Extracellular vesicles overexpressing pd-l1 / tnfr1, construction method and application thereof in rheumatoid arthritis

Abstract

The application provides an extracellular vesicle overexpressing PD-L1 / TNFR1, a construction method and application thereof in rheumatoid arthritis. In the application, a lentivirus expression vector of PD-L1 / TNFR1 is constructed, the lentivirus expression vector is transfected into a packaging cell HEK293T to produce lentivirus overexpressing PD-L1 / TNFR1, then the lentivirus is used to infect HEK293T cells, and a cell line stably expressing PD-L1 / TNFR1 is obtained through puromycin screening; the high expression PD-L1 / TNFR1 EVs secreted by the cell line stably expressing PD-L1 / TNFR1 are extracted by ultracentrifugation, and it is confirmed through NTA, TEM, FCM, WB and IF that the EVs co-expressing PD-L1 / TNFR1 are successfully constructed. The PD-L1 / TNFR1 EVs can be effectively gathered to the lesion site of RA, because PD-L1 and TNFR1 are co-expressed on the surface of the EVs, PD-L1 and TNFR1 can be simultaneously and efficiently delivered to the inflammation site of arthritis. Firstly, TNFR1 on the surface of the EVs can effectively remove the inflammatory factor TNF-alpha at the lesion site, secondly, PD-L1 on the surface of the EVs can interact with activated T cells in the RA microenvironment to inhibit the activity of the T cells, and finally the purpose of synergistically improving RA is achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to extracellular vesicles overexpressing PD-L1 / TNFR1, their construction methods, and their application in rheumatoid arthritis. Background Technology

[0002] Respiratory rheumatoid arthritis (RA) is a chronic, systemic, predominantly inflammatory autoimmune disease characterized by synovial inflammation, cartilage and bone destruction, and joint dysfunction. With in-depth research into the pathogenesis of RA, targeted biological therapy and targeted small molecule therapy have made significant progress in recent years. Early TNF-α inhibitors, including monoclonal antibodies (such as Infliximab) and receptor fusion proteins (such as Etanercept), have shown significant efficacy in reducing joint swelling and improving patients' quality of life. Subsequent studies found that while some patients respond well to TNF-α inhibitors, a considerable proportion still experience initial or secondary non-response. To address these issues, second- and third-generation TNF-α inhibitors have continuously optimized their molecular structures, improving stability and efficacy. Besides TNF-α, other pro-inflammatory factors and signaling pathways have also become targets of targeted therapy research. IL-6 receptor antagonists (such as Tocilizumab) and IL-1 receptor antagonists (such as Anakinra) have significantly improved the clinical symptoms of patients with refractory RA by blocking key inflammatory factor signaling. In recent years, JAK inhibitors (such as tofacitinib, baricitinib, and upadacitinib), as a class of small-molecule oral drugs, have provided new treatment options for refractory rheumatoid arthritis (RA) by interfering with the JAK-STAT signaling pathway and inhibiting the expression of various pro-inflammatory factors. Although these drugs can effectively improve RA symptoms, the search for drugs with fewer side effects and higher bioavailability remains of great importance.

[0003] Innovations in biomaterials and drug delivery systems have provided new avenues for targeted therapy. Novel biomaterials such as nanomaterials, liposomes, and extracellular vesicles have been introduced into targeted drug delivery systems. These systems enable targeted drug delivery, stable release, and protection of bioactivity, significantly improving treatment efficiency and reducing systemic side effects and drug tolerance. Although biomaterials and drug delivery systems have made significant progress in RA treatment research, several challenges remain. These include large-scale production and standardization, long-term in vivo safety assessment of materials, and optimizing delivery strategies for different patient characteristics. Summary of the Invention

[0004] The purpose of this invention is to address some of the challenges that still exist in existing research on RA treatment.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for constructing extracellular vesicles overexpressing PD-L1 / TNFR1, characterized by comprising the following steps:

[0007] S1: A lentiviral expression vector for PD-L1 / TNFR1 was constructed and transfected into HEK293T packaging cells to generate lentiviruses that highly express PD-L1 / TNFR1. HEK293T cells were then infected with the lentiviruses that highly express PD-L1 / TNFR1, and cell lines that stably express PD-L1 / TNFR1 were obtained by selection with puromycin. At the same time, three lentiviral expression vectors that overexpress PD-L1, TNFR1, and TNFR1 / PD-L1 were constructed using the same method, and three cell lines that stably express PD-L1, TNFR1, and TNFR1 / PD-L1 were obtained, respectively.

[0008] S2: Ultracentrifugation was used to extract EVs that highly expressed PD-L1 / TNFR1 secreted by cells that stably expressed PD-L1 / TNFR1. At the same time, PD-L1 EVs, TNFR1 EVs and TNFR1 / PD-L1 EVs were extracted as controls.

[0009] Preferably, the specific steps of S1 are as follows:

[0010] (1) Plasmids PLVX-Flag-PD-L1, PLVX-Flag-TNFR1, PLVX-Flag-TNFR1-PD-L1, PLVX-Flag-PD-L1-TNFR1 and psPAX2 and pMD2.G from the lentivirus delivery system were co-transfected into HEK293T cells. After culturing for 24 h, the medium was replaced with fresh DMEM. After 48 h and 72 h, the cell culture supernatant was collected and filtered through a 0.45 μm filter to obtain three lentiviruses carrying PD-L1, TNFR1, TNFR1 / PD-L1 and PD-L1 / TNFR1, respectively.

[0011] (2) Infect HEK293T cells with good growth status and confluence of 30% to 50% with the prepared lentivirus, add 1 mL of virus solution to each well; at the same time, add Polybrene to increase the infection efficiency. After 12 h of virus infection, discard the supernatant, add new DMEM to complete the culture and continue to culture. After 48 h, discard the supernatant.

[0012] (3) Add 2 mL of culture medium containing puromycin and screen for one week. Expand and passage the screened cells, and add 0.5 μg / mL of puromycin to maintain resistance. After continuous screening and passage for 3 generations, HEK293T cell lines that stably express PD-L1, TNFR1, TNFR1 / PD-L1 and PD-L1 / TNFR1 are obtained.

[0013] Preferably, the mass ratio of PLVX-Flag-PD-L1-TNFR1, psPAX2, and pMD2.G in S1 is 3:1:1, and the mass ratio of PLVX-Flag-PD-L1, PLVX-Flag-TNFR1, PLVX-Flag-TNFR1-PD-L1 to psPAX2 and pMD2.G as controls is also 3:1:1.

[0014] Preferably, the specific steps of S2 are as follows:

[0015] (1) When the cell density reaches 80%, replace it with DMEM medium and collect the cell supernatant after culturing for 24 h.

[0016] (2) Centrifuge to remove cells, large particles and debris, small organelle fragments and larger impurities from the supernatant. Collect the supernatant and filter it with a 0.22 μm sterile filter.

[0017] (3) The filtered supernatant was centrifuged at 150,000 g for 2 h, the supernatant was removed, the precipitate was retained, the precipitate was resuspended in 200 μL PBS to collect EVs, the concentration was detected and then stored at -80 ℃ for later use.

[0018] (4) The extraction methods for PD-L1 EVs, TNFR1 EVs and TNFR1 / PD-L1 EVs are as described above.

[0019] Preferably, the centrifugation in step (2) of S2 is performed in four stages: first, centrifugation at 500 g for 5 min to remove cells; then, centrifugation at 1000 g for 10 min to remove large particles and debris; and then centrifugation at 16500 g for 30 min to remove small organelle debris and larger impurities. Finally, centrifugation at 150000 g for 2 h is performed, the supernatant is discarded, the precipitate is retained, and the precipitate is resuspended in 200 μL PBS to collect EVs.

[0020] Preferably, in S1, a single-chain polypeptide composed of PD-L1 and TNFR1 is first designed, and the single-chain polypeptide composed of PD-L1 and TNFR1 is fused with the transmembrane domain gene of platelet-derived growth factor receptor (PDGFR). A (GGGGS)4 flexible linker is also inserted between PD-L1 and TNFR1, and a (GGGGS)4 flexible linker is also inserted in the middle of the single-chain polypeptide composed of TNFR1 / PD-L1.

[0021] This application also provides the use of overexpressing PD-L1 / TNFR1 EVs in the preparation of drugs for treating rheumatoid arthritis, wherein the overexpressing PD-L1 / TNFR1 EVs are obtained by the above-described construction method.

[0022] Compared with the prior art, this application has the following beneficial effects:

[0023] In this application, HEK293T cells stably expressing PD-L1 / TNFR1 were constructed using lentiviral transfection. Simultaneously, HEK293T cells stably expressing PD-L1, TNFR1, and TNFR1 / PD-L1 were constructed as controls. EVs were then extracted using ultracentrifugation, and the successful construction of dual-expressing PD-L1 / TNFR1 EVs was confirmed by particle size analysis (NTA), transmission electron microscopy (TEM), flow cytometry (FCM), Western blotting (WB), and immunofluorescence (IF).

[0024] Secondly, this application demonstrates through specific experiments the regulatory effect of dual-expression PD-L1 / TNFR1 EVs on the secretion level of pro-inflammatory factors in T cells and their scavenging effect on the inflammatory factor TNF-α. Furthermore, animal experiments have shown that PD-L1 / TNFR1 EVs can significantly reduce the production of pro-inflammatory cytokines while increasing the expression of anti-inflammatory cytokines, effectively improving synovial inflammation and reducing cartilage damage in CIA mice, demonstrating great potential for clinical treatment of RA. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of PD-L1 / TNFR1 EVs treating CIA mice according to one embodiment of the present invention;

[0026] Figure 2Characterization of a cell line stably expressing PD-L1 / TNFR1 according to one embodiment of the present invention. (A) Molecular design for generating the PD-L1 / TNFR1 domain fusion protein. (B) Flow cytometry analysis showing the expression of PD-L1, TNFR1, PD-L1 / TNFR1, and TNFR1 / PD-L1 on HEK293T cells. (C) Western blotting showing the expression levels of PD-L1, TNFR1, PD-L1 / TNFR1, and TNFR1 / PD-L1 on HEK293T cells. (D) Immunofluorescence characterization of the expression of PD-L1, TNFR1, PD-L1 / TNFR1, and TNFR1 / PD-L1 on HEK293T cells. (Red fluorescence represents PD-L1, green fluorescence represents TNFR1, and blue fluorescence represents DAPI).

[0027] Figure 3 This invention describes the characterization of extracellular vesicles of PD-L1 / TNFR1 in one embodiment, wherein (A) NTA analysis shows the diameter of extracellular vesicles; (B) Western blotting shows the expression levels of CD63, CD81, PD-L1, and TNFR1 on extracellular vesicles; and (C) TEM is used to observe the morphology of extracellular vesicles.

[0028] Figure 4 This invention illustrates the clearance of TNF-α by PD-L1 / TNFR1 EVs in vitro and their interaction with PD-1 and T cells in one embodiment. Specifically: (A) The clearance efficiency of PD-L1 / TNFR1 EVs against TNF-α at an initial cytokine concentration of 5 ng / mL (n = 3). (B) The interaction between TNF-α and different concentrations of Control EVs, PD-L1 EVs, TNFR1 EVs, PD-L1 / TNFR1 EVs, and TNFR1 / PD-L1 EVs (n = 3). (C) The interaction of PD-L1 / TNFR1 EVs with PD-1 (5 ng / mL) (n = 3). (D) The interaction between the five groups of EVs and PD-1 (5 ng / mL) (n = 3). (E) The interaction between TNFR1 / PD-L1 EVs (red) and activated splenic T cells (green) was verified by immunofluorescence staining. (F) Flow cytometry analysis of EVs binding to T cells. (G) Study on the inhibitory effects of Control EVs, PD-L1 EVs, TNFR1 EVs, PD-L1 / TNFR1 EVs, and TNFR1 / PD-L1 EVs (100 ug / mL) on T cell (CFSE-labeled) proliferation. (H) Flow cytometry analysis of spleen CD4 in different treatment groups. + CD3 +T cell percentage. (I, J, K) Cytokines released by T cells in the culture medium from different treatment groups were measured using an ELISA kit (n = 3).

[0029] Figure 5 This invention illustrates the biodistribution of PD-L1 / TNFR1 EVs in CIA mice. Specifically, (A) in vivo fluorescence imaging of CIA mice at different time points after intravenous injection of different EVs; (B) imaging of major organs (heart, liver, spleen, lungs, kidneys) and fore and hind legs 24 h later; (C) fluorescence intensity analysis of major organs (n=3); and (D) flow cytometry analysis of the binding of PD-L1 / TNFR1 EVs to spleen T cells in vivo.

[0030] Figure 6 This invention uses the ink footprint experiment to evaluate the motor function of CIA mice. (A) Footprint images of mice used in the ink footprint experiment. (B) Achilles tendon function index (AFI) graph, a statistical graph of the distance to the opposite foot (TOF), footprint length factor (PLF), foot span expansion factor (TSF), and middle toe width factor (ITF). (C) Statistical graph of forelimb grip strength in CIA mice after treatment. The lower right image shows the hindlimb grip strength statistical graph of CIA mice after treatment.

[0031] Figure 7 This invention monitors the therapeutic efficacy of PD-L1 / TNFR1 EVs on CIA mice. (A) Schematic diagram of the establishment of the CIA mouse model and treatment regimen. (B) Hind paw images of each group. (C) Infrared thermography images of the hind paws of each group. (D) Changes in body weight of each group. (E) Changes in paw thickness of each group. (F) Average joint score of each group. (G) Micro-CT images of mouse foot and ankle reconstruction. (H, I, J, K) Trabecular bone thickness (Tb.Th), bone mineral density (BMD), bone surface area / bone volume ratio (BS / BV), and bone volume fraction (BV / TV) (n=3).

[0032] Figure 8 This is a tissue section of the mouse knee joint used in this invention. (A) Representative images of H&E staining of the ankle joints of mice from different treatment groups and (B) Safranin O staining. Scale bars: 400 μm (top), 100 μm (bottom). (C) Immunohistochemical detection of CD3 in the synovial tissue. + Relative expression of T cells (scale bar, 100 μm). (D) Immunofluorescence analysis of the expression of IL-10, TNF-α, IL-1β and IL-6 in the joint tissues of mice in each group;

[0033] Figure 9To illustrate the immunomodulatory effects of PD-L1 / TNFR1 EVs on CIA mice in this invention, flow cytometry analysis was performed on IFN-γ levels in the spleen (A, B) and serum (C, D). + CD4 + T cells and CD25 + FoxP3 + T cell expression (n=3);

[0034] Figure 10 This study evaluates the biosafety of PD-L1 / TNFR1 EVs in this invention. (A) HE staining of major organs in CIA mice receiving different treatments (scale bar, 200 μm). (B) Complete blood count of mice in the PBS and PD-L1 / TNFR1 EVs groups. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to specific embodiments.

[0036] Please see Figure 1 , Figure 1 The process of constructing PD-L1 / TNFR1 overexpressing EVs was demonstrated, as well as their therapeutic effect on RA by inhibiting the activity of activated T cells, clearing the inflammatory factor TNF-α, and regulating the inflammatory microenvironment at the lesion site.

[0037] In this application, the constructed lentiviral expression vector was first transfected into HEK293T packaging cells to generate lentiviruses that highly express PD-L1 / TNFR1. HEK293T cells were then infected with this virus, and cell lines stably expressing PD-L1 / TNFR1 were obtained through puromycin selection. Using the same method, PD-L1, TNFR1, and TNFR1 / PD-L1 cell lines were constructed.

[0038] Subsequently, ultracentrifugation was used to extract EVs (extracellular substances) that highly expressed PD-L1, TNFR1, PD-L1 / TNFR1, and TNFR1 / PD-L1. After PD-L1 / TNFR1 EVs were injected into mice via the tail vein, the EVs exhibited an ELVIS effect, targeting and accumulating at the sites of joint inflammation in the limbs. PD-L1 and TNFR1 served as experimental controls.

[0039] Among them, TNFR1 on the surface of EVs can clear TNF-α in the inflammatory microenvironment, while PD-L1 inhibits T cell activation by binding to PD-1 on the surface of T cells, thereby reducing the release of T cell pro-inflammatory factors such as TNF-α and IFN-γ, thus alleviating abnormal proliferation and inflammatory infiltration of synovial tissue, restoring cartilage and reducing bone erosion, and producing a therapeutic effect on CIA model mice.

[0040] The above content will be explained in conjunction with specific verification experiments:

[0041] Research subjects:

[0042] DBA / 1 mice, male, 6 weeks old, weighing 20 ± 2 g; C57BL / 6 mice, female, 6 weeks old, weighing 20 ± 2 g, were purchased from the Experimental Animal Center of Nantong University. The purchases were approved by the Animal Ethics Committee of Nantong University (S20250210-013).

[0043] Experimental instruments, reagents, and their sources:

[0044]

[0045]

[0046] Example 1: Construction of extracellular vesicles expressing both PD-L1 and TNFR1

[0047] S1: Construction of HEK293T cell line stably expressing PD-L1 / TNFR1

[0048] Please see Figure 2 A. The transmembrane domain of the human platelet-derived growth factor receptor (PDGFR) is a proven and effective tool for displaying mammalian membrane proteins, and it was used as a fusion partner to express target proteins on vesicle membranes. To ensure co-expression of PD-L1 and TNFR1 on the same vesicle, a single-chain polypeptide composed of PD-L1 and TNFR1 was designed and fused to the transmembrane domain gene of PDGFR. To enhance the flexibility between the proteins, a (GGGGS)4 linker was inserted between PD-L1 and TNFR1. Considering the significant impact of protein orientation on the physicochemical properties and biological functions of the expressed proteins, two fusion proteins were constructed: PD-L1 / TNFR1 and TNFR1 / PD-L1.

[0049] The specific steps are as follows:

[0050] (1) Plasmids PLVX-Flag-PD-L1, PLVX-Flag-TNFR1, PLVX-Flag-PD-L1-TNFR1, PLVX-Flag-TNFR1-PD-L1 (0.75 μg), psPAX2 (0.25 μg), and pMD2.G (0.25 μg) from the lentiviral packaging system were co-transfected into HEK293T cells (6 × 10⁻⁶ cells / year). 5Cells (with a confluence of 70%-80% at transfection) were cultured for 24 hours and then replaced with fresh DMEM medium. The cell culture supernatant was collected after 48 hours and 72 hours and filtered through a 0.45 μm filter to obtain lentiviruses carrying PD-L1, TNFR1, PD-L1 / TNFR1, and TNFR1 / PD-L1.

[0051] (2) Infect HEK293T cells with good growth status and confluence of 30% to 50% with the prepared lentivirus, add 1 mL of virus solution to each well; add Polybrene (final concentration of 5 μg / mL) at the same time; after infection, discard the supernatant and replace it with fresh DMEM complete medium for further culture. After 48 h, subsequent screening or detection can be carried out.

[0052] (3) Add 2 mL of culture medium containing puromycin (1 μg / mL) to screen for 5 days. Expand and passage the screened cells, and use 0.5 μg / mL of puromycin to maintain resistance. After continuous screening and passage for 3 generations, HEK293T cell lines that stably express PD-L1, TNFR1, PD-L1 / TNFR1 and TNFR1 / PD-L1 were obtained.

[0053] S2: Extraction of EVs by ultracentrifugation

[0054] (1) When the cell density is about 80%, switch to DMEM medium and collect the cell supernatant after culturing for 24 h.

[0055] (2) Centrifuge at 500 g for 5 min to remove cells, then centrifuge at 1000 g for 10 min to remove large particles and debris, and then centrifuge at 16500 g for 30 min to remove small organelle debris and larger impurities. Collect the supernatant and filter it with a 0.22 μm sterile filter.

[0056] (3) After filtration, the supernatant was centrifuged at 150,000 g for 2 h, the supernatant was removed, the precipitate was retained, the precipitate was resuspended in 200 μL PBS to collect EVs, the concentration was detected and then stored at -80 ℃ for later use.

[0057] Example 2: Characterization of HEK293T cell line stably expressing PD-L1 / TNFR1

[0058] 2.1: Flow Cytometry Identification of Stable Cell Lines

[0059] (1) Collect cells, centrifuge at 1500 rpm for 5 min, collect the precipitate, wash twice with PBS, and resuspend in 200 μL of PBS containing 1% FBS;

[0060] (2) Seal with FcBlock for 30 min;

[0061] (3) Add APC-CD274 mouse antibody and PE-CD120 mouse antibody at the same time, and incubate on ice in the dark for 1 h;

[0062] (4) Centrifuge at 1500 rpm for 5 min, wash twice with PBS, and resuspend the cells in 100 μL of PBS;

[0063] (5) Analyze the samples by flow cytometry.

[0064] The results are as follows Figure 2 As shown in B, flow cytometry confirmed that PD-L1 and TNFR1 were successfully co-expressed in HEK293T cells transfected with PD-L1 / TNFR1 or TNFR1 / PD-L1 plasmids, and also verified cell lines that expressed PD-L1 or TNFR1 alone.

[0065] 2.2 Western blot identification of stable cell lines

[0066] (1) Collect cells, place the well plate on an ice box, remove cell supernatant, wash with PBS, add 100 μL RIPA lysis buffer to each well of the 6-well plate, lyse on ice for 30 min, collect protein, and measure protein concentration using a spectrophotometer.

[0067] (2) Add loading buffer according to the volume ratio of protein solution: 5×loading buffer = 4:1, mix well, denature at 100 ℃ for 5 min, cool and store at -80 ℃.

[0068] (3) Prepare the separating gel of the required concentration, seal with isopropanol and press the separating gel evenly. After the separating gel solidifies, add the stacking gel and quickly insert the comb. After solidification, subsequent experiments can be carried out.

[0069] (4) Install the gel on the electrophoresis tank to check for leaks. After the leak test is completed, pour the prepared and mixed electrophoresis solution between the two glass plates, pull out the comb, and add the sample with an equal amount of protein and marker.

[0070] (5) First set the V to 80 V to run the stacking gel, then run the separating gel at 120 V. Stop when the bromophenol blue reaches the bottom.

[0071] (6) Cut the PVDF membrane to a suitable size and then put it into methanol for activation. Then immerse it in pre-cooled transfer buffer and soak for 5 min to equilibrate. Gently shake to remove the residual methanol on the membrane. Cover the PVDF membrane with the gel, then put the clamp into the tank, add the pre-cooled transfer solution, and perform the transfer at 300 mA.

[0072] (7) After the PVDF was transferred, it was blocked with blocking solution, and then primary antibodies (anti-mouse CD274 antibody and anti-rabbit CD120a antibody) were added and incubated overnight at 4 °C.

[0073] (8) Discard the primary antibody, then wash with TBST, then add the corresponding secondary antibody (goat anti-mouse secondary antibody, goat anti-rabbit secondary antibody) dilution solution and incubate at room temperature for 2 h, then wash with TBST.

[0074] (9) Prepare ECL luminescent solution at a ratio of 1:1, image it using a gel imaging system, acquire the image, and analyze the gray value using ImageJ.

[0075] Please see Figure 2 C. Consistent with the results of flow cytometry analysis, the Western blot results also showed that PD-L1 and TNFR1 were co-expressed at high levels in the transfected cells, while cells expressing only PD-L1 or TNFR1 showed only PD-L1 or TNFR1.

[0076] 2.3 Immunofluorescence identification of stable cell lines

[0077] (1) Plating: Place pre-sterilized cell spreaders into a 24-well plate, then seed the cells into the well plate and place it in an incubator for 24 h.

[0078] (2) Fixation: Carefully remove the cell supernatant, add 200 μL of immunofluorescence fixative and fix for 15 min, then wash with PBS.

[0079] (3) Permeation: Add 0.1% TritonX-100 to permeate for 15 min, then wash with PBS.

[0080] (4) Blocking: Add immunofluorescence blocking solution and block for 30 min, then discard the blocking solution.

[0081] (5) Antibody incubation: Add 50 μL of diluted primary antibody (anti-mouse CD274 (PD-L1) antibody, anti-rabbit CD120a (TNFR1) antibody), incubate overnight at 4 °C, warm to 30 min the next day, discard the primary antibody, wash with PBS, add secondary antibody (Alexa Fluor 647 labeled goat anti-mouse IgG, Alexa Fluor 488 labeled goat anti-rabbit IgG), incubate at room temperature in the dark for 2 h;

[0082] (6) Nucleus staining and mounting: Add 20 μL of a solution containing DAPI and antiquenching agent to the sample, and then carefully place the small round slice containing cells onto the glass slide with tweezers, ensuring that no air bubbles are generated.

[0083] (7) Taking pictures: Adjust the appropriate light source according to the wavelength of the excitation light of the fluorescent dye used, and acquire images under an inverted fluorescence microscope.

[0084] Please see Figure 2 D. The co-localization of PD-L1 (Alexa Fluor 647-labeled goat anti-mouse IgG), TNFR1 (Alexa Fluor 488-labeled goat anti-rabbit IgG), and DAPI nuclear staining demonstrated the expression and localization of PD-L1 and TNFR1 in HEK293T cells.

[0085] Based on the results of the above verification experiments, it is demonstrated that cells co-expressing PD-L1 and TNFR1 were successfully prepared in this application.

[0086] Example 3: Characterization of EVs

[0087] 3.1 NTA characterization of EVs

[0088] The extracted EVs were diluted with sterile PBS to an appropriate concentration (1×10⁻⁶). 7 ~1×10 9 (particles / mL). After sample dilution, detection was performed. The Brownian motion trajectory of EVs was automatically analyzed using NTA software to calculate the particle size distribution and concentration of EVs. Measurements were repeated three times, and the results were averaged. Please refer to [link to relevant documentation]. Figure 3 A,NTA results show that the average particle size of the various EVs constructed in this application is approximately 100-150 nm.

[0089] 3.2 Western Blot characterization of EVs

[0090] The EVs precipitate was resuspended in 100 μL of RIPA lysis buffer and lysed on ice for 30 min. The protein concentration was measured using a spectrophotometer. The mixture was centrifuged at 12000 g for 15 min, and the supernatant was collected. Then, 1 / 4 volume of 5× loading buffer was added, mixed, and denatured at 95 °C for 5 min. After cooling on ice, the mixture was used for subsequent experiments. Primary antibodies (CD63, CD81, CD120a, CD274) were added, and after washing, secondary antibodies were added and incubated at room temperature for 1 h. After washing, the mixture was developed.

[0091] Please see Figure 3 Western blotting (WB) analysis further confirmed the presence of EV marker proteins. CD63 and CD81, which are not expressed in cells, were significantly expressed in EVs. Calnexin was present in cells but absent in EVs, confirming that the extracted extracellular vesicles conformed to the standard extracellular vesicle protein expression characteristics. In extracellular vesicles from cell lines stably expressing PD-L1, TNFR1, TNFR1 / PD-L1, and PD-L1 / TNFR1, the intensity of the target bands for PD-L1 and TNFR1 was significantly enhanced.

[0092] 3.3 Transmission electron microscopy characterization of EVs

[0093] (1) Take 10 μL of EVs suspension and drop it onto the front side of the copper mesh carbon film of the transmission electron microscope, and dry it at room temperature.

[0094] (2) After drying, the EVs were negatively stained with 10 μL of 2% phosphotungstic acid solution to enhance the contrast of the membrane structure. The EVs were then dried at room temperature.

[0095] (3) Observe the prepared sample with a transmission electron microscope (Talos, USA) and take images with an appropriate magnification.

[0096] The results are as follows Figure 3 As shown in Figure C, various EVs exhibit a typical bilayer membrane structure with a diameter of approximately 50–150 nm, and are uniform and intact in morphology. These results demonstrate that this application successfully prepared extracellular vesicles overexpressing PD-L1 / TNFR1.

[0097] Example 4: Interaction of EVs with PD-1 and TNF-α

[0098] (1) Mouse PD-1, TNF-α and PD-L1 / TNFR1 EVs were mixed with DMEM containing 10% FBS and incubated at 37 °C for 2 h. The supernatant was then collected after centrifugation at 16500 g for 10 min.

[0099] (2) The concentration of remaining PD-1 in the supernatant was quantified using an ELISA kit. The supernatant sample was added to a plate with coated antibody (100 μL per well) and incubated at room temperature for 2 h. After washing three times, the diluted detection antibody was added to all wells (100 μL per well) and incubated at room temperature for 1 h. After washing, 100 μL of avidin-HRP was added to each well and incubated at room temperature for 30 min. After washing three times, 100 μL of TMB was added to each well and incubated at room temperature for 8 min. 100 μL of stop solution was added to each well. Finally, the plate was read at 450 nm and the binding kinetics curve was plotted using Graphpad Prism8.

[0100] (3) In order to compare the interaction of Control EVs, PD-L1 EVs, TNFR1 EVs, TNFR1 / PD-L1 EVs, and PD-L1 / TNFR1 EVs with PD-1 and TNF-α, 5 ng / mL of recombinant mouse PD-1 and TNF-α were mixed with different EVs (100 μg / mL), and then measured using the above method. The results are expressed as the percentage of the remaining PD-1 or TNF-α relative to the total amount.

[0101] Please see Figure 4A. When the initial concentration of TNF-α is 5 ng / mL, it can effectively remove TNF-α, and the corresponding IC50 value (half-maximum inhibitory concentration) is 1.84 μg / mL. Figure 4 B shows that PD-L1 / TNFR1 EVs exhibited significantly higher TNF-α clearance efficiency than Control EVs, primarily due to the binding effect of highly expressed TNFR1 on TNF-α. Additionally, please refer to [link to relevant documentation]. Figure 4 C, when the initial concentration of PD-1 is 5 ng / mL, its corresponding IC50 value is 43.32 μg / mL. Figure 4 The results showed that the interaction between PD-L1 / TNFR1 EVs and PD-1 was significantly higher than that of Control EVs, mainly due to the recognition of PD-1 by PD-L1.

[0102] Example 5: Interaction between EVs and T cells

[0103] 5.1 Extraction and culture of T cells

[0104] (1) Preparation of spleen single-cell suspension: Select healthy C57BL / 6 mice, remove the spleen, temporarily store it in 1640 medium containing 10% FBS, grind the spleen until there are no red clumps, filter the single-cell suspension with a mesh, and centrifuge at 700 g for 5 min.

[0105] (2) Red blood cell removal: After centrifugation, discard the supernatant and add 8 mL of red blood cell lysis buffer to the resulting cell pellet for resuspending. Incubate at room temperature for 10 min to lyse the red blood cells. Then add 5 mL of complete culture medium to terminate the lysis reaction, and centrifuge at 700g for 5 min, discarding the supernatant. Next, wash the cell pellet three times with PBS solution, and finally adjust the cell concentration to 1×10⁻⁶ cells / mL with PBS. 8 cells / mL, to be used in subsequent experiments.

[0106] (3) T cells were isolated from spleen single-cell suspension using the EasySep™ mouse T cell isolation kit (Stemcell);

[0107] a. Add 20 μL / mL of FcR blocker to the cell suspension;

[0108] b. Continue adding 50 μL / mL of Isolation Cocktail;

[0109] c. Mix thoroughly and incubate at room temperature for 10 min;

[0110] d. RapidSpheres TMAnd to ensure the particles are evenly dispersed, add RapidSpheres. TM Add 75 μL / mL to the sample, mix and incubate at room temperature for 2.5 min, then transfer to a sterile flow cytometer.

[0111] e. Place the flow cytometer containing the sample into a magnet and incubate at room temperature for 3 minutes. Gently aspirate the clear portion with a pipette; this is the purified and isolated T cells.

[0112] (4) T cell culture: CD28 (2 μg / mL), IL-2 (10 ng / mL), β-mercaptoethanol (2 nM) and sodium pyruvate (10 mM) were added to 1640 medium containing 10% FBS. The cells were resuspended and the T cell density was adjusted to 1×10⁻⁶ cells / mL. 6 Cells / mL were seeded in wells coated with CD3 (5 μg / mL) overnight at 4 °C and incubated for 48 h to obtain activated T cells.

[0113] 5.2 T cell uptake of EVs

[0114] (1) Activated T cells were injected with 1×10 6 Cells were seeded at a density of cells / mL into 24-well plates and incubated for 24 h.

[0115] (2) Label EVs with DiD (10 μM), wash with PBS and collect by ultracentrifugation, add to well plate at a final concentration of 100 μg / mL and incubate for 24 h;

[0116] (3) After washing with PBS, Fc Block was added to remove non-specific antibodies, then anti-TCR beta-APC antibody was added and incubated for 30 min. After washing with PBS 3 times, the cell uptake was finally assessed by flow cytometry.

[0117] Please see Figure 4 F. Flow cytometry analysis showed that EVs can bind to T cells, and T cells can take up EVs, especially when EVs express PD-L1 on their surface, this interaction is more significant.

[0118] 5.3 Colocalization of PD-L1 (red) on EVs and PD-1 (green) on activated T cells

[0119] (1) DiD-labeled PD-L1 EVs, PD-L1 / TNFR1 EVs, TNFR1 / PD-L1 EVs and activated T cells were co-incubated for 24 h;

[0120] (2) The cells were fixed with 4% PFA for 30 min, blocked at room temperature for 1 h, stained with PD-1 antibody overnight at 4 ℃, washed, and incubated with secondary antibody Alexa Fluor 488 goat anti-mouse IgG for 1 h. The cells were then washed three times with PBS.

[0121] (3) After adding an anti-fluorescence quencher containing DAPI, observe and photograph using a confocal laser scanning microscope (ZEISS, Germany).

[0122] Please see Figure 4 E. Confocal microscopy revealed that extracellular vesicles (red) overexpressing PD-L1 could colocalize with PD-1 (green) on the surface of activated spleen T cells. The fluorescence of EVs and T cells highly overlapped on the cell membrane, forming a clear yellow colocalization signal region, indicating their interaction.

[0123] 5.4 Flow cytometry verification of the effect of EVs on T cell proliferation

[0124] (1) Adjust the cell density to 1×10⁻⁶ cells using 1 mL of CFDASE cell labeling solution. 6 Approximately [number] cells / mL;

[0125] (2) Add 1 mL of CFDASE stock solution, mix well, and incubate at 37 °C for 10 min;

[0126] (3) Add 10 mL of 1640 complete cell culture medium and mix well;

[0127] (4) After centrifugation, add 5 mL of complete cell culture medium to wash;

[0128] (5) Add 5 mL of complete cell culture medium and incubate at 37 °C for 5 min to promote the residence of CFDASE in the cells and the entry of unreacted CFDASE into the complete cell culture medium.

[0129] (6) Then, the cells can be cultured according to the normal culture method of T cells described above. After 48 h of cell activation, EVs of each group are added, and cell proliferation is detected by flow cytometry after 48 h.

[0130] The results are as follows Figure 4 As shown in G, the proportion of dividing cells stained by CFSE decreased in all EV treatment groups, indicating that both TNF-α clearance and restoration of the PD-1 / PD-L1 inhibitory axis can inhibit T cell proliferation. Among them, the PD-L1 / TNFR1 EVs group showed the most significant results. Therefore, in subsequent animal experiments, we used the PD-L1 / TNFR1 EVs group instead of the TNFR1 / PD-L1 EVs group.

[0131] The above results demonstrate that simultaneously clearing TNF-α and restoring the PD-1 / PD-L1 inhibitory axis is the most effective way to inhibit T cell activation.

[0132] 5.5 Effects of PD-L1 / TNFR1 EVs on T cell proliferation

[0133] The effects of EVs on CD4 were investigated using flow cytometry. + CD3 + T-cell immunomodulatory effects were observed, with PD-L1 / TNFR1EVs significantly reducing CD4 levels. + CD3 + The proportion of T cells ( Figure 4 H).

[0134] 5.6 Detection of cytokines using ELISA

[0135] (1) Collect the supernatant of each group of EVs and activated T cells after co-culturing for 48 h in the above experiment, centrifuge at 1500 g for 10 min at room temperature, and collect the supernatant.

[0136] (2) Add 100 μL of the sample to be tested into the reaction well and incubate at 37 °C for 1 h;

[0137] (3) Add 100 μL of enzyme-labeled antibody, incubate at 37 °C for 1 h, and wash 3 times;

[0138] (4) Add 100 μL of TMB substrate solution and incubate at 37 °C for 10 min;

[0139] (5) Add 50 μL of stop solution;

[0140] (6) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of each well at 450 nm.

[0141] Finally, this application measured the concentration of cytokines released by T cells in the culture medium. Figure 4 PD-L1 / TNFR1 EVs reduce the production of inflammatory cytokines (IL-1β, TNF-α, INF-γ). In summary, these results indicate that PD-L1 / TNFR1 EVs alter T cells to an immunosuppressive state and inhibit the production of inflammatory cytokines.

[0142] Example 6: Establishment and Treatment of CIA Model

[0143] (1) Mice were randomly divided into six groups: Normal group, PBS group, Control EVs group, PD-L1 EVs group, TNFR1 EVs group, and PD-L1 / TNFR1 EVs group. One week later, the five groups other than the Normal group were used to establish the model. At the time of the first immunization, bovine type II collagen (2 mg / mL) and complete Freund's adjuvant (2 mg / mL) were mixed 1:1 on ice until they did not disperse when dropped on water. Type II collagen emulsion was injected into the muscle at the base of the tail of the mice at multiple points, 100 μL per mouse, and this was recorded as day 0 of immunization.

[0144] (2) 21 days after the initial immunization, a booster immunization was performed. Bovine type II collagen and incomplete Freund's adjuvant were mixed 1:1 on ice and 100 μL was injected into the base of the tail of each mouse.

[0145] (3) The arthritis index was assessed every 3 days, and the thickness of the hind paw pads of mice was measured using calipers. CIA model score: 0 points: no symptoms; 1 point: swelling and redness of a single phalanx; 2 points: redness and swelling of two or more phalanxes; 3 points: obvious stiffness of the phalanx joints; 4 points: swelling and redness of the entire paw, including the ankle joint. After scoring the paws of the four limbs separately, the scores of each paw were added together to obtain the joint inflammation score, with a maximum total score of 16 points.

[0146] (4) After the CIA mice were successfully modeled, they were injected via the tail vein. The PBS group was injected with 0.1 mL of sterile PBS, while the other four EVs groups were injected with 100 μg / mL of EVs. This was done twice a week for a total of 3 weeks.

[0147] Example 7: Study on the biological distribution of EVs in CIA mice

[0148] (1) Add DiD fluorescent dye to four groups of EVs at 100 μg / mL, incubate overnight at 4 °C with shaking in the dark, and centrifuge to remove unbound dye;

[0149] (2) Four groups of DiD-labeled EVs were injected into mice via the tail vein, with the fluorescent dye DID serving as a control;

[0150] (3) Mice were anesthetized with isoflurane gas and images were taken at 1 h, 3 h, 6 h, 12 h and 24 h using a small animal in vivo imaging system (IVIS);

[0151] Please see Figure 5In mice with CIA, EVs labeled with DID were injected via the tail vein. After 1 h, 3 h, 6 h, 12 h, and 24 h, the fluorescence signal in the inflamed joints of the DID-labeled CIA mice was negligible after 1 h. However, in the PD-L1, TNFR1, and PD-L1 / TNFR1 EVs groups, strong fluorescence signals were observed in the inflamed joints after 1 h. The fluorescence intensity gradually increased at the inflamed joints over time, with a duration of up to 24 h.

[0152] (4) After 24 hours of imaging, the mice were sacrificed, and their paws and major organs (heart, liver, spleen, lungs and kidneys) were collected for in vitro imaging. Fluorescence intensity analysis was performed on these parts.

[0153] The results are as follows Figure 5 B and Figure 5 As shown in Figure C, 24 h after injection, organ fluorescence intensity indicated that EVs were mostly enriched in the liver and spleen, and the fluorescence at the joints of the fore and hind paws in the EVs group was significantly higher than that in the free DID group. This demonstrates that EVs have a good ability to target inflamed joint sites and are highly advantageous in targeted therapy for inflammatory diseases.

[0154] To investigate the interaction between PD-L1 / TNFR1 EVs and splenic T cells in CIA mice, this application also analyzed their co-localization using flow cytometry. First, splenic T cells were isolated, and then the cells were stained to label a specific receptor (CD3) on the T cell surface. (See [link to relevant documentation]). Figure 5 D. Flow cytometry results showed that 35.2% of T cells bound to PD-L1 / TNFR1 EVs, indicating that PD-L1 / TNFR1 EVs can effectively bind to splenic T cells. This result provides further evidence for the role of PD-L1 / TNFR1 EVs in mediating immune regulation.

[0155] Example 8: Analysis of Mouse Motion Ability Using Ink Footprints

[0156] (1) Prepare a flat experimental channel using cardboard, and lay a dry white absorbent paper on it to collect footprint imprints. Ensure the experimental environment is quiet and the lighting is moderate to minimize interference with the mice.

[0157] (2) Carefully remove the mouse, apply ink evenly to the soles of the mouse's feet, and gently guide the mouse from the entrance of the passage into the white paper area.

[0158] (3) After the mouse walks across the white paper area, its ink-stained feet will leave continuous footprint lines on the white paper. Remove the white paper and move it to a ventilated place to dry.

[0159] (4) After the ink dries, measure the motion parameters such as step length, stride length, and step frequency to analyze the motion ability and coordination of the experimental subjects.

[0160] Please see Figure 6 A and Figure 6 B. Through analysis of the number of footprints, stride length, and gait symmetry, mice treated with PD-L1 EVs, TNFR1 EVs, and PD-L1 / TNFR1 EVs showed significant improvements in gait symmetry and stride length. Compared with the control group, the mice's grip strength and motor ability were significantly improved. Among the four treatment groups, the PD-L1 / TNFR1 EVs group showed the best recovery of motor function.

[0161] In addition, this application also includes fore and hind paw gripping force tests to assess the mouse's paw strength; please refer to [link to relevant documentation]. Figure 6 C and Figure 6 The bottom right figure shows that the grip strength of the fore and hind paws of mice treated with PD-L1 / TNFR1 EVs was significantly enhanced, indicating that the reduction of joint inflammation improved limb function and strength in the mice. The combined results of the two experiments demonstrate that overexpression of PD-L1 / TNFR1 EVs can effectively alleviate joint inflammation in CIA mice, improve their motor function and limb strength, and provide strong experimental evidence for the application of EVs in the treatment of rheumatoid arthritis.

[0162] Example 9: Therapeutic effect of EVs in CIA mice

[0163] Please see Figure 7 A. First, mice that were successfully induced by CIA were intravenously injected twice a week with PBS, Control EVs, PD-L1 EVs, TNFR1 EVs, and PD-L1 / TNFR1 EVs, and clinical scores were performed and the thickness of the hind paw pads of the CIA mice was measured with calipers.

[0164] Three weeks after treatment, mice in each group were photographed and subjected to infrared thermography. Then, the hind paw joints of the mice were collected for micro-computed tomography (micro-CT) analysis. Hind paws from different groups were fixed in 4% paraformaldehyde solution for 24 hours, and then scanned and analyzed using a micro-CT scanner and imaging system.

[0165] Please see Figure 7 B. The PBS and Control EVs treatment groups showed natural disease progression and severe swelling of the paws. Compared with the PBS and Control EVs groups, the PD-L1, TNFR1, and PD-L1 / TNFR1 EVs treatment groups had milder symptoms, showing reduced inflammation and swelling.

[0166] Since the typical characteristics of joint inflammation are redness, swelling, pain, and elevated temperature, this application uses an infrared thermal scanning camera to measure the temperature of the hind paw joints of CIA mice. Please refer to [link to relevant documentation]. Figure 7 C. Compared with the normal group mice (25.3 ℃), the ankle joint temperature of CIA-inflamed mice was significantly higher (32.3 ℃). Compared with the PBS and Control EVs treatment groups, the ankle joint temperature of the PD-L1 EVs treatment group (27.2 ℃), TNFR1 EVs treatment group (29.0 ℃), and PD-L1 / TNFR1 EVs group (26.9 ℃) was significantly lower, with the PD-L1 / TNFR1 EVs group showing the lowest temperature.

[0167] Furthermore, monitoring of mouse weight showed that the weight of the treated group gradually returned to normal. Figure 7 D). Simultaneously, the thickness measurement of the mouse's hind paw was quantified ( Figure 7 E), and the severity of arthritis in mice was assessed by scoring all paw joints. Figure 7 F). The arthritis score in the PD-L1 / TNFR1 EVs treatment group was significantly lower than that in the PBS group, showing the most significant effect among the three EVs treatment groups, indicating a good therapeutic effect.

[0168] Finally, please see Figure 7 G. In this application, the distal tibia, ankle joint, and paw of the right hind limb of mice were preserved, and micro-CT scans and 3D reconstructions were performed after removing excess soft tissue. Results showed that the ankle and toe joints of mice in the PBS group suffered the most severe bone destruction, exhibiting moth-eaten changes. The Control EVs group showed no therapeutic effect, while the PD-L1 EVs, TNFR1 EVs, and PD-L1 / TNFR1 EVs groups all showed varying degrees of improvement, with the PD-L1 / TNFR1 EVs group showing the most significant reduction in bone damage. Data analysis indicated that PD-L1 / TNFR1 EVs significantly improved trabecular bone thickness (Tb.Th), bone mineral density (BMD), bone surface area / bone volume ratio (BS / BV), and bone volume fraction (BV / TV). Figure 7 HK).

[0169] Example 10: Mouse tissue sections and staining

[0170] 10.1 Preparation of Paraffin Sections

[0171] (1) Sample processing: After the CIA mice were euthanized, the heart, liver, spleen, lungs, kidneys and joints were removed. After removing the skin and flesh of the hind limbs, they were soaked in 4% paraformaldehyde solution and stored for 48 h.

[0172] (2) Joint decalcification: Wash the tissue three times with PBS for 20 min each time. Then, transfer the tissue to 20 times its volume of EDTA decalcification solution and place it in a 4 ℃ refrigerator. Change the decalcification solution once a week. When the needle tip can easily penetrate the bone tissue, it indicates that the decalcification process is complete.

[0173] (3) Dehydration: Dehydration is carried out step by step according to the ethanol gradient (from low concentration to high concentration);

[0174] (4) Transparency: The tissue was transparentized by sequentially using a mixture of xylene and ethanol, followed by xylene;

[0175] (5) Wax impregnation: The tissue is sequentially immersed in soft wax 1, soft wax 2, hard wax 1, and hard wax 2 for treatment;

[0176] (6) Embedding: Take a tissue sample and place it in an embedding mold, pour in preheated and melted paraffin, and then cool and solidify it.

[0177] (7) Sectioning: After the solidified tissue wax block is properly trimmed, it is fixed on the microtome for continuous cutting, and the section thickness is set to 5 μm;

[0178] (8) Mounting: Place the cut tissue wax slide in a water bath to flatten it, adjust the position of the slide appropriately, and then remove it. Then place it on a glass slide to air dry naturally for later use.

[0179] 10.2 Hematoxylin-eosin (HE) staining

[0180] (1) Dewaxing paraffin sections to water: First, immerse the sections in two environmentally friendly dewaxing solutions in turn, each for 20 minutes. Then, soak the sections in anhydrous ethanol for 5 minutes, then transfer them to 75% alcohol for 5 minutes, and finally rinse with ddH2O.

[0181] (2) Pretreatment: Place the slides in high-resolution constant staining pretreatment solution for 1 min;

[0182] (3) Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 5 min, then rinse with ddH2O, differentiate with differentiation solution, rinse with ddH2O again, and finally use blueing solution for blueing treatment and rinse with running water.

[0183] (4) Eosin staining: Dehydrate the sections in 95% alcohol for 1 min, then immerse them in eosin staining solution for 15 s;

[0184] (5) Dehydration and mounting: Treat with ethanol and xylene for 5 min each according to the gradient from low to high concentration, and finally mount with neutral resin;

[0185] (6) Microscopic photography: Take pictures under a microscope and collect images for analysis.

[0186] Please see Figure 8 A, H&E staining results showed that in the PBS group, inflammatory cell infiltration in the joint was significantly increased, synovial hyperplasia and thickening were severe, and there was significant articular cartilage damage. In contrast, in the PD-L1 / TNFR1 EVs treatment group, the joint cavity was almost completely restored, the cartilage interface was clearly visible, inflammatory cells were reduced, and synovial inflammation was significantly alleviated.

[0187] 10.3 Safranin O–Fast Green Staining

[0188] (1) Deplasticization of tissue sections to water: First, place the tissue sections in ethylene glycol ethyl ether acetate I and treat them at 37 ℃ for 6 h; then replace with ethylene glycol ethyl ether acetate II and continue incubation at 37 ℃ until the next day; then place the sections in ethylene glycol ethyl ether acetate III and incubate at room temperature for 15 min, then transfer them to ethylene glycol ethyl ether acetate IV and continue incubation at room temperature for 15 min to complete the deplasticization process. Subsequently, soak them in 100% ethanol I and ethanol II for 10 min each, then soak them in 95%, 90%, and 80% ethanol for 10 min each, and finally wash with ddH2O;

[0189] (2) Fast Green staining: Immerse the sections in Fast Green staining solution for 3 min, then rinse with water to remove excess staining solution. If the color is light, counterstain; if the color is dark, use differentiation solution for differentiation treatment;

[0190] (3) Safranin staining: After staining the tissue sections in safranin staining solution, dehydrate them three times with anhydrous ethanol gradient to remove excess staining solution.

[0191] (4) Clearing and mounting: Clear the dehydrated sections with xylene solution for 5 min, then add neutral resin to cover the tissue and complete the mounting operation;

[0192] (5) Microscope photography: Take pictures under a microscope and collect images for analysis.

[0193] Please see Figure 8B. Safranin O staining showed that the levels of glycosaminoglycans (GAG), a component of the cartilage matrix, were higher in the PD-L1, TNFR1, and PD-L1 / TNFR1 groups than in the PBS and Control EVs groups, suggesting effective reversal of cartilage damage. Importantly, the PD-L1 / TNFR1 group had the largest area of ​​Safranin O staining positivity, closer to the normal group, indicating an effective therapeutic effect in CIA mice. These results suggest that PD-L1 / TNFR1 EVs can alleviate synovial inflammation and reduce cartilage destruction in CIA mice, demonstrating great potential for clinical treatment of RA.

[0194] 10.4 Immunohistochemistry (IHC)

[0195] (1) Bake the slices at 65 °C for 2 h, then put them in xylene solution (3 times, 10 min each time), then put them in anhydrous ethanol twice for 10 min each, then put them in 95% ethanol, 85% ethanol and 75% ethanol for 5 min each, and finally put them in ddH2O for 10 min to complete dewaxing and dehydration.

[0196] (2) Prepare 1× antigen retrieval solution and put it in a pressure cooker to boil. Then put the slice into the antigen retrieval solution and boil for another 10 minutes after boiling. Take it out and cool it to room temperature.

[0197] (3) After washing with ddH2O for 10 min, draw circles around the tissue with a hydrophobic pen;

[0198] (4) H2O2 was added to the tissue and incubated in the dark for 10 min. The tissue was washed twice with TBST for 5 min each time and then blocked with blocking solution for 1 h.

[0199] (5) Add CD3 primary antibody directly and incubate overnight in a humidified box at 4 °C in the dark;

[0200] (6) Gently shake off the primary antibody, wash 3 times with TBST for 5 min each time, add secondary antibody, incubate at room temperature for 1 h, wash 3 times with TBST for 5 min each time;

[0201] (7) Prepare DAB solution for staining for 30 s, observe the color under a microscope. If the color is too light, the staining time can be extended appropriately. Wash with TBST 3 times, 5 min each time.

[0202] (8) Hematoxylin dye was dropped onto the tissue for 1 min, washed with ddH2O for 2 min, placed in differentiation solution for 3 s, and the nuclear staining effect was observed under a microscope to confirm. TBST was used to wash 3 times, 5 min each time.

[0203] (9) The slides were placed in 75% ethanol, 85% ethanol and 95% ethanol for 5 min each, then in anhydrous ethanol for 10 min, and finally in xylene for 10 min. Then the mounting medium was directly dripped onto the slides for mounting, and the slides were placed in a fume hood to air dry.

[0204] (10) Microscopic observation, image acquisition, staining and scoring.

[0205] Please see Figure 8 C. Immunohistochemical analysis showed that CD3 was present in the synovium of CIA mice. + T cell aggregation was significant. In mice treated with PD-L1 EVs, TNFR1 EVs, and PD-L1 / TNFR1 EVs, CD3... + T cell infiltration was significantly reduced, with the most significant reduction observed in the PD-L1 / TNFR1 EVs treatment group.

[0206] 10.5 Immunofluorescence (IF) of paraffin sections

[0207] (1) After dewaxing, antigen retrieval, H2O2 incubation and blocking as described above, add primary antibodies (IL-10, TNF-α, IL-1β and IL-6), place the slides in a humidified chamber and incubate overnight at 4 ℃.

[0208] (2) Wash the slides three times with TBST for 5 minutes each time, and gently shake to remove unbound primary antibody.

[0209] (3) Add fluorescently labeled secondary antibody, incubate at room temperature in the dark for 1 h, and wash with TBST 3 times in the dark for 10 min each time after incubation.

[0210] (7) Add DAPI-containing anti-fluorescence quenching mounting medium to the slide, cover with a coverslip, and after the slide dries for a while, it can be observed with a Thunder fluorescence microscope.

[0211] (8) Observe the fluorescence signal and acquire images at an appropriate excitation wavelength, and save the images for subsequent analysis.

[0212] Please see Figure 8Compared with the Normal group, the expression of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) was significantly increased in mice in the PBS and Control EVs groups, indicating that these cytokines play an important role in the pathogenesis of RA. The Control EVs group did not significantly alter the expression of these pro-inflammatory factors. The PD-L1, TNFR1, and PD-L1 / TNFR1 groups all showed decreased expression levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), while IL-10 expression was significantly increased. Among these, the PD-L1 / TNFR1 EVs group exhibited the strongest anti-inflammatory effect. These results indicate that injection of PD-L1 / TNFR1 EVs can significantly reduce the production of pro-inflammatory cytokines while increasing the expression of anti-inflammatory cytokines, thus having a positive impact on the treatment of RA.

[0213] Given the crucial role of Th17 / Treg cell balance in RA pathology, this application further investigated the effects of PD-L1 / TNFR1 EVs on these two effector cell types. To assess changes in Th17 and Treg cell populations in the blood and spleen of CIA mice after treatment, this application used flow cytometry to analyze CD4+ cells. + IL-17 + cell( Figure 9 A, C) and CD4 + FoxP3 + Cells in CD4 + The proportion of T cells ( Figure 9 (B, D). Compared with the PBS group, mice in the PD-L1 EVs group, TNFR1 EVs group, and PD-L1 / TNFR1 EVs group all showed CD4+ expression after treatment. + IL-17 + Cellular significant downregulation and CD4 + FoxP3 + The upregulation of cellularity indicated a decrease in the proportion of Th17 cells and an increase in the proportion of Treg cells, with the most significant changes observed in the PD-L1 / TNFR1 EVs group. These results suggest that overexpression of PD-L1 / TNFR1 EVs can effectively restore the Th17 / Treg imbalance associated with arthritis, thereby having a positive impact on the treatment of RA.

[0214] Example 11: Biosafety Evaluation of EVs

[0215] While achieving significant therapeutic effects, the potential toxicity and biosafety issues of EVs also require attention. To assess the biosafety of EVs, we examined HE-stained tissue sections of the heart, liver, spleen, lungs, and kidneys of mice in each treatment group after treatment. The results showed no significant differences between the organ tissue sections of each treatment group and the normal group. Figure 10 A). In addition, we performed a complete blood count (CBC) on blood samples from mice in the PD-L1 / TNFR1 EVs group and the control group (…). Figure 10 (B) The results showed that there were no statistically significant differences between the two groups in the number of red blood cells, white blood cells, hemoglobin, platelets, neutrophils, lymphocytes, and monocytes. These results indicate that PD-L1 / TNFR1 EVs treatment has no significant toxic side effects and has good biocompatibility.

[0216] In summary, this study experimentally verified the role of PD-L1 / TNFR1 EVs in T-cell immunomodulation, suggesting that they can correct the immune imbalance in RA by inhibiting excessive T-cell activation and clearing inflammatory factors at the lesion site. Furthermore, the extracellular vesicles expressing both PD-L1 and TNFR1 constructed in this application not only inhibit joint inflammation and reverse tissue damage but also possess immunomodulatory effects. This combined therapy provides a more reliable and comprehensive treatment option for RA. Moreover, PD-L1 / TNFR1 EVs exhibit good biocompatibility and show promising application potential in the clinical treatment of RA.

Claims

1. A method for constructing extracellular vesicles overexpressing PD-L1 / TNFR1, characterized in that: Includes the following steps: S1: A lentiviral expression vector for PD-L1 / TNFR1 was constructed and transfected into HEK293T packaging cells to generate lentiviruses that highly express PD-L1 / TNFR1. HEK293T cells were then infected with the lentiviruses that highly express PD-L1 / TNFR1, and cell lines that stably express PD-L1 / TNFR1 were obtained by selection with puromycin. At the same time, three lentiviral expression vectors that overexpress PD-L1, TNFR1, and TNFR1 / PD-L1 were constructed using the same method, and three cell lines that stably express PD-L1, TNFR1, and TNFR1 / PD-L1 were obtained, respectively. S2: Ultracentrifugation was used to extract EVs that highly expressed PD-L1 / TNFR1 secreted by cells that stably expressed PD-L1 / TNFR1. At the same time, PD-L1 EVs, TNFR1 EVs and TNFR1 / PD-L1 EVs were extracted as controls.

2. The method for constructing extracellular vesicles overexpressing PD-L1 / TNFR1 according to claim 1, characterized in that: The specific steps of S1 are as follows: Plasmids PLVX-Flag-PD-L1, PLVX-Flag-TNFR1, PLVX-Flag-TNFR1-PD-L1, PLVX-Flag-PD-L1-TNFR1 and psPAX2, along with pMD2.G from the lentiviral delivery system, were co-transfected into HEK293T cells. After 24 h of culture, the medium was replaced with fresh DMEM. Cell culture supernatant was collected at 48 h and 72 h and filtered through a 0.45 μm filter to obtain three lentiviruses carrying PD-L1, TNFR1, TNFR1 / PD-L1, and PD-L1 / TNFR1, respectively. The prepared lentivirus was used to infect HEK293T cells with good growth and confluence of 30%–50%. 1 mL of virus solution was added to each well. Polybrene was added at the same time to increase the infection efficiency. After 12 h of virus infection, the supernatant was discarded, and fresh DMEM was added for further culture. The supernatant was discarded after 48 h. Add 2 mL of culture medium containing puromycin and select for one week. Expand and passage the selected cells, and add 0.5 μg / mL of puromycin to maintain resistance. After continuous selection and passage for 3 generations, HEK293T cell lines that stably express PD-L1, TNFR1, TNFR1 / PD-L1 and PD-L1 / TNFR1 were obtained.

3. The method for constructing extracellular vesicles overexpressing PD-L1 / TNFR1 according to claim 2, characterized in that: In S1, the mass ratio of PLVX-Flag-PD-L1-TNFR1, psPAX2, and pMD2.G is 3:1:1, and the mass ratio of PLVX-Flag-PD-L1, PLVX-Flag-TNFR1, PLVX-Flag-TNFR1-PD-L1 to psPAX2 and pMD2.G, which serve as controls, is also 3:1:

1.

4. The method for constructing extracellular vesicles overexpressing PD-L1 / TNFR1 according to claim 1, characterized in that: The specific steps of S2 are as follows: When the cell density reaches 80%, switch to DMEM-containing medium and culture for 24 h, then collect the cell supernatant. Centrifugation was used to remove cells, large particles and debris, small organelle fragments and larger impurities from the supernatant. The supernatant was then collected and filtered through a 0.22 μm sterile filter. The filtered supernatant was centrifuged at 150,000 g for 2 h, the supernatant was removed, the precipitate was retained, the precipitate was resuspended in 200 μL PBS to collect EVs, the concentration was detected and then stored at -80 ℃ for later use. The extraction methods for PD-L1 EVs, TNFR1 EVs, and TNFR1 / PD-L1 EVs are as described above.

5. The method for constructing extracellular vesicles overexpressing PD-L1 / TNFR1 according to claim 4, characterized in that: In step (2) of S2, the centrifugation is divided into four times. First, centrifuge at 500 g for 5 min to remove cells. Then, centrifuge at 1000 g for 10 min to remove large particles and debris. Then, centrifuge at 16500 g for 30 min to remove small organelle debris and larger impurities. Finally, centrifuge at 150000 g for 2 h, remove the supernatant, keep the precipitate, and resuspend the precipitate in 200 μL PBS to collect EVs.

6. The method for constructing extracellular vesicles overexpressing PD-L1 / TNFR1 according to claim 1, characterized in that: In S1, a single-chain polypeptide composed of PD-L1 and TNFR1 was first designed. The single-chain polypeptide composed of PD-L1 and TNFR1 was fused with the transmembrane domain gene of platelet-derived growth factor receptor (PDGFR). A (GGGGS)4 flexible linker was also inserted between PD-L1 and TNFR1. A (GGGGS)4 flexible linker was also inserted in the middle of the single-chain polypeptide composed of TNFR1 / PD-L1.

7. An EV that overexpresses PD-L1 / TNFR1, characterized in that: It is constructed using the construction method described in any one of claims 1-6.

8. The application of PD-L1 / TNFR1 overexpression EVs in the preparation of drugs for treating rheumatoid arthritis, characterized in that: The overexpressed PD-L1 / TNFR1 EVs were constructed using any one of the construction methods of claims 1-6.

Images