Cell membrane modified nano composite material as well as preparation method and application thereof

Abstract

The invention discloses a cell membrane modified nano composite material as well as a preparation method and application thereof. The nanocomposite sequentially comprises a membrane structure outer layer, a piezoelectric material middle layer and a magnetostrictive material core structure from outside to inside, and the outer surface of the membrane structure contains protein molecules. Under the stimulation of an external magnetic field, the nano composite material disclosed by the invention can regulate Th1 / Th2 balance, promote Th2 cell polarization and enhance T cell proliferation, so that T cell mediated autoimmune response is relieved, host immune homeostasis is maintained, and the nano composite material has a wide application prospect in treatment of arthritis.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a cell membrane modified nano composite material and a preparation method and application thereof. Background Art

[0002] T helper cells can polarize into Th1 and Th2 subsets under various conditions. Unlike the pathogenic role of the Th1 subset in chronic autoimmune diseases such as diabetes, multiple sclerosis, and rheumatoid arthritis, Th2 cells are protective. Therefore, a successful deviation from a Th1-dominant response to a Th2-dominant response may have clinical benefits for patients with autoimmune diseases. However, there is no particularly effective therapeutic strategy to achieve a stable conversion of Th1 to Th2 in vivo.

[0003] Magnetic fields are essential factors for many physiological processes in living organisms. + How T cells respond to magnetic fields and whether external magnetic fields can modulate T cell-mediated autoimmune diseases remain largely unknown.

[0004] The information in the background technology is only for illustrating the general background of the present invention and should not be regarded as admitting or suggesting in any form that such information constitutes the prior art known to a person skilled in the art. Summary of the invention

[0005] In order to solve at least some of the technical problems in the above-mentioned prior art, the present invention constructs a nanocomposite material which includes, from the outside to the inside, an outer layer of a membrane structure, an intermediate layer of a piezoelectric material, and a core structure of a magnetostrictive material, and the outer surface of the membrane structure contains protein molecules targeting T cells. The nanocomposite material of the present invention can adjust the Th1 / Th2 balance and reduce the autoimmune reaction mediated by T cells, so it can be used to treat arthritis. Specifically, the present invention includes the following contents.

[0006] The first aspect of the present invention provides the use of a nanocomposite material in the preparation of a drug for treating arthritis, wherein the nanocomposite material comprises, from the outside to the inside, an outer layer of a membrane structure, an intermediate layer of a piezoelectric material, and a core structure of a magnetostrictive material, and the outer surface of the membrane structure contains protein molecules.

[0007] In certain embodiments, according to the use of the nanocomposite material of the present invention in the preparation of a drug for treating arthritis, the membrane structure is derived from a biological membrane or an artificially synthesized membrane structure.

[0008] In certain embodiments, according to the use of the nanocomposite material of the present invention in the preparation of a drug for treating arthritis, the protein molecule is a binding molecule capable of binding to the surface of T cells.

[0009] In certain embodiments, according to the use of the nanocomposite material of the present invention in the preparation of a drug for treating arthritis, the T cell surface comprises at least one of the following antigens: CD1, CD2, CD3, CD5, CD7, CD8, CD16, CD25, CD26, CD27, CD28, CD30, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD126, CD150, CD153, CD154, CD161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX 40. GITR, LAG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6 and CCR7.

[0010] In certain embodiments, according to the use of the nanocomposite material of the present invention in the preparation of a drug for treating arthritis, the protein molecule is a protein that can target CD4 + MHC II complex of T cells.

[0011] In certain embodiments, according to the use of the nanocomposite material of the present invention in the preparation of a drug for treating arthritis, the membrane structure is a cell membrane separated from an immune cell.

[0012] In certain embodiments, according to the use of the nanocomposite material of the present invention in the preparation of a medicament for treating arthritis, the cell membrane comprises a cell membrane isolated from a dendritic cell, a B lymphocyte and / or a macrophage.

[0013] In certain embodiments, according to the use of the nanocomposite material described in the present invention in the preparation of a drug for treating arthritis, the piezoelectric material is selected from at least one of ferrite, niolate, titanate, silicate and aluminate materials, and / or selected from at least one of polyvinylidene fluoride, polyesters, polymethyl methacrylate, nylon, polyvinyl chloride, poly-L-lactide, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), vinylidene fluoride / trifluoroethylene copolymer, vinylidene fluoride / tetrafluoroethylene copolymer, polyvinylidene fluoride-hexafluoropropylene and polydimethylsiloxane materials; the magnetostrictive material is selected from ferrite materials.

[0014] A second aspect of the present invention provides a method for preparing a nanocomposite material, comprising:

[0015] (1) preparing a magnetostrictive piezoelectric nanocomposite material, wherein the magnetostrictive piezoelectric nanocomposite material comprises a piezoelectric material intermediate layer and a magnetostrictive material core structure;

[0016] (2) The magnetostrictive piezoelectric nanocomposite material is encapsulated in a film material, wherein the outer surface of the film material contains protein molecules.

[0017] The third aspect of the present invention provides a nanocomposite material obtained by the preparation method described in the second aspect of the present invention.

[0018] The fourth aspect of the present invention provides a method for regulating Th1 / Th2 cell balance in vitro, comprising the step of contacting the nanocomposite material of the present invention with isolated cells in the presence of a magnetic field of 0.01-300 mT.

[0019] The nanocomposite material of the present invention can adjust the Th1 / Th2 balance, promote Th2 cell polarization and enhance T cell proliferation under the stimulation of an external magnetic field, thereby alleviating T cell-mediated autoimmune reactions and maintaining host immune homeostasis, and can be used to treat arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The preparation and characterization of DC@CFO / BFO nanocomposites are shown, where a is a representative high-resolution transmission electron microscopy image of DC@CFO / BFO nanocomposites; b and c are free radicals ·OH and ·O2 generated by CFO / BFO nanoparticles under ultrasound, respectively. - Electron paramagnetic resonance (EPR) images of CFO / BFO nanoparticles; d and e are free radicals ·OH and ·O2 generated by CFO / BFO nanoparticles, respectively. - f is the electron paramagnetic resonance image of the DC membrane; f is the flow cytometric analysis of the surface markers of bone marrow-derived dendritic cells (BMDC); g and h are the particle sizes of CFO / BFO nanoparticles and DC@CFO / BFO nanocomposites; i is the zeta potential of CFO / BFO nanoparticles and DC@CFO / BFO nanocomposites; j is the glycoprotein levels on the DC membrane, CFO / BFO nanoparticles and DC@CFO / BFO nanocomposites measured by ELISA.

[0021] Figure 2 The biosafety evaluation of DC@CFO / BFO nanocomposites is shown, where ac is the separation of initial CD4 +T cells were incubated with DC@CFO / BFO nanocomposites for 24 h in the presence or absence of a magnetic field, and the viability of T cells was determined by CCK-8 at an absorbance wavelength of 450 nm; d and e were intravenously injected with FITC-labeled DC@CFO / BFO nanocomposites (10 mg / kg) into mice, and the in vivo distribution of DC@CFO / BFO was evaluated by bioluminescence imaging; fu was C57BL / 6 mice injected intravenously with 50 mg / kg of DC@CFO / BFO nanocomposites every day and stimulated with a magnetic field (1.5 mT) for 30 minutes every day. Seven days after injection, the mouse serum was collected and a blood routine test was performed. RBC denotes red blood cells, WBC denotes white blood cells, PLT denotes platelets, HGB denotes hemoglobin, MCV denotes mean corpuscular volume, and HCT denotes hematocrit.

[0022] Figure 3 The DC@CFO / BFO treated and magnetic field stimulated T cell transcriptional analysis is shown, where a is the CD4 + PCA analysis of T cell transcripts; b is CD4 + Heat map of gene expression of T cells; c is the volcano plot analysis of pairwise comparison of transcriptional analysis between the DC@CFO / BFO+M group (DC@CFO / BFO and magnetic field treatment) and the DC@CFO / BFO group.

[0023] Figure 4 The results show that DC@CFO / BFO nanocomposites promote Th2 cell polarization and proliferation under external magnetic field stimulation, where ae is a naive T cell (CD4 + CD25 - CD62L hi CD44 lo ) were cultured under various polarization conditions and in the presence or absence of DC@CFO / BFO nanocomposites and magnetic field treatment, and then stimulated with PMA and ionomycin for another 5 h; f is the flow cytometric analysis of IFNγ, IL4, IL17A, FOXP3 and CD25 staining in T cells (to assess differentiation efficiency) after activation of CD4 T cells with anti-CD3 / CD28 antibodies. + T cells and induced them to differentiate into Th2 cells, and then these cells were treated with DC@CFO / BFO nanocomposites in the presence or absence of magnetic field stimulation. Under Th2 differentiation conditions, CD4 + GSEA analysis of genes expressed in T cells, ES represents enrichment score, NES represents normalized enrichment score; g represents activation of CD4 with antibodies against CD3 / CD28+ T cells and induced them to differentiate into Th2 cells, and then treated these cells with DC@CFO / BFO nanocomposites in the presence or absence of magnetic field stimulation, showing that in the presence or absence of DC@CFO / BFO nanocomposites and magnetic field stimulation, CD4 + Heat map of genes expressed in T cells related to “T helper 2 cell differentiation”; h and i are activation of CD4 with antibodies against CD3 / CD28, respectively. + T cells and induced them to differentiate into Th2 cells, and then these cells were treated with DC@CFO / BFO nanocomposites in the presence or absence of magnetic field stimulation to perform CD4 + Flow cytometric analysis of CFSE dilution staining and Ki-67 expression in T cells. MFI represents mean fluorescence intensity.

[0024] Figure 5 The magnetoelectric nanocomposite improves arthritis under external magnetic field stimulation, wherein, a is a schematic diagram of DC@CFO / BFO nanocomposite treatment in CIA mouse model, DBA / 1 mice were intradermally injected with bovine type II collagen (CII) emulsified in Freund's complete adjuvant or Freund's incomplete adjuvant on day 0 and day 21, 10 mg / kg of DC@CFO / BFO nanocomposite was intravenously injected into the mice, and a 1.5 mT magnetic field was applied for 30 minutes at the designated time points after nanocomposite injection; b is the arthritis score of arthritic animals evaluated every other day after the onset of the disease, with a maximum clinical score of 16 points; c is a representative image of ankle joints from different treatment groups, with a scale bar of 200 μm; d is a measurement of paw thickness of arthritic mice from different treatment groups; e is a micro-CT analysis of bone mineral density (BMD) of ankle joints from different treatment groups; f is a measurement of proteoglycan loss in ankle joints based on safranin-O / fast green staining images; g and h are flow cytometric analysis of CD4 in the spleen of mice from different treatment groups, respectively. + IFNγ + Cells and CD4 + IL4 + The proportion of cells; i and j are flow cytometric analysis of CD4 + IFNγ + Cells and CD4 + IL4 + k and l were flow cytometric analysis of CD4 + IFNγ + Cells and CD4 + IL4 +m is to obtain mouse serum and measure anti-type 2 collagen (anti-CII) specific antibodies by ELISA; n is to obtain mouse serum and evaluate inflammatory factors.

[0025] Figure 6 shows that CD4 + Flow cytometric analysis of T cell subsets, where a is the CD4 + Flow cytometric analysis of T cell ratio; b CD4 + Flow cytometric analysis of T cell ratio; c is CD4 + Flow cytometric analysis of T cell proportions.

[0026] Figure 7 The T cell subset analysis and serum inflammation assessment of arthritis mice are shown, where a and b are CD4 + IL17A + Cells and CD4 + FOXP3 + c and d were flow cytometric analysis of the proportion of CD4 + IL17A + Cells and CD4 + FOXP3 + e and f were flow cytometry analysis of CD4 + IL17A + Cells and CD4 + FOXP3 + g was to obtain mouse serum and evaluate inflammatory factors.

[0027] Figure 8 Figure 3 shows the inhibition of arthritis in mice by Th2 cells treated with magnetoelectric nanocomposites, where a is a schematic diagram of the administration of Th2 cells pretreated with DC@CFO / BFO nanocomposites in a CIA mouse model. Bovine type II collagen (CII) emulsified in Freund's complete adjuvant or Freund's incomplete adjuvant was intradermally injected into DBA / 1 mice on days 0 and 21, and CD4 was activated with antibodies against CD3 / CD28. +T cells were induced to differentiate into Th2 cells, treated with DC@CFO / BFO nanocomposites (10 μg / mL) and stimulated with a magnetic field (1.5 mT) for 30 min, and Th2 cells pretreated with DC@CFO / BFO nanocomposites were intravenously injected into arthritic mice; b, the arthritis scores of arthritic animals were evaluated every other day after the onset of the disease, and the maximum clinical score for each animal was 16 points; c, representative images of ankle joints from different treatment groups, with a scale bar of 200 μm; d, measurement of paw thickness of arthritic mice from different treatment groups; e, micro-CT analysis of bone mineral density (BMD) of ankle joints from different treatment groups; f, proteoglycan loss in ankle joints; g, obtaining mouse serum and quantifying anti-type 2 collagen (anti-CII)-specific antibodies by ELISA. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0031] use

[0032] One aspect of the present invention provides the use of a nanocomposite material in the preparation of a drug for treating arthritis, wherein the nanocomposite material comprises, from the outside to the inside, an outer layer of a membrane structure, an intermediate layer of a piezoelectric material, and a core structure of a magnetostrictive material, wherein the outer surface of the membrane structure contains protein molecules.

[0033] The nanocomposite material of the present invention can promote Th2 cell polarization and enhance T cell proliferation under external magnetic field stimulation, so it can reduce T cell-mediated autoimmune response, thereby maintaining host immune homeostasis. It should be understood that Th2 cells pretreated with nanocomposite materials or Th2 cells containing nanoparticles (cobalt ferrite-bismuth ferrite) are also within the protection scope of the present invention. Based on this, the present invention further provides the use of Th2 cells pretreated with nanocomposite materials or Th2 cells containing cobalt ferrite-bismuth ferrite in the preparation of a drug for treating arthritis.

[0034] In the present invention, arthritis includes but is not limited to degenerative arthritis (such as but not limited to osteoarthritis, etc.), autoimmune arthritis (such as but not limited to rheumatoid arthritis, rheumatoid arthritis, etc.), infectious arthritis (such as but not limited to Neisseria gonococcal arthritis, non-gonococcal arthritis, etc.), etc. In a preferred embodiment, the arthritis is autoimmune arthritis, especially arthritis caused by T cells.

[0035] In the present invention, the source of the membrane structure is not particularly limited, as long as its surface contains protein molecules that can bind to the surface of T cells. Examples of membrane structures include, but are not limited to, naturally occurring biological membranes or artificially synthesized membrane structures. In a preferred embodiment, the biological membrane is a cell membrane separated from immune cells, examples of which include, but are not limited to, a cell membrane separated from dendritic cells, B lymphocytes or macrophages. In a preferred embodiment, the biological membrane is a cell membrane of dendritic cells.

[0036] In the present invention, the T cell surface contains at least one of the following antigens: CD1, CD2, CD3, CD5, CD7, CD8, CD16, CD25, CD26, CD27, CD28, CD30, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD126, CD150, CD153, CD154, CD161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, L AG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6 and CCR7.

[0037] Those skilled in the art are familiar with how to make the outer surface of the cell membrane contain or express a protein that can target and bind to CD4 +In a preferred embodiment, the protein molecule is a protein molecule that can target CD4 + MHC II complex of T cells.

[0038] In the present invention, the piezoelectric material is selected from at least one of ferrite, niolate, titanate, silicate and aluminate materials, and / or selected from at least one of polyvinylidene fluoride, polyester, polymethyl methacrylate, nylon, polyvinyl chloride, poly-L-lactide, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), vinylidene fluoride / trifluoroethylene copolymer, vinylidene fluoride / tetrafluoroethylene copolymer, polyvinylidene fluoride-hexafluoropropylene and polydimethylsiloxane materials. In a preferred embodiment, the piezoelectric material intermediate layer is bismuth ferrite.

[0039] In the present invention, the magnetostrictive material is selected from ferrite materials, and the ferrite materials have the following structure: N a Fe 1-a O b , N includes at least one of Co, Mn, Zn and Ni, a is a number ranging from 0.25 to 0.75, and b is 3. In a preferred embodiment, the magnetostrictive material is cobalt ferrite.

[0040] It is understandable that the magnetostrictive piezoelectric nanocomposite material of the present invention may or may not contain the element doped therein. When containing the element doped therein, the element doped therein includes but is not limited to at least one of carbon, nitrogen, phosphorus, sulfur, silicon, aluminum, iron, titanium, nickel, manganese, copper, silver and zinc, thereby improving the piezoelectric property and / or magnetostrictive property of the magnetostrictive piezoelectric nanocomposite material.

[0041] In the present invention, the size of the nanocomposite material is 100-500nm, preferably 100-450nm, also preferably 100-400nm, further preferably 100-350nm, more preferably 100-300nm, more preferably 100-250nm, for example 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250nm.

[0042] In the present invention, external magnetic field stimulation refers to treating the nanocomposite particles under the condition of a magnetic field of 0.01-300 mT. The magnetic field strength is also preferably 0.1-200 mT, further preferably 0.5-100 mT, more preferably 0.5-50 mT, and most preferably 0.5-10 mT, such as 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 mT. In a preferred embodiment, the magnetic field is applied at a frequency of once a day, 15-30 min each time, for 3 consecutive days at the above magnetic field strength.

[0043] Preparation method

[0044] One aspect of the present invention provides a method for preparing a nanocomposite material, comprising:

[0045] (1) preparing a magnetostrictive piezoelectric nanocomposite material, wherein the magnetostrictive piezoelectric nanocomposite material comprises a piezoelectric material intermediate layer and a magnetostrictive material core structure;

[0046] (2) The magnetostrictive piezoelectric nanocomposite material is encapsulated in a film material, wherein the outer surface of the film material contains protein molecules.

[0047] The step (1) of the present invention is to prepare a core structure of a magnetostrictive material and an intermediate layer of a piezoelectric material.

[0048] In a specific embodiment, the preparation of the core structure of the magnetostrictive material in step (1) of the present invention comprises: dissolving FeCl3, CoCl2 and CTAB in deionized water, wherein the molar ratio of FeCl3, CoCl2 and CTAB is (2-6):1:(3-9), preferably (2-5):1:(3-8), and further preferably (2-4):1:(3-6), for example, 2:1:3, 2:1:4, 2:1:5, 2:1:6, 3:1:3, 3:1:4, 3:1:5, 3:1:6, 4:1:3, 4:1:4, 4:1:5, 4:1:6, and then, adding NaOH to the above solution under stirring and ultrasonic dispersion, wherein the molar ratio of FeCl3 to NaOH is 1:(20-100), preferably 1:(30-90), more preferably 1:(40-80), for example 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, and then the resulting solution is transferred to a sealed hydrothermal reactor and heated at 150-250°C, preferably 160-240°C, more preferably 170-230°C, for example 170°C, 180°C, 190°C, 200°C, 210°C, 220°C, 230°C for 12-36h, preferably 14-34h, also preferably 16-32h, more preferably 18-30h, for example 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30h. The obtained black powder is then washed with deionized water and ethanol, and then dried overnight at 80-110°C, preferably 85-105°C, and also preferably 90-100°C, such as 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, and 100°C to obtain the core structure of the magnetostrictive material, i.e., cobalt ferrite (CFO) nanoparticles.

[0049] In a preferred embodiment, the preparation of the piezoelectric material intermediate layer of step (1) of the present invention comprises: dissolving Bi(NO3)·5H2O and Fe(NO3)·9H2O in ethylene glycol to prepare a bismuth ferrite (BFO) precursor, wherein the molar ratio of Bi(NO3)·5H2O to Fe(NO3)·9H2O is (1-5):1, for example, 1:1, 2:1, 3:1, 4:1, 5:1, and then drying CF The CFO nanoparticles are dispersed in the BFO precursor solution and ultrasonically treated for 1-5 h (e.g., 1, 2, 3, 4, 5 h), wherein the molar ratio of the CFO nanoparticles to the BFO precursor is (1-5):1, e.g., 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, and then the solution is dried for 6-20 h, preferably 8-18 h, and also preferably 10-16 h, e.g., 10, 11 , 12, 13, 14, 15, 16h, and then the dried powder is heated to 500-700°C, preferably 520-680°C, more preferably 540-660°C, for example 540°C, 550°C, 560°C, 570°C, 580°C, 590°C, 600°C, 610°C at a heating rate of 5-15°C / min, preferably 7-13°C / min, for example 7, 8, 9, 10, 11, 12, 13°C / min. , 620°C, 630°C, 640°C, 650°C, and 660°C for 1-5h, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5h. Finally, the annealed powder is ultrasonically treated for 15-45min, preferably 20-40min, and also preferably 25-35min, for example, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35min, and filtered through a filter membrane.

[0050] In certain embodiments, the membrane material described in step (2) of the present invention is an artificially synthesized membrane structure, and the artificially synthesized membrane structure can be prepared by methods known in the art, without particular limitation, examples of which include but are not limited to self-assembly method, template method, charge induction method, etc.

[0051] In certain embodiments, the membrane material in step (2) of the present invention is a cell membrane, and the cell membrane is obtained by cell culture, cell separation, or cell lysis.

[0052] In a specific embodiment, the method for preparing the cell membrane comprises: first isolating bone marrow from mouse femur and tibia, and inducing dendritic cell (DC) differentiation using 10-30 ng / ml, preferably 12-28 ng / ml, and preferably 14-26 ng / ml, such as 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 ng / ml recombinant granulocyte-macrophage colony stimulating factor (GM-CSF) and 1-20 ng / ml, preferably 2-18 ng / ml, and preferably 4-16 ng / ml, and more preferably 6-14 ng / ml, such as 6, 7, 8, 9, 10, 11, 12, 13, 14 ng / ml recombinant interleukin-4, and then sorting DC (CD11c + MHC II + The cells are then lysed, and the lysate is ultrasonicated for 1-10 min, preferably 2-9 min, and more preferably 3-8 min, such as 3, 4, 5, 6, 7, 8 min, to collect the cell membrane. The lysis method is not particularly limited, and the sorted dendritic cells can be frozen and thawed in liquid nitrogen for several cycles (preferably 1-5 cycles, such as 1, 2, 3, 4, 5 cycles) to achieve the lysis.

[0053] In a preferred embodiment, step (2) of the present invention comprises continuously passing the membrane material through a 300-500nm, preferably 320-480nm, also preferably 340-460nm, more preferably 360-440nm, for example 360, 370, 380, 390, 400, 410, 420, 430, 440nm porous membrane (for example, a polycarbonate porous membrane), and then extruding the membrane material through a 100-300nm, preferably 120-280nm, also preferably 140-260nm, more preferably 160-240nm, for example 160, 170, 180, 190, 200, 210, 220, 230, 240nm porous membrane to the magnetostrictive piezoelectric nanocomposite material obtained in step (1).

[0054] Methods for regulating Th1 / Th2 cell balance in vitro

[0055] In one aspect of the present invention, a method for regulating the balance of Th1 / Th2 cells in vitro is provided, which comprises the step of contacting the nanocomposite material of the present invention with isolated cells in the presence of a magnetic field of 0.01-300 mT. The isolated cells are T cells, in particular CD4 + T cells.

[0056] The present invention further provides a method for promoting T cell differentiation into Th2 cell subsets in vitro, comprising the step of contacting the nanocomposite material of the present invention with isolated cells in the presence of a magnetic field of 0.01-300 mT.

[0057] Example 1

[0058] The preparation and performance analysis of DC@CFO / BFO nanocomposites are exemplarily shown below.

[0059] 1. Experimental Materials

[0060] DBA / 1 mice were purchased from Weitong Lihua Laboratory Animal Technology Co., Ltd. All animals were housed and maintained under specific pathogen-free conditions. All animal experiments were performed according to protocols approved by the Ethics Committee of Peking University Health Science Center.

[0061] 2. Preparation method

[0062] 1. Preparation of CFO / BFO

[0063] (1) Preparation of core-structured CFO nanoparticles: 0.092 M FeCl3, 0.046 M CoCl2, and 0.14 M CTAB were dissolved in deionized water. Then, 6 M NaOH was added to the above solution under thorough mechanical stirring and ultrasonic dispersion, and the resulting solution was transferred to a sealed polytetrafluoroethylene hydrothermal reactor and heated at 200 ° C for 24 hours. The black powder was then washed with deionized water and ethanol and then dried at 95 ° C overnight.

[0064] (2) Preparation of core-shell CFO / BFO nanoparticles: 0.011 M Bi(NO3)·5H2O and 0.01 M Fe(NO3)·9H2O were dissolved in ethylene glycol to prepare a BFO precursor, and then 0.15 g of dried CFO nanoparticles were dispersed into 100 mL of the BFO precursor solution and ultrasonically treated for 3 hours. The solution was then dried overnight, and the dried powder was subsequently heated to 600°C at a heating rate of 10°C / min and annealed for 2.5 hours. Finally, the annealed powder was ultrasonically treated for 30 minutes and filtered through a 0.22 μm filter membrane to obtain magnetoelectric core-shell CFO / BFO nanoparticles.

[0065] 2. Preparation of DC@CFO / BFO nanocomposites

[0066] Preparation of DC membrane modified CFO / BFO nanocomposites: First, bone marrow from mouse femur and tibia was isolated, and DC differentiation was induced using 20 ng / ml recombinant GM-CSF and 10 ng / ml recombinant IL4. DCs (CD11c + MHC II +cells), lysed by freezing and thawing in liquid nitrogen for 3 cycles, and the lysate was sonicated for 5 min by a bath sonicator to collect the cell membranes, which were then continuously extruded through 400 nm and then 200 nm polycarbonate porous membranes onto the magnetoelectric core-shell CFO / BFO nanoparticles to obtain the DC@CFO / BFO nanocomposite.

[0067] 3. Performance Analysis Method

[0068] 1. Characterization of CFO / BFO Nanoparticles and DC@CFO / BFO Nanocomposites

[0069] The surface morphology of CFO / BFO nanoparticles was characterized by scanning electron microscopy (SEM). The structures of core-shell CFO / BFO nanoparticles and DC film-coated CFO / BFO composites were observed using high-resolution transmission electron microscopy (TEM). The composition of CFO / BFO nanoparticles was determined using energy dispersive X-ray spectroscopy (EDS) spectroscopy or X-ray diffraction spectroscopy with TEM. Piezoelectric response force microscopy (PFM) measurements were recorded by a commercial atomic force microscope equipped with a ferroelectric testing system. The measurements were performed with a vibrating sample magnetometer (VSM). The nanocomposites were placed in ddH2O or DMSO with or without magnetic field stimulation or ultrasonic treatment for ·OH and ·O2 - Detection. 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was added to the solution and the reactive species generated by the nanocomposite were detected by EPR technique. The size and surface potential of the nanocomposite were measured by the instrument in an aqueous environment at room temperature.

[0070] 2. Immunoblotting Analysis

[0071] Cells were lysed with RIPA lysis buffer supplemented with a protease inhibitor cocktail and subjected to SDS-PAGE. The antibodies used in this example are as follows: anti-CD11b (abcam, ab133357, 1: 1000), anti-β-catenin (abcam, ab32572, 1: 5000), anti-HDAC1 (Santa Cruz Biotechnology, sc-81598, 1: 1000), anti-puromycin (Merck-millipore, MABE341, 1: 1000) and anti-GAPDH (RayAntibody, RM2002, 1: 5000).

[0072] 3. Sample Preparation and Flow Cytometry Analysis

[0073] Isolate immune cells from the spleen or lymph nodes by grinding the tissue in PBS containing FBS and then filtering through a cell strainer.

[0074] To analyze immune cells in the joints, the ankle was cut from above the heel to the middle of the paw, minced and incubated with occasional mixing in DMEM medium containing collagenase A. The dissociated cells were then washed and filtered through a cell strainer.

[0075] To analyze the surface proteins of DC@CFO / BFO nanocomposites, DC@CFO / BFO nanocomposites were incubated with specific antibodies at room temperature and analyzed by flow cytometry.

[0076] To detect the cellular uptake of the materials, CFO / BFO nanoparticles and DC@CFO / BFO nanocomposites were mixed with the same mass of FITC aqueous solution and then stirred. Magnetic separation was performed and washed three times with deionized water to remove excess FITC. The FITC-coupled materials were incubated with mouse naive T cells or spleen cells, and then FITC was detected by flow cytometry. + cell.

[0077] To detect T cell proliferation, isolated T cells were stained with CFSE at room temperature, and then induced to activate and proliferate, and then collected and subsequently analyzed by flow cytometry.

[0078] To analyze cell surface marker expression (CD45, CD4, CD8, CD25, B220, CD11b, Ly6G, CD11c, MHC II, F4 / 80), cells were incubated with specific antibodies at room temperature. To stain intracellular markers (IFNγ, IL4, IL17A), cells were treated with a cocktail of protein transport inhibitors, then fixed and permeabilized, followed by staining with specific antibodies. To detect nuclear protein expression (Ki-67, FOXP3), cells were fixed and permeabilized, followed by staining with specific antibodies. The antibodies used included: anti-CD45 (eBioscience, 30-F11), anti-CD4 (BioLegend, GK1.5), anti-CD8 (eBioscience, 53-6.7), anti-CD25 (BioLegend, 3C7), anti-B220 (BioLegend, RA3-6B2), anti-CD11b (BioLegend, M1 / 70), anti-Ly6G (BioLegend, RB6-8C5), anti-CD11c (BioLegend, N418), anti-F4 / 80 (BioLegend, BM8), anti-MHC II (BioLegend, IA / IE)(M5 / 114.15.2), anti-IFNγ (BioLegend, XMG1.2), anti-IL4 (eBioscience, 11B11), anti-IL17A (eBioscience, eBio17B7), anti-Ki-67 (BioLegend, 16A8) and anti-FOXP3 (eBioscience, FJK-16s).

[0079] 4. In vivo imaging of DC@CFO / BFO nanocomposites

[0080] To study the in vivo distribution of the nanocomposite, FITC-labeled DC@CFO / BFO nanocomposite (10 mg / kg) was intravenously injected into C57BL / 6 mice. The mouse heart, liver, kidney, lung, and spleen were isolated at the indicated time points, and then bioluminescence imaging was performed using an instrument. Images were analyzed using software.

[0081] 5. Biosafety Assessment

[0082] For in vitro cytotoxicity assays, naive mouse CD4 + T cells were exposed to a series of concentrations of the nanocomposites for 24 h, and the viability of T cells was assessed by Cell Counting Kit-8.

[0083] For in vivo biosafety assessment, C57BL / 6 mice were administered high concentration (50 mg / kg) of DC@CFO / BFO nanocomposites daily and subjected to magnetic field stimulation for 30 minutes. Seven days after injection, blood biochemistry and routine blood tests were performed, and histological analysis of major organs was performed.

[0084] 6. In vitro T cell differentiation

[0085] Isolation of mouse naive CD4 from peripheral lymph nodes by flow cytometry + T cells (CD4 + CD25 - CD62L hi CD44 lo ), followed by activation with 2μg / ml plate-bound anti-CD3 antibody and 1μg / ml anti-CD28 antibody. For Th1 polarization, 10μg / mL anti-IL4 antibody and 10ng / mL IL-12 were used. For Th2 polarization, 10μg / mL anti-IFN-γ antibody and 20ng / mL IL4 were used. For Th17 polarization, 20ng / mL IL-6, 5ng / mL TGF-β, 10μg / mL anti-IFN-γ antibody and 10μg / mL anti-IL4 antibody were used. For iTreg cell differentiation, 1ng / mL TGF-β and 4ng / mL IL-2, 10μg / mL anti-IFN-γ antibody and 10μg / mL anti-IL4 antibody were used. T cells were incubated with 10μg / mL DC@CFO / BFO nanocomposites and treated with a 1.5mT magnetic field for 30 minutes every day. After 48 or 72 hours of culture, the cells were collected for flow cytometry analysis.

[0086] IV. Experimental Results

[0087] 1. Preparation and characterization of cell membrane-coated magnetoelectric nanocomposites

[0088] In this example, a DC@CFO / BFO nanocomposite material was synthesized, including a magnetostrictive cobalt (II) ferrite CoFe2O4 (CFO) core and a piezoelectric bismuth (III) ferrite BiFeO3 (BFO) shell (CFO / BFO), and the surface morphology and core-shell structure of the film-modified CFO / BFO nanocomposite material were confirmed by high-resolution transmission electron microscopy. Figure 1 In addition, the oxide species (·OH and ·O2) generated by the CFO / BFO nanocomposites in magneto-electrocatalysis were detected using EPR spectroscopy under an external magnetic field. - Moreover, due to the piezoelectric properties of BFO, magnetoelectric CFO / BFO nanocomposites and BFO nanoparticles can generate ·OH and ·O2 in the presence of external ultrasound. -The characteristic signal of ROS is not triggered by nanoparticles alone. Figure 1 These findings therefore elucidate the role of the magnetic field in inducing magnetostrictive strain in the CFO core and initiating potential polarization in the BFO shell, thereby demonstrating efficient magnetoelectric conversion.

[0089] To achieve CD4 + To investigate T cell targeting specificity, bone marrow-derived dendritic cells were selected as the basis for the study by manipulating their major histocompatibility complex (MHC) II expression to bind to CD4 + Binding of T cell receptors (TCR) on T cells. Mouse bone marrow cells were first isolated and induced to differentiate into dendritic cells using GM-CSF and IL4. CD11c + MHC II + Fluorescence activated cell sorting (FACS) of cells and collection of cell membranes ( Figure 1 f). Then, membrane-modified magnetoelectric nanocomposites (DC@CFO / BFO) were prepared by repeatedly extruding freshly extracted BMDC membranes onto the prepared CFO / BFO nanoparticles. As revealed by TEM, a transparent cell membrane layer of about 10 nm was observed on the surface of the membrane-modified CFO / BFO nanomaterials ( Figure 1 a). Dynamic light scattering experiments showed that the size of the modified DC@CFO / BFO composite material was about 170nm ( Figure 1 g and h). Then the zeta potential measurement showed that the surface charge of DC@CFO / BFO nanocomposites was less negative than that of CFO / BFO nanoparticles ( Figure 1 In addition, through the quantification of glycoproteins, it was found that the glycoprotein content on the DC@CFO / BFO nanocomposites was about 90% of that in the free BMDC membrane, indicating that the membrane had the correct orientation when modified onto the nanoparticles ( Figure 1 j). It can be seen that the present embodiment successfully prepared the dendritic cell membrane modified magnetoelectric nanocomposite material, which exhibited excellent magnetic field responsiveness and magnetostrictive piezoelectric properties.

[0090] 2. Biodistribution characteristics and safety evaluation of DC film-coated magnetoelectric nanocomposites

[0091] First, a standard cell counting kit-8 (CCK-8) and mouse naive CD4 + The cytotoxicity of DC@CFO / BFO nanocomposites with or without magnetic field stimulation was evaluated in T cells and it was found that DC@CFO / BFO nanocomposites exhibited good biocompatibility at concentrations below 100 μg / mL ( Figure 2 In order to maintain CD4 +The high activity of T cells, DC@CFO / BFO nanocomposites at a concentration of 10 μg / mL were selected for subsequent studies. + T cell uptake of nanocomposites. FITC-labeled DC@CFO / BFO or CFO / BFO nanoparticles were incubated with naive mouse CD4 + The DC membrane accelerated T cell phagocytosis of the nanocomposite, which began at approximately 2 h and peaked at 8 h. After 12 h of co-incubation of the material with mouse spleen cells, most of the DC@CFO / BFO nanocomposites were engulfed by CD4 + T cells, while the majority of bare CFO / BFO nanoparticles were taken up by macrophages. Thus, these data suggest that modifying DC membranes on CFO / BFO nanoparticles increases their targeting specificity and promotes CD4 + T cell uptake.

[0092] To investigate the biodistribution characteristics of the nanocomposites in vivo, FITC-labeled DC@CFO / BFO nanocomposites were administered to C57BL / 6 mice via intravenous injection. In vivo imaging showed that the nanocomposites were enriched in the liver, kidney, lung, and spleen and remained in the body for 4 days after injection ( Figure 2 d and e). Consistent with the low cytotoxicity in vitro, supplementation of high concentration (50 mg / kg) of DC@CFO / BFO nanocomposites with or without magnetic field stimulation had little effect on murine blood cells 7 days after injection ( Figure 2 In addition, the DC@CFO / BFO nanocomposite had negligible effects on the functional parameters of the liver and kidneys, and no significant abnormalities were observed in major organs by histological analysis. Therefore, the data of this example verified the excellent safety of the DC@CFO / BFO nanocomposite in vivo.

[0093] 3. DC@CFO / BFO nanocomposites promote Th2 cell polarization and proliferation under external magnetic field

[0094] In order to further study the DC@CFO / BFO nanocomposites in CD4 + Role in T cell activation and differentiation, activation of CD4 with anti-CD3 and anti-CD28 antibodies under various helper T cell polarization conditions + Flow cytometry analysis showed that in T cells treated with the external magnetic field (DC@CFO / BFO nanocomposite), the production of IL4 was enhanced, while the production of IFN-γ was reduced, indicating that the combination of magnetoelectric nanocomposite and magnetic field promoted Th2 differentiation and inhibited Th1 differentiation ( Figure 4In addition, T cells produced less IL17A in the DC@CFO / BFO group and the DC@CFO / BFO+M group, and the proportion of induced Treg cells (iTreg) in all groups did not differ significantly ( Figure 4 a, d and e).

[0095] To more precisely characterize the role of magnetoelectric nanocomposites in Th2 polarization, RNA sequencing was performed to analyze the transcriptome of T cells treated with magnetoelectric nanocomposites and magnetic fields under Th2 differentiation conditions.

[0096] Principal component analysis (PCA) and differentially expressed genes (DEGs) analysis showed that activated T cells in the presence or absence of DC@CFO / BFO nanocomposites exhibited similar gene expression patterns under magnetic field-free conditions ( Figure 3 However, T cells in the DC@CFO / BFO+M group were clearly distinguished from T cells without nanocomposite or magnetic field stimulation by their transcriptomes ( Figure 3 a and b). Subsequent GO enrichment showed that genes related to “DNA replication”, “T cell differentiation”, and “T cell proliferation” were over-represented among the significantly up-regulated genes in T cells under Th2 cell polarization conditions (vector group and DC@CFO / BFO group) relative to naive T cells, indicating favorable T cell polarization induction.

[0097] Subsequently, this example analyzed the overall gene expression changes of T cells treated with DC@CFO / BFO in the presence or absence of a magnetic field, and found that after DC@CFO / BFO+M treatment, there were 898 upregulated genes and 1703 downregulated genes in T cells ( Figure 3 c). Consistent with previous data, upregulated genes were enriched in “Type II immune response” and “Th2 cell differentiation” compared with T cells in the DC@CFO / BFO group, reflecting the stimulatory effect of DC@CFO / BFO treatment and concomitant magnetic field stimulation on Th2 cell polarization ( Figure 4 f and g). In addition, DC@CFO / BFO nanocomposites promoted signaling pathways associated with T cell proliferation when loaded with a magnetic field. Subsequent flow cytometry analysis also confirmed that under Th2 cell polarization conditions, CD4 + T cells underwent more cell divisions in response to DC@CFO / BFO+M treatment, as detected by CFSE dilution assay and Ki-67 staining ( Figure 4 h and i). Taken together, these findings provide evidence that treatment with magnetoelectric nanocomposites combined with magnetic field stimulation promoted the induction of Th2 cell polarization and enhanced T cell proliferation in vitro.

[0098] Example 2

[0099] The following exemplifies the application of DC@CFO / BFO nanocomposites in arthritis.

[0100] 1. Experimental Methods

[0101] 1. Collagen-induced arthritis (CIA) model

[0102] 100 μ g of bovine type II collagen (CII) was injected intradermally into DBA / 1 mice, and the collagen was emulsified in Freund's complete adjuvant containing 4 mg / ml heat-killed mycobacteria. On the 21st day, a reinforcing emulsion prepared by CII and Freund's incomplete adjuvant was administered intradermally near the main injection site. At the specified time point, 10 mg / kg of DC@CFO / BFO nanocomposite was intravenously injected into mice, and 1.5 mT magnetic field was loaded every day for 30 minutes after the nanocomposite was injected. Clinical evaluation of arthritis animals was performed every other day after the onset of the disease, and the maximum clinical score of each animal was 16 points.

[0103] 2. Enzyme-linked immunosorbent assay (ELISA)

[0104] ELISA for glycoproteins on cell membranes or nanoparticles was performed according to the manufacturer's instructions.

[0105] Type 2 collagen-specific antibodies (anti-CII) in serum collected from immunized mice were measured by ELISA. Serum samples were added to plates pre-coated with 10 μg / mL of CII, followed by goat anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA.

[0106] 3. Serum inflammatory cytokine detection

[0107] Murine serum samples were collected from CIA mice, and inflammatory cytokines were measured according to the manufacturer's instructions.

[0108] 4. Histological Analysis

[0109] For histological evaluation of arthritis in mice, mouse hind paws and femurs were fixed with paraformaldehyde, decalcified in EDTA solution, dehydrated in graded alcohols, paraffin embedded and tissue sections were used for histological evaluation. H&E staining, Safranin-O / fast green and toluidine blue staining were performed to score the histopathology of inflammatory arthritis. Images were acquired using a microscope.

[0110] 5. RNA Sequencing

[0111] To investigate the role of magnetoelectric nanocomposites in T cell-mediated responses, CD4 +T cells were incubated with magnetoelectric nanocomposites and subjected to magnetic field treatment under Th2 differentiation conditions. Total RNA was then purified using magnetic beads linked to poly-T oligonucleotides. RNA-seq libraries were constructed using a library preparation kit and reads were sequenced on the Illumina platform. Clean data were obtained by removing reads containing adapters and poly-N and low-quality reads from the raw data according to the manufacturer's instructions. Clean reads were aligned to the reference genome Hisat2 based on the gene model annotation file. The fragments per kilobase per million mapped reads (FPKM) were calculated for each gene based on the length of the gene and the number of reads mapped to the gene. The gene sets from the RNAseq data were analyzed for overlap with the curated datasets in MSigDB.

[0112] 6. Quantitative Proteomic Analysis

[0113] Isolation of CD4 + T cells were then incubated with the magnetoelectric nanocomposites under Th2 differentiation conditions and magnetic field treatment. 100 μg of protein samples from each subject were subjected to LC-MS / MS analysis.

[0114] 7. Non-targeted metabolomics analysis

[0115] Isolation of CD4 + T cells were then incubated with magnetoelectric nanocomposites under Th2 differentiation conditions and magnetic field treatment. To extract the metabolites of the T cells, the cell pellets were resuspended in methanol (precooled to -80°C) and frozen in liquid nitrogen. These quenched cells were then thawed and vortexed. After centrifugation, the supernatant was transferred to a new tube and lyophilized under vacuum. The dried samples were then reconstituted in methanol and incubated. The samples were then centrifuged and the supernatant was used for LC-MS / MS analysis.

[0116] 8. Statistical Analysis

[0117] Statistical analysis was performed using software. The differences between the two groups were analyzed using a two-tailed Student's t-test. P < 0.05 was considered significant.

[0118] 2. Experimental Results

[0119] Considering CD4 + T cells play a key role in regulating the host's autoimmune response. This example further evaluated the anti-inflammatory effect of DC@CFO / BFO nanocomposites under repeated magnetic fields. This example explored the therapeutic application of magnetoelectric nanocomposites in a collagen-induced arthritis (CIA) model and monitored the dynamic pathological process ( Figure 5 a). After disease onset, arthritis scores in DC@CFO / BFO+M-treated mice were significantly reduced ( Figure 5 b). The data showed that DC@CFO / BFO nanocomposite treatment alleviated tissue swelling caused by local inflammation under external magnetic field stimulation ( Figure 5 c and d). Micro-CT showed that the joint structure of the DC@CFO / BFO+M group was well preserved and the bone density around the ankle joint was restored ( Figure 5 c and e). Subsequent histological analysis of ankle joint injuries showed that treatment of arthritis in mice with DC@CFO / BFO nanocomposites under magnetic field stimulation significantly reduced the extent of synovitis and the overall destruction of cartilage and bone ( Figure 5 c and f).

[0120] It is worth noting that in the spleen of mice in the DC@CFO / BFO+M group ( Figure 5 g and h), draining lymph nodes (dLN) ( Figure 5 i and j) and ankle joints ( Figure 5 In k and l), it was consistently observed that the frequency of IL4-producing cells was higher, while IFNγ + As shown by flow cytometry analysis, DC@CFO / BFO treatment also slightly reduced IL17A in CIA mice. + T cells and increased FOXP3 + In addition, humoral anti-type 2 collagen (anti-CII)-specific antibody detection and serum cytokine profile showed that DC@CFO / BFO+M inhibited the production of pathogenic anti-CII IgG and pro-inflammatory cytokines ( Figure 5 m and n).

[0121] In order to further verify the finding that the anti-inflammatory effect of the magnetoelectric nanocomposite material is mainly reflected in the stimulation of Th2 cell polarization, this example used the magnetoelectric nanocomposite material to treat the initial CD4 + T cells and subjected them to magnetic field stimulation under Th2 differentiation conditions. These cells were then transferred into mice after the onset of arthritis ( Figure 8 a). By arthritis score ( Figure 8 b) Gross observation and histological analysis ( Figure 8 The DC@CFO / BFO nanocomposite and magnetic field pretreatment of T cells significantly reduced the incidence and severity of arthritis in CIA mice. Lower levels of anti-CII antibodies were detected in the DC@CFO / BFO+M group, indicating the inhibitory effect of magnetoelectric nanocomposite on the development of arthritis ( Figure 8 g). Taken together, these data therefore demonstrate the anti-arthritic efficacy of magnetoelectric nanocomposites under the action of an external magnetic field.

[0122] Conclusion

[0123] The DC membrane-modified magnetoelectric nanocomposite material of the present invention can regulate the Th1 / Th2 balance and promote the proliferation of Th2 cells, thereby improving the autoimmune response mediated by T cells and maintaining the host immune homeostasis. The present invention enhances the T cell targeting specificity of magnetoelectric particles through DC membrane modification, and can be used for arthritis caused by T cells.

[0124] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a nanocomposite material in preparing a drug for treating arthritis, characterized in that: The nanocomposite material comprises, from outside to inside, an outer layer of a membrane structure, an intermediate layer of a piezoelectric material and a core structure of a magnetostrictive material, wherein the outer surface of the membrane structure contains protein molecules.

2. Use of the nanocomposite material according to claim 1 in preparing a drug for treating arthritis, characterized in that: The membrane structure is derived from a biological membrane or an artificially synthesized membrane structure.

3. Use of the nanocomposite material according to claim 1 in preparing a drug for treating arthritis, characterized in that: The protein molecule is a binding molecule capable of binding to the surface of T cells.

4. Use of the nanocomposite material according to claim 3 in preparing a drug for treating arthritis, characterized in that: The T cell surface contains at least one of the following antigens: CD1, CD2, CD3, CD5, CD7, CD8, CD16, CD25, CD26, CD27, CD28, CD30, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD126, CD150, CD153, CD1 54. CD161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LA G3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR, CCR1, CCR2, CCR4, CCR6 and CCR7.

5. Use of the nanocomposite material according to claim 1 in preparing a drug for treating arthritis, characterized in that: The protein molecule is capable of targeting CD4 + MHC II complex of T cells.

6. Use of the nanocomposite material according to claim 1 in preparing a drug for treating arthritis, characterized in that: The membrane structure is a cell membrane separated from immune cells.

7. Use of the nanocomposite material according to claim 6 in preparing a drug for treating arthritis, characterized in that: The cell membranes include cell membranes isolated from dendritic cells, B lymphocytes and / or macrophages.

8. A method for preparing a nanocomposite material, characterized in that: include: (1) preparing a magnetostrictive piezoelectric nanocomposite material, wherein the magnetostrictive piezoelectric nanocomposite material comprises a piezoelectric material intermediate layer and a magnetostrictive material core structure; (2) The magnetostrictive piezoelectric nanocomposite material is encapsulated in a film material, wherein the outer surface of the film material contains protein molecules.

9. A nanocomposite material, characterized in that: It is obtained according to the preparation method according to claim 8.

10. A method for regulating Th1 / Th2 cell balance in vitro, characterized in that: The method comprises the step of contacting the nanocomposite material according to claim 9 with isolated cells in the presence of a magnetic field of 0.01-300 mT.

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