CN120324602A - A bispecific cell membrane nanoparticle and its preparation method and application - Google Patents

Abstract

The present invention belongs to the field of drug preparation, and specifically relates to a bispecific cell membrane nanoparticle and a preparation method and application thereof, wherein the preparation method comprises the following steps: primary CD4 ⁺ T cells are induced into regulatory T cells in vitro using TGF-β and rapamycin; distearoylphosphatidylethanolamine-polyethylene glycol-active ester is reacted with anti-CD4 antigen binding fragment to obtain DP-aCD4; regulatory T cells are pre-incubated with free aCD4, and then 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 AP-IA@aCD4Tn are prepared by a liposome extruder. The bispecific cell membrane nanoparticles of the present invention can efficiently induce the production of antigen-specific regulatory T cells after in vivo administration, thereby achieving accurate and efficient treatment and prevention of rheumatoid arthritis.

Description

Bispecific cell membrane nanoparticle and preparation method and application thereof

Technical Field

The invention belongs to the field of medicine preparation, and in particular relates to a bispecific cell membrane nanoparticle, and a preparation method and application thereof.

Background

Rheumatoid arthritis (Rheumatoid arthritis, RA) is an autoimmune disease characterized mainly by inflammatory joint synovial and cartilage destruction caused by an incorrect attack of the immune system on normal tissues. Clinically, anti-inflammatory small molecule drugs and biological agents are generally used for treating RA in a systemic way, but due to short half-life period and poor targeting property of the drugs, the curative effect is insufficient, and the nonspecific inhibition of the immune system is easy to cause serious toxic and side effects. The immune tolerance therapy can accurately treat RA, and is one of the most leading RA treatment strategies at present. Induction of antigen-specific regulatory T cells (Regulatory T cells, tregs) production is central to immune tolerance therapies, which currently deliver antigens to antigen presenting cells (ANTIGEN PRESENTING CELL, APC) of immune organs in general, and dendritic cells (DENDRITIC CELL, DC) in particular, induce T cell tolerance. However, the regulatory T cell induction efficiency of the current strategy is still insufficient, and the curative effect is still required to be further improved. Therefore, there is a need to provide a method with high induction efficiency and better therapeutic effect for regulatory T cells.

Disclosure of Invention

The invention provides a bispecific cell membrane nanoparticle and a preparation method and application thereof, wherein the bispecific cell membrane nanoparticle can specifically bind a CD80/86 costimulatory molecule of a dendritic cell and a CD4 receptor of a CD4 ⁺ T cell simultaneously through a surface membrane protein CTLA4 and an anti-CD 4 antigen binding fragment to form a cell pair of the dendritic cell-CD 4 ⁺ T cell, so that the interaction between an antigen presenting cell and the CD4 ⁺ T cell is promoted, and meanwhile, the bispecific cell membrane nanoparticle releases antigen peptide and immunosuppression, so that antigen-specific regulatory T cells are efficiently generated, a better immunoregulation effect is achieved, and the accurate and efficient treatment and prevention of rheumatoid arthritis are realized.

The technical scheme provided by the invention is as follows:

A method for preparing bispecific cell membrane nanoparticles comprises the following steps of inducing primary CD4 ⁺ T cells into regulatory T cells in vitro by adopting TGF-beta and rapamycin, enabling distearoyl phosphatidylethanolamine-polyethylene glycol-active ester to react with an anti-CD 4 antigen binding fragment to obtain DP-aCD4, pre-incubating free anti-CD 4 antigen binding fragment of the regulatory T cells, co-incubating the free anti-CD 4 antigen binding fragment with DP-aCD4 to obtain aCD 4-regulatory T cells, mixing the aCD 4-regulatory T cells with antigen peptide and immunosuppressant, and preparing the bispecific cell membrane nanoparticles by a liposome extruder.

Further, the molar ratio of distearoyl phosphatidylethanolamine-polyethylene glycol-active ester to the anti-CD 4 antigen binding fragment is 2-20:1.

Further, distearoyl phosphatidylethanolamine-polyethylene glycol-active ester and an anti-CD 4 antigen binding fragment react for 10-14 hours under the condition of pH 8-9 to obtain DP-aCD4.

Further, the antigen peptide is one or more of citrullinated peptide, type II collagen, glucose-6-phosphate isomerase peptide, heat shock protein peptide and RA33 peptide.

Further, the immunosuppressant comprises one or more of rapamycin, everolimus, temsirolimus, tacrolimus and cyclosporine A.

Further, the ratio of the antigenic peptide to aCD 4-regulatory T cells is 5 to 20. Mu.g, 1X 10 ⁷ cells, and the ratio of the immunosuppressant to aCD 4-regulatory T cells is 1to 4. Mu.g, 1X 10 ⁷ cells.

The invention also provides a bispecific cell membrane nanoparticle which is prepared by adopting the preparation method.

The invention also provides application of the bispecific cell membrane nanoparticle in preparation of immunosuppressive drugs.

Further, the immunosuppressant drug is a drug for treating and/or preventing rheumatoid arthritis.

Further, the bispecific cell membrane nanoparticle is used to induce antigen-specific regulatory T cells.

Further, the bispecific cell membrane nanoparticle is used for alleviating inflammation, cartilage degradation, synovial fibrosis and bone erosion caused by rheumatoid arthritis.

The invention also provides application of the bispecific cell membrane nanoparticle in preparation of medicaments for promoting antigen presentation.

The bispecific nanoparticle is prepared by extracting CD4 ⁺ T cells, inducing the CD4 ⁺ T cells into regulatory T cells in vitro by using TGF-beta and rapamycin, reacting DSPE-PEG ₂₀₀₀ -NHS with an anti-CD 4 antigen binding fragment to synthesize DSPE-PEG ₂₀₀₀ -aCD4 (DP-aCD 4), incubating the regulatory T cells with the anti-CD 4 antigen binding fragment, shielding CD4 receptors on the cell surface, incubating the regulatory T cells with the DP-aCD4 to prepare aCD 4-regulatory T cells (aCD 4-T _regs), uniformly mixing the aCD 4-regulatory T cells with antigen peptide (ANTIGEN PEPTID, AP) and immunosuppressant (Immunosuppressive agents, IA), and continuously extruding the mixture by a liposome extruder to prepare the bispecific nanoparticle AP-IA@aCD4Tn.

Advantageous effects

The innovative advantage of the bispecific nano-delivery system is its unique dual targeting mechanism, which efficiently induces immune tolerance by synergistically modulating the critical cellular interaction network in the immune microenvironment. The DC-CD4 engagement system efficiently induces the production of regulatory T cells by simultaneously targeting DC and CD4 ⁺ T cells, by physically drawing in the spatial distance between the two, and by reconstructing the formation of an "immune synapse" in a local microenvironment. The dual-targeting mediated intercellular bridging effect can remarkably enhance the co-stimulatory signaling efficiency of DC to T cells, and when the AP loaded in the nanoparticle is efficiently presented by DC, the differentiation direction of the CD4 ⁺ T cells is reprogrammed into antigen-specific regulatory T cells under the micro-environment regulation and control of IA. Meanwhile, the co-stimulation signal transduction efficiency of DC and T cells is obviously enhanced through the double-targeting mediated cell bridging effect, and the limitation that the traditional single-target therapy is difficult to coordinate cell differentiation is broken through. The cascade amplification strategy based on the natural immune regulation mechanism avoids the systemic immune suppression risk while improving the induction efficiency of the regulatory T cells, and finally remodels immune homeostasis through factors such as TGF-beta/IL-10 secreted by the regulatory T cells, thereby realizing efficient and accurate treatment and prevention of the rheumatoid arthritis.

Drawings

FIG. 1 is a schematic diagram of the preparation process and action mechanism (created by using Biorender. Com) of bispecific cell membrane nanoparticles according to an embodiment of the present invention, wherein a in FIG. 1 is a synthetic route, b in FIG. 1 is an action mechanism diagram;

FIG. 2 is a graph of in vitro induced flow results (n=3) of regulatory T cells in FIG. 2, CTLA4 expression levels (n=3) of regulatory T cells in FIG. 2, binding efficiency (n=3) of DP-aCD4 prepared by modifying different molar ratios of DSPE-PEG ₂₀₀₀ (DP) with nanoparticles on CD4 ⁺ T cells in FIG. 2, encapsulation efficiency and drug loading (n=3) of nanoparticles in FIG. 2, release curve (n=3) of nanoparticles in FIG. 2, data expressed as mean+ -SD, and unpaired two-tailed T test for statistical analysis;

FIG. 3 is a representation of a bispecific cell membrane nanoparticle, wherein FIG. 3a is a copolymer Jiao Biaozheng nanoparticle, FIG. 3b is an electron microscope image of the nanoparticle, FIG. 3c is the particle size and potential (n=3) of the nanoparticle, FIG. 3d is the particle size (n=3) of the nanoparticle under different storage conditions, FIG. 3e is the activity (n=3) of the membrane protein of the nanoparticle under different storage conditions, FIG. 3 f is a copolymer Jiao Tu of the nanoparticle combined with CD4 ⁺ T cells, FIG. 3g is a copolymer Jiao Tu of the nanoparticle ingested by bone marrow-derived dendritic cells (BMDC), FIG. 3h is a copolymer image of antigen presented to the cell membrane surface after the nanoparticle is ingested by the BMDC cells, data are expressed as mean+ -SD, and statistical analysis uses unpaired double tail T test;

FIG. 4 shows the immunosuppression of DC cells by bispecific cell membrane nanoparticles, wherein a in FIG. 4 shows the expression levels of CD80 and PD-L1 (n=5) after co-incubation of BMDC with nanoparticles, b in FIG. 4 shows the expression levels of mRNA TNF- α and TGF- β (n=5) in BMDC, c in FIG. 4 shows the levels of TNF- α and TGF- β (n=5) in the incubation liquid, and data are expressed as mean+ -SD, and statistical analysis uses One-way analysis (One-way analysis) and graph-based post-hoc test (Tukey post-test);

FIG. 5 shows the immunosuppression of CD4 ⁺ T cells by bispecific cell membrane nanoparticles, wherein a in FIG. 5 is the expression level of CTLA4 and PD-1 (n=5) after co-incubation of CD4 ⁺ T cells with nanoparticles, b in FIG. 5 is the levels of TNF- α and TGF- β in the incubation medium (n=5), c in FIG. 5 is the levels of TNF- α and TGF- β in CD4 ⁺ T cells mRNA (n=5), data expressed as mean+ -SD, and statistical analysis using One-way analysis of variance (One-way ANOVA) and basal post-hoc assay (Tukey post-assay);

FIG. 6 shows the bridging capacity of the DC-CD4 system, wherein FIG. 6 a is a schematic representation of CII-RM@aCD4Tn mediated binding of CD4 ⁺ T cells to DC (created using Biorender. Com), FIG. 6 b is CII-RM@Tn or CII-RM@aCD4Tn co-cultured with BMDC-CD4 ⁺ T cells for 1 hour, flow cytometry analyzes cell pair formation (n=3), FIG. 6 c is an immunofluorescent staining image of Inguinal Lymph Node (iLNs) after injection of CII-RM@aCD4Tn (intradermal), bottom-row photographs are magnified images of top-row rectangular areas (secondary cortical areas), scale bar: 100 μm (top row), 20 μm (bottom row), blue: DAPI, red: CD11c, green: CD4; yellow: cy 5-I-RM@aCDTn, and statistical unparalleled data are shown as statistical data for statistical unparalleled pairs;

FIG. 7 shows the biodistribution of CII-RM@aCD4Tn, wherein a in FIG. 7 is the fluorescence imaging of the main organ (aLN is the axillary lymph node, iLN is the inguinal lymph node) after i.d. injection of Cy5-CII-RM@aCD4Tn in the mouse, b in FIG. 7 is the fluorescence intensity analysis of the main organ (n=5), c in FIG. 7 is the proportional distribution of the fluorescence intensities in the organs, d in FIG. 7 is the fluorescence imaging of the mouse at different time points after injection of the drug, e in FIG. 7 is the curve of the fluorescence intensity of the injection area over time (n=5), f in FIG. 7 is the area under the curve of the fluorescence intensity (n=5), and data are expressed as mean.+ -. SD for statistical analysis using unpaired two-tailed t-test;

FIG. 8 is an in vivo toxicity assessment of a DC-CD4 bridging system, wherein FIG. 8a is an experimental scheme for DBA/1 mouse safety assessment, FIG. 8 b is alanine Aminotransferase (ALT), aspartate Aminotransferase (AST) and albumin levels (n=5) in serum, FIG. 8 c is a blood routine test result (n=5), data are expressed as mean+ -SD, and statistical analysis uses One-way analysis of variance (One-way ANOVA) and a graph-based post-hoc test (Tukey post-hoc test);

FIG. 9 is a hematoxylin-eosin (H & E) stained image of a major organ, scale: 100 μm, data expressed as mean.+ -. SD, statistical analysis using One-way analysis of variance (One-way ANOVA) and graph-based post-hoc test (Tukey post-hoc test);

FIG. 10 shows the in vivo induction of regulatory T cell production by OVA-RM@aCD4Tn, wherein FIG. 10 a shows a FACS representative flow chart of the intracellular Foxp3 ⁺ regulatory T cells (T _regs) of CD25 ⁺ in lymph nodes of OT-II mice, FIG. 10 b shows the percentage of the intracellular Foxp3 ⁺ cells and the absolute number of regulatory T cells (n=5) of CD25 ⁺ in lymph nodes, FIG. 10 c shows a FACS representative flow chart of the intracellular CD25 ⁺ Foxp3⁺ regulatory T cells of CD4 ⁺ in spleen of OT-II mice, FIG. 10 d shows the percentage of the intracellular CD25 ⁺ Foxp3⁺ cells and the absolute number of regulatory T cells (n=5) of CD4 ⁺ in spleen, data expressed as mean.+ -. SD, and statistical analysis uses One-way variance analysis (One-way ANOVA) and a graph-based post-hoc test (Tukey post-test);

FIG. 11 shows an in vivo induction of antigen-specific immunosuppression by OVA-RM@aCD4Tn, wherein a in FIG. 11 shows a cluster thermal map (n=3) of gene transcription expression associated with spleen CD4 ⁺ T cells of mice after the administration of different nanoparticles, a representative enzyme-linked immunospot-mediated immunoblotter (ELISPOT) spot-map (n=5) of tumor necrosis factor-alpha (TNF-alpha) cells after the stimulation of spleen cells with chicken ovalbumin peptide _323-339（OVA_323-339) or type II collagen peptide (CII _259-272), b in FIG. 11 shows the number of TNF-alpha cells after the antigen stimulation of spleen cells (n=5), c in FIG. 11 shows a cluster thermal map (n=3) of gene transcription expression associated with spleen CD4 ⁺ T cells after the administration of different nanoparticles, d in FIG. 11 shows a signal path analysis of a Beijing gene and a genomic encyclopedia (TnGY) of a differential expression gene between an OVA group and an OVA-RM group, and a statistical analysis is expressed by mean value.+ -. SD;

FIG. 12 shows the treatment effect of CII-RM@aCD4Tn on collagen-induced arthritis (CIA) mice, wherein a in FIG. 12 is an experimental plan for evaluating the early treatment effect of CII-RM@aCD4Tn, b in FIG. 12 is an arthritis scoring curve (n=6), c in FIG. 12 is a weight change curve (n=6), d in FIG. 12 is a paw swelling degree measurement (n=6) on day 42, e in FIG. 12 is a motor ability assessment (n=6) on day 42 using a rod (Rotarod) experimental system, data are expressed as mean.+ -. SD, and statistical analysis uses a Two-way ANOVA (Two-way ANOVA) and a Tukey post-test (b and c), or a One-way ANOVA and a Tukey post-test (d and e);

FIG. 13 shows the therapeutic effect of CII-RM@aCD4Tn on collagen-induced arthritis (CIA) mice, wherein, in FIG. 13, a and d are representative images of ankle H & E stained tissue sections (a) and histopathological evaluation of synovitis scores (d) (n=6), in FIG. 13, b and E are representative images of ankle interleukin-6 (IL-6) immunohistochemical staining (b) and synovial IL-6 stained area measurement (E) (n=6), in FIG. 13, c and f are representative images of ankle Marsone (Masson) staining (c) and synovial fibrotic area measurement (f) (n=f), and data are expressed as mean.+ -. SD, and statistical analysis uses One-way ANOVA and post-hoc inspection (Tukey post hoc inspection);

FIG. 14 shows the therapeutic effect of CII-RM@aCD4Tn on collagen-induced arthritis (CIA) mice, wherein, in FIG. 14, a and d are representative images (a) of ankle safranine O-fast green (SO-FG) staining and synovial cartilage thickness measurement (d) (n=6), in FIG. 14, b is a miniature-CT representative image of ankle on day 42, in FIG. 14, c is a representative image of ankle coronal section, red box (1X 1 mm) is a region of interest (ROI) of tibial cancellous bone, arrow indicates cancellous bone region of tibia (red), fibula (yellow) and talus (green), scale: 1mm, in FIG. 14, e is bone volume fraction (n=6) of ROI region, data is expressed as mean+ -SD, and statistical analysis uses One-way ANOVA and post-matrix test (Tukey post-test);

FIG. 15 shows immunofluorescent staining of CD4 ⁺ CD25⁺ Foxp3⁺ cells in the synovium, wherein FIG. 15 a shows immunofluorescent staining image of the synovium, scale bar 200 μm, blue DAPI, red CD4, green CD25, yellow Foxp3, FIG. 15 b shows Foxp3 ⁺ cell staining area (n=6) in the synovium, FIG. 15 c shows CD4 ⁺ CD25⁺ Foxp3⁺ cell staining area (n=6) in the synovium, data expressed as mean.+ -. SD, statistical analysis using One-way analysis of variance (One-way ANOVA) and graph-based post-test (Tukey post-test);

FIG. 16 shows a flow-through analysis of regulatory T cells in lymph nodes and spleen, wherein FIG. 16a shows a FACS representative flow-through graph of Foxp3 ⁺ regulatory T cells in CD25 ⁺ cells in lymph nodes, FIG. 16 b shows a percentage of Foxp3 ⁺ cells in CD25 ⁺ cells in lymph nodes and absolute number of regulatory T cells (n=6), FIG. 16 c shows a FACS representative flow-through graph of CD25 ⁺ Foxp3⁺ regulatory T cells in CD4 ⁺ cells in spleen, FIG. 16 d shows a percentage of CD25 ⁺ Foxp3⁺ cells in CD4 ⁺ cells in spleen and absolute number of regulatory T cells (n=6), data expressed as mean.+ -. SD, and statistical analysis uses One-way ANOVA and post-basal test (Tukey post-test);

FIG. 17 shows serum and synovial cytokine analyses, wherein FIG. 17 a shows the levels of interleukin-6 (IL-6), interleukin-1 beta (IL-1 beta), interferon-gamma (IFN-gamma), interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) (n=6) in serum, FIG. 17 b shows the levels of IL-6, IL-1 beta, IFN-gamma, IL-10 and TGF-beta (n=6) in synovial membranes, data are expressed as mean.+ -. SD, and statistical analysis uses One-way ANOVA (One-way ANOVA) and graph-based post-hoc test (Tukey post-test);

FIG. 18 shows the prophylactic effect of CII-RM@aCD4Tn in CIA mice, wherein FIG. 18 a shows the experimental plan of the prophylactic treatment effect of CII-RM@aCD4Tn, FIG. 18 b shows the arthritis score curve (n=6), FIG. 18 c shows the weight change curve (n=6), FIG. 18 d shows the 42 th paw thickness measurement (n=6), FIG. 18 e shows the exercise ability assessment (n=6) of CIA mice on day 42 using the Rotarod system, FIG. 18 f shows the heat map analysis (n=6) of pro-inflammatory and anti-inflammatory cytokines in serum and synovial membranes, data expressed as mean.+ -. SD, statistical analysis using Two-factor analysis of variance (Two-way ANOVA) and the basis post-hoc test (Tukey post-hoc) (b and c), or One-way ANOVA and the basis post-hoc test (Tukey post-hoc test) (d and e);

FIG. 19 shows a flow chart of regulatory T cells in lymph nodes and spleen, wherein FIG. 19 a shows a FACS representative flow chart of Foxp3 ⁺ regulatory T cells in CD25 ⁺ cells in lymph nodes, FIG. 19 b shows a percentage of Foxp3 ⁺ cells in CD25 ⁺ cells in lymph nodes and absolute number of regulatory T cells (n=6), FIG. 19 c shows a FACS representative flow chart of CD25 ⁺ Foxp3⁺ regulatory T cells in CD4 ⁺ cells in spleen, FIG. 19 d shows a percentage of CD25 ⁺ Foxp3⁺ cells in CD4 ⁺ cells in spleen and absolute number of regulatory T cells (n=6), data are expressed as mean.+ -. SD, statistical analysis is performed using or single-way ANOVA and post-basal test (Tukey post-test).

Detailed Description

The above-described aspects are further described below with reference to the accompanying drawings and specific embodiments.

Example 1

1. Reagent(s)

DSPE-PEG ₂₀₀₀ -NHS (DP-NHS, distearoyl phosphatidylethanolamine-polyethylene glycol-active ester) was purchased from Shanghai Biotechnology Co., ltd (Ponsure Biotech). Rapamycin (RM) was purchased from aladine. FITC-rapamycin, FITC-NHS, cy3-NHS and Cy5-NHS were purchased from Chongqing technology. Phorbol ester 12-myristate 13-acetate (PMA) was purchased from Ai Bokang (Abcam). Both DiO, diR, DAPI, BCA protein quantification kit and coomassie brilliant blue rapid staining solution were purchased from bi yun. Type II Collagen (CII), complete Freund's Adjuvant (CFA), incomplete Freund's Adjuvant (IFA) and type II collagen assay kit were purchased from Kang Derui s (Chondrex) in the united states. ELISA kits for mouse TNF-alpha, TGF-beta, IL-6, IL-10, IL-1 beta and IFN-gamma, flow absolute count microspheres (Count BrightTM) and fixable green dead cell dyes were purchased from Siemens technology. The mouse TNF-alpha and IFN-gamma ELISPOT kits were purchased from Daidae as biotechnology. Diphtheria toxin was purchased from sigma-aldrich (SIGMA ALDRICH). Mouse CD4 ⁺ CD25⁺ T cells and CD4 ⁺ T cell sorting kit and matched magnetic beads were purchased from canadian stem cell technology limited (STEMCELL).

Recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) and mouse IL-2, IL-7 and TGF-beta were purchased from Peprotech, pipetech, U.S.A. Anti-mouse CD3 (CLONE: 17A 2) and CD28 (CLONE: D665) antibodies were purchased from Bayer (Bio X Cell). Mouse anti-CD 4 antigen binding fragments (aCD 4, CAT: PFBL-034) were purchased from Craba (Crabtive Biolabs) U.S.A. Recombinant CD4 protein was purchased from american metaplasia technology (MedChemExpress). IL-6 antibodies for immunohistochemistry were purchased from Bai Sheng (BioLegend). Antibodies to CD4-PE, CD25-FITC, foxp3-APC, CD11c-FITC for immunohistochemical staining were purchased from BioLegend, U.S.A. Flow assay 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 antibodies were purchased from the hundred technology (BioLegend). MHC II molecule I-Ab-APC monoclonal antibodies (for flow-through) were purchased from Innova (Invitrogen) in the United states.

2. A mouse

Female C57BL/6 mice and male DBA/1 mice of 6-8 weeks old were purchased from the Kavens (Cavens) laboratory animal center. OT-II (C57 BL/6Smoc-Igs2em1 (CD 2-TCRa (OT-II) -CD2-TCRb (OT-II)) Smoc) mice were purchased from Jackson laboratories. All animals are fed in an SPF-level environment, the temperature is 22+/-2 ℃, the humidity is 45-55%, and the illumination period is 12 hours and the brightness is alternate. All protocols involving mice were approved by the laboratory animal ethics committee of the university of su and strictly adhere to ethics guidelines.

All data are expressed as mean ± standard deviation, and statistical analysis was performed using data processing software (GRAPHPAD PRISM, version 10.1). The comparison between groups uses unpaired two-tailed t-test, one-way analysis of variance (ANOVA) combined with the basis of the graph (Tukey) post-hoc test, or two-way ANOVA combined with Tukey post-hoc test.

3. In vitro induction of differentiation of CD4 ⁺ T cells into regulatory T cells

CD4 ⁺ T cells were isolated from mouse spleen and Lymph Nodes (LNs) by magnetic bead negative sorting. The specific steps are dissecting groin, armpit, mesenteric lymph node and spleen after euthanasia of mouse CO ₂, mechanically dispersing, sieving with 70 μm sieve, and centrifuging at 4deg.C 600 Xg for 5 min. Erythrocytes were removed with ACK lysate at room temperature followed by purification of CD4 ⁺ T cells according to mouse CD4 ⁺ T cell sorting kit instructions. Isolated CD4 ⁺ T cells were seeded in 6-well plates activated with anti-CD 3 (1. Mu.g/mL) and CD28 (2. Mu.g/mL) coated antibodies in RPMI-1640 medium containing 10% Fetal Bovine Serum (FBS), 1% penicillin/streptomycin, 2mM glutamine, 20 ng/mL IL-2 and 20 ng/mL IL-7. Addition of TGF-beta (10 ng/mL) and RM (50 nM) induced regulatory T cell differentiation. After 3-5 days of culture, foxp3, CD25 and CTLA-4 expression were examined by flow cytometry (BD Aria III) to verify regulatory T cell induction efficiency.

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

Nanovesicles (Tns) derived from regulatory T cells were prepared by mechanical extrusion by centrifugation of regulatory T cells washed and resuspended in PBS (1X 10 ⁷ cells/mL), extruded sequentially through 5 μm, 1 μm, 400 nm and 200 nm polycarbonate membranes (AVESTIN LF-1 extruder, 20 rounds of each membrane), and finally ultracentrifuged (4 ℃ C. 100,000Xg 2 hours) to collect Tns. CII-RM@Tn was prepared by mixing regulatory T cells (1X 10 ⁷ cells/mL) with bovine type II collagen (CII, 20. Mu.g/mL) and RM (4. Mu.g/mL) and pressing (method same Tn). Construction of an aCD 4-regulatory T cell (aCD 4-T _regs) based on DSPE-PEG platform DP-NHS was reacted with an anti-CD 4 antigen binding fragment (anti-CD 4 Fab fragment, aCD 4) in different molar ratios (molar ratio 0:1 to 20:1) at pH 8.5 for 12 hours and unbound DP-NHS was removed by ultrafiltration to give DP-aCD4. After preincubation of the regulatory T cells with free aCD4 (300. Mu.g/million T _regs), the cells were incubated with DP-aCD4 (50. Mu.g/million T _regs) and the aCD 4-regulatory T cells (aCD 4-T _regs) were collected by centrifugation. And then wrapping the CII and the RM according to a Tn method to obtain the CII-RM@aCD4Tn.

5. CII-RM@aCD4Tn characterization

The membrane insertion efficiency was examined by flow cytometry and confocal microscopy using DP-aCD4-Cy3 synthesized in different molar ratios incubated with regulatory T cells. FITC-RM, cy3-Collagen, cy5-aCD4 were co-assembled as CII-RM@aCD4Tn and confocal imaged. Morphology was observed by Transmission Electron Microscopy (TEM), and particle size and potential were measured by dynamic light scattering (Zetasizer). The CII encapsulation efficiency and RM content were determined by collagen ELISA kit and HPLC (mobile phase: 10 mM ammonium acetate/acetonitrile=25:75), respectively, by optimizing the feed ratio of CII to RM (CII 5-20. Mu.g/mL, RM 1-4. Mu.g/mL). Drug release experiments the release was monitored by dialysis (MWCO 800 kda,37 ℃) over 24 hours. CII-RM@aCD4Tn was measured for particle size after storage at-80℃to 25℃for 0-21 days. The biological activity of the membrane proteins was detected by incubating the stored samples with dye-labeled anti-CD 25, CTLA4, PD-1 antibodies and recombinant CD4 for 15 minutes at room temperature, and then measuring the Fluorescence Intensity (FI) of the supernatant after ultracentrifugation.

Bone marrow-derived dendritic cells (BMDCs) were evaluated for CII-RM@aCD4Tn uptake. BMDCs from male DBA mice bone marrow was extracted by rinsing the femur and tibia with PBS. After removal of the erythrocytes by ACK lysis buffer, the cell suspension was filtered through a 70 μm screen to remove debris. The collected cells were cultured in RPMI 1640 medium and 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 day 3 and BMDCs was harvested on day 7 for subsequent experiments.

To assess Nanoparticle (NPs) uptake and antigen presentation, CII was labeled as FITC. BMDCs (1X 10 ⁶ cells/well, 6 well plate) were incubated with CII-RM@aCD4Tn or free drug (CII and RM), respectively, for 12 or 24 hours. After incubation BMDCs was washed twice with PBS and the nuclei and cell membranes were stained with DAPI and DiO, respectively. Prior to confocal imaging, cells were fixed with 4% paraformaldehyde and resuspended in anti-fluorescence quenching coverslipping.

Experimental results:

CD4 ⁺ T cells isolated from spleen and LNs were successfully induced as regulatory T cells and showed high levels of CD25 and Foxp3 expression (fig. 2 a). In addition, the critical immunoregulatory marker molecule CTLA4 was also significantly upregulated following stimulation (b in fig. 2). To optimize binding of CII-RM@aCD4Tn to CD4 ⁺ T cells, different molar ratios of DSPE-PEG-NHS (DP) to DP-aCD4 of the anti-CD 4 Fab protein (aCD 4) were synthesized. DP-aCD4 binds to the highest level of CD4 ⁺ T cells at a DP:aCD4 molar ratio of 8, however, excessive DP modification resulted in inactivation of aCD4, thereby reducing the targeting of CII-RM@aCD4Tn to CD4 ⁺ T cells (c in 2). Thus, 8:1 was chosen as the optimal ratio to ensure high CD4 ⁺ T cell binding capacity of CII-RM@aCD4Tn. Encapsulation efficiency and drug loading of CII and RM are shown in figure 2 d. CII-RM@aCD4Tn achieves sustained drug release over 24 hours (e in 2). CII-RM@aCD4Tn carrying CII and RM, modified aCD4 was successfully prepared (FIG. 3 a). Transmission Electron Microscopy (TEM) examination showed that the prepared nanoparticles were spherical in shape, structurally complete, and had a particle size of about 130 nm a (b in fig. 3). CII-RM@aCD4Tn has a hydrated diameter of 165.7 nm and a Zeta potential of-16.1 mV (c in FIG. 3). Long-term stability evaluation showed no significant changes in particle size and biological function after storage for 3 weeks at-80 ℃ for CII-rm@adcd4tn (fig. 3d and e). Confocal results showed that CII-rm@adcd 4tn was effective in targeting CD4 ⁺ T cells (f in fig. 3).

To evaluate the role of Tn in enhancing the efficacy of DCs in CII and RM delivery, FITC-labeled free drug or CII-rm@adcd 4Tn was incubated with BMDCs for 12 hours. The results showed that while CII and RM were taken up by all cells, tn-loaded drug intake was significantly higher than free drug (g in fig. 3). To investigate antigen (CII) presentation on the surface of DCs, FITC-labeled CII was incubated with BMDCs for 24 hours. As a result, CII-RM@aCD4Tn was found to significantly promote antigen presentation (h in FIG. 3).

Example 2

Immunosuppression of DC and CD4 ⁺ T cells by CII-RM@aCD4Tn

BMDCs after 48 hours of co-culture with different nanoparticles, LPS (50 ng/mL) was stimulated for 24 hours and CD80 and PD-L1 expression was detected in a flow-through manner. ELISA detects TNF-alpha and TGF-beta secretion. qRT-PCR analysis of TNF- α and TGF- β mRNA levels (primers see Table 1).

TABLE 1

CD4 ⁺ T cells were co-cultured with each type of nanoparticle for 48 hours and stimulated with PMA (20 ng/mL) for 6 hours. Inactivation of CD4 ⁺ T cells (iCD 4) was induced by the addition of 10 ng/mL IL-10 and 10 ng/mL TGF-beta for 72 hours. Expression of CTLA4 and PD-1 was detected by flow cytometry, while levels of TNF- α and TGF- β in the cells and culture supernatant were determined by qRT-PCR and ELISA, respectively.

Experimental results:

Tolerogenic DCs (tDCs) play a key role in maintaining immune homeostasis and preventing autoimmune responses. BMDCs after 48 hours incubation with the different formulations, LPS was used for 24 hours and tested for activation markers and secreted cytokines. Mature BMDCs (mDCs) showed high levels of CD80, while CII-RM@aCD4Tn was significantly reduced after treatment (FIG. 4 a). Interestingly, BMDCs exhibited higher PD-L1 levels after CII-RM@aCD4Tn treatment (FIG. 4, a). In addition, the group of mDCs had the highest expression level of the pro-inflammatory cytokine TNF- α and its mRNA, while the anti-inflammatory cytokine TGF- β and its mRNA was the lowest (b and c in fig. 4) compared to the other groups. CII-RM@aCD4Tn treatment significantly inhibited TNF- α protein and its mRNA expression, while significantly upregulating TGF- β (b and c in FIG. 4).

Inactivated CD4 ⁺ T cells (iCD 4 s) are critical in immunomodulation and autoimmune prophylaxis and have therapeutic potential in the fields of autoimmune diseases, allergies, organ transplantation, etc. To assess whether CII-rm@adcd 4tn could induce iCD4s, CD4 ⁺ T cells were incubated with them for 48 hours followed by stimulation with PMA for 6 hours. Flow cytometry analysis showed increased expression of CTLA4 and PD-1 after CII-rm@agc4tn treatment (a in fig. 5) compared to mature CD4 ⁺ T cells (mCD 4 s), indicating successful induction of the regulatory phenotype. In addition, CII-RM@aCD4Tn treated cells exhibited significantly increased levels of TGF- β mRNA and cytokines, while TNF- α expression was significantly reduced (FIGS. 5 b and c).

In conclusion, CII-RM@aCD4Tn can effectively promote the differentiation of tDCs and iCD4s, enhance the secretion of anti-inflammatory cytokines and inhibit the pro-inflammatory reaction.

Example 3

1. Bridging of CD4 ⁺ T cells with DC by CII-RM@aCD4Tn

BMDCs (1X 10 ⁶) and CD4 ⁺ T cells (1X 10 ⁶) were incubated with CII-RM@Tn or CII-RM@aCD4Tn (37 ℃ C., 1 hour) and evaluated for DC-CD4 binding capacity. 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@adcd4tn with DC-CD4 in vivo, cy 5-labeled CII-rm@adcd4tn was injected intradermally into the bilateral inguinal of male DBA/1 mice. After 24 hours, mice were sacrificed and Inguinal Lymph Nodes (iLNs) were frozen and sectioned and stained with fluorescent-labeled CD11c and CD4 antibodies. The slice is imaged by a panoramic scanner (Pannoramic MID-250FLASH-DESK,3 DHISTECH).

2. In vivo distribution of CII-RM@aCD4Tn

Cy 5-labeled CII-RM@aCD4Tn was injected intradermally into the unilateral or bilateral dorsum of male DBA/1 mice, respectively, at the same total dose. Mice were sacrificed 24 hours later and IVIS biopsies were taken from heart, liver, spleen, lung, kidney, axillary lymph nodes (aLNs) and iLNs. To follow drug release in vivo, cy 5-labeled CII in combination with RM or CII-rm@agc4tn was injected into the right inguinal of mice and the injection site FI was monitored by IVIS for 7 consecutive days.

Experimental results:

The CD4 molecule on CD4 ⁺ T cells was a stable biomarker with the CD80/86 co-stimulatory molecule on DC, making it an ideal target for CII-RM@aCD4Tn binding (FIG. 6 a). When CD4 ⁺ T cells were mixed with BMDCs in the same ratio, CII-RM@Tn or CII-RM@aCD4Tn was added, respectively, and incubated for 1 hour. FACS analysis showed that both cells of the CII-RM@Tn treated group remained dispersed in the culture broth, while the CII-RM@aCD4Tn treated group effectively aggregated CD4 ⁺ T cells and BMDCs into cell pairs (b in FIG. 6). To test its binding capacity in vivo, DBA/1 mice were injected intradermally with CII-RM@aCD4Tn on their ventral side, 24 hours later, iLN were taken for immunofluorescence section analysis. The results indicate that CII-rm@adcd 4tn is not only able to target DCs (CD 11c ⁺), but also to bind efficiently to CD4 ⁺ T cells and promote their interaction in the lymph node collateral cortex region (fig. 6 c). These data demonstrate that CII-RM@aCD4Tn has dual targeting capability, can bind to CD4 ⁺ T cells and DC at the same time, and can effectively promote intercellular bridging in vitro and in vivo.

To assess the lymph node targeting ability of CII-rm@adc4tn, intradermal injections were performed at different locations ventrally of DBA/1 mice. After single-sided (left or right) injection, fluorescence signals appear predominantly on the same side iLN and aLN, with less accumulation in the major organs (a-c in fig. 7). After double-sided injection, the fluorescence intensities of iLN and aLN account for 60% of the total fluorescence intensity (c in fig. 7). These results indicate that intradermal injection on the ventral side of both sides is effective in delivering CII-RM@aCD4Tn to the systemic lymph nodes, potentially producing good immunomodulatory effects. In vivo imaging at different time points showed that CII-rm@adcd4tn could persist near the injection site for at least 1 week, while free drug (cii+rm) rapidly diffused within 4 days (d and e in fig. 7). Quantitative analysis showed a 2.8-fold increase in area under the fluorescence intensity curve within 1 week for the CII-RM@aCD4Tn group (f in FIG. 7) compared to the free drug group, highlighting its prolonged retention capacity.

Example 4

In vivo safety assessment of CII-RM@aCD4Tn

Male DBA/1 mice were randomly divided into 6 groups (6 per group) with the remaining five groups receiving 150 μg CII+20 μg RM (G3), 1 mg Tn (G4), 1 mg aCD4Tn (G5), 1.2 mg CII-RM@Tn nanoparticle (G6) and 1.2 mg CII-RM@aCD4Tn nanoparticle (G7), respectively. Groups were injected intradermally on both sides of the back of mice on day 0 and day 3. Blood from each group of mice was collected on day 7 for liver function detection and whole blood cell count. After euthanasia of mice, heart, liver, spleen, lung and kidney tissues were collected and analyzed for pathological abnormalities by hematoxylin-eosin (H & E) staining.

Experimental results:

safety evaluation showed that the biochemical analysis of blood showed no significant change in the average of alanine Aminotransferase (ALT), aspartate Aminotransferase (AST) and albumin levels for each group (b in fig. 8), and that the blood routine test results were all within normal physiological ranges (c in fig. 8). In addition, histological examination of the major organs did not reveal signs of tissue injury or inflammation (fig. 9). These results demonstrate that CII-rm@adc4tn has good biosafety without causing cytotoxicity or organ toxicity.

Example 5

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

Nanoparticles encapsulating OVA _323-339 were injected intradermally into the dorsal bilateral of OT-II mice. After 4 days, aLNs, iLNs and spleen were taken and analyzed for regulatory T cell ratios by flow cytometry (addition of 2. Mu.L of counting beads and staining for CD45, CD4, CD25 and Foxp 3). In addition, IFN-. Gamma.and TNF-. Alpha.secreting cells were detected by ELISPOT by inoculating spleen cells at 1X 10 ⁶/well to ELISPOT plates, stimulating with 10. Mu.g/mL OVA _323-339 (specific antigen) or CII (non-specific antigen), respectively, for 24 hours, collecting the supernatant and quantifying cytokines by ELISA, and counting the number of secreting cells according to manufacturer's instructions.

2. RNA sequencing and differential Gene enrichment analysis

Total RNA was extracted from spleen lymphocytes and a sequencing library was constructed based on the effective concentration and the amount of data targeted and sequenced (Illumina NovaSeq platform). Effective sequencing data were quantified at the transcriptional level by HTSeq (differential analysis using edder (Robinson MD et al 2010) to correct for p-values (padj) <0.05 as a significance standard). The mainstream hierarchical clustering method converts log10 (fpkm+1) values into normalized values and performs clustering. Gene Ontology (GO) enrichment analysis using a kit (ClusterProfiler) screened for significant enrichment pathways with p-value < 0.05. The gene set of the Gene Set Enrichment Analysis (GSEA) was derived from the KEGG database and by comparing the ordered list of differentially expressed genes from the two sets of samples, it was checked whether the pre-set gene set was significantly enriched at the top or bottom. RNA sequencing and gene enrichment analysis services were performed by Lichuan Biotechnology Co.

Experimental results:

In view of the effective induction of tDCs and iCD4s by CII-RM@aCD4Tn, we further studied the ability of this system to induce regulatory T cells. Since OT-II transgenic mice derived CD4 ⁺ T cells were highly responsive to ovalbumin 323-339 peptide (OVA _323-339), we first synthesized OVA _323-339 loaded nanoparticles. Immunosuppression was assessed in OT-II mice, with significant increases in both the proportion and absolute number of regulatory T cells in LNs and spleen following ventral intradermal injection of OVA-RM@aCD4Tn (FIGS. 10 a-d). Wherein, the spleen was 8.4-fold increased in regulatory T cells in the OVA-RM@aCD4Tn group compared to the PBS group, and 1.4-fold increased compared to the OVA-RM@Tn group (a-d in FIG. 10).

To further investigate whether this immunosuppression is antigen-specific, we isolated spleen cells from mice in different experimental groups and stimulated with OVA _323-339 (related antigen) or CII (unrelated antigen) in vitro. The results showed a 60.4% decrease in TNF-. Alpha.secreting cells upon OVA _323-339 stimulation following OVA _323-339 treatment compared to the OVA-RM@Tn group, whereas no TNF-. Alpha.secreting cells were observed upon CII stimulation (FIGS. 11 a and b).

To elucidate the mechanism of OVA-rm@agc4tn mediated immunosuppression, we performed transcriptome analysis on CD4 ⁺ T cells isolated from spleen 4 days after administration of OT-II mice. After OVA-RM@aCD4Tn treatment, pro-inflammatory factors (IL-6, TNF, MMP9, IL-1β and IL-17A) were significantly down-regulated, while anti-inflammatory markers (IL-10, foxp3, TGF-. Beta.1, CTLA4, IL1 RN) were significantly up-regulated (FIG. 11 c). KEGG signaling pathway analysis further showed that this system regulates CD4 ⁺ T cell function through TGF- β, TNF and T cell receptor signaling pathways and is closely related to autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes, etc. (d in fig. 11). In conclusion, the OVA-RM@aCD4Tn has better immunoregulatory capability than the OVA-RM@Tn, and shows potential application value in autoimmune disease treatment.

Example 6

1. Therapeutic Effect of CII-RM@aCD4Tn in CIA mouse model

To establish the CIA mouse model, male DBA/1 mice were injected intradermally with 100 μl of CII emulsion (CII: cfa=1:1) on both sides of the caudal base. After 21 days, boost was performed with CII and IFA. Subsequently, mice were randomly divided into seven groups of 6 mice each. One group was a healthy control group (G1), and the other six groups were respectively 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).

Therapeutic intervention was performed from day 22 to day 28 (3 injections, 1 every 3 days) by intradermal injection on both sides of the back of mice, each at a dose of 200 μl. Arthritis scores and body weight changes were recorded every 3 days. The arthritis scoring criteria were 0 score without erythema and swelling, 1 score with erythema and mild swelling localized to the tarsal or ankle joint, 2 score with erythema and mild swelling extending from the ankle joint to the tarsal bone, 3 score with erythema and moderate swelling extending from the ankle joint to the metatarsal joint, 4 score with erythema and severe swelling affecting the ankle joint, foot and toe, or limb rigidity. Each mouse was scored for four limbs, with the top score for individual mice being 16 points. On day 42, foot thickness was measured and a rotarod test was performed.

2. Rotating rod experiment

Mice were assessed for motor coordination using a computerized rotarod (ZL-200 c, bestcell). Mice were placed on stationary bars daily for 2-3 minutes to reduce stress 2 days prior to the experiment. On day 41, mice were spun at 15 rpm for 3 minutes and drop times were recorded, followed by an increase in spin from 4 rpm to 20rpm and critical speed at drop was recorded. The experiment was repeated on day 42.

3. Inflammatory joint tissue pathology and immunohistochemical analysis

The end point of the experiment was stained with hematoxylin-eosin (H & E), safranin O-fast green (SO-FG) and Masson (Masson) from the posterior ankle of the mice. H & E sections were semi-quantitatively scored (0-3 score) by three pathologists according to three indicators of synovial hyperplasia, inflammatory cell infiltration, etc., total score of 0-1 for no synovitis, 2-4 for low grade synovitis, and 5-9 for high grade synovitis. The tibial and talar cartilage thickness was measured by software (Image J, version 1.53 c) under SO-FG staining and Masson (Masson) staining quantified synovial fibrosis area. The hindpaw bone parameters (tube current 385 μa, tube voltage 65 kV, pixel size 17.76 μm) were analyzed using a microcomputer tomography (Micro-CT, skyScan 1276), three-dimensional images were reconstructed by software (micrometers) and bone volume fraction (BV/TV), bone trabecular thickness (tb.th), bone trabecular number (tb.n), bone trabecular separation (tb.sp), structural Model Index (SMI) and bone density (BMD) were calculated. IL-6 immunohistochemical analysis Using software (Image J) quantitative synovial positive area.

Experimental results:

To assess the early therapeutic potential of this system, different nanoformulations were administered on days 22, 25 and 28, respectively, to treat collagen-induced arthritis (CIA) mice (fig. 12 a). At the end of the experiment, the severity of arthritis was assessed by blindly scoring paw swelling and redness. Mice receiving CII-rm@adc4tn treatment had significantly lower arthritis scores than PBS group, indicating significant reduction in inflammation and edema (b in fig. 12). In addition, the body weight of mice in the RA group was greatly reduced due to disease progression and severe swelling, and the body weight loss of mice in the CII-rm@adcdtn group was significantly reduced (c in fig. 12). Paw thickness measurements demonstrated that the CII-rm@adcdtn group showed the best therapeutic effect among all treatment groups (d in fig. 12). Subsequently, we performed behavioral assessment on the treated mice by the rotarod system to examine their motor coordination and balance ability. The CII-rm@adcdtn group of mice showed significant improvement over the two training sessions, including a decrease in the frequency of falls from the rotating lever and an increase in the rotational speed before falls (e in fig. 12).

To further evaluate the pathological damage of the ankle joint, histological analysis was performed. In hematoxylin-eosin (H & E) -stained tissue sections, the joint cavities of CII-rm@adcd 4tn group mice were almost completely restored, the joint interfaces were clear, no apparent synovitis or degenerative changes of articular cartilage, while RA group exhibited severe pathological changes including massive inflammatory cell infiltration, synovial hyperplasia, pannus formation and cartilage erosion (fig. 13 a and d). Pannus formation is one of the features of pathological changes in RA. In immunohistochemical stained sections, the CII-RM@aCD4Tn group mice had minimal IL-6 distribution in the joints and synovium, and the affected areas were minimal (FIGS. 13 b and e). Analysis of Masson staining showed extensive fibrosis in the synovial tissue of mice in the RA group, whereas the area of fibrosis in the CII-RM@aCD4Tn group was only 12.0% and was 94.2% less than in the RA group (c and f in FIG. 13). Furthermore, safranin O-fast green (SO-FG) staining showed that the cartilage structure of the CII-RM@aCD4Tn group remained intact, with a cartilage thickness of 135.5 μm, approaching the healthy group level (FIGS. 14 a and d).

Since bone erosion is a critical factor affecting disease severity and poor prognosis, we used three-dimensional microct imaging to assess bone integrity. In the RA group, ankle skeletal structure exhibited severe bone erosion, bone surface roughness, and trabecular injury to the tibia, fibula, and talus. In contrast, the CII-rm@adcd4tn group showed significantly superior bone remodeling capacity, and the microct parameters of ROI quantitative analysis were significantly improved (b, c and e in fig. 14), indicating that bone lesions were effectively repaired.

Overall, these results demonstrate that CII-rm@adcdtn can significantly alleviate inflammation, cartilage degradation, synovial fibrosis and bone erosion in a CIA mouse model, exhibit powerful therapeutic effects, help slow RA progression and improve joint function.

Example 7

Mouse paw sections, distribution of regulatory T cells in synovium was analyzed by Image J software after staining with CD4, CD25, foxp3 antibodies. ELISA kits were used to detect IL-6, IL-1. Beta., IFN-. Gamma., IL-10 and TGF-. Beta.levels in serum and synovium. The number of regulatory T cells and their duty cycle in spleen and LNs were analyzed by flow cytometry.

Experimental results:

One of the pathological features of RA is that activated T cells and macrophages infiltrate the synovium of multiple joints and secrete a range of pro-inflammatory cytokines including IFN- γ, IL-6 and IL-1β. These cytokines continue to activate inflammatory responses, leading to synovial hyperplasia and secondary bone erosion. In contrast, immunoregulatory cells (especially regulatory T cells) play a critical role in maintaining immune homeostasis by secreting anti-inflammatory cytokines such as IL-10 and TGF-beta. Immunofluorescence analysis showed a significant increase in the proportion of regulatory T cells (CD 4 ⁺ CD25⁺ Foxp3⁺) in synovium after CII-rm@adcd 4tn treatment (fig. 15 a-c).

Furthermore, we further analyzed regulatory T cell populations in LNs and spleen by flow cytometry. Also, the number of regulatory T cells increased significantly after CII-RM@aCD4Tn treatment, indicating that they have a systemic immunomodulatory effect (a-d in FIG. 16). At the same time, CII-RM@aCD4Tn treatment significantly reduced the levels of pro-inflammatory cytokines (IL-6, IL-1β and IFN- γ) in serum and synovium, while significantly up-regulating secretion of IL-10 and TGF- β (FIG. 17). These results indicate that CII-rm@adc4tn helps to restore systemic immune homeostasis by promoting the production of regulatory T cells, transitioning the inflammatory microenvironment to an immunoregulatory state, thereby significantly reducing the levels of pro-inflammatory cytokines.

Example 8

Prevention effect verification of CII-RM@aCD4Tn on CIA mice

To assess its prophylactic potential, DBA/1 mice were given NPs treatment prior to CIA induction. Male DBA/1 mice were randomly divided into seven groups of 6. One group was a healthy control group (G1), and the other six groups were respectively 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). NPs were co-injected twice on day-21 and day-14, followed by CIA induction on day 0 and day 21. Arthritis scores and weight changes were monitored every 3 days and foot thickness was measured and a rotarod test was performed on day 42. In addition, ELISA kits were used to detect IL-6, IL-1. Beta., TNF-. Alpha., IFN-. Gamma., IL-10 and TGF-. Beta.levels in serum and synovial tissue, and the number and duty cycle of regulatory T cells in spleen and LNs were analyzed by flow cytometry.

Experimental results:

to investigate the prophylactic effect of CII-rm@adcd 4tn, mice were injected 3 and 2 weeks prior to CIA induction, respectively (fig. 18 a). After CII-RM@aCD4Tn treatment, the mice had normal appearance with no signs of redness or swelling and had body weight recovered almost to healthy levels (b-d in FIG. 18). In addition, mice in the CII-rm@adct4tn group showed the strongest locomotor ability in all experimental groups (e in fig. 18), indicating that they have excellent protective effect on joint function. The levels of pro-inflammatory cytokines were significantly reduced in CII-rm@adcd 4tn treated mice, while the anti-inflammatory cytokine levels were significantly increased (f in fig. 18). In addition, the number and proportion of regulatory T cells in the LNs and spleen of mice in the CII-RM@aCD4Tn group were the highest in all experimental groups (a-d in FIG. 19). These results demonstrate that the DC-CD4 bridging system has a powerful prophylactic effect in preventing RA progression, highlighting its feasibility as a vaccine-based immunomodulation strategy.

In this study, we developed a DC-CD4 bridged tolerogenic nanosystem (AP-IA@aCD4Tn) for the efficient induction of antigen-specific regulatory T cells for the treatment of rheumatoid arthritis. The platform co-delivers antigenic peptides and immunosuppressants via bispecific nanoparticles while acting on dendritic cells (via CTLA4-CD 80/86) and CD4 ⁺ T cells (via anti-CD 4 antibodies), achieving spatial coupling of DCs to T cells, thereby promoting formation of a tolerogenic microenvironment. In a collagen-induced arthritis mouse model, nanoparticles CII-RM@aCD4Tn loaded with type II collagen (collagen II) and rapamycin (rapamycin) are constructed, regulatory T cells in lymph nodes and spleen are remarkably amplified after intradermal injection, the expression of pro-inflammatory cytokines (such as TNF-alpha and IL-6) is inhibited, and the level of anti-inflammatory mediators (such as TGF-beta and IL-10) is improved, so that pathological manifestations of synovitis, cartilage destruction, bone erosion and the like are effectively relieved. These findings suggest that DC-CD4 bridged tolerogenic nanosystems are promising as an accurate and durable autoimmune disease treatment strategy. Future work should focus on its clinical transformations and its use in other antigen-driven diseases, paving the way for revolutionary immunotherapy.

Claims (10)

1. A preparation method of the bispecific cell membrane nanoparticle is characterized by comprising the following steps of inducing primary CD4 ⁺ T cells into regulatory T cells in vitro by adopting TGF-beta and rapamycin, enabling distearoyl phosphatidylethanolamine-polyethylene glycol-active ester to react with an anti-CD 4 antigen binding fragment to obtain DP-aCD4, pre-incubating free anti-CD 4 antigen binding fragment of the regulatory T cells, co-incubating the free anti-CD 4 antigen binding fragment with DP-aCD4 to obtain aCD 4-regulatory T cells, mixing the aCD 4-regulatory T cells with antigen peptide and immunosuppressant, and preparing the bispecific cell membrane nanoparticle by a liposome extruder.

2. The method for preparing the bispecific cell membrane nanoparticle according to claim 1, wherein the molar ratio of distearoyl phosphatidylethanolamine-polyethylene glycol-active ester to the anti-CD 4 antigen-binding fragment is 2-20:1.

3. The method for preparing the bispecific cell membrane nanoparticle according to claim 1, wherein distearoyl phosphatidylethanolamine-polyethylene glycol-active ester and the anti-CD 4 antigen-binding fragment react for 10-14 h under the condition of pH 8-9 to obtain DP-aCD4.

4. The method of claim 1, wherein the antigenic peptide is one or more of citrullinated peptide, type II collagen, glucose-6-phosphate isomerase peptide, heat shock protein peptide, RA33 peptide.

5. The method of preparing bispecific cell membrane nanoparticles according to claim 1, wherein the immunosuppressant comprises one or more of rapamycin, everolimus, temsirolimus, tacrolimus, cyclosporine a.

6. The method for preparing the bispecific cell membrane nanoparticle according to claim 1, wherein the ratio of the antigenic peptide to the aCD 4-regulatory T cells is 5-20 μg/1×10 ⁷ cells, and the ratio of the immunosuppressant to the aCD 4-regulatory T cells is 1-4 μg/1×10 ⁷ cells.

7. A bispecific cell membrane nanoparticle prepared by the method of any one of claims 1-6.

8. Use of the bispecific cell membrane nanoparticle of claim 7 for the preparation of an immunosuppressive drug.

9. The use according to claim 8, wherein the immunosuppressive drug is a drug for the treatment and/or prevention of rheumatoid arthritis.

10. The use according to claim 9, wherein the bispecific cell membrane nanoparticle is for inducing antigen-specific regulatory T cells.