, and inhibition of respiratory chain complex activity, resulting in a precipitous drop in the cellular energy reserve, ATP. This severe energy metabolic imbalance prevents chondrocytes from maintaining the highly complex synthesis and homeostasis of the extracellular matrix, initiating apoptosis and releasing matrix-degrading enzymes, ultimately leading to irreversible cartilage erosion.

[0003] Mitochondrial transplantation therapy involves directly implanting cultured healthy mitochondria into damaged cells or tissues. It is emerging as a promising treatment approach. Previous studies have shown that exogenous mitochondria can be internalized by recipient cells to enhance ATP production and restore redox homeostasis, demonstrating great promise in the treatment of mitochondrial diseases. However, for cartilage tissue, the dense extracellular matrix structure of chondrocytes limits drug penetration. Exogenous mitochondria, due to their micrometer-scale particle size, diffuse slowly in synovial fluid and struggle to effectively penetrate the nanoporous matrix barrier of cartilage. Furthermore, their particle size restricts their internalization and uptake by cells, resulting in low bioavailability and severely limiting their therapeutic efficacy.

[0004] Chinese patent CN114908046B discloses a method for preparing mitochondrial membrane vesicles, including the following steps: S1: extracting mitochondria from mouse embryonic fibroblast cell line 3T3; S2: extracting the outer mitochondrial membrane; S3: sonicating the outer mitochondrial membrane, and then extruding it multiple times in a polycarbonate porous membrane to obtain empty mitochondrial membrane vesicles; S4: encapsulating CuS with the mitochondrial membrane vesicles. However, this method does not contain active mitochondrial components.

[0005] Chinese patent CN120227353A discloses an engineered mitochondria, its preparation method, and its application, including: constructing immortalized mesenchymal stem cells; isolating and extracting mitochondria from the immortalized mesenchymal stem cells; pretreating the immortalized mesenchymal stem cells with IFN-γ and TNF-α to induce apoptosis, and extracting engineered apoptotic extracellular vesicle membranes; mixing the mitochondria with the engineered apoptotic extracellular vesicle membranes, and obtaining engineered mitochondria with a surface coated with the engineered apoptotic extracellular vesicle membranes after sonication and centrifugation. However, the particle size of these engineered mitochondria is 200-500 nm, and their ability to penetrate dense cartilage matrix is ​​limited.

[0006] Therefore, improving the utilization efficiency of mitochondria in cartilage has become a key issue that must be addressed in clinical translation. Summary of the Invention

[0007] The purpose of this invention is to overcome the problems of physical barriers and low bioavailability in existing technologies, thereby providing a mitochondrial-derived biomimetic vesicle, its preparation method, and its application.

[0008] The objective of this invention can be achieved through the following technical solutions: In a first aspect, the present invention provides a method for preparing mitochondrial-derived biomimetic vesicles, comprising the following steps: S1: Mitochondria were isolated and extracted from bone marrow mesenchymal stem cells; S2: Place the mitochondria in a PBS solution containing the drug to obtain a PBS solution containing the mitochondria and the drug. Use a liposome extruder to pass the PBS solution containing the mitochondria and the drug through a polycarbonate membrane to obtain a filtrate. Then, centrifuge the filtrate using an ultrafiltration tube and collect the retentate in the inner tube to obtain mitochondrial-derived biomimetic vesicles.

[0009] Furthermore, the specific steps in step S1 are as follows: a: Select third-generation bone marrow mesenchymal stem cells cultured in vitro, add mitochondrial separation reagent, homogenize the cells, and obtain a homogenate solution; b: Centrifuge the homogenate at low speed, collect the supernatant, and then centrifuge at high speed. The resulting precipitate is the mitochondria.

[0010] Further, preferably, the conditions for low-speed centrifugation in b are: 4°C, 500-700×g centrifugation for 10-30 minutes; The conditions for high-speed centrifugation are: 4℃, centrifugation at 10000-12000×g for 10-30 minutes.

[0011] Further, the drug mentioned in step S2 is dihydromyricetin (DMY) at a concentration of 100-300 μM.

[0012] Further, in step S2, the mass-to-volume ratio of mitochondria to the drug-containing PBS solution is 100 μg: 600-1000 μl; preferably, the mass-to-volume ratio of mitochondria to the drug-containing PBS solution is 100 μg: 800 μl.

[0013] Furthermore, in step S2, the material is passed through polycarbonate films of 400 nm and 200 nm in sequence.

[0014] Furthermore, the centrifugation conditions in S2 are 2000-4000g and 4℃.

[0015] Furthermore, the particle size of the mitochondrial-derived biomimetic vesicles is 100-200 nm.

[0016] Secondly, the present invention provides a mitochondrial-derived biomimetic vesicle, which is prepared using the above-described method for preparing mitochondrial-derived biomimetic vesicles.

[0017] Thirdly, the present invention provides the application of mitochondrial-derived biomimetic vesicles in the preparation of drugs for treating osteoarthritis.

[0018] Compared with the prior art, the present invention has the following beneficial effects: Compared to pure mitochondria, the mitochondrial-derived biomimetic vesicles prepared in this invention have a particle size of <200nm, which is beneficial for cartilage penetration and cellular uptake. Furthermore, the mitochondrial-derived biomimetic vesicles retain the homologous targeting ability of mitochondria, enabling precise delivery of drugs to the functionally impaired mitochondrial network.

[0019] Furthermore, in vitro experiments have demonstrated that the mitochondrial-derived biomimetic vesicles prepared in this invention can downregulate the expression of the catabolic marker MMP13 and promote the expression of anabolism markers (COL2 and ACAN) and glycosaminoglycans. Moreover, under inflammation-induced conditions, ATP content in chondrocytes decreases and ROS increases abnormally, while DMY-BMMVs increase ATP production and reduce inflammation-induced ROS. Attached Figure Description

[0020] Figure 1 The images show transmission electron microscopy (TEM) images and diameter statistics of biomimetic vesicles; where A is the TEM image of BMMVs and DMY-BMMVs, and B is the average diameter statistics of MT, BMMVs, and DMY-BMMVs.

[0021] Figure 2 WB diagram of mitochondrial-associated proteins of BMMVs and DMY-BMMVs.

[0022] Figure 3 The image shows the UV absorbance scans of DMY, BMMV, and DMY-BMMV.

[0023] Figure 4The results of the cartilage matrix penetration tests for MT, BMMV, and DMY-BMMV are shown in Figure 1; where A is a flowchart, B is the result in 3D chondrocyte spheroids, and C is the result in rat articular cartilage explants.

[0024] Figure 5 The graph shows the cellular uptake detection results of MT, BMMV, and DMY-BMMV, and the subcellular localization results after DMY-BMMV uptake; where A is the flow cytometry result, with fluorescence intensity on the x-axis and cell number on the y-axis; B is the statistical graph of cell positivity rate after uptake in each group; and C is the colocalization map of DMY-BMMV and various organelles (endoplasmic reticulum, lysosome, mitochondria).

[0025] Figure 6 The image shows the results of the detection of chondrocyte extracellular matrix improvement; where A is the expression of COL2 and ACAN genes detected by qRT-PCR, and B is the staining of the extracellular matrix by Safranin O.

[0026] Figure 7 The image shows the results of improved mitochondrial function in chondrocytes; where A represents intracellular ATP content and B represents intracellular ROS level.

[0027] Figure 8 This is a flowchart of the preparation process for DMY-BMMVs. Detailed Implementation

[0028] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0029] Example 1 The preparation process of mitochondrial-derived biomimetic vesicles (DMY-BMMVs) is as follows: Figure 8 As shown, it includes the following steps: S1: Primary bone marrow mesenchymal stem cells (BMSCs) were selected from in vitro culture and seeded in αMEM medium (Yuanpei-L510KJ) containing 10% fetal bovine serum (Gibco-10099141C) and 1% penicillin antibiotics (Gibco 15140-122). The medium was changed every 2-3 days. When the cells reached 80-90% confluence, they were passaged. The third generation cells were used for experiments: after washing with PBS, the cells were collected and homogenized using a mitochondrial separation reagent (Beyotime-C3601) (Dounce homogenizer, 30 cycles) to obtain the homogenate.

[0030] S2: Centrifuge the homogenate at 4℃ and 600×g for 10 minutes, take the supernatant, and then centrifuge at 4℃ and 11,000×g for 10 minutes. The resulting precipitate is the mitochondria.

[0031] S3: Place 100 μg of mitochondria in 800 μl of 200 μM dihydromyricetin (DMY) in PBS solution, and gently pass it through a liposome extruder (pressure less than 100 psi) sequentially through a 400 nm and a 200 nm polycarbonate membrane to obtain filtrate. Then, centrifuge at 3000 g and 4 °C using a 10 kDa ultrafiltration tube, and collect the inner tube retentate and the tube permeate separately. The inner tube retentate is DMY-BMMVs, and the tube permeate is free DMY.

[0032] Comparative Example 1 A blank biomimetic mitochondrial membrane vesicle (BMMV) is prepared in a manner that differs from that in Example 1: the mitochondria (100 μg) obtained in step S2 of Example 1 are resuspended in 800 μl of DMY-free PBS solution and then gently extruded sequentially through 400 nm and 200 nm polycarbonate membranes using a liposome extruder (pressure less than 100 psi) to obtain blank BMMVs without drug loading.

[0033] Comparative Example 2 An intact mitochondrial (MT) is prepared in a manner that differs from that of Example 1 in that the mitochondrial (100 μg) obtained in step S2 of Example 1 is resuspended in PBS solution (800 μl) for later use, without subsequent gradient extrusion and DMY loading steps.

[0034] The DMY-BMMVs prepared in Example 1, the BMMVs prepared in Comparative Example 1, and the intact mitochondria (MTs) prepared in Comparative Example 2 were characterized.

[0035] (1) Transmission electron microscopy images of vesicles The morphology of DMY-BMMVs and BMMVs was analyzed by transmission electron microscopy; the particle size of DMY-BMMVs, BMMVs and MT was analyzed by dynamic light scattering.

[0036] The results are as follows Figure 1 As shown in A and B, compared to purified mitochondrial MT, the average diameter of DMY-BMMVs and BMMVs obtained by using a liposome extruder is reduced to <200 nm, which is beneficial for cartilage penetration and cellular uptake.

[0037] (2) Western blot verification of mitochondrial extraction The prepared samples (DMY-BMMVs, BMMVs, MT) were added to RIPA lysis buffer and lysed on ice for 30 minutes. After lysis, the samples were centrifuged at 12,000×g for 10-15 minutes at 4°C, and the supernatant was collected as the protein sample. 5×SDS loading buffer was added, mixed, and then heated in a 95°C metal bath for 5 minutes to denature the protein. SDS-PAGE electrophoresis was then performed, with 15 μg of the denatured protein loaded and electrophoresed until the bromophenol blue indicator reached the bottom of the gel. The protein was then transferred to a PVDF membrane using a wet transfer method. After transfer, the membrane was blocked with 5% skim milk blocking buffer at room temperature for 1 hour, and then incubated overnight at 4°C with primary antibody (TOMM20: Wanleibio-WL03626; ND1: Abcam-ab181848; SOD2: Cell Signaling Technology-D3X8F; β-actin: Servicebio-GB15003). The following day, the membrane was washed three times with TBST for 10 minutes each time, then incubated with HRP-labeled secondary antibody (Wanleibio-WLA023) at room temperature for 1 hour, washed three more times with TBST, and finally developed with ECL chemiluminescent substrate and photographed.

[0038] The results are as follows Figure 2 As shown, the biomimetic vesicles (DMY-BMMVs and BMMVs) effectively preserved mitochondrial-related proteins (such as TOMM20, ND1, and SOD2), and compared with β-actin, mitochondrial-related proteins were reduced in whole-cell lysate (total) and cytosol (Cytosol), indicating that the extraction of mitochondria was successful (the small amount of actin was a residue on the mitochondrial surface).

[0039] (3) Verify drug loading The samples (DMY-BMMVs, BMMVs, and free DMY) were scanned across the entire wavelength range of 200-400 nm using a multi-functional microplate reader. The results are as follows: Figure 3 As shown, DMY-BMMVs exhibit a characteristic absorption peak at 324 nm, consistent with free DMY, while blank BMMVs show no absorption at this wavelength. This indicates successful DMY drug loading.

[0040] Example 3 verifies cartilage penetration ability 1) Construction of a three-dimensional chondrocyte spheroid model: Culture conditions: 37°C, 5% CO2 Culture medium: High glucose-DMEM medium (source culture-L110KJ) containing 10% fetal bovine serum (Gibco-10099141C) and 1% penicillin antibiotics (Gibco 15140-122). Rat primary chondrocytes were cultured at 5 × 10⁶ cells per well. 5Cells were seeded at a density of [number] cells per well in a low-adsorption U-shaped bottom 96-well plate and centrifuged at 400 × g for 5 minutes. After incubation at 37°C and 5% CO2 for 72 hours, chondrocyte spheroids with a diameter of approximately 400 μm were formed. 2) Construction of a rat articular cartilage explant model: Culture conditions: 37°C, 5% CO2 Culture medium: High glucose-DMEM medium (source culture-L110KJ) containing 10% fetal bovine serum (Gibco-10099141C) and 1% penicillin antibiotics (Gibco 15140-122). Small pieces of cartilage were excised from the knee joints of 12-week-old SD rats and cultured at 37°C and 5% CO2 for 72 hours.

[0041] 3) Intact mitochondria (MT), BMMVs, and DMY-BMMVs labeled with red fluorescent markers: Prepare a 5 μM working solution of the Dil fluorescent probe (red) using PBS. Add mitochondria (MT), BMMVs and DMY-BMMVs and incubate at 37°C for 30 minutes in the dark. Then, use a 10 kDa ultrafiltration tube to centrifuge and ultrafilter, and wash three times with PBS to remove excess Dil.

[0042] 4) Verify that biomimetic mitochondrial vesicles can overcome the physical barrier of cartilage matrix. Red fluorescently labeled intact mitochondria, BMMVs, and DMY-BMMVs were co-incubated with a three-dimensional chondrocyte spheroid model for 24 h or with a rat articular cartilage explant model at 37°C and 5% CO2 for 72 h, respectively. Figure 4 A) Fluorescence distribution was observed using a confocal microscope, and the results are as follows: Figure 4 As shown in B and C, simple mitochondria are only attached to the surface, while biomimetic vesicles penetrate deep into the layers of both models, confirming that nano-sized biomimetic mitochondrial vesicles can overcome the physical barrier of the cartilage matrix.

[0043] Example 4 verifies subcellular localization after DMY-BMMV uptake. After co-incubating chondrocytes with red fluorescently labeled (Dil fluorescent probes) MT, BMMV, and DMY-BMMVs for 24 h, the cells were washed with PBS to remove untaken portions and collected for flow cytometry analysis.

[0044] The results are as follows Figure 5 As shown in A and B, the nanoscale biomimetic vesicles of BMMV vesicles can be efficiently taken up by cells and are visible within 6 hours.

[0045] Further subcellular colocalization analysis showed that internalized DMY-BMMVs were primarily colocalized with mitochondria, rather than the endoplasmic reticulum or lysosomes. Figure 5 C). This indicates that BMMVs retain the homologous targeting ability of mitochondria, enabling precise drug delivery to the dysfunctional mitochondrial network.

[0046] Example 5 verifies the improvement of mitochondrial function 1) Verify the restoration of extracellular matrix homeostasis in inflammatory chondrocytes An inflammatory chondrocyte model was established by inducing primary chondrocytes with IL-1β at a final concentration of 10 ng / ml for 24 h (culture conditions: 37°C, 5% CO2, culture medium: high glucose-DMEM medium (source culture-L110KJ) containing 10% fetal bovine serum (Gibco-10099141C) and 1% penicillin antibody (Gibco15140-122)). BMMVs prepared in Comparative Example 1, MT prepared in Comparative Example 2, and DMY-BMMV prepared in Example 1 were added at a final concentration of 25 μg / ml. Blank groups (healthy chondrocytes, Blank group) and control groups (no addition, IL-1β group) were also set up. The cells were cultured using a 400 nm pore size Transwell co-culture system (upper chamber for MT, BMMVs, or DMY-BMMV, lower chamber for chondrocytes). After 48 h, chondrocytes were collected, total RNA was extracted, and chondrocyte anabolic biomarkers were detected by qRT-PCR. After 120 hours, the cells were fixed with 4% paraformaldehyde for 1 hour, washed three times with PBS, and then stained with safranin O to assess the synthesis and deposition of glycosaminoglycans.

[0047] The results are as follows Figure 6 As shown in Figures A and B, qRT-PCR revealed that IL-1β stimulation significantly downregulated chondrocyte anabolic markers (COL2 and ACAN). Safranin O staining showed a significant decrease in glycosaminoglycan content in the extracellular matrix after IL-1β stimulation (staining was weakened). Both BMMVs and DMY-BMMVs significantly reversed these pathological changes, with DMY-BMMVs exhibiting the most significant therapeutic effect. This indicates the restoration of extracellular matrix homeostasis in inflamed chondrocytes.

[0048] 2) Detect ATP content Intracellular ATP levels were detected using an ATP assay kit (Beyotime-S0026) following the manufacturer's instructions. In short, chondrocytes from different treatment groups were gently aspirated of the culture medium, and lysed with 200 µL of lysis buffer per well in a 6-well plate. The cells were centrifuged at 12,000 × g for 5 min at 4°C, and the supernatant was collected. A standard curve was prepared by serially diluting ATP standards. 20 µL of sample was added to each 100 µL working solution chamber, mixed thoroughly, and immediately measured using a microplate reader to detect the chemiluminescence value. ATP levels were calculated based on the standard curve.

[0049] The results are as follows Figure 7 As shown in Figure A, inflammation-induced ATP content in chondrocytes decreases, while DMY-BMMVs increase ATP production, indicating improved mitochondrial function.

[0050] 3) Detect ROS levels Chondrocytes were incubated in serum-free, high-glucose DMEM medium (source culture - L110KJ) containing 10µM DCFH-DA at 37°C in the dark for 30 minutes. Cells were washed three times with PBS to remove unbound probes. Fluorescence imaging was performed immediately using a fluorescence microscope for observation and recording.

[0051] The results are as follows Figure 7 As shown in Figure B, inflammation-induced chondrocyte ROS levels are abnormally elevated, while DMY-BMMVs reduce inflammation-induced ROS, indicating improved mitochondrial function.

[0052] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing mitochondrial-derived biomimetic vesicles, characterized in that, Includes the following steps: S1: Mitochondria were isolated and extracted from bone marrow mesenchymal stem cells; S2: Place the mitochondria in a PBS solution containing the drug to obtain a PBS solution containing the mitochondria and the drug. Use a liposome extruder to pass the PBS solution containing the mitochondria and the drug through a polycarbonate membrane to obtain a filtrate. Then, centrifuge the filtrate using an ultrafiltration tube and collect the retentate in the inner tube to obtain mitochondrial-derived biomimetic vesicles.

2. The method for preparing mitochondrial-derived biomimetic vesicles according to claim 1, characterized in that, The specific steps in step S1 are as follows: a: Select third-generation bone marrow mesenchymal stem cells cultured in vitro, add mitochondrial separation reagent, homogenize the cells, and obtain a homogenate solution; b: Centrifuge the homogenate at low speed, collect the supernatant, and then centrifuge at high speed. The resulting precipitate is the mitochondria.

3. The method for preparing mitochondrial-derived biomimetic vesicles according to claim 2, characterized in that, b. The conditions for low-to-medium speed centrifugation are: 4℃, 500-700×g centrifugation for 10-30 minutes; The conditions for high-speed centrifugation are: 4℃, centrifugation at 10000-12000×g for 10-30 minutes.

4. The method for preparing mitochondrial-derived biomimetic vesicles according to claim 1, characterized in that, The drug mentioned in step S2 is dihydromyricetin, with a concentration of 100-300 μM.

5. The method for preparing mitochondrial-derived biomimetic vesicles according to claim 1, characterized in that, In step S2, the mass-to-volume ratio of mitochondria to the drug-containing PBS solution is 100 μg: 600-1000 μl.

6. The method for preparing mitochondrial-derived biomimetic vesicles according to claim 1, characterized in that, In step S2, the material is passed sequentially through polycarbonate membranes with pore sizes of 400 nm and 200 nm.

7. The method for preparing mitochondrial-derived biomimetic vesicles according to claim 1, characterized in that, The centrifugation conditions in S2 are 2000-4000g and 4℃.

8. The method for preparing mitochondrial-derived biomimetic vesicles according to claim 1, characterized in that, The mitochondrial-derived biomimetic vesicles have a particle size of 100-200 nm.

9. A mitochondrial-derived biomimetic vesicle, characterized in that, It was prepared using the method for preparing mitochondrial-derived biomimetic vesicles as described in any one of claims 1-8.

10. The use of the mitochondrial-derived biomimetic vesicles as described in claim 9 in the preparation of a medicament for treating osteoarthritis.

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