A method for targeted modification of Expi293 cell exosomes to deliver siMMP13

Abstract

The present invention discloses a method for delivering siMMP13 to chondrocytes by targeting peptide-modified Expi293 cell exosomes, comprising the following steps: separating and purifying Expi293 cell exosomes, labeling Expi293 cell exosomes, coupling DSPE-CAP polypeptide with Expi293 cell exosomes in a targeted manner, and then loading siMMP13 into the targeted modified Expi293 cell exosomes by electroporation. The present invention targets and modifies Expi293 cell exosomes to deliver siMMP13 through a chemical modification scheme, has a simple preparation method, high targeting, good aggregation effect at damaged parts, and realizes efficient drug release. Targeted delivery of siMMP13 can inhibit the metabolic decomposition of cartilage matrix, thereby inhibiting the progression of osteoarthritis, and is a drug delivery system with high safety, high targeting, and a wide range of applications.

Description

Technical Field

[0001] The present invention relates to a method for targeted modification of exosomes, and in particular to a method for targeted modification of Expi293 cell exosomes for delivery of siMMP13. Background Art

[0002] Exosomes are small membrane vesicles that can be secreted by a variety of cells. Their diameter is between 30-150nm. The contents are a variety of non-coding RNAs, proteins, and nucleic acids. They release their contents by binding to receptor cells to exert different biological functions. Exosomes are stable in nature and have a long half-life in the body. They can cross biological barriers to reach target cells and be taken up by receptor cells in different ways. They can act as biomarkers or drug delivery carriers. They can also be used in combination with traditional viral vectors to form a more powerful gene therapy tool. As donor cells for exosomes, Expi293 cells have a short proliferation cycle and can be cultured in serum-free high-density suspension. They can provide a large amount of stable exosomes to meet the needs of clinical trials.

[0003] Matrix metalloproteinase 13 (MMP13) is a key enzyme for the cleavage of type II collagen and a major decomposition factor involved in cartilage degradation. In situ injection of siRNA targeting MMP13 into joint tissue reduces the production of MMP13 protein and the expression of pro-inflammatory genes in joint tissue, thereby inhibiting the degeneration of articular cartilage and the formation of osteophytes, achieving the effect of treating osteoarthritis.

[0004] CN113018265A provides a method for encapsulating miRNA-140 in targeted exosomes for treating osteoarthritis. It is obtained by transfecting CAP-lamp2b recombinant plasmid into mouse dendritic cells to obtain cartilage-targeted exosomes, and encapsulating miRNA-140 into targeted exosomes by electroporation. However, the targeted exosomes obtained by cell engineering have high requirements for cell culture technology and equipment, and the production process is numerous and time-consuming. CN113274421A discloses an anti-inflammatory exosome preparation for knee joint treatment, a preparation method and an application. However, when adipose mesenchymal stem cells cultured in vitro age or differentiate, the exosomes obtained therefrom will also have corresponding functional reductions. In addition, the culture cost of adherent cells is high, the yield of exosomes is low, and there is the possibility of serum contamination during cell culture. Summary of the invention

[0005] Based on the above background technology, the technical problem to be solved by the present invention is to provide a method for targeted modification of Expi293 cell exosomes to deliver siMMP13, that is, utilizing the property of distearoylphosphatidylethanolamine (DSPE) as a lipid that can be inserted into the lipid bilayer membrane of exosomes, inserting affinity peptide (CAP) lipid polypeptide into the surface of the exosome membrane, and establishing an Expi293 cell exosome system so that it has a cartilage-targeted delivery effect.

[0006] The first aspect of the present invention provides a method for targeted modification of Expi293 cell exosomes, the method comprising the following steps:

[0007] (1) Preparation of DSPE-CAP: connecting DSPE and the affinity peptide CAP that has a targeting effect on chondrocytes;

[0008] (2) Preparation of chondrocyte-targeted exosomes DSPE-CAP-Exo: incubate the exosomes of Expi293 cells with the DSPE-CAP described in step (1) to collect the chondrocyte-targeted exosomes DSPE-CAP-Exo component.

[0009] The affinity peptide CAP has the sequence shown as DWRVIIPPRPSAC.

[0010] Preferably, before step (1), the method may further include step (a) extracting and characterizing exosomes from Expi293 cells;

[0011] Preferably, the step (a) specifically comprises: obtaining Expi293 cell exosomes from Expi293 cells;

[0012] Preferably, step (2) specifically comprises: incubating the exosomes of Expi293 cells with the DSPE-CAP described in step (1), removing the DSPE-CAP polypeptide not bound to the exosome membrane by a SEC column, and collecting the liquid of components 4-12 to obtain the exosomes targeting chondrocytes;

[0013] Preferably, the pH of the incubation is 7-9, more preferably 8;

[0014] Preferably, the incubation temperature is 4°C-40°C, more preferably 25-30°C;

[0015] Preferably, the incubation time is 10 minutes to 16 hours;

[0016] Preferably, the incubation is at 4°C for 16 hours or the incubation is at 30°C for 30 minutes.

[0017] The second aspect of the present invention provides a chondrocyte-targeted exosome DSPE-CAP-Exo, which is prepared according to the method for targeted modification of Expi293 cell exosomes described in the first aspect.

[0018] The third aspect of the present invention provides a method for constructing exosomes that target chondrocytes and knock down MMP13, the method comprising electroporating siRNA targeting MMP13 into the chondrocyte-targeting exosome DSPE-CAP-Exo described in the second aspect, so that the siRNA is loaded onto the exosomes.

[0019] Preferably, the exosomes DSPE-CAP-Exo are added to the electroporation buffer, and siMMP13 is added at the same time, mixed, and electroporated; then, the free siMMP13 that is not loaded into the exosomes is removed by SEC column, and the 4-12 components are collected to obtain the exosomes loaded with siMMP13.

[0020] Preferably, the electrotransfer buffer is 1 mM EDTA;

[0021] Preferably, a GenePulser Xcell electroporator (Biorad) is used, and the electroporation voltage parameters are set to 450 V and 120 ms;

[0022] Preferably, the exosome aggregates formed during the electroporation process can be vigorously blown by a pipette and then placed at 22°C for 2 hours.

[0023] The fourth aspect of the present invention provides an exosome DSPE-CAP-Exo / siMMP13 that targets chondrocytes and knocks down MMP13, which is prepared according to the exosome construction method described in the third aspect.

[0024] The fifth aspect of the present invention provides the use of the exosomes DSPE-CAP-Exo targeting chondrocytes as described in the second aspect or the exosomes DSPE-CAP-Exo / siMMP13 targeting chondrocytes and knocking down MMP13 as described in the fourth aspect in the preparation of a drug for treating osteoarthritis.

[0025] The sixth aspect of the present invention provides a pharmaceutical composition for treating osteoarthritis, which comprises the exosomes DSPE-CAP-Exo targeting chondrocytes as described in the second aspect or the exosomes DSPE-CAP-Exo / siMMP13 targeting chondrocytes and knocking down MMP13 as described in the fourth aspect.

[0026] Compared with the prior art, the present invention has the following beneficial effects: the present invention realizes the therapeutic potential of siRNA drugs, and efficiently and targetedly delivers drugs for the treatment of osteoarthritis; the Expi293 cells in the present invention are adapted to serum-free high-density suspension culture, and can be used for large-scale and standardized production of exosomes, thus solving the batch differences of exosomes from the source; compared with the targeted exosomes obtained by genetically engineering cells, the method of coupling exosomes with targeting peptides by DSPE used in the present invention is simple, and the loading efficiency of the targeting peptides is high, and the biosafety is high; the exosomes of the present invention can be used to treat osteoarthritis, can significantly inhibit the expression of MMP13, slow down the cartilage damage process in the osteoarthritis animal model, play a cartilage protection role, and have a positive effect on the treatment of osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Flow chart of the construction of a novel exosome delivery system for Expi293 cells using the chemical modification method;

[0028] Figure 2 Identification and characterization of the targeted delivery system of Expi293 cell exosomes: (A) Western blot detection of related protein expression after Expi293 cell exosome modification (B) Nanoparticle size analyzer detection of the particle size of Expi293 cell exosomes before and after modification (C) Electron microscopy identification of the morphology of Expi293 cell exosomes before and after modification;

[0029] Figure 3 This is a graph showing the efficiency of DSPE-CAP-Exo targeting chondrocytes;

[0030] Figure 4 This is a diagram for evaluating the knockdown effect of siMMP13 in chondrocytes;

[0031] Figure 5 This is a diagram showing the efficiency of DSPE-CAP-Exo targeted delivery of siMMP13 to chondrocytes and the knockdown effect;

[0032] Figure 6 To construct osteoarthritis in vitro and evaluate the effect of DSPE-CAP-Exo-siMMP13;

[0033] Figure 7 This is a diagram showing the effect of drug-loaded exosomes on cell phenotype;

[0034] Figure 8 This is the in vivo metabolic evaluation diagram of DSPE-CAP-Exo;

[0035] Fig. 9 This is a diagram for evaluating the tissue targeting effect of DSPE-CAP-Exo;

[0036] Fig.10HE staining of visceral organs (heart, liver, spleen, lung, and kidney) of DSPE-CAP-Exo;

[0037] Fig.11 This is the DSPE-CAP-Exo liver and kidney biochemical assay diagram;

[0038] Fig.12 This is a diagram of the in vivo therapeutic effect of DSPE-CAP-Exo / siMMP13. DETAILED DESCRIPTION

[0039] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with the examples, which are only used to explain the present invention and do not limit the scope of protection. The instruments, equipment, consumables and reagents used are all commercially available products unless otherwise specified.

[0040] Example 1

[0041] Step 1: Obtaining Expi293 exosomes

[0042] Expi293 cells were inoculated into a 250ml culture flask at 1E+06 cells / ml. When the density reached 4E+06 cells / ml, the supernatant was collected. Centrifuge at 500g for 10min at 4℃ to remove the cell precipitate. Collect the supernatant and centrifuge at 3000g for 20min at 4℃ to remove cell debris. Collect the supernatant and centrifuge at 10000g for 30min at 4℃. Collect the supernatant and pass it through a 0.22um filter membrane. Centrifuge the filtered supernatant at 130000g for 70min at 4℃ and discard the supernatant. Add 36ml PBS to the white precipitate at the bottom and mix well. Centrifuge at 130000g for 70min at 4℃, discard the supernatant, resuspend the exosomes with about 500ul PBS, and collect and place in a -80℃ refrigerator for later use.

[0043] Step 2: Cartilage-targeted modification of Expi293 exosomes to obtain DSPE-CAP-Exo

[0044] First, weigh 5 mg of DSPE-PEG-Mal (purchased from Merck) and add 200 ul of anhydrous ethanol to dissolve it; at the same time, use 1 ml of Tris•HCl with a pH value of 6.8-7.4 to dissolve the CAP-Csy peptide (DWRVIIPPRPSAC, synthesized by Nanjing Peptide Industry Co., Ltd.), add it to the DSPE solution, and use Tris•HCl solution to make up the system to 5 mL. Wrap the reaction tube with tin foil and place it on a shaker to mix overnight. The next day, transfer the solution to the dialysis membrane, tie the two ends of the dialysis membrane with rubber bands, and after confirming that it is correct, put it into a large beaker filled with 2L of deionized water and an appropriate amount of anhydrous ethanol, and place the beaker on a magnetic stirrer to stir overnight to obtain the DSPE-CAP polypeptide.

[0045] Secondly, 100 ug of purified exosomes were incubated with DSPE-CAP peptide (0.23 ug / mL) at 37°C for 30 min, and the DSPE-CAP peptide that was not bound to the exosome membrane was removed by SEC column. Fractions 4-12 were collected to obtain DSPE-CAP peptide-modified cartilage-targeted exosomes (DSPE-CAP-Exo).

[0046] Example 2

[0047] Experimental process: (A) Western blot detection of related protein expression after exosome modification in Expi293 cells

[0048] 100ul of SDS-PAGE protein loading buffer (1x) (biosharp) was added to Expi293 cells or Expi293 cell exosomes, and after brief vortexing, the proteins were lysed at 95°C for 10min. The protein concentrations extracted from cells and exosomes were determined using Nano Drop. Protein lysates (20 μg) of Expi293 cells and their exosomes were taken, protein markers and protein samples were added to each lane, and proteins were separated using 15% Bis-Tris gel (Bio-Rad), and electrophoresed at 190V constant voltage for 60min. The protein gel was then electrotransferred to a 0.2-μm PVDF (Milipore) membrane, and the membrane was transferred at 275mA constant flow for 70min. 1% BSA was added for blocking, incubated on a shaker at room temperature for 1 hour, and then incubated overnight at 4°C in the primary antibody diluted in 1% BSA blocking buffer. The primary antibodies used were as follows: Anti-CD9 (ab134045, Abcam), Anti-CD81 (ab92726, Abcam), Anti-HSP70 (ab9485, Abcam), Anti-Calnexin antibody. Afterwards, rinse in 1xTBST for 6 times, 5 min each time, and then put the PVDF membrane into the secondary antibody diluted in 1% BSA and incubate at room temperature for 1 hour. After the incubation, rinse in 1xTBST for 6 times, 5 min each time, at room temperature (RT), and add developer for exposure. Figure 2 As shown in A, WB detected the expression of CD9, CD81, HSP70, and CANX in Expi293 cells and Expi293 cell exosomes.

[0049] (B) Nanoparticle size analyzer was used to detect the particle size of Expi293 cell exosomes before and after modification.

[0050] The Nanocoulter instrument was used to detect nanovesicles. For each sample, the number of particles in the diluent was detected before the test. If the number of particles recorded in 60s was less than 5, 200ul was added to the detection well after the exosomes were diluted, and the size of 100 particles was recorded. The results are shown in Figure 2As shown in B, the diameter of engineered exosomes DSPE-CAP-Exo is slightly larger than that of Exo.

[0051] (C) Electron microscopy was used to detect the morphology of exosomes before and after transformation.

[0052] 1% glutaraldehyde was used to fix the exosomes. 5-10ul of the exosome solution was added to the copper mesh and adsorbed at room temperature for about 10 minutes. The excess liquid was carefully removed with filter paper. Then 10ul of 2% phosphotungstic acid solution (pH=6.5) was added to the copper mesh. The exosomes were stained at room temperature for 2 minutes. The excess staining solution was carefully removed with filter paper. The copper mesh was dried at room temperature. The images were imaged by TEM (FEI, Hillsboro, OR, USA) with an observation voltage of 120kV. Figure 2 C shows representative TEM images of Exo and DSPE-CAP-Exo.

[0053] Example 3

[0054] Experimental process: The targeting effect of the obtained cartilage-targeted exosomes (DSPE-CAP-Exo) was verified by in vitro experiments. answer In vitro cell experiments used DiI-labeled exosomes to compare the affinity effects of CAP-targeted exosomes (DSPE-CAP-Exo) and non-targeted exosomes (Exo) on chondrocytes. The specific steps are as follows:

[0055] Exosomes (Exo) isolated from Expi293 cells / exosomes with cartilage targeting (DSPE-CAP-Exo) were incubated with 1 mM DiI dye at a volume ratio of 400:1 at 37° in the dark for 30 min. The free unbound dye was removed by SEC column to obtain DiI-labeled exosomes.

[0056] A cell slide was laid on the bottom of a 12-well plate, and 8w rat chondrocytes were added to each well. The plate was incubated at 37°C overnight. 20ug Exo / DSPE-CAP-Exo was added to the 12-well plate. After 6, 8, and 12h, the cell culture medium was discarded. The plate was washed 3 times with PBS and fixed with 4% paraformaldehyde for 20min. The plate was washed 3 times with PBS, 500ul DMEM was added and a drop of Hochest stain was added. The plate was incubated at 37°C for 30min. A drop of anti-quenching reagent was added to the slide, the slide was fixed, and the efficiency of cell uptake of exosomes was detected using confocal FV1000. The results are shown in Figure 2. Figure 3 As shown, in the DSPE-CAP-Exo treatment group, a significant DiI signal was observed, while a small amount of DiI signal was observed in the Exo treatment group, indicating that the CAP sequence can more efficiently guide exosomes to specifically bind and enter chondrocytes.

[0057] Example 4

[0058] In vitro experiments verified the knockdown effect of siMMP13 on MMP13 in rat chondrocytes.

[0059] Step 1: Digest rat chondrocytes and prepare single cell suspension, count them, and seed them in a 12-well plate at 6w cells / well. Transfection is performed when the cell confluence reaches 50%-70%.

[0060] Step 2: Prepare the siMMP13-Lipofectamine 2000 complex as follows:

[0061] a: Add 2.5ul, 5ul, and 7.5ul of siMMP13 (20uM) into 100 μl of serum-free and dual antibody-free DMEM culture medium, and mix gently;

[0062] b: Add 2 μl Lipofectamine 2000 (Invitrogen) into 100 μl serum-free and dual antibody-free DMEM medium, mix gently and incubate at room temperature for 5 minutes;

[0063] c: Add solution a to solution b, pipette gently to mix (200ul in total), and incubate at room temperature for 20 minutes.

[0064] Step 3: Remove the old culture medium in the dish, wash twice with PBS, and replace with DMEM medium without double antibody containing 10% fetal bovine serum, 1 ml per well;

[0065] Step 4: Evenly add 200 μl of siMMP13-Lipofectamine 2000 complex (i.e., ab mixture) to each well and shake gently;

[0066] Step 5: Total RNA was extracted from cells using Trizol reagent (Invitrogen), and RNA was quantified to 1ug for reverse transcription (Nanjing Novizan Biotechnology Co., Ltd., MR101-02). 2X Universal SYBR GreenFast qPCR Mix (ABclonal, RM21203) was used, and GAPDH was used as the internal control. The 2-ΔΔCt method was used to calculate the relative expression of RNA and quantify the expression of mRNA. The results showed that the siMMP13 sequence we screened had a good knockdown effect ( Figure 4 ).

[0067] Example 5

[0068] The targeted delivery and intracellular function of the obtained siMMP13 in chondrocytes were verified by in vitro experiments. In vitro cell experiments used FAM-labeled siMMP13 and DiI-labeled exosomes to explore whether the chondrocyte-targeted exosomes (DSPE-CAP-Exo) obtained in Example 1 delivered siMMP13 to chondrocytes more effectively, and to detect whether the delivered siMMP13 played a role in chondrocytes. The specific steps are as follows:

[0069] The exosomes DSPE-CAP-Exo and Exo obtained in Example 1 were loaded with FAM-labeled siMMP13 by electroporation. The exosomes were added to 100ul electroporation buffer (1 mM EDTA), and 2ug of siMMP13 was added at the same time. They were gently mixed and placed in a 100ul electroporation tube. GenePulser Xcell electroporator (Biorad) was used, and the electroporation voltage parameters were set to 450 V and 120ms, and the electroporation was performed once. Immediately put it on ice for 5 minutes. The exosome aggregates formed during the electroporation process can be vigorously blown by a pipette, and then placed at 22°C for 2 hours. The free siMMP13 not loaded into the exosomes was removed by SEC column, and the 4-12 components were collected to obtain exosomes loaded with siMMP13 (DSPE-CAP-Exo / siMMP13 and Exo / siMMP13).

[0070] A cell slide was laid on the bottom of a 12-well plate, and 8w rat chondrocytes were added to each well and incubated overnight at 37°C. The prepared DSPE-CAP-Exo / siMMP13 and Exo / siMMP13 were added to the cells at 4h and 6h, respectively, and the cell culture medium was discarded. Wash with PBS three times and fix with 4% paraformaldehyde for 20min. Wash with PBS three times, add 500ul DMEM and drop a drop of Hochest stain, and incubate at 37°C for 30min. Drop a drop of anti-quenching reagent on the slide, fix the slide, and use confocal FV1000 to detect the efficiency of cell uptake of exosomes. The experimental results are as follows: Figure 5 As shown in A, significant FAM and DiI signals were observed in the DSPE-CAP-Exo / siMMP13-treated group, but very few in the Exo / siMMP13-treated group. The two signals overlapped, suggesting that si-MMP13 and exosomes colocalized in chondrocytes.

[0071] Next, we tested whether the siMMP13 delivered in chondrocytes was functional. 1.5×105 cells were seeded in a 6-well plate per well and placed in a 37°C incubator overnight. The prepared DSPE-CAP-Exo / siMMP13 and Exo / siMMP13 were added to the cells. After 24 hours, RNA was collected and the expression level of MMP13 gene was detected by RT-qPCR. After 48 hours, protein was collected to detect the expression level of MMP13 protein. Figure 5 As shown in Figures B and 5C, the DSPE-CAP-Exo / siMMP13 group treatment was able to effectively reduce the intracellular MMP13 level. In contrast, the efficiency of knocking down MMP13 in the non-targeted exosome Exo / siMMP13 treatment group was low.

[0072] This study designed siRNA sequences and primer sequences required for qPCR

[0073]

[0074] Example 6

[0075] Inducing osteoarthritis model in vitro, exploring the DSPE-CAP-Exo / siMMP13 prepared in Example 5 in vitro model Based on the effect of knocking down MMP13.

[0076] Rat chondrocytes were plated at 8E+04 cells / well and treated with 50ng / ml Rat-IL-1β (SinoBiological). After 24h, the expression of MMP13, COL2A1 and Aggrecan genes was detected. The experimental results showed that after IL-1β treatment of cells, the expression of COL2A1 and Aggrecan genes decreased, and the expression of MMP13 gene increased. At the same time, CCK8 results showed that after IL-1β treatment of chondrocytes, cell viability decreased, which confirmed that the in vitro osteoarthritis model (OA) was successfully established ( Figure 6 A and 6B). Based on the successful construction of the in vitro OA model, the prepared DSPE-CAP-Exo / siMMP13 and Exo / siMMP13 were added to the cells, and the MMP13 knockdown effect was detected by qPCR combined with WB. The results showed that based on the successful induction of the in vitro OA model, compared with Exo / siMMP13, the DSPE-CAP-Exo / siMMP13 group treatment was more effective in reducing the expression of MMP13 in chondrocytes ( Figure 6 C and 6D).

[0077] Example 7

[0078] Through in vitro experiments, Study on the cytotoxicity of DSPE-CAP-Exo / siMMP13 prepared in Example 5 and its improvement Functions of inflammatory cells. Experimental process: Rat chondrocytes were plated at 8E+04 cells / well and treated with 50ng / ml Rat-IL-1β (SinoBiological) for 24h. The prepared DSPE-CAP-Exo / siMMP13 and Exo / siMMP13 were co-cultured with chondrocytes for 24h to detect the effect on the cell phenotype of chondrocytes. The results are as follows: Figure 7 As shown in AC, after IL-1β treatment of chondrocytes, cell migration ability and cell viability decreased, and the level of cell apoptosis increased. However, this effect can be effectively reversed by co-culturing chondrocytes with DSPE-CAP-Exo / siMMP13 and Exo / siMMP13. At the same time, compared with the lipo-treated group, the DSPE-CAP-Exo / siMMP13 and Exo / siMMP13-treated groups were less toxic to cells and could more effectively restore cell migration ability and cell viability.

[0079] Example 8

[0080] Experimental process: The targeting effect of DSPE-CAP-Exo was verified through in vivo experiments. In vivo experiments used DiR-labeled DSPE-CAP-Exo, a fluorescent dye that highlights the lipid membrane of exosomes. DiR-labeled DSPE-CAP-Exo was injected into rat joints via intra-articular injection, and its distribution in rats was monitored by animal live imaging at 4h, D1, D2, D3, D4, D5, and D6 after administration ( Figure 8 A). The fluorescence signal of DiR showed that after intra-articular injection, Exo without CAP showed a wider distribution and spread to other parts of the body ( Figure 8 BC). After D6, the main organ Exo was found to be enriched in the kidneys in addition to the joint cavity. However, DSPE-CAP-Exo remained in the joint cavity. Therefore, chondrocyte targeting limited the labeled exosomes after intra-articular injection without significantly spreading to other organs, showing the effect of targeted delivery in clinical trials ( Fig. 9 The rats were dissected on day 6, and HE staining showed that DiR, CAP, DiR+Exo, and DSPE-CAP-Exo were non-toxic to major organs ( Fig.10 ), blood biochemical test showed that different treatment groups had no effect on the renal function of rats, but had slight damage to the liver function ( Fig.11 ).

[0081] Example 9

[0082] Evaluation of the effects of DSPE-CAP-Exo / siMMP13 in animal experiments. The DSPE-CAP-Exo / siMMP13 obtained in Example 5 was injected into the bone joint cavity of the arthritis animal model. Among them, regarding the arthritis animal model, this project selected 6-week-old SD rats and constructed a knee OA model using medial meniscus instability (DMM) surgical modeling. Since the DMM surgical model has become the gold standard for studying the onset and progression of post-traumatic osteoarthritis (OA), we adopted this model and developed OA rats.

[0083] DMM surgical approach:

[0084] Under sterile conditions, the medial approach to the knee joint was used, and a 5mm longitudinal incision was made 2mm medial to the patella and tibial plateau. The subcutaneous muscles and soft tissues were bluntly separated, and the joint capsule was opened with ophthalmic scissors. The infrapatellar fat pad was bluntly separated. After the patella was dislocated, the medial meniscus tibial ligament could be directly seen with the naked eye. The medial meniscus tibial ligament was cut with a microsurgery knife, the joint cavity was flushed with saline, the patella was repositioned, the damaged ligament was sutured and repaired, and then the joint capsule was closed, and the joint capsule, muscles, fascia, and skin were sutured layer by layer.

[0085] OA treatment effect detection: Tissue sections were used to detect cartilage defects. Two weeks after DMM surgery, PBS, Exo / siMMP13, and DSPE-CAP-Exo / siMMP13 were injected into the joint cavity of osteoarthritis model rats through the knee joints. Four weeks after drug injection, the rats were euthanized by carbon dioxide, and the bilateral knee joints were obtained. After fixation with 4% paraformaldehyde for 48 hours, the samples were placed in EDTA alkaline solution for decalcification for two months. The samples were dehydrated, embedded, and sliced ​​for H&E staining ( Fig.12 A), Toluidine blue staining ( Fig.12 B) and safranin fast green staining ( Fig.12 C). The results suggest that DSPE-CAP-Exo / siMMP13 has a better therapeutic effect on OA compared with PBS and Exo / siMMP13 (see Fig.12 AC).

[0086] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for constructing exosomes that target chondrocytes and knock down MMP13, characterized in that: The siRNA targeting MMP13 is electrotransferred into the exosome DSPE-CAP-Exo targeting chondrocytes, so that the siRNA is loaded onto the exosome; the sequence of the siRNA is shown as GGUCUGGUCUGAUGUGACA; The preparation method of the chondrocyte-targeted exosome DSPE-CAP-Exo comprises the following steps: (1) Preparation of DSPE-CAP: connecting DSPE and an affinity peptide CAP having a targeting effect on chondrocytes; the sequence of the affinity peptide CAP is shown as DWRVIIPPRPSAC; (2) Incubate the Expi293 cell exosomes with the DSPE-CAP described in step (1) to collect the chondrocyte-targeted exosome DSPE-CAP-Exo component.

2. The method for constructing exosomes targeting chondrocytes and knocking down MMP13 according to claim 1, characterized in that: Before step (1), the method further includes step (a) extraction and characterization of exosomes from Expi293 cells.

3. The method for constructing exosomes targeting chondrocytes and knocking down MMP13 according to claim 2, characterized in that: The step (a) specifically includes: obtaining the Expi293 cell exosomes from Expi293 cells.

4. The method for constructing exosomes targeting chondrocytes and knocking down MMP13 according to claim 1, characterized in that: Step (2) specifically includes: incubating the exosomes of Expi293 cells with the DSPE-CAP described in step (1), removing the DSPE-CAP polypeptide not bound to the exosome membrane through a SEC column, and obtaining exosomes targeting chondrocytes.

5. The method for constructing exosomes targeting chondrocytes and knocking down MMP13 according to claim 4, characterized in that: The pH of the incubation is 7-9.

6. The method for constructing exosomes targeting chondrocytes and knocking down MMP13 according to claim 5, characterized in that: The pH of the incubation was 8.

7. The method for constructing exosomes targeting chondrocytes and knocking down MMP13 according to claim 4, characterized in that: The incubation temperature is 4°C-40°C.

8. The method for constructing exosomes targeting chondrocytes and knocking down MMP13 according to claim 7, characterized in that: The incubation temperature is 25-30°C.

9. The method for constructing exosomes targeting chondrocytes and knocking down MMP13 according to claim 4, characterized in that: The incubation time is from 10 minutes to 16 hours.

10. The method for constructing exosomes targeting chondrocytes and knocking down MMP13 according to claim 4, characterized in that: The incubation condition is 4° C. for 16 hours or the incubation condition is 30° C. for 30 minutes.

11. The method for constructing exosomes targeting chondrocytes and knocking down MMP13 according to claim 1, characterized in that: The exosomes DSPE-CAP-Exo were added to the electroporation buffer, and siRNA targeting MMP13 was added at the same time, mixed, and electroporated; then the free siRNA not loaded into the exosomes was removed by SEC column to obtain the exosomes loaded with siRNA targeting MMP13.

12. The method for constructing exosomes targeting chondrocytes and knocking down MMP13 according to claim 11, characterized in that: The electroporation buffer is 1 mM EDTA.

13. The method for constructing exosomes targeting chondrocytes and knocking down MMP13 according to claim 11, characterized in that: GenePulser Xcell electroporator was used, and the electroporation voltage parameters were set to 450 V and 120 ms.

14. The method for constructing exosomes targeting chondrocytes and knocking down MMP13 according to claim 11, characterized in that: The exosome aggregates formed during the electroporation process were vigorously blown away by a pipette and then placed at 22°C for 2 h.

15. An exosome DSPE-CAP-Exo / siMMP13 targeting chondrocytes and knocking down MMP13, characterized in that: It is prepared according to the method for constructing exosomes targeting chondrocytes and knocking down MMP13 according to any one of claims 1 to 14. 16 . Use of the exosome DSPE-CAP-Exo / siMMP13 targeting chondrocytes and knocking down MMP13 as claimed in claim 15 in the preparation of a drug for treating osteoarthritis. 17 . A pharmaceutical composition for treating osteoarthritis, comprising the exosome DSPE-CAP-Exo / siMMP13 targeting chondrocytes and knocking down MMP13 as claimed in claim 15 .

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