Preparation method of sTREM2-loaded exosome microspheres and application of sTREM2-loaded exosome microspheres in treatment of osteoarthritis

Abstract

The invention provides a preparation method of microspheres loaded with sTREM2 exosomes and an application of the microspheres loaded with the sTREM2 exosomes in treatment of osteoarthritis (OA). After the sTREM2 exosome is prepared, the sTREM2 exosome is loaded by adopting biocompatible microspheres, and then modification is carried out to enable the microspheres to carry positive charges, so that exosome internalization is promoted. According to the invention, the sTREM2 is loaded in the exosome for the first time and is prepared into the methacrylic acid esterified hyaluronic acid microspheres (HAMA), so that local administration and continuous release of the sTREM2 are realized, and the concentration and bioavailability of the sTREM2 in the joint are improved; through PLL modification, the microspheres are positively charged, so that the binding capacity with the surfaces of cartilage cells is enhanced, and the cellular uptake efficiency and the treatment effect are improved. The S-Exo (at) HAMA (at) PLL microspheres provided by the invention have good biocompatibility and safety, have long preservation time and stable drug release effect in vivo, and can significantly relieve OA symptoms and inhibit cartilage matrix degradation and synovial inflammation.

Description

Technical Field

[0001] The present invention belongs to the technical field of osteoarthritis treatment, and relates to a drug for treating osteoarthritis, specifically to a hyaluronic acid microsphere-loaded sTREM2 exosome and its preparation method, and also relates to its application in the treatment of osteoarthritis. Background Art

[0002] Osteoarthritis (OA) is a chronic degenerative joint disease characterized by articular cartilage degeneration, synovial inflammation, and bone changes. Its pathogenesis is complex, involving processes such as chondrocyte degeneration, chronic inflammation, joint space narrowing, and subchondral bone changes. [1] Currently, the treatment of OA still mainly relies on non-steroidal anti-inflammatory drugs (NSAIDs) and local drug injections, but these treatment methods can only relieve symptoms and cannot prevent the progression of the disease. [4-5] .

[0003] Traditional drug delivery methods (such as oral administration and intravenous injection) have problems such as low drug bioavailability, gastrointestinal side effects, and cardiovascular risks. [6-7] Especially when injecting drugs into the joint, due to the rapid clearance effect of synovial capillaries and the existence of the cartilage barrier, it is difficult for drugs to stay in the joint cavity for a long time, which limits the treatment effect. [7-8] .

[0004] Exosomes (Exo) are small vesicles secreted by various cell types, carrying proteins, RNA, and other active molecules, capable of transmitting signals between cells, regulating inflammatory responses, and promoting tissue repair. [12-13] In the treatment of osteoarthritis, exosomes can relieve inflammation and improve cartilage repair by regulating the polarization state of macrophages.

[15] However, although exosomes, as important mediators of intercellular communication, play an important role in tissue repair, they face problems such as low drug delivery efficiency and insufficient targeting in clinical applications.

[11] , the inefficient delivery and degradation of exosomes in the joint are still a major challenge in their application, such as (1) the low local delivery efficiency of exosomes and poor stability in the joint cavity; (2) existing treatment methods cannot effectively prevent the progression of OA and only play a role in relieving symptoms; (3) there are drug side effects during the treatment process, and frequent drug administration is required, increasing the treatment burden on patients. Summary of the Invention

[0005] The present invention is directed to the above problems and provides a microsphere loaded with sTREM2 exosomes (S-EXOs), a preparation method thereof, and an application in the treatment of osteoarthritis. Injecting the microsphere into the joints of OA mice aims to regulate the immune response in the joints, promote cartilage repair, and slow down the progression of osteoarthritis (OA) by targeted delivery of S-EXOs. This technology combines the biocompatibility of hyaluronic acid microspheres, the immunomodulatory effect of S-EXOs, and an innovative microsphere structure design to solve the problems of low drug delivery efficiency and poor persistence of therapeutic effects in existing OA treatments.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] I. Product composition:

[0008] The core product of the present invention is a complex of microspheres loaded with sTREM2 exosomes, which mainly includes the following components:

[0009] 1. Microspheres

[0010] Prepared from biocompatible materials such as hyaluronic acid (HAMA), chitosan, sodium alginate, etc.

[0011] Function: As a drug carrier, it provides the delivery function to support exosomes, and at the same time improves the residence time and stability of exosomes in the joint cavity.

[0012] In a preferred embodiment of the present invention, hyaluronic acid microspheres (HAMA microspheres) are used as the drug carrier. High-molecular-weight hyaluronic acid provides higher biological stability and is more suitable for sustained-release delivery; the surface can be modified with targeting molecules (sTrem2) to enhance the targeting ability; it has good biocompatibility. Therefore, hyaluronic acid (HA) microspheres as a drug carrier can provide long-term drug release, promote local treatment, and at the same time have good biocompatibility and biodegradability. In addition, hyaluronic acid microspheres can maintain the stability of exosomes through the property of water absorption and swelling, avoiding rapid clearance in the joints.

[0013] 2. sTREM2 exosomes

[0014] Function: sTREM2 (Triggering Receptor Expressed on Myeloid Cells 2) is an innate immune receptor. By regulating macrophage polarization, it can polarize from the pro-inflammatory M1 type to the anti-inflammatory M2 type, thereby reducing the inflammatory response in the joints and promoting cartilage repair.

[0015] Function: sTREM2 improves its stability and local delivery efficiency in the joint cavity by binding to exosomes loaded with hyaluronic acid microspheres. The innovation of sTREM2 in this invention is that it can precisely regulate the polarization and inflammatory response of macrophages through the exosome delivery system, solving the problem of immune response disorder in OA.

[0016] 3. Positive charge modification

[0017] The microspheres are given a positive charge, thereby increasing their affinity to the surface of chondrocytes. Since chondrocytes are usually negatively charged, the modification of the positive charge on the surface of the microspheres can increase the adsorption capacity of the microspheres and promote the internalization of exosomes.

[0018] Select other positively charged molecules or polymers as modifiers, such as poly-lysine (PLL), polyethyleneimine (PEI), etc.

[0019] In a preferred embodiment of the present invention, polylysine (PLL) is selected as a modifier. The modification of polylysine can effectively enhance the binding force between microspheres and chondrocytes, ensuring efficient delivery of drugs at the target site.

[0020] 2. Preparation process

[0021] The preparation method of the sTREM2-loaded exosome microspheres of the present invention is summarized as follows: after preparing sTREM2 exosomes, biocompatible microspheres are used for loading, and then the microspheres are modified so that they carry a positive charge to promote internalization of the exosomes.

[0022] The preparation steps are as follows:

[0023] A. Preparation of sTREM2 exosomes

[0024] The preparation method is as follows: exosomes are collected using synovial mesenchymal stem cells, and sTREM2 protein is incubated with the exosomes. The specific steps are as follows:

[0025] (1-1) Cultivate cells using a high-concentration exosome-specific culture medium (preferably FBS culture medium in which exosomes have been removed by ultracentrifugation) and collect the culture fluid;

[0026] (1-2) Exosome isolation: centrifugation at 300 × g for 10 min to remove cells, centrifugation at 2000 × g for 20 min to remove cell debris, and ultracentrifugation at 100,000 × g for 70 min to precipitate exosomes;

[0027] (1-3) Exosome washing: resuspend the pellet with PBS and repeat the ultracentrifugation step to remove impurities;

[0028] (1-4) Exosome storage: Resuspend the exosomes in an appropriate amount of PBS and store at -80°C.

[0029] The incubation temperature of sTREM2 protein with exosomes is 37 °C.

[0030] B. Microsphere loading

[0031] Prepare micron-sized microspheres using biocompatible materials, then mix sTREM2 exosomes with the microspheres and incubate at 37 °C for 1 - 2 hours to promote exosome adsorption or encapsulation. By adjusting the ratio of exosome concentration to hyaluronic acid microspheres, ensure a high loading efficiency, and then separate the unloaded exosomes by centrifugation or dialysis.

[0032] Among them, the biocompatible material is as described above and is selected from any one of hyaluronic acid, chitosan, and sodium alginate;

[0033] The diameter of the micron-sized microspheres is 1 - 10 μm, which is suitable for injection into local tissues or as a sustained-release carrier, or is mostly used for local inflammation treatment or soft tissue filling. The preparation method is as follows: Prepare a solution with a concentration of 1 - 3 mg / mL, add a cross-linking agent, and form microspheres under stirring. In a preferred embodiment of the present invention, use a cross-linking agent (such as glutaraldehyde or EDC / NHS) to react with hyaluronic acid and form HA microspheres under stirring. During the reaction process, optimize the microsphere size by adjusting the reaction conditions (such as pH, temperature).

[0034] C. Positive charge modification

[0035] Select a positively charged molecule or polymer as a modifier. After preparing a solution with a concentration of 0.01 - 0.1% (w / v), add the microspheres loaded with exosomes into it, gently stir or rotate to mix evenly, carry out the modification reaction at room temperature for 30 minutes to 1 hour, then remove the unbound modifier by centrifugation or dialysis, and wash with sterile PBS.

[0036] The results of SEM, TEM, and NTA identification and observation show that exosomes can effectively transport sTREM2 protein; XPS analysis of hyaluronic acid microspheres loaded with sTREM2 exosomes shows that the main components of the synthesized material are carbon, oxygen, and nitrogen.

[0037] The results of the endocytosis experiment showed that the hyaluronic acid microspheres loaded with sTREM2 exosomes could be effectively endocytosed by target cells (chondrocytes, BMDMs, RAW246.7), and it was time-dependent. The longer the time, the better the endocytosis effect. The results of the hemolysis experiment showed that the hemolytic properties of the hyaluronic acid microspheres loaded with sTREM2 exosomes at different concentrations were good, and it was concentration-dependent, ensuring their blood compatibility in biomedical applications. By detecting the expression of macrophage polarization markers, it was shown that the hyaluronic acid microspheres loaded with sTREM2 exosomes regulated the polarization of macrophages into M1 type (pro-inflammatory) or M2 type (anti-inflammatory), providing a mechanistic basis for their application in immunomodulation and the treatment of inflammatory diseases. At the same time, by analyzing the expression levels of different markers, the potential role of the hyaluronic acid microspheres loaded with sTREM2 exosomes in macrophage function remodeling was clarified.

[0038] Based on the experimental results, the present invention also provides the use of the above-mentioned microspheres loaded with sTREM2 exosomes in the preparation of a therapeutic drug for osteoarthritis.

[0039] In addition, the present invention further provides a pharmaceutical composition for treating osteoarthritis, comprising an active ingredient and a pharmaceutically acceptable excipient, wherein the active ingredient is selected from the above-mentioned microspheres loaded with sTREM2 exosomes.

[0040] In terms of the drug form, the pharmaceutical composition of the present invention can be selected from injection preparations, perfusion preparations, oral preparations, etc., and a local injection preparation is preferred. When administering the drug, in addition to local injection, other administration methods can also be considered, such as intra-articular perfusion, oral administration, etc. (although oral administration may face problems such as reduced bioavailability, it may be used as an alternative in some cases).

[0041] This pharmaceutical composition can be used alone or in combination with other therapeutic drugs for osteoarthritis.

[0042] The innovation of the technical solution of the present invention is as follows:

[0043] The present invention first loaded sTREM2 into exosomes and prepared them into hyaluronic acid microspheres, realizing the local administration and sustained release of sTREM2, and improving its concentration and bioavailability in the joint.

[0044] Through PLL modification, the microspheres were positively charged, enhancing their binding ability to the surface of chondrocytes and improving the cell uptake efficiency and therapeutic effect.

[0045] The S-Exo@HAMA@PLL microspheres provided by the present invention have good biocompatibility and safety, have a long preservation time and a stable drug release effect in vivo, and can significantly relieve OA symptoms, inhibit cartilage matrix degradation and synovial inflammation.

[0046] The technical solution of the present invention has the following advantages:

[0047] Social benefits: By improving the OA treatment strategy, it is possible to reduce the treatment frequency of patients, reduce drug side effects, reduce medical costs, improve the quality of life of patients, and reduce the social and economic burden of the disease.

[0048] Economic benefits: It reduces drug waste and side effects in OA treatment and lowers the overall cost of treatment.

[0049] Technical benefits: The present invention provides an innovative exosome delivery system, which significantly improves the stability and delivery efficiency of exosomes in joints, thereby more effectively regulating the immune response and promoting cartilage repair. Brief Description of the Drawings

[0050] Figure 1 Shows the AEM observation results of the material;

[0051] Figure 2 Shows the TEM and NTA identification of hyaluronic acid microspheres loaded with sTREM2 exosomes. Among them, the upper figure is SMCs-Exo, and the lower figure is SMCs-Exo-sTREM2.

[0052] Figure 3 Shows the results of XPS analysis of hyaluronic acid microspheres loaded with sTREM2 exosomes.

[0053] Figure 4 Shows the endocytosis of chondrocytes to hyaluronic acid microspheres loaded with sTREM2 exosomes.

[0054] Figure 5 Shows the endocytosis of RAW264.7 to hyaluronic acid microspheres loaded with sTREM2 exosomes.

[0055] Figure 6 Shows the endocytosis of BMDM to hyaluronic acid microspheres loaded with sTREM2 exosomes.

[0056] Figure 7 Shows the hemolysis of hyaluronic acid microspheres loaded with sTREM2 exosomes at different concentrations.

[0057] Figure 8 Shows the effect of hyaluronic acid microspheres loaded with sTREM2 exosomes on macrophage polarization. Detailed Description of the Invention

[0058] The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include a detailed description of traditional methods, such methods are well known to those of ordinary skill in the art and are described in many publications.

[0059] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to persons skilled in the art. In addition, any methods and materials similar or equivalent to those described may be applied to the present invention, and the preferred methods and materials described in the specific embodiments are for illustrative purposes only.

[0060] Example 1 Preparation of S-Exo@HAMA@PLL Microspheres

[0061] 1. Extraction of Exosomes

[0062] (1) Cell culture:

[0063] Culture cells expressing sTREM2. It is recommended to use a special medium for high-concentration exosomes (such as a medium obtained by ultracentrifuging FBS to remove exosomes), and collect the culture solution.

[0064] (2) Isolation of exosomes:

[0065] a) Centrifuge to remove cell debris and large particulate matter:

[0066] Centrifuge at 300×g for 10 minutes to remove cells.

[0067] Centrifuge at 2000×g for 20 minutes to remove cell debris.

[0068] b) Extract exosomes by ultracentrifugation:

[0069] Centrifuge at 100,000×g for 70 minutes to precipitate exosomes.

[0070] c) Wash exosomes:

[0071] Resuspend the precipitate with PBS and repeat the ultracentrifugation step to remove impurities.

[0072] d) Storage:

[0073] Resuspend the exosomes in an appropriate amount of PBS and store at -80°C.

[0074] 2. Loading sTREM2 Exosomes onto Hyaluronic Acid Microspheres

[0075] (1) Preparation of hyaluronic acid microspheres:

[0076] Solution preparation: Prepare a hyaluronic acid solution (1–3 mg / mL).

[0077] Crosslinking reaction: Use a crosslinking agent (such as glutaraldehyde or EDC / NHS) to react with hyaluronic acid to form microspheres under stirring. During the reaction, optimize the microsphere size by adjusting the reaction conditions (such as pH, temperature).

[0078] The particle size of hyaluronic acid microspheres is preferably 1-10 µm, which is suitable for injection into local tissues or as a sustained-release carrier, or is mostly used for local inflammation treatment or soft tissue filling.

[0079] (2)sTREM2-loaded exosomes:

[0080] Co-incubation: Mix sTREM2 exosomes with hyaluronic acid microspheres and incubate at 37°C for 1-2 hours to promote exosome adsorption or encapsulation.

[0081] Optimization conditions: Adjust the ratio of exosome concentration to hyaluronic acid microspheres to ensure high loading efficiency.

[0082] Separation and purification: Separate unloaded exosomes by centrifugation or dialysis.

[0083] 3. Polylysine modification

[0084] (1)Solution preparation: Prepare a 0.01–0.1% (w / v) polylysine solution.

[0085] (2)Modification process:

[0086] Surface modification: Add hyaluronic acid microspheres loaded with exosomes to the polylysine solution, gently stir or rotate to mix evenly, and react at room temperature for 30 minutes to 1 hour.

[0087] Washing: Remove unbound polylysine by centrifugation or dialysis, and wash with sterile PBS.

[0088] Confirm the quality and content of exosomes through techniques such as electron microscopy and nanoparticle tracking analysis (NTA). The results are as Figure 1 and Figure 2 shown. Hyaluronic acid microspheres (HAMA), microspheres loaded with sTREM2 exosomes (S-Exo&HAMA), and the final S-Exo@HAMA@PLL microspheres are all round. The particle size of sTREM2 exosomes is concentrated around 100 nm. The particle size distribution range of sTREM2-loaded exosomes becomes slightly wider, within the range of 100-200 nm, and the number of particles per milliliter of solution also decreases accordingly. This shows that the method of the present invention can ensure the quality and quantity of exosomes, ensure their effective transport of sTREM2 protein, and enable them to play an immunomodulatory role in the joint.

[0089] The main components of the synthesized material (S-Exo@HAMA@PLL microspheres) were analyzed by X-ray photoelectron spectroscopy (XPS), and the results showed carbon, oxygen, and nitrogen ( Figure 3 ). This indicates that after PLL modification, it helps to improve the affinity between the microspheres and chondrocytes, enhance the internalization efficiency of exosomes, and ensure that exosomes can stay in the joint cavity for a long time.

[0090] Example 2: S-Exo@HAMA@PLL microspheres have excellent internalization effect in chondrocytes

[0091] The material of hyaluronic acid microspheres loaded with sTREM2 exosomes was co-incubated with chondrocytes, BMDMs, and RAW246.7. Serum-free medium was selected as the culture medium to avoid interference of serum components with endocytosis. The experiment was carried out in a culture environment of 37 °C and 5% CO 2 to observe the endocytosis effect at the same time with incubation times of 6 and 24 h. DiI fluorescence-labeled exosomes were used in the experiment.

[0092] The results are shown in Figures 4 - 6 . Hyaluronic acid microspheres loaded with sTREM2 exosomes can be effectively internalized by target cells and show time-dependence. The longer the incubation time, the better the internalization effect. This experiment provides data support for exploring its delivery efficiency and downstream effects in cells. Through endocytosis research, the biological activity of the material delivering sTREM2 exosomes can be verified, providing a theoretical basis for its application in immunomodulation and arthritis treatment.

[0093] Example 3: Different concentrations of hyaluronic acid microspheres loaded with sTREM2 exosomes have excellent hemolysis performance

[0094] Hyaluronic acid microspheres loaded with sTREM2 exosomes at different concentrations (12.5, 25, 50, and 100 μg / mL) were respectively co-incubated with a certain volume of fresh anticoagulated whole blood at 37 °C for 3 hours. PBS treatment was set as the positive control. After incubation, centrifugation was performed to separate, and the supernatant was collected and the color change was observed. Further, the hemolysis rate was measured by a spectrophotometer, and the absorbance (OD value) of the supernatant was detected at a wavelength of 540 nm.

[0095] The results are shown in Figure 7 . The hemolytic properties of hyaluronic acid microspheres loaded with sTREM2 exosomes at different concentrations are not significantly different, ensuring their blood compatibility in biomedical applications. By measuring the hemolysis rate, its concentration-dependent hemolytic characteristics can be judged, providing data support for subsequent dose selection and safety assessment.

[0096] Example 4: Hyaluronic acid microspheres loaded with sTREM2 exosomes have a polarization regulation effect on macrophages

[0097] Macrophages (RAW264.7 and BMDM) were cultured and treated with hyaluronic acid microspheres loaded with sTREM2 exosomes. After treatment, total cellular RNA was extracted, cDNA was generated by reverse transcription, and the mRNA expression levels of polarization-related markers, including M1-type polarization markers (CD86, iNOS) and M2-type polarization markers (CD163, IL-10), were detected by qRT-PCR. The experiments included an untreated group, a hyaluronic acid microsphere control group, and a blank vector exosome group to verify the specificity and effect of the experimental treatment.

[0098] The results are as Figure 8 shown. By detecting the expression of macrophage polarization markers, hyaluronic acid microspheres loaded with sTREM2 exosomes promoted the transformation of macrophages from the M1 type (pro-inflammatory) to the M2 type (anti-inflammatory), providing a mechanistic basis for their application in immunomodulation and the treatment of inflammatory diseases, and also clarifying their potential role in macrophage function remodeling.

[0099] The references cited in the background art are as follows:

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[0103] [4] Pelletier JP, Martel-Pelletier J, Rannou F, Cooper C. Efficacy and safety of oral NSAIDs and analgesics in the management of osteoarthritis: evidence from real-life setting trials and surveys. Semin Arthritis Rheum. 2016;45(4 Suppl):S22–S27. doi:10.1016 / j.semarthrit.2015.11.009

[0104] [5] Alcaraz MJ, Guillén MI, Ferrándiz ML. Emerging therapeutic agents in osteoarthritis. Biochem Pharmacol. 2019;165:4–16. doi:10.1016 / j.bcp.2019.02.034

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[10] Glyn-Jones S, Palmer AJ, Agricola R, et al. Osteoarthritis. Lancet. 2015;386(9991):376–387. doi:10.1016 / s0140-6736(14)60802-3

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[15] Fang C, Zhong R, Lu S, Yu G, Liu Z, Yan C, Gao J, Tang Y, Wang Y, Zhao Q, Feng X. TREM2 promotes macrophage polarization from M1 to M2 and suppresses osteoarthritis through the NF-κB / CXCL3 axis. Int J Biol Sci. 2024 Mar 11;20(6):1992-2007. doi: 10.7150 / ijbs.91519. PMID: 38617547; PMCID: PMC11008261.

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[16] Hui Yuan, Pengcheng Xiao, Bruno Sarmento, Gang Chen, Wenguo Cui, Synovial Lubrication Factors‐Recruiting Biomimetic Polyphenolized Hyaluronic Acid for Promoting Cartilage Repair. Advanced Functional Materials, 2024. doi: 10.1002 / adfm.202406668.

[0116] The parts not described in this invention are the same as the prior art or are implemented using the prior art. The applicant declares that this invention uses the above embodiments to illustrate the detailed method of this invention, but this invention is not limited to the above detailed method, that is, it does not mean that this invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement to this invention, the equivalent substitution of each raw material of the products of this invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and the public disclosure scope of this invention.

Claims

1. A method for preparing exosome microspheres loaded with sTREM2, characterized in that: The method comprises the following steps: after preparing sTREM2 exosomes, biocompatible microspheres are used for loading, and then the microspheres are modified so that they carry a positive charge to promote the internalization of the exosomes.

2. The method for preparing sTREM2-loaded exosome microspheres according to claim 1, characterized in that: The preparation steps are as follows: A. Preparation of sTREM2 exosomes The preparation was carried out by the following method: exosomes were collected using synovial mesenchymal stem cells, and sTREM2 protein was incubated with the exosomes; B. Microsphere loading Micrometer-sized microspheres were prepared using biocompatible materials, and then sTREM2 exosomes were mixed with the microspheres and incubated at 37°C to promote exosome adsorption or encapsulation; C. Positive charge modification Select positively charged molecules or polymers as modifiers, prepare a solution of corresponding concentration, add the microspheres loaded with exosomes, gently stir or rotate to mix, carry out the modification reaction at room temperature, and then remove the unbound modifiers.

3. The preparation method according to claim 2, characterized in that: in, In step A, the steps of exosome extraction are as follows: (1-1) Cultivating synovial mesenchymal stem cells using a high-concentration exosome-specific culture medium and collecting the culture fluid; (1-2) Exosome isolation: centrifugation at 300 × g for 10 min to remove cells, centrifugation at 2000 × g for 20 min to remove cell debris, and ultracentrifugation at 100,000 × g for 70 min to precipitate exosomes; (1-3) Exosome washing: resuspend the pellet with PBS and repeat the ultracentrifugation step to remove impurities; (1-4) Exosome storage: resuspend the exosomes in an appropriate amount of PBS and store at -80°C. The conditions for incubation of sTREM2 protein and exosomes were as follows: the co-incubation was performed at a temperature of 37°C.

4. The preparation method according to claim 3, characterized in that: in, The high-concentration exosome-specific culture medium is selected from a culture medium obtained by removing exosomes from FBS by ultracentrifugation.

5. The preparation method according to claim 2, characterized in that: in, In step B, the biocompatible material is selected from any one of hyaluronic acid, chitosan, and sodium alginate; The diameter of the micron-sized microspheres is 1-10 μm, and the preparation method is as follows: a solution with a concentration of 1-3 mg / mL is prepared, a cross-linking agent is added, and microspheres are formed under stirring; The co-incubation time of sTREM2 exosomes and microspheres was 1 to 2 hours.

6. The preparation method according to claim 2, characterized in that: in, In step C, the modifier is selected from poly-lysine or polyethyleneimine, and the w / v concentration is 0.01–0.1%; The modification reaction time at room temperature is 30 minutes to 1 hour; Remove unbound modifier by centrifugation or dialysis and wash with sterile PBS.

7. A microsphere loaded with sTREM2 exosomes, characterized in that The method is prepared by any one of claims 1 to 6.

8. Use of the microspheres loaded with sTREM2 exosomes according to claim 7 in the preparation of drugs for the treatment of osteoarthritis.

9. A pharmaceutical composition for treating osteoarthritis, characterized in that: It comprises an active component and a pharmaceutically acceptable excipient, wherein the active component is selected from the microspheres loaded with sTREM2 exosomes as described in claim 7.

10. The pharmaceutical composition for treating osteoarthritis according to claim 9, characterized in that: The pharmaceutical composition is used in combination with other osteoarthritis therapeutic drugs.

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