Construction and Application of Core-Shell Structure Nanoparticles for Targeted Elimination of Antigen-Specific T Cells

Abstract

The present invention provides the construction and application of core-shell structured nanoparticles for targeted elimination of antigen-specific T cells. The inner core of the core-shell structured nanoparticles is a polymer nanoparticle loaded with tacrolimus, and the outer shell is coated with a cationic lipid membrane for loading antigens, with the characteristic of programmed drug release. After the drug-loaded nanoparticles are taken up by dendritic cells, the outer-layer antigens are rapidly released, processed by DCs and presented in the form of antigen peptide-major histocompatibility complex, which recognizes and couples with the T cell receptor, thereby targeting antigen-specific T cells; the inner core FK506 is slowly released later, and can be transmitted to T cells through cell-cell interactions to achieve specific T cell clone elimination. The antigen on the outer layer of the nanoparticles of the present invention can be a protein, polypeptide or nucleic acid sequence encoding an antigenic epitope, and FK506 can be replaced by other immunosuppressants. This nano drug delivery system can play a role in diseases caused by overactive T cell immunity.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceuticals and relates to the construction and application of a core-shell structured nanoparticle for targeted elimination of antigen-specific T cells. It mainly constructs a drug-loaded core-shell structured nanoparticle for alleviating the body damage caused by abnormal activation of T cells, as well as the pharmaceutical applications of the nanoparticle in autoimmune diseases such as type I diabetes, multiple sclerosis, rheumatoid arthritis, and inflammatory vasculitis, and allogeneic organ transplantation. Background Art

[0002] Due to the scarcity of syngeneic grafts, allogeneic organ transplantation has gradually become the main means of clinical application. However, allogeneic grafts are recognized as invasive "non-self" substances by the immune system of the organ recipient and thus suffer from immune attack and rejection. Overactive cellular immunity is one of the main inducements of allogeneic transplantation rejection. Among them, effector T cells expand vigorously, generating a large number of allogeneic antigen-specific T cell clones to attack the graft, resulting in inflammation, tissue damage, and even transplantation failure [Front Immunol 7,582(2016)]; while memory T cells can mediate chronic transplantation rejection, which is closely related to the poor prognosis of patients [Sci Adv 8,eabk0270(2022)].

[0003] To alleviate T cell-mediated rejection (TCMR) and prolong graft survival time, organ recipients are usually required to take immunosuppressants lifelong. Tacrolimus (FK506) is a representative of the second-generation (non-steroidal) immunosuppressants and is a calcium-dependent phosphatase inhibitor (CNI) approved by the FDA, which is clinically used for the postoperative control of solid organ transplantation such as heart, liver, and kidney. FK506 interferes with the calcineurin-nuclear factor of activated T cells (NFAT) pathway, thereby inhibiting the activation and proliferation of T cells and restricting TCMR [Biochim Biophys Acta 1498,1-18(2000)]. However, the application of FK506 is limited by its narrow therapeutic window and many side effects [Kidney Int 67,2440-2447(2005)], which are mainly caused by the non-specific immunosuppressive effect of FK506 on broad-spectrum T cells [Nat Biotechnol 37,238-251(2019)]. In fact, patients taking FK506 for a long time have systemic immune impairment, leading to an increased susceptibility to opportunistic infections and malignancies [N Engl J Med351,2715-2729(2004)][Clin Transplant 22,372-377(2008)]. Therefore, delivering FK506 to allogeneic graft antigen-specific T cells may improve transplantation outcomes and reduce off-target toxicity.

[0004] Dendritic cells (DCs) play an important role in regulating allogeneic immune responses [Nat Rev Immunol 4, 24-34 (2004)]. Mature DCs can trigger the full activation of allogeneic graft-specific T cells through the cell surface: 1) presenting allogeneic antigen peptide-major histocompatibility complex molecules (pMHC), interacting with specific T cell receptors (TCRs), which serves as the first signal mediating T cell activation; 2) expressing B7 ligands, such as co-stimulatory molecules like CD80 and CD86, which trigger the second signal for T cell activation after binding to the receptor CD28 on the T cell surface; 3) secreting inflammatory cytokines that promote lymphocyte activity to induce the third signal for T cell activation. In contrast, tolerogenic DCs express MHC molecules on the surface, but the co-stimulatory signals are weaker than the co-inhibitory signals [Genome Biol 6, 223 (2005)], and the secretion of immunostimulatory cytokines is insufficient [Nat Rev Immunol 7, 610-621 (2007)]. These tolerogenic DCs, used alone or in combination with other immunosuppressants and / or immunomodulators, can promote T cell exhaustion, regulatory T cell (Treg) expansion, and type II CD4 helper T (Th2) cell polarization, and have been shown to effectively reduce allograft rejection. However, this treatment modality has insufficient antigen specificity and there are certain safety concerns [Diabetes Care 34, 2026-2032 (2011)][Int Immunopharmacol 21, 247-254 (2014).

[0005] Currently, the strategies targeting allogeneic antigen-specific T cells mainly include: 1. Using soluble pMHC monomers or multimers presenting antigen-specific immunodominant epitopes to directly recognize and conjugate T cells expressing antigen-specific TCRs [Neurology 54, 1414-1420 (2000)] [Nat Immunol 3, 383-391 (2002)]; 2. Using tolerogenic DNA vaccines [Sci Transl Med 5, 191ra182 (2013)], antigen proteins, antigen peptides [J Exp Med 149, 758-773 (1979)], antigen-conjugated apoptotic cells [J Immunol 191, 5341-5346 (2013)] or antigen-loaded microparticles / nanoparticles [ACS Nano 8, 2148-2160 (2014)] to indirectly target allogeneic antigen-specific T cells through antigen-presenting cells (APCs). Based on this, we propose that pMHC-TCR-mediated selective intercellular conjugation may be used to target specific T cells, and the close interaction between immune cells may promote the transfer of drugs from DCs presenting specific antigens to allogeneic antigen-responsive T cells, thereby avoiding the inhibition of broad-spectrum T cells and reducing related risks. Summary of the Invention

[0006] One object of the present invention is to provide a method for constructing a core-shell structured nanoparticle for targeting and eliminating antigen-specific T cells, and the specific steps are as follows:

[0007] (1) Construction of the core of the immunosuppressant-loaded nanoparticle:

[0008] The core-shell structured nanocarrier of the present invention uses poly(lactic-co-glycolic acid) (PLGA) nanoparticles as the hydrophobic core, which can encapsulate the immunosuppressant FK506, and the drug-lipid ratio is 1:10 - 1:5000 (w / w).

[0009] The PLGA core can be replaced by polymer materials such as poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(orthoester) (POE), poly(methyl methacrylate) (PMMA), ethylene-vinyl acetate copolymer (EVA), etc., or inorganic materials such as mesoporous silica (MSN).

[0010] FK506 can be replaced by other immunosuppressants that inhibit T cell activation, proliferation or function, including drugs such as cyclosporine, rapamycin, glucocorticoids or mycophenolate mofetil.

[0011] (2) Coating a cationic lipid membrane on the outer layer of the immunosuppressant-loaded nanoparticle core:

[0012] In step (1), a lipid membrane containing cationic lipids is coated on the surface of the immunosuppressant-loaded nanoparticle core, where the cationic lipids account for 10%-90% of the total lipid mass.

[0013] The structural composition of the lipid membrane includes cationic lipids, helper lipids, structural lipids, and PEGylated lipids. Among them, the cationic lipids can be selected from (2,3-dioleoyl-propyl) trimethylammonium chloride (DOTAP), 1,2-dimethyl-3-trimethylammonium-propane (DMTAP), 1,2-stearoyl-3-trimethylammonium-propane (DSTAP), dimethyldioctadecylammonium (DDA), or 3β-[N-(N’,N’-dimethylaminoethyl) carbamoyl] cholesterol (DC-Chol); the helper lipids can be selected from phosphatidylcholine, such as distearoylphosphatidylcholine (DSPC), or phosphatidylethanolamine, such as 1,2-diacyl-sn-glycero-3-phosphoethanolamine (DOPE); the structural lipids can be selected from cholesterol.

[0014] (3) Preparation of the core-shell structured nanoparticles co-loaded with antigen and immunosuppressant:

[0015] The core-shell structured nanoparticles with the core loaded with immunosuppressant and the shell coated with a positively charged lipid membrane are prepared by a one-step method or a multi-step method.

[0016] The preparation of the core-shell structured nanoparticles loaded with immunosuppressant according to the present invention can be completed in one step by microfluidic technology, or completed in two steps by nanoprecipitation plus lipid membrane extrusion coating.

[0017] The loading of antigen on the outer layer of the core-shell structured nanoparticles can be achieved through the electrostatic interaction and van der Waals force between the positively charged lipid membrane and the negatively charged antigen, or through structural modification of the antigen to promote the bonding between the antigen and the outer lipid membrane. Among them, the form of the antigen can be protein, polypeptide, nucleic acid sequence encoding an antigenic epitope, or immune complex.

[0018] The drug-loaded core-shell structured nanoparticles according to the present invention have the property of programmed drug release, that is, the outer antigen is rapidly released, and the inner immunosuppressant is slowly released later.

[0019] The second object of the present invention is to provide the application of the said nanocarrier in the preparation of drugs for alleviating allogeneic organ transplantation and autoimmune diseases. These diseases or pathological conditions are caused by abnormal activation of T cells, and the relevant antigenic epitopes have been identified and recognized. The present invention can be used to prevent or treat diseases or pathological conditions related to antigen-specific T cell activation.

[0020] The core-shell structured nanoparticles co-loaded with antigen and immunosuppressant constructed in the present invention mainly act directly on APCs, especially DCs. After the drug-loaded nanoparticles are taken up by DCs into the cells, the outer antigen is rapidly released, which is processed by DCs and presented in the form of pMHC for recognizing and conjugating antigen-specific T cells expressing specific TCRs.

[0021] The core-shell structured nanoparticles co-loaded with antigen and immunosuppressant constructed in the present invention act indirectly on T cells through DCs. After the drug-loaded nanoparticles are taken up by DCs, the FK506 loaded in the core is slowly released later, which can be transferred to T cells through cell-cell interaction for targeted elimination of T cell clones.

[0022] The drug-loaded core-shell structured nanoparticles described in the present invention can be administered directly, acting through passive targeting of DCs in the body, or treating ex vivo induced differentiated homologous DCs in vitro, and then adoptively transferring the DCs treated with the preparation into the patient's body for direct action.

[0023] The drug-loaded core-shell structured nanoparticles described in the present invention can be used to relieve immunopathological conditions caused by abnormal activation of antigen-specific T cells, including allogeneic organ transplantation and autoimmune diseases such as type I diabetes, multiple sclerosis, rheumatoid arthritis, and small vessel vasculitis, and have good prospects for production and application. Description of the Drawings

[0024] Figure 1 It is the phenotypic changes of BMDCs detected by flow cytometry after treatment with different drug-loaded liposomes for 24 h, that is, the changes in the expression of surface molecules CD80 / CD86 (A), MHC I (B), and MHC II (C) (n = 4).

[0025] Figure 2 It is the fluorescence images (B) of Cy3-OVA-Lipo uptake by BMDCs and DC2.4 after pretreatment with FK506-Lipo or Blank-Lipo for 12 h (A) and the semi-quantitative results (C) of Cy3 fluorescence signals (n = 4), scale bar, 200 μm.

[0026] Figure 3 It is a schematic diagram of the preparation of core-shell structured nanoparticles by microfluidics.

[0027] Figure 4 It is the particle size, polydispersity index (PDI) (A) and surface potential (B) of different nanoparticles detected by dynamic light scattering method (n = 3), the core-shell structure of the nanoparticles observed by transmission electron microscopy (C), and the semi-quantification of the core size and shell thickness of the nanoparticles (D, n = 86 for Blank-NPs; n = 56 for FK506-NPs).

[0028] Figure 5 It is the detection of the particle size and PDI changes of different nanoparticles stored in Milli-Q water at 4 °C for 28 days by dynamic light scattering method (n = 3).

[0029] Figure 6 It is the detection of the loading of OVA by Blank-NPs and FK506-NPs by 10% SDS-PAGE combined with rapid silver staining method (A), and the gray value (B) of the OVA protein band (white dotted box) and the encapsulation efficiency of OVA (C) were calculated by analyzing the pictures with the image processing software Image J.

[0030] Figure 7 It is the release of OVA and FK506 within 48 h in vitro, which were detected by BCA method and high performance liquid chromatography respectively (n = 3).

[0031] Figure 8 It is the expression of co-stimulatory molecules CD80 and CD86 (A), MHC I and MHC II molecules (B) by BMDCs after treatment with different preparations for 24 h (n = 4).

[0032] Figure 9 It is the result of immunoblotting to investigate the expression of transcription factors STAT1 and STAT3 by BMDCs after treatment with FK506-NPs and OVA@FK506-NPs for 24 h, with untreated cells as the control.

[0033] Figure 10 It is the uptake results of nanoparticles and antigen OVA by BMDCs (A) or DC2.4 cells (B) after treatment with different preparations for 2, 6, 10, 24 or 48 h (n = 4).

[0034] Figure 11 It is the confocal laser microscopy to observe the co-localization of intracellular cell nuclei, antigen, lysosomes and nanoparticles (A) and the co-localization coefficient of nanoparticles and lysosomes (B) in BMDCs or DC2.4 cells after treatment with OVA@FK506-NPs for 8 h (n = 3). Scale bar, 25 μm.

[0035] Figure 12 It is the confocal method to observe the transfer of OVA@FK506-NPs from BMDCs to adjacent (view 1 and 2) rather than distant (view 3 and 4) lymphocytes.

[0036] Figure 13 It is the detection of PD-1 in lymphocyte populations by flow cytometry after co-incubating homologous SPLCs with BMDCs treated with different preparations for 4 days + CD8 T cells (A) and PD-1 + CD4 T cells (B) ratio (n = 4).

[0037] Figure 14 The levels of IFN-γ (A), TNF-α (B), and IL-4 (C) in the supernatant of the co-culture system of SPLCs and BMDCs were detected by ELISA (n = 4).

[0038] Figure 15 The proportions of PD-1+ CD8 T cells (A) and PD-1+ CD4 T cells (B) in activated and resting (with or without α-CD3 / -28Abs stimulation) SPLCs treated with different preparations for 4 days were detected by flow cytometry (n = 4).

[0039] Figure 16 Flow cytometry was used to detect the expression of MHC II molecules and H2K 257-264 / SIINFEKL in BMDCs treated with culture medium, FK506-NPs, OVA@FK506-NPs, or OVA b @FK506-NPs for 24 h.

[0040] Figure 17 Flow cytometry was used to analyze the total number of lymphocytes (A), the proportion of OVA tetramer-positive CD8 T cells (B), and the number of OVA tetramer-positive CD8 T cells (C) in SPLCs after co-incubation with BMDCs under different treatment conditions for 4 days (n = 6).

[0041] Figure 18 The expression levels of IFN-γ (A), IL-10 (B), and IL-4 (C) in the supernatant of the co-culture system of SPLCs derived from OT-I and different BMDCs were detected by ELISA (n = 4).

[0042] Figure 19 Flow cytometry was used to analyze the changes in the memory phenotypes of CD8 T cells in SPLCs under different treatment conditions (n = 6). Resting SPLCs were co-incubated with different BMDCs for 4 days (A) or directly treated with different preparations for 4 days after activation with α-CD3 / -28Abs (B), and then the proportions of naive T cells (Tnaive, CD44-CD62L+), effector memory T cells (Tem, CD44+CD62L-), and central memory T cells (Tcm, CD44+CD62L+) in the CD8 T cell population were analyzed.

[0043] Figure 20It is the acquisition and purification of antigens from the full-thickness skin tissue on the backs of BALB / c and C57BL / 6 mice. The fat and tissue clumps in the skin tissue lysate (STL) were removed by centrifugation (8000 rpm, 5 min) twice to obtain a clear and transparent STL.

[0044] Figure 21 It is the quantification of the antigen concentration in the skin tissue lysate (STL) by the BCA method (n = 3).

[0045] Figure 22 It is the detection of the distribution of protein antigens in STL with different sample loading amounts according to molecular weight by 10% SDS-PAGE combined with rapid silver staining method (A), and the Image J image processing software was used to analyze the pictures to calculate the gray values of the protein bands (B).

[0046] Figure 23 It is the detection of the ability of NPs and FK506-NPs (i.e., F-NPs) to load different STLs by 10% SDS-PAGE combined with rapid silver staining method (A), the Image J image processing software was used to analyze the pictures, calculate the gray values of the protein bands (B, D), and the encapsulation efficiency of the nanoparticles for STL (C, E).

[0047] Figure 24 It is a schematic diagram of allogeneic skin transplantation modeling and drug administration in mice.

[0048] Figure 25 It is the graft rejection scoring of recipient mice during the experiment (A) and the morphology of the transplanted skin grafts at 2 days and 13 days after transplantation surgery (B).

[0049] Figure 26 It is the observation of the histopathology of the grafted skin and kidneys of recipient mice in each group by H&E section.

[0050] Figure 27 It is the change in body weight of recipient mice in each group during the experiment.

[0051] Figure 28 It is the analysis of the levels of IL-2, IFN-γ, IL-4, and IL-10 in the sera (A) and spleens (B) of recipient mice in each group by ELISA method (n = 4).

[0052] Figure 29 It is the analysis of CD8 T cells, CD4 T cells, and CD4 Treg cells in the spleens (A) and draining lymph nodes (B) of recipient mice in each group by flow cytometry (n = 4).

[0053] Figure 30Flow cytometry was used to analyze the immune responses of recipient mice in each group to unrelated antigen stimulation after drug administration. On the 13th day after transplantation surgery, the responses of recipient mice in each group to the stimulation of the unrelated antigen OVA were detected (A). Twenty-four hours after immunizing the mice with OVA, peripheral blood of the mice was isolated for flow cytometry analysis of the proportion of OVA-specific CD8 T cells (B-C) (n = 3). Detailed implementation mode

[0054] The present invention will be further described in conjunction with the accompanying drawings and implementation examples.

[0055] Example 1 Determination of the optimal administration sequence of FK506 and antigen (Ag)

[0056] Bone marrow was isolated from the femurs and tibias of adult C57BL / 6 mice and stimulated in vitro with interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) for six days to induce the differentiation of bone marrow cells into myeloid dendritic cells (BMDCs), which were used as immature DCs (imDCs). Meanwhile, cationic liposomes were used to prepare OVA-Lipo loaded with the model antigen ovalbumin OVA and FK506-Lipo loaded with the immunosuppressant, and blank liposome Blank-Lipo was used as a formulation control. The acquisition of imDCs and the preparation of drug-loaded liposomes refer to our previous research basis [Adv Healthc Mater 10, e2001934 (2021)][Biomaterials 272, 120757 (2021)].

[0057] The pharmacological effects of drugs are closely related to their release behavior. To induce tolerogenic, rather than immunostimulatory, DCs, we investigated the effects of different liposomes on the phenotypic maturation of DCs and screened the optimal administration sequence of FK506 and Ag. imDCs were treated with cell culture medium (negative control), lipopolysaccharide (LPS, 100 ng / mL, positive control), Blank-Lipo, FK506-Lipo (10 nM FK506), OVA-Lipo (10 μg / mL OVA), simultaneous administration of Ag and FK506 (OVA-Lipo plus FK506-Lipo), administration of Ag first followed by FK506 (OVA-Lipo+FK506-Lipo), and administration of FK506 first followed by Ag (FK506-Lipo+OVA-Lipo). Among them, the administration interval for the sequential administration groups was 12 h. After 24 h, the DCs were collected and the changes in cell phenotypes were analyzed by flow cytometry.

[0058] The results showed (see Figure 1) Upon LPS stimulation, DCs were significantly activated, and the expression of co-stimulatory molecules CD80 / 86 and MHC I / II molecules was upregulated. In contrast, the expression of co-stimulatory molecules in DCs was hardly affected by Blank-Lipo and FK506-Lipo, but could be induced and upregulated by OVA-Lipo. In addition, different liposomes had different effects on the expression of MHC molecules in DCs. Compared with Blank-Lipo, FK506-Lipo and OVA-Lipo induced an increase in MHC I and a decrease in MHC II. These results indicate that OVA delivered by this vector can induce the activation of DCs and subsequent antigen presentation. In contrast, FK506 may inhibit the activation of DCs while promoting MHC class I antigen presentation. Therefore, a drug delivery system co-loading and appropriately releasing FK506 and OVA may induce the presentation of specific antigens, especially tolerogenic DCs presenting MHC class I antigens, by blocking co-stimulatory signals and upregulating MHC class I antigen presentation. On the other hand, we found that the phenotypic maturation (tolerogenic or immunostimulatory) of DCs mainly depends on the nature of the drug they initially contact. Specifically, the pharmacological effects of OVA-Lipo+FK506-Lipo were similar to those of OVA-Lipo, while the pharmacological effects of FK506-Lipo+OVA-Lipo were similar to those of FK506-Lipo. At the same time, the activation pattern of DCs in the OVA-Lipo plus FK506-Lipo group was intermediate between the other two dosing regimens.

[0059] Next, we investigated the effect of FK506 pretreatment on antigen uptake by DCs. Cy3-OVA-Lipo was prepared using OVA chemically bonded with the fluorescent dye Cy3 as a model antigen. imDCs were stimulated with FK506-Lipo and Blank-Lipo respectively. After 12 h, free liposomes were washed away with PBS, and Cy3-OVA-Lipo was added and cultured for another 12 h. Subsequently, the cells were rinsed with PBS, fluorescence microscopy was used to take fluorescence photographs, and semi-quantitative analysis of the signal intensity of Cy3 was performed. The results showed that in both DC cell lines (BMDCs and DC2.4), pretreatment with FK506 inhibited antigen uptake by the cells (see Figure 2 ). Therefore, releasing the antigen first and then releasing FK506 may be a more appropriate drug release pattern.

[0060] Example 2 Preparation and Physicochemical Property Characterization of Core-Shell Structure Nanoparticles Ag@FK506-NPs Co-Loading Antigen and Immunosuppressant

[0061] Repeated drug administration is cumbersome and not recommended clinically. Preparations that co-deliver different drugs and have a programmed drug release pattern in a single delivery can be used as a more convenient alternative. We prepared core-shell structured nanoparticles Ag@FK506-NPs co-loading antigen and immunosuppressant and their control nanoparticles by microfluidic technology. Specifically, PLGA and FK506 (30:1, w / w) were dissolved in acetonitrile (acetonitrile phase), and DOTAP, DOPE, DSPE-PEG 2000 and cholesterol (50:20:1.5:28.5, mol / mol) were dissolved in ethanol (ethanol phase). Milli-Q water phase was used to focus on the acetone phase to form drug-PLGA nanoparticles (i.e., FK506-PLGA core). Then, the suspension of FK506-PLGA nanoparticles and the ethanol phase were made to form a convection to achieve the lipid membrane coating on the surface of the nanoparticle core (i.e., lipid shell). Subsequently, the organic solvent was removed by rotary evaporation under reduced pressure, the free drug was removed by ultrafiltration, the nanoparticles were collected and resuspended with Milli-Q water to obtain core-shell structured nanoparticles with FK506 loaded in the core and coated with a cationic lipid membrane on the shell, named FK506-NPs. During the preparation, the flow rate ratio of each phase was controlled by a microfluidic device to ensure the uniform morphology of the preparation. Next, using OVA as a model antigen, OVA and FK506-NPs were co-incubated at room temperature for 2 h, and OVA was loaded on the surface of the core-shell structured nanoparticles through the electrostatic interaction and hydrophilic-hydrophobic interaction between OVA and the cationic lipid shell to obtain OVA@FK506-NPs. Control nanoparticles, including Blank-NPs (without FK506 and OVA) and OVA@NPs (without FK506), were prepared in a similar manner (see Figure 3 ).

[0062] The results showed that the prepared nanoparticles had a high encapsulation efficiency (EE%) of FK506 (73.0 ± 0.48%, analyzed by ultra-high performance liquid chromatography), were uniformly spherical, had a particle size of about 150 nm before antigen adsorption, carried about 50 mV positive charge on the surface, and had a typical core-shell structure (see Figure 4 ) and good storage stability (see Figure 5 ). After co-incubation of the nanoparticles with OVA for 2 h, the particle size increased by about 50 nm, and the surface potential decreased to about -10 mV. An obvious antigen adsorption layer on the surface of the nanoparticles was visible in the transmission electron microscope (TEM) image. The 10% SDS-PAGE results showed that about 20 - 30% of OVA could be loaded by the nanoparticles (see Figure 6 ). Next, we investigated the in vitro release patterns of OVA and FK506 in OVA@FK506-NPs. The results showed that most of OVA was released around 4 h and was basically completely released until 48 h; while FK506 showed a slow but constant release behavior (see Figure 7). These results indicate that our carrier has a two-stage release pattern, that is, the antigen adsorbed in the outer layer is released rapidly, while FK506 encapsulated in the PLGA core is released slowly.

[0063] Example 3 Investigation of phenotypic changes of DCs stimulated by Ag@FK506-NPs in vitro

[0064] ImDCs were treated with Blank-NPs, FK506-NPs (10 nM FK506) or OVA-NPs (10 μg / mL OVA), and the culture medium treatment group and LPS stimulation group were used as negative and positive controls, respectively. Phenotypic changes of DCs were analyzed 24 hours later (see Figure 8 ), compared with the liposome-based drug delivery system, the effects of FK506 based on core-shell structured nanoparticles on DCs maturation are roughly similar, namely: FK506 limits the expression of costimulatory molecules and MHC II molecules of DCs and promotes the expression of MHC class I molecules; while OVA can promote the expression of MHC class I molecules, it cannot effectively stimulate DCs activation (CD80 / 86, MHC II) under the delivery of this carrier. However, the use of OVA@NPs has the risk of inducing immune activation. For example, the uptake of OVA@NPs by mature DCs or DCs stimulated by inflammatory signals will lead to immune system activation, while the use of FK506-NPs alone cannot guarantee antigen-specific tolerance of DCs. At the same time, OVA@FK506-NPs can simultaneously provide DCs with costimulatory blockade and MHC class I molecule-restricted antigen presentation. Compared with FK506-NPs, OVA@FK506-NPs induce increased expression of intracellular immunosuppressive transcription factors STAT1 and STAT3 in DCs (see Figure 9 ), which may be because under this administration method, the antigen and FK506 are released sequentially, resulting in the asynchrony of the first and second signals of DCs activating T cells, promoting the tolerogenic maturation of cells.

[0065] We then investigated the interaction between DCs and T cells. The results of in vitro cell uptake experiments showed that (see Figure 10 ), DCs (BMDCs and DC2.4) under the stimulation of nanoparticles, the intracellular accumulation and uptake of the carrier continued to increase with time, reaching a peak at 24h and decreasing at 48h, indicating that there is cell metabolism or excretion of the carrier; while the uptake of antigen continued to increase within 48h, which may be related to the different release and distribution behaviors of antigen and carrier in cells. Confocal results show (see Figure 11) After 8 h of administration, some antigens dissociated from the carrier, were released into the cells and escaped lysosomal degradation, which might be beneficial for MHC class I presentation. A large amount of the carrier co-localized with lysosomes. Studies have shown that cells can exocytose the internalized nanoparticles or microparticles out of the cells [Small 9, 697-704 (2013)], which might be beneficial for DCs to transfer FK506 to neighboring T cells. Subsequently, we treated BMDCs with OVA@FK506-NPs for 8 h, washed the cells with PBS, and then co-incubated these DCs with homologous splenic lymphocytes (SPLCs) for 24 h, and took fluorescence confocal pictures. The results showed (see Figure 12 ), red fluorescence signals of the carrier were visible in the intracellular of SPLCs adjacent to DCs (especially lymphocytes forming immune synapses). On the contrary, the green fluorescence signals representing OVA remained in DCs and were not transferred to SPLCs. At the same time, under this experimental method, the fluorescence signals were hardly visible in T cells far away from DCs. These results indicate that after being internalized by DCs, the antigens adsorbed on the surface of the nanoparticles are rapidly released, and the nanocore containing FK506 might be transferred to neighboring T cells in a contact-dependent manner (TCR coupling); or after entering lysosomes, it is exocytosed out of the cells through lysosomal exocytosis and passively diffuses to neighboring T cells in a non-contact-dependent manner (i.e., diffusion / distance dependence), realizing immunosuppressive regulation of specific T cells.

[0066] Example 4 Investigation of the effect of DCs treated with Ag@FK506-NPs on the phenotype and quantity of antigen-specific T cells

[0067] The BMDCs pretreated with the above different nanoparticles for 24 h were co-incubated with SPLCs in vitro for 4 days, and then the phenotypic changes of T cells were analyzed. The results showed (see Figure 13), FK506 had little effect on the expression of the activation marker PD-1 in T cells (including CD4 T cells and CD8 T cells) (FK506-NPs vs Blank-NPs), while antigen stimulation could significantly increase the expression of PD-1 in T cells (FK506-NPs vs OVA@NPs). Notably, OVA@FK506-NPs restricted the activation effect of antigen stimulation (i.e., OVA@NPs) on CD8 T cells, but not on CD4 T cells, indicating that these nanoparticles co-loaded with antigen and FK506 mainly acted on CD8 T cells. This might be because under this treatment, DCs highly expressed MHC class I molecules, and at the same time, the carrier antigen could be partially released into the cytoplasm, thus promoting the cross-presentation of exogenous antigens by DCs [J Control Release 341, 184 - 205 (2022)]. Meanwhile, compared with OVA@NPs, the levels of IFN-γ and TNF-α in the culture systems of the FK506-NPs and OVA@FK506-NPs treatment groups were lower, while the level of IL-4 was higher (see Figure 14 ), indicating that the immune activation functions of these T cells, especially CD8 T cells, were restricted under immunosuppressive treatment. However, for lymphocytes activated by non-specific stimulation with anti-CD3 / CD28 antibodies (α-CD3 / -28Abs), the treatment with OVA@FK506-NPs showed an inhibitory effect on the expression of PD-1 in both CD4 T cells and CD8 T cells, and different treatments had less effect on resting lymphocytes (see Figure 15 ), suggesting that DCs might mediate the regulation of antigen-specific CD8 T cells.

[0068] Next, we investigated the effect of Ag@FK506-NPs on antigen-specific CD8 T cells with the help of DCs, and prepared OVA 257-264 @FK506-NPs loaded with specific OVA polypeptides in the same way. The TCR of CD8 T cells in OT-I mice could specifically recognize H-2K b (i.e., MHC class I molecule)-restricted OVA 257-264 (SIINFEKL). DCs were stimulated with FK506-NPs, OVA@FK506-NPs, and OVA 257-264 @FK506-NPs for 24 h respectively, and then the DCs were co-incubated with SPLCs derived from OT-I for 4 days. The results showed (see Figure 16 ) that compared with the untreated group and the FK506-NPs group, under the stimulation of Ag@FK506-NPs, especially in the OVA 257-264 @FK506-NPs treatment group, the expression of MHC II molecules on DCs was significantly downregulated, while H2K b / SIINFEKL was significantly upregulated, indicating that this antigen delivery method could indeed induce DCs to present antigen-specific pMHC. Further, after co-incubating these DCs with T cells, Ag@FK506-NPs, especially OVA 257-264 @FK506-NPs, significantly inhibited the total lymphocyte count, and this effect was more significant on CD8 T cells expressing specific TCRs, indicating that antigen-specific T cell clearance was indeed induced, and this clearance effect was positively correlated with the matching degree of the dominant epitopes of the delivered antigen (see Figure 17 ). In addition, in the culture system, the expression of IFN-γ in the drug administration groups containing FK506 was inhibited, while in the Ag@FK506-NPs group, there was also a significant increase in the expression of immunosuppressive cytokines IL-10 and IL-4 (see Figure 18 ), indicating that an immunosuppressive and / or tolerogenic microenvironment was successfully established.

[0069] The presence of memory cells is an important obstacle to the long-term survival of allogeneic grafts. Therefore, we also investigated the effect of nanoparticles on the memory phenotype of T cells under different T cell activation states, including SPLCs activated antigen-specifically depending on DCs, SPLCs activated non-specifically under α-CD3 / -28Abs stimulation, and untreated SPLCs. The results showed (see Figure 19 ) that the effects of different formulations on the memory phenotype of CD8 T cells were different with or without the participation of DCs. FK506-NPs increased the proportion of memory cells (central memory Tcm and effector memory Tem), which was restricted by the introduction of antigens, especially Tem. These results were more obvious under T cell activation (rather than resting) conditions, indicating that our drug delivery strategy could inhibit memory generation while restricting the effector activation of T cells, perhaps better promoting anti-transplant rejection.

[0070] Example 5 Isolation and Encapsulation of Mouse Primary Skin Antigens

[0071] Skin tissue lysate (STL) contains a large number of known or unknown antigens that can simultaneously become target targets; diverse MHC-recognizing epitope peptides can activate innate immune responses and acquired immune responses. Therefore, we first isolated and purified the full-thickness skin antigens from the backs of donor BALB / c mice and control donor C57BL / 6 mice. STL was obtained by the repeated freeze-thaw method (-80 °C to 37 °C) combined with the ultrasonic treatment method (power 30%, probe ultrasound for 2 min, work for 2 s and pause for 3 s, 3 - 4 times, treated on ice), and the soluble antigen was purified by centrifugation (centrifuged twice to remove the floating fat in the upper layer and the precipitated tissue blocks in the lower layer, and the clear middle layer was taken) (see Figure 20),The protein concentration was quantified by the BCA method and obtained as follows: the STL protein concentration of BALB / c (Bal) was 5.06 ± 0.36 μg / μL; the STL protein concentration of C57BL / 6 (C57) was 4.59 ± 0.40 μg / μL (see Figure 21 ). The distribution of antigens in STL according to molecular weight and the ability of nanoparticles to load STL antigens were detected by SDS-PAGE whole-cell protein electrophoresis. The results showed that proteins with a wide molecular weight distribution of 10 - 100 kD were displayed after electrophoresis of STL at different dilution multiples (see Figure 22 ), and our carriers NPs and FK506-NPs (i.e., F-NPs) could load STL. The encapsulation efficiency for Bal STL was about 35 - 40%, and the encapsulation efficiency for C57 STL was 15 - 25% (the different encapsulation efficiencies for skin antigens of different mouse species may be related to the different compositions of skin tissues) (see Figure 23 ), which could be used for subsequent experiments.

[0072] Example 6 Investigation of the inhibition of allogeneic skin transplantation rejection in mice by Ag@FK506-NPs-treated DCs

[0073] Mouse xenogeneic skin transplantation is non-vascularized, and the humoral immune response can be disregarded, which is often used to observe the acute rejection reaction mediated by T cells after surgery. We used BALB / c as the donor and C57BL / 6 as the recipient to construct a full-thickness skin transplantation model on the back of mice. The recipient mice were administered drugs starting 2 days before transplantation, once every 4 days, for a total of 4 times. Saline (A#), 10^6 Bal@FK506-NPs DCs (B#, 80.402 pg FK506 per mouse), or Free FK506 (C#, 2 μg FK506 per mouse) were subcutaneously injected near the bilateral inguinal lymph nodes (LNs). The mice were sacrificed on the 13th day after surgery for analysis (see Figure 24 ).

[0074] Although the dosage of FK506 in the DCs-based administration group (B#) was only 1 / 25000 of that in the free drug group (C#), this treatment group showed better anti-transplantation rejection effects, with lower transplantation rejection scores during the experiment and higher graft survival rates on the 13th day after transplantation (40% for A#, 100% for B#, and 70% for C#) (see Figure 25 ), and the histopathological conditions of the transplanted skin flaps were better (the least inflammatory cell infiltration and subcutaneous bleeding) (see Figure 26 ). In addition, B# had higher biosafety. CNIs still have acute and chronic nephrotoxicity clinically. The experimental results showed that compared with the saline group (A#) and the adoptive DCs group (B#), inflammatory cell infiltration was visible in the kidneys of the free drug group (C#) (see Figure 26), while the body weight of the mice was relatively high (see Figure 27 ), which may be related to metabolic abnormalities caused by impaired renal function. In addition, in B# and C#, the levels of cytokines IL-2 and IFN-γ in the serum and spleen were both inhibited, indicating that systemic inflammation was alleviated; on the other hand, the DCs-based drug administration group (B#) showed the best ability to promote the expression of IL-4 and IL-10, indicating the promotion of the establishment of systemic immunosuppression (see Figure 28 ).

[0075] Next, we examined the immune cells in the secondary lymphoid organs of the recipient mice at the end of the experiment, namely, the spleen and the draining lymph nodes (dLNs). The results showed (see Figure 29 ) that different treatments had no significant effect on the numbers of CD8 T cells, total CD4 T cells, and DCs in the spleen; while in the dLNs, the experimental groups, especially group B#, significantly inhibited CD8 T cells, and there was no difference in the ability of group B# and C# to inhibit total CD4 T cells, indicating that using DCs as an indirect carrier of drugs might improve the bioavailability of drugs through the natural dLN tropism of DCs and their close communication with T cells, and at the same time this drug delivery method had a greater impact on CD8 T cells. In addition, in the spleen and dLNs, the proportion of CD4 Tregs in group B# was higher, which could further promote the transplantation effect by inducing a tolerogenic microenvironment.

[0076] Example 7 Investigation of the Inhibition of Antigen-Specific T Cells by Ag@FK506-NPs-Treated DCs in Vivo

[0077] In another group of experiments, the transplanted recipient mice were restimulated with OVA on the 13th day after surgery (100 μg OVA per mouse, subcutaneously immunized near the inguinal lymph nodes), and the peripheral blood of the mice was collected 24 h later, and the proportion of OVA-specific CD8 T cells was analyzed by flow cytometry. The results showed (see Figure 30 ) that free FK506 (C#) significantly inhibited OVA-specific CD8 T cells, perhaps because of the non-selective pharmacological inhibition of free drugs on broad-spectrum T cells, while Bal@FK506-NPs DCs (B#) could retain the immune response of the body to OVA restimulation, which might be attributed to its antigen-specific T cell clearance ability. Generally speaking, our treatment strategy can use DCs as an indirect targeted drug delivery system to achieve the clearance of antigen-specific T cells, especially antigen-specific CD8 T cells, thereby avoiding the toxic side effects caused by damage to broad-spectrum T cell clones.

Claims

1. A method for constructing a core-shell structured nanoparticle for targeted elimination of antigen-specific T cells, characterized in that, It is achieved through the following solutions: (1)Construction of the immunosuppressant-loaded nanoparticle core: Using poly (lactic-co-glycolic acid) nanoparticles as the hydrophobic core to encapsulate the immunosuppressant tacrolimus, where the drug-lipid mass ratio of tacrolimus to poly (lactic-co-glycolic acid) is 1:10 - 1:5000; (2)Coating the outer layer of the immunosuppressant-loaded nanoparticle core with a cationic lipid membrane: Coating the surface of the immunosuppressant-loaded nanoparticle core in step (1) with a lipid membrane containing cationic lipids, where the cationic lipids account for 10% - 90% of the total lipid mass; The lipid membrane is selected from cationic lipids, co-lipids, structural lipids, and PEGylated lipids. The cationic lipid is selected as (2,3-dioleoyl-propyl) trimethyl ammonium chloride, the co-lipid is selected as dioleoyl phosphatidylethanolamine, the structural lipid is selected as cholesterol, and the PEGylated lipid is selected as 1,2-diacyl-sn-glycero-3-phosphoethanolamine; (3)Preparation of the core-shell structured nanoparticles co-loaded with antigen and immunosuppressant: The core-shell structured nanoparticles with the immunosuppressant loaded in the core and coated with a positively charged lipid membrane on the surface are prepared by a one-step method or a multi-step method, and then the antigen is loaded on the particle surface to obtain the core-shell structured nanoparticles co-loaded with antigen and immunosuppressant.

2. The construction method according to claim 1, characterized in that, In step (1), tacrolimus can be replaced with cyclosporine, rapamycin, glucocorticoid, or mycophenolate mofetil.

3. The construction method according to claim 1, characterized in that, In step (3), the preparation of the core-shell structured nanoparticles loaded with immunosuppressant is completed in one step by microfluidic technology, or completed in two steps by nanoprecipitation plus lipid membrane extrusion coating.

4. The construction method according to claim 1, characterized in that, The antigen loaded on the outer layer of the core-shell structured nanoparticles in step (3) is antigen protein, antigen polypeptide, or immune complex containing antigen.

5. The construction method according to claim 1, characterized in that, In step (3), the loading of the antigen on the outer layer of the core-shell structured nanoparticles is achieved through the electrostatic interaction and van der Waals force between the positively charged lipid membrane and the negatively charged antigen, or through the structural modification of the antigen to promote the bonding between the antigen and the outer lipid membrane.

6. The construction method according to claim 1, characterized in that, In step (3), the core-shell structured nanoparticles co-loaded with antigen and immunosuppressant have a programmed drug release mode of rapid release of the outer antigen and delayed sustained release of the inner immunosuppressant.

7. Application of the core-shell structured nanoparticle prepared by the method according to claim 1 in the preparation of drugs for alleviating allogeneic organ transplantation and autoimmune diseases, characterized in that, The core-shell structured nanoparticles act directly on antigen-presenting cells and indirectly act on T cells through APCs to prevent or treat diseases or pathological conditions related to the activation of antigen-specific T cells.

8. The application according to claim 7, characterized in that, The disease is caused by the damage of the body caused by the abnormal activation of T cells, where the antigen epitopes related to the disease have been recognized and approved, and the clinical diagnosis is type I diabetes, multiple sclerosis, rheumatoid arthritis, or inflammatory vasculitis.

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