WO2023134122A1 - 清除促炎因子和抑制t细胞活化的细胞膜包被纳米诱饵及其制备方法与应用 - Google Patents

清除促炎因子和抑制t细胞活化的细胞膜包被纳米诱饵及其制备方法与应用 Download PDF

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WO2023134122A1
WO2023134122A1 PCT/CN2022/100876 CN2022100876W WO2023134122A1 WO 2023134122 A1 WO2023134122 A1 WO 2023134122A1 CN 2022100876 W CN2022100876 W CN 2022100876W WO 2023134122 A1 WO2023134122 A1 WO 2023134122A1
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nds
prm
nano
cells
nanobait
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French (fr)
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殷黎晨
侯梦滢
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苏州大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the invention belongs to the field of biomaterials and medical technology, in particular to a cell membrane-coated nano-bait for removing proinflammatory factors and inhibiting T cell activation, and a preparation method and application thereof.
  • T cells The destruction of immune tolerance to self-antigens represented by the continuous activation of T cells is the root cause of autoimmune diseases.
  • the subsequent dysregulated inflammatory response can lead to the progression of autoimmune diseases, resulting in autoimmune damage to organs throughout the body.
  • the activation of T cells is mediated by co-stimulatory signaling molecules, and programmed death receptor-1 (PD-1) is an important co-stimulatory signaling molecule that provides inhibitory signals to the activation of T cells.
  • PD-1 programmed death receptor-1
  • mPD-1 membrane-bound PD-1
  • sPD-1 free soluble PD-1
  • mPD-1 PD-1/PD-L1 inhibitory axis
  • activated CD4 + T cells differentiate into T helper cells (Th1 and Th17) under the action of pro-inflammatory factors such as IL-6 and IL-1 ⁇ , and these two CD4 + T cell subsets with pro-inflammatory phenotype are It is considered to be the main mediator of the pathogenesis of autoimmune diseases.
  • Th1 cells secrete IFN- ⁇ to activate macrophages and make them produce more pro-inflammatory factors.
  • Th17 cells secrete TNF- ⁇ and IL-6, up-regulate the expression of adhesion molecules on vascular endothelial cells, and promote the adhesion and infiltration of more immune cells. Therefore, activated CD4 + T cells and their secreted pro-inflammatory factors form a positive cycle, which continuously aggravates the inflammatory response.
  • small molecule immunosuppressants are widely used to treat autoimmune diseases, however, they often have side effects such as nephrotoxicity, hepatotoxicity and myelosuppression.
  • biologics represented by monoclonal antibodies and recombinant cytokine receptors are also widely used, which block inflammatory signals by binding to pro-inflammatory factors.
  • these protein drugs are often challenged by immunogenicity, poor hydrolytic stability, high cost, and antibody resistance after repeated administration.
  • this single-target approach is often insufficient to halt or reverse the progression of autoimmune diseases involving complex genetic backgrounds and the involvement of multiple pro-inflammatory factors. Therefore, the development of immunomodulatory drugs with low toxicity and multiple targets is an urgent requirement for the treatment of autoimmune diseases.
  • the present invention provides a cell membrane-coated nano-bait for removing pro-inflammatory factors and inhibiting T cell activation, as well as its preparation method and application.
  • the nanobait can effectively bind and remove highly expressed pro-inflammatory factors, and can inhibit the activation of T cells at the same time. After systemic administration, it has long-term blood circulation. It has great potential in the clinical treatment of inflammatory autoimmune diseases.
  • the first object of the present invention is to provide a nano-bait, comprising a nano-core and a macrophage cell membrane expressing PD-L1 coated with the nano-core;
  • the nano-core is selected from polymer nanoparticles and/or inorganic nanoparticles; the particle size of the nano-core is 50-200nm.
  • the macrophages are selected from natural macrophages, induced differentiation macrophages or genetically engineered macrophages.
  • Natural macrophages macrophages extracted from bone marrow, peripheral blood, peritoneal cavity, tumor-associated macrophages, or established macrophage cell lines RAW 264.7 cells, ANA-1 cells, J774A.1 cells and THP-1 cells, etc. .
  • the shape of the nano-bait is spherical, cubic, conical, cylindrical, prism-shaped, pyramid-shaped, or other regular or irregular shapes
  • the particle size range of the nano-bait is 1nm ⁇ 10 ⁇ m; further, the particle size ranges from 10 nm to 5 ⁇ m, and from 500 nm to 1 ⁇ m.
  • the polymer in the polymer nanoparticles is selected from the group consisting of polylactic-co-glycolic acid (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone ( One or more of PCL), polylysine, polyglutamic acid, polybutylcyanoacrylate (PBCA), chitosan and gelatin.
  • PLGA polylactic-co-glycolic acid
  • PLA polylactic acid
  • PGA polyglycolic acid
  • PCL polycaprolactone
  • PCL polylysine
  • polyglutamic acid polybutylcyanoacrylate
  • PBCA polybutylcyanoacrylate
  • gelatin chitosan
  • the polymer in the polymer nanoparticles is selected from polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), polyglycolic acid (PGA) or polycaprolactone ( One or more of PCL).
  • PLGA polylactic acid-glycolic acid copolymer
  • PLA polylactic acid
  • PGA polyglycolic acid
  • PCL polycaprolactone
  • the inorganic nanoparticles are selected from nanoparticles prepared from any one of gold, silicon, iron and copper; the nano-cores are negatively charged.
  • the nano-core is prepared by the following methods: nano-precipitation method, emulsified solvent volatilization method, ion gel method, direct dissolution method, dialysis method, emulsification method, media grinding method, high-pressure homogeneous method, supercritical fluid method, emulsified solvent diffusion method or solid reverse micellar solution method.
  • the macrophages are selected from RAW 264.7 cells, human bone marrow mononuclear or human bone marrow macrophages, or human peripheral blood mononuclear cells or peripheral blood macrophages, ANA-1 cells, One or more of J774A.1 cells and THP-1 cells.
  • the macrophages are selected from RAW 264.7 cells; the macrophages are stimulated by IFN- ⁇ .
  • the macrophage cell membrane expresses macrophage-specific surface markers selected from PD-L1, LFA-1, MAC-1, IL-1R, IFN- ⁇ R, TNFR and IL- One or more of 6R.
  • the macrophage cell membrane maintains or retains the original natural structural integrity (such as primary structure, secondary structure, tertiary structure or quaternary structure integrity) or activity (such as Binding activity, receptor activity, signal transduction pathway activity).
  • original natural structural integrity such as primary structure, secondary structure, tertiary structure or quaternary structure integrity
  • activity such as Binding activity, receptor activity, signal transduction pathway activity
  • the macrophage cell membrane is obtained by lysing and separating components of macrophages
  • the lysis method includes: ultrasonic lysis, enzymatic lysis, chemical lysis, homogenate lysis and/or hypotonic swelling Lysis; the component separation includes: centrifugation (for example, stepwise centrifugation), precipitation, filtration, magnetic beads, and chromatographic separation.
  • the mass ratio of the cell membrane to the nano-nucleus is 1:100-1:0.1; further, the mass ratio of the cell membrane to the nano-nucleus is 1:80-1:20; Further, the mass ratio of the cell membrane to the nano-nucleus is 1:64 ⁇ 1:4; further, the mass ratio of the cell membrane to the nano-nucleus is 1:10.
  • the second object of the present invention is to provide the preparation method of the nano-bait, which includes the following steps: by applying an external force, the macrophage cell membrane expressing PD-L1 wraps the nano-core to form the nano-bait; The method is selected from sonication, mechanical co-extrusion, electroporation or heating.
  • macrophage cell membranes and nanonuclei expressing PD-L1 are encapsulated by co-ultrasound, and the ultrasonic conditions are: ultrasonic frequency 50-150W, time 1-5min.
  • the preparation method of the nanobait is as follows: macrophages are lysed by ultrasound, and cell membranes are obtained by stepwise centrifugation; the obtained cell membranes and nanonuclei are ultrasonically obtained to obtain nanobaits.
  • the nanobait of the present invention has one or more of the following characteristics selected from the following group: (1) has the ability to specifically bind and remove pro-inflammatory factors (TNF- ⁇ , IL-6, IL -1 ⁇ , IFN- ⁇ ); (2) has the ability to specifically bind and clear the pro-inflammatory factor PD-1; (3) compared with the nano-decoys (RM NDs) encapsulated in natural macrophage cell membranes, has Ability to inhibit T cell proliferation; (4) Prolonged half-life in vivo (e.g., circulation half-life over 10 hours) compared with empty nanonuclei; (5) Enhanced targeting ability to inflammatory sites compared with RM NDs.
  • the third object of the present invention is to provide a pharmaceutical composition, which includes the nanobait and a pharmaceutically or physiologically acceptable carrier.
  • the carrier includes excipients, disintegrants, diluents, lubricants, binders, wetting agents, flavoring agents, suspending agents, surfactants and preservatives.
  • the carrier may contain liquids such as water, saline, glycerol and ethanol.
  • auxiliary substances in these carriers such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffering substances, and the like.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably, the pH is about 6-8; the dosage form of the drug It is tablet, capsule, soft capsule, granule, pill, oral liquid, emulsion, dry suspension, dry extract or injection; Injection, oral, topical, intramuscular, intradermal, rectal, inhalation, etc.
  • the fourth object of the present invention is to provide a kit, which includes any one of the nanobait or the pharmaceutical composition.
  • the fifth object of the present invention is to provide the application of the nanobait, the pharmaceutical composition or the kit in the preparation of drugs for the prevention and/or treatment of inflammatory autoimmune diseases.
  • the inflammatory autoimmune disease includes rheumatoid arthritis; inflammatory bowel disease, type I diabetes, systemic lupus erythematosus or systemic sclerosis.
  • the nano-decoy coated with cell membrane inherits the surface pro-inflammatory factor receptors of macrophages, and directly removes pro-inflammatory factors through receptor recognition, avoiding potential adverse reactions.
  • the nanobait coated with cell membrane inherits the PD-L1 on the surface of macrophages, binds to T cells through receptor recognition, and directly inhibits T cell activation.
  • the introduction of nano-cores (such as PLGA) restricts the flow of membrane components and greatly improves the serum stability of nano-baits. 4. By inheriting the surface-specific proteins of macrophages, the nanobait avoids endocytosis by macrophages, thereby prolonging blood circulation. 5.
  • the nanobait By inheriting the surface-specific protein of macrophages, the nanobait combines with the adhesion molecules expressed by the cells at the inflammatory site to achieve targeting and adhesion to the inflammatory site. 6.
  • the nanobait of the present application has the advantages of long circulation, high neutralization efficiency of pro-inflammatory factors, high T cell activation inhibition efficiency, and high safety.
  • Figure 1 Schematic diagram of the preparation process of the PRM NDs nanobait of the present invention (Figure 1A); and the pro-inflammatory factor and PD-L1 protein bands of RM, PRM and PRM NDs under Western blot analysis ( Figure 1B).
  • Figure 2 The morphology of PRM NDs under the transmission electron microscope of the present invention.
  • Figure 3 Hydration particle size and potential of PLGA, PRM NVs and PRM NDs of the present invention.
  • Figure 4 The particle size of PRM NVs and PRM NDs of the present invention at different time points in PBS.
  • Figure 5 Adsorption curves of PRM NDs of the present invention adsorbing different concentrations of pro-inflammatory factors in vitro.
  • Figure 6 The relative concentration of pro-inflammatory factors after the action of PRM NDs and RM NDs of the present invention (reflecting the relative removal of pro-inflammatory factors).
  • Figure 7 After the PRM NDs of the present invention interact with RM NDs, the remaining pro-inflammatory factors stimulate cells to produce adhesion molecules (reflecting the degree of cell activation).
  • Figure 8 After the PRM NDs and RM NDs of the present invention act on the remaining IL-1 ⁇ to stimulate cell apoptosis.
  • Figure 9 Adsorption curves of different concentrations of PD-1 adsorbed by PRM NDs of the present invention in vitro.
  • Figure 10 The relative PD-1 concentration after the interaction between PRM NDs and RM NDs of the present invention (reflecting the relative removal of PD-1).
  • Figure 11 Changes in cell viability after co-incubation of CD4 + T cells isolated from the spleen of the present invention with PRM NDs or RM NDs.
  • Figure 12 Changes in the proliferation of CD4 + T cells isolated from the spleen of the present invention after co-incubation with PRM NDs or RM NDs.
  • Figure 13 MAC-1 and LFA-1 protein bands of RM, PRM and PRM NDs analyzed by Western blot of the present invention.
  • Figure 14 Fluorescent images of DiD PRM NDs or DiD RM NDs of the present invention incubated with activated endothelial cells, chondrocytes, and intestinal epithelial cells.
  • Figure 15 Flow cytometry analysis of the present invention for the level of NDs adhered to cells after incubation of DiD PRM NDs or DiD RM NDs with activated endothelial cells, chondrocytes, and intestinal epithelial cells.
  • Figure 16 In vivo circulation time of intravenously injected DiD PRM NDs of the present invention.
  • Figure 17 In vivo fluorescence imaging images of intravenously injected DiD PRM NDs targeting inflammatory joints of the present invention.
  • Figure 18 Protein levels of pro-inflammatory factors and PD-1 in serum of ZIA mice after PRM NDs treatment of the present invention.
  • Figure 19 Protein levels of pro-inflammatory factors and PD-1 in the synovium of ZIA mice after PRM NDs treatment of the present invention.
  • Figure 20 The ratio of Th1 and Th17 cells in the spleen of ZIA mice after PRM NDs treatment of the present invention.
  • Figure 21 Hematoxylin-eosin, safranin-fast green, immunohistochemical staining images, histological scoring and cartilage thickness measurement of ZIA mouse knee joints after treatment with PRM NDs of the present invention.
  • Figure 22 Typical micro-computed tomography (micro-CT) images of the microstructure of ZIA mouse knee joint bone tissue after treatment with PRM NDs of the present invention, as well as bone volume fraction (BV/TV) and trabecular bone number (Tb. N).
  • micro-CT micro-computed tomography
  • Figure 23 Changes in relative body weight of UC mice after treatment with PRM NDs of the present invention.
  • Figure 24 Changes in disease activity index of UC mice after PRM NDs treatment of the present invention.
  • Figure 25 Representative colon pictures and colon length statistics of UC mice treated with PRM NDs of the present invention.
  • Figure 26 Protein levels of pro-inflammatory factors in the colon of UC mice after PRM NDs treatment of the present invention.
  • Figure 27 Hematoxylin-eosin staining images of colon tissue sections of UC mice after PRM NDs treatment of the present invention.
  • Figure 28 Keratin 18 immunofluorescent staining images of colon tissue sections of UC mice after PRM NDs treatment of the present invention.
  • Figure 29 Representative hematological parameters of C57/BL6 mice treated with PBS or PRM NDs of the present invention.
  • Figure 30 Representative and biochemical parameters of C57/BL6 mice treated with PBS or PRM NDs of the present invention.
  • Figure 31 H&E staining images of major organs of C57/BL6 mice after PBS or PRM NDs treatment of the present invention.
  • “*” indicates p ⁇ 0.05
  • "**” indicates p ⁇ 0.01
  • "***” indicates p ⁇ 0.001.
  • the present application provides a cell membrane-coated nanobait, which can specifically remove pro-inflammatory factors to avoid activation of T cells by pro-inflammatory factors, and bind to PD-1 on the surface of T cells to inhibit the activation of T cells.
  • PRM NDs nanobaits have a longer circulation time in the body, which solves the shortcomings of protein drugs (such as antibody drugs) used in the prior art such as short blood circulation time.
  • macrophages treated with IFN- ⁇ are lysed by ultrasound, and PRM is obtained by stepwise centrifugation; then PRM and PLGA nano-nuclei are ultrasonically combined for 2 minutes (100W) to obtain PRM NDs nanobaits.
  • PRM and PLGA nano-nuclei are ultrasonically combined for 2 minutes (100W) to obtain PRM NDs nanobaits.
  • this nanobait has efficient and stable clearance of pro-inflammatory factors, and can bind to PD-1 on the surface of T cells to inhibit the activation of T cells.
  • ZIA Zymosan A-induced arthritis
  • UC dextran sodium sulfate-induced ulcerative colitis
  • nanobait and “cell membrane-coated/encapsulated nanobait/material”, used interchangeably, refer to an artificially synthesized nanomaterial comprising a nanonucleus and a cell membrane surrounding it, which may have a camouflaged biomimetic The function of trapping and removing unfavorable factors or components in the system.
  • macrophage refers to cells generated from hematopoietic stem cells in the bone marrow, mainly including monocytes and macrophages. Macrophages in this application can be genetically engineered to overexpress on their surface to help improve pro-inflammatory factor clearance and PD-1 binding.
  • cell membrane refers to a naturally occurring biological membrane obtained from macrophages or their organelles, or a modified or altered membrane with all or part of the biological activity of macrophages.
  • the cell membrane used in this application can be the cell membrane obtained from the macrophages of this application, which can be separated, remove some components (such as lipids, sugar chains) and/or add some components (such as overexpressed PD-L1 , other cell surface antigens).
  • nanocore refers to any nanoparticle having nanoscale dimensions that can be used to support cell membranes of the present application.
  • Materials that can be used to prepare the nano-decoy nano-core of the present application include but are not limited to polymer nanomaterials or inorganic nanomaterials, such as polylactic-co-glycolic acid (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), poly Caprolactone (PCL), polylysine, polyglutamic acid, polybutylcyanoacrylate (PBCA), chitosan, gelatin; gold, silicon, iron, copper, etc.
  • the nanobait of the present application can have various suitable shapes, such as sphere, cube, cone, cylinder, prism, pyramid, or other regular or irregular shapes.
  • the size of the nanobait in the present application can be 1 nanometer to 10 micrometers or any value or range of values therebetween, such as 10 nanometers to 5 micrometers, 500 nanometers to 1 micrometer and so on.
  • the application also provides a method for preparing the nanobait of the application, the method comprising:
  • A providing a nano-core
  • B providing the cell membrane of a macrophage expressing PD-L1
  • C wrapping the cell membrane onto the nano-core to form the nano-bait.
  • the nano-cores of the present application can be prepared from raw materials by various methods known in the art (such as using nano-precipitation method), and can also be purchased directly from various suppliers.
  • the nano-core can have an opposite potential to the cell membrane to form a charge attraction to further stabilize the nano-bait.
  • Macrophage cell membranes can be obtained by cell lysis and separation, for example, the lysis includes: ultrasonic lysis, enzymatic lysis, chemical lysis, homogenate lysis and/or hypotonic swelling lysis; the separation includes: centrifugation (for example, stepwise centrifugation) , precipitation, filtration, magnetic beads, chromatographic separation.
  • the cells can be harvested, cultured, engineered, etc. to obtain macrophages with the desired number and functions.
  • Cell membranes should have a certain structural integrity and retain desired functions, and be able to partially or fully envelop the nanocore.
  • the cell membrane can completely wrap the nano-core to increase the stability of the nano-bait.
  • the cell membrane has a size greater than or equal to the surface area of the nanocore.
  • functional molecules such as functional epitopes and receptors on the cell membrane surface of macrophages are retained on the cell membrane.
  • the coating of the nano-core by the cell membrane can be realized by applying external force.
  • sound waves such as ultrasonic method
  • mechanical force such as mechanical co-extrusion
  • electrical energy electro energy
  • thermal energy thermo energy
  • the method of the present application may include: lysing macrophages by sonication, and obtaining cell membranes by stepwise centrifugation; sonicating the obtained cell membranes together with nanonuclei to obtain nanobaits.
  • the present application also provides a medicament, a pharmaceutical composition or a kit, which contains an effective amount of the nanobait of the present application or the composition of components (a) and (b), and a pharmaceutically acceptable carrier.
  • active substance or “active substance of the present application” are used interchangeably to refer to a nanobait or a combination of components (a) and (b).
  • the composition of components (a) and (b) may comprise independently stored components (a) and component (b) and optional carrier, before use, component (a ) and component (b) are mixed with an optional carrier and prepared into a nanobait drug for prevention and/or treatment.
  • the medicament can be used to prevent and/or treat autoimmune diseases with overexpression of proinflammatory cytokines and activation of T cells.
  • the active substances of the present application and products containing the active substances can be used to prevent and/or treat rheumatoid arthritis and ulcerative colitis caused by overexpression of pro-inflammatory factors and overactivation of T cells, for example.
  • the terms “comprising” or “comprising” include “comprising”, “consisting essentially of”, and “consisting of”.
  • the term “pharmaceutically acceptable” ingredient is a substance suitable for use in humans and/or animals without undue adverse side effects such as toxicity, irritation and allergic reactions, ie having a reasonable benefit/risk ratio.
  • the term “effective amount” refers to an amount that can produce functions or activities on humans and/or animals and that can be accepted by humans and/or animals.
  • the term “pharmaceutically acceptable carrier” refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • Suitable vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
  • Pharmaceutically acceptable carriers in compositions can contain liquids such as water, saline, glycerol and ethanol.
  • auxiliary substances in these carriers such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffering substances, and the like.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8.
  • unit dosage form refers to the preparation of the composition of the present application into a dosage form required for single administration for the convenience of administration, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, capsules , Sustained release agent. It is to be understood that the effective dosage of the active substances employed may vary with the severity of the subject to be administered or treated. The specific situation is determined according to the individual conditions of the subject (such as the subject's weight, age, physical condition, desired effect), which is within the scope of judgment of a skilled physician.
  • the composition of the present application can be solid (such as granules, tablets, freeze-dried powder, suppositories, capsules, sublingual tablets) or liquid (such as oral liquid) or other suitable shapes.
  • the route of administration can be adopted: intravenous injection, intraperitoneal injection, intralesional injection, oral administration, local administration, intramuscular, intradermal, rectal, inhalation and other methods.
  • the composition of the present application may also contain other active substances for improving and treating diseases related to osteoclast hyperactivity or hyperfunction.
  • the other active substances are selected from the group consisting of commonly used clinical osteoclast inhibitors, antibiotics, antitumor agents, anti-inflammatory agents and the like.
  • the nanobait of the present application can also be combined with other drugs and treatment methods, such as chemotherapy, radiotherapy, phototherapy, cryotherapy, surgery, cell therapy, transplantation and the like.
  • a cell membrane-coated nanobait is provided, the structure of which is PRM NDs, wherein PRM is the cell membrane of a macrophage expressing PD-L1, and PLGA is a nanonucleus.
  • the preparation method of the nanobait coated with PRM NDs cell membrane may include, after the macrophage is stimulated by IFN- ⁇ (50ng/mL) for 48 hours, the macrophage is lysed by ultrasound, The PRM was obtained by centrifugation step by step; then the PRM and PLGA nano-nuclei were ultrasonicated for 2 minutes (100W) to obtain the PRM NDs nanobait.
  • the cell membranes of the macrophages are separated by ultrasonication and centrifugation.
  • RAW 264.7 cells were suspended in homogenization buffer containing 20 mM Tris ⁇ HCl (pH 7.5), 10 mM KCl, 75 mM sucrose, 2 mM MgCl 2 and protease/phosphatase inhibitors.
  • the suspension was disrupted with a JY 92-IIN homogenizer (75W), and then the supernatant was collected by centrifugation at 20,000g for 25 minutes, and the cell membrane was collected by centrifugation of the supernatant at 100,000g for 35 minutes.
  • the protein content of the collected cell membranes was determined using a BCA kit. Membranes containing about 5 mg of membrane proteins can be extracted from 3 x 107 RAW 264.7 cells.
  • the PLGA nano-core is prepared by volatilization of acetone. Specifically, 1 mL of acetone dissolved with PLGA (10 mg/mL) was added dropwise to 2 mL of deionized water, and then the mixture was stirred in the open air until the acetone evaporated completely.
  • the PRM NDs nanobait is prepared by ultrasonic method. Specifically, the PRM and PLGA nano-cores were sonicated for 2 minutes with a bath sonicator (Fisher Scientific FS30D, 100W) at a mass ratio of 1:10. As an advantage, the PRM NDs nanobait of this application is composed of FDA-approved PLGA and biologically derived cell membrane, which has excellent biocompatibility and safety.
  • the application further discloses the application of the above-mentioned cell membrane-coated nano-bait in the preparation of anti-postmenopausal osteoporosis drugs.
  • RAW 264.7 cells were stimulated by IFN- ⁇ (50ng/mL) for 48 hours, they were suspended in a solution containing 20mM Tris ⁇ HCl (pH 7.5), 10mM KCl, 75mM sucrose, 2mM MgCl 2 and a piece of protease/phosphatase inhibitor in the homogenization buffer.
  • the suspension was disrupted with a JY 92-IIN homogenizer (75W), and then the supernatant was collected by centrifugation at 20,000g for 25 minutes, and the cell membrane was collected by centrifugation of the supernatant at 100,000g for 35 minutes.
  • the protein content of the collected cell membranes was determined using a BCA kit.
  • Membranes containing about 5 mg of membrane proteins can be extracted from 3 ⁇ 10 7 RAW 264.7 cells.
  • PRM and PLGA nanonuclei were sonicated with a bath sonicator (Fisher Scientific FS30D, 100W) at a mass ratio of 1:10 for 2 minutes to obtain PRM NDs nanobaits.
  • a bath sonicator Fisher Scientific FS30D, 100W
  • Embodiment 1 preparation and characterization of PRM NDs nanobait
  • the cell membrane-coated nanocomposite-PRM NDs nanobait of the present application was prepared according to the process described in Figure 1A. The preparation steps are as follows:
  • membrane material mouse monocyte/macrophage-like cells RAW 264.7 (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number SCSP-5036, the medium is DMEM containing 10% FBS, 37 ° C, 5% CO 2 ) stimulated by IFN- ⁇ (50ng/mL) for 48 hours, then suspended in 20mM Tris ⁇ HCl (pH 7.5), 10mM KCl, 75mM sucrose, 2mM MgCl 2 and protease/phosphatase inhibitors (purchased from Pierce, Cat. No. A32953, each tablet dissolved in 10 mL of solution) in homogenization buffer.
  • DMEM containing 10% FBS, 37 ° C, 5% CO 2
  • IFN- ⁇ 50ng/mL
  • 20mM Tris ⁇ HCl pH 7.5
  • 10mM KCl 75mM sucrose
  • 2mM MgCl 2 protease/phosphatase inhibitors
  • the cells in the suspension were disrupted with a JY 92-IIN homogenizer (75W), and then the supernatant was collected by centrifugation at 20000g for 25 minutes, and the cell membrane was collected by centrifugation of the supernatant at 100000g for 35 minutes.
  • the protein content of the collected cell membranes was determined using the BCA kit.
  • Membrane material containing about 5 mg of membrane protein can be extracted from about 3 ⁇ 10 7 RAW 264.7 cells.
  • the expressions of pro-inflammatory factor receptors and PD-L1 on the surface of RM, PRM and PRM NDs were detected by Western blot.
  • the concentration of IFN ⁇ R, TNFR, IL-6R, IL-1R and PD-L1 primary antibody is 1:1000, and the concentration of HRP-labeled secondary antibody is 1:500, see Figure 1 for details.
  • the expressions of proinflammatory receptors (TNFR, IL-6R, IL-1R, IFN ⁇ R) and PD-L1 on PRM were significantly higher than those on RM.
  • the PRM coated on the nano-core has basically the same expression of surface markers as the uncoated PRM, suggesting that it may have similar membrane functions to natural cells.
  • the nanobait is in the form of a spherical structure and has a recognizable clear membrane structure.
  • the hydrodynamic size and zeta potential of the nanobaits were determined using a Zetasizer Nano ZS90 (Malvern Instruments, Ltd., UK). See Figure 3 for details. As shown in Figure 3, the hydrodynamic size of the nanobait is about 115.4 nm, and the zeta potential is about -45.6 mV.
  • a stable nanobait containing PLGA nano-cores and PD-L1-expressed macrophage cell membranes effectively wrapping the nano-cores was prepared by the method of the present application, which can provide surface proteins similar to corresponding natural cells on the surface, Examples include cytokine-binding receptors and PD-L1.
  • Example 2 In vitro clearance of pro-inflammatory factors by PRM NDs and inhibition of cell activation and apoptosis mediated by pro-inflammatory factors
  • PRM NDs can effectively eliminate TNF- ⁇ , IL-6, IL-1 ⁇ and IFN- ⁇ , and the IC50 (half maximal inhibitory concentration) is 666.4, 164.8, 1410.7 and 90.5 ⁇ g, respectively PLGA/mL.
  • the present invention studies the ability of PRM NDs to inhibit the activation of vascular endothelial cells, chondrocytes and intestinal epithelial cells induced by proinflammatory factors in vitro by measuring the expression level of ICAM-1.
  • Co-incubate pro-inflammatory factors (TNF- ⁇ , IL-6, IL-1 ⁇ or IFN- ⁇ , 100ng/mL) with RM NDs or PRM NDs (2mg PLGAmL -1 ) in cell culture medium containing 10% FBS . The mixture was incubated at 37 °C for 2 h and then centrifuged at 16,000 g for 15 min to remove NDs.
  • HUVEC cells human umbilical vein endothelial cells
  • C28/I2 cells human chondrocytes
  • Caco-2 cells human cloned colon adenocarcinoma cells that are structurally and functionally similar to intestinal cells
  • the expression level of ICAM-1 was determined by Western blot. See Figure 7 for details, as shown in Figure 7, when co-cultured with PRM NDs, the upregulation of ICAM-1 in HUVEC, C28/I2 and Caco-2 cells was inhibited, and PRM NDs showed stronger inhibition than RM NDs effect.
  • the PRM NDs of the present application can effectively inhibit cytokine-induced cell activation, because higher receptor levels on PRM can clear more cytokines.
  • RM NDs or PRM NDs (2 mg PLGA/mL) were incubated with IL-1 ⁇ (100 ng/mL) in cell culture medium containing 10% FBS at 37 °C for 2 h, and then the mixture was centrifuged at 16,000 g for 15 min to NDs were removed, and the supernatant was incubated with C28/I2 cells for 48 hours. Cells were then digested with 0.25% EDTA/trypsin and washed with PBS. Cells were then stained with Annexin V-FITC and analyzed by flow cytometry.
  • Example 3 The effect of PRM NDs on PD-1 scavenging and its effect on CD4 + T cells
  • PRM NDs To investigate the clearance efficiency of PRM NDs on PD-1, these recombinant PD-1 (8 ng/mL) were mixed with PRM NDs in DMEM containing 10% fetal bovine serum at a final concentration (0-2 mg PLGA/mL). After incubation at 37°C for 2 h, centrifuge at 16100 g for 15 min, and use an ELISA kit to measure the concentration of PD-1 in the supernatant. Binding kinetic profiles were plotted with Graphpad Prism 8. See Figure 9 for details. As shown in Figure 9, PRM NDs can effectively remove PD-1, with an IC50 (half maximum inhibitory concentration) of 106.7 ⁇ g PLGA/mL.
  • IC50 half maximum inhibitory concentration
  • PD-1 (2ng/mL) was mixed with RM NDs or PRM NDs (2mg PLGA/mL) in DMEM containing 10% FBS Mix and incubate at 37°C for 2 hours. It was then centrifuged at 16100g for 10 minutes. The concentration of PD-1 in the supernatant was quantified using an ELISA kit. See Figure 10 for details. As shown in Figure 10, PRM NDs exhibited high PD-1 clearance efficiency, while empty carrier PLGA NPs and RM NDs had little binding effect. The above results collectively indicate that PRM NDs have the ability to clear PD-1.
  • CD4 + T cells were first isolated from the spleen of healthy mice, and then the cells were treated with plate-bound anti-CD3 (1 ⁇ g/mL), anti-CD28 (5 ⁇ g/mL) and IL-2 (10 ng/mL) for 48 hours to make them activation. Then, the activated CD4 + T cells (1 ⁇ 10 6 ) were incubated with PRM NDs or RM NDs in cell culture medium containing 10% fetal calf serum at different PLGA concentrations for 48 hours.
  • PRM NDs could effectively inhibit the proliferation of CD4 + T cells, and the proportion of cell division was significantly reduced after CFSE staining. Furthermore, the viability of CD4 + T cells was significantly reduced after treatment with PRM NDs. However, RM NDs had negligible effects on the activity and proliferation of CD4 + T cells due to the minimal amount of PD-L1 on the membrane of untreated macrophages. Thus, the PRM NDs of the present application can inhibit T cell activation by effectively restoring the PD-1/PD-L1 inhibitory axis, thereby inhibiting T cell proliferation.
  • Embodiment 4 cell adhesion experiment of PRM NDs
  • LFA-1 and MAC-1 on the surface of RM, PRM and PRM NDs were detected by Western blot.
  • the concentration of LFA-1 and MAC-1 primary antibody was 1:1000, and the concentration of HRP-labeled secondary antibody was 1:500. See Figure 13 for details. As shown in Figure 13, after IFN- ⁇ treatment, the expression of LFA-1 and MAC-1 on PRM was significantly higher than that on RM.
  • the present invention studies the adhesion ability of PRM NDs and RM NDs to inflammatory cells by laser confocal microscopy and flow cytometry.
  • DiD was encapsulated in PLGA NPs as described above, followed by further coating of cell membranes on DiD PLGA NPs.
  • HUVEC cells were seeded in 12-well plates (1 ⁇ 10 5 cells/well) and cultured overnight. The cells were treated with LPS (300ng/mL) for 6h, washed with PBS, and blocked with 1% BSA for 1h.
  • DiD PLGA NPs DiD RBCM NDs, DiD RM NDs and DiD PRM NDs
  • DMEM 0.2 mg PLGAmL ⁇ 1
  • FBS fetal bovine serum
  • DiD PRM NDs showed higher binding affinity to inflammatory HUVEC, C28/I2 and Caco-2 cells than DiD RM NDs and DiD RBCM NDs, as shown in the confocal laser scanning microscope images, which indicated that Consistent with higher levels of LFA-1 and MAC-1 on PRM than on RM and RBCM.
  • Flow cytometry analysis further confirmed that DiD PRM NDs adhered to inflammatory cells more readily than other tested NDs.
  • ZIA mice were injected intravenously with RM NDs or PRM NDs, and the accumulation of NDs at inflamed joints was observed by a small animal in vivo imager.
  • Zymosan A Zymosan A, 20 ⁇ L, 15 mg/mL
  • sterile PBS (20 ⁇ L) was injected intra-articularly into the right posterior knee joint as a sham-operated control.
  • DiD RM NDs or DiD PRM NDs were injected intravenously at a dose of 15 mg DiD PLGA/kg. Mice were anesthetized at predetermined time points and fluorescently imaged by a small animal intravital fluorescence imager.
  • FIG. 17 the left rear knee joint of mice injected intravenously with DiD PRM NDs showed stronger fluorescence intensity than mice injected with DiD RM NDs.
  • the fluorescence intensity of knee joints treated with DiD PRM NDs peaked at 4 hours after injection, which was 3.6 times higher than that of joints treated with DiD RM NDs. In contrast, almost no fluorescent signal was observed in the right posterior knee joint.
  • mice Male C57/BL6 mice were intra-articularly injected with zymosan A (Zymosan A, 20 ⁇ L, 15 mg/mL) to establish a ZIA mouse model. After modeling, RM NDs or PRM NDs were injected intravenously at a dose of 75 mg PLGA/kg every 2 days, and an equal volume of PBS was injected as a negative control. On day 7, mice were sacrificed, and peripheral blood was collected and centrifuged at 500 g for 10 minutes at 4°C to extract serum. ELISA kits were used to quantify the levels of pro-inflammatory factors and PD-1 in serum.
  • the remaining cells were washed with RPMI and incubated in RPMI containing 10% FBS, 12-myristate 13-acetate (50 ng/mL), ionomycin (750 ng/mL) and GolgiStop (1 ⁇ L/mL) at 37 Incubate at °C for 5 hours. Then incubated with PE-anti-mouse CD4 (1:100) on ice for 20 minutes, washed with PBS, resuspended in fix/permeabilize solution, and subsequently treated with Alexa 488-anti-mouse IL-17A (1:100) or APC-anti-mouse IFN- ⁇ (1:100) were further stained, and finally the cells were analyzed by flow cytometry.
  • micro-CT Stemscan 1176
  • Skyscan 1176 was used to scan the left rear knee joint specimen of ZIA mice.
  • Obtain a high-resolution scanning image (9-20mm) (resolution: 8.8mm, source voltage: 50kV, source current: 500mA, rotation step size: 0.7U).
  • Data sets were reconstructed using CT analyzer software (Skyscan) to obtain 3D images of femoral tissue and measure morphometric parameters. Bone erosion from micro-CT scans was calculated based on reconstructed data and an in-house written Fiji script.
  • Regions of interest (ROIs) in trabecular bone were selected for analysis of the following morphometric parameters, including: (1) bone volume density ratio (BV/TV), (2) trabecular number (Tb.N). See Figure 22 for details. As shown in Figure 22, after treatment with PRM NDs, the surface bone erosion of ZIA mice was significantly reduced, and the bone mass density (BV/TV) basically returned to normal levels. In addition, the destruction of trabecular bone microstructure in ZIA mice was largely inhibited after PRM NDs treatment, and the number of trabecular bones (Tb.N) was also restored to the level of normal mice. These results suggest that PRM NDs can effectively prevent bone loss during arthritis.
  • BV/TV bone volume density ratio
  • Tb.N trabecular number
  • Embodiment 9 PRM NDs treatment on the relieving effect of ulcerative colitis
  • the present invention further investigated the efficacy of PRM NDs on ulcerative colitis (UC), an important category of inflammatory bowel disease (IBD), by feeding mice with 3% DSS water for 0-7 days, and at 2 , 4 and 6 days intravenous injection of PBS, RM NDs or PRM NDs.
  • UC ulcerative colitis
  • IBD inflammatory bowel disease
  • Shortened colon length is a classic symptom of DSS-induced UC. See Figure 25 for details. As shown in Figure 25, DSS feeding significantly shortened the colon length of mice in the PBS group to about 4 cm, while the colon length of mice in the PRM NDs group returned to about 6.4 cm, which was similar to that of normal mice. Furthermore, as shown in Figure 26, PRM NDs significantly reduced the levels of pro-inflammatory factors in the colon, including TNF- ⁇ , IFN- ⁇ , IL-1 ⁇ , and IL-6, verifying the potent anti-inflammatory effect of PRM NDs on UC .
  • Embodiment ten the in vivo biocompatibility of PRM NDs nanobait
  • PBS 200 ⁇ L
  • PRM NDs 75 mg PLGA/kg, 200 ⁇ L
  • PBS 200 ⁇ L
  • PRM NDs 75 mg PLGA/kg, 200 ⁇ L
  • Blood and major organs were collected. Hematological evaluations were performed on a Cobas 501 automated hematology analyzer (Roche, USA). See Figure 29 for details. The levels of serum biochemical parameters were measured using BC-5380 automatic chemical analyzer (Mindray, China). See Figure 30 for details. Major organs were fixed in 10% formalin, embedded in paraffin, cross-sectioned at 8 ⁇ m thickness, stained with H&E, and observed by whole-mount transmission light microscopy. See Figure 31 for details.

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Abstract

一种清除促炎因子和抑制T细胞活化的细胞膜包被纳米诱饵及其制备方法与应用。所述纳米诱饵包括：纳米核和包被所述纳米核的程序性死亡配体1(PD-L1)表达的巨噬细胞细胞膜。所述纳米诱饵可为PRM NDs，其中PRM为PD-L1表达的巨噬细胞细胞膜，PLGA为纳米核。所述纳米诱饵在治疗存在促炎因子高表达的和T细胞过度活化的自身免疫疾病中的应用。所述纳米诱饵可有效地清除类风湿性关节炎和炎症性肠病中高表达的促炎因子，同时能够抑制T细胞的过度激活，并且在全身施用后具备长时间的血液循环，在类风湿性关节炎和炎症性肠病的临床治疗中具有极大的潜力。

Description

清除促炎因子和抑制T细胞活化的细胞膜包被纳米诱饵及其制备方法与应用 技术领域
本发明属于生物材料和医学技术领域，尤其是指清除促炎因子和抑制T细胞活化的细胞膜包被纳米诱饵及其制备方法与应用。
背景技术
以T细胞持续激活为代表的对自身抗原的免疫耐受被破坏，是自身免疫性疾病的根本原因。随后失调的炎症反应会导致自身免疫性疾病的进展，从而对全身器官的造成自身免疫性损害。T细胞的激活是由共刺激信号分子介导的，程序性死亡受体-1(PD-1)是一种重要的共刺激信号分子，其对T细胞的激活提供抑制性信号。在正常的生理状态下，组织表达的程序性死亡配体1(PD-L1)与T细胞上的膜结合型PD-1(mPD-1)结合，传递抑制信号，从而抑制T细胞的激活，维持外周的免疫耐受。然而，在自身免疫性疾病的病理条件下，过度产生的游离可溶型PD-1(sPD-1)与mPD-1竞争结合PD-L1，从而导致PD-1/PD-L1抑制轴的缺失，最终导致T细胞不受控制的激活。然后，活化的CD4 +T细胞在IL-6和IL-1β等促炎因子的作用下分化为T辅助细胞(Th1和Th17)，这两种具有促炎症表型的CD4 +T细胞亚群被认为是自身免疫性疾病发病的主要媒介。特别是Th1细胞，其通过分泌IFN-γ以激活巨噬细胞并使其产生更多的促炎因子。Th17细胞则通过分泌TNF-α和IL-6，上调血管内皮细胞上粘附分子的表达，促进更多免疫细胞的粘附和浸润。因此，活化的CD4 +T细胞及其分泌的促炎因子形成一个正向循环，不断加剧炎症反应。
在临床上，小分子免疫抑制剂被广泛用于治疗自身免疫性疾病，然而，它们往往有副作用，如肾脏毒性、肝脏毒性和骨髓抑制。此外，以单克隆抗体和重组细胞因子受体为代表的生物制剂也被广泛采用，它们通过与促炎因子结合来阻断炎症信号。然而，这些蛋白质药物常常受到免疫原性、水解稳定性差、成本高和重复给药后的抗体抗性的挑战。此外，这种单一靶点的方式往往不足以阻止或逆转涉及复杂遗传背景和多种促炎因子参与的自身免疫性疾病的进展。因此，开发低毒性、多靶点的免疫调节药物是治疗自身免疫性疾病的迫切要求。
因此，本领域中亟需研发一种新型的更安全和更有效并且可以同时抑制CD4 +T细胞激活和清除促炎因子的药物以用于管理自身免疫性疾病。
发明内容
为解决上述技术问题，本发明提供了一种清除促炎因子和抑制T细胞活化的细胞膜包被纳米诱饵及其制备方法与应用。其中纳米诱饵能有效结合并清除高表达的促炎因子， 同时能够抑制T细胞的活化，在全身施用后具备长时间的血液循环，在促炎因子高表达和T细胞过度活化相关疾病(尤其是炎性自身免疫性疾病)的临床治疗中具有极大的潜力。
本发明的第一个目的在于提供一种纳米诱饵，包括纳米核和包被所述纳米核的PD-L1表达的巨噬细胞细胞膜；
其中，所述纳米核选自聚合物纳米颗粒和/或无机纳米颗粒；所述纳米核的粒径为50-200nm。
所述巨噬细胞选自天然巨噬细胞、诱导分化形成的巨噬细胞或经基因工程化改造的巨噬细胞。天然的巨噬细胞：骨髓、外周血、腹腔提取的巨噬细胞、肿瘤相关巨噬细胞，或者建成的巨噬细胞系RAW 264.7细胞、ANA-1细胞、J774A.1细胞和THP-1细胞等。
在本发明的一个实施例中，所述纳米诱饵形态为球形、立方体、圆锥形、圆柱形、棱柱形、棱锥形、或其它规则或不规则的形状，所述纳米诱饵的粒径范围为1nm～10μm；进一步的，粒径范围为10nm～5μm，500nm～1μm。
在本发明的一个实施例中，所述聚合物纳米颗粒中聚合物选自聚乳酸-羟基乙酸共聚物(PLGA)、聚乳酸(PLA)、聚乙醇酸(PGA)、聚己酸内酯(PCL)、聚赖氨酸、聚谷氨酸、聚氰基丙烯酸正丁酯(PBCA)、壳聚糖和明胶中的一种或多种。
在本发明的一个实施例中，所述聚合物纳米颗粒中聚合物选自聚乳酸-羟基乙酸共聚物(PLGA)、聚乳酸(PLA)、聚乙醇酸(PGA)或聚己酸内酯(PCL)中的一种或多种。
在本发明的一个实施例中，所述无机纳米颗粒选自由金、硅、铁和铜中任一种物质制备得到的纳米颗粒；所述纳米核带负电。
在本发明的一个实施例中，所述纳米核通过以下方法制备得到：纳米沉淀法、乳化溶剂挥发法、离子凝胶法、直接溶解法、透析法、乳化法、介质研磨法、高压均质法、超临界流体法、类乳化溶剂扩散法或固态反相胶束溶液法。
在本发明的一个实施例中，所述巨噬细胞选自RAW 264.7细胞、人骨髓单核或人骨髓巨噬细胞，或人外周血单核细胞或外周血巨噬细胞、ANA-1细胞、J774A.1细胞和THP-1细胞中的一种或多种。
在本发明的一个实施例中，所述巨噬细胞选自RAW 264.7细胞；所述巨噬细胞经IFN-γ刺激。
在本发明的一个实施例中，所述巨噬细胞细胞膜表达巨噬细胞特异性表面标志物选自PD-L1、LFA-1、MAC-1、IL-1R、IFN-γR、TNFR和IL-6R中的一种或多种。
在本发明的一个实施例中，所述巨噬细胞细胞膜维持或保留细胞膜原有的天然结构完整性(例如一级结构、二级结构、三级结构或四级结构完整性)或活性(例如结合活性、受体活性、信号传导通路活性)。
在本发明的一个实施例中，所述巨噬细胞细胞膜的通过巨噬细胞的裂解和组分分离得到，裂解方法包括：超声裂解、酶裂解、化学裂解、匀浆裂解和/或低渗溶胀裂解；所述组分分离包括：离心(例如逐级离心)、沉淀、过滤、磁珠、层析分离。
在本发明的一个实施例中，所述细胞膜与纳米核的质量比为1:100～1:0.1；进一步的，所述细胞膜与所述纳米核的质量比为1:80～1:20；进一步的，所述细胞膜与所述纳米核的质量比为1:64～1:4；进一步的，所述细胞膜与所述纳米核的质量比为1:10。
本发明的第二个目的在于提供所述的纳米诱饵的制备方法，包括以下步骤：通过施加外力使得PD-L1表达的巨噬细胞细胞膜包裹纳米核，形成所述纳米诱饵；所述施加外力的方法选自声波法、机械共挤压、电穿孔法或加热法。
在本发明的一个实施例中，PD-L1表达的巨噬细胞细胞膜与纳米核通过共同超声实现包裹，所述超声条件：超声频率50～150W，时间1～5min。
在本发明的一个实施例中，所述的纳米诱饵的制备方法如下所示：通过超声裂解巨噬细胞，并通过逐级离心得到细胞膜；将所得细胞膜与纳米核共同超声，得到纳米诱饵。
在本发明的一个实施例中，本发明所述纳米诱饵具有选自下组的一个或多个如下特征：(1)具有特异性结合并清除促炎因子(TNF-α,IL-6,IL-1β,IFN-γ)的能力；(2)具有特异性结合并清除促炎因子PD-1的能力；(3)与天然巨噬细胞细胞膜包载的纳米诱饵(RM NDs)相比，具有抑制T细胞增殖的能力；(4)与空载纳米核相比，具有延长的体内半衰期(例如循环半衰期超过10小时)；(5)与RM NDs相比，具有增强的炎症部位靶向能力。
本发明的第三个目的在于提供一种药物组合物，所述药物组合物包括所述的纳米诱饵，以及药学上或生理学上可接受的载体。
在本发明的一个实施例中，所述载体包括赋形剂、崩解剂、稀释剂、润滑剂、粘合剂、湿润剂、矫味剂、助悬剂、表面活性剂和防腐剂中的一种或多种；所述载体可含有液体，如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如填充剂、崩解剂、润滑剂、助流剂、泡腾剂、润湿剂或乳化剂、矫味剂、pH缓冲物质等。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地，pH约为6-8；所述药物的剂型为片剂、胶囊剂、软胶囊剂、颗粒剂、丸剂、口服液、乳剂、干混悬剂、干浸膏剂或注射剂；所述药物的给药途径可采用：静脉注射、腹腔注射、病灶内注射、口服、局部给药、肌内、皮内、直肠、吸入等方式。
本发明的第四个目的在于提供一种试剂盒，所述试剂盒包括任一项所述纳米诱饵或所述的药物组合物。
本发明的第五个目的在于提供所述的纳米诱饵、所述的药物组合物或所述的试剂盒在制备预防或/和治疗炎性自身免疫性疾病药物中的应用。
在本发明的一个实施例中，所述炎性自身免疫性疾病包括类风湿性关节炎；炎症性肠病、I型糖尿病、系统性红斑狼疮或系统性硬化病。
本发明的上述技术方案相比现有技术具有以下优点：
1.细胞膜包被的纳米诱饵继承了巨噬细胞的表面促炎因子受体，通过受体识别作用直接清除促炎因子，避免了潜在的不良反应。2.细胞膜包被的纳米诱饵继承了巨噬细胞的表 面PD-L1，通过受体识别作用与T细胞结合，直接抑制T细胞活化。3.纳米核(例如PLGA)的引入限制了膜组分的流动，大大提高了纳米诱饵的血清稳定性。4.纳米诱饵通过继承巨噬细胞的表面特异性蛋白，避免被巨噬细胞内吞，从而延长了血液循环。5.纳米诱饵通过继承巨噬细胞的表面特异性蛋白，与炎症部位细胞表达的粘附分子结合，实现了对炎症部位的靶向和粘附。6.相比于现行自身免疫病治疗药物，本申请的纳米诱饵具备长循环、高促炎因子中和效率、高T细胞活化抑制效率、高安全性等优势。
附图说明
为了使本发明的内容更容易被清楚的理解，下面根据本发明的具体实施例并结合附图，对本发明作进一步详细的说明，其中
图1：本发明PRM NDs纳米诱饵制备流程示意图(图1A)；以及Western blot分析下RM、PRM和PRM NDs的促炎因子和PD-L1蛋白条带(图1B)。
图2：本发明透射电镜下的PRM NDs形貌。
图3：本发明PLGA、PRM NVs和PRM NDs的水合粒径及电位。
图4：本发明PRM NVs和PRM NDs在PBS中不同时间点时的粒径。
图5：本发明PRM NDs在体外吸附不同浓度促炎因子的吸附曲线。
图6：本发明PRM NDs与RM NDs作用后的相对促炎因子浓度(反映促炎因子相对去除量)。
图7：本发明PRM NDs与RM NDs作用后剩余促炎因子刺激细胞产生黏附分子(反映细胞活化程度)。
图8：本发明PRM NDs与RM NDs作用后剩余IL-1β刺激细胞凋亡。
图9：本发明PRM NDs在体外吸附不同浓度PD-1的吸附曲线。
图10：本发明PRM NDs与RM NDs作用后的相对PD-1浓度(反映PD-1相对去除量)。
图11：本发明脾脏分离的CD4 +T细胞与PRM NDs或RM NDs共孵育后，细胞的活力变化。
图12：本发明脾脏分离的CD4 +T细胞与PRM NDs或RM NDs共孵育后，细胞的增殖变化。
图13：本发明Western blot分析下RM、PRM和PRM NDs的MAC-1和LFA-1蛋白条带。
图14：本发明 DiDPRM NDs或 DiDRM NDs与活化的内皮细胞、软骨细胞、肠上皮细胞孵育后的荧光图像。
图15：本发明流式细胞术分析 DiDPRM NDs或 DiDRM NDs与活化的内皮细胞、软骨细胞、肠上皮细胞孵育后粘附在细胞上的NDs水平。
图16：本发明静脉注射 DiDPRM NDs的体内循环时间。
图17：本发明静脉注射 DiDPRM NDs靶向炎症关节的活体荧光成像图。
图18：本发明PRM NDs处理后ZIA小鼠的血清中促炎因子和PD-1的蛋白水平。
图19：本发明PRM NDs处理后ZIA小鼠的滑膜中促炎因子和PD-1的蛋白水平。
图20：本发明PRM NDs处理后ZIA小鼠的脾脏中Th1和Th17细胞的比例。
图21：本发明PRM NDs处理后ZIA小鼠膝关节苏木素-伊红、番红-固绿、免疫组化染色图像以及组织学评分和软骨厚度测量。
图22：本发明PRM NDs处理后ZIA小鼠膝关节骨组织微结构的典型微计算机断层扫描(micro-CT)图像以及CT定量的骨体积分数(BV/TV)、骨小梁数(Tb.N)。
图23：本发明PRM NDs处理后UC小鼠的相对体重的变化。
图24：本发明PRM NDs处理后UC小鼠的疾病活动指数的变化。
图25：本发明PRM NDs处理后UC小鼠的结肠代表性图片和结肠长度统计。
图26：本发明PRM NDs处理后UC小鼠的结肠中促炎因子的蛋白水平。
图27：本发明PRM NDs处理后UC小鼠的结肠组织切片的苏木素-伊红染色图像。
图28：本发明PRM NDs处理后UC小鼠的结肠组织切片的角蛋白18免疫荧光染色图像。
图29：本发明PBS或PRM NDs处理后C57/BL6小鼠的代表性血液学参数。
图30：本发明PBS或PRM NDs处理后C57/BL6小鼠的代表性和生化参数。
图31：本发明PBS或PRM NDs处理后C57/BL6小鼠的主要器官的H&E染色图像。各图中，“*”表示p<0.05，“**”表示p<0.01，“***”表示p<0.001。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
本申请中提供了一种细胞膜包被的纳米诱饵，其能够特异性清除促炎因子从而避免促炎因子激活T细胞，并与T细胞表面PD-1结合从而抑制T细胞的活化。尤其是，PRM NDs纳米诱饵在体内具备较长的循环时间，解决了现有技术使用的蛋白质药物(例如抗体药物)血液循环时间短等缺点。
在本申请的一些具体实施方式中，首先将经IFN-γ处理的巨噬细胞通过超声进行裂解，并通过逐级离心得到PRM；再将PRM与PLGA纳米核共同超声2分钟(100W)，得到PRM NDs纳米诱饵。相关实验表明，这种纳米诱饵具备高效而稳定的促炎因子清除能力，并能结合与T细胞表面PD-1结合从而抑制T细胞的活化。在Zymosan A诱导的关节炎(ZIA)和葡聚糖硫酸钠诱导的溃疡性结肠炎(UC)小鼠模型中，使用尾静脉注射方式将PRM NDs纳米诱饵注入小鼠体内，成功下调异常增高的促炎因子和sPD-1，并显著抑制T细胞活化。
纳米诱饵
如本文所用，术语“纳米诱饵”、“细胞膜包被/包裹的纳米诱饵/材料”、可互换使用，是指包含纳米核以及包裹其外的细胞膜的人工合成纳米材料，其可具有伪装仿生以诱捕并去除系统内不利因子或成分的功能。
如本文所用，术语“巨噬细胞”是指由骨髓中的造血干细胞生成的细胞，主要包括单核细胞和巨噬细胞。本申请中的巨噬细胞可经过遗传工程化改造，以在其表面过表达有助于提高促炎因子清除和PD-1结合。
如本文所用，术语“细胞膜”是指获自巨噬细胞或其细胞器的天然存在的生物膜，或经过修饰、改变的具有巨噬细胞全部或部分生物活性的膜。用于本申请的细胞膜可为获自本申请巨噬细胞的细胞膜，其可经分离、去除部分组分(例如脂质、糖链)和/或添加部分组分(例如过表达的PD-L1、其他细胞表面抗原)。
如本文所用，术语“纳米核”是指具有纳米级尺寸的可用于支持本申请细胞膜的任何纳米颗粒。可用于制备本申请纳米诱饵纳米核的材料包括但不限于高分子纳米材料或无机纳米材料，例如聚乳酸-羟基乙酸共聚物(PLGA)、聚乳酸(PLA)、聚乙醇酸(PGA)、聚己酸内酯(PCL)、聚赖氨酸、聚谷氨酸、聚氰基丙烯酸正丁酯(PBCA)、壳聚糖、明胶；金、硅、铁、铜等。
根据所用材料或方法的不同，本申请的纳米诱饵可具有各种适合的形状，例如球形、立方体、圆锥形、圆柱形、棱柱形、棱锥形、或其它规则或不规则的形状。本申请纳米诱饵的尺寸可为1纳米～10微米或其间的任何数值或数值范围，例如10纳米～5微米，500纳米～1微米等。
纳米诱饵的制备方法
本申请中还提供了制备本申请纳米诱饵的方法，所述方法包括：
(A)提供纳米核；(B)提供PD-L1表达的巨噬细胞的细胞膜；(C)使所述细胞膜包裹到所述纳米核上，形成所述纳米诱饵。
本申请的纳米核可采用本领域中已知的各种方法，从原料制得(例如采用纳米沉淀法)，也可直接购自各供应商。纳米核可与细胞膜具有相反的电势，以形成电荷吸引进一步稳定纳米诱饵。
巨噬细胞细胞膜可通过细胞裂解和分离获得，例如所述裂解包括：超声裂解、酶裂解、化学裂解、匀浆裂解和/或低渗溶胀裂解；所述分离包括：离心(例如逐级离心)、沉淀、过滤、磁珠、层析分离。在获得细胞膜之前可对细胞进行采集、培养、工程化改造等处理，以获得具有所需数量和功能的巨噬细胞。
细胞膜应具有一定的结构完整性和保留所需的功能，并能够部分或完全包裹纳米核。优选细胞膜能够完全包裹纳米核以增加纳米诱饵的稳定性。在一些实施方式中，细胞膜具有大于或等于纳米核表面积的尺寸。在一些实施方式中，细胞膜上保留了巨噬细胞细胞膜表面的功能性表位、受体等功能性分子。
细胞膜对纳米核的包被可通过外力的施加实现。例如，可采用声波(例如超声法)、机械力(如机械共挤压)、电能(电穿孔法)、热能(加热法)等方式实现包裹。在一些实施方式中，本申请的方法可包括：通过超声裂解巨噬细胞，并通过逐级离心得到细胞膜；将所得细胞膜与纳米核共同超声，得到纳米诱饵。
药物、药物组合物或试剂盒
本申请还提供了一种药物、药物组合物或试剂盒，其中含有有效量的本申请的纳米诱饵或组分(a)和(b)的组合物，以及药学上可接受的载体。如本文所用，术语“活性物质”或“本申请的活性物质”可互换使用，是指纳米诱饵或组分(a)和(b)的组合物。其中，组分(a)和(b)的组合物中可包含独立存放的组分(a)和组分(b)以及可任选的载剂，在临用前，可将组分(a)和组分(b)与可任选的载剂混合并制备成可供预防和/或治疗用的纳米诱饵药物。在一些实施方案中，所述药物可用于预防和/或治疗促炎因子过度表达及T细胞活化的自身免疫性疾病。例如，本申请的活性物质、包含所述活性物质的产品可用于预防和/或治疗促炎因子过度表达和T细胞活化过度活化导致的类风湿性关节炎和溃疡性结肠炎，例如。
如本文所用，术语“含有”或“包括”包括了“包含”、“基本上由……构成”、和“由……构成”。如本文所用，术语“药学上可接受的”成分是适用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)的，即有合理的效益/风险比的物质。如本文所用，术语“有效量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。如本文所用，术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。该术语指这样一些药剂载体：它们本身并不是必要的活性成分，且施用后没有过分的毒性。合适的载体是本领域普通技术人员所熟知的。在《雷明顿药物科学》(Remington’s Pharmaceutical Sciences，Mack Pub.Co.，N.J.1991)中可找到关于药学上可接受的赋形剂的充分讨论。
在组合物中药学上可接受的载体可含有液体，如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如填充剂、崩解剂、润滑剂、助流剂、泡腾剂、润湿剂或乳化剂、矫味剂、pH缓冲物质等。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地，pH约为6-8。如本文所用，术语“单位剂型”是指为了服用方便，将本申请的组合物制备成单次服用所需的剂型，包括但不限于各种固体剂(如片剂)、液体剂、胶囊剂、缓释剂。应理解，所用活性物质的有效剂量可随待施用或治疗的对象的严重程度而变化。具体情况根据对象的个体情况(例如对象体重、年龄、身体状况、所需达到的效果)来决定，这在熟练医师可以判断的范围内。
本申请的组合物，可以为固态(如颗粒剂、片剂、冻干粉、栓剂、胶囊、舌下含片)或液态(如口服液)或其它合适的形状。给药途径可采用：静脉注射、腹腔注射、病灶内注射、口服、局部给药、肌内、皮内、直肠、吸入等方式。此外，本申请的组合物中还可含有用于改善和治疗破骨细胞过多或功能亢进相关疾病的其它活性物质。例如，所述的其它活性物质选自下组：临床常用破骨细胞抑制剂、抗生素、抗肿瘤剂、抗炎剂等。本申请的纳米诱饵还可以与其它药物和治疗手段联合，例如化疗、放疗、光疗、冷冻疗法、手术、细胞疗法、移植等。
具体示例
本部分中提供了本申请的一些具体实施方式，应理解这些示例并不用于限制本申请的保护范围，而仅为了帮助理解本申请。
在本申请的一些具体实施方式中，提供了一种细胞膜包被的纳米诱饵，其结构为PRM NDs，其中PRM为PD-L1表达的巨噬细胞的细胞膜，PLGA为纳米核。
在本申请的一些具体实施方式中，PRM NDs细胞膜包被的纳米诱饵的制备方法可包括，巨噬细胞经IFN-γ(50ng/mL)刺激48小时后，将巨噬细胞通过超声进行裂解，并通过逐级离心得到PRM；再将PRM与PLGA纳米核共同超声2分钟(100W)，得到PRM NDs 纳米诱饵。
在本申请的一些具体实施方式中，巨噬细胞经IFN-γ(50ng/mL)刺激48小时后，巨噬细胞的细胞膜通过超声和离心进行分离。具体的，将RAW 264.7细胞悬浮于含有20mM Tris·HCl(pH 7.5)，10mM KCl，75mM蔗糖，2mM MgCl 2和蛋白酶/磷酸酶抑制剂的匀浆缓冲液中。用JY 92-IIN匀浆器(75W)破碎悬浮液，然后以20000g离心25分钟收集上清液，并对上清液以100000g离心35分钟收集细胞膜。使用BCA试剂盒测定收集的细胞膜的蛋白质含量。含有约5mg膜蛋白的膜可以从3×10 7个RAW 264.7细胞中提取。
在本申请的一些具体实施方式中，PLGA纳米核通过丙酮挥发进行制备。具体的，将溶有PLGA(10mg/mL)的1mL丙酮滴加到2mL的去离子水中，然后将混合物在露天搅拌直至丙酮完全蒸发。
在本申请的一些具体实施方式中，PRM NDs纳米诱饵通过超声法制备。具体的，将PRM和PLGA纳米核以1:10的质量比用浴超声波仪(Fisher Scientific FS30D，100W)超声处理2分钟。作为一个优点，本申请的PRM NDs纳米诱饵由FDA批准的PLGA和生物来源的细胞膜组成，具有优秀的生物相容性和安全性。
本申请进一步公开了上述细胞膜包被的纳米诱饵在制备抗绝经后骨质疏松药物中的应用。
作为具体示例，本申请制备PRM NDs纳米诱饵的方法如图1A所示。具体制备方法举例为：
(1)将RAW 264.7细胞经IFN-γ(50ng/mL)刺激48小时后，悬浮于含有20mM Tris·HCl(pH 7.5)，10mM KCl，75mM蔗糖，2mM MgCl 2和一片蛋白酶/磷酸酶抑制剂的匀浆缓冲液中。用JY 92-IIN匀浆器(75W)破碎悬浮液，然后以20000g离心25分钟收集上清液，并对上清液以100000g离心35分钟收集细胞膜。使用BCA试剂盒测定收集的细胞膜的蛋白质含量。含有约5mg膜蛋白的膜可以从3×10 7RAW 264.7细胞中提取。
(2)将溶有PLGA(10mg/mL)的1mL丙酮滴加到2mL的去离子水中，然后将混合物在露天搅拌直至丙酮完全蒸发，得到PLGA纳米核。
(3)将PRM和PLGA纳米核以1:10的质量比用浴超声波仪(Fisher Scientific FS30D，100W)超声处理2分钟，得到PRM NDs纳米诱饵。
实施例
下面结合具体实施例，进一步阐述本申请。应理解，这些实施例仅用于说明本申请而不用于限制本申请的范围。本领域技术人员可对本申请做出适当的修改、变动，这些修改和变动都在本申请的范围之内。
下列实施例中未注明具体条件的实验方法，可采用本领域中的常规方法，例如参考《分子克隆实验指南》(第三版，纽约，冷泉港实验室出版社，New York：Cold Spring Harbor Laboratory Press，1989)、《动物细胞培养》(Animal Cell Culture，R.I.Freshney编著，1987)或按照供应商所建议的条件。DNA的测序方法为本领域常规的方法，也可由商业公司提供测试。
除非另外说明，否则百分比和份数按重量计算。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本申请方法中。文中所述的较佳实施方法与材料仅作示范之用。
所有数据均以平均值±标准差表示，并使用Student’s t-test进行统计分析。在*p<0.05时判断两组之间差异显著，在**p<0.01和***p<0.001时差异非常显著。
实施例一、PRM NDs纳米诱饵的制备和表征
按照图1A中所述流程制备本申请的细胞膜包被纳米复合物-PRM NDs纳米诱饵。制备步骤如下：
(1)膜材料的制备：将小鼠单核/巨噬细胞样细胞RAW 264.7(购自中国科学院细胞库，目录号SCSP-5036，培养基为含有10％FBS的DMEM，37℃，5％CO 2)经IFN-γ(50ng/mL)刺激48小时后悬浮于含有20mM Tris·HCl(pH 7.5)、10mM KCl、75mM蔗糖、2mM MgCl 2和蛋白酶/磷酸酶抑制剂(购自Pierce，货号A32953，每片溶于10mL溶液中)的匀浆缓冲液中。用JY 92-IIN匀浆器(75W)破碎悬浮液中的细胞，然后以20000g离心25分钟收集上清液，并对上清液以100000g离心35分钟收集细胞膜。使用BCA试剂盒测定所收集细胞膜的蛋白质含量。可从约3×10 7个RAW 264.7细胞中提取出含有约5mg膜蛋白的膜材料。
(2)纳米核的制备：将溶有聚乳酸-羟基乙酸共聚物-PLGA(所用PLGA购自Sigma-Aldrich，目录号719900，酸封端,lactide:glycolide 50:50,分子量38000-54000)10mg/mL)的1mL丙酮滴加到2mL的去离子水中，然后将混合物在露天搅拌直至丙酮完全蒸发，得到PLGA纳米核。
(3)将PRM和PLGA纳米核以预设的质量比(1:50～1:2)在去离子水中用浴超声波仪(Fisher Scientific FS30D，100W)超声处理2分钟，得到PRM NDs纳米诱饵。
结果显示，经IFN-γ处理后，细胞膜RAW264.7细胞表面表达的PD-L1和促炎因子受体显著上调。在RAW：PLGA的质量比为1:50-2:1时可得到均一的纳米诱饵，而在1:10质量比时，纳米诱饵具备完整的包膜和合适的粒径。所得纳米诱饵在制备后可在室温保存三天而不发生沉淀或粒径改变。
如下对巨噬细胞膜和所得细胞膜包被纳米复合物进行表征：
利用Western blot检测RM、PRM和PRM NDs表面促炎因子受体及PD-L1的表达。IFNγR、TNFR、IL-6R、IL-1R和PD-L1一抗的浓度为1:1000，HRP标记的二抗的浓度为1:500，具体参见图1。如图1所示，经IFN-γ处理后，PRM上的促炎因子受体(TNFR、IL-6R、IL-1R、IFNγR)及PD-L1的表达明显高于RM。且包裹在纳米核上的PRM具有与未包被的PRM基本相同的表面标志物表达，提示其与天然细胞可具有相似的膜功能。
用乙酸双氧铀(0.2wt％)染色后，使用透射电子显微镜(TEM，TECNAI G2，FEI，US)观察PRM NDs纳米诱饵的形态。具体参见图2。如图2所示，纳米诱饵呈球状结构，并具有可辨识的清晰膜结构。
使用Zetasizer Nano ZS90(Malvern Instruments，Ltd.，UK)测定纳米诱饵的流体动力学尺寸和zeta电位。具体参见图3。如图3所示，纳米诱饵的流体动力学尺寸约为115.4nm，zeta电位约为-45.6mV。
通过测量PRM NDs在PBS中的粒径来评估纳米诱饵的稳定性。具体参见图4。如图4所示，PRM NVs的粒径随时间变化发生明显变化，而PRM NDs粒径基本维持不变。该结果表明刚性PLGA纳米核限制了包裹其外的膜组分的流动，使得整个PRM NDs在PBS中具有改善的稳定性。
综上，通过本申请的方法制得了包含PLGA纳米核和有效包裹该纳米核的PD-L1表达的巨噬细胞细胞膜的稳定纳米诱饵，其能够在表面上提供与相应天然细胞类似的表面蛋白，例如细胞因子结合受体和PD-L1。
实施例二、PRM NDs对促炎因子的体外清除作用和对促炎因子介导的细胞活化和细胞凋亡的抑制作用
为探讨PRM NDs对TNF-α、IL-1β、IL-6和IFN-γ的清除效率，将这些重组因子(8ng/mL)与PRM NDs在含10％胎牛血清的DMEM中混合，最终浓度(0～2mg PLGA/mL)。在37℃孵育2h后，16100g离心15min，使用ELISA试剂盒测定上清液中各促炎因子的浓度。用Graphpad Prism 8绘制结合动力学剖面。具体参见图5，如图5所示，PRM NDs可以有效地清除TNF-α、IL-6、IL-1β和IFN-γ，IC50(半数最大抑制浓度)分别为666.4、164.8、1410.7和90.5μg PLGA/mL。
为了比较PLGA NPs、RBCM NDs、RM NDs、PRM NDs等和PRM NVs的清除能力，将TNF-α、IL-1β、IL-6或IFN-γ(2ng/mL)与RM NDs或PRM NDs(2mg PLGA/mL)在含10％FBS的DMEM中混合，在37℃温育2小时。然后以16100g离心10分钟。使用ELISA试剂盒定量上清液中各因子的浓度。具体参见图6，如图6所示，PRM NDs展现出明显高于RM NDs的清除效率，而空载体PLGA NPs和RBCM NDs纳米诱饵则几乎没有清除效果。以上结果共同表明，PRM NDs比RM NDs具备更强力的促炎因子清除效果，且PRM NDs额外具备清除PD-1的能力。
本发明通过测定ICAM-1的表达水平，研究了PRM NDs在体外抑制促炎因子诱导的血管内皮细胞、软骨细胞和肠道上皮细胞活化的能力。将促炎因子(TNF-α、IL-6、IL-1β或IFN-γ，100ng/mL)与RM NDs或PRM NDs(2mg PLGAmL -1)在含10％FBS的细胞培养基中进行共孵育。混合物在37℃下孵育2小时，然后在16,000g下离心15分钟以去除NDs。然后，收集上清液并与HUVEC细胞(人脐静脉内皮细胞)、C28/I2细胞(人软骨细胞)和Caco-2细胞(人类克隆的结肠腺癌细胞，其结构和功能与肠细胞相似)孵育6小时，然后通过Western blot测定ICAM-1的表达水平。具体参见图7，如图7所示，当与PRM NDs共同培养时，HUVEC、C28/I2和Caco-2细胞中ICAM-1的上调被禁止，且PRM NDs显示出比RM NDs更强的抑制作用。由此，本申请的PRM NDs可有效抑制细胞因子诱导的细胞激活，因为PRM上更高的受体水平可以清除更多的细胞因子。
通过流式细胞术检测了PRM NDs对IL-1β介导的C28/I2细胞凋亡的抑制能力。将RM NDs或PRM NDs(2mg PLGA/mL)与IL-1β(100ng/mL)在含10％FBS的细胞培养液中在37℃下孵育2小时，然后将混合物在16,000g下离心15分钟以去除NDs，将上清液与C28/I2细胞孵育48小时。随后将细胞用0.25％EDTA/胰蛋白酶消化，用PBS清洗。随后细胞经Annexin V-FITC染色，并进行流式细胞仪分析。具体参见图8，如图8所示，用IL-1β处理48小时后，C28/I2细胞的凋亡率为25.0％，而与PRM NDs共同培养后，凋亡率降低到14.5％，此外PRM NDs显示出比RM NDs更强的抗凋亡作用，这与PRM NDs更高的IL-1β清除效率相一致。由此，本申请的PRM NDs可以有效消除促炎因子从而缓解炎症引起的细胞活化，并保护细胞免受凋亡。
实施例三、PRM NDs对PD-1的清除作用及对CD4 +T细胞的影响
为探讨PRM NDs对PD-1的清除效率，将这些重组PD-1(8ng/mL)与PRM NDs在含 10％胎牛血清的DMEM中混合，最终浓度(0～2mg PLGA/mL)。在37℃孵育2h后,16100g离心15min,使用ELISA试剂盒测定上清液中PD-1的浓度。用Graphpad Prism 8绘制结合动力学剖面。具体参见图9，如图9所示，PRM NDs可以有效地清除PD-1，IC50(半数最大抑制浓度)为106.7μg PLGA/mL。
为了比较空载体PLGA NPs、RBCM NDs、RM NDs、PRM NDs和PRM NVs的清除能力，将PD-1(2ng/mL)与RM NDs或PRM NDs(2mg PLGA/mL)在含10％FBS的DMEM中混合，在37℃温育2小时。然后以16100g离心10分钟。使用ELISA试剂盒定量上清液中PD-1的浓度。具体参见图10，如图10所示，PRM NDs展现出高效的PD-1清除效率，而空载体PLGA NPs和RM NDs则几乎没有结合效果。以上结果共同表明，PRM NDs具备清除PD-1的能力。
为了评估PRM NDs纳米诱饵对CD4 +T细胞增殖的抑制作用。首先从健康小鼠的脾脏中分离得到CD4 +T细胞，随后用板结合anti-CD3(1μg/mL),anti-CD28(5μg/mL)和IL-2(10ng/mL)处理细胞48h使其活化。然后，将活化的CD4 +T细胞(1×10 6)与PRM NDs或RM NDs在含10％胎牛血清的细胞培养液中按不同PLGA浓度孵育48h。细胞活力使用CellTiter-Lumi TMPlus荧光检测试剂盒测试，结果以未接受NDs处理的对照细胞的存活率百分比表示。具体参见图11。在平行实验中，为了评估CD4+T细胞的增殖，将活化的CD4 +T细胞(1×10 6)用CFSE(5μM)在37℃下染色20分钟，并通过加入5倍体积的含10％FBS的冷培养基以停止反应。将细胞与PRM NDs或RM NDs(100μg PLGA mL -1)在含10％FBS的细胞培养基中培养48小时，随后用流式细胞术进行分析。具体参见图12。结果显示，PRM NDs可以有效抑制CD4 +T细胞的增殖，CFSE染色后细胞分裂比例明显降低。此外，经PRM NDs治疗后，CD4 +T细胞的活力显著降低。然而，RM NDs对CD4 +T细胞的活性和增殖的影响微不足道，这是由于未处理的巨噬细胞膜上的PD-L1数量极少。由此，本申请的PRM NDs可通过有效恢复PD-1/PD-L1抑制轴抑制T细胞激活，从而抑制T细胞增殖。
实施例四、PRM NDs的细胞粘附实验
利用Western blot检测RM、PRM和PRM NDs表面LFA-1和MAC-1水平表达。LFA-1和MAC-1一抗的浓度为1:1000，HRP标记的二抗的浓度为1:500。具体参见图13。如图13所示，经IFN-γ处理后，PRM上的LFA-1和MAC-1的表达明显高于RM。
本发明通过激光共聚焦显微镜和流式细胞术研究了PRM NDs和RM NDs对炎症细胞的粘附能力。如上述所述将DiD封装在PLGA NPs中，随后在 DiDPLGA NPs上进一步涂上细胞膜。将HUVEC细胞接种于12孔板(1×10 5个细胞/孔)，培养过夜。细胞用LPS处理(300ng/mL)6h后用PBS冲洗，并用1％BSA封闭1h。随后用各种 DiDPLGA NPs( DiDRBCM NDs、 DiDRM NDs和 DiDPRM NDs)与细胞在包含10％FBS的DMEM(0.2mg PLGAmL -1)中在4℃下共孵育1h。随后细胞用PBS洗涤并用流式细胞仪分析。具体参见图14。在平行研究中，用4％多聚甲醛固定细胞，用DAPI(5μg/mL)对细胞核进行染色，并用激光共聚焦显微镜观察。具体参见图15。 DiDRBCM NDs因其缺乏靶向炎症的特定膜蛋白作为对照。结果显示，正如共聚焦激光扫描显微镜图像所显示的， DiDPRM NDs(红色)比 DiDRM NDs和 DiDRBCM NDs对炎性HUVEC、C28/I2和Caco-2细胞显示出更高的结合亲和力，这与PRM上的LFA-1和MAC-1水平高于RM和RBCM的情况一致。流式细胞仪分析进一步证实， DiDPRM NDs 比其他测试的NDs更容易粘附在炎症细胞上。这些结果共同表明，IFN-γ处理可以提高RM上的LFA-1和MAC-1水平，从而增强NDs与炎症细胞的结合能力。由此，本申请的PRM NDs可以增加跨越炎症血管的迁移，并加强它们在炎症微环境中的保留。
实施例五、PRM NDs的体内半衰期和炎症组织靶向的研究
为了证明PRM NDs具备较长的体内循环时间，对PRM NDs在静脉注射后的药代动力学进行研究。雄性C57/BL6小鼠(6-8周，18-20g，购自上海斯莱克实验动物有限责任公司，以每笼四只饲养于洁净室中，随意饮水，12:12小时光暗循环，温度25±1℃。动物实验方案由苏州大学机构动物护理和使用委员会审查和批准)以15mg  DiDPLGA/kg的剂量静脉注射 DiDPLGA NPs或 DiDPRM NDs。在预定的时间点收集血液，并通过分光荧光法(λ ex＝644nm，λ em＝663nm)确定血浆中的 DiDPLGA的含量。计算循环半衰期(t 1/2)。具体参见图16，如图16所示，PRM NDs显示比PLGA(t 1/2＝3.11小时)显著延长的血液循环时间，其t 1/2为12.04小时。该结果提示本申请的PRM NDs纳米诱饵在体内具有较长的循环半衰期，相较于循环半衰期较短的药物(例如蛋白质类药物)，能更有效发挥其治疗作用。
为了证明PRM NDs对炎症组织的靶向能力，对ZIA小鼠进行RM NDs或PRM NDs静脉注射，通过小动物活体成像仪观察炎症关节处的NDs聚集。对雄性C57/BL6小鼠左后膝关节关节腔内注射酵母聚糖A(Zymosan A，20μL，15mg/mL)，建立ZIA小鼠模型，右后膝关节关节腔内注射无菌PBS(20μL)作为假手术对照。诱导后的第一天，以15mg  DiDPLGA/kg的剂量静脉注射 DiDRM NDs或 DiDPRM NDs。在预定的时间点将小鼠麻醉，并通过小动物活体荧光成像仪进行荧光成像。具体参见图17，如图17所示，静脉注射 DiDPRM NDs的小鼠左后膝关节比注射 DiDRM NDs的小鼠显示出更强的荧光强度。经 DiDPRM NDs处理的膝关节荧光强度在注射后4小时达到峰值，是经 DiDRM NDs处理的关节的3.6倍。相比之下，在右后膝关节几乎没有观察到荧光信号。这些结果表明， DiDPRM NDs在发炎的关节中具有更强的聚集性，这与它们与发炎的内皮细胞和软骨细胞的结合亲和力一致。
实施例六、PRM NDs的体内抗炎和免疫抑制能力
在体内检测PRM NDs对类风湿性关节炎相关生化指标的改善。对雄性C57/BL6小鼠进行关节腔内注射酵母聚糖A(Zymosan A，20μL，15mg/mL)，建立ZIA小鼠模型。建模后，每2天以75mg PLGA/kg的剂量静脉注射RM NDs或PRM NDs，并注射等体积PBS做为阴性对照。在第7天，处死小鼠，收集外周血并在4℃以500g离心10分钟以提取血清。使用ELISA试剂盒定量血清中的促炎因子和PD-1水平。具体参见图18，如图18所示，PRM NDs处理的ZIA小鼠中的血清促炎因子浓度相较于RM NDs组明显降低。这与PRM NDs体外较强的促炎因子吸附能力一致。
进一步检测关节滑膜中促炎因子和PD-1的表达水平。将滑膜组织分离并用RIPA裂解液裂解并匀浆，用ELISA试剂盒测得匀浆液中各因子的水平。结具体参见图19，如图19所示，在PRM NDs处理后，滑膜组织中的促炎因子和PD-1均得到大幅度降低，而RM NDs只能降低促炎因子，且不如PRM NDs高效。
同时通过流式细胞术分析小鼠脾脏内CD4 +T细胞亚群来评估PRM NDs的免疫抑制功能。诱导后第7天，牺牲小鼠，收获脾脏，通过细胞过滤器研磨，并分散在含10％FBS的RPMI中，形成单细胞悬浮液。在700g离心5分钟后，收集细胞颗粒，用ACK裂解缓冲液重新悬浮以去除红细胞。剩下的细胞用RPMI清洗，并在含有10％FBS、12-肉豆蔻酸 13-醋酸酯(50ng/mL)、离子霉素(750ng/mL)和GolgiStop(1μL/mL)的RPMI中在37℃下培养5小时。然后用PE-anti-mouse CD4(1:100)在冰上孵育20分钟，用PBS洗涤，重新悬浮在固定/渗透液中，随后用Alexa
Figure PCTCN2022100876-appb-000001
488-anti-mouse IL-17A(1:100)或APC-anti-mouse IFN-γ(1：100)进一步染色，最后用流式细胞术对细胞进行分析。具体参见图20，如图20所示，在PRM NDs处理后，Th1和Th17细胞的占比明显降低，且低于RM NDs治疗组，突出了PRM NDs的抑制CD4 +T细胞活化的功能。
实施例七、PRM NDs对骨组织的组织学影响
对PRM NDs处理后的ZIA小鼠的膝关节骨组织进行组织学分析。在Zymosan诱导后第7天(给药方案同实施例六)，处死小鼠，收获膝关节组织，固定在10％福尔马林缓冲液中，然后在脱钙溶液(14％EDTA)中于室温孵育1个月以进行脱钙。然后，将组织包埋在石蜡中，以8μm的厚度横切片。对切片进行苏木精-伊红、番红-固绿、免疫组化染色，并对切片进行组织学评分和软骨厚度的测量。具体参见图21，如图21所示，PRM NDs治疗后，滑膜增厚和粒细胞浸润明显受到抑制，组织学评分降低。PRM NDs(～124m)处理后软骨厚度(红色)明显高于PBS(～88m)或RM NDs(～103m)处理后。且PRM NDs治疗明显减少了软骨内IL-6的表达。
实施例八、PRM NDs治疗对骨组织的影像学影响
通过micro-CT进一步分析关节完整性的恢复。在Zymosan诱导后的第7天(给药方案同实施例六)，使用micro-CT(Skyscan 1176)扫描ZIA小鼠的左后膝关节标本。获得高分辨率扫描图(9-20mm)(分辨率：8.8mm，源电压：50kV，源电流：500mA，旋转步长：0.7U)。使用CT分析仪软件(Skyscan)重建数据集以获得股骨组织的3D图像并测量形态测量参数。基于重建数据和内部编写的Fiji脚本计算micro-CT扫描的骨侵蚀。该程序确定骨表面和骨内部空间，并填充骨表面的孔隙。选择小梁骨中的感兴趣区域(ROI)用于分析以下形态测量参数，包括：(1)骨体积密度比(BV/TV)，(2)骨小梁数(Tb.N)。具体参见图22，如图22所示，经PRM NDs治疗后，ZIA小鼠表面骨侵蚀明显减轻，骨量密度(BV/TV)基本恢复到正常水平。此外，PRM NDs治疗后ZIA小鼠骨小梁微观结构的破坏受到很大程度的抑制，骨小梁数量(Tb.N)也恢复到正常小鼠的水平。这些结果表明PRM NDs可以有效地防止关节炎期间的骨丢失。
以上试验证据有力证明了PRM NDs纳米诱饵对于类风湿性关节炎治疗存在巨大潜力。
实施例九、PRM NDs治疗对溃疡性结肠炎的缓解作用
本发明进一步研究了PRM NDs对溃疡性结肠炎(UC)的疗效，UC是炎症性肠病(IBD)的一个重要类别，在0-7天内用3％的DSS水喂养小鼠，在第2、4和6天静脉注射PBS、RM NDs或PRM NDs。具体参见图23，如图23所示，在经历DSS诱导的UC小鼠中注意到持续的体重下降，然而，在用PRM NDs治疗后，体重下降被明显抑制。通过计算疾病活动指数(DAI)进一步监测疾病的进展，该指数由体重减轻指数、粪便出血指数和粪便稠度指数之和表示。具体参见图24，如图24所示，PBS处理的小鼠显示出明显高于正常小鼠的DAI，而PRM NDs处理则明显降低了DAI。
结肠长度缩短是DSS诱导的UC的一个典型症状。具体参见图25，如图25所示，DSS喂养使得PBS组小鼠的结肠长度明显缩短至约4厘米，而PRM NDs组小鼠得结肠长度恢复到约6.4厘米，与正常小鼠相似。此外，如图26所示，PRM NDs明显降低了结肠中促 炎因子的水平，包括TNF-α、IFN-γ、IL-1β和IL-6，验证了PRM NDs对UC的强效抗炎作用。此外，如图27所示，结肠切片的组织学分析进一步显示，PRM NDs明显减轻了坏死细胞增多、粘膜损伤、隐窝结构破坏和炎症细胞浸润的情况。最后，结肠上皮的完整性是通过细胞角蛋白18(CK18)的免疫荧光染色来评估的，细胞角蛋白18是一种上皮细胞的标志物，可以在凋亡细胞中被酶解。具体参见图28，如图28所示，经PRM NDs治疗的小鼠的结肠组织中，CK18的表达水平大大高于PBS处理的结肠组织，表明结肠上皮细胞的完整性得到改善。以上结果均证明了PRM NDs增强的促炎因子清除能力可以与对T细胞激活的抑制作用合作，扭转炎症微环境，防止结肠损伤。
实施例十、PRM NDs纳米诱饵的体内生物相容性
将PBS(200μL)或PRM NDs(75mg PLGA/kg，200μL)静脉内注射到雄性C57/BL6小鼠中，给药方案同实施例六。收集血液和主要器官(心、肝、脾、肺和肾)。在Cobas501自动血液分析仪(Roche，USA)上进行血液学评估。具体参见图29。使用BC-5380自动化学分析仪(Mindray，China)测定血清生化参数水平。具体参见图30。将主要器官固定在10％福尔马林中，包埋在石蜡中，以8μm的厚度横切片，用H&E染色，并通过整体透视光学显微镜观察。具体参见图31。
结果显示，PRM NDs处理的小鼠在代表性血液学参数和生化参数上没有异常。在H&E染色的主要器官横截面中，未检测到坏死、炎症、水肿或其他病理症状。这些结果表明全身给药后PRM NDs具有良好的生物相容性，具有高安全性。

Claims (14)

  1. 一种纳米诱饵，其特征在于，包括纳米核和包被所述纳米核的PD-L1表达的巨噬细胞细胞膜；
    其中，所述纳米核选自聚合物纳米颗粒和/或无机纳米颗粒；
    所述巨噬细胞选自天然巨噬细胞、诱导分化形成的巨噬细胞或经基因工程化改造的巨噬细胞。
  2. 根据权利要求1所述的纳米诱饵，其特征在于，所述聚合物纳米颗粒中聚合物选自聚乳酸-羟基乙酸共聚物、聚乳酸、聚乙醇酸、聚己酸内酯、聚赖氨酸、聚谷氨酸、聚氰基丙烯酸正丁酯、壳聚糖和明胶中的一种或多种。
  3. 根据权利要求1所述的纳米诱饵，其特征在于，所述无机纳米颗粒选自由金、硅、铁和铜中任一种物质制备得到的纳米颗粒。
  4. 根据权利要求1所述的纳米诱饵，其特征在于，所述纳米核带负电。
  5. 根据权利要求1所述的纳米诱饵，其特征在于，所述巨噬细胞选自RAW 264.7细胞、人骨髓单核细胞、人骨髓巨噬细胞、人外周血单核细胞、外周血巨噬细胞、ANA-1细胞、J774A.1细胞和THP-1细胞中的一种或多种。
  6. 根据权利要求1所述的纳米诱饵，其特征在于，所述巨噬细胞细胞膜表达巨噬细胞特异性表面标志物选自PD-L1、LFA-1、MAC-1、IL-1R、IFNγR、TNFR和IL-6R中的一种或多种。
  7. 根据权利要求1所述的纳米诱饵，其特征在于，所述巨噬细胞细胞膜与纳米核的质量比为1:100～1:0.1。
  8. 权利要求1-7中任一项所述的纳米诱饵的制备方法，其特征在于，包括以下步骤：通过施加外力使得PD-L1表达的巨噬细胞细胞膜包裹纳米核，形成所述纳米诱饵。
  9. 根据权利要求8所述的制备方法，其特征在于，所述施加外力的方法选自声波法、机械共挤压、电穿孔法或加热法。
  10. 根据权利要求8所述的制备方法，其特征在于，所述PD-L1表达的巨噬细胞细胞膜与纳米核通过共同超声实现包裹，所述超声条件：超声频率50～150W，超声时间1～5min。
  11. 一种药物组合物，其特征在于，所述药物组合物包括权利要求1-7任一项所述 的纳米诱饵，以及药学上或生理学上可接受的载体。
  12. 一种试剂盒，其特征在于，所述试剂盒包括权利要求1-7中任一项所述纳米诱饵或权利要求11所述的药物组合物。
  13. 权利要求1-7任一项所述的纳米诱饵、权利要求11所述的药物组合物或权利要求12所述的试剂盒在制备预防或/和治疗炎性自身免疫性疾病药物中的应用。
  14. 根据权利要求13所述的应用，其特征在于，所述炎性自身免疫性疾病包括类风湿性关节炎、炎症性肠病、I型糖尿病、系统性红斑狼疮或系统性硬化病。
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