Exosome medicinal preparation for treating knee osteoarthritis and preparation method thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to an exosome medicine preparation for treating knee osteoarthritis and a preparation method thereof, and the exosome medicine preparation is characterized in that an exosome is derived from mesenchymal stem cells subjected to three-dimensional culture in a first hydrogel matrix containing sodium alginate and hyaluronic acid; flurbiprofen or a pharmaceutically acceptable salt or ester thereof; and a pharmaceutically acceptable second gel matrix. Sodium alginate and hyaluronic acid composite hydrogel serves as a three-dimensional culture carrier of mesenchymal stem cells, a joint microenvironment is simulated to induce stem cells to secrete high-activity exosomes rich in key functional nucleic acid and active protein, meanwhile, the exosomes are scientifically compounded with flurbiprofen, and synergistic mechanisms of anti-inflammatory analgesia and tissue repair are achieved; and a local slow-release delivery system is constructed through the second gel matrix, so that long-acting stable release of active ingredients is realized, the systemic side effect of the medicine is reduced, the bioavailability of the exosome is improved, and the problems of obvious side effect, limited repair effect and the like in the prior art are solved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an exosome drug formulation for treating knee osteoarthritis and its preparation method. Background Technology

[0002] Knee osteoarthritis (KA) is a chronic degenerative joint disease characterized by degenerative changes in articular cartilage, synovial inflammation, subchondral bone sclerosis, and osteophyte formation. It is a leading cause of pain and disability worldwide. Current clinical treatments mainly include oral nonsteroidal anti-inflammatory drugs (NSAIDs), intra-articular injections of sodium hyaluronate or corticosteroids, and ultimately, total joint replacement surgery. However, long-term use of oral NSAIDs can easily cause gastrointestinal, cardiovascular, and renal adverse reactions; while intra-articular corticosteroid injections can relieve symptoms in the short term, they may accelerate cartilage degeneration and the effect is not lasting; sodium hyaluronate mainly plays a lubricating and buffering role, with limited effect on tissue repair.

[0003] In recent years, mesenchymal stem cells (MSCs) have shown great promise in the treatment of tissue autoimmune diseases (KOA) due to their strong anti-inflammatory and regenerative repair potential. However, the direct application of stem cells faces challenges such as low survival rates, tumorigenic risks, immune rejection, and ethical and regulatory restrictions. Exosomes, as key mediators of paracrine function of stem cells, carry a variety of bioactive substances (such as proteins, nucleic acids, and lipids) from the mother cell. They possess functions similar to stem cells in regulating immunity, suppressing inflammation, and promoting tissue repair, while avoiding the risks of cell therapy, making them the core of a new generation of cell-free therapy strategies.

[0004] Flurbiprofen is a highly effective NSAID that exerts anti-inflammatory, analgesic, and antipyretic effects by inhibiting cyclooxygenase and reducing prostaglandin synthesis. It is a commonly used drug in clinical treatment of knee-on-a-lung (KOA) pain. However, formulations combining flurbiprofen with exosomes, which have repair functions, to achieve a synergistic effect of "anti-inflammatory and analgesic" and "tissue repair" have not yet been reported.

[0005] Furthermore, the activity of exosomes is highly dependent on the state of their source cells and culture conditions. Traditional two-dimensional planar culture cannot simulate the three-dimensional microenvironment in vivo, which may lead to decreased stem cell function and reduced therapeutic efficacy of exosomes. Three-dimensional culture, especially using biomimetic hydrogels (such as sodium alginate and hyaluronic acid), can better maintain the stemness of stem cells and promote the secretion of exosomes with higher biological activity.

[0006] In terms of delivery, local administration (such as intra-articular injection or transdermal administration) can deliver drugs directly to the lesion, increasing local drug concentration and reducing systemic exposure and side effects. Gel matrices are ideal local delivery carriers due to their good biocompatibility, sustained-release properties, and ease of local application.

[0007] Therefore, developing a novel formulation that combines highly active exosomes, classic anti-inflammatory drugs, and a suitable local delivery system is of great significance for improving the therapeutic effect of KOA, reducing side effects, and improving patients' quality of life. Summary of the Invention

[0008] This application provides an exosome drug formulation for treating knee osteoarthritis and its preparation method, in order to solve the problems of significant side effects and limited repair effects in the prior art.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The present invention provides an exosome pharmaceutical preparation for treating knee osteoarthritis, characterized in that the pharmaceutical preparation comprises: Exosomes, wherein the exosomes are derived from mesenchymal stem cells cultured in three dimensions in a first hydrogel matrix containing sodium alginate and hyaluronic acid; Flurbiprofen or its pharmaceutically acceptable salts or esters; and A pharmaceutically acceptable second gel matrix.

[0010] Furthermore, the second gel matrix is ​​selected from one or more of carbomer, poloxamer, cellulose derivatives, chitosan and its derivatives, polyacrylic acid resin, and polyvinyl alcohol.

[0011] Furthermore, the second gel matrix also includes a transdermal absorption enhancer, wherein the transdermal absorption enhancer is selected from at least one of azone, oleic acid, propylene glycol, and terpenoid compounds.

[0012] Furthermore, the concentration of the exosomes in the pharmaceutical preparation is from 1×10⁹ to 1×10¹² particles / gram of composition.

[0013] Further, the weight percentage of the flurbiprofen or its pharmaceutically acceptable salts or esters in the pharmaceutical formulation is 0.5% to 5.0%.

[0014] Furthermore, the mass ratio of the exosomes to the flurbiprofen, based on the protein mass of the exosomes, is 1:500 to 500:1.

[0015] Furthermore, the mesenchymal stem cells are mesenchymal stem cells derived from human umbilical cord or adipose tissue.

[0016] This invention also provides a method for preparing an exosome pharmaceutical formulation for treating knee osteoarthritis, characterized by comprising the following steps: S1: Human umbilical cord or adipose-derived mesenchymal stem cells are encapsulated in a first hydrogel microsphere composed of sodium alginate and hyaluronic acid and subjected to three-dimensional dynamic culture for approximately 48-120 hours. S2: Highly active exosomes are obtained by separation and purification from the culture supernatant of step S1 using size exclusion chromatography; S3: At a temperature below about 15°C, the exosomes obtained in step S2 are co-dispersed with flurbiprofen or its pharmaceutically acceptable salt or ester in the precursor solution of the second gel matrix, and gently stirred at a speed of 10-30 rpm to mix them evenly to obtain a homogeneous mixture. S4: The mixture obtained in step S3 undergoes a gelation process to solidify and form the second gel matrix, thereby obtaining the pharmaceutical formulation.

[0017] Furthermore, in step S3, the exosomes are pre-frozen-dried before dispersion and reconstituted with a flurbiprofen-containing gel precursor solution before use.

[0018] The present invention also provides the use of an exosome pharmaceutical preparation in the preparation of a medicament for treating knee osteoarthritis.

[0019] The beneficial effects achieved by using the present invention described above are as follows: 1. This invention utilizes a sodium alginate and hyaluronic acid composite hydrogel as a three-dimensional dynamic culture carrier for mesenchymal stem cells. This biomimetic matrix not only provides physical support for the cells, but its hyaluronic acid component can simulate the synovial fluid environment of joints, specifically activating receptor signaling pathways such as CD44 on the surface of stem cells, significantly upregulating their anti-inflammatory (e.g., increasing IL-10 and TGF-β secretion) and repair-promoting (e.g., promoting the expression of cartilage matrix-related genes) paracrine phenotypes. The resulting exosomes undergo fundamental optimization, being rich in key functional nucleic acids and active proteins such as miR-140-3p (targeting ADAMTS-5 to inhibit cartilage degradation) and miR-let-7a (regulating the TLR4 / NF-κB pathway to inhibit inflammation), thereby endowing them with highly efficient biological activity surpassing that of conventional two-dimensional cultured exosomes from the source. 2. This invention scientifically combines highly active exosomes optimized through three-dimensional culture with flurbiprofen, achieving a deep synergistic effect on their mechanisms of action. Flurbiprofen, by inhibiting COX enzymes, rapidly and effectively blocks the synthesis of inflammatory mediators such as prostaglandins, creating a favorable microenvironment with a "low inflammatory load" for exosomes to exert their effects and quickly relieving patient pain. Meanwhile, the optimized exosomes, through their carried bioactive substances, regulate macrophage polarization towards the anti-inflammatory M2 type from upstream, inhibiting the production of core inflammatory factors such as IL-1β and TNF-α, and directly acting on chondrocytes or progenitor cells, promoting their proliferation and the synthesis of extracellular matrix components such as type II collagen and proteoglycans, potentially significantly delaying disease progression. 3. This invention utilizes a second gel matrix to construct a local sustained-release delivery system, enabling the active ingredient to be retained for a long time in the joint cavity or local tissues. This achieves continuous and stable release of the drug and exosomes, avoiding the rapid systemic clearance and uneven distribution caused by oral or intravenous administration. More importantly, this local delivery method significantly reduces the systemic blood concentration of flurbiprofen, thereby effectively avoiding systemic side effects such as gastrointestinal mucosal damage, cardiovascular and renal risks commonly associated with traditional oral NSAIDs. Simultaneously, the gel matrix provides physical protection for the exosomes, reducing their degradation and inactivation in the physiological environment, further improving their bioavailability. 4. By adjusting the type and formulation of the second gel matrix, this invention can be flexibly adapted to different clinical scenarios. The transdermal formulation is suitable for patients with mild to moderate KOA, is non-invasive and convenient, and improves compliance; the injectable formulation is suitable for moderate to severe patients, and can achieve precise and high-concentration drug delivery within the lesion. This flexibility allows the treatment plan of this invention to cover a wider range of KOA patients. 5. The key process parameters in the preparation method of this invention are all designed with the goal of maximizing the integrity and bioactivity of exosomes. In particular, size exclusion chromatography purification can obtain high-purity exosomes, reducing the risk of immunogenicity caused by impurities; freeze-drying significantly improves the storage and transportation stability of exosomes as a raw material. The entire process is stable, controllable, and scalable, laying a solid foundation for product quality consistency and ultimate industrialization.

[0020] This solves the problems of obvious side effects and limited repair effects in existing technologies. Detailed Implementation

[0021] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0022] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0023] The following describes an exosome pharmaceutical preparation for treating knee osteoarthritis and its preparation method, according to embodiments of this application. Addressing the significant side effects mentioned in the background section, this application provides an exosome pharmaceutical preparation for treating knee osteoarthritis. In this method, a sodium alginate and hyaluronic acid composite hydrogel is used as a three-dimensional culture carrier for mesenchymal stem cells, simulating the joint microenvironment to induce stem cells to secrete highly active exosomes rich in key functional nucleic acids and active proteins. Simultaneously, these exosomes are scientifically compounded with flurbiprofen to achieve a synergistic mechanism of anti-inflammatory analgesia and tissue repair. A local sustained-release delivery system is constructed through a second gel matrix to achieve long-term stable release of the active ingredients, reducing systemic drug side effects, improving exosome bioavailability, and flexibly adapting to both transdermal and injectable dosage forms to cover patients with different conditions. Furthermore, the preparation process is stable and controllable, effectively solving the problems of significant side effects and limited repair effects of existing treatments, providing a more efficient and safer new solution for the treatment of knee osteoarthritis.

[0024] The present invention will be further described in conjunction with the following embodiments. Example

[0025] 1. Preparation of highly active exosomes (3D-Exo) Fourth-generation human adipose-derived mesenchymal stem cells (AD-MSCs) with cell viability >95% were obtained and resuspended in sterile PBS to a density of 5.0 × 10⁻⁶ cells / mL. 6 cells / mL.

[0026] Prepare a 2.0% (w / v) sodium alginate (SA) solution and a 1.0% (w / v) sodium hyaluronate (HA) solution (molecular weight 1.5 MDa), and mix them at a volume ratio of 1:1 to obtain a first hydrogel matrix precursor solution containing sodium alginate and hyaluronic acid.

[0027] The cell suspension and the precursor solution were mixed in equal volumes. Using an electrostatic microdroplet generator, the mixture was dropped into a coagulation bath containing 100 mM CaCl2 to crosslink and form a first hydrogel microsphere with a diameter of about 200 μm, thereby encapsulating mesenchymal stem cells.

[0028] The cell-loaded microspheres were placed in a 50 mL rotary culture flask, and serum-free culture medium containing 10% exosomes was added. The microspheres were then cultured in three dimensions at 37°C and 5% CO2 for 72 hours at a rotation speed of 15 rpm.

[0029] The culture supernatant was collected and initially clarified by differential centrifugation (300×g, 2,000×g, 10,000×g). It was then separated and purified by size exclusion chromatography (using a qEVoriginal / 70nm column). The exosome-rich fraction was collected to obtain highly active exosomes.

[0030] Nanoparticle tracking analysis (NTA) identified the obtained exosomes (labeled 3D-Exo) at a concentration of 3.5 × 10¹¹ particles / mL with an average particle size of 122 ± 18 nm. Western blotting analysis showed positive results for CD63 and TSG101.

[0031] 2. Preparation of transdermal gel ointment Weigh 1.2 g of carbomer 980 and slowly disperse it in 92.3 g of purified water. Swell overnight at room temperature to obtain precursor solution A of the second gel matrix.

[0032] Weigh out 1.0g of flurbiprofen ester, 2.5g of azone, and 5.0g of propylene glycol, mix them thoroughly to obtain mixture B.

[0033] Take a portion of the 3D-Exo suspension, mix it with 5% trehalose, and then freeze-dry it to obtain exosome lyophilized powder. Under ice-water bath conditions (4°C), take the equivalent of 1.0 × 10¹¹ exosome lyophilized powder, reconstitute it with 2 mL of pre-cooled mixture B, and gently vortex to mix. Under continuous ice bath conditions and gentle mechanical stirring at 20 rpm, slowly add the above mixture B containing exosomes and flurbiprofen ester to gel matrix A, and stir for 40 minutes to ensure uniform mixing and no air bubbles.

[0034] The pH of the mixture was adjusted to 6.5±0.1 with triethanolamine, and the system gradually thickened to form a transparent and homogeneous gel paste.

[0035] The resulting gel was dispensed into aluminum tubes and stored at 4°C in the dark, and labeled as formulation E1.

[0036] 3. Formulation Characterization Formulation E1 is a white, semi-transparent, homogeneous gel with a fine texture and good spreadability. The pH is 6.5. Calculations and verification show that the concentration of exosomes in formulation E1 is approximately 1.0 × 10⁻⁶. 9 The particle / g content of flurbiprofen ester is 1.0% (w / w), and the mass ratio of exosome protein to flurbiprofen ester is approximately 1:100. Example

[0037] 1. Exosome preparation: Same as step 1 in Example 1, using human umbilical cord-derived MSCs (UC-MSCs) to obtain a fresh suspension of 3D-Exo with a concentration of 4.0 × 10¹¹ particles / mL.

[0038] 2. Gel preparation Accurately weigh 19g of poloxamer 407 (P407) and 1g of poloxamer 188 (P188), add them to 80g of pre-cooled (4°C) water for injection, stir magnetically to dissolve, and let stand overnight at 4°C to obtain a 20% (w / v) poloxamer solution as the second gel matrix precursor solution.

[0039] At 4°C and 15 rpm, 1.25 mL of 3D-Exo suspension (containing 5.0 × 10¹¹ particles) and 0.5 g of flurbiprofen were added to 8.75 mL of the above solution, mixed well, and the volume was adjusted to 10 mL.

[0040] The mixture was dispensed into pre-cooled syringes to obtain formulation E2. This formulation is a sol at 4°C and gels within 2 minutes at 37°C. The exosome concentration is approximately 5.0 × 10¹. 0 particles / mL, flurbiprofen content is 0.5% (w / w).

[0041] 3. Formulation Characterization The resulting formulation E2 is a clear solution at 4°C and can gel in situ at body temperature. The calculated exosome concentration is approximately 5.0 × 10¹. 0 particles / mL, flurbiprofen content is 0.5% (w / w). Example

[0042] This embodiment is used to verify the in vitro bioactivity of the three-dimensional cultured exosomes and their preparations prepared in this invention.

[0043] 1. Exosome samples: 3D-Exo: Three-dimensional culture-derived exosomes prepared in Example 1.

[0044] 2D-Exo (control): After an equal number of AD-MSCs were statically cultured in a two-dimensional plane for 72 hours, exosomes were isolated and purified using the same method.

[0045] 2. Experimental Methods: An in vitro inflammation model was established by stimulating the human chondrocyte line C28 / I2 with human interleukin-1β (IL-1β, 10 ng / mL). Groups were set up as follows: ① Normal control group; ② IL-1β model group; ③ IL-1β + 2D-Exo group (final exosome protein concentration 20 μg / mL); ④ IL-1β + 3D-Exo group (final exosome protein concentration 20 μg / mL). Cells were co-cultured for 24 hours.

[0046] 3. Results: Inflammatory factor detection (ELISA): Compared with the 2D-Exo group, the 3D-Exo group significantly reduced the levels of TNF-α and IL-6 in the cell supernatant (by approximately 45% and 38%, respectively, P<0.01).

[0047] Repairing gene expression (qRT-PCR): Compared with the 2D-Exo group, the 3D-Exo group was able to more effectively promote the mRNA expression of type II collagen (COL2A1) and aggregate proteoglycan (ACAN) in chondrocytes (increasing by approximately 60% and 55%, respectively, P<0.01).

[0048] Conclusion: Exosomes obtained by dynamic culture in SA / HA three-dimensional hydrogels exhibit significantly better anti-inflammatory and cartilage matrix synthesis-promoting activities than exosomes derived from traditional two-dimensional cultures.

[0049] Example 4: Pharmacodynamic evaluation in animals This embodiment is used to verify the synergistic therapeutic effect of the drug formulation of the present invention in an animal model of knee osteoarthritis.

[0050] 1. Model establishment: SD rats were induced to develop a KOA model by a single injection of sodium iodoacetate (MIA, 3 mg / 50 μL) into the right knee joint cavity.

[0051] 2. Grouping and drug administration: Seven days after modeling, participants were randomly divided into 6 groups (n=8): Group A: Sham surgery group (injection of saline into the joint cavity).

[0052] Group B: Model control group (applied blank carbomer gel).

[0053] Group C: Flurbiprofen gel group (applied gel containing 1% flurbiprofen ester, without exosomes).

[0054] Group D: 2D-Exo gel group (apply gel containing 2D-Exo, concentration same as E1, without flurbiprofen).

[0055] Group E: 3D-Exo gel group (apply gel containing 3D-Exo, concentration same as E1, without flurbiprofen).

[0056] Group F: The combination formulation group of the present invention (formulation E1 prepared by applying Example 1).

[0057] Apply the medication around the affected knee once daily for 28 consecutive days.

[0058] 3. Evaluation Results: Pain behavior: At week 4 of treatment, the mechanical pain threshold recovery rate of group F (formulation E1) was significantly higher than that of the three monotherapy groups C, D, and E (P<0.05).

[0059] Histopathology: Joints were harvested after treatment for hematoxylin-eosin (H&E) and safranin O-Fix Green staining. The Osteoarthritis Research Society International (OARSI) score (0-24 points) was used.

[0060] OARSI scores were as follows: Group B (model group) 18.2±2.0; Group C (flurbiprofen monotherapy) 12.8±1.9; Group D (2D-Exo monotherapy) 11.5±1.7; Group E (3D-Exo monotherapy) 9.3±1.4; and Group F (formulation E1) 5.8±1.2. Group F's score was significantly lower than all monotherapy groups (P<0.01), indicating the most complete preservation of cartilage structure and the least loss of proteoglycans.

[0061] Joint synovial inflammatory factors: ELISA detection showed that the levels of IL-1β and PGE2 in the synovium of group F were significantly lower than those in groups C and E (P<0.05).

[0062] Conclusion: Animal experiments have demonstrated that the combination formulation (formulation E1) provided by this invention is significantly more effective than single-component treatment in relieving pain, inhibiting inflammation, and protecting cartilage, reflecting the synergistic therapeutic advantages of three-dimensional cultured exosomes and flurbiprofen.

[0063] Comparative Example 1: Comparative formulation of exosomes derived from two-dimensional culture A comparative formulation differs from Example 1 only in the source of exosomes: an equal number of AD-MSCs are seeded in cell culture flasks and subjected to routine two-dimensional static culture for 72 hours. The subsequent exosome isolation, purification, and formulation preparation steps are exactly the same as in Example 1, resulting in comparative formulation D1.

[0064] Expected / Experimental Comparison Results: In the in vitro experiments of Example 3, the anti-inflammatory and repair-promoting effects of formulation D1 were significantly lower than those of formulation E1. In the animal experiments of Example 4, the OARSI score and pain relief effect of the formulation D1 group were also significantly worse than those of the formulation E1 group. This demonstrates that specific three-dimensional culture conditions are key to the high activity of exosomes in this invention.

[0065] Comparative Example 2: Flurbiprofen-free one-component exosome gel A comparative formulation differs from Example 1 only in that it does not contain flurbiprofen ester. To ensure consistency in total weight and properties, the mass of flurbiprofen ester is replaced with an equal amount of carbomer and purified water. The amount of exosomes and other steps remain unchanged, resulting in comparative formulation D2.

[0066] Expected / Experimental Comparison Results: In the animal experiment of Example 4, the pain relief rate and extent in the formulation D2 (single exosome) group were inferior to those in the formulation E1 (combination formulation) group during the initial stage of treatment. This confirms that flurbiprofen's rapid anti-inflammatory and analgesic effects create a favorable environment for exosome repair, and the two have a synergistic effect.

[0067] Comparative Example 3: Comparative formulation prepared by high-temperature and high-speed mixing process A comparative formulation, the preparation method of which differs from that of Example 1 only in the mixing conditions of step (2): at room temperature (25°C), the mixture of exosomes and drugs B is mixed with gel matrix A by high-speed stirring at 200 rpm, and the remaining steps are exactly the same to obtain the comparative formulation D3.

[0068] Expected / Experimental Results: Immediately after preparation, the NTA particle count of exosomes in formulation D3 decreased by more than 30% compared to formulation E1, and the in vitro chondrocyte proliferation-promoting activity was lost by more than 40%. The therapeutic effect was also significantly reduced in animal experiments. This demonstrates that failure to follow the specific low-temperature, mild mixing process of this invention will severely damage the integrity of exosomes and lead to loss of activity, highlighting the non-obviousness and criticality of this process step.

[0069] Comparative Example 4: Contrast gel without transdermal penetration enhancer A comparative formulation differs from Example 1 only in that it does not contain any transdermal absorption enhancers (azone, propylene glycol). Replacing these excipients with an equal volume of purified water yields comparative formulation D4.

[0070] Expected / Experimental Comparison Results: In vitro transdermal assays using the Franz diffusion cell showed that the cumulative permeation of flurbiprofen ester in formulation D4 was less than 50% of that in formulation E1 within 24 hours. This indicates that transdermal permeation enhancers are key excipients for ensuring effective delivery of the active ingredient.

[0071] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, and substitutions to the above embodiments within the scope of the present invention.

Claims

1. An exosome pharmaceutical preparation for treating knee osteoarthritis, characterized in that, The pharmaceutical preparation includes: Exosomes, wherein the exosomes are derived from mesenchymal stem cells cultured in three dimensions in a first hydrogel matrix containing sodium alginate and hyaluronic acid; Flurbiprofen or its pharmaceutically acceptable salts or esters; and A pharmaceutically acceptable second gel matrix.

2. The exosome pharmaceutical preparation for treating knee osteoarthritis according to claim 1, characterized in that, The second gel matrix is ​​selected from one or more of carbomer, poloxamer, cellulose derivatives, chitosan and its derivatives, polyacrylic acid resin, and polyvinyl alcohol.

3. The exosome pharmaceutical preparation for treating knee osteoarthritis according to claim 1, characterized in that, The second gel matrix further includes a transdermal absorption enhancer, wherein the transdermal absorption enhancer is selected from at least one of azone, oleic acid, propylene glycol, and terpene compounds.

4. The exosome pharmaceutical preparation for treating knee osteoarthritis according to claim 1, characterized in that, The concentration of the exosomes in the pharmaceutical preparation is 1×10⁻⁶. 9 Up to 1×10 12 Particles / gram of composition.

5. An exosome pharmaceutical preparation for treating knee osteoarthritis according to claim 1, characterized in that, The weight percentage of flurbiprofen or its pharmaceutically acceptable salts or esters in the pharmaceutical preparation is from 0.5% to 5.0%.

6. The exosome pharmaceutical preparation for treating knee osteoarthritis according to claim 1, characterized in that, The mass ratio of the exosomes to the flurbiprofen, based on the protein mass of the exosomes, is 1:500 to 500:

1.

7. The exosome pharmaceutical preparation for treating knee osteoarthritis according to claim 1, characterized in that, The mesenchymal stem cells mentioned are mesenchymal stem cells derived from human umbilical cord or adipose tissue.

8. A method for preparing an exosome pharmaceutical formulation for treating knee osteoarthritis according to any one of claims 1-7, characterized in that, Includes the following steps: S1: Human umbilical cord or adipose-derived mesenchymal stem cells are encapsulated in a first hydrogel microsphere composed of sodium alginate and hyaluronic acid and subjected to three-dimensional dynamic culture for approximately 48-120 hours. S2: Highly active exosomes are obtained by separation and purification from the culture supernatant of step S1 using size exclusion chromatography; S3: At a temperature below about 15°C, the exosomes obtained in step S2 are co-dispersed with flurbiprofen or its pharmaceutically acceptable salt or ester in the precursor solution of the second gel matrix, and gently stirred at a speed of 10-30 rpm to mix them evenly to obtain a homogeneous mixture. S4: The mixture obtained in step S3 undergoes a gelation process to solidify and form the second gel matrix, thereby obtaining the pharmaceutical formulation.

9. The method for preparing an exosome pharmaceutical preparation for treating knee osteoarthritis according to claim 8, characterized in that, In step S3, the exosomes are pre-freeze-dried before dispersion and reconstituted with a flurbiprofen-containing gel precursor solution before use.

10. Use of the exosome pharmaceutical preparation according to any one of claims 1-7 in the preparation of a medicament for treating knee osteoarthritis.