Methods to induce fibroblasts to exhibit an immunosuppressive phenotype

Abstract

This invention belongs to the field of biomedicine and relates to a method for inducing fibroblasts to exhibit an immunosuppressive phenotype. Specifically, this invention relates to a method for inducing fibroblasts to exhibit an immunosuppressive phenotype, comprising the following steps: treating fibroblasts with (1) IFN-γ or (2) IFN-γ and rapamycin. This invention also relates to cell membrane vesicle nanoparticles and their uses. The cell membrane vesicles or cell membrane-encapsulated nanoparticles of this invention can effectively treat or prevent arthritis, especially rheumatoid arthritis, and have good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a method for inducing fibroblasts to exhibit an immunosuppressive phenotype. This invention also relates to cell membrane vesicle nanoparticles and their uses. Specifically, the cell membrane vesicles are cell membrane vesicles of synovial fibroblasts. Background Technology

[0002] Rheumatoid arthritis (RA) is a common autoinflammatory disease characterized by persistent synovitis in multiple joints, ultimately leading to bone destruction. (1) It is reported that the incidence of rheumatoid arthritis (RA) in adults is approximately 0.5-1.0%, and it is one of the leading causes of decreased labor capacity and disability. (2) The pathogenesis of rheumatoid arthritis (RA) is complex, and there is currently no cure. Persistent inflammation is the core of RA pathogenesis; therefore, controlling inflammation is the cornerstone of RA treatment. Although recent years have seen significant improvements in RA treatment outcomes due to in-depth research and the emergence of various new targeted drugs, including biologic DMARDs (bDMARDs) such as TNF-α and IL-6 inhibitors, and targeted synthetic DMARDs (tsDMARDs) such as JAK inhibitors, the development of various new targeted therapies has greatly improved the treatment outcomes of RA. (3) However, even with adequate dosage and duration of existing medications, more than 10% of RA patients still experience progressive inflammation, ultimately leading to joint destruction and severely impacting their quality of life. (4) Meanwhile, the widespread immunosuppression caused by long-term use of existing drugs also poses risks to patients, such as infection and tumors. Therefore, exploring new, safer, and more effective targets for inflammation control is of great significance for improving the prognosis of RA patients. (5) .

[0003] Fibroblast-like synoviocytes (FLS) are the main cells in the inflamed synovium of rheumatoid arthritis (RA). Recent studies have revealed that FLS are an important factor in the persistence of RA inflammation. (6) RA-FLS promotes abnormal activation of T cells in the joints, exhibiting a "pro-inflammatory phenotype," characterized by high expression of IL-6 (an important inflammatory factor that promotes Th17 differentiation and is involved in the pathogenesis of RA). (7) It is the main source of intra-articular IL-6 in RA inflammation. (8) Promote T cell survival (9) They also play other roles. However, in tumors and physiological inflammation, fibroblasts also exhibit an immunosuppressive phenotype that inhibits activated T cells, manifested by high expression of PD-L1, FASL, IDO, etc., participating in inflammation relief, maintenance of peripheral immune tolerance, and tumor immune escape. (10,11) Previous studies have shown that inflammatory factors have a regulatory effect on fibroblast phenotype. (12) .

[0004] Cell membrane-coated nanoparticles are an emerging technology in the field of nanomedicine in recent years, providing novel and highly targeted therapeutic strategies for complex diseases. (13,14) By encapsulating the cell membrane, various known and unknown molecules from the membrane surface are completely transferred to the surface of nanoparticles, mimicking the characteristics of the source cell and modulating multiple target sites. The encapsulated cell membrane can homologously fuse with the source cell, achieving targeted drug delivery. (15) In rheumatoid arthritis (RA), studies have already utilized the cell membranes of activated macrophages, platelets, and endothelial cells to create vesicles, encapsulate nanoparticles, and evaluate their therapeutic potential in animal models. (16-18) .

[0005] Currently, there is still a need to develop new treatments for RA. Summary of the Invention

[0006] Through in-depth research and creative work, the inventors have developed a method for inducing fibroblasts to exhibit an immunosuppressive phenotype, and obtained treated synovial fibroblasts. Using the treated synovial fibroblasts, the inventors extracted cell membrane vesicles and further prepared cell membrane-encapsulated nanoparticles. The inventors surprisingly discovered that the cell membrane vesicles or cell membrane-encapsulated nanoparticles can effectively treat or prevent arthritis, especially rheumatoid arthritis, and have promising application prospects. Therefore, the following invention is provided:

[0007] One aspect of the present invention relates to a method for inducing fibroblasts to exhibit an immunosuppressive phenotype, comprising the following steps:

[0008] Fibroblasts were treated with (1) IFN-γ or (2) IFN-γ and rapamycin.

[0009] In some embodiments of the present invention, the method for inducing fibroblasts to exhibit an immunosuppressive phenotype, wherein the final concentration of IFN-γ in (1) or (2) is 50-150 ng / ml, preferably 80-120 ng / ml, 90-110 ng / ml or 95-105 ng / ml, more preferably 100 ng / ml.

[0010] In some embodiments of the present invention, the method for inducing fibroblasts to exhibit an immunosuppressive phenotype includes a final concentration of rapamycin of 5-15 μM / L, preferably 8-12 μM / L or 9-11 μM / L, and more preferably 10 μM / L.

[0011] In some embodiments of the present invention, the method for inducing fibroblasts to exhibit an immunosuppressive phenotype includes a treatment time of 6-48 hours, preferably 12-36 hours or 18-30 hours, and more preferably 24 hours.

[0012] In some embodiments of the present invention, the method for inducing fibroblasts to exhibit an immunosuppressive phenotype, wherein the fibroblasts are synovial fibroblasts; preferably, the synovial fibroblasts are mammalian synovial fibroblasts, more preferably human synovial fibroblasts or mouse synovial fibroblasts.

[0013] In some embodiments of the present invention, the method for inducing fibroblasts to exhibit an immunosuppressive phenotype is a method for treating fibroblasts (e.g., synovial fibroblasts).

[0014] Another aspect of the present invention relates to a fibroblast obtained by the method described in any one of the present invention for inducing fibroblasts to exhibit an immunosuppressive phenotype. The obtained synovial fibroblast is an immunosuppressive synovial fibroblast. Preferably, the fibroblast is a synovial fibroblast; more preferably, the synovial fibroblast is a mammalian synovial fibroblast, more preferably a human synovial fibroblast or a mouse synovial fibroblast.

[0015] Another aspect of the invention relates to a cell membrane vesicle obtained from the synovial fibroblasts of the present invention.

[0016] Another aspect of the present invention relates to a cell membrane-encapsulated nanoparticle, which is obtained by encapsulating nanoparticles with cell membrane vesicles of the present invention.

[0017] In some embodiments of the present invention, the cell membrane-encapsulated nanoparticles are polylactic acid-glycolic acid copolymer nanoparticles.

[0018] In some embodiments of the present invention, the cell membrane-encapsulated nanoparticles have an average particle size of 80-200 nm.

[0019] Another aspect of the invention relates to a pharmaceutical composition comprising cell membrane vesicles of the invention or cell membrane-encapsulated nanoparticles as described in any one of the invention, and one or more pharmaceutically acceptable excipients.

[0020] Another aspect of the invention relates to the use of the cell membrane vesicles of the invention or the cell membrane-encapsulated nanoparticles of the invention in the preparation of medicaments for the treatment or prevention of arthritis, particularly rheumatoid arthritis.

[0021] Unconstrained by theoretical limitations, FLS exhibits high plasticity. In vitro induced immunosuppressive phenotype-FLS may alter its phenotype in the in vivo inflammatory microenvironment. Furthermore, in vitro IFN-γ-induced immunosuppressive phenotype-FLS also secretes high levels of IL-6. Since the molecules exerting inhibitory effects are primarily located on the cell membrane surface, to fully utilize the anti-inflammatory effects of the immunosuppressive phenotype-FLS, prevent its phenotype from converting to a pro-inflammatory phenotype-FLS in RA-inflamed joints, and simultaneously avoid the pro-inflammatory effects of its high-level IL-6 secretion, selecting the cell membrane of in vitro IFN-γ-induced immunosuppressive phenotype-FLS and fabricating cell membrane-encapsulated nanoparticles may achieve therapeutic goals.

[0022] The inventors added IFN-γ-immunosuppressive phenotype-FLS cell membranes to activated T cells in vitro and used IFN-γ-immunosuppressive phenotype-FLS cell membranes to encapsulate nanoparticles in a collagen-induced arthritis (CIA) animal model of rheumatoid arthritis. Results showed that the IFN-γ-FLS cell membranes effectively retained their immunosuppressive characteristics and significantly inhibited the proliferation of activated T cells and the production of cytokines (especially IL-17A). Figure 3D ); IFN-γ-FLS cell membrane-encapsulated nanoparticles can effectively target inflamed joints and alleviate arthritis in the CIA model.

[0023] In this invention, the term "pro-inflammatory phenotype-FLS" (pro-inflammatory synovial fibroblasts) refers to synovial fibroblasts that secrete pro-inflammatory cytokines such as IL-6 within inflamed joints. This group of cells can promote the activation and differentiation of T cells, especially the differentiation of Th17 cells, and promote the progression of inflammation.

[0024] In this invention, the term "immunosuppressive phenotype-FLS" (immunosuppressive synovial fibroblasts) refers to a portion of intra-articular synovial fibroblasts that exhibit the regulation of co-inhibitory molecules on their membrane cell surface, enabling them to interact with activated T cells, inhibit T cell proliferation and activation, and thus exert an anti-inflammatory effect.

[0025] Beneficial effects of the invention

[0026] Immunosuppressive phenotype-FLS plays an important role in promoting arthritis remission. The anti-inflammatory effect of IFN-γ-immunosuppressive phenotype-FLS cell membrane-encapsulated nanoparticles makes them a promising new treatment strategy for RA. Their joint-targeting properties provide a feasible means to further utilize these nanoparticles to carry drugs that may reverse the RA-pro-inflammatory phenotype-FLS, i.e., immunosuppressive phenotype-FLS. Attached Figure Description

[0027] Figure 1 IFN-γ induces an immunosuppressive phenotype in FLS. Figure 1The results showed that IFN-γ stimulation of FLS upregulation can suppress T cell co-inhibitory molecules, such as CD274, FASL, and Galectin-9, while promoting the expression and secretion of IL-6. This manifests as an inhibitory phenotype of FLS.

[0028] Figure 2 Rapamycin promotes the induction of the IFN-γ immunosuppressive phenotype.

[0029] Figure 3A Electron microscopy morphology of cell membrane vesicles was extracted after IFN-γ stimulation and after co-stimulation with IFN-γ and rapamycin.

[0030] Figure 3B IFN-γ stimulation and co-stimulation with IFN-γ and rapamycin followed by FLS extraction of cell membrane vesicles can inhibit T cell proliferation in vitro, and CFSE shows proliferating T cells.

[0031] Figure 3C IFN-γ stimulation and FLS-extracted cell membrane vesicles after co-stimulation with IFN-γ and rapamycin can inhibit the proportion of T cell proliferation in vitro.

[0032] Figure 3D IFN-γ stimulation and co-stimulation with IFN-γ and rapamycin followed by FLS extraction of cell membrane vesicles can inhibit the function of T cells in secreting cytokines, especially IL-17A, in vitro.

[0033] Figure 4A Collagen-induced arthritis (CIA) model: FIRN treatment significantly reduced the clinical disease score in mice with arthritis.

[0034] Note: FIRN: Cell membrane vesicles encapsulated in nanoparticles extracted from cells with an inhibitory phenotype stimulated by rapamycin in combination with IFN-γ.

[0035] Figure 4B FIRN treatment significantly reduced toe thickness in CIA mice.

[0036] Figure 4C The appearance of the toes in mice treated with FIRN and methotrexate, and magnetic resonance and pseudo-color imaging of the toes showed that FIRN treatment significantly reduced inflammation of the toes.

[0037] Figure 4D Statistical graph of average signal intensity of FIRN and methotrexate-treated mouse paw magnetic resonance imaging.

[0038] Figure 4E Imaging images of the toe bones of mice treated with FIRN and methotrexate. The results showed that FIRN treatment significantly reduced bone destruction in arthritic mice.

[0039] Figure 4F Bone section staining of mice treated with FIRN and methotrexate showed that the positive control group exhibited extensive lymphocyte infiltration at the joints and significant cartilage destruction. The FIRN treatment group significantly reduced lymphocyte infiltration and cartilage destruction.

[0040] Note: Safranin O staining (tomato red staining) and toluidine blue staining (blue staining) show cartilage tissue.

[0041] Figure 4G Statistical graph of cartilage content in mice treated with FIRN and methotrexate. Detailed Implementation

[0042] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0043] Mouse synovial fibroblasts were purchased from Qingqi (Shanghai) Biotechnology Development Co., Ltd., catalog number: BFN60810489; they were cultured in a cell culture medium (DMEM high glucose medium containing 10% fetal bovine serum) in an incubator.

[0044] Example 1: Experiment on IFN-γ stimulation of mouse synovial fibroblasts

[0045] Mouse synovial fibroblasts (FLS) were stimulated with a final concentration of 100 ng / ml of IFN-γ (FLS cell confluence was approximately 80%, and the density was approximately 1*102). 6 Cells were collected (number per ml) and RNA was extracted after 24 hours. Changes in relevant functional genes in FLS were detected by RT-PCR.

[0046] The results are as follows Figure 1 As shown.

[0047] The results showed that IFN-γ upregulated T-cell-suppressing molecules in FLS, such as PD-L1 (CD274), galectin-9, FASL, and IDO; it had little or no effect on T-cell-activating molecules, such as CD80, CD86, ICOS-L, CD40, GITR-L, CD70, and OX-40L, or even reduced them; it upregulated the expression levels of inflammatory cytokines IL-6 and TNF-α; and it reduced the expression levels of extracellular matrix (ECM) production-related genes, such as α-SMA, collagen I, collagen III, collagen IV, and collagen V, as well as tissue damage-related genes, such as MMP3, MMP9, and MMP13. These results indicate that IFN-γ primarily induces the immune regulatory function of FLS and inhibits its role in extracellular matrix homeostasis.

[0048] Example 2: Rapamycin (RAPA) and IFN-γ co-stimulation of mouse synovial fibroblasts

[0049] FLS cells were stimulated with IFN-γ at a final concentration of 100 ng / ml and rapamycin at a concentration of 10 μM / L (cell density as in Example 1 above). After 24 hours, cells were collected and RNA was extracted. The expression of molecules such as PD-L1, which inhibit the phenotype, was detected by RT-PCR.

[0050] The results are as follows Figure 2 As shown.

[0051] The results showed that the combination of IFN-γ and rapamycin could further promote IFN-γ stimulation and upregulate co-inhibitory molecules, while also promoting the expression and secretion of IL-6, resulting in an FLS-inhibiting phenotype. Furthermore, compared with IFN-γ stimulation alone in Example 1, the upregulation of co-inhibitory molecules such as PD-L1 was more significant when IFN-γ and rapamycin were used in combination.

[0052] Example 3: Extraction of cell membrane vesicles

[0053] Untreated flaccid cells (FLS), FLS stimulated with IFN-γ for 24 hours, and FLS stimulated with IFN-γ + rapamycin for 24 hours were collected using a cell scraper. The supernatant was discarded by centrifugation, and the cell pellet was placed at -80°C for 30 min, then at room temperature for 30 min, repeating this freeze-thaw cycle three times. Cells were resuspended in Tris buffer containing PMSF and dissolved by sonication. The cell lysate was sterilized by passing through a 0.22 μm filter before adding Tris buffer. Centrifugation was performed at 4°C for 10 min at 2000 rpm, then at 4000 rpm for 10 min, and finally at 5000 rpm for 10 min. The supernatant was collected, and the pellet was resuspended in 20 mL of Tris buffer (pH 7.4) for 10 min at 2000 rpm, then at 4000 rpm for 10 min, and finally at 5000 rpm for 10 min. The supernatant was collected again, and the supernatants from both centrifugations were combined into a 50 mL centrifuge tube and centrifuged at 12000 rpm for 70 min. Discard the supernatant, resuspend the collected precipitate in PBS, quantify using the BCA method, aliquot and store at low temperature.

[0054] Example 4: Preparation and Characterization of Membrane-Coated Nanoparticles

[0055] Polylactic-co-glycolic acid (PLGA) was dissolved in dimethylformamide (DMF) and prepared to a concentration of 20 mg / mL using an ultrasonic method; a 5% PVP solution was prepared as a surfactant and excipient.

[0056] Under uniform stirring conditions, PLGA solution was slowly added dropwise to an aqueous solution containing 2% PVP, and stirring was continued for 6 hours in the dark. The resulting liquid solution was placed in a dialysis bag with a molecular weight of 10 kDa and dialyzed overnight in water at 4°C, with the dialysate changed three times during the process. PLGA nanoparticles were obtained. A suitable amount of the obtained PLGA nanoparticles were subjected to transmission electron microscopy (TEM) to observe their morphology, and their particle size and potential were measured using a particle size analyzer. The particle size of the PLGA nanoparticles was 100-150 nm.

[0057] A suitable amount of cell membrane vesicles and PLGA nanoparticles obtained in Example 3 (the mass ratio of cell membrane to PLGA nanoparticles was 3:1) were ultrasonicated for 5 minutes in a water bath. Then, they were sequentially extruded through 400 nm and 200 nm polycarbonate membranes using a membrane extruder to obtain cell membrane-encapsulated nanoparticles. A suitable amount of cell membrane-encapsulated nanoparticles were observed under an electron microscope, and their particle size and potential were measured using a particle size analyzer.

[0058] The results are as follows Figure 3AAs shown in the figure. The results show that the extracted cell membrane vesicles have a complete membrane envelope structure, and the vesicle size is about 100-200 nm, which meets the requirements of nanoparticles.

[0059] Example 5: Activation of T cells

[0060] Mouse (C57 / B6) spleen cells were collected, ground, filtered, and prepared into a single-cell suspension. Red blood cells were removed using erythrocyte lysis buffer. A mixture of spleen cells and a biotin-labeled negative selection antibody cocktail (Miltenyi Biotec, 130-095-130) was incubated in MACS buffer for 30 minutes. Unbound antibodies were removed by centrifugation, and Strep-magnetic beads were added. The suspension was then purified using a Mini Macs sorting system. T cells. Purified T cells were resuspended in 1 mL PBS, and 1 μL of CFSE dye (Thermo CellTrace) was added to the cell suspension. TM CFSE Cell Proliferation Kit (for flowcytometry, C34554) was used to incubate T cells at 37°C for 15 minutes. The reaction was terminated with 1640 medium containing 10% FBS, and the cells were centrifuged to obtain CFSE-stained T cells. T cells were then activated using a CD3 / CD28 beads activation kit (Thermo-gibco, 11452D).

[0061] Example 6: CFSE staining and nanoparticle-stimulated culture, FACS detection of T cell proliferation

[0062] 1. The activated and CFSE-stained T cells (obtained in Example 5) were resuspended in 1640 medium containing 10% FBS.

[0063] 2. Cell membrane vesicle nanoparticles (prepared in Example 4) were added to activated and CFSE-stained T cells at a final concentration of 10 ng / ml, with 2*10 nanoparticles per well. 5 The medium was prepared at a density of 200 μl and seeded onto a 96-well cell culture plate for co-culture.

[0064] 3. T cells were collected at different time points (24 hours, 48 ​​hours, 72 hours, and 96 hours). The T cells were in suspension; cells were collected from three wells in a 96-well plate by agitation. The proportion of CFSE decay (the proportion of CFSE decay represents the proportion of proliferating cells) was detected by flow cytometry, along with the proportion of proliferating T cells and their generation number.

[0065] The results are as follows Figures 3B to 3D As shown.

[0066] The results showed that cell membrane vesicle nanoparticles extracted from cells with IFN-γ and rapamycin combined with IFN-γ-stimulated inhibitory phenotypes could significantly inhibit the activation and function of T cells in vitro, and the effect of rapamycin combined with IFN-γ was better than that of IFN-γ alone.

[0067] Example 7: Preparation and Evaluation of Collagen-Induced Arthritis (CIA) Model

[0068] A collagen-induced arthritis animal model was established using 6-8 week old DBA / 1J male rats weighing approximately 20g. Bovine type II collagen and complete Freund's adjuvant were completely emulsified on ice. Mice were immunized via multiple intradermal injections at the base of the tail and back (100μg / 100μl / mouse). The initial immunization was recorded as day 0. On day 21, a booster immunization was administered (at the same dose) using bovine type II collagen and incomplete Freund's adjuvant, completely emulsified on ice.

[0069] Thirty-five days after model induction, mice were treated twice a week with membrane-coated nanoparticles (prepared in Example 4, 20 μg / time / mouse). The adjuvant control group and positive control group received equal doses of PBS, while the drug control group received methotrexate (MTX). Each group contained more than seven mice. The adjuvant control group consisted of mice that were not immunized with bovine type II collagen, i.e., mice without induced CIA.

[0070] The degree of swelling in the mouse paws was measured using calipers, and scores were given based on the redness and swelling of the paws and the number of inflamed joints. The scoring criteria for the paws were as follows: 0 points was normal; 0.5 points was mild redness and swelling in only one toe; 2 points was redness and moderate swelling in two joints (pad, joint, and 2-5 toes); 3 points was redness and severe swelling in the entire paw; and 4 points was redness and swelling accompanied by deformity of the paw joints.

[0071] The results are as follows Figures 4A to 4E As shown.

[0072] The results showed that rapamycin combined with IFN-γ-stimulated inhibitory phenotype cell-derived nanoparticles encapsulated in cell membrane vesicles (FIRN) effectively relieved CIA inflammation and inhibited bone destruction, and showed better efficacy compared with the methotrexate treatment group.

[0073] Example 8: H&E staining and safranin O and toluidine blue staining

[0074] Bone tissue from the paws of mice in the positive control group, adjuvant control group, methotrexate group, and FIRN treatment group (Example 7) was decalcified and dehydrated (immersion in EDTA decalcification solution, followed by gradient alcohol dehydration), embedded, and sectioned. After dewaxing, sections were stained with hematoxylin for 1-3 minutes and rinsed with running water. Differentiation was performed with 1% hydrochloric acid-alcohol solution, followed by a brief rinse with running water. The sections were then rinsed with tap water to achieve a blue staining. After satisfactory staining under a microscope, sections were stained with eosin for 0.5-1 minutes, rinsed with water, dehydrated with gradient alcohol, and mounted. For Safranin O staining (Solarbio, G1371), bone tissue sections were stained with freshly prepared Weigert stain for 3 minutes, differentiated with acidic ethanol for 15 seconds, washed with distilled water for 10 minutes, immersed in Fast Green stain for 5 minutes, washed with distilled water for 1 minute, stained with Safranin O for 1 minute, washed with distilled water for 1 minute, dehydrated, and mounted. Toluidine blue staining (Solarbio, G3663): Bone tissue sections were stained with 0.1% toluidine blue for 10 minutes, washed with distilled water for 5 minutes, dehydrated with alcohol, and mounted.

[0075] The results are as follows Figures 4F to 4G The results showed that the adjuvant control group mice had intact joint surfaces, no lymphocyte infiltration, and intact cartilage surfaces; the positive control group mice had a large number of lymphocyte infiltrations in the joints, pannus formation, and cartilage erosion and destruction. In the treatment group, lymphocyte infiltration at the joints was significantly reduced, no obvious cartilage destruction was observed, and the treatment effect of FIRN was superior to that of the MTX treatment group and other treatment groups.

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[0095] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof.

Claims

1. The use of cell membrane vesicle-encapsulated nanoparticles in the preparation of drugs for the treatment or prevention of rheumatoid arthritis, wherein, The cell membrane vesicles were extracted from synovial fibroblasts. The synovial fibroblasts mentioned are immunosuppressed synovial fibroblasts. The synovial fibroblasts were obtained by inducing an immunosuppressive phenotype in fibroblasts, which included the following steps: treating the fibroblasts with IFN-γ and rapamycin. The cell membrane vesicles are the active ingredient in the drug.

2. The use according to claim 1, wherein, The synovial fibroblasts mentioned are mammalian synovial fibroblasts.

3. The use according to claim 2, wherein, The synovial fibroblasts are human synovial fibroblasts or mouse synovial fibroblasts.

4. The use according to claim 1, wherein, The final concentration of IFN-γ is 50-150 ng / ml.

5. The use according to claim 1, wherein, The final concentration of IFN-γ is 80-120 ng / ml.

6. The use according to claim 1, wherein, The final concentration of IFN-γ was 100 ng / ml.

7. The use according to claim 1, wherein, The final concentration of rapamycin is 5-15 μM.

8. The use according to claim 1, wherein, The final concentration of rapamycin is 8-12 μM.

9. The use according to claim 1, wherein, The final concentration of rapamycin was 10 μM.

10. The use according to claim 1, wherein, The processing time is 6-48 hours.

11. The use according to claim 1, wherein, The processing time is 12-36 hours.

12. The use according to claim 1, wherein, The processing time is 24 hours.

13. The use according to claim 1, wherein, The nanoparticles are polylactic acid-hydroxyacetic acid copolymer nanoparticles.

14. The use according to claim 1, wherein, The average particle size of the nanoparticles is 80-200 nm.

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