Bispecific cell membrane nanoparticle as well as preparation method and application thereof

Abstract

The invention belongs to the field of medicine preparation, and particularly relates to bispecific cell membrane nanoparticles and a preparation method and application thereof, and the preparation method comprises the following steps: carrying out in-vitro induction on primary CD4 + T cells by adopting TGF-beta and rapamycin to obtain regulatory T cells; enabling the distearoyl phosphatidyl ethanolamine-polyethylene glycol-active ester to react with the anti-CD4 antigen binding fragment, so as to obtain DP-aCD4; pre-incubating free aCD4 by the regulatory T cell, and co-incubating with DP-aCD4 to obtain an aCD4-regulatory T cell; the aCD4-regulatory T cells are mixed with an antigen peptide and an immunosuppressive agent, and the bispecific cell membrane nanoparticles AP-IA (at) aCD4Tn are prepared through a liposome extruder. After in-vivo administration of the bispecific cell membrane nanoparticles, generation of antigen-specific regulatory T cells can be efficiently induced, and accurate and efficient treatment and prevention of rheumatoid arthritis are realized.

Description

Technical Field

[0001] The present invention belongs to the field of drug preparation, and particularly relates to a bispecific cell membrane nanoparticle and a preparation method and application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammatory synovium and cartilage destruction of joints, which is caused by the immune system mistakenly attacking normal tissues. Clinically, anti-inflammatory small molecule drugs and biological agents are usually used systemically to treat RA. However, due to the short half-life and poor targeting of the drugs, the therapeutic effect is insufficient, and non-specific inhibition of the immune system is likely to cause serious toxic and side effects. Immune tolerance therapy can precisely treat RA and is one of the most advanced RA treatment strategies. Inducing the generation of antigen-specific regulatory T cells (Tregs) is the core of immune tolerance therapy. Existing therapies usually deliver antigens to antigen-presenting cells (APCs) in immune organs, especially dendritic cells (DCs), to induce T cell tolerance. However, the efficiency of inducing regulatory T cells in current strategies is still insufficient, and the therapeutic effect needs to be further improved. Therefore, there is an urgent need to provide a method with high efficiency in inducing regulatory T cells and good therapeutic effect. Summary of the Invention

[0003] The present invention provides a bispecific cell membrane nanoparticle and a preparation method and application thereof. The bispecific cell membrane nanoparticle can specifically bind to the CD80 / 86 co-stimulatory molecule and CD4 of dendritic cells through the surface membrane protein CTLA4 and the antigen-binding fragment of anti-CD4 antigen + and the CD4 receptor of CD4 T cells, forming a dendritic cell-CD4 T cell pair, promoting the interaction between antigen-presenting cells and CD4 T cells. At the same time, the bispecific cell membrane nanoparticle releases antigen peptides and immunosuppressants, efficiently generates antigen-specific regulatory T cells, has a good immunomodulatory effect, and realizes precise and efficient treatment and prevention of rheumatoid arthritis. + T cells, promoting the interaction between antigen-presenting cells and CD4 + T cells. At the same time, the bispecific cell membrane nanoparticle releases antigen peptides and immunosuppressants, efficiently generates antigen-specific regulatory T cells, has a good immunomodulatory effect, and realizes precise and efficient treatment and prevention of rheumatoid arthritis.

[0004] The technical solution provided by the present invention is as follows:

[0005] A preparation method of a bispecific cell membrane nanoparticle, comprising the following steps: primary CD4 +T cells are induced into regulatory T cells in vitro using TGF-β and rapamycin; distearoyl phosphatidylethanolamine-polyethylene glycol-activated ester is reacted with the antigen-binding fragment of anti-CD4 to obtain DP-aCD4; after pre-incubating regulatory T cells with the free antigen-binding fragment of anti-CD4, they are co-incubated with DP-aCD4 to obtain aCD4-regulatory T cells; aCD4-regulatory T cells are mixed with antigen peptides and immunosuppressants, and bispecific cell membrane nanoparticles are prepared using a liposome extruder.

[0006] Furthermore, the molar ratio of distearoyl phosphatidylethanolamine-polyethylene glycol-activated ester to the antigen-binding fragment of anti-CD4 is 2-20:1.

[0007] Furthermore, distearoyl phosphatidylethanolamine-polyethylene glycol-activated ester and the antigen-binding fragment of anti-CD4 are reacted under the conditions of pH 8-9 for 10-14 h to obtain DP-aCD4.

[0008] Furthermore, the antigen peptide is one or more of citrullinated peptide, type II collagen, glucose-6-phosphate isomerase peptide, heat shock protein peptide, RA33 peptide.

[0009] Furthermore, the immunosuppressant includes one or more of rapamycin, everolimus, temsirolimus, tacrolimus, cyclosporine A.

[0010] Furthermore, the ratio of antigen peptide to aCD4-regulatory T cells is 5-20 μg: 1 × 10 7 cells; the ratio of immunosuppressant to aCD4-regulatory T cells is 1-4 μg: 1 × 10 7 cells.

[0011] The present invention also provides a bispecific cell membrane nanoparticle prepared by the above preparation method.

[0012] The present invention also provides the application of the above bispecific cell membrane nanoparticle in the preparation of immunosuppressive drugs.

[0013] Furthermore, the immunosuppressive drug is a drug for treating and / or preventing rheumatoid arthritis.

[0014] Furthermore, the bispecific cell membrane nanoparticle is used to induce antigen-specific regulatory T cells.

[0015] Furthermore, the bispecific cell membrane nanoparticle is used to relieve inflammation, cartilage degradation, synovial fibrosis and bone erosion caused by rheumatoid arthritis.

[0016] The present invention also provides the application of the above bispecific cell membrane nanoparticle in the preparation of drugs for promoting antigen presentation.

[0017] Preparation of bispecific nanoparticles: Extract CD4 + T cells and induce them into regulatory T cells in vitro using TGF-β and rapamycin; React DSPE-PEG 2000 -NHS with the anti-CD4 antigen-binding fragment to synthesize DSPE-PEG 2000 -aCD4 (DP-aCD4); Co-incubate regulatory T cells with the anti-CD4 antigen-binding fragment to shield the CD4 receptor on the cell surface, and then co-incubate with DP-aCD4 to prepare aCD4-regulatory T cells (aCD4-T regs ); Mix aCD4-regulatory T cells with antigen peptide (Antigen peptid, AP) and immunosuppressive agents (Immunosuppressive agents, IA), and continuously extrude through a liposome extruder to prepare the bispecific nanoparticle AP-IA@aCD4Tn.

[0018] Beneficial effects

[0019] The innovative advantage of the bispecific nanodelivery system lies in its unique dual-targeting mechanism, which efficiently induces immune tolerance by coordinately regulating the key cell interaction network in the immune microenvironment. The DC-CD4 bridging system targets both DC and CD4 + T cells, physically shortening the spatial distance between them, reconstructing the formation of "immune synapses" in the local microenvironment, and efficiently inducing the generation of regulatory T cells. The cell bridging effect mediated by dual targeting can significantly enhance the co-stimulatory signal transduction efficiency of DC to T cells. When the AP loaded in the nanoparticles is efficiently presented by DC, under the microenvironment regulation created by IA, the differentiation direction of CD4 + T cells is reprogrammed into antigen-specific regulatory T cells. At the same time, the cell bridging effect mediated by dual targeting significantly enhances the co-stimulatory signal transduction efficiency between DC and T cells, breaking through the limitation of traditional single-target therapies in coordinating cell differentiation. This cascade amplification strategy based on the natural immune regulation mechanism improves the induction efficiency of regulatory T cells while avoiding the risk of systemic immunosuppression, and finally reshapes the immune homeostasis through factors such as TGF-β / IL-10 secreted by regulatory T cells, achieving efficient and precise treatment and prevention of rheumatoid arthritis. Brief description of the drawings

[0020] Figure 1 Schematic diagram of the preparation process and mechanism of action of the bispecific cell membrane nanoparticles of the present invention's embodiment (created using Biorender.com); where Figure 1 a is the synthetic route; Figure 1 b is the mechanism of action diagram;

[0021] Figure 2 For the preparation and characterization of bispecific cell membrane nanoparticles; among them Figure 2 a in it is the flow cytometry result diagram of in vitro induction of regulatory T cells (n = 3); Figure 2 b in it is the CTLA4 expression level of regulatory T cells (n = 3); Figure 2 c in it is the nanoparticles modified with different molar ratios of DSPE-PEG 2000 (DP) The binding efficiency of DP-aCD4 prepared to CD4 + T cells (n = 3); Figure 2 d in it is the encapsulation efficiency and drug loading of the nanoparticles (n = 3); Figure 2 e in it is the release curve of the nanoparticles (n = 3); The data are expressed as mean ± SD, and the statistical analysis uses unpaired two-tailed t-test;

[0022] Figure 3 For the characterization of bispecific cell membrane nanoparticles; among them Figure 3 a in it is the confocal characterization of the nanoparticles; Figure 3 b in it is the electron micrograph of the nanoparticles; Figure 3 c in it is the particle size and zeta potential of the nanoparticles (n = 3); Figure 3 d in it is the particle size of the nanoparticles under different storage conditions (n = 3); Figure 3 e in it is the activity of the membrane protein of the nanoparticles under different storage conditions (n = 3); Figure 3 f in it is the nanoparticles and CD4 + Confocal image of the binding of T cells; Figure 3 g in it is the confocal image of the uptake of nanoparticles by bone marrow-derived dendritic cells (BMDC); Figure 3 h in it is the confocal image of the presentation of antigen to the cell membrane surface after BMDC cells take up the nanoparticles; The data are expressed as mean ± SD, and the statistical analysis uses unpaired two-tailed t-test;

[0023] Figure 4 For the immunosuppression of bispecific cell membrane nanoparticles on DC cells; among them Figure 4 a in it is the expression levels of CD80 and PD-L1 after co-incubation of BMDC with the nanoparticles (n = 5); Figure 4 b in it is the expression levels of mRNA TNF-α and TGF-β in BMDC (n = 5); Figure 4 c in it is the levels of TNF-α and TGF-β in the incubation solution (n = 5); The data are expressed as mean ± SD, and the statistical analysis uses one-way ANOVA and Tukey post hoc test;

[0024] Figure 5 For the immunosuppression of bispecific cell membrane nanoparticles on CD4 + T cells; among themFigure 5 where a is CD4 + Expression levels of CTLA4 and PD-1 after co-incubation of T cells with nanoparticles (n = 5); Figure 5 where b is the levels of TNF-α and TGF-β in the incubation solution (n = 5); Figure 5 where c is CD4 + Levels of TNF-α and TGF-β mRNA in T cells (n = 5); Data are expressed as mean ± SD, and statistical analysis was performed using one-way ANOVA and Tukey's post hoc test;

[0025] Figure 6 is the bridging ability of the DC-CD4 system; among which, Figure 6 where a is the schematic diagram of CII-RM@aCD4Tn-mediated CD4 + T cell binding to DC (created using Biorender.com); Figure 6 where b is the analysis of cell pair formation by flow cytometry after co-culturing CII-RM@Tn or CII-RM@aCD4Tn with BMDC-CD4 + T cells for 1 hour (n = 3); Figure 6 where c is the immunofluorescence staining image of inguinal lymph nodes (iLNs) after intradermal (i.d.) injection of CII-RM@aCD4Tn in mice; The bottom row of photos is the magnified image of the top row rectangular area (paracortical area); Scale bar: 100 μm (top row), 20 μm (middle row), 5 μm (bottom row); Blue: DAPI; Red: CD11c; Green: CD4; Yellow: Cy5-CII-RM@aCD4Tn; Data are expressed as mean ± SD, and statistical analysis was performed using unpaired two-tailed t-test;

[0026] Figure 7 is the biodistribution of CII-RM@aCD4Tn; among which, Figure 7 where a is the fluorescence imaging of major organs 12 hours after i.d. injection of Cy5-CII-RM@aCD4Tn in mice (aLN is axillary lymph node, iLN is inguinal lymph node); Figure 7 where b is the fluorescence intensity analysis of major organs (n = 5); Figure 7 where c is the proportional distribution of fluorescence intensity in each organ; Figure 7 where d is the fluorescence imaging of mice at different time points after drug injection; Figure 7 where e is the curve of fluorescence intensity change over time in the injection area (n = 5); Figure 7 where f is the area under the fluorescence intensity curve (n = 5); Data are expressed as mean ± SD, and statistical analysis was performed using unpaired two-tailed t-test;

[0027] Figure 8 In vivo toxicity assessment of the DC-CD4 bridging system; wherein Figure 8 a in it is the experimental protocol for the safety assessment of DBA / 1 mice; Figure 8 b in it is the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and albumin in serum (n = 5); Figure 8 c in it is the results of blood routine tests (n = 5); Data are expressed as mean ± SD, and statistical analysis is performed using one-way ANOVA and Tukey's post hoc test;

[0028] Figure 9 Are hematoxylin-eosin (H&E) staining images of major organs; Scale bar: 100 μm; Data are expressed as mean ± SD, and statistical analysis is performed using one-way ANOVA and Tukey's post hoc test;

[0029] Figure 10 OVA-RM@aCD4Tn induces the generation of regulatory T cells in vivo; wherein, Figure 10 a in it is CD25 in the lymph nodes of OT-II mice + Intracellular Foxp3 + Regulatory T cells (T regs ) representative flow cytometry plots of FACS; Figure 10 b in it is CD25 in the lymph nodes + Intracellular Foxp3 + Percentage of cells and absolute number of regulatory T cells (n = 5); Figure 10 c in it is CD4 in the spleen of OT-II mice + Intracellular CD25 + Foxp3 + Representative flow cytometry plots of FACS of regulatory T cells; Figure 10 d in it is CD4 in the spleen + Intracellular CD25 + Foxp3 + Percentage of cells and absolute number of regulatory T cells (n = 5); Data are expressed as mean ± SD, and statistical analysis is performed using one-way ANOVA and Tukey's post hoc test;

[0030] Figure 11 OVA-RM@aCD4Tn induces antigen-specific immunosuppression in vivo; wherein, Figure 11 a in it is that OT-II mice are pre-treated with different nanoparticles, and then spleen cells are extracted and treated with chicken ovalbumin peptide 323-339(OVA 323-339 ) or type II collagen peptide (CII 259-272 ), representative enzyme-linked immunosorbent spot (ELISPOT) dot plots of tumor necrosis factor-α (TNF-α)-secreting cells after stimulation (n = 5); Figure 11 In b, the number of TNF-α-secreting cells in splenocytes after antigen stimulation (n = 5); Figure 11 In c, a cluster heatmap of transcriptional expression of spleen CD4 + T cell-related genes in mice after administration of different nanoparticles (n = 3); Figure 11 In d, Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis of differentially expressed genes between the OVA group and the OVA-RM@aCD4Tn group; Data are expressed as mean ± SD, and statistical analysis was performed using one-way analysis of variance (One-way ANOVA) and Tukey's post hoc test;

[0031] Figure 12 Therapeutic effect of CII-RM@aCD4Tn on collagen-induced arthritis (CIA) mice; Among them, Figure 12 In a, the experimental protocol for evaluating the early therapeutic effect of CII-RM@aCD4Tn; Figure 12 In b, the arthritis score curve (n = 6); Figure 12 In c, the body weight change curve (n = 6); Figure 12 In d, paw swelling measurement on day 42 (n = 6); Figure 12 In e, evaluation of the motor ability of CIA mice using a Rotarod experimental system on day 42 (n = 6); Data are expressed as mean ± SD, and statistical analysis was performed using two-way analysis of variance (Two-way ANOVA) and Tukey's post hoc test (b and c), or one-way analysis of variance (One-way ANOVA) and Tukey's post hoc test (d and e);

[0032] Figure 13 Therapeutic effect of CII-RM@aCD4Tn on collagen-induced arthritis (CIA) mice; Among them, Figure 13 In a and d, representative images of hematoxylin and eosin (H&E) stained tissue sections of the ankle joint (a) and histopathological evaluation of synovitis score (d) (n = 6); Figure 13 In b and e, representative images of immunohistochemical staining of interleukin-6 (IL-6) in the ankle joint (b) and measurement of the synovial IL-6 staining area (e) (n = 6); Figure 13In (c) and (f), representative images of Masson staining of the ankle joint and measurement of synovial fibrosis area (f) are shown (n = f); data are presented as mean ± SD, and statistical analysis was performed using one-way ANOVA and Tukey's post hoc test;

[0033] Figure 14 This shows the therapeutic effect of CII-RM@aCD4Tn on collagen-induced arthritis (CIA) mice; among them, Figure 14 In (a) and (d), representative images of safranin O-fast green (SO-FG) staining of the ankle joint and measurement of synovial cartilage thickness (d) are shown (n = 6); Figure 14 In (b), representative micro-CT images of the ankle joint on day 42 are shown; Figure 14 In (c), representative images of the coronal section of the ankle joint are shown; the red square (1×1×1 mm) is the region of interest (ROI) of the tibial cancellous bone; the arrows indicate the cancellous bone regions of the tibia (red), fibula (yellow), and talus (green); scale bar: 1 mm; Figure 14 In (e), the bone volume fraction of the ROI region is shown (n = 6); data are presented as mean ± SD, and statistical analysis was performed using one-way ANOVA and Tukey's post hoc test;

[0034] Figure 15 This shows immunofluorescence staining of CD4 + CD25 + Foxp3 + regulatory T cells in the synovium; among them, Figure 15 In (a), immunofluorescence staining images of the synovium are shown; scale bar: 200 μm; blue: DAPI; red: CD4; green: CD25; yellow: Foxp3; Figure 15 In (b), the stained area of Foxp3 + cells in the synovium is shown (n = 6); Figure 15 In (c), the stained area of CD4 + CD25 + Foxp3 + cells in the synovium is shown (n = 6); data are presented as mean ± SD, and statistical analysis was performed using one-way ANOVA and Tukey's post hoc test;

[0035] Figure 16 This shows flow cytometry analysis of regulatory T cells in the lymph nodes and spleen; among them, Figure 16 In (a), a representative FACS flow cytometry plot of CD25 + intracellular Foxp3 + regulatory T cells in the lymph nodes is shown; Figure 16where b is CD25 in lymph nodes + intracellular Foxp3 + percentage of cells and absolute number of regulatory T cells (n = 6); Figure 16 where c is CD4 in the spleen + intracellular CD25 + Foxp3 + Representative FACS flow cytometry plot of regulatory T cells; Figure 16 where d is CD4 in the spleen + intracellular CD25 + Foxp3 + percentage of cells and absolute number of regulatory T cells (n = 6); Data are expressed as mean ± SD, and statistical analysis was performed using one-way ANOVA and Tukey's post hoc test;

[0036] Figure 17 For serum and synovial cytokine analysis; among them, Figure 17 where a is the levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), interferon-γ (IFN-γ), interleukin-10 (IL-10), and transforming growth factor-β (TGF-β) in serum (n = 6); Figure 17 where b is the levels of IL-6, IL-1β, IFN-γ, IL-10, and TGF-β in synovium (n = 6); Data are expressed as mean ± SD, and statistical analysis was performed using one-way ANOVA and Tukey's post hoc test;

[0037] Figure 18 For the preventive effect of CII-RM@aCD4Tn in CIA mice; among them, Figure 18 where a is the experimental protocol for the preventive treatment effect of CII-RM@aCD4Tn; Figure 18 where b is the arthritis score curve (n = 6); Figure 18 where c is the body weight change curve (n = 6); Figure 18 where d is the paw thickness measurement on day 42 (n = 6); Figure 18 where e is the assessment of motor ability of CIA mice using the Rotarod system on day 42 (n = 6); Figure 18 where f is the heat map analysis of pro-inflammatory and anti-inflammatory cytokines in serum and synovium (n = 6); Data are expressed as mean ± SD, and statistical analysis was performed using two-way ANOVA and Tukey's post hoc test (b and c), or one-way ANOVA and Tukey's post hoc test (d and e);

[0038] Figure 19 For flow cytometry analysis of regulatory T cells in lymph nodes and spleen; wherein, Figure 19 In a, CD25 in lymph nodes + Intracellular Foxp3 + Representative FACS flow cytometry plot of regulatory T cells; Figure 19 In b, CD25 in lymph nodes + Intracellular Foxp3 + Percentage of cells and absolute number of regulatory T cells (n = 6); Figure 19 In c, CD4 in spleen + Intracellular CD25 + Foxp3 + Representative FACS flow cytometry plot of regulatory T cells; Figure 19 In d, CD4 in spleen + Intracellular CD25 + Foxp3 + Percentage of cells and absolute number of regulatory T cells (n = 6); Data are expressed as mean ± SD, and statistical analysis was performed using one-way ANOVA and Tukey's post hoc test. Detailed implementation manners

[0039] The above solution will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0040] Example 1

[0041] 1. Reagents

[0042] DSPE-PEG 2000-NHS (DP-NHS, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-succinimidyl succinate]) was purchased from Ponsure Biotech Co., Ltd. (Shanghai, China). Rapamycin (RM) was purchased from Aladdin. FITC-rapamycin, FITC-NHS, Cy3-NHS and Cy5-NHS were purchased from Yuansi Technology Co., Ltd. (Chongqing, China). Phorbol 12-myristate 13-acetate (PMA) was purchased from Abcam. DiO, DiR, DAPI, BCA Protein Assay Kit and Coomassie Brilliant Blue Rapid Staining Solution were all purchased from Beyotime. Type II collagen (CII), Complete Freund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IFA) and Type II Collagen Detection Kit were purchased from Chondrex, Inc. (USA). ELISA kits for mouse TNF-α, TGF-β, IL-6, IL-10, IL-1β and IFN-γ, Count Bright™ Absolute Counting Beads and Fixable Viability Dye were purchased from Thermo Fisher Scientific. ELISPOT kits for mouse TNF-α and IFN-γ were purchased from Dakewe Biotech Co., Ltd. Diphtheria toxin was purchased from Sigma Aldrich. Mouse CD4 + CD25 + T cells and CD4 + T cell sorting kits and matching magnetic beads were purchased from STEMCELL Technologies Inc. (Canada).

[0043] Recombinant murine granulocyte - macrophage colony - stimulating factor (GM - CSF), murine IL - 2, IL - 7 and TGF - β were purchased from Peprotech (USA). Anti - murine CD3 (CLONE: 17A2) and CD28 (CLONE: D665) antibodies were purchased from Bio X Cell. Murine anti - CD4 antigen - binding fragment (aCD4, CAT: PFBL - 034) was purchased from Creative Biolabs (USA). Recombinant CD4 protein was purchased from MedChemExpress (USA). IL - 6 antibody for immunohistochemistry was purchased from BioLegend. CD4 - PE, CD25 - FITC, Foxp3 - APC, CD11c - FITC antibodies used for immunohistochemical staining were purchased from BioLegend (USA). Antibodies for flow cytometry including CD25 - PE, CD25 - APC, CD45 - FITC, CTLA4 - APC - Cy7, PD - 1 - PE, LAG - 3 - FITC, Foxp3 - APC, Foxp3 - APC - Cy7, Eα / MHCII - APC, CD80 - PE, CD4 - APC, CD11c - PE, CD40 - APC, MHC II - PE, CD86 - APC, CD127 - APC, PD - L1 - PE were all purchased from BioLegend. Monoclonal antibody of MHC II molecule I - Ab - APC (for flow cytometry) was purchased from Invitrogen (USA).

[0044] 2. Mice

[0045] Female C57BL / 6 mice and male DBA / 1 mice at 6 - 8 weeks of age were purchased from Cavens Experimental Animal Center. OT - II (C57BL / 6Smoc - Igs2em1(CD2 - TCRa(OT - II)-CD2 - TCRb(OT - II))Smoc) mice were purchased from Jackson Laboratory. All animals were housed in an SPF - level environment at a temperature of 22 ± 2°C, humidity of 45 - 55%, and a 12 - hour light - dark cycle. All experimental protocols involving mice were approved by the Laboratory Animal Ethics Committee of Soochow University and strictly adhered to ethical guidelines.

[0046] All data were expressed as mean ± standard deviation and statistical analysis was performed using data - processing software (GraphPad Prism, version 10.1). Unpaired two - tailed t - test, one - way analysis of variance (ANOVA) combined with Tukey's post - hoc test or two - way ANOVA combined with Tukey's post - hoc test were used for comparison between groups.

[0047] 3. In vitro induction of CD4+ T cells differentiate into regulatory T cells

[0048] Isolation of CD4 T cells from mouse spleen and lymph nodes (LNs) by magnetic bead negative sorting + T cells. Specific steps: After CO2 euthanasia, the mice were dissected and the inguinal, axillary, and mesenteric lymph nodes and spleen were obtained. After mechanical dispersion, they were filtered through a 70 μm filter and centrifuged at 4°C, 600×g for 5 minutes. Red blood cells were removed with ACK lysis buffer at room temperature, and then the mouse CD4 + T cell sorting kit instructions for purification of CD4 + T cells. Isolated CD4 + T cells were seeded in 6-well plates and activated with antibodies coated with anti-CD3 (1 μg / mL) and CD28 (2 μg / mL) in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, 2 mM glutamine, 20 ng / mL IL-2, and 20 ng / mL IL-7. TGF-β (10 ng / mL) and RM (50 nM) were added to induce regulatory T cell differentiation. After 3-5 days of culture, the expression of Foxp3, CD25, and CTLA-4 was detected by flow cytometry (BD Aria III) to verify the efficiency of regulatory T cell induction.

[0049] 4. Preparation of Tn, CII-RM@Tn, and CII-RM@aCD4Tn

[0050] Regulatory T cell-derived nanovesicles (Tn) were prepared by mechanical extrusion: regulatory T cells were washed by centrifugation and then resuspended in PBS (1×10 7 cells / mL), and then squeezed through 5 μm, 1 μm, 400 nm, and 200 nm polycarbonate membranes (Avestin LF-1 extruder, 20 times for each membrane), and finally ultracentrifuged (100,000 × g for 2 h at 4 °C) to collect Tn. 7 cells / mL) were mixed with bovine type II collagen (CII, 20 μg / mL) and RM (4 μg / mL) and extruded (the same method as Tn). aCD4-regulatory T cells (aCD4-T) based on DSPE-PEG platform regs ) Construction: DP-NHS with different molar ratios (molar ratio 0:1 to 20:1) was reacted with anti-CD4 antigen binding fragment (anti-CD4 Fab fragment, aCD4), and the reaction was carried out at pH 8.5 for 12 hours. Unbound DP-NHS was removed by ultrafiltration to obtain DP-aCD4. Regulatory T cells were pre-incubated with free aCD4 (300 μg / million T regs), and then incubated with DP-aCD4 (50 μg / million T regs ), and aCD4-regulatory T cells (aCD4-T regs ) were collected by centrifugation. Subsequently, CII and RM were wrapped by the Tn method to obtain CII-RM@aCD4Tn.

[0051] 5. Characterization of CII-RM@aCD4Tn

[0052] DP-aCD4-Cy3 synthesized with different molar ratios was incubated with regulatory T cells, and the membrane insertion efficiency was detected by flow cytometry and confocal microscopy. FITC-RM, Cy3-Collagen, and Cy5-aCD4 were co-assembled into CII-RM@aCD4Tn and confocal imaging was performed. The morphology was observed by transmission electron microscopy (TEM), and the particle size and potential were measured by a dynamic light scattering instrument (Zetasizer). The feeding ratio of CII and RM was optimized (CII 5 - 20 μg / mL, RM 1 - 4 μg / mL), and the encapsulation efficiency of CII and the content of RM were measured by a collagen ELISA kit and HPLC (mobile phase: 10 mM ammonium acetate / acetonitrile = 25:75), respectively. The drug release experiment was monitored by the dialysis method (MWCO 800 kDa, 37 °C) for the release amount within 24 hours. The particle size of CII-RM@aCD4Tn was detected after storage at -80 °C to 25 °C for 0 - 21 days. The biological activity of membrane proteins was detected by incubating the stored samples with dye-labeled anti-CD25, CTLA4, PD-1 antibodies and recombinant CD4 at room temperature for 15 minutes, and then measuring the fluorescence intensity (FI) of the supernatant after ultracentrifugation.

[0053] The uptake of CII-RM@aCD4Tn by bone marrow-derived dendritic cells (BMDCs) was evaluated. BMDCs were derived from male DBA mice, and the bone marrow was extracted by flushing the femurs and tibias with PBS. After removing red blood cells with ACK lysis buffer, the cell suspension was filtered through a 70 μm sieve to remove debris. The collected cells were cultured in RPMI 1640 medium supplemented with 10% (v / v) fetal bovine serum (FBS), 1% (v / v) penicillin-streptomycin, and 20 ng / mL GM-CSF to promote differentiation. Fresh medium was changed on the 3rd day, and BMDCs were harvested on the 7th day for subsequent experiments.

[0054] To evaluate the uptake and antigen presentation of nanoparticles (NPs), CII was labeled with FITC. BMDCs (1×10 6Cells / well, 6-well plate) were incubated with CII-RM@aCD4Tn or free drugs (CII and RM) for 12 or 24 hours. After incubation, BMDCs were washed twice with PBS and stained with DAPI and DiO for nuclei and cell membranes, respectively. Before confocal imaging, cells were fixed with 4% paraformaldehyde and resuspended in anti-fluorescence quenching mounting medium.

[0055] Experimental results:

[0056] CD4 + T cells isolated from the spleen and LNs were successfully induced into regulatory T cells and showed high levels of CD25 and Foxp3 expression ( Figure 2 in a). In addition, the key immunomodulatory marker molecule CTLA4 was also significantly upregulated after stimulation ( Figure 2 in b). To optimize the binding of CII-RM@aCD4Tn to CD4 + T cells, DP-aCD4 with different molar ratios of DSPE-PEG-NHS (DP) to anti-CD4 Fab protein (aCD4) was synthesized. DP-aCD4 had the highest binding level to CD4 + T cells at a DP:aCD4 molar ratio of 8. However, excessive modification of DP would lead to the inactivation of aCD4, thus reducing the targeting of CII-RM@aCD4Tn to CD4 + T cells (in c of 2). Therefore, 8:1 was selected as the optimized ratio to ensure the high CD4 + T cell binding ability of CII-RM@aCD4Tn. The encapsulation efficiency and drug loading of CII and RM are shown in Figure 2 d. CII-RM@aCD4Tn could achieve sustained drug release within 24 hours (in e of 2). CII-RM@aCD4Tn loaded with CII and RM and modified with aCD4 was successfully prepared ( Figure 3 in a). Transmission electron microscopy (TEM) detection showed that the prepared nanoparticles were spherical, with a complete structure and a particle size of about 130 nm ( Figure 3 in b). CII-RM@aCD4Tn had a hydrated diameter of 165.7 nm and a Zeta potential of -16.1 mV ( Figure 3 in c). Long-term stability assessment showed that there were no significant changes in the particle size and biological function of CII-RM@aCD4Tn after storage at -80 °C for 3 weeks ( Figure 3 in d and e). Confocal results showed that CII-RM@aCD4Tn could effectively target CD4 + T cells ( Figure 3 in f).

[0057] To evaluate the role of Tn in enhancing the delivery efficiency of CII and RM to DCs, FITC-labeled free drug or CII-RM@aCD4Tn was co-incubated with BMDCs for 12 h. The results showed that although all cells took up CII and RM, the uptake of Tn-loaded drugs was significantly higher than that of free drugs ( Figure 3 in g). To study the antigen (CII) presentation on the surface of DCs, FITC-labeled CII was co-incubated with BMDCs for 24 h. It was found that CII-RM@aCD4Tn significantly promoted antigen presentation ( Figure 3 in h).

[0058] Example 2

[0059] Immunosuppressive effect of CII-RM@aCD4Tn on DCs and CD4 + T cells

[0060] After BMDCs were co-cultured with different nanoparticles for 48 h, they were stimulated with LPS (50 ng / mL) for 24 h, and the expressions of CD80 and PD-L1 were detected by flow cytometry. The secretions of TNF-α and TGF-β were detected by ELISA. The mRNA levels of TNF-α and TGF-β were analyzed by qRT-PCR (primers are shown in Table 1).

[0061] Table 1

[0062]

[0063] CD4 + T cells were co-cultured with various types of nanoparticles for 48 h and stimulated with PMA (20 ng / mL) for 6 h. Inactivated CD4 + T cells (iCD4) were induced by adding 10 ng / mL IL-10 and 10 ng / mL TGF-β for 72 h. The expressions of CTLA4 and PD-1 were detected by flow cytometry, and the levels of TNF-α and TGF-β in cells and culture medium supernatants were determined by qRT-PCR and ELISA, respectively.

[0064] Experimental results:

[0065] Tolerogenic DCs (tDCs) play a key role in maintaining immune homeostasis and preventing autoimmune reactions. After BMDCs were incubated with different preparations for 48 h, they were activated with LPS for 24 h, and their activation markers and secreted cytokines were detected. Mature BMDCs (mDCs) showed high levels of CD80, which were significantly reduced after treatment with CII-RM@aCD4Tn ( Figure 4 in a). Interestingly, after treatment with CII-RM@aCD4Tn, BMDCs showed a higher level of PD-L1 ( Figure 4In a). In addition, compared with other groups, the pro-inflammatory cytokine TNF-α and its mRNA expression levels were the highest in the mDCs group, while the anti-inflammatory cytokine TGF-β and its mRNA expression levels were the lowest ( Figure 4 In b and c). CII-RM@aCD4Tn treatment could significantly inhibit the expression of TNF-α protein and its mRNA, while significantly up-regulating TGF-β ( Figure 4 In b and c).

[0066] Inactivated CD4 + T cells (iCD4s) are crucial in immune regulation and autoimmune prevention and have therapeutic potential in areas such as autoimmune diseases, allergies, and organ transplantation. To evaluate whether CII-RM@aCD4Tn could induce iCD4s, CD4 + T cells were co-incubated with it for 48 hours and then stimulated with PMA for 6 hours. Flow cytometry analysis showed that compared with mature CD4 + T cells (mCD4s), after treatment with CII-RM@aCD4Tn, the expression of CTLA4 and PD-1 increased ( Figure 5 In a), indicating successful induction of a regulatory phenotype. In addition, the cells treated with CII-RM@aCD4Tn showed significantly increased TGF-β mRNA and cytokine levels, while the TNF-α expression was significantly decreased ( Figure 5 In b and c).

[0067] In summary, CII-RM@aCD4Tn can effectively promote the differentiation of tDCs and iCD4s, enhance the secretion of anti-inflammatory cytokines, and at the same time inhibit the pro-inflammatory response.

[0068] Example 3

[0069] 1. The bridging effect of CII-RM@aCD4Tn on CD4 + T cells and DCs

[0070] BMDCs (1×10 6 ) and CD4 + T cells (1×10 6(0) Co-incubate with CII-RM@Tn or CII-RM@aCD4Tn (37 °C, 1 hour) to evaluate the DC-CD4 binding ability. Subsequently, the cells were fixed at room temperature for 30 minutes and stained with fluorescently labeled CD4 and CD11c antibodies, and the DC-CD4 cell pairs were detected by flow cytometry. To visualize the interaction of CII-RM@aCD4Tn with DC-CD4 in vivo, Cy5-labeled CII-RM@aCD4Tn was injected intradermally into the bilateral inguinal regions of male DBA / 1 mice. After 24 hours, the mice were sacrificed, and the inguinal lymph nodes (iLNs) were taken for cryosectioning and stained with fluorescently labeled CD11c and CD4 antibodies. The sections were imaged using a panoramic scanner (Pannoramic MID-250FLASH-DESK, 3DHISTECH).

[0071] 2. In vivo distribution of CII-RM@aCD4Tn

[0072] Cy5-labeled CII-RM@aCD4Tn was injected intradermally into the unilateral or bilateral backs of male DBA / 1 mice at the same total dose. After 24 hours, the mice were sacrificed, and the heart, liver, spleen, lung, kidney, axillary lymph nodes (aLNs), and iLNs were taken for IVIS in vivo imaging. To track the in vivo drug release, Cy5-labeled CII combined with RM or CII-RM@aCD4Tn was injected into the right inguinal region of the mice, and the FI at the injection site was continuously monitored by IVIS for 7 days.

[0073] Experimental results:

[0074] CD4 + The CD4 molecule on T cells and the CD80 / 86 co-stimulatory molecule on DCs are stable biomarkers, making it an ideal target for CII-RM@aCD4Tn binding ( Figure 6 in a). When CD4 + T cells were mixed with BMDCs at the same ratio and then incubated with CII-RM@Tn or CII-RM@aCD4Tn for 1 hour respectively. FACS analysis showed that the two types of cells in the CII-RM@Tn treatment group were still dispersed in the culture medium, while the CII-RM@aCD4Tn treatment group effectively aggregated CD4 + T cells and BMDCs to form cell pairs ( Figure 6 in b). To detect its in vivo binding ability, CII-RM@aCD4Tn was injected intradermally into the ventral side of DBA / 1 mice. After 24 hours, iLN was taken for immunofluorescence section analysis. The results showed that CII-RM@aCD4Tn could not only target DCs (CD11c + ), but also effectively bind to CD4 +T cells and promote their interaction in the paracortical area of lymph nodes ( Figure 6 c). These data confirm that CII-RM@aCD4Tn has dual targeting ability and can bind to CD4 + T cells and DCs simultaneously, effectively promoting cell-cell bridging both in vitro and in vivo.

[0075] To evaluate the lymph node targeting ability of CII-RM@aCD4Tn, intradermal injections were performed at different positions on the ventral side of DBA / 1 mice. After unilateral (left or right) injection, the fluorescence signal mainly appeared in the ipsilateral iLN and aLN, with less accumulation in the major organs ( Figure 7 a-c). After bilateral injection, the fluorescence intensity in iLN and aLN accounted for 60% of the total fluorescence intensity ( Figure 7 c). These results indicate that bilateral intradermal injection on the ventral side can effectively deliver CII-RM@aCD4Tn to systemic lymph nodes, thus potentially producing good immunomodulatory effects. In vivo imaging at different time points showed that CII-RM@aCD4Tn could persist near the injection site for at least 1 week, while the free drug (CII + RM) rapidly diffused within 4 days ( Figure 7 d and e). Quantitative analysis showed that the area under the fluorescence intensity curve within 1 week in the CII-RM@aCD4Tn group was 2.8-fold higher than that in the free drug group ( Figure 7 f), highlighting its prolonged retention ability.

[0076] Example 4

[0077] In vivo safety evaluation of CII-RM@aCD4Tn

[0078] Male DBA / 1 mice were randomly divided into 6 groups (6 mice per group): healthy group (G1), and the other five groups received 150 μg CII + 20 μg RM (G3), 1 mg Tn (G4), 1 mg aCD4Tn (G5), 1.2 mg CII-RM@Tn nanoparticles (G6), and 1.2 mg CII-RM@aCD4Tn nanoparticles (G7), respectively. Intradermal injections were performed bilaterally on the backs of the mice on day 0 and day 3. On day 7, blood samples were collected from each group of mice for liver function tests and complete blood cell counts. After the mice were euthanized, heart, liver, spleen, lung, and kidney tissues were collected and analyzed for pathological abnormalities by hematoxylin-eosin (H&E) staining.

[0079] Experimental results:

[0080] The safety assessment showed that blood biochemical analysis indicated no significant changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin levels in each group ( Figure 8In b), the results of the blood routine tests were all within the normal physiological range ( Figure 8 In c). In addition, histological examinations of the major organs revealed no signs of tissue damage or inflammation ( Figure 9 ). These results confirmed that CII-RM@aCD4Tn had good biosafety and did not cause cytotoxicity or organ toxicity.

[0081] Example 5

[0082] 1. In vitro and in vivo induction of antigen-specific regulatory T cells

[0083] The nanoparticles encapsulating OVA 323-339 were intradermally injected bilaterally into the back of OT-II mice. Four days later, aLNs, iLNs, and spleens were collected, and the proportion of regulatory T cells was analyzed by flow cytometry (adding 2 μL of counting beads and staining for CD45, CD4, CD25, and Foxp3). In addition, IFN-γ and TNF-α secreting cells were detected by ELISPOT: Splenocytes were seeded at 1×10 6 / well into an ELISPOT plate and stimulated with 10 μg / mL OVA 323-339 (specific antigen) or CII (nonspecific antigen) for 24 hours. The supernatant was collected and the cytokines were quantified by ELISA, and the number of secreting cells was counted according to the manufacturer's instructions.

[0084] 2. RNA sequencing and differential gene enrichment analysis

[0085] Total RNA was extracted from splenic lymphocytes, and a sequencing library was constructed according to the effective concentration and the target data volume and sequenced (Illumina NovaSeq 6000 platform). The effective sequencing data were quantified at the transcriptional level by HTSeq (differential analysis was performed using edgeR (Robinson MD et al., 2010), with an adjusted p-value (padj) < 0.05 as the significance criterion). The mainstream hierarchical clustering method was used to convert the log10(FPKM + 1) values into standardized values and perform clustering. Gene Ontology (GO) enrichment analysis was performed using the toolkit (ClusterProfiler), and significantly enriched pathways with a p-value < 0.05 were screened. The gene sets for gene set enrichment analysis (GSEA) were derived from the KEGG database, and by comparing the ranked lists of differentially expressed genes in two groups of samples, it was tested whether the preset gene sets were significantly enriched at the top or bottom. The RNA sequencing and gene enrichment analysis services were completed by Lianchuan Biotechnology Co., Ltd.

[0086] Experimental results:

[0087] Given that CII-RM@aCD4Tn can effectively induce tDCs and iCD4s, we further investigated the ability of this system to induce regulatory T cells. Since CD4 + T cells derived from OT-II transgenic mice are highly responsive to ovalbumin 323-339 peptide (OVA 323-339 ), we first synthesized OVA 323-339 -loaded nanoparticles. To evaluate the immunosuppressive effect in OT-II mice, after intraperitoneal injection of OVA-RM@aCD4Tn, the proportion and absolute number of regulatory T cells in LNs and spleen were significantly increased ( Figure 10 a-d). Among them, compared with the PBS group, the number of regulatory T cells in the spleen of the OVA-RM@aCD4Tn group increased by 8.4-fold, and increased by 1.4-fold compared with the OVA-RM@Tn group ( Figure 10 a-d).

[0088] To further investigate whether this immunosuppression is antigen-specific, we isolated splenocytes from mice in different experimental groups and stimulated them in vitro with OVA 323-339 (relevant antigen) or CII (irrelevant antigen). The results showed that compared with the OVA-RM@Tn group, the number of TNF-α secreting cells decreased by 60.4% after treatment with OVA-RM@aCD4Tn under OVA 323-339 stimulation, while no TNF-α secreting cells were observed under CII stimulation ( Figure 11 a and b).

[0089] To clarify the immunosuppressive mechanism mediated by OVA-RM@aCD4Tn, we performed transcriptome analysis on CD4 + T cells isolated from the spleen of OT-II mice 4 days after administration. After treatment with OVA-RM@aCD4Tn, pro-inflammatory factors (IL-6, TNF, MMP9, IL-1β and IL-17A) were significantly down-regulated, while anti-inflammatory markers (IL-10, Foxp3, TGF-β1, CTLA4, IL1RN) were significantly up-regulated ( Figure 11 c). KEGG signaling pathway analysis further showed that this system regulates the function of CD4 + T cells through TGF-β, TNF and T cell receptor signaling pathways, and is closely related to autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, and type 1 diabetes ( Figure 11 d). In summary, OVA-RM@aCD4Tn has better immunomodulatory ability than OVA-RM@Tn, showing potential application value in the treatment of autoimmune diseases.

[0090] Example 6

[0091] 1. Therapeutic effect of CII-RM@aCD4Tn in CIA mouse model

[0092] To establish a CIA mouse model, male DBA / 1 mice were intradermally injected with 100 μL of CII emulsion (CII: CFA = 1:1) on both sides of the tail base. After 21 days, they were boost-immunized with CII and IFA. Subsequently, the mice were randomly divided into seven groups of 6 mice each. Among them, one group was the healthy control group (G1), and the other six groups were the CIA group (G2), 150 μg CII + 20 μg RM group (G3), 1 mg Tn group (G4), 1 mg aCD4Tn group (G5), 1.2 mg CII-RM@Tn group (G6), and 1.2 mg CII-RM@aCD4Tn group (G7).

[0093] Therapeutic intervention was carried out from day 22 to day 28 (a total of 3 injections, once every 3 days), by intradermal injection on both sides of the mouse back, with a dose of 200 μL per injection for each mouse. The arthritis score and body weight change were recorded every 3 days. The arthritis scoring criteria were as follows: 0 points, no erythema and swelling; 1 point, erythema and mild swelling limited to the tarsus or ankle joint; 2 points, erythema and mild swelling extended from the ankle joint to the tarsus; 3 points, erythema and moderate swelling extended from the ankle joint to the metatarsophalangeal joint; 4 points, erythema and severe swelling involved the ankle joint, foot and toes, or limb rigidity occurred. Each limb of each mouse was scored, and the highest score for a single mouse was 16 points. On day 42, the foot thickness was measured and a rotarod test was performed.

[0094] 2. Rotarod test

[0095] The computerized rotarod apparatus (ZL-200c, Bestcell) was used to evaluate the motor coordination ability of mice. Before the experiment, the mice were placed on the stationary rod for 2 - 3 minutes every day for 2 consecutive days to reduce stress. On day 41, the mice were rotated at a constant speed of 15 revolutions per minute for 3 minutes, and the number of falls was recorded; then the rotational speed was increased from 4 revolutions per minute to 20 revolutions per minute, and the critical speed at the time of falling was recorded. The experiment was repeated on day 42.

[0096] 3. Histopathology and immunohistochemistry analysis of inflamed joint tissues

[0097] At the experimental endpoint, the hind ankle joints of the mice were taken for hematoxylin-eosin (H&E), safranin O-fast green (SO-FG), and Masson staining. For the H&E sections, three pathologists semi-quantitatively scored (0-3 points) according to three indicators including synovial hyperplasia and inflammatory cell infiltration. A total score of 0-1 points indicated no synovitis, 2-4 points indicated low-grade synovitis, and 5-9 points indicated high-grade synovitis. The thickness of the tibial and talar cartilage was measured by software (Image J, version 1.53c) under SO-FG staining, and the area of synovial fibrosis was quantified by Masson staining. Micro-computed tomography (Micro-CT, SkyScan 1276) was used to analyze the hind paw bone parameters (tube current 385 μA, tube voltage 65 kV, pixel size 17.76 μm). Three-dimensional images were reconstructed by software (Mimics) and bone volume fraction (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), structure model index (SMI), and bone mineral density (BMD) were calculated. IL-6 immunohistochemical analysis was used to quantify the area of positive synovial regions by software (Image J).

[0098] Experimental results:

[0099] To evaluate the early treatment potential of the system, different nanotherapeutics were administered on days 22, 25, and 28 to treat collagen-induced arthritis (CIA) mice ( Figure 12 as shown in a). At the experimental endpoint, the severity of arthritis was evaluated by blinded scoring of paw swelling and redness. The arthritis scores of the mice treated with CII-RM@aCD4Tn were significantly lower than those of the PBS group, indicating a significant reduction in inflammation and edema ( Figure 12 as shown in b). In addition, due to disease progression and severe swelling, the body weights of the mice in the RA group decreased significantly, and the weight loss of the mice in the CII-RM@aCD4Tn group was significantly alleviated ( Figure 12 as shown in c). The results of paw thickness measurements confirmed that the CII-RM@aCD4Tn group showed the best therapeutic effect among all treatment groups ( Figure 12 as shown in d). Subsequently, we performed a behavioral assessment of the treated mice using a rotarod system to detect their motor coordination and balance ability. The mice in the CII-RM@aCD4Tn group showed significant improvement during the two training sessions, including a reduced frequency of falling off the rotating rod and an increased rotational speed before falling ( Figure 12 as shown in e).

[0100] To further evaluate the pathological damage of the ankle joint, histological analysis was performed. In the histological sections stained with hematoxylin and eosin (H&E), the joint cavity of the mice in the CII-RM@aCD4Tn group was almost completely restored, the joint interface was clear, and there were no obvious synovitis or degenerative changes in articular cartilage. In contrast, the RA group showed severe pathological changes, including massive infiltration of inflammatory cells, synovial hyperplasia, pannus formation, and cartilage erosion ( Figure 13 a and d in Figure 13 ). Pannus formation is one of the characteristic pathological changes of RA. In the sections stained immunohistochemically, the distribution of IL-6 in the joints and synovium of the mice in the CII-RM@aCD4Tn group was the least, and the affected area was the smallest ( Figure 13 b and e in Figure 14 ). Masson staining analysis showed that there was extensive fibrosis in the synovial tissue of the mice in the RA group, while the fibrotic area in the CII-RM@aCD4Tn group was only 12.0%, a 94.2% reduction compared with the RA group ( c and f in

[0101] Figure 14 ). In addition, the results of safranin O-fast green (SO-FG) staining showed that the cartilage structure in the CII-RM@aCD4Tn group was well preserved, and the cartilage thickness was 135.5 μm, close to the level of the healthy group (

[0102] a and d in

[0103] Example 7

[0104] Slices of mouse paws were stained with antibodies against CD4, CD25, and Foxp3 to analyze the distribution of regulatory T cells in the synovium, and then analyzed by Image J software. ELISA kits were used to detect the levels of IL-6, IL-1β, IFN-γ, IL-10, and TGF-β in the serum and synovium. Flow cytometry was used to analyze the number and proportion of regulatory T cells in the spleen and LNs.

[0105] Experimental results:

[0106] One of the pathological features of RA is the infiltration of activated T cells and macrophages into the synovium of multiple joints and the secretion of a series of pro-inflammatory cytokines, including IFN-γ, IL-6, and IL-1β. These cytokines continuously activate the inflammatory response, leading to synovial hyperplasia and secondary bone erosion. In contrast, immunoregulatory cells (especially regulatory T cells) play a key role in maintaining immune homeostasis by secreting anti-inflammatory cytokines such as IL-10 and TGF-β. Immunofluorescence analysis showed that after treatment with CII-RM@aCD4Tn, the proportion of regulatory T cells (CD4 + CD25 + Foxp3 + )in the synovium increased significantly ( Figure 15 a-c in

[0107] ). In addition, we further analyzed the regulatory T cell population in LNs and spleens by flow cytometry. Similarly, after treatment with CII-RM@aCD4Tn, the number of regulatory T cells increased significantly, indicating its systemic immunomodulatory effect ( Figure 16 a-d in Figure 17 ). At the same time, treatment with CII-RM@aCD4Tn significantly reduced the levels of pro-inflammatory cytokines (IL-6, IL-1β, and IFN-γ) in serum and synovium, while significantly upregulating the secretion of IL-10 and TGF-β (

[0108] Example 8

[0109] Verification of the preventive effect of CII-RM@aCD4Tn on CIA mice

[0110] To evaluate its preventive potential, DBA / 1 mice were treated with NPs before CIA induction. Male DBA / 1 mice were randomly divided into seven groups of six mice each. Among them, one group was the healthy control group (G1), and the remaining six groups were the CIA group (G2), the 150 μg CII + 20 μg RM group (G3), the 1 mg Tn group (G4), the 1 mg aCD4Tn group (G5), the 1.2 mg CII-RM@Tn group (G6), and the 1.2 mg CII-RM@aCD4Tn group (G7). NPs were injected twice on days -21 and -14, followed by CIA induction on days 0 and 21. Arthritis scores and body weight changes were monitored every 3 days, and foot thickness and rotarod test were measured on day 42. In addition, ELISA kits were used to detect the levels of IL-6, IL-1β, TNF-α, IFN-γ, IL-10, and TGF-β in serum and synovial tissues, and flow cytometry was used to analyze the number and proportion of regulatory T cells in the spleen and LNs.

[0111] Experimental results:

[0112] To investigate the preventive effect of CII-RM@aCD4Tn, mice were injected 3 weeks and 2 weeks before CIA induction ( Figure 18 in a). After treatment with CII-RM@aCD4Tn, the appearance of the mice's paws was normal, without signs of redness or swelling, and their body weight almost recovered to the healthy level ( Figure 18 in b-d). In addition, the mice in the CII-RM@aCD4Tn group showed the strongest motor ability among all experimental groups ( Figure 18 in e), indicating its excellent protective effect on joint function. The levels of pro-inflammatory cytokines in the mice treated with CII-RM@aCD4Tn were significantly reduced, while the levels of anti-inflammatory cytokines were significantly increased ( Figure 18 in f). In addition, the number and proportion of regulatory T cells in the LNs and spleen of the mice in the CII-RM@aCD4Tn group were the highest among all experimental groups ( Figure 19 in a-d). These results indicate that the DC-CD4 bridging system has a strong preventive effect in preventing the progression of RA, highlighting the feasibility of its use as a vaccine-based immunomodulatory strategy.

[0113] In this study, we developed a DC-CD4 bridging tolerance nanosystem (AP-IA@aCD4Tn) for efficient induction of antigen-specific regulatory T cells to treat rheumatoid arthritis. This platform co-delivers antigen peptides and immunosuppressants through bispecific nanoparticles, acting simultaneously on dendritic cells (through CTLA4-CD80 / 86) and CD4 +T cells (via anti-CD4 antibody) achieved spatial coupling of DCs and T cells, thereby promoting the formation of a tolerogenic microenvironment. In a murine model of collagen-induced arthritis, nanoparticles CII-RM@aCD4Tn loaded with type II collagen (collagen II) and rapamycin were constructed. After intradermal injection, they significantly expanded regulatory T cells in the lymph nodes and spleen, inhibited the expression of pro-inflammatory cytokines (such as TNF-α, IL-6), and increased the levels of anti-inflammatory mediators (such as TGF-β, IL-10), thus effectively alleviating pathological manifestations such as synovitis, cartilage destruction, and bone erosion. These findings indicate that the DC-CD4 bridged tolerogenic nanosystem has the potential to serve as a precise and durable autoimmune disease treatment strategy. Future work should focus on its clinical translation and its application in other antigen-driven diseases to pave the way for transformative immunotherapy.

Claims

1. A preparation method of a bispecific cell membrane nanoparticle, characterized in that, It includes the following steps: Primary CD4 + T cells were induced into regulatory T cells in vitro using TGF-β and rapamycin; distearoylphosphatidylethanolamine-polyethylene glycol-activated ester was reacted with the anti-CD4 antigen-binding fragment to obtain DP-aCD4; after pre-incubating regulatory T cells with the free anti-CD4 antigen-binding fragment, they were co-incubated with DP-aCD4 to obtain aCD4-regulatory T cells; aCD4-regulatory T cells were mixed with antigen peptides and immunosuppressants, and bispecific cell membrane nanoparticles were prepared using a liposome extruder.

2. The preparation method of the bispecific cell membrane nanoparticles according to claim 1, wherein The molar ratio of distearoyl phosphatidylethanolamine - polyethylene glycol - active ester to anti - CD4 antigen - binding fragment is 2 - 20:

1.

3. The preparation method of the bispecific cell membrane nanoparticles according to claim 1, characterized in that, Distearoyl phosphatidylethanolamine - polyethylene glycol - active ester and anti - CD4 antigen - binding fragment react under the condition of pH 8 - 9 for 10 - 14 h to obtain DP - aCD4.

4. The preparation method of the bispecific cell membrane nanoparticles according to claim 1, characterized in that, The antigen peptide is one or more of citrullinated peptide, type II collagen, glucose - 6 - phosphate isomerase peptide, heat shock protein peptide, RA33 peptide.

5. The preparation method of the bispecific cell membrane nanoparticles according to claim 1, characterized in that, The immunosuppressant includes one or more of rapamycin, everolimus, temsirolimus, tacrolimus, cyclosporin A.

6. The preparation method of the bispecific cell membrane nanoparticles according to claim 1, characterized in that, The ratio of antigen peptide to aCD4 - regulatory T cells is 5 - 20 μg: 1 × 10 7 cells; the ratio of immunosuppressant to aCD4 - regulatory T cells is 1 - 4 μg: 1 × 10 7 cells.

7. A bispecific cell membrane nanoparticle, characterized in that, It is prepared by the preparation method described in any one of claims 1 - 6.

8. Use of the bispecific cell membrane nanoparticles described in claim 7 in the preparation of immunosuppressive drugs.

9. The application according to claim 8, wherein The immunosuppressive drug is a drug for treating and / or preventing rheumatoid arthritis.

10. The application according to claim 9, wherein The bispecific cell membrane nanoparticles are used to induce antigen - specific regulatory T cells.

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