WO2022235125A1 - Lipid-biopolymer nanoparticles having antibody and autoantigen bound to surface thereof, and use thereof - Google Patents

Lipid-biopolymer nanoparticles having antibody and autoantigen bound to surface thereof, and use thereof Download PDF

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Publication number
WO2022235125A1
WO2022235125A1 PCT/KR2022/006518 KR2022006518W WO2022235125A1 WO 2022235125 A1 WO2022235125 A1 WO 2022235125A1 KR 2022006518 W KR2022006518 W KR 2022006518W WO 2022235125 A1 WO2022235125 A1 WO 2022235125A1
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nanoparticles
lipid
antibody
autoantigen
bound
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PCT/KR2022/006518
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French (fr)
Korean (ko)
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오유경
박진원
비엣 르쿠옥
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서울대학교 산학협력단
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Priority claimed from KR1020220056180A external-priority patent/KR20220152512A/en
Publication of WO2022235125A1 publication Critical patent/WO2022235125A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • 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

Definitions

  • the present invention relates to a lipid-biopolymer nanoparticle to which an antibody and an autoantigen are bound to a surface, a method for preparing the same, and a use thereof, and the like.
  • the present invention claims priority based on Korean Patent Application No. 10-2021-0058377 filed on May 6, 2021 and Korean Patent Application No. 10-2022-0056180, filed on May 6, 2022, The contents disclosed in the specification and drawings are incorporated herein by reference.
  • Autoimmune diseases are diseases in which an abnormal immune response occurs to normal body tissues or cells. Representatively, Rheumatoid arthritis, Inflammatory Bowel Disease, and Diabetes Mellitus type 1 ), and multiple sclerosis, which not only threatens the quality of life and life of the patient, but also causes great economic and emotional loss to the patient's family as well as society.
  • autoimmune diseases The etiology of most of these autoimmune diseases is unclear, and it is known that various genetic and environmental factors act in a complex way, and each patient has a different etiology. Therefore, in the treatment of autoimmune diseases, rather than achieving a cure by excluding the underlying factors of the patient, appropriate surgery and drug treatment are performed according to the patient's condition and symptoms, thereby alleviating the symptoms and delaying the progression of the disease, thereby improving the patient's life. The main objective is to improve the quality.
  • the etiology of autoimmune diseases is unclear, but as a result, the patient's immune system is characterized by an excessive immune response against abnormal tissues and cells. Attempts have been made to improve the patient's symptoms by inhibiting the response.
  • drug therapy has a number of problems, one of which is that the patient must be treated with the drug for a long period of time, or almost for the rest of his life.
  • Drug therapy for autoimmune diseases is symptomatic and not a fundamental treatment, so to relieve symptoms, you must take drugs continuously.
  • long-term drug use may not only put a psychological burden on the patient, but may also impair the quality of life due to drug interactions and side effects.
  • the currently used drug therapy lowers the systemic immune function, making the patient vulnerable to other infectious diseases and adversely affecting hematopoiesis.
  • antigen-specific immune tolerance is achieved using nanoparticles of a vaccine formulation by encapsulating autoantigens and immunosuppressive agents such as rapamycin in highly biocompatible polymers such as poly(lactic-co-glycolic acid) (PLGA).
  • PLGA poly(lactic-co-glycolic acid)
  • the immune system of our body can be largely divided into innate immune system and adaptive immune system, and the biggest difference between the two is specificity for antigen.
  • the former induces a non-specific but rapid immune response, and the latter shows a specific but delayed immune response.
  • the smooth mediation of these two immune responses maintains an effective and long-term immune response.
  • cells that play a role as mediators of the innate immune response and the adaptive immune response are antigen-presenting cells, and representatively, there are dendritic cells, macrophages, and B cells. . Therefore, it is effective to selectively modulate antigen-presenting cells to induce effective antigen-specific immune tolerance.
  • nanoparticles as a means to regulate the function of antigen-presenting cells in the body and suppress excessive immune responses to autoantigens.
  • nanoparticles capable of effectively performing the above functions without causing side effects in the body have not yet been discovered.
  • fullerene nanoparticles, platinum nanoparticles, manganese nanoparticles, etc. have been studied, but they exhibit toxicity in vivo and have poor biocompatibility.
  • the present invention has been devised to solve the above problems, and the surface is coated with a lipid membrane encapsulated with an immunomodulatory agent, and an autoantigen and antigen-presenting cell-specific antibody are bound to the surface of the biopolymer nanoparticles (antioxidants). This was completed by confirming that the nanoparticles) can prevent, improve, and treat autoimmune diseases by inhibiting excessive immune responses and inducing immune tolerance to autoantigens.
  • an object of the present invention is biopolymer nanoparticles; a lipid film coating the surface of the nanoparticles; an autoantigen bound to the surface of the lipid membrane; And an antigen-presenting cell-specific antibody or fragment thereof bound to the surface of the lipid membrane, to provide an antibody and an autoantigen bound to the surface of the lipid-biopolymer nanoparticles.
  • Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of autoimmune diseases comprising the lipid-biopolymer nanoparticles bound to the surface of the antibody and the autoantigen as an active ingredient.
  • Another object of the present invention is to provide a food composition for the prevention or improvement of autoimmune diseases comprising the lipid-biopolymer nanoparticles bound to the surface of the antibody and autoantigen as an active ingredient.
  • Another object of the present invention is to provide a method for preparing lipid-biopolymer nanoparticles, wherein the antibody and the autoantigen are bound to the surface.
  • the present invention is antioxidant nanoparticles; a lipid film coating the surface of the nanoparticles; an autoantigen bound to the surface of the lipid membrane; And it provides an antigen-presenting cell-specific antibody or fragment thereof bound to the lipid membrane surface, wherein the antibody and the autoantigen are bound to the surface of the lipid-antioxidant nanoparticles.
  • the antioxidant nanoparticles may be one or more selected from the group consisting of polydopamine nanoparticles, tannin nanoparticles, and cerium oxide nanoparticles, but is not limited thereto.
  • the lipid membrane may be pegylated, but is not limited thereto.
  • the lipid membrane is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), phosphorylglycerol (PG) , phosphocholine (PC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide), cholesterol (cholesterol), 1,2-dioleoyl-sn -glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and at least one selected from the group consisting of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) may be included, but is not limited thereto.
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-
  • the molar ratio of DPPC: DPPG may be 1 to 10: 1, but is not limited thereto.
  • the molar ratio of the DPPC: DPPG: DSPE-PEG2000-maleimide may be 100 to 1000: 10 to 500: 1, but is not limited thereto.
  • the antigen-presenting cell-specific antibody or fragment thereof may be an antibody or fragment thereof that specifically binds to dendritic cells or macrophages, but is not limited thereto.
  • the antigen-presenting cell-specific antibody or fragment thereof is CD80, CD86, CD123, CD303, CD304, CD68, CD11b, CD11c, BDCA-1, DC-SIGN, MHCII, F4/80, It may bind to one or more proteins selected from the group consisting of CD206, and CSF1-R, but is not limited thereto.
  • the antigen-presenting cell-specific antibody or fragment thereof is IgG, Fab', F(ab') 2 , Fab, Fv, recombinant IgG (rIgG), single chain Fv (scFv), and It may be at least one selected from the group consisting of diabody, but is not limited thereto.
  • the autoantigen may be a protein capable of inducing an autoimmune disease in an individual, a fragment thereof, or a variant thereof, but is not limited thereto.
  • the autoantigen is collagen, insulin, insulin B chain, proinsulin, myelin protein, myelin basic protein, myelin proteolipid protein, myelin oligodendrocytes It may be a protein derived from one or more selected from the group consisting of myelin oligodendrocyte glycoprotein, Hsp60, and Hsp65, a fragment thereof, or a variant thereof, but is not limited thereto.
  • the autoantigen; And the antigen-presenting cell-specific antibody or fragment thereof may have a thiol group or may be modified to have a thiol group, but is not limited thereto.
  • the lipid membrane comprises a lipid having a maleimide group, and the autoantigen; And the antigen-presenting cell-specific antibody or fragment thereof may be bound to a lipid membrane through binding of a thiol group and a maleimide group, but is not limited thereto.
  • the nanoparticles may further include an immunosuppressant, but is not limited thereto.
  • the immunosuppressant may be encapsulated in the lipid membrane, but is not limited thereto.
  • the immunosuppressive agent may be one or more selected from the group consisting of corticosteroids (Glucocorticoids), calcineurin inhibitors, antimetabolites, mTOR inhibitors, and vitamin D3, but is not limited thereto. .
  • corticosteroids Glucocorticoids
  • calcineurin inhibitors calcineurin inhibitors
  • antimetabolites mTOR inhibitors
  • vitamin D3 vitamin D3
  • the nanoparticles may have a diameter of 50 to 200 nm, but is not limited thereto.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of autoimmune diseases, comprising the lipid-antioxidant nanoparticles bound to the surface of the antibody and the autoantigen as an active ingredient.
  • the present invention provides the use of the antibody and the autoantigen bound to the surface of the lipid-antioxidant nanoparticles for the prevention or treatment of autoimmune diseases.
  • the present invention provides a use of the antibody and the autoantigen bound to the surface of the lipid-antioxidant nanoparticles to induce immune tolerance to the autoantigen.
  • the present invention provides a method for preventing or treating an autoimmune disease, comprising administering the antibody and the autoantigen bound to the surface of the lipid-antioxidant nanoparticles to an individual in need thereof.
  • the present invention provides a method for inducing immune tolerance to an autoantigen, comprising administering the antibody and the autoantigen bound to the surface of the lipid-antioxidant nanoparticles to a subject in need thereof.
  • the present invention provides the use of the lipid-antioxidant nanoparticles bound to the surface of the antibody and the autoantigen for the preparation of a medicament for the prevention or treatment of autoimmune diseases.
  • the present invention provides the use of the antibody and the lipid-antioxidant nanoparticles bound to the surface of the autoantigen for the preparation of an agent for inducing immune tolerance to the autoantigen.
  • the present invention provides a kit for preventing or treating autoimmune diseases, comprising the pharmaceutical composition.
  • the present invention provides a food composition for the prevention or improvement of autoimmune diseases, comprising the lipid-antioxidant nanoparticles bound to the surface of the antibody and the autoantigen as an active ingredient.
  • the food composition includes a health functional food composition.
  • the present invention provides a health functional food for preventing or improving autoimmune diseases, comprising the lipid-antioxidant nanoparticles bound to the surface of the antibody and autoantigen as an active ingredient.
  • the lipid-antioxidant nanoparticles to which the antibody and the autoantigen are bound to the surface may satisfy one or more characteristics selected from the group consisting of, but are not limited thereto:
  • the lipid-antioxidant nanoparticles bound to the surface of the antibody and autoantigen may target lymph nodes or spleen, but are not limited thereto.
  • the autoimmune disease is rheumatoid arthritis, juvenile rheumatoid arthritis, systemic scleroderma, adult Still's disease, systemic lupus erythematosus, atopic dermatitis, Behcet's disease, multiple sclerosis, systemic sclerosis, Sjogren's disease Syndrome, primary biliary cirrhosis, celiac disease, inflammatory bowel disease, type 1 diabetes, autoimmune hemolytic anemia, Goodpasture syndrome, Graves disease, Hashimoto's thyroiditis, hyperthyroidism, myasthenia gravis, pemphigus, vasculitis, encephalomyelitis, pituitary, Vitiligo, asthma, primary biliary cirrhosis, optic nerve myelitis, pemphigus vulgaris, irritable bowel disease, Crohn's disease, colitis, ulcerative colitis, psoriasis, cardiomyopathy, mya
  • the present invention comprises the steps of (S1) producing antioxidant nanoparticles by inducing self-assembly of a biopolymer in a basic environment;
  • the antibody and autoantigen of claim 1 comprising the step of reacting the surface-coated antioxidant nanoparticles with an autoantigen and an antigen-presenting cell-specific antibody or fragment thereof with the surface of the lipid membrane-bound lipid-antioxidation
  • a method for preparing nanoparticles is provided.
  • the biopolymer may be one or more selected from the group consisting of polydopamine, tannin, and cerium oxide, but is not limited thereto.
  • the lipid membrane may be one in which an immunosuppressant is encapsulated, but is not limited thereto.
  • Lipid-antioxidant nanoparticles according to the present invention have autoantigens and antigen-presenting cell-specific antibodies modified on the surface to deliver the autoantigen specifically to antigen-presenting cells, and the surface is coated with a lipid membrane encapsulated with an immunomodulatory agent. Immune tolerance to autoantigens can be induced.
  • the nanoparticles according to the present invention are administered to an animal model of autoimmune disease, they effectively target lymph nodes and spleen, which are immune organs, and effectively inhibit excessive immune activation by antigen-presenting cells, thereby preventing, delaying, and It has been shown to be curable. Therefore, the nanoparticles according to the present invention are expected to be usefully utilized for the prevention or treatment of various autoimmune diseases, including encephalomyelitis.
  • LDPN Lipid-antioxidant nanoparticles
  • PN antioxidant nanoparticles
  • AbaLDPN-MOG pegylated lipid layers
  • PN antioxidant nanoparticles
  • LDPN lipid-antioxidant nanoparticles
  • LDPN-MOG autoantigen-modified lipid-antioxidant nanoparticles
  • AbaLDPN-MOG antibody and autoantigen-modified lipid-antioxidant nanoparticles
  • Figure 6 is flow cytometry the cell binding capacity of the nanoparticles after each treatment of CD80/86-expressing dendritic cells (dendritic cells) with nanoparticles modified with CD80/86-specific recombinant antibody or nanoparticles not modified with the antibody. It is the result of comparison by analysis.
  • Each of the nanoparticles was labeled with fluorescence (Cy5), and the degree of cell binding of the nanoparticles was confirmed through the fluorescence intensity of the cells.
  • FIG. 10 is a diagram showing an in vitro experiment for confirming the interaction inhibitory effect between dendritic cells and T cells of the nanoparticles according to the present invention.
  • the well plate was coated with a dendritic cell surface protein, and the nanoparticles of the present invention and T cells were treated together.
  • FIG. 11 is a result confirming the degree of inhibition of the interaction between dendritic cells and T cells through the IL-2 level in the culture medium when the nanoparticles according to the present invention are treated with T cells under the experimental conditions of FIG. 10 . According to the present invention, it was confirmed that the surface-modified lipid-antioxidant nanoparticles with the antibody block the interaction between dendritic cells and T cells to inhibit IL-2 secretion of T cells.
  • helper T cells CD3 and CD4 double positive; CD4 + T cells
  • cytotoxic T cells by splenocytes when the nanoparticles according to the present invention were treated together with T cells under the experimental conditions of FIG. (CD3 and CD8 double positive; CD8 + T cells) It is the result of confirming that expansion (expansion) is suppressed.
  • 15 is a diagram showing the mechanism of induction of immune-tolerance dendritic cells by nanoparticles according to the present invention.
  • 16 and 17 are results of confirming the distribution of nanoparticles in lymph nodes, which are target organs, by time after subcutaneous administration of the nanoparticles according to the present invention to mice. It was confirmed that the nanoparticles on the surface of which the recombinant antibody was modified had increased lymph node distribution and more continuously accumulated compared to the nanoparticles on which the antibody was not modified.
  • 20A shows an experimental schedule of an autoimmune disease animal model for confirming the humoral immunosuppressive effect of nanoparticles according to the present invention.
  • nanoparticles according to the present invention effectively inhibited the production of autoantibodies (anti-OVA IgG) in the serum of the animal model in an autoimmune disease model using ovalbumin. From this, it can be seen that the nanoparticles of the present invention have an effect of inhibiting ovalbumin-specific humoral immune response.
  • FIG. 21A to 21C are experimental schedules for confirming the prevention (FIG. 21A), progression inhibition (FIG. 21B), and therapeutic effect (FIG. 21C) of the nanoparticles according to the present invention for autoimmune disease (encephalomyelitis).
  • Figures 22a to 22c show changes in body weight over time and clinical trials after administering the nanoparticles according to the present invention to mice with encephalomyelitis for the purpose of preventing (FIG. 22A), inhibiting progression (FIG. 22B), or treating (FIG. 22C) of an autoimmune disease. It is the result of follow-up of adverse symptoms.
  • the nanoparticles according to the present invention were shown to improve the clinical symptoms of the subject and control the body weight to a normal level.
  • FIG. 23a to 23c are after administration of the nanoparticles according to the present invention to mice with encephalomyelitis for the purpose of preventing (FIG. 23A), inhibiting progression (FIG. 23B), or treating (FIG. 23C) of an autoimmune disease, dendritic cells (CD11c + MHCII) + ), macrophages (CD11b + F4/80 + ), helper T cells (CD4 + T cells or helper T cells; CD3 + CD4 + ), and cytotoxic T cells (CD8 + T cells or cytotoxic T cells; CD3 + CD8 + ) is the result of confirming the degree of invasion of the central nervous system. Regulatory T cells were analyzed using CD25 + FoxP3 + as a marker in helper T cells.
  • FIG. 24A to 24C show that when the nanoparticles according to the present invention are administered to mice with encephalomyelitis for the purpose of preventing (FIG. 24A), inhibiting progression (FIG. 24B), or treating (FIG. 24C) of an autoimmune disease, the ratio of regulatory T cells is It is the result of confirming the increase. In particular, it was also confirmed that the nanoparticles can inhibit the proportion of helper T cells expressing IFN- ⁇ or IL-17A that can damage tissues ( FIG. 24A ).
  • 25A to 25C are after administration of the nanoparticles according to the present invention to mice with encephalomyelitis for the purpose of preventing (FIG. 25A), inhibiting progression (FIG. 25B), or treating (FIG. 25C) of an autoimmune disease, cellularity by nanoparticles It is the result of confirming the ability to regulate the immune response. In mice administered with the nanoparticles according to the present invention, it was confirmed that the number of IFN- ⁇ splenocytes activated by the autoantigen (MOG peptide) decreased.
  • MOG peptide autoantigen
  • 26 is a result of confirming the invasion of immune cells into the central nervous system (myelin myelin) by immunofluorescent tissue method after administering the nanoparticles according to the present invention to an animal model of encephalomyelitis for the purpose of inhibiting disease progression.
  • FIG. 27 is a representative view showing the structure of the nanoparticles according to the present invention and the effect of inducing immune tolerance on autoantibodies thereof.
  • the antioxidant nanoparticles according to the present invention have a surface coated with a lipid membrane encapsulated with an immunomodulatory agent, and the surface is modified with autoantigens and antigen-presenting cell-specific antibodies through the lipid membrane.
  • the present invention provides antioxidant nanoparticles; a lipid film coating the surface of the nanoparticles; an autoantigen bound to the surface of the lipid membrane; and an antigen-presenting cell-specific antibody or fragment thereof bound to the surface of the lipid membrane, an antibody and an autoantigen bound to the surface of the lipid-antioxidant nanoparticles.
  • antioxidant nanoparticles refers to nanoparticles having an antioxidant function, and more preferably, nanoparticles capable of trapping active oxygen.
  • the antioxidant nanoparticles according to the present invention are biopolymer-based biopolymer nanoparticles, and may be nanoparticles containing a biopolymer or composed of a biopolymer.
  • a biopolymer is a natural polymer generated from the cells of a living organism or a polymer artificially synthesized by mimicking it, and is characterized by having excellent biocompatibility, biodegradability, and/or sustainability.
  • the nanoparticles are composed of biopolymers, even when administered in vivo, they have excellent biocompatibility and may not cause side effects such as toxicity.
  • Antioxidant nanoparticles according to the present invention preferably polydopamine nanoparticles, tannin nanoparticles, and characterized in that at least one selected from the group consisting of cerium oxide nanoparticles, most preferably polydopamine (polydopamine) nanoparticles can
  • autoimmune diseases refers to diseases caused by the immune system attacking normal cells or normal tissues inside, not by an external antigen. That is, the autoimmune disease refers to a disease caused by the activation of an excessive immune response to an autoantigen. Accordingly, in the present invention, autoimmune diseases include, without limitation, diseases that can be improved/treated by suppressing immune responses to autoantigens as well as diseases caused by an excessive immune response.
  • the autoimmune disease according to the present invention means a disease that can be induced by the production of autoantibodies against the autoantigen modified on the surface of the nanoparticles according to the present invention, and is not limited to specific diseases, but preferably Rheumatoid arthritis, juvenile rheumatoid arthritis, systemic scleroderma, adult Still's disease, systemic lupus erythematosus, atopic dermatitis, Behcet's disease, multiple sclerosis, systemic sclerosis, Sjogren's syndrome, primary biliary cirrhosis, celiac disease, inflammatory bowel disease, Type 1 diabetes, autoimmune hemolytic anemia, Goodpasture's syndrome, Graves' disease, Hashimoto's thyroiditis, hyperthyroidism, myasthenia gravis, pemphigus, vasculitis, encephalomyelitis, pituitary gland, vitiligo, asthma, primary biliary cirrhos
  • the antioxidant nanoparticles according to the present invention are preferably prepared by self-polymerization of a biopolymer under basic conditions, and are not limited to specific shapes or sizes.
  • the antioxidant nanoparticles may have a diameter of 50 to 200 nm, 50 to 150 nm, 50 to 100 nm, 100 to 200 nm, or 150 to 200 nm, but is not limited thereto.
  • the nanoparticles according to the present invention are characterized in that the surface is coated with a lipid membrane.
  • the present inventors have confirmed that when the nanoparticles are coated with a lipid membrane containing a lipid of a specific composition ratio, the stability of the nanoparticles is improved, and the nanoparticles can be stably bound to antibodies and autoantigens.
  • the lipid membrane may be pegylated.
  • the lipid membrane may include a lipid having a maleimide group.
  • the lipid membrane is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), phosphorylglycerol (PG), phosphocholine (PC) , 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide), cholesterol (cholesterol), 1,2-dioleoyl-sn-glycero-3- It may include at least one selected from the group consisting of phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).
  • DOPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • DOPE 1,
  • the 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol may be 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol), but limited thereto doesn't happen
  • the lipid membrane may include 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG).
  • DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • DPPG 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol
  • the molar ratio of DPPC: DPPG is 1 to 10: 1, 1 to 8: 1, 1 to 5: 1, 1-3: 1, 1.5 to 10: 1, 1.5 to 8: 1, 1.5 to 5: 1, 1.5 to 3 : 1, 2 to 10 : 1, 2 to 8 : 1, 2 to 6 : 1, 2 to 4 : 1, or 2 to 3 : 1, but is not limited thereto.
  • the lipid membrane is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), and 1,2-disteroyl-sn -glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide) may be included.
  • DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • DPPG 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol
  • DSPE-PEG2000-maleimide 1,2-disteroyl-sn -glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000]
  • the molar ratio of the DPPC: DPPG: DSPE-PEG2000-maleimide is 100 to 1000: 10 to 500: 1, 100 to 1000: 50 to 500: 1, 200 to 1000: 100 to 500: 1, 300 to 1000: 100 to 500: 1,500 to 1000: 200 to 500: 1, 600 to 1000: 200 to 500: 1, 300 to 900: 100 to 500: 1, 300 to 900: 200 to 500: 1, 300 to 800: 200 to 500: 1, 400 to 800: 200 to 400: 1, 500 to 800: 200 to 400: 1, or 600 to 800: 250 to 350: 1, but is not limited thereto.
  • the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine is represented by Formula 1 below, and the 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) is represented by Formula 2 , the phosphoglycerol (PG) is represented by Formula 3, the phosphocholine (PC) is represented by Formula 4, and the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol) )-2000] (DSPE-PEG2000-maleimide) may be represented by the following Chemical Formula 5, but is not limited thereto.
  • DPPG 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol
  • PG phosphoglycerol
  • PC phosphocholine
  • the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol) )-2000] DSPE
  • nanoparticles according to the present invention are characterized in that the surface is modified (bound, conjugated, supported, or attached) with an antigen-presenting cell-specific antibody or fragment thereof.
  • antibodies refers to specific protein molecules capable of specifically reacting with and binding to a specific antigen or epitope region thereof, and immunoglobulin molecules having antigen-binding ability (e.g., monoclonal antibodies, polyclonal antibodies, etc.), fragments of the immunoglobulin molecules (eg, IgG, Fab', F(ab') 2 , Fab, Fv, recombinant IgG (rIgG), single chain Fv (scFv), or diabodies, etc.) and the like.
  • immunoglobulin molecules having antigen-binding ability e.g., monoclonal antibodies, polyclonal antibodies, etc.
  • fragments of the immunoglobulin molecules eg, IgG, Fab', F(ab') 2 , Fab, Fv, recombinant IgG (rIgG), single chain Fv (scFv), or diabodies, etc.
  • the immunoglobulin molecule has a heavy chain and a light chain, each heavy and light chain comprising a constant region (region) and a variable region, wherein the light and heavy chain variable regions are capable of binding to an epitope of an antigen; a region “complementarity determining region (CDR)”; and four “framework regions” (FRs).
  • the CDRs of each chain are called sequentially CDR1, CDR2, CDR3, typically starting from the N-terminus, and are also identified by the chain in which the specific CDR is located.
  • a complete antibody has a structure with two full-length light chains and two full-length heavy chains, each light chain linked to the heavy chain by a disulfide bond.
  • the antibody may be an animal-derived antibody, a mouse-human chimeric antibody, a humanized antibody, or a human antibody.
  • antibody fragment refers to a functional fragment of the antibody capable of exhibiting the function of the antibody.
  • the antibody according to the present invention is an antibody that specifically binds to an antigen-presenting cell.
  • the antibody is a concept including a recombinant protein that performs a function similar to the antibody. That is, it is sufficient that the antigen-presenting cell-specific antibody according to the present invention is a recombinant protein capable of specifically binding to the antigen-presenting cell-specific protein, and does not necessarily have the structure of the antibody. More preferably, the antigen-presenting cell-specific antibody according to the present invention is an antibody capable of binding to a protein specifically expressed in the antigen-presenting cell.
  • the term “antigen presenting cells (APCs)” refers to a group of cells that process and present antigens so that specific lymphocytes such as T cells can recognize them. do. Natural types of APC include dendritic cells, macrophages, Langerhans cells, and B cells.
  • the antigen-presenting cell-specific antibody or fragment thereof is an antibody or fragment thereof that specifically binds to dendritic cells or macrophages. That is, the antigen-presenting cell-specific antibody or fragment thereof is an antibody or fragment thereof that binds to a protein specifically expressed in dendritic cells or macrophages.
  • the antigen-presenting cell-specific antibody or fragment thereof is sufficient as long as an antibody or fragment thereof capable of specifically binding to dendritic cells or macrophages is not limited to a specific type, and natural antibodies as well as artificially synthesized antibodies (recombinant antibody) can all be used.
  • the antibody or fragment thereof is selected from the group consisting of CD80, CD86, CD123, CD303, CD304, CD68, CD11b, CD11c, BDCA-1, DC-SIGN, MHCII, F4/80, CD206, and CSF1-R It is capable of binding to one or more selected proteins.
  • the antigen-presenting cell-specific antibody or fragment thereof according to the present invention is an antibody or fragment thereof that specifically binds to CD80 and/or CD86. More preferably, the antigen-presenting cell-specific antibody or fragment thereof according to the present invention is a recombinant protein (eg, antibody) or fragment thereof that specifically binds to CD80 and/or CD86, comprising the amino acid sequence of SEQ ID NO: 1 Or, more preferably, it may consist of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto, and variants of the amino acid sequence are included within the scope of the present invention.
  • the cell-penetrating peptide according to the present invention is a functional equivalent of a polypeptide constituting it, for example, some amino acid sequence of the polypeptide is modified by deletion, substitution or insertion, It is a concept including variants capable of functionally the same action as the polypeptide.
  • the recombinant protein (eg, antibody) or fragment thereof that specifically binds to CD80 and/or CD86 is 70% or more, more preferably 80% or more, even more preferably the amino acid sequence of SEQ ID NO: 1 may comprise an amino acid sequence having at least 90% sequence homology, most preferably at least 95% sequence homology.
  • % sequence homology for a polypeptide is determined by comparing two optimally aligned sequences to a comparison region, and a portion of the sequence of the polypeptide in the comparison region is a reference sequence (additional or additional may include additions or deletions (ie, gaps) compared to not including deletions).
  • autoantigens refers to an antigen of the subject itself that stimulates the immune system of the subject to produce autoantibodies. That is, autoantigens are inducers of autoimmune diseases, and autoimmune diseases are induced when an adaptive immune response to the autoantigens occurs. Autoantigens are not targeted by the immune system under normal conditions, but may act as antigens due to lack of immune resistance due to immune or environmental factors. Specific types of autoantigens are not limited, and proteins, peptides, enzyme complexes, ribonucleoproteins, DNA, phospholipids, and the like may all function as autoantigens. The specific type of autoantigen may vary depending on the type of autoimmune disease.
  • MAG Myelin Oligodendrocyte Glycoprotein
  • the types of autoantigens that can cause each autoimmune disease are known in the art (see, for example, Korean Patent Application Laid-Open No. 10-2020-0079507).
  • the autoantigen according to the present invention is sufficient as long as it stimulates the immune system to produce autoantibodies and can induce autoimmune diseases, and is not limited to specific types.
  • the autoantigen bound to the nanoparticles according to the present invention is one capable of inducing an autoimmune disease targeted by the nanoparticles (prevention, improvement, or treatment) by inducing the production of autoantibodies.
  • a person skilled in the art can select an appropriate autoantigen according to the type of autoimmune disease desired or according to the subject to which the nanoparticles will be administered, referring to known knowledge in the art.
  • the autoantigen according to the present invention may be a protein capable of inducing an autoimmune disease in an individual, a fragment thereof, or a variant thereof, but is not limited thereto.
  • the autoantigen is collagen, insulin, insulin B chain, proinsulin, myelin protein, myelin basic protein, myelin proteolipid protein, myelin oligodendrocyte glycoprotein (myelin oligodendrocyte glycoprotein), Hsp60, and may be a protein derived from one or more selected from the group consisting of Hsp65, a fragment thereof, or a variant thereof, but is not limited thereto.
  • the autoantigen; And the antigen-presenting cell-specific antibody or fragment thereof may have a thiol group or may be modified to have a thiol group.
  • the thiol group includes a free thiol group.
  • the thiol group is an autoantigen; And it may be bound to the amine group of the antigen-presenting cell-specific antibody or fragment thereof. That is, the thiol group is an autoantigen; And among the amino acids constituting the antigen-presenting cell-specific antibody or fragment thereof, it may be bound to an amino acid having a primary amine group, such as Lysine (Lys) or Arigine (Arg).
  • the thiol group may be present in the constant region of the light chain of the antibody or fragment thereof, but is not limited thereto.
  • the autoantigen; And antigen-presenting cell-specific antibody or fragment thereof may bind to the lipid membrane of nanoparticles through the thiol group, but is not limited thereto.
  • the bonding may be made through bonding of the thiol group and the maleimide group of the lipid membrane, but is not limited thereto.
  • the nanoparticles according to the present invention are characterized in that they further comprise an immunomodulatory agent, preferably an immunosuppressant agent.
  • the immunomodulatory agent may be encapsulated (bound, captured, or supported) on the lipid membrane coating the surface of the nanoparticles or inside the lipid membrane.
  • Immunosuppressants are drugs that suppress immune function to prevent the immune system from damaging healthy cells or tissues. It is mainly administered to prevent transplant rejection in patients undergoing organ transplantation or stem cell transplantation, and is also used to treat autoimmune diseases. That is, the immunosuppressive agent may induce immune tolerance of the immune system to the autoantigen.
  • the immunosuppressive agent is sufficient as long as it can suppress immune function, and is not limited to specific types, but specific examples include corticosteroids (Glucocorticoids), calcineurin (Calcineurine) inhibitors, antimetabolites, and mTOR inhibitors; and the like.
  • the immunosuppressive agent is dexamethasone, betamethasone, prednisone, prednisolone, methylprednisolone, triamcinolone A, deflazacort. Cyclosporine A), Tacrolimus, MPA (Mycophenolic acid), MMF (Mycophenolate mofetil), Azathioprine, Mizoribine, Everolimus, Rapamycin, Retinoic acid ( Retinoic acid), and may be selected from vitamin D3, but is not limited thereto.
  • the present invention provides a pharmaceutical composition for preventing, improving, or treating autoimmune diseases, comprising the lipid-antioxidant nanoparticles bound to the surface of the antibody and autoantigen according to the present invention as an active ingredient.
  • Such treatment includes inhibiting disease progression of an autoimmune disease.
  • the prevention, improvement, or therapeutic effect of the nanoparticles for autoimmune diseases is achieved by the immunosuppressive effect of the nanoparticles or the immune tolerance inducing effect on the autoantigens.
  • the lipid-antioxidant nanoparticles bound to the surface of the antibody and autoantigen may satisfy one or more characteristics selected from the group consisting of:
  • the nanoparticles when administered to a subject, they can target the lymph node or spleen, that is, migrate to the lymph node or spleen to exert the above effect.
  • Inhibiting the interaction between the antigen-presenting cell and the T cell means inhibiting the activation of the T cell by the antigen-presenting cell, preferably, the activation of the cytotoxic T cell. Furthermore, this includes induction of tolerogenic dendritic cells by the nanoparticles according to the present invention.
  • the (b) suppressing the tissue invasion of immune cells preferably means inhibiting the tissue invasion of dendritic cells, macrophages, and cytotoxic T cells.
  • the nanoparticles are for the purpose of preventing, improving, or treating encephalomyelitis, it means inhibiting the central nervous system invasion of the immune cells.
  • the (e) inflammatory cytokine may be one or more selected from the group consisting of TNF- ⁇ , IL-1 ⁇ , and IL-6, but may include without limitation as long as it is a cytokine that can induce inflammation. That is, the nanoparticles according to the present invention can inhibit the production and secretion of the inflammatory cytokines by activated immune cells.
  • the autoantibody of (f) may preferably be an autoantibody against an autoantigen supported on the nanoparticles. That is, the nanoparticles according to the present invention perform a function of inhibiting the generation of autoantibodies against the autoantigen by inducing immune tolerance against the antigen-presenting cell-specific delivery of the autoantigen.
  • the present invention provides a pharmaceutical composition for immunomodulation, comprising, as an active ingredient, the lipid-antioxidant nanoparticles bound to the surface of the antibody and the autoantigen according to the present invention.
  • the immunomodulation includes the use of immunosuppression against the autoantigen and the use of inducing immune tolerance against the autoantigen.
  • the content of the nanoparticles in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc., for example, 0.0001 to 99.9% by weight, or 0.001 to 50% by weight based on the total weight of the composition may be, but is not limited thereto.
  • the content ratio is a value based on the dry amount from which the solvent is removed.
  • the pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.
  • the excipient may be, for example, at least one selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a humectant, a film-coating material, and a controlled-release additive.
  • the pharmaceutical composition according to the present invention can be prepared according to a conventional method, respectively, in powders, granules, sustained-release granules, enteric granules, liquids, eye drops, elsilic, emulsions, suspensions, spirits, troches, fragrances, and limonaade.
  • tablets, sustained release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusates, Warnings, lotions, pasta, sprays, inhalants, patches, sterile injection solutions, or external preparations such as aerosols can be formulated and used, and the external preparations are creams, gels, patches, sprays, ointments, warning agents , lotion, liniment, pasta, or cataplasma.
  • Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.
  • water diluted hydrochloric acid, diluted sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (Twinester), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc.
  • water diluted hydrochloric acid, diluted sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (Twinester), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone,
  • sucrose solution other sugars or sweeteners may be used, and if necessary, a fragrance, colorant, preservative, stabilizer, suspending agent, emulsifier, thickening agent, etc. may be used.
  • Purified water may be used in the emulsion according to the present invention, and if necessary, an emulsifier, preservative, stabilizer, fragrance, etc. may be used.
  • the suspending agent according to the present invention includes distilled water, aqueous glucose solution, acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906 , a suspending agent such as HPMC 2910 may be used, and if necessary, a surfactant, preservative, stabilizer, colorant, and fragrance may be used.
  • the injection according to the present invention includes distilled water for injection, glucose aqueous solution, 0.9% sodium chloride injection, Ringel injection, dextrose injection, dextrose + sodium chloride injection, PEG (PEG), lactated Ringel injection, ethanol, propylene glycol, non-volatile solvents such as oil-sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; Solubilizing aids such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tweens, nijeongtinamide, hexamine, and dimethylacetamide; Weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, buffers such as album
  • the suppository according to the present invention includes cacao fat, lanolin, witepsol, polyethylene glycol, glycerogelatin, methyl cellulose, carboxymethyl cellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, Lecithin, Lanet Wax, Glycerol Monostearate, Tween or Span, Imhausen, Monolene (Propylene Glycol Monostearate), Glycerin, Adeps Solidus, Butyrum Tego -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydroxote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydro Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium, A, AS, B, C, D, E, I, T, Massa-MF, Masupol, Masupol-15, Neos
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract, for example, starch, calcium carbonate, sucrose ) or lactose, gelatin, etc.
  • excipients for example, starch, calcium carbonate, sucrose ) or lactose, gelatin, etc.
  • lubricants such as magnesium stearate talc are also used.
  • Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc.
  • various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.
  • Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • the nanoparticles according to the present invention are dispersed in distilled water or isotonic solution and administered.
  • the isotonic solution is not limited to a specific type, and may include without limitation as long as it is isotonic with the body fluid of the subject to be administered, but is preferably selected from aqueous glucose solution and physiological saline (NaCl solution).
  • aqueous glucose solution and physiological saline (NaCl solution).
  • NaCl solution physiological saline
  • the concentration (w/w%) of the aqueous glucose solution may be 4 to 6%, and most preferably, the concentration may be 5%, but is not limited thereto.
  • the physiological saline concentration (w/w%) may be 0.5 to 1%, and most preferably, the concentration may be 0.9%, but is not limited thereto.
  • composition according to the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and type of the patient's disease; Sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined.
  • the nanoparticles according to the present invention based on the weight of the antioxidant nanoparticles, 1 to 100 mg / kg, 1 to 90 mg / kg, 1 to 80 mg / kg, 1 to 70 mg relative to the individual weight /kg, 1-60 mg/kg, 1-50 mg/kg, 1-40 mg/kg, 1-30 mg/kg, 1-20 mg/kg, 1-10 mg/kg, 1-5 mg/kg kg, 10-100 mg/kg, 20-100 mg/kg, 30-100 mg/kg, 40-100 mg/kg, 10-80 mg/kg, 10-60 mg/kg, 20-60 mg/kg , 30 to 60 mg / kg, or may be administered in a dose of 40 to 60 mg / kg, but is not limited thereto.
  • the nanoparticles may be administered multiple times until the desired effect is achieved as well as a single administration.
  • the administration method of the nanoparticles according to the present invention may vary depending on the purpose.
  • the nanoparticles according to the present invention when the nanoparticles according to the present invention are administered for the purpose of preventing autoimmune diseases, the nanoparticles may be administered in advance in the absence of symptoms of the disease, and the nanoparticles according to the present invention inhibit the progression of autoimmune diseases
  • the nanoparticles may be administered immediately after the disease is identified.
  • the nanoparticles according to the present invention are administered for the purpose of treating an autoimmune disease, the nanoparticles may be administered immediately after symptoms of the autoimmune disease appear.
  • the pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount capable of obtaining the maximum effect with a minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention pertains.
  • the pharmaceutical composition of the present invention may be administered to an individual by various routes. All modes of administration can be envisaged, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal (intrathecal) injection, sublingual administration, buccal administration, rectal insertion, vaginal It can be administered according to internal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, skin administration, transdermal administration, and the like.
  • the pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with several related factors such as the disease to be treated, the route of administration, the patient's age, sex, weight, and the severity of the disease.
  • “individual” means a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses, cattle, etc. means the mammals of
  • administration means providing a predetermined composition of the present invention to a subject by any suitable method.
  • prevention means any action that suppresses or delays the onset of a target disease
  • treatment means that the target disease and its metabolic abnormalities are improved or It means any action that is advantageously changed
  • improvement means any action that reduces a parameter related to a desired disease, for example, the degree of a symptom by administration of the composition according to the present invention.
  • the "improvement” includes “inhibiting the progression of a disease”.
  • the present invention provides a food composition for preventing or improving autoimmune diseases, comprising the nanoparticles according to the present invention as an active ingredient.
  • the food composition includes a health functional food composition. That is, the main object of the present invention is to provide a functional dietary and pharmaceutical composition comprising the nanoparticles as an active ingredient.
  • the biopolymer nanoparticles of the present invention When the nanoparticles of the present invention are used as food additives, the biopolymer nanoparticles can be added as they are or used together with other foods or food ingredients, and can be appropriately used according to a conventional method.
  • the mixed amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment).
  • the nanoparticles of the present invention may be added in an amount of 15% by weight or less, or 10% by weight or less based on the raw material.
  • the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.
  • the health beverage composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, as in a conventional beverage.
  • the above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol.
  • natural sweeteners such as taumatine and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used.
  • the proportion of the natural carbohydrate is generally about 0.01-0.20 g, or about 0.04-0.10 g per 100 mL of the composition of the present invention.
  • the composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, It may contain a carbonation agent used for carbonated beverages, and the like.
  • the composition of the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The proportion of these additives is not critical, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.
  • health functional food is the same term as food for special health use (FoSHU), and refers to foods with high medical and medical effects processed to efficiently exhibit bioregulatory functions in addition to nutritional supply. Meaning, the food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, pills, etc. to obtain a useful effect in the prevention or improvement of autoimmune diseases.
  • the health functional food of the present invention can be prepared by a method commonly used in the art, and during the manufacture, it can be prepared by adding raw materials and components commonly added in the art.
  • unlike general drugs there are no side effects that may occur when taking the drug for a long period of time by using food as a raw material, and it can be excellent in portability.
  • the present invention provides a kit for preventing, improving, or treating autoimmune diseases, comprising the nanoparticles according to the present invention.
  • the “kit” is not limited to a specific form or type, and a kit of a type commonly used in the art may be used.
  • the kit according to the present invention may further include a storage solution of the nanoparticles, an administration tool, a suspension for administration, etc. in addition to the nanoparticles, and may further include instructions on the characteristics of the nanoparticles, manufacturing methods, etc. can
  • the present invention comprises the steps of (S1) producing antioxidant nanoparticles by inducing self-assembly of a biopolymer in a basic environment;
  • the antibody and autoantigen of claim 1 comprising the step of reacting the surface-coated antioxidant nanoparticles with an autoantigen and an antigen-presenting cell-specific antibody or fragment thereof with the surface of the lipid membrane-bound lipid-antioxidation
  • a method for preparing nanoparticles is provided.
  • the biopolymer in step (S1) is as described above.
  • the biopolymer may be one or more selected from the group consisting of polydopamine, tannin, and cerium oxide, but is not limited thereto.
  • the step (S1) may specifically include the following steps:
  • the biopolymer solution may be preferably selected from the group consisting of polydopamine solution, tannin solution, and cerium oxide solution, preferably polydopamine aqueous solution, more preferably dopamine hydrochloride. It may be a solution.
  • the solvent of the biopolymer solution may be distilled water, but is not limited thereto.
  • the basic solution is used for the purpose of inducing self-assembly of the biopolymer in a basic environment.
  • the specific type of the basic solution is not limited, but may be preferably selected from sodium hydroxide solution, potassium hydroxide solution, calcium hydroxide solution, and the like.
  • the biopolymer solution is titrated to pH 5 to 10, pH 6 to 10, pH 7 to 10, pH 8 to 10, pH 9 to 10, or pH 9.5 to 10 with a basic solution.
  • a basic solution may be, but is not limited thereto.
  • the self-assembly of the step (S1-2) may be accomplished by magnetically stirring the biopolymer solution.
  • the magnetic stirring may be carried out at 10 to 35 °C, 10 to 30 °C, 10 to 27 °C, 15 to 30 °C, 20 to 30 °C, 20 to 27 °C, or 23 to 27 °C, 1 to 40 hours, 1 to 35 hours, 1 to 30 hours, 1 to 25 hours, 5 to 35 hours, 10 to 35 hours, 15 to 35 hours, 15 to 30 hours, 20 to 30 hours, or 20 to 25 hours may be performed, but is not limited thereto.
  • step (S1) of the manufacturing method may optionally further include the following steps after the step (S1-2):
  • the washing of step (S1-3) can be made by suspending the biopolymer nanoparticles in distilled water and then centrifuging, but is not limited thereto, and the nanoparticle washing method known in the art can be used without limitation.
  • the nanoparticle washing method known in the art can be used without limitation.
  • the filtration of step (S1-4) may be made using a polycarbonate filter, but is not limited thereto.
  • the biopolymer nanoparticles may be stored in a suspended state in distilled water, but is not limited thereto.
  • the lipid membrane is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), phosphorylglycerol (PG), phosphocholine ( PC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide), cholesterol (cholesterol), 1,2-dioleoyl-sn-glycero- It may include at least one selected from the group consisting of 3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).
  • DOPC 3-phosphocholine
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanol
  • the lipid membrane is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), and 1,2-disteroyl-sn- It may be prepared by mixing glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide) in the molar ratio described above, dissolving it in an organic solvent and concentrating under reduced pressure.
  • DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • DPPG 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol
  • 1,2-disteroyl-sn- It may be prepared by mixing glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide) in the
  • the organic solvent may be any one or more selected from the group consisting of chloroform, hexane, ethyl acetate, methanol, dichloromethane, carbon tetrachloride, benzene, DMSO and DMF, preferably chloroform and methanol solution,
  • the chloroform and methanol solution is chloroform: methanol (v/v) 1 to 10: 1, 1 to 8: 1, 1 to 6: 1, 1 to 5: 1, 2 to 10: 1, 3 to 10: 1, 2 to 8: 1, 3 to 7: 1, or may be mixed in a ratio of 3 to 6: 1, but is not limited thereto.
  • the lipid membrane and the antioxidant nanoparticles have a weight ratio (w/w) of the lipid membrane: antioxidant nanoparticles of 1 to 20: 27, 1 to 18: 27, 1 to 16: 27, 1 to 14: 27, 1 to 12: 27, 1 to 10: 27, 1 to 8: 27, 1 to 6: 27, or 1 to 4: 27, but is not limited thereto, and the nanoparticles may be coated with the lipid. It is enough to have
  • the lipid membrane may be an immunomodulatory agent, more preferably an immunosuppressive agent encapsulated therein.
  • the immunosuppressant may be encapsulated in the lipid membrane by dissolving each lipid component in an organic solvent during the preparation of the lipid membrane and adding together when mixing.
  • the immunosuppressive agent has a weight ratio (w/w) of the immunosuppressant: antioxidant nanoparticles (preferably polydopamine nanoparticles) of 0.0001 to 0.01: 1, 0.0001 to 0.01: 1, 0.001 to 0.01: 1, or 0.001 to 0.005:1 may be added, but is not limited thereto.
  • step (S3) reacting the antigen-presenting cell-specific antibody or fragment thereof with the nanoparticles is an autoantigen in the suspension of nanoparticles obtained from step (S2);
  • a solution of antigen-presenting cell-specific antibody or fragment thereof may be added and stirred.
  • the stirring may be performed at 10 to 35 °C, 10 to 30 °C, 10 to 27 °C, 15 to 30 °C, 20 to 30 °C, 20 to 27 °C, or 23 to 27 °C, 1 to 30 hours, 1 to 25 hours, 1 to 20 hours, 1 to 15 hours, 5 to 30 hours, 10 to 25 hours, 10 to 20 hours, or 10 to 15 hours, but is not limited thereto.
  • the antigen-presenting cell-specific antibody or fragment thereof has a weight ratio (w/w) of the antibody or fragment: antioxidant nanoparticles (preferably polydopamine nanoparticles) of 0.01 to 1:1, 0.01 to 0.9:1, 0.01 to 0.8:1, 0.01 to 0.7:1, 0.01 to 0.6:1, 0.05 to 1:1, 0.07 to 1:1, 0.09 to 1:1, 0.1 to 1:1, 0.2 to 1:1, 0.3 to 1:1, 0.2 to 0.8:1, 0.2 to 0.6:1, or 0.4 to 0.6:1 may be added, but is not limited thereto.
  • antioxidant nanoparticles preferably polydopamine nanoparticles
  • the autoantigen has a weight ratio (w/w) of the autoantigen: antioxidant nanoparticles (preferably polydopamine nanoparticles) of 0.01 to 1:1, 0.02 to 1:1, 0.05 to 1 : 1, 0.07 to 1:1, 0.09 to 1:1, 0.01 to 0.8:1, 0.01 to 0.6:1, 0.01 to 0.5:1, 0.01 to 0.3:1, 0.05 to 0.3:1, or 0.09 to 0.2: 1 may be added, but is not limited thereto.
  • antioxidant nanoparticles preferably polydopamine nanoparticles
  • the preparation method includes the autoantigen;
  • the method may further include modifying the antigen-presenting cell-specific antibody or fragment thereof to have a thiol group.
  • the method of adding the thiol group is known in the art, but it may be preferably carried out by adding a traut reagent to a solution of the autoantigen or antibody and reacting.
  • Antioxidant nanoparticles (hereinafter, “AbaLDPN-MOG”) were prepared whose surface was coated with a lipid layer containing an immune tolerance-inducing drug, and autoantigens and recombinant antibodies were bound to the surface of the lipid layer.
  • the overall manufacturing process is shown in FIG. 1 .
  • Polydopamine nanoparticles were synthesized through self-polymerization of dopamine under alkaline conditions.
  • dopamine hydrochloride was dissolved in 25 mL of triple distilled water (TDW) to prepare a final concentration of 2 mg/mL, and then the dopamine hydrochloride aqueous solution was titrated to pH 9.68 using 1N sodium hydroxide solution, Magnetic stirring was carried out at room temperature (25 °C) for 24 hours.
  • TDW triple distilled water
  • a black polydopamine nanoparticle precipitate was collected by centrifugation at 13,500 g for 20 minutes, and washed with triple distilled water until the supernatant became transparent.
  • the polydopamine nanoparticles were filtered through a 450 nm polycarbonate filter and stored at 4° C. as a suspension in distilled water.
  • PN or antioxidant nanoparticles polydopamine nanoparticles.
  • 1,2-dipalmitoyl-sn-glycero-3-phosphocholine DPPC
  • 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol DPPG
  • 1,2-disteroyl-sn-glycero-3-phosphoethanolamine -N-[maleimide(polyethyleneglycol)-2000] DSPE-PEG2000-maleimide
  • DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • DPPG 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol
  • DSPE-PEG2000-maleimide 1,2-disteroyl-sn-glycero-3-phosphoethanolamine -N-[maleimide(polyethyleneglycol)-2000]
  • DSPE-PEG2000-maleimide 1,2-disteroyl-sn-glycero-3-phosphoethanolamine -N-[maleimide(poly
  • 1,2-dipalmitoyl-sn-glycero-3-phosphocholine DPPC
  • 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol DPPG
  • 1,2-disteroyl-sn-glycero-3-phosphoethanolamine -N-[maleimide(polyethyleneglycol)-2000] DSPE-PEG2000-maleimide
  • DSPE-PEG2000-maleimide 1,2-disteroyl-sn-glycero-3-phosphoethanolamine -N-[maleimide(polyethyleneglycol)-2000]
  • DSPE-PEG2000-maleimide 1,2-disteroyl-sn-glycero-3-phosphoethanolamine -N-[maleimide(polyethyleneglycol)-2000]
  • DSPE-PEG2000-maleimide 1,2-disteroyl-sn-glycero-3-phosphoethanolamine -N-[maleimide(polyethyleneg
  • the prepared lipid thin film was hydrated by adding the polydopamine aqueous solution prepared in Example 1-1 or an isotonic 5% glucose aqueous solution.
  • the drug-encapsulated lipid-antioxidant nanoparticles (LDPN) were stored at 4°C.
  • Autoantigen myelin oligodendrocyte glycoprotein, MOG
  • traut reagent was added to a final concentration of 2 mg/mL to 10 mg/mL of an autoantigen not containing a thiol group, reacted at 25° C. for about 1 hour, followed by thiolation and purification. Autoantigens that already have a thiol group can be used immediately.
  • the purified autoantigen was treated with a lipid-antioxidant nanoparticle suspension and reacted with nanoparticles at 25 °C for 12 hours, and centrifuged at 13,500 g for 20 minutes to bind autoantigen-bound lipid-antioxidant nanoparticles (LDPN).
  • LDPN autoantigen-bound lipid-antioxidant nanoparticles
  • Recombinant antibody and autoantigen were added to the nanoparticles prepared in Example 1-3 and vigorously stirred.
  • Recombinant antibody (10 mg/mL; CD80/86-specific antibody) and autoantigen (10 mg/mL) were reacted under traut reagent 2 mg/mL conditions, followed by thiolation and purification.
  • a recombinant protein Abatacept (Orencia, Bristol-Myers Squibb; BMS) was used as the CD80/86-specific antibody.
  • Abatacept is a protein in which human cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is fused with the Fc portion of human IgG1, and can specifically bind to CD80 and CD86.
  • CTL-4 cytotoxic T-lymphocyte-associated antigen 4
  • Abatacept has the form of a homodimer, and the monomer is known to have a 357 amino acid sequence (SEQ ID NO: 1).
  • Belatacept (Nulojix, BMS) may be used.
  • Autoantigens or antibodies that already have a thiol group can be used immediately. Purified autoantigens and antibodies were treated with lipid-antioxidant nanoparticle suspension to react with nanoparticles at 25° C.
  • AbaLDPN-MOG lipid-bound autoantigen and recombinant antibody- Antioxidant nanoparticles
  • polydopamine nanoparticles (control) prepared by the method of Example 1-1 and nanoparticles prepared by the method of Examples 1-3, 1-4, or 1-5 were prepared, and 10% fetal calf
  • serum fetal bovine serum
  • serum containing 100 units/ml penicillin the size of AbaLDPN-MOG was measured using a dynamic light scattering method (ELS8000 instrument, Photal, Osaka, Japan).
  • ELS8000 instrument Photal, Osaka, Japan
  • the zeta potential in aqueous solution was measured using laser Doppler electrophoresis, and it was confirmed that there was no significant difference between the respective compositions.
  • the immunomodulatory drug (dexamethasone) encapsulated in AbaLDPN-MOG prepared in Example 1 and the autoantigen bound to the nanoparticles were quantified using high performance liquid chromatography (HPLC, Agilent).
  • nanoparticles are dispersed in methanol, and all drugs encapsulated in the lipid layer are precipitated by ultrasonic dispersion for 30 minutes, followed by centrifugation at 27,000 x g for 20 minutes to dissolve the drug. Only the supernatant was analyzed.
  • the mobile phase was prepared in a composition of tertiary distilled water: acetonitrile in a volume ratio of 70:30, and the detection wavelength was 254 nm.
  • a C18 reverse-phase chromatography column (C18 reverse-phase HPLC column, Phenomenex) was used for the column, and was carried out at 25°C.
  • Example 1 the nanoparticles prepared in Example 1 were freeze-dried, and Fourier transformation-infrared spectroscopy (FT-IR) (FT/IR-400, JASCO) was performed to confirm the lipid thin film coating of the nanoparticles. did. Infrared spectroscopy was measured using attenuated total reflection (ATR). As a result, it was confirmed that a strong peak appeared at 2900 to 3000 cm -1 . This is due to the CH stretching vibration that many fatty acids of phospholipids have. In the case of Raman spectroscopy (LabRAM HR. Evolution, HORIBA), freeze-dried nanoparticles were used similarly to the above and analyzed using a 532 nm laser. As a result, it was confirmed that the D band and G band, which are the characteristics of the polymer compound having aromatic rings, appeared in common in all compositions (FIG. 4).
  • FT-IR Fourier transformation-infrared spectroscopy
  • Example 1 The particle size and shape of AbaLDPN prepared in Example 1 were evaluated, and in the case of elemental analysis, energy dispersive X-ray spectroscopy (EDS) mounted on a transmission electron microscope was used.
  • EDS energy dispersive X-ray spectroscopy
  • the analysis results are shown in FIG. 5 . Specifically, as a result of transmission electron microscopy, it was confirmed that the shape of the particle was similar to the result of size analysis of the nanoparticles using the dynamic light scattering method. In addition, as a result of elemental analysis using EDS, it was confirmed that the detection of carbon, nitrogen, and oxygen, which are major constituent elements of antioxidant nanoparticles, was the most dominant.
  • the detection of phosphorus refers to the phospholipid layer, and the detection of sulfur is by a protein bound to the surface.
  • the binding ability of the LDPN nanoparticles and AbaLDPN-MOG nanoparticles prepared in Example 1 to antigen-presenting cells expressing CD80/86 was confirmed by flow cytometry, confocal microscopy, and transmission electron microscopy, and under ex vivo conditions. Selectivity for target cells was evaluated.
  • the binding ability of LDPN and AbaLDPN-MOG prepared in Example 1 to CD80/86-expressing dendritic cells was confirmed by flow cytometry and confocal microscopy.
  • the nanoparticles were surface-modified with a recombinant antibody capable of specifically binding to CD80/86 according to Example 1.
  • spleen-derived dendritic cells from C57BL/6 (8 weeks old) were obtained using flow cytometry (FACS Aria Sorting).
  • flow cytometry FACS Aria Sorting
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • IL-4 interleukin-4
  • IMDM Iscove's Modified Dulbecco's Medium
  • dendritic cells were treated with Cy5-labeled nanoparticles as polydopamine to a concentration of 400 ⁇ g/mL. After 1 hour, the nanoparticles are removed, and the Cy5 fluorescence intensity of the dendritic cells is checked by flow cytometry.
  • the experimental group treated with AbaLDPN-MOG nanoparticles on the surface of which the antibody was modified exhibited higher fluorescence intensity than the experimental group treated with LDPN-MOG.
  • the above results show that the nanoparticle modification of the recombinant antibody increases the binding capacity of the nanoparticles to dendritic cells.
  • dendritic cells and nanoparticles were cultured in the same manner as above, and cells from which nanoparticles were removed were fixed in 10% formalin/PBS (v/v) for 1 hour. Afterwards, nuclei were stained with Hoechst at 2 ⁇ g/mL for 15 minutes and observed under a microscope.
  • the binding ability of LDPN and AbaLDPN-MOG prepared in Example 1 to CD80/86-expressing dendritic cells was confirmed by transmission electron microscopy.
  • the nanoparticles were surface-modified with a recombinant antibody capable of specifically binding to CD80/86 according to Example 1.
  • nanoparticles were treated to be 400 ⁇ g/mL as polydopamine. After 1 h, each cell was collected and fixed with Karnovsky's solution for 2 h, then washed 3 times with cold 0.05 M sodium carcodylate buffer, and the pellet was washed with 1% osmium tetroxide at 4 °C for 2 h. (osmium tetroxide) was post-fixed. The fixed pellet was washed three times with cold triple distilled water, then stained with 0.5% uranyl acetate at 4°C overnight, and ethanol (30%, 50%, 70%, 80%, 90% and 100%). 3) was dehydrated.
  • the in vitro conditions of FIG. 10 mimic the interaction between dendritic cells and T cells, and are implemented by coating a 96-well plate with anti-CD3 antibody (aCD3Ab) and CD80 Fc fusion protein (CD80 Fc).
  • ACD3Ab and CD80 Fc were each prepared in PBS at a concentration of 10 ⁇ g/mL, covered with a well plate, and stored at 4°C for 12 hours. After 12 hours, when the coating of the well plate is completed, after washing once with PBS, spleen cells prepared from C57BL/6 (8 weeks old) were seeded at a density of 3 ⁇ 10 6 cells per well, and the nanoparticles were 400 as polydopamine. It was processed so that it might become microgram/mL. After 48 hours, the medium in which the cells were cultured was obtained, and IL-2 was detected by performing ELISA (R&D systems, DY402-05) according to the manufacturer's protocol.
  • the group treated with nanoparticles (LDPN-MOG or AbaLDPN-MOG) containing the immunomodulatory drug dexamethasone had IL-2 secretion by T cells compared to the group treated with dexamethasone and antigen directly (Dexa+MOG). was remarkably suppressed (FIG. 11).
  • AbaLDPN-MOG nanoparticles whose surface was modified with a dendritic cell-specific antibody inhibited IL-2 secretion of T cells more effectively than LDPN-MOG on which the antibody was not modified.
  • the antibody-modified lipid-antioxidant nanoparticles according to the present invention effectively block the interaction between T cells and dendritic cell surface proteins coated on a well plate, thereby inhibiting T cell activation. .
  • the nanoparticles according to the present invention inhibited IL-2 secretion of T cells more effectively compared to CTLA-4 Fc used as a positive control.
  • nanoparticles according to the present invention The effect of inhibiting the interaction between dendritic cells and T cells was evaluated through the proliferation and division levels of T cells in the in vitro environment shown in FIG. 10 .
  • the experiment was carried out under the same conditions as in FIG. 10, and similarly, aCD3Ab and CD80 Fc were coated on a 96 well plate and implemented. Cells were washed once with PBS before seeding.
  • Splenocytes were prepared by staining with carboxyfluorescein succinimidyl ester (CFSE).
  • CFSE carboxyfluorescein succinimidyl ester
  • the nanoparticles according to the present invention significantly inhibited the division and proliferation of T cells compared to the control groups (untreated control group, MOG-treated group, dexamethasone and MOG-treated group).
  • control groups untreated control group, MOG-treated group, dexamethasone and MOG-treated group.
  • AbaLDPN-MOG nanoparticles whose surface was modified with a dendritic cell-specific antibody inhibited the division and proliferation of T cells more effectively than LDPN-MOG on which the antibody was not modified.
  • Analysis of T cell expansion index through fluorescence intensity was performed with flowJo v10 program.
  • CD3 and CD4 double positive cells meaning helper T cells; and CD3 and CD8 double-positive cells, which mean cytotoxic T cells, respectively, were analyzed, and it was confirmed that AbaLDPN-MOG nanoparticles whose surface was modified with a dendritic cell-specific antibody effectively inhibited both types of T cells.
  • splenocytes were treated with nanoparticles corresponding to 400 ⁇ g/mL as polydopamine for 1 hour, and the cells were cultured for 72 hours with the nanoparticles removed. After activating the cells by treating the cultured splenocytes with 1 ⁇ g/mL LPS, the cells were checked by flow cytometry 6 hours later. In order to analyze the protein present in the cytoplasm, it was treated with LPS at a concentration of 5 ⁇ g/mL with brefeldin A to prevent the leakage of the protein into the cytoplasm. Fluorescent antibody staining of cells was performed according to the protocol of the corresponding product (BioLegend 424401).
  • CD11c and MHCII Only double-positive cells of CD11c and MHCII were selected from spleen cells, and the mean fluorescence intensity of fluorescence for their target proteins was analyzed.
  • the cells treated with the antioxidant-nanoparticles according to the present invention decreased the levels of CD80, CD86, and MHCII, which are cell membrane proteins related to the activation of dendritic cells, and the inflammatory cytokine TNF- It was confirmed that the secretion of ⁇ , IL-1 ⁇ , and IL-6 was also suppressed. In particular, the effect was more pronounced in cells treated with lipid-antioxidant nanoparticles (AbaLDPN-MOG), the surface of which was modified with a recombinant antibody.
  • the above results show that the nanoparticles according to the present invention can induce immune-tolerance dendritic cells by effectively blocking T cell activation by CD80, CD86, and MHCII in dendritic cells (FIG. 15).
  • Lymph nodes and spleen which are secondary immune organs, are the places where antigen-presenting cells, including dendritic cells, are abundantly distributed, and are major target organs for the nanoparticles for immunomodulation according to the present invention.
  • the present inventors confirmed through preclinical tomographic imaging, flow cytometry and immunofluorescence histology that the nanoparticles were targeted to the secondary immune organs of the animal model when the nanoparticles were subcutaneously injected into an animal model.
  • Cy5-labeled nanoparticles were injected subcutaneously in the lumbar spine of C57BL/6 (8 weeks old). Nanoparticles were administered as polydopamine at 50 mg/kg, and when 6, 24, 48, and 72 hours had elapsed after administration, Cy5 fluorescence intensity in lymph nodes was measured.
  • the spleen-targeting ability of the recombinant antibody (CD80/86-specific recombinant antibody) and autoantigen-modified lipid-antioxidant nanoparticles prepared according to Example 1 on the surface was confirmed by analyzing dendritic cells in the lymph nodes.
  • the nanoparticles according to the present invention were administered to mice in the same manner as in Experimental Example 6-2, and then lymph nodes of the mice were isolated and confirmed by flow cytometry.
  • lymph node targeting ability of the recombinant antibody (CD80/86-specific recombinant antibody) and the lipid-antioxidant nanoparticles modified on the surface of the autoantigen was confirmed by immunofluorescence histology.
  • the lymph nodes of the mouse were isolated and histological analysis was performed using a confocal microscope and an Automated Multimodal Tissue Analysis System (PerkinElmer, Vectra). did.
  • the isolated lymph nodes were fixed in 10% formalin/PBS (v/v) for 24 hours, and then the cryoprotection process was performed in 30% sucrose solution for 24 hours.
  • the tissue embedding medium was OCT compound tissue-TEK, and was stored at -80 °C after processing using liquid nitrogen.
  • the cryosection slide was prepared using a cryosection (Leica), and the thickness of the tissue section was 5 ⁇ m.
  • the efficacy of the nanoparticles according to the present invention was confirmed in an animal model of autoimmune disease using ovalbumin (OVA).
  • OVA ovalbumin
  • the effects of nanoparticles on the humoral immune response were evaluated by analyzing the concentration of autoantibodies in serum, and the modulating effect of the cellular immune response was evaluated by ELISPOT.
  • An autoimmune disease animal model (12 weeks old) using C57BL/6 was constructed so that an immune response to an autoantigen (ovalbumin, OVA), not a pathogenic antigen, occurs.
  • OVA autoantigen
  • CFA complete freund's adjuvant
  • OVA invivogen
  • the concentration of the autoantibody in the body of the animal model was measured.
  • the nanoparticles of the present invention (AbaLDPN-OVA) loaded with an autoantigen (AbaLDPN-OVA) were subcutaneously injected three times with an interval of 7 days, and the tail of the individual every week from the time point 14 days elapsed from the start date of induction of the autoimmune disease model. 10 ⁇ L of blood was obtained from the vein. Blood was well diluted in 5 mM EDTA/PBS aqueous solution containing 10% FBS, and then centrifuged at 2,000 g for 10 minutes to separate red blood cells. The separated serum samples were diluted according to the sensitivity of the ELISA, and ELISA was performed using an OVA-coated well plate.
  • the level of autoantibody (anti-OVA IgG) in the mouse administered with the nanoparticles of the present invention was significantly reduced compared to other controls, in particular, the recombinant antibody was modified on the surface of the lipid - It was confirmed that the level of autoantibodies in the mice receiving the antioxidant nanoparticles decreased more significantly than the mice receiving the nanoparticles in which the antibody was not modified.
  • the above results show that the nanoparticles according to the present invention can effectively suppress the humoral immune response for a long period of time despite continuous antigen exposure, and that the humoral immunosuppressive effect of the nanoparticles surface-modified with antibodies and autoantigens is particularly excellent. back it up
  • the efficacy of the nanoparticles according to the present invention was evaluated using an experimental autoimmune encephalomyelitis (EAE) model. Efficacy evaluation was made by confirming the degree of central nervous system invasion and demyelination of immune cells, including changes in the subject's clinical symptoms (clinical score) and body weight.
  • EAE experimental autoimmune encephalomyelitis
  • the experimental autoimmune encephalomyelitis (EAE) model is an experimental animal model widely used in the study of multiple sclerosis in humans. Fragment peptides 35 to 55 of myelin oligodendrocyte glycoprotein (MOG) present in the nervous system of healthy individuals ( It is a model constructed by inducing an autoimmune response to MOG 35-55 ).
  • MOG myelin oligodendrocyte glycoprotein
  • MOG 35-55 MOG peptide
  • CFA Heat inactivated M. tuberculosis, 4 mg/mL
  • PTX pertussis toxin
  • the administration schedule differed depending on the experimental purpose (prevention, progression inhibition, and treatment of autoimmune diseases).
  • the nanoparticles were administered twice at intervals of 1 week from 2 weeks before inducing the disease model (FIG. 21a).
  • the nanoparticles were administered twice at intervals of 1 week (FIG. 21b), and when symptoms began to appear when the purpose of the treatment of autoimmune disease was to start, the subjects with a clinical score of 0.5 were randomly selected, and the control group and the experimental group , and the nanoparticles were administered by subcutaneous injection 4 times every 3 days to 50 mg/kg as polydopamine ( FIG. 21c ).
  • mice treated with the lipid-antioxidant nanoparticles effectively improved clinical symptoms and normally controlled body weight, and in particular, AbaLDPN-MOG on which the recombinant antibody was modified on the surface of LDPN-MOG was not treated. It was confirmed that it showed more encouraging results compared to that ( FIGS. 22a to 22c ).
  • Antioxidant according to the present invention The ability to improve the central nervous system invasion of immune cells by nanoparticles was evaluated by flow cytometry.
  • nanoparticles were administered to the autoimmune disease model in the same manner as in Experimental Example 8-1, and after 28 days from the start date of the experiment, the spinal cord was extracted from each individual.
  • the spinal cord was isolated into single cells using 1 mg/mL collagenase (Sigma-Aldrich) and a cell strainer (40 ⁇ m) (SPL), and was used after hemolysis of red blood cells in hypotonic buffer.
  • Each immune cell was basically selected to express CD45, CD11c + MHCII + for dendritic cells, CD11b + F4/80 + for macrophages, CD3 + CD4 + for helper T cells, and cytotoxic T
  • CD3 + CD8 + and in the case of regulatory T cells, CD25 + FoxP3 + in helper T cells were analyzed as markers.
  • helper T cells they were analyzed to commonly label CD3 + CD4 + and express either IFN- ⁇ or IL-17A. The labeling method was performed in the same manner as the flow cytometry method.
  • FIGS. 23A-23C The results are shown in FIGS. 23A-23C.
  • the antioxidant nanoparticles according to the present invention were administered for the purpose of prevention, progression inhibition, or treatment of autoimmune diseases, it was confirmed that the invasion of immune cells into the central nervous system was reduced compared to the untreated control group.
  • the effect was more pronounced in the AbaLDPN-MOG administration group than in the LDPN-MOG administration group. More specifically, AbaLDPN-MOG inhibited invasion into the central nervous system most effectively for dendritic cells, macrophages, and cytotoxic T cells, and LDPN-MOG and AbaLDPN-MOG showed similar effects on helper T cells.
  • FIGS. 24a to 24c shows that the antioxidant nanoparticles according to the present invention have an effect of increasing the ratio of regulatory T cells capable of regulating an excessive immune response.
  • the ratio of helper T cells expressing IFN- ⁇ or IL-17A, which can damage tissues was most significantly decreased in the experimental group administered with AbaLDPN-MOG.
  • Antioxidant according to the present invention The ability of nanoparticles to modulate antigen-specific cellular immune response of immune cells was evaluated using ELISPOT.
  • nanoparticles were administered to the autoimmune disease model in the same manner as in Experimental Example 8-1, and splenocytes were extracted from the subjects after 28 days from the start date of the experiment, under the treatment of 5 ⁇ g/mL MOG peptide.
  • ELISPOT (BD, Mouse IFN- ⁇ ELISPOT Set, 551083) was performed. Specific experiments were carried out according to the protocol of the product.
  • the nanoparticles according to the present invention When the antioxidant nanoparticles according to the present invention are administered for the purpose of preventing, inhibiting progression, or treating autoimmune diseases, the nanoparticles activate the splenocytes of each animal model by autoantigen (MOG peptide) to produce IFN- ⁇ . It was confirmed that secretion was reduced. That is, the nanoparticles can effectively inhibit the cellular immune response of immune cells activated by the MOG peptide, and in particular, in the case of lipid-antioxidant nanoparticles modified with recombinant antibody, lipid-antioxidant nanoparticles that are not modified with recombinant antibody It has been shown to more effectively modulate the immune response.
  • MOG peptide autoantigen
  • Antioxidant according to the present invention The ability of the nanoparticles to improve the central nervous system invasion of immune cells and the inhibitory effect of demyelination was analyzed using immunofluorescence histology.
  • nanoparticles were administered to the autoimmune disease model in the same manner as in Experimental Example 8-1, and after 28 days from the start date of the experiment, the spinal cord was extracted from each individual. The extracted spinal cord was treated in the same manner as in Experimental Example 6-3, and immunofluorescence histology was performed.
  • CD45 is a marker of immune cells that invaded the central nervous system
  • MBP myelin basic protein
  • mice receiving the antioxidant nanoparticles according to the present invention showed a remarkably reduced central nervous system invasion of immune cells compared to the untreated control group. appear.
  • the above results show that the antioxidant nanoparticles according to the present invention can protect the myelin sheath in an animal model of encephalomyelitis and regulate the immune response by blocking the invasion of the central nervous system by immune cells.
  • the lipid-antioxidant nanoparticles to which the antibody and the autoantigen according to the present invention are bound to the surface effectively target the spleen or lymph node when administered to a subject, bind specifically to antigen-presenting cells, and are encapsulated in the lipid membrane. It was confirmed that not only effectively delivered the immunosuppressive agent, but also induces immune tolerance to autoantigens ( FIG. 27 ). That is, the nanoparticles according to the present invention can inhibit the generation of autoantibodies against autoantigens, suppress hyperactivity of immune cells, and suppress invasion of normal tissues by immune cells, thus preventing and treating various autoimmune diseases. It is expected to be used for this purpose.
  • Lipid-antioxidant nanoparticles according to the present invention have autoantigens and antigen-presenting cell-specific antibodies modified on the surface to deliver the autoantigen specifically to antigen-presenting cells, and the surface is coated with a lipid membrane encapsulated with an immunomodulatory agent. Immune tolerance to autoantigens can be induced.
  • the nanoparticles according to the present invention are administered to an animal model of autoimmune disease, they effectively target lymph nodes and spleen, which are immune organs, and effectively inhibit excessive immune activation by antigen-presenting cells, thereby preventing, delaying, and It has been shown to be curable. Therefore, the nanoparticles according to the present invention are expected to be usefully utilized for the prevention or treatment of various autoimmune diseases, including encephalomyelitis.

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Abstract

Lipid-antioxidizing nanoparticles according to the present invention have an autoantigen and an antigen-presenting cell-specific antibody modified on the surface thereof, and thus are able to deliver autoantigens specifically to antigen-presenting cells, and are coated on the surface with a lipid membrane encapsulating an immunomodulator, and thus can induce immunotolerance to autoantigens. Particularly, it was confirmed that the nanoparticles according to the present invention, when administered to animal models of autoimmune diseases, can effectively target the lymph nodes and spleen, which are immune organs, to effectively inhibit excessive immune activation by antigen-presenting cells and thereby prevent, delay, and treat the autoimmune diseases. Therefore, the nanoparticles according to the present invention are expected to be effectively utilized for preventing or treating various autoimmune diseases including encephalomyelitis.

Description

항체 및 자가항원이 표면에 결합된 지질-생체고분자 나노입자 및 이의 용도Lipid-biopolymer nanoparticles bound to surfaces of antibodies and autoantigens and uses thereof
본 발명은 항체 및 자가항원이 표면에 결합된 지질-생체고분자 나노입자, 이의 제조방법, 및 이의 용도 등에 관한 것이다. 본 발명은 2021년 5월 6일 출원된 한국특허출원 제10-2021-0058377호 및 2022년 5월 6일 출원된 한국특허출원 제10-2022-0056180호에 기초한 우선권을 주장하며, 해당 출원들의 명세서 및 도면에 개시된 내용은 본 출원에 원용된다.The present invention relates to a lipid-biopolymer nanoparticle to which an antibody and an autoantigen are bound to a surface, a method for preparing the same, and a use thereof, and the like. The present invention claims priority based on Korean Patent Application No. 10-2021-0058377 filed on May 6, 2021 and Korean Patent Application No. 10-2022-0056180, filed on May 6, 2022, The contents disclosed in the specification and drawings are incorporated herein by reference.
자가면역질환 (Autoimmune diseases)은 정상적인 신체 조직이나 세포에 대해 비정상적인 면역반응이 일어나는 질환으로 대표적으로 류마티스성 관절염 (Rheumatoid arthritis), 염증성 장질환 (Inflammatory bowel disease), 제1형 당뇨병 (Diabetes Mellitus type 1), 및 다발성 경화증 (Multiple sclerosis) 등을 포함하며 이는 환자의 삶의 질과 생명을 위협할 뿐만이 아니라 환자의 가족은 물론, 사회적으로도 큰 경제적, 정서적 손실을 야기한다.Autoimmune diseases are diseases in which an abnormal immune response occurs to normal body tissues or cells. Representatively, Rheumatoid arthritis, Inflammatory Bowel Disease, and Diabetes Mellitus type 1 ), and multiple sclerosis, which not only threatens the quality of life and life of the patient, but also causes great economic and emotional loss to the patient's family as well as society.
이러한 자가면역질환의 대부분은 병인이 불분명한데, 다양한 유전적 및 환경적 요인이 복합적으로 작용하여 발생하는 것으로 알려져 있으며 환자 개개인 마다 다른 병인을 가진다. 따라서 자가면역질환의 치료는 환자의 기저 요인을 배제하여 완치를 달성하는 것 보다는 환자의 상태 및 증상에 따라 적절한 수술 및 약물치료를 수행하여, 증상을 완화하고 질환의 진행을 지연시켜 환자의 삶의 질을 개선하려는 방향을 주요 목적으로 하고 있다. The etiology of most of these autoimmune diseases is unclear, and it is known that various genetic and environmental factors act in a complex way, and each patient has a different etiology. Therefore, in the treatment of autoimmune diseases, rather than achieving a cure by excluding the underlying factors of the patient, appropriate surgery and drug treatment are performed according to the patient's condition and symptoms, thereby alleviating the symptoms and delaying the progression of the disease, thereby improving the patient's life. The main objective is to improve the quality.
자가면역질환의 병인은 불명확하나, 결과적으로 환자의 면역계가 비정상적인 조직 및 세포에 대하여 과잉 면역반응을 보이는 것을 특징으로 하므로, 주로 소염진통제 및 스테로이드를 포함하여 다양한 면역억제제를 사용해 우리 몸의 전신적인 면역반응을 저해함으로써 환자의 증상을 개선하는 시도들이 이루어지고 있다. The etiology of autoimmune diseases is unclear, but as a result, the patient's immune system is characterized by an excessive immune response against abnormal tissues and cells. Attempts have been made to improve the patient's symptoms by inhibiting the response.
하지만, 약물치료요법은 다양한 문제점을 갖고 있는데, 그 중 하나는 환자가 장기간, 혹은 거의 평생 동안 약물치료를 받아야 한다는 점이다. 특히 젊은 나이의 환자에게 자가면역질환이 발생한 경우, 이와 같은 문제가 더욱 대두될 수밖에 없다. 자가면역질환의 약물치료요법은 대증요법에 맞춰져 있으며 근본적인 치료는 아니므로 증상을 완화시키기 위해서는 지속적으로 약물을 복용하여야 한다. 그러나 장기적인 약물 복용은 환자의 심리적 부담감을 줄 뿐만이 아니라 약물상호작용 및 부작용 등에 의해 삶의 질을 저해할 수 있다. 또한 현재 사용되는 약물요법은 전신적인 면역기능을 저하시키므로, 환자를 기타 감염질환에 취약하게 만들며 조혈작용에도 악영향을 미친다. However, drug therapy has a number of problems, one of which is that the patient must be treated with the drug for a long period of time, or almost for the rest of his life. In particular, when an autoimmune disease occurs in a young patient, such a problem is inevitable. Drug therapy for autoimmune diseases is symptomatic and not a fundamental treatment, so to relieve symptoms, you must take drugs continuously. However, long-term drug use may not only put a psychological burden on the patient, but may also impair the quality of life due to drug interactions and side effects. In addition, the currently used drug therapy lowers the systemic immune function, making the patient vulnerable to other infectious diseases and adversely affecting hematopoiesis.
따라서 자가면역질환을 치료하는데 있어 자가항원 (self-antigen)에 대한 선택적인 면역반응만을 억제하여 약물요법의 효율을 극대화하고, 약물요법이 갖는 한계점을 극복하려는 시도가 이루어지고 있다. 관련 선행 기술로는 poly(lactic-co-glycolic acid) (PLGA)와 같은 생체적합성이 높은 고분자 내에 자가항원과 Rapamycin과 같은 면역억제제를 봉입하여 백신 제형의 나노입자를 이용하여 항원 특이적 면역 관용을 유발한 연구가 있다 (Proceedings of the National Academy of Sciences of the United States of America 112(2) (2015) E156-E165). 또한, 임상에서도 제1형 당뇨의 경우 프로인슐린 (Proinsulin) 또는 다발성 경화증의 경우 미엘린 염기성 단백질 (Myelin basic protein)과 같은 자가면역질환을 유발 할 수 있는 자가항원을 투여하여, 자가항원에 대한 면역관용을 유도하는 전략들이 시도되고 있다.Therefore, in the treatment of autoimmune diseases, attempts are being made to maximize the efficiency of drug therapy by suppressing only the selective immune response to self-antigens, and to overcome the limitations of drug therapy. In the related prior art, antigen-specific immune tolerance is achieved using nanoparticles of a vaccine formulation by encapsulating autoantigens and immunosuppressive agents such as rapamycin in highly biocompatible polymers such as poly(lactic-co-glycolic acid) (PLGA). There are studies that have prompted (Proceedings of the National Academy of Sciences of the United States of America 112(2) (2015) E156-E165). In clinical practice, autoantigens that can induce autoimmune diseases, such as proinsulin in the case of type 1 diabetes or myelin basic protein in the case of multiple sclerosis, are administered to achieve immune tolerance against autoantigens. Strategies to induce
한편, 우리 몸의 면역체계는 크게 선천성 면역체계 (Innate immune system)와 적응 면역 (Adaptive immune system)으로 나눌 수 있는데, 이 둘의 가장 큰 차이점은 항원에 대한 특이성이다. 전자는 비특이적이지만 신속한 면역반응을 유도하며 후자는 특이적이지만 지연된 면역반응을 보인다. 이 2가지 면역반응의 원활한 매개로 효과적이고 장기적인 면역반응이 유지된다. 이때 선천성 면역반응과 적응 면역반응의 매개자 역할을 하는 세포가 항원제시세포 (Antigen-presenting cells) 이며, 대표적으로 수지상세포 (Dendritic cells), 대식세포 (Macrophages) 및 B 세포 (B cells) 등이 있다. 따라서, 효과적인 항원 특이적 면역관용을 유도하기 위해서는 항원제시세포를 선택적으로 조절하는 것이 효율적이다.On the other hand, the immune system of our body can be largely divided into innate immune system and adaptive immune system, and the biggest difference between the two is specificity for antigen. The former induces a non-specific but rapid immune response, and the latter shows a specific but delayed immune response. The smooth mediation of these two immune responses maintains an effective and long-term immune response. At this time, cells that play a role as mediators of the innate immune response and the adaptive immune response are antigen-presenting cells, and representatively, there are dendritic cells, macrophages, and B cells. . Therefore, it is effective to selectively modulate antigen-presenting cells to induce effective antigen-specific immune tolerance.
한편, 체내에서 항원제시세포의 기능을 조절하고 자가항원에 대한 과잉의 면역반응을 억제하기 위한 수단으로 나노입자를 사용하려는 시도들이 이루어지고 있다. 그러나, 많은 생체 유래 또는 합성 나노물질들이 발견 및 개발되고 있음에도 불구하고, 체내에서 부작용을 일으키지 않으면서 위와 같은 기능을 효과적으로 수행할 수 있는 나노입자는 아직까지 발굴되지 않고 있다. 그 예로 프러렌 나노입자, 백금 나노입자, 망간 나노입자 등이 연구되고 있으나, 이들은 생체내 독성을 나타내며 생체적합성이 좋지 않은 문제가 있다. On the other hand, attempts have been made to use nanoparticles as a means to regulate the function of antigen-presenting cells in the body and suppress excessive immune responses to autoantigens. However, despite the discovery and development of many bio-derived or synthetic nanomaterials, nanoparticles capable of effectively performing the above functions without causing side effects in the body have not yet been discovered. For example, fullerene nanoparticles, platinum nanoparticles, manganese nanoparticles, etc. have been studied, but they exhibit toxicity in vivo and have poor biocompatibility.
따라서, 생체적합성이 높으면서도 항원제시세포에 특이적으로 면역조절제를 전달하여 자가항원에 대한 면역관용을 유도할 수 있는 나노전달체의 개발이 시급한 실정이다.Therefore, there is an urgent need to develop a nanocarrier capable of inducing immune tolerance to autoantigens by delivering an immunomodulatory agent specifically to antigen-presenting cells while having high biocompatibility.
본 발명은 상기와 같은 문제점을 해결하기 위해 안출된 것으로서, 면역조절제 등이 봉입된 지질막으로 표면이 코팅되고, 상기 표면에 자가항원 및 항원제시세포-특이적 항체가 결합된 생체고분자 나노입자 (항산화 나노입자)가 과잉의 면역반응을 저해하고 자가항원에 대한 면역관용을 유도함으로써 자가면역질환 등을 예방, 개선, 및 치료할 수 있음을 확인하여 완성된 것이다.The present invention has been devised to solve the above problems, and the surface is coated with a lipid membrane encapsulated with an immunomodulatory agent, and an autoantigen and antigen-presenting cell-specific antibody are bound to the surface of the biopolymer nanoparticles (antioxidants). This was completed by confirming that the nanoparticles) can prevent, improve, and treat autoimmune diseases by inhibiting excessive immune responses and inducing immune tolerance to autoantigens.
따라서, 본 발명의 목적은 생체고분자 나노입자; 상기 나노입자 표면을 코팅하는 지질막; 상기 지질막 표면에 결합된 자가항원; 및 상기 지질막 표면에 결합된 항원제시세포 특이적 항체 또는 이의 단편을 포함하는, 항체 및 자가항원이 표면에 결합된 지질-생체고분자 나노입자를 제공하는 것이다.Accordingly, an object of the present invention is biopolymer nanoparticles; a lipid film coating the surface of the nanoparticles; an autoantigen bound to the surface of the lipid membrane; And an antigen-presenting cell-specific antibody or fragment thereof bound to the surface of the lipid membrane, to provide an antibody and an autoantigen bound to the surface of the lipid-biopolymer nanoparticles.
본 발명의 다른 목적은 상기 항체 및 자가항원이 표면에 결합된 지질-생체고분자 나노입자를 유효성분으로 포함하는 자가면역질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다. Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of autoimmune diseases comprising the lipid-biopolymer nanoparticles bound to the surface of the antibody and the autoantigen as an active ingredient.
본 발명의 다른 목적은 상기 항체 및 자가항원이 표면에 결합된 지질-생체고분자 나노입자를 유효성분으로 포함하는 자가면역질환의 예방 또는 개선용 식품 조성물을 제공하는 것이다. Another object of the present invention is to provide a food composition for the prevention or improvement of autoimmune diseases comprising the lipid-biopolymer nanoparticles bound to the surface of the antibody and autoantigen as an active ingredient.
본 발명의 또 다른 목적은 상기 항체 및 자가항원이 표면에 결합된 지질-생체고분자 나노입자의 제조방법을 제공하는 것이다.Another object of the present invention is to provide a method for preparing lipid-biopolymer nanoparticles, wherein the antibody and the autoantigen are bound to the surface.
그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 본 발명이 속하는 기술 분야의 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical task to be achieved by the present invention is not limited to the tasks mentioned above, and other tasks not mentioned may be clearly understood by those of ordinary skill in the art to which the present invention belongs from the following description. There will be.
본 발명은 항산화 나노입자; 상기 나노입자 표면을 코팅하는 지질막; 상기 지질막 표면에 결합된 자가항원; 및 상기 지질막 표면에 결합된 항원제시세포 특이적 항체 또는 이의 단편을 포함하는, 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자를 제공한다.The present invention is antioxidant nanoparticles; a lipid film coating the surface of the nanoparticles; an autoantigen bound to the surface of the lipid membrane; And it provides an antigen-presenting cell-specific antibody or fragment thereof bound to the lipid membrane surface, wherein the antibody and the autoantigen are bound to the surface of the lipid-antioxidant nanoparticles.
본 발명의 일 구현예에서, 상기 항산화 나노입자는 폴리도파민 나노입자, 탄닌 나노입자, 및 세륨 산화물 나노입자로 이루어진 군에서 선택된 하나 이상일 수 있으나, 이에 한정되지 않는다.In one embodiment of the present invention, the antioxidant nanoparticles may be one or more selected from the group consisting of polydopamine nanoparticles, tannin nanoparticles, and cerium oxide nanoparticles, but is not limited thereto.
본 발명의 다른 구현예에서, 상기 지질막은 페길화된 것일 수 있으나, 이에 한정되지 않는다.In another embodiment of the present invention, the lipid membrane may be pegylated, but is not limited thereto.
본 발명의 또 다른 구현예에서, 상기 지질막은 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), phosphorylglycerol (PG), phosphocholine (PC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide), 콜레스테롤 (cholesterol), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 및 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)로 이루어진 군에서 선택된 하나 이상을 포함할 수 있으나, 이에 한정되지 않는다.In another embodiment of the present invention, the lipid membrane is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), phosphorylglycerol (PG) , phosphocholine (PC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide), cholesterol (cholesterol), 1,2-dioleoyl-sn -glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and at least one selected from the group consisting of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) may be included, but is not limited thereto.
본 발명의 또 다른 구현예에서, 상기 DPPC : DPPG의 몰비율은 1 내지 10 : 1 일 수 있으나, 이에 한정되지 않는다.In another embodiment of the present invention, the molar ratio of DPPC: DPPG may be 1 to 10: 1, but is not limited thereto.
본 발명의 또 다른 구현예에서, 상기 DPPC : DPPG : DSPE-PEG2000-maleimide의 몰비율은 100 내지 1000 : 10 내지 500 : 1 일 수 있으나, 이에 한정되지 않는다.In another embodiment of the present invention, the molar ratio of the DPPC: DPPG: DSPE-PEG2000-maleimide may be 100 to 1000: 10 to 500: 1, but is not limited thereto.
본 발명의 또 다른 구현예에서, 상기 항원제시세포 특이적 항체 또는 이의 단편은 수지상세포 또는 대식세포에 특이적으로 결합하는 항체 또는 이의 단편일 수 있으나, 이에 한정되지 않는다.In another embodiment of the present invention, the antigen-presenting cell-specific antibody or fragment thereof may be an antibody or fragment thereof that specifically binds to dendritic cells or macrophages, but is not limited thereto.
본 발명의 또 다른 구현예에서, 상기 항원제시세포 특이적 항체 또는 이의 단편은 CD80, CD86, CD123, CD303, CD304, CD68, CD11b, CD11c, BDCA-1, DC-SIGN, MHCII, F4/80, CD206, 및 CSF1-R로 이루어진 군에서 선택된 하나 이상의 단백질에 결합하는 것일 수 있으나, 이에 한정되지 않는다.In another embodiment of the present invention, the antigen-presenting cell-specific antibody or fragment thereof is CD80, CD86, CD123, CD303, CD304, CD68, CD11b, CD11c, BDCA-1, DC-SIGN, MHCII, F4/80, It may bind to one or more proteins selected from the group consisting of CD206, and CSF1-R, but is not limited thereto.
본 발명의 또 다른 구현예에서, 상기 항원제시세포 특이적 항체 또는 이의 단편은 IgG, Fab', F(ab')2, Fab, Fv, 재조합 IgG (rIgG), 단일쇄 Fv (scFv), 및 디아바디 (diabody)로 이루어진 군에서 선택된 하나 이상일 수 있으나, 이에 한정되지 않는다.In another embodiment of the present invention, the antigen-presenting cell-specific antibody or fragment thereof is IgG, Fab', F(ab') 2 , Fab, Fv, recombinant IgG (rIgG), single chain Fv (scFv), and It may be at least one selected from the group consisting of diabody, but is not limited thereto.
본 발명의 또 다른 구현예에서, 상기 자가항원은 개체에서 자가면역질환을 유도할 수 있는 단백질, 이의 단편, 또는 이의 변이체일 수 있으나, 이에 한정되지 않는다.In another embodiment of the present invention, the autoantigen may be a protein capable of inducing an autoimmune disease in an individual, a fragment thereof, or a variant thereof, but is not limited thereto.
본 발명의 또 다른 구현예에서, 상기 자가항원은 콜라겐, 인슐린, 인슐린 B 사슬, 프로인슐린, 미엘린 단백질, 미엘린 염기성 단백질 (myelin basic protein), 미엘린 프로테오리피드 단백질 (myelin proteolipid protein), 미엘린 희소돌기아교세포 당단백질 (myelin oligodendrocyte glycoprotein), Hsp60, 및 Hsp65로 이루어진 군에서 선택된 하나 이상으로부터 유래된 단백질, 이의 단편, 또는 이의 변이체일 수 있으나, 이에 한정되지 않는다.In another embodiment of the present invention, the autoantigen is collagen, insulin, insulin B chain, proinsulin, myelin protein, myelin basic protein, myelin proteolipid protein, myelin oligodendrocytes It may be a protein derived from one or more selected from the group consisting of myelin oligodendrocyte glycoprotein, Hsp60, and Hsp65, a fragment thereof, or a variant thereof, but is not limited thereto.
본 발명의 또 다른 구현예에서, 상기 자가항원; 및 항원제시세포 특이적 항체 또는 이의 단편은 티올기를 갖는 것이거나, 티올기를 갖도록 변형된 것일 수 있으나, 이에 한정되지 않는다.In another embodiment of the present invention, the autoantigen; And the antigen-presenting cell-specific antibody or fragment thereof may have a thiol group or may be modified to have a thiol group, but is not limited thereto.
본 발명의 또 다른 구현예에서, 상기 지질막은 말레이미드기를 갖는 지질을 포함하고, 상기 자가항원; 및 항원제시세포 특이적 항체 또는 이의 단편은 티올기 및 말레이미드기의 결합을 통해 지질막에 결합되는 것일 수 있으나, 이에 한정되지 않는다. In another embodiment of the present invention, the lipid membrane comprises a lipid having a maleimide group, and the autoantigen; And the antigen-presenting cell-specific antibody or fragment thereof may be bound to a lipid membrane through binding of a thiol group and a maleimide group, but is not limited thereto.
본 발명의 또 다른 구현예에서, 상기 나노입자는 면역억제제를 더 포함할 수 있으나, 이에 한정되지 않는다.In another embodiment of the present invention, the nanoparticles may further include an immunosuppressant, but is not limited thereto.
본 발명의 또 다른 구현예에서, 상기 면역억제제는 상기 지질막에 봉입되어 있을 수 있으나, 이에 한정되지 않는다.In another embodiment of the present invention, the immunosuppressant may be encapsulated in the lipid membrane, but is not limited thereto.
본 발명의 또 다른 구현예에서, 상기 면역억제제는 부신피질호르몬 (Glucocorticoids), 칼시뉴린 (Calcineurine) 억제제, 항대사제, mTOR 억제제, 및 비타민 D3로 이루어진 군에서 선택된 하나 이상일 수 있으나, 이에 한정되지 않는다.In another embodiment of the present invention, the immunosuppressive agent may be one or more selected from the group consisting of corticosteroids (Glucocorticoids), calcineurin inhibitors, antimetabolites, mTOR inhibitors, and vitamin D3, but is not limited thereto. .
본 발명의 또 다른 구현예에서, 상기 나노입자는 직경이 50 내지 200 nm일 수 있으나, 이에 한정되지 않는다.In another embodiment of the present invention, the nanoparticles may have a diameter of 50 to 200 nm, but is not limited thereto.
또한, 본 발명은 상기 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자를 유효성분으로 포함하는, 자가면역질환의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of autoimmune diseases, comprising the lipid-antioxidant nanoparticles bound to the surface of the antibody and the autoantigen as an active ingredient.
또한, 본 발명은 상기 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자의 자가면역질환의 예방 또는 치료 용도를 제공한다. 또한, 본 발명은 상기 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자의 자가항원에 대한 면역관용 유도 용도를 제공한다.In addition, the present invention provides the use of the antibody and the autoantigen bound to the surface of the lipid-antioxidant nanoparticles for the prevention or treatment of autoimmune diseases. In addition, the present invention provides a use of the antibody and the autoantigen bound to the surface of the lipid-antioxidant nanoparticles to induce immune tolerance to the autoantigen.
또한, 본 발명은 상기 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자를 이를 필요로 하는 개체에 투여하는 단계를 포함하는, 자가면역질환의 예방 또는 치료방법을 제공한다. 또한, 본 발명은 상기 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자를 이를 필요로 하는 개체에 투여하는 단계를 포함하는, 자가항원에 대한 면역관용 유도 방법을 제공한다.In addition, the present invention provides a method for preventing or treating an autoimmune disease, comprising administering the antibody and the autoantigen bound to the surface of the lipid-antioxidant nanoparticles to an individual in need thereof. In addition, the present invention provides a method for inducing immune tolerance to an autoantigen, comprising administering the antibody and the autoantigen bound to the surface of the lipid-antioxidant nanoparticles to a subject in need thereof.
또한, 본 발명은 자가면역질환의 예방 또는 치료용 약제의 제조를 위한 상기 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자의 용도를 제공한다. 또한, 본 발명은 자가항원에 대한 면역관용 유도제의 제조를 위한 상기 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자의 용도를 제공한다.In addition, the present invention provides the use of the lipid-antioxidant nanoparticles bound to the surface of the antibody and the autoantigen for the preparation of a medicament for the prevention or treatment of autoimmune diseases. In addition, the present invention provides the use of the antibody and the lipid-antioxidant nanoparticles bound to the surface of the autoantigen for the preparation of an agent for inducing immune tolerance to the autoantigen.
또한, 본 발명은 상기 약학적 조성물을 포함하는, 자가면역질환의 예방 또는 치료용 키트를 제공한다.In addition, the present invention provides a kit for preventing or treating autoimmune diseases, comprising the pharmaceutical composition.
또한, 본 발명은 상기 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자를 유효성분으로 포함하는, 자가면역질환의 예방 또는 개선용 식품 조성물을 제공한다. 상기 식품 조성물은 건강기능식품 조성물을 포함한다.In addition, the present invention provides a food composition for the prevention or improvement of autoimmune diseases, comprising the lipid-antioxidant nanoparticles bound to the surface of the antibody and the autoantigen as an active ingredient. The food composition includes a health functional food composition.
또한, 본 발명은 상기 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자를 유효성분으로 포함하는, 자가면역질환의 예방 또는 개선용 건강기능식품을 제공한다.In addition, the present invention provides a health functional food for preventing or improving autoimmune diseases, comprising the lipid-antioxidant nanoparticles bound to the surface of the antibody and autoantigen as an active ingredient.
본 발명의 일 구현예에서, 상기 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자는 하기로 이루어진 군에서 선택된 하나 이상의 특징을 만족할 수 있으나, 이에 한정되지 않는다:In one embodiment of the present invention, the lipid-antioxidant nanoparticles to which the antibody and the autoantigen are bound to the surface may satisfy one or more characteristics selected from the group consisting of, but are not limited thereto:
(a) 항원제시세포 및 T 세포의 상호작용을 억제함;(a) inhibit the interaction of antigen-presenting cells and T cells;
(b) 면역세포의 조직 침습을 억제함;(b) inhibiting tissue invasion of immune cells;
(c) 보조 T 세포, 세포독성 T 세포, 수지상세포, 및 대식세포로 이루어진 군에서 선택된 하나 이상의 수준 또는 활성을 감소시킴;(c) reducing the level or activity of one or more selected from the group consisting of helper T cells, cytotoxic T cells, dendritic cells, and macrophages;
(d) 조절 T 세포의 수준 또는 활성을 증가시킴;(d) increasing the level or activity of regulatory T cells;
(e) 염증성 사이토카인의 수준 또는 활성을 억제함; 및(e) inhibiting the level or activity of an inflammatory cytokine; and
(f) 자가항체의 생성을 억제함.(f) Inhibits the production of autoantibodies.
본 발명의 다른 구현예에서, 상기 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자는 림프절 또는 비장을 표적화 (targeting)할 수 있으나, 이에 한정되지 않는다.In another embodiment of the present invention, the lipid-antioxidant nanoparticles bound to the surface of the antibody and autoantigen may target lymph nodes or spleen, but are not limited thereto.
본 발명의 또 다른 구현예에서, 상기 자가면역질환은 류마티스 관절염, 소아 류마티스 관절염, 전신성 경피증, 성인 스틸씨병, 전신 홍반성 루푸스, 아토피 피부염, 베체트병 (Behcet's disease), 다발성 경화증, 전신성 경화증, 쇼그렌 증후군, 원발성 담즙성 경변증, 셀리악병, 염증성 장질환, 제1형 당뇨병,　자가면역성 용혈성 빈혈증, 굿파스쳐 증후군, 그레이브스 병, 하시모토 갑상선염, 감상선 항진증, 중증근무력증, 천포창, 혈관염, 뇌척수염, 뇌하수체염, 백반증, 천식, 원발성 담즙성 간경변, 시신경 척수염, 심상성 천포창 (pemphigus vulgaris), 과민성 장질환, 크론병, 대장염, 궤양성 대장염, 건선, 심근병증, 중증 근무력증 (myasthenia gravis), 결절성 다발동맥염, 길랑바레 증후군, 및 강직성 척추염 (ankylosing spondylitis)으로 이루어진 군에서 선택된 하나 이상일 수 있으나, 이에 한정되지 않는다.In another embodiment of the present invention, the autoimmune disease is rheumatoid arthritis, juvenile rheumatoid arthritis, systemic scleroderma, adult Still's disease, systemic lupus erythematosus, atopic dermatitis, Behcet's disease, multiple sclerosis, systemic sclerosis, Sjogren's disease Syndrome, primary biliary cirrhosis, celiac disease, inflammatory bowel disease, type 1 diabetes, autoimmune hemolytic anemia, Goodpasture syndrome, Graves disease, Hashimoto's thyroiditis, hyperthyroidism, myasthenia gravis, pemphigus, vasculitis, encephalomyelitis, pituitary, Vitiligo, asthma, primary biliary cirrhosis, optic nerve myelitis, pemphigus vulgaris, irritable bowel disease, Crohn's disease, colitis, ulcerative colitis, psoriasis, cardiomyopathy, myasthenia gravis, polyarteritis nodosa, Guillain It may be at least one selected from the group consisting of Barre's syndrome, and ankylosing spondylitis, but is not limited thereto.
또한, 본 발명은 (S1) 염기성 환경에서 생체고분자의 자가조립을 유도하여 항산화 나노입자를 제조하는 단계;In addition, the present invention comprises the steps of (S1) producing antioxidant nanoparticles by inducing self-assembly of a biopolymer in a basic environment;
(S2) 상기 항산화 나노입자의 현탁액으로 지질막을 수화시켜 지질막으로 표면이 코팅된 항산화 나노입자를 제조하는 단계; 및(S2) hydrating the lipid membrane with the suspension of the antioxidant nanoparticles to prepare antioxidant nanoparticles whose surface is coated with the lipid membrane; and
(S3) 상기 지질막으로 표면이 코팅된 항산화 나노입자를 자가항원, 및 항원제시세포 특이적 항체 또는 이의 단편과 반응시키는 단계를 포함하는, 제1항의 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자의 제조방법을 제공한다.(S3) The antibody and autoantigen of claim 1 comprising the step of reacting the surface-coated antioxidant nanoparticles with an autoantigen and an antigen-presenting cell-specific antibody or fragment thereof with the surface of the lipid membrane-bound lipid-antioxidation A method for preparing nanoparticles is provided.
본 발명의 일 구현예에서, 상기 생체고분자는 폴리도파민, 탄닌, 및 세륨 산화물로 이루어진 군에서 선택된 하나 이상일 수 있으나, 이에 한정되지 않는다.In one embodiment of the present invention, the biopolymer may be one or more selected from the group consisting of polydopamine, tannin, and cerium oxide, but is not limited thereto.
본 발명의 다른 구현예에서, 상기 지질막은 면역억제제가 봉입되어 있는 것일 수 있으나, 이에 한정되지 않는다.In another embodiment of the present invention, the lipid membrane may be one in which an immunosuppressant is encapsulated, but is not limited thereto.
본 발명에 따른 지질-항산화 나노입자는 자가항원 및 항원제시세포 특이적 항체가 표면에 수식되어 있어 항원제시세포 특이적으로 자가항원을 전달할 수 있고, 표면은 면역조절제가 봉입된 지질막으로 코팅되어 있어 자가항원에 대해 면역관용을 유도할 수 있다. 특히 본 발명에 따른 나노입자는 자가면역질환 동물모델에 투여하였을 때 면역기관인 림프절 및 비장을 효과적으로 타겟팅하여 항원제시세포들에 의한 과잉의 면역 활성화를 효과적으로 억제함으로써, 자가면역질환을 예방, 지연, 및 치료할 수 있음이 확인되었다. 따라서, 본 발명에 따른 나노입자는 뇌척수염 등을 포함한 다양한 자가면역질환의 예방 내지 치료를 위해 유용히 활용될 것으로 기대된다.Lipid-antioxidant nanoparticles according to the present invention have autoantigens and antigen-presenting cell-specific antibodies modified on the surface to deliver the autoantigen specifically to antigen-presenting cells, and the surface is coated with a lipid membrane encapsulated with an immunomodulatory agent. Immune tolerance to autoantigens can be induced. In particular, when the nanoparticles according to the present invention are administered to an animal model of autoimmune disease, they effectively target lymph nodes and spleen, which are immune organs, and effectively inhibit excessive immune activation by antigen-presenting cells, thereby preventing, delaying, and It has been shown to be curable. Therefore, the nanoparticles according to the present invention are expected to be usefully utilized for the prevention or treatment of various autoimmune diseases, including encephalomyelitis.
도 1은 본 발명에 따른, 항체 및 자가항원으로 수식된 지질-항산화 나노입자의 모식도이다. 항산화 나노입자 (PN)의 표면을 약물을 함유한 지질막으로 코팅하여 지질-항산화 나노입자 (LDPN)를 형성하고, 페길화된 지질층 표면에 자가항원만 수식시키거나 (LDPN-MOG), 자가항원 및 항체를 모두 수식시켜 (AbaLDPN-MOG) 제조하였다.1 is a lipid-modified lipid-antioxidant nanoparticle according to the present invention and an antibody and an autoantigen. Lipid-antioxidant nanoparticles (LDPN) are formed by coating the surface of antioxidant nanoparticles (PN) with a drug-containing lipid film, and only autoantigens are modified on the surface of pegylated lipid layers (LDPN-MOG), autoantigens and All of the antibodies were modified (AbaLDPN-MOG) and prepared.
도 2는 항산화 나노입자 (PN); 지질-항산화 나노입자 (LDPN); 자가항원 수식 지질-항산화 나노입자 (LDPN-MOG); 및 항체 및 자가항원 수식된 지질-항산화 나노입자 (AbaLDPN-MOG)의 혈청 내에서의 입자의 크기와 제타전위 (ξ-potential)를 측정한 것이다. 입자 크기는 동적광산란법 (dynamic light scattering)을 이용하여 측정했으며 제타전위는 레이저 도플러 전기영동법 (laser doppler electrophoresis)을 이용하여 측정했다. 입자 간에 크기 및 제타전위의 유의미한 차이가 없는 것으로 나타났다.2 shows antioxidant nanoparticles (PN); lipid-antioxidant nanoparticles (LDPN); autoantigen-modified lipid-antioxidant nanoparticles (LDPN-MOG); And antibody and autoantigen-modified lipid-antioxidant nanoparticles (AbaLDPN-MOG) in the serum of the particle size and zeta potential (ξ-potential) was measured. The particle size was measured using dynamic light scattering and the zeta potential was measured using laser doppler electrophoresis. There was no significant difference in size and zeta potential between particles.
도 3은 각 나노입자에 봉입된 면역조절 약물의 양과 나노입자의 표면에 수식된 자가항원의 양을 고성능 액체크로마토그래피 (HPLC)를 통해 확인한 결과이다.3 is a result of confirming the amount of the immunomodulatory drug encapsulated in each nanoparticle and the amount of the autoantigen modified on the surface of the nanoparticle through high performance liquid chromatography (HPLC).
도 4는 분광학적 분석을 통해 각 나노입자들의 지질 코팅 여부 및 화학적 작용기의 성질을 확인한 결과이다.4 is a result of confirming whether or not the lipid coating of each nanoparticles and the properties of chemical functional groups through spectroscopic analysis.
도 5는 투과전자현미경으로 각 나노입자의 형태 (morphology)를 관찰하고, 에너지 분산형 X-선 분광법을 통해 원소분석을 수행한 결과이다. 원소분석 결과, 폴리도파민 나노입자의 주요 원소인 탄소 (C), 질소 (N), 산소 (O)가 검출됐고, 인지질 유래의 인 (P), 단백질 유래의 황 (S)이 검출됐다.5 is a result of observing the morphology of each nanoparticle with a transmission electron microscope, and performing elemental analysis through energy dispersive X-ray spectroscopy. As a result of elemental analysis, carbon (C), nitrogen (N), and oxygen (O), which are the main elements of polydopamine nanoparticles, were detected, phosphorus (P) derived from phospholipids, and sulfur (S) derived from proteins were detected.
도 6은 CD80/86을 발현하는 수지상세포 (dendritic cell)에 CD80/86-특이적 재조합 항체로 수식된 나노입자 또는 상기 항체로 수식되지 않은 나노입자를 각각 처리한 후 나노입자의 세포 결합능을 유세포분석으로 비교한 결과이다. 각 나노입자는 형광 (Cy5)으로 표지하였으며, 세포의 형광 강도를 통해 나노입자의 세포 결합 정도를 확인했다. Figure 6 is flow cytometry the cell binding capacity of the nanoparticles after each treatment of CD80/86-expressing dendritic cells (dendritic cells) with nanoparticles modified with CD80/86-specific recombinant antibody or nanoparticles not modified with the antibody. It is the result of comparison by analysis. Each of the nanoparticles was labeled with fluorescence (Cy5), and the degree of cell binding of the nanoparticles was confirmed through the fluorescence intensity of the cells.
도 7은 수지상세포에 CD80/86-특이적 재조합 항체로 수식된 나노입자 또는 상기 항체로 수식되지 않은 나노입자를 각각 처리한 후 나노입자의 세포 결합능을 공초점현미경으로 확인한 결과이다.7 is a result of confirming the cell-binding ability of the nanoparticles by confocal microscopy after treating dendritic cells with nanoparticles modified with CD80/86-specific recombinant antibody or nanoparticles not modified with the antibody, respectively.
도 8은 수지상세포에 CD80/86-특이적 재조합 항체로 수식된 나노입자 또는 항체로 수식되지 않은 나노입자를 각각 처리한 후 나노입자의 세포 결합 정도를 투과전자현미경으로 확인한 결과이다.8 is a result of confirming the degree of cell binding of the nanoparticles with a transmission electron microscope after treating dendritic cells with nanoparticles modified with CD80/86-specific recombinant antibody or nanoparticles not modified with the antibody, respectively.
도 9는 CD80/86-특이적 재조합 항체로 수식된 나노입자가 내포작용을 통해 수지상세포 내부로 도입되어 리소좀으로 이동하는 것을 공초점현미경으로 관찰한 결과이다.9 is a result of observation with a confocal microscope that nanoparticles modified with CD80/86-specific recombinant antibody are introduced into dendritic cells through endocytosis and migrate to lysosomes.
도 10은 본 발명에 따른 나노입자의 수지상세포 및 T 세포간 상호작용 저해효과를 확인하기 위한 in vitro 실험을 나타낸 그림이다. 웰 플레이트에는 수지상세포 표면단백질을 코팅하고, 본 발명의 나노입자 및 T 세포를 함께 처리하였다.10 is a diagram showing an in vitro experiment for confirming the interaction inhibitory effect between dendritic cells and T cells of the nanoparticles according to the present invention. The well plate was coated with a dendritic cell surface protein, and the nanoparticles of the present invention and T cells were treated together.
도 11은 도 10의 실험 조건에서 본 발명에 따른 나노입자를 T 세포와 함께 처리하였을 때 수지상세포 및 T 세포의 상호작용 억제 정도를 배양액 내 IL-2 수준을 통해 확인한 결과이다. 본 발명에 따른, 항체로 표면이 수식된 지질-항산화 나노입자가 수지상세포 및 T 세포간 상호작용을 차단하여 T 세포의 IL-2 분비를 억제하는 것을 확인하였다. 11 is a result confirming the degree of inhibition of the interaction between dendritic cells and T cells through the IL-2 level in the culture medium when the nanoparticles according to the present invention are treated with T cells under the experimental conditions of FIG. 10 . According to the present invention, it was confirmed that the surface-modified lipid-antioxidant nanoparticles with the antibody block the interaction between dendritic cells and T cells to inhibit IL-2 secretion of T cells.
도 12 및 도 13은 도 10의 실험 조건에서 본 발명에 따른 나노입자를 T 세포와 함께 처리하였을 때 비장세포에 의한 보조 T 세포 (CD3 및 CD4 이중 양성; CD4+ T cells) 및 세포독성 T 세포 (CD3 및 CD8 이중 양성; CD8+ T cells) 확장 (expansion)이 억제된 것을 확인한 결과이다.12 and 13 show helper T cells (CD3 and CD4 double positive; CD4 + T cells) and cytotoxic T cells by splenocytes when the nanoparticles according to the present invention were treated together with T cells under the experimental conditions of FIG. (CD3 and CD8 double positive; CD8 + T cells) It is the result of confirming that expansion (expansion) is suppressed.
도 14는 본 발명에 따른 나노입자에 의한 수지상세포 세포막 단백질 CD80, CD86, 및 MHCII의 수준 감소; 및 염증성 사이토카인인 TNF-α, IL-1β, 및 IL-6의 분비 억제 효과를 확인한 결과이다. 14 is a reduction in the levels of dendritic cell membrane proteins CD80, CD86, and MHCII by nanoparticles according to the present invention; and inflammatory cytokines TNF-α, IL-1β, and IL-6 secretion inhibitory effect.
도 15는 본 발명에 따른 나노입자에 의한 면역관용 수지상세포의 유도 기작을 나타낸 그림이다.15 is a diagram showing the mechanism of induction of immune-tolerance dendritic cells by nanoparticles according to the present invention.
도 16 및 17은 본 발명에 따른 나노입자를 마우스에 피하투여한 후, 시간별로 표적기관인 림프절에서의 나노입자의 분포를 확인한 결과이다. 재조합 항체가 표면에 수식된 나노입자는 항체가 수식되지 않은 나노입자에 비해 림프절 분포가 증가하고, 더 지속적으로 축적되는 것을 확인하였다.16 and 17 are results of confirming the distribution of nanoparticles in lymph nodes, which are target organs, by time after subcutaneous administration of the nanoparticles according to the present invention to mice. It was confirmed that the nanoparticles on the surface of which the recombinant antibody was modified had increased lymph node distribution and more continuously accumulated compared to the nanoparticles on which the antibody was not modified.
도 18은 본 발명에 따른 나노입자 (Cy5 표지됨)를 마우스에 피하투여한 후, 림프절 세포집단 중 Cy5 양성인 수지상세포의 비율을 확인한 결과이다. 재조합 항체가 표면에 수식된 나노입자는 항체가 수식되지 않은 나노입자에 비해 림프절 내의 수지상세포에 더 효과적으로 결합하는 것을 확인하였다.18 is a result of confirming the ratio of Cy5-positive dendritic cells in the lymph node cell population after subcutaneous administration of the nanoparticles (Cy5-labeled) according to the present invention to mice. It was confirmed that the nanoparticles on the surface of which the recombinant antibody was modified bind more effectively to dendritic cells in the lymph nodes compared to the nanoparticles in which the antibody is not modified.
도 19는 본 발명에 따른 나노입자 (Cy5 표지됨)를 마우스에 피하투여한 후, 면역형광조직법으로 마우스의 림프절 조직을 분석하여 나노입자의 분포 (Cy5 형광신호)를 확인한 이미지이다.19 is an image confirming the distribution of nanoparticles (Cy5 fluorescence signal) by subcutaneously administering the nanoparticles (Cy5-labeled) according to the present invention to mice, and then analyzing the lymph node tissue of the mouse by immunofluorescence histology.
도 20a은 본 발명에 따른 나노입자의 체액성 면역 억제 효과를 확인하기 위한 자가면역질환 동물모델 실험 스케쥴을 나타낸 것이다. 20A shows an experimental schedule of an autoimmune disease animal model for confirming the humoral immunosuppressive effect of nanoparticles according to the present invention.
도 20b는 오브알부민 (Ovalbumin)을 이용한 자가면역질환 모델에서 본 발명에 따른 나노입자가 상기 동물모델의 혈청 내 자가항체 (anti-OVA IgG)의 생성을 효과적으로 저해한 것을 확인한 결과이다. 이로부터 본 발명의 나노입자가 오브알부민 특이적 체액성 면역반응을 억제하는 효과가 있음을 알 수 있다.20B is a result confirming that the nanoparticles according to the present invention effectively inhibited the production of autoantibodies (anti-OVA IgG) in the serum of the animal model in an autoimmune disease model using ovalbumin. From this, it can be seen that the nanoparticles of the present invention have an effect of inhibiting ovalbumin-specific humoral immune response.
도 21a 내지 21c는 본 발명에 따른 나노입자의 자가면역질환 (뇌척수염)의 예방 (도 21a), 진행 억제 (도 21b), 및 치료 효과 (도 21c)를 확인하기 위한 실험 스케쥴이다.21A to 21C are experimental schedules for confirming the prevention (FIG. 21A), progression inhibition (FIG. 21B), and therapeutic effect (FIG. 21C) of the nanoparticles according to the present invention for autoimmune disease (encephalomyelitis).
도 22a 내지 22c는 본 발명에 따른 나노입자를 자가면역질환의 예방 (도 22a), 진행 억제 (도 22b), 또는 치료 (도 22c) 목적으로 뇌척수염 마우스에 투여한 후 시간에 따른 체중 변화 및 임상적 증상을 추적관찰한 결과이다. 모든 동물모델에서, 본 발명에 따른 나노입자가 개체의 임상적 증상을 개선하고 체중을 정상 수준으로 조절하는 것으로 나타났다.Figures 22a to 22c show changes in body weight over time and clinical trials after administering the nanoparticles according to the present invention to mice with encephalomyelitis for the purpose of preventing (FIG. 22A), inhibiting progression (FIG. 22B), or treating (FIG. 22C) of an autoimmune disease. It is the result of follow-up of adverse symptoms. In all animal models, the nanoparticles according to the present invention were shown to improve the clinical symptoms of the subject and control the body weight to a normal level.
도 23a 내지 23c는 본 발명에 따른 나노입자를 자가면역질환의 예방 (도 23a), 진행 억제 (도 23b), 또는 치료 (도 23c) 목적으로 뇌척수염 마우스에 투여한 후, 수지상세포 (CD11c+ MHCII+), 대식세포 (CD11b+ F4/80+), 보조 T 세포 (CD4+ T 세포 또는 조력 T 세포; CD3+ CD4+), 및 세포독성 T 세포 (CD8+ T 세포 또는 cytotoxic T cells; CD3+ CD8+)의 중추신경계 침습 정도를 확인한 결과이다. 조절 T 세포의 경우 조력 T 세포 내에서 CD25+ FoxP3+를 마커로 하여 분석하였다.23a to 23c are after administration of the nanoparticles according to the present invention to mice with encephalomyelitis for the purpose of preventing (FIG. 23A), inhibiting progression (FIG. 23B), or treating (FIG. 23C) of an autoimmune disease, dendritic cells (CD11c + MHCII) + ), macrophages (CD11b + F4/80 + ), helper T cells (CD4 + T cells or helper T cells; CD3 + CD4 + ), and cytotoxic T cells (CD8 + T cells or cytotoxic T cells; CD3 + CD8 + ) is the result of confirming the degree of invasion of the central nervous system. Regulatory T cells were analyzed using CD25 + FoxP3 + as a marker in helper T cells.
도 24a 내지 24c는 본 발명에 따른 나노입자를 자가면역질환의 예방 (도 24a), 진행 억제 (도 24b), 또는 치료 (도 24c) 목적으로 뇌척수염 마우스에 투여하였을 때, 조절 T 세포의 비율이 증가하는 것을 확인한 결과이다. 특히, 상기 나노입자는 조직을 손상시킬 수 있는 IFN-γ 또는 IL-17A를 발현하는 보조 T세포의 비율을 억제할 수 있다는 것도 확인되었다 (도 24a).24A to 24C show that when the nanoparticles according to the present invention are administered to mice with encephalomyelitis for the purpose of preventing (FIG. 24A), inhibiting progression (FIG. 24B), or treating (FIG. 24C) of an autoimmune disease, the ratio of regulatory T cells is It is the result of confirming the increase. In particular, it was also confirmed that the nanoparticles can inhibit the proportion of helper T cells expressing IFN-γ or IL-17A that can damage tissues ( FIG. 24A ).
도 25a 내지 25c는 본 발명에 따른 나노입자를 자가면역질환의 예방 (도 25a), 진행 억제 (도 25b), 또는 치료 (도 25c) 목적으로 뇌척수염 마우스에 투여한 후, 나노입자에 의한 세포성 면역반응 조절 능력을 확인한 결과이다. 본 발명에 따른 나노입자를 투여 받은 마우스에서, 자가항원 (MOG peptide)에 의해 활성화된 IFN-γ 비장세포의 수가 감소한 것을 확인하였다.25A to 25C are after administration of the nanoparticles according to the present invention to mice with encephalomyelitis for the purpose of preventing (FIG. 25A), inhibiting progression (FIG. 25B), or treating (FIG. 25C) of an autoimmune disease, cellularity by nanoparticles It is the result of confirming the ability to regulate the immune response. In mice administered with the nanoparticles according to the present invention, it was confirmed that the number of IFN-γ splenocytes activated by the autoantigen (MOG peptide) decreased.
도 26은 본 발명에 따른 나노입자를 뇌척수염 동물모델에 질병 진행 억제 목적으로 투여한 후, 면역형광조직법으로 면역세포의 중추신경계 (미엘린 수초) 침습을 확인한 결과이다.26 is a result of confirming the invasion of immune cells into the central nervous system (myelin myelin) by immunofluorescent tissue method after administering the nanoparticles according to the present invention to an animal model of encephalomyelitis for the purpose of inhibiting disease progression.
도 27은 본 발명에 따른 나노입자의 구조 및 이의 자가항체에 대한 면역관용 유도 효과를 나타낸 대표도이다.27 is a representative view showing the structure of the nanoparticles according to the present invention and the effect of inducing immune tolerance on autoantibodies thereof.
본 발명에 따른 항산화 나노입자는 표면이 면역조절제가 봉입된 지질막으로 코팅되고, 상기 지질막을 통해 자가항원 및 항원제시세포 특이적 항체로 표면이 수식된 것으로서, 생체 내 투여시 비장 및 림프절 내에 존재하는 항원제시세포에 특이적으로 결합하고, 자가항원에 대한 면역관용을 유도하여, 자가항원에 대한 과잉의 면역반응으로 일어나는 자가면역질환을 예방, 개선, 지연, 및 치료할 수 있다. The antioxidant nanoparticles according to the present invention have a surface coated with a lipid membrane encapsulated with an immunomodulatory agent, and the surface is modified with autoantigens and antigen-presenting cell-specific antibodies through the lipid membrane. By specifically binding to antigen-presenting cells and inducing immune tolerance to autoantigens, it is possible to prevent, improve, delay, and treat autoimmune diseases caused by an excessive immune response to autoantigens.
따라서, 본 발명은 항산화 나노입자; 상기 나노입자 표면을 코팅하는 지질막; 상기 지질막 표면에 결합된 자가항원; 및 상기 지질막 표면에 결합된 항원제시세포 특이적 항체 또는 이의 단편을 포함하는, 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자를 제공하는 것을 목적으로 한다.Accordingly, the present invention provides antioxidant nanoparticles; a lipid film coating the surface of the nanoparticles; an autoantigen bound to the surface of the lipid membrane; and an antigen-presenting cell-specific antibody or fragment thereof bound to the surface of the lipid membrane, an antibody and an autoantigen bound to the surface of the lipid-antioxidant nanoparticles.
본 발명에 있어서, “항산화 나노입자 (antioxidant nanoparticles)”는 항산화 기능을 갖는 나노입자로서 더 바람직하게는 활성산소를 포촉할 수 있는 나노입자를 의미한다. 바람직하게는, 본 발명에 따른 항산화 나노입자는 생체고분자-기반의 생체고분자 나노입자 (biopolymer nanoparticles)로서, 생체고분자를 포함하거나, 생체고분자로 이루어진 나노입자일 수 있다. 생체고분자란 살아있는 유기체의 세포로부터 생성되는 천연 고분자 내지 이를 모방하여 인공적으로 합성된 고분자로서, 우수한 생체적합성, 생분해성, 및/또는 지속성 (sustainability) 등을 갖는 것을 특징으로 한다. 본 발명의 일 구현예에서, 상기 나노입자는 생체고분자로 구성되어 있어, 생체 내 투여하더라도 생체적합성이 우수하고, 독성 등의 부작용을 일으키지 않을 수 있다. 본 발명에 따른 항산화 나노입자는, 바람직하게는 폴리도파민 나노입자, 탄닌 나노입자, 및 세륨 산화물 나노입자로 이루어진 군에서 선택된 하나 이상인 것을 특징으로 하며, 가장 바람직하게는 폴리도파민 (polydopamine) 나노입자일 수 있다. In the present invention, “antioxidant nanoparticles” refers to nanoparticles having an antioxidant function, and more preferably, nanoparticles capable of trapping active oxygen. Preferably, the antioxidant nanoparticles according to the present invention are biopolymer-based biopolymer nanoparticles, and may be nanoparticles containing a biopolymer or composed of a biopolymer. A biopolymer is a natural polymer generated from the cells of a living organism or a polymer artificially synthesized by mimicking it, and is characterized by having excellent biocompatibility, biodegradability, and/or sustainability. In one embodiment of the present invention, since the nanoparticles are composed of biopolymers, even when administered in vivo, they have excellent biocompatibility and may not cause side effects such as toxicity. Antioxidant nanoparticles according to the present invention, preferably polydopamine nanoparticles, tannin nanoparticles, and characterized in that at least one selected from the group consisting of cerium oxide nanoparticles, most preferably polydopamine (polydopamine) nanoparticles can
본 발명에 있어서, “자가면역질환 (autoimmune diseases)”은 면역계가 외부 항원이 아닌 내부의 정상 세포 내지 정상 조직을 공격하여 발생하는 질환을 의미한다. 즉, 자가면역질환은 자가항원에 대한 과도한 면역반응의 활성화로 인해 발생하는 질환을 의미한다. 따라서, 본 발명에 있어서 자가면역질환은, 과잉 면역반응으로 인한 질환뿐만 아니라 자가항원에 대한 면역반응을 억제함으로써 개선/치료될 수 있는 질환을 제한 없이 포함한다. 즉, 본 발명에 따른 자가면역질환은 본 발명에 따른 나노입자의 표면에 수식된 자가항원에 대한 자가항체의 생성으로 유도될 수 있는 질환을 의미하고, 구체적인 질환으로 한정되는 것은 아니나, 바람직하게는 류마티스 관절염, 소아 류마티스 관절염, 전신성 경피증, 성인 스틸씨병, 전신 홍반성 루푸스, 아토피 피부염, 베체트병 (Behcet's disease), 다발성 경화증, 전신성 경화증, 쇼그렌 증후군, 원발성 담즙성 경변증, 셀리악병, 염증성 장질환, 제1형 당뇨병,　자가면역성 용혈성 빈혈증, 굿파스쳐 증후군, 그레이브스 병, 하시모토 갑상선염, 감상선 항진증, 중증근무력증, 천포창, 혈관염, 뇌척수염, 뇌하수체염, 백반증, 천식, 원발성 담즙성 간경변, 시신경 척수염, 심상성 천포창 (pemphigus vulgaris), 과민성 장질환, 크론병, 대장염, 궤양성 대장염, 건선, 심근병증, 중증 근무력증 (myasthenia gravis), 결절성 다발동맥염, 길랑바레 증후군, 및 강직성 척추염 (ankylosing spondylitis) 등으로부터 선택될 수 있다.In the present invention, "autoimmune diseases" refers to diseases caused by the immune system attacking normal cells or normal tissues inside, not by an external antigen. That is, the autoimmune disease refers to a disease caused by the activation of an excessive immune response to an autoantigen. Accordingly, in the present invention, autoimmune diseases include, without limitation, diseases that can be improved/treated by suppressing immune responses to autoantigens as well as diseases caused by an excessive immune response. That is, the autoimmune disease according to the present invention means a disease that can be induced by the production of autoantibodies against the autoantigen modified on the surface of the nanoparticles according to the present invention, and is not limited to specific diseases, but preferably Rheumatoid arthritis, juvenile rheumatoid arthritis, systemic scleroderma, adult Still's disease, systemic lupus erythematosus, atopic dermatitis, Behcet's disease, multiple sclerosis, systemic sclerosis, Sjogren's syndrome, primary biliary cirrhosis, celiac disease, inflammatory bowel disease, Type 1 diabetes, autoimmune hemolytic anemia, Goodpasture's syndrome, Graves' disease, Hashimoto's thyroiditis, hyperthyroidism, myasthenia gravis, pemphigus, vasculitis, encephalomyelitis, pituitary gland, vitiligo, asthma, primary biliary cirrhosis, optic neuromyelitis, vulgaris pemphigus vulgaris, irritable bowel disease, Crohn's disease, colitis, ulcerative colitis, psoriasis, cardiomyopathy, myasthenia gravis, polyarteritis nodosa, Guillain-Barré syndrome, and ankylosing spondylitis, etc. can
본 발명에 따른 항산화 나노입자는 바람직하게는 생체고분자가 염기성 조건하에서 자가중합 (self-polymerization)되어 제조된 것을 특징으로 하며, 구체적인 형태나 크기에 제한 없다. 바람직하게는, 상기 항산화 나노입자는 직경이 50 내지 200 nm, 50 내지 150 nm, 50 내지 100 nm, 100 내지 200 nm, 또는 150 내지 200 nm 일 수 있으나, 이에 한정되는 것은 아니다.The antioxidant nanoparticles according to the present invention are preferably prepared by self-polymerization of a biopolymer under basic conditions, and are not limited to specific shapes or sizes. Preferably, the antioxidant nanoparticles may have a diameter of 50 to 200 nm, 50 to 150 nm, 50 to 100 nm, 100 to 200 nm, or 150 to 200 nm, but is not limited thereto.
본 발명에 따른 나노입자는 표면이 지질막으로 코팅된 것을 특징으로 한다. 본 발명자들은 상기 나노입자를 특정 조성비의 지질을 포함하는 지질막으로 코팅하였을 때 나노입자의 안정성이 향상되고, 상기 나노입자가 항체 및 자가항원과 안정적으로 결합할 수 있음을 확인하였다. 바람직하게는, 상기 지질막은 페길화된 것일 수 있다. 또한, 상기 지질막은 말레이미드기 (maleimide group)을 갖는 지질을 포함할 수 있다. 바람직하게는, 상기 지질막은 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), phosphorylglycerol (PG), phosphocholine (PC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide), 콜레스테롤 (cholesterol), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 및 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)로 이루어진 군에서 선택된 하나 이상을 포함할 수 있다. 바람직하게는, 상기 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG)는 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol)일 수 있으나, 이에 한정되지 않는다.The nanoparticles according to the present invention are characterized in that the surface is coated with a lipid membrane. The present inventors have confirmed that when the nanoparticles are coated with a lipid membrane containing a lipid of a specific composition ratio, the stability of the nanoparticles is improved, and the nanoparticles can be stably bound to antibodies and autoantigens. Preferably, the lipid membrane may be pegylated. In addition, the lipid membrane may include a lipid having a maleimide group. Preferably, the lipid membrane is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), phosphorylglycerol (PG), phosphocholine (PC) , 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide), cholesterol (cholesterol), 1,2-dioleoyl-sn-glycero-3- It may include at least one selected from the group consisting of phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). Preferably, the 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) may be 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol), but limited thereto doesn't happen
바람직하게는, 상기 지질막은 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) 및 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG)를 포함할 수 있다. 상기 DPPC : DPPG의 몰비율은 1 내지 10 : 1, 1 내지 8 : 1, 1 내지 5 : 1, 1 내지 3 : 1, 1.5 내지 10 : 1, 1.5 내지 8 : 1, 1.5 내지 5 : 1, 1.5 내지 3 : 1, 2 내지 10 : 1, 2 내지 8 : 1, 2 내지 6 : 1, 2 내지 4 : 1, 또는 2 내지 3 : 1 일 수 있으나, 이에 한정되는 것은 아니다. Preferably, the lipid membrane may include 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG). The molar ratio of DPPC: DPPG is 1 to 10: 1, 1 to 8: 1, 1 to 5: 1, 1-3: 1, 1.5 to 10: 1, 1.5 to 8: 1, 1.5 to 5: 1, 1.5 to 3 : 1, 2 to 10 : 1, 2 to 8 : 1, 2 to 6 : 1, 2 to 4 : 1, or 2 to 3 : 1, but is not limited thereto.
더욱 바람직하게는, 상기 지질막은 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 및 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide)를 포함할 수 있다. 상기 DPPC : DPPG : DSPE-PEG2000-maleimide의 몰비율은 100 내지 1000 : 10 내지 500 : 1, 100 내지 1000 : 50 내지 500 : 1, 200 내지 1000 : 100 내지 500 : 1, 300 내지 1000 : 100 내지 500 : 1, 500 내지 1000 : 200 내지 500 : 1, 600 내지 1000 : 200 내지 500 : 1, 300 내지 900 : 100 내지 500 : 1, 300 내지 900 : 200 내지 500 : 1, 300 내지 800 : 200 내지 500 : 1, 400 내지 800 : 200 내지 400 : 1, 500 내지 800 : 200 내지 400 : 1, 또는 600 내지 800 : 250 내지 350 : 1 일 수 있으나, 이에 한정되는 것은 아니다.More preferably, the lipid membrane is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), and 1,2-disteroyl-sn -glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide) may be included. The molar ratio of the DPPC: DPPG: DSPE-PEG2000-maleimide is 100 to 1000: 10 to 500: 1, 100 to 1000: 50 to 500: 1, 200 to 1000: 100 to 500: 1, 300 to 1000: 100 to 500: 1,500 to 1000: 200 to 500: 1, 600 to 1000: 200 to 500: 1, 300 to 900: 100 to 500: 1, 300 to 900: 200 to 500: 1, 300 to 800: 200 to 500: 1, 400 to 800: 200 to 400: 1, 500 to 800: 200 to 400: 1, or 600 to 800: 250 to 350: 1, but is not limited thereto.
바람직하게는, 상기 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)는 하기 화학식 1로 표시되고, 상기 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG)는 하기 화학식 2로 표시되고, 상기 phosphoglycerol (PG)는 화학식 3으로 표시되고, 상기 phosphocholine (PC)는 화학식 4로 표시되고, 상기 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG2000-maleimide)는 하기 화학식 5로 표시될 수 있으나, 이에 한정되는 것은 아니다.Preferably, the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) is represented by Formula 1 below, and the 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) is represented by Formula 2 , the phosphoglycerol (PG) is represented by Formula 3, the phosphocholine (PC) is represented by Formula 4, and the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol) )-2000] (DSPE-PEG2000-maleimide) may be represented by the following Chemical Formula 5, but is not limited thereto.
[화학식 1][Formula 1]
Figure PCTKR2022006518-appb-img-000001
Figure PCTKR2022006518-appb-img-000001
[화학식 2][Formula 2]
Figure PCTKR2022006518-appb-img-000002
Figure PCTKR2022006518-appb-img-000002
[화학식 3][Formula 3]
Figure PCTKR2022006518-appb-img-000003
Figure PCTKR2022006518-appb-img-000003
[화학식 4][Formula 4]
Figure PCTKR2022006518-appb-img-000004
Figure PCTKR2022006518-appb-img-000004
[화학식 5][Formula 5]
Figure PCTKR2022006518-appb-img-000005
Figure PCTKR2022006518-appb-img-000005
본 발명에 따른 나노입자는 표면이 항원제시세포 특이적 항체 또는 이의 단편으로 수식 (결합, 접합, 담지, 또는 부착)된 것을 특징으로 한다. The nanoparticles according to the present invention are characterized in that the surface is modified (bound, conjugated, supported, or attached) with an antigen-presenting cell-specific antibody or fragment thereof.
본 발명에 있어서, "항체 (antibodies)"란 특정 항원 또는 그의 에피토프 부위와 특이적으로 반응하여 결합할 수 있는 특이적인 단백질 분자를 의미하며, 항원과 결합능력을 가지는 면역글로불린 분자 (예를 들어, 단일클론 항체, 다클론 항체 등), 상기 면역글로불린 분자의 단편 (예를 들어, IgG, Fab', F(ab')2, Fab, Fv, 재조합 IgG (rIgG), 단일쇄 Fv (scFv), 또는 디아바디(diabody) 등) 등이 이에 포함된다. 특히, 상기 면역글로불린 분자는 중쇄 및 경쇄를 가지며 각각의 중쇄 및 경쇄는 불변영역(부위) 및 가변영역을 포함하고, 상기 경쇄 및 중쇄 가변 영역은 항원의 에피토프에 결합할 수 있는, 3개의 다변가능한 영역인 "상보성 결정영역(complementarity determining region, CDR)"; 및 4개의 "구조 영역(framework region, FR)"을 포함한다. 각각의 사슬의 CDR은 전형적으로 N-말단으로부터 시작하여 순차적으로 CDR1, CDR2, CDR3로 불리우고, 또한 특정 CDR이 위치하고 있는 사슬에 의해서 식별된다. 완전한 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 갖는 구조이며 각각의 경쇄는 중쇄와 이황화 결합으로 연결되어 있다. 상기 항체는 동물 유래 항체, 마우스-인간 키메릭 항체(chimeric antibody), 인간화 항체(humanized antibody), 또는 인간 항체일 수 있다. 본 발명에 있어서 “항체의 단편”이란 상기 항체의 기능을 발휘할 수 있는 상기 항체의 기능성 단편을 의미한다.In the present invention, "antibodies" refers to specific protein molecules capable of specifically reacting with and binding to a specific antigen or epitope region thereof, and immunoglobulin molecules having antigen-binding ability (e.g., monoclonal antibodies, polyclonal antibodies, etc.), fragments of the immunoglobulin molecules (eg, IgG, Fab', F(ab') 2 , Fab, Fv, recombinant IgG (rIgG), single chain Fv (scFv), or diabodies, etc.) and the like. In particular, the immunoglobulin molecule has a heavy chain and a light chain, each heavy and light chain comprising a constant region (region) and a variable region, wherein the light and heavy chain variable regions are capable of binding to an epitope of an antigen; a region “complementarity determining region (CDR)”; and four “framework regions” (FRs). The CDRs of each chain are called sequentially CDR1, CDR2, CDR3, typically starting from the N-terminus, and are also identified by the chain in which the specific CDR is located. A complete antibody has a structure with two full-length light chains and two full-length heavy chains, each light chain linked to the heavy chain by a disulfide bond. The antibody may be an animal-derived antibody, a mouse-human chimeric antibody, a humanized antibody, or a human antibody. In the present invention, "antibody fragment" refers to a functional fragment of the antibody capable of exhibiting the function of the antibody.
본 발명에 따른 항체는 항원제시세포에 특이적으로 결합하는 항체이다. 다만, 본 발명에서 상기 항체는 항체와 유사한 기능을 수행하는 재조합 단백질을 포함하는 개념이다. 즉, 본 발명에 따른 항원제시세포 특이적 항체는, 항원제시세포 특이적 단백질에 특이적으로 결합할 수 있는 재조합 단백질이면 충분하고, 반드시 항체의 구조를 가져야 하는 것은 아니다. 보다 바람직하게는, 본 발명에 따른 항원제시세포 특이적 항체는 항원제시세포에서 특이적으로 발현되는 단백질에 결합할 수 있는 항체이다. 본 명세서에서 사용되는 용어, “항원제시세포 (antigen presenting cells, APCs)”란 T 세포 등과 같은 특정 림프구 (lymphocytes)가 인식할 수 있도록 항원 (antigen)을 가공 및 제시 (presenting)하는 세포 그룹을 지칭한다. 자연적인 APC의 종류로는 수지상세포, 대식세포, 랑게르한스세포, B 세포 등이 있다. 바람직하게는, 상기 항원제시세포 특이적 항체 또는 이의 단편은 수지상세포 또는 대식세포에 특이적으로 결합하는 항체 또는 이의 단편이다. 즉, 상기 항원제시세포 특이적 항체 또는 이의 단편은 수지상세포 또는 대식세포에서 특이적으로 발현되는 단백질에 결합하는 항체 또는 이의 단편이다. 따라서, 상기 항원제시세포 특이적 항체 또는 이의 단편은 수지상세포 또는 대식세포에 특이적으로 결합할 수 있는 항체 또는 이의 단편이면 충분하고 구체적인 종류에 한정되는 것은 아니며, 천연 항체는 물론 인공적으로 합성된 항체 (재조합 항체)가 모두 사용될 수 있다. 바람직하게는, 상기 항체 또는 이의 단편은 CD80, CD86, CD123, CD303, CD304, CD68, CD11b, CD11c, BDCA-1, DC-SIGN, MHCII, F4/80, CD206, 및 CSF1-R로 이루어진 군에서 선택된 하나 이상의 단백질에 결합할 수 있다. 가장 바람직하게는, 본 발명에 따른 항원제시세포 특이적 항체 또는 이의 단편은 CD80 및/또는 CD86에 특이적으로 결합하는 항체 또는 이의 단편이다. 더욱 바람직하게는, 본 발명에 따른 항원제시세포 특이적 항체 또는 이의 단편은 CD80 및/또는 CD86에 특이적으로 결합하는 재조합 단백질 (예컨대, 항체) 또는 이의 단편으로서, 서열번호 1의 아미노산 서열을 포함하거나, 더욱 바람직하게는 서열번호 1의 아미노산 서열로 이루어진 것일 수 있으나 이에 제한되지 않으며, 상기 아미노산 서열의 변이체가 본 발명의 범위 내에 포함된다. 즉, 본 발명에 따른 세포 투과성 펩타이드는 이를 구성하는 폴리펩타이드의 작용성 등가물, 예를 들어, 폴리펩타이드의 일부 아미노산 서열이 결실 (deletion), 치환 (substitution) 또는 삽입 (insertion)에 의해 변형되었지만, 상기 폴리펩타이드와 기능적으로 동일한 작용을 할 수 있는 변이체 (variants)를 포함하는 개념이다. 예를 들어, 상기 CD80 및/또는 CD86에 특이적으로 결합하는 재조합 단백질 (예컨대, 항체) 또는 이의 단편은 서열번호 1의 아미노산 서열과 70% 이상, 더욱 바람직하게는 80% 이상, 더 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 아미노산 서열을 포함할 수 있다. 예를 들면, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 또는 100%의 서열 상동성을 갖는 폴리펩타이드를 포함한다. 폴리펩타이드에 대한 “서열 상동성의 %”는 두 개의 최적으로 배열된 서열과 비교 영역을 비교함으로써 확인되며, 비교 영역에서의 폴리펩타이드의 서열의 일부는 두 서열의 최적 배열에 대한 참고 서열(추가 또는 삭제를 포함하지 않음)에 비해 추가 또는 삭제(즉, 갭)를 포함할 수 있다.The antibody according to the present invention is an antibody that specifically binds to an antigen-presenting cell. However, in the present invention, the antibody is a concept including a recombinant protein that performs a function similar to the antibody. That is, it is sufficient that the antigen-presenting cell-specific antibody according to the present invention is a recombinant protein capable of specifically binding to the antigen-presenting cell-specific protein, and does not necessarily have the structure of the antibody. More preferably, the antigen-presenting cell-specific antibody according to the present invention is an antibody capable of binding to a protein specifically expressed in the antigen-presenting cell. As used herein, the term “antigen presenting cells (APCs)” refers to a group of cells that process and present antigens so that specific lymphocytes such as T cells can recognize them. do. Natural types of APC include dendritic cells, macrophages, Langerhans cells, and B cells. Preferably, the antigen-presenting cell-specific antibody or fragment thereof is an antibody or fragment thereof that specifically binds to dendritic cells or macrophages. That is, the antigen-presenting cell-specific antibody or fragment thereof is an antibody or fragment thereof that binds to a protein specifically expressed in dendritic cells or macrophages. Accordingly, the antigen-presenting cell-specific antibody or fragment thereof is sufficient as long as an antibody or fragment thereof capable of specifically binding to dendritic cells or macrophages is not limited to a specific type, and natural antibodies as well as artificially synthesized antibodies (recombinant antibody) can all be used. Preferably, the antibody or fragment thereof is selected from the group consisting of CD80, CD86, CD123, CD303, CD304, CD68, CD11b, CD11c, BDCA-1, DC-SIGN, MHCII, F4/80, CD206, and CSF1-R It is capable of binding to one or more selected proteins. Most preferably, the antigen-presenting cell-specific antibody or fragment thereof according to the present invention is an antibody or fragment thereof that specifically binds to CD80 and/or CD86. More preferably, the antigen-presenting cell-specific antibody or fragment thereof according to the present invention is a recombinant protein (eg, antibody) or fragment thereof that specifically binds to CD80 and/or CD86, comprising the amino acid sequence of SEQ ID NO: 1 Or, more preferably, it may consist of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto, and variants of the amino acid sequence are included within the scope of the present invention. That is, the cell-penetrating peptide according to the present invention is a functional equivalent of a polypeptide constituting it, for example, some amino acid sequence of the polypeptide is modified by deletion, substitution or insertion, It is a concept including variants capable of functionally the same action as the polypeptide. For example, the recombinant protein (eg, antibody) or fragment thereof that specifically binds to CD80 and/or CD86 is 70% or more, more preferably 80% or more, even more preferably the amino acid sequence of SEQ ID NO: 1 may comprise an amino acid sequence having at least 90% sequence homology, most preferably at least 95% sequence homology. For example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85 on %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence Polypeptides with homology are included. The “% sequence homology” for a polypeptide is determined by comparing two optimally aligned sequences to a comparison region, and a portion of the sequence of the polypeptide in the comparison region is a reference sequence (additional or additional may include additions or deletions (ie, gaps) compared to not including deletions).
본 발명에 있어서, “자가항원 (self-antigens 또는 autoantigens)”은 개체의 면역계가 자가항체 (autoantibodies)를 생성하도록 자극하는, 해당 개체 자체의 항원을 의미한다. 즉, 자가항원은 자가면역질환의 유도원으로서, 자가항원에 대해 적응 면역 반응이 일어날 때 자가면역질환이 유발된다. 자가항원은 정상적인 상태에서는 면역체계의 표적이 되지 않지만, 면역적인 요인이나 환경적 요인에 의해 면역적 내성이 결여되면서 항원으로 작용할 수 있다. 자가항원의 구체적인 종류에는 제한이 없고, 단백질, 펩타이드, 효소 복합체, 리보핵단백질 (ribonucleoprotein), DNA, 인지질 등이 모두 자가항원으로 기능할 수 있다. 자가항원의 구체적인 종류는 자가면역질환의 종류에 따라 달라질 수 있다. 예를 들어, 자가면역질환 중 하나인 뇌척수염은 미엘린 희소돌기아교세포 당단백질 (Myelin Oligodendrocyte Glycoprotein, MOG)이 자가항원으로 작용하는 것으로 알려져 있다. 각 자가면역질환을 유발할 수 있는 자가항원의 종류는 당업계에 공지되어 있다 (예를 들어, 대한민국 공개특허공보 제10-2020-0079507호 참조). In the present invention, "self-antigens (or autoantigens)" refers to an antigen of the subject itself that stimulates the immune system of the subject to produce autoantibodies. That is, autoantigens are inducers of autoimmune diseases, and autoimmune diseases are induced when an adaptive immune response to the autoantigens occurs. Autoantigens are not targeted by the immune system under normal conditions, but may act as antigens due to lack of immune resistance due to immune or environmental factors. Specific types of autoantigens are not limited, and proteins, peptides, enzyme complexes, ribonucleoproteins, DNA, phospholipids, and the like may all function as autoantigens. The specific type of autoantigen may vary depending on the type of autoimmune disease. For example, in encephalomyelitis, one of the autoimmune diseases, it is known that Myelin Oligodendrocyte Glycoprotein (MOG) acts as an autoantigen. The types of autoantigens that can cause each autoimmune disease are known in the art (see, for example, Korean Patent Application Laid-Open No. 10-2020-0079507).
즉, 본 발명에 따른 자가항원은 면역계가 자가항체를 생성하도록 자극하는 것으로서, 자가면역질환을 유도할 수 있는 것이라면 충분하고, 구체적인 종류로 한정되는 것은 아니다. 가장 바람직하게는, 본 발명에 따른 나노입자에 결합된 자가항원은, 자가항체의 생성을 유도함으로써 상기 나노입자가 목적 (예방, 개선, 또는 치료)으로 하는 자가면역질환을 유도할 수 있는 것이다. 당업자는 목적하는 자가면역질환의 종류에 따라, 혹은 상기 나노입자를 투여 받을 개체에 따라 당업계의 공지된 지식을 참고하여 적절한 자가항원을 선택할 수 있다. 바람직하게는, 본 발명에 따른 자가항원은 개체에서 자가면역질환을 유도할 수 있는 단백질, 이의 단편, 또는 이의 변이체일 수 있으나, 이에 한정되지 않는다. 일 구현예에서, 상기 자가항원은 콜라겐, 인슐린, 인슐린 B 사슬, 프로인슐린, 미엘린 단백질, 미엘린 염기성 단백질 (myelin basic protein), 미엘린 프로테오리피드 단백질 (myelin proteolipid protein), 미엘린 희소돌기아교세포 당단백질 (myelin oligodendrocyte glycoprotein), Hsp60, 및 Hsp65로 이루어진 군에서 선택된 하나 이상으로부터 유래된 단백질, 이의 단편, 또는 이의 변이체일 수 있으나, 이에 한정되는 것은 아니다.That is, the autoantigen according to the present invention is sufficient as long as it stimulates the immune system to produce autoantibodies and can induce autoimmune diseases, and is not limited to specific types. Most preferably, the autoantigen bound to the nanoparticles according to the present invention is one capable of inducing an autoimmune disease targeted by the nanoparticles (prevention, improvement, or treatment) by inducing the production of autoantibodies. A person skilled in the art can select an appropriate autoantigen according to the type of autoimmune disease desired or according to the subject to which the nanoparticles will be administered, referring to known knowledge in the art. Preferably, the autoantigen according to the present invention may be a protein capable of inducing an autoimmune disease in an individual, a fragment thereof, or a variant thereof, but is not limited thereto. In one embodiment, the autoantigen is collagen, insulin, insulin B chain, proinsulin, myelin protein, myelin basic protein, myelin proteolipid protein, myelin oligodendrocyte glycoprotein (myelin oligodendrocyte glycoprotein), Hsp60, and may be a protein derived from one or more selected from the group consisting of Hsp65, a fragment thereof, or a variant thereof, but is not limited thereto.
바람직하게는, 상기 자가항원; 및 항원제시세포 특이적 항체 또는 이의 단편은 티올기 (thiol group)를 갖는 것이거나, 티올기를 갖도록 변형된 것일 수 있다. 상기 티올기는 자유 티올기를 포함한다. 상기 티올기는 자가항원; 및 항원제시세포 특이적 항체 또는 이의 단편의 amine group에 결합되어 있을 수 있다. 즉, 상기 티올기는 자가항원; 및 항원제시세포 특이적 항체 또는 이의 단편을 구성하는 아미노산 중에서도 Lysine (Lys) 또는 Arigine (Arg) 등과 같이 1차 amine group을 갖는 아미노산에 결합되어 있을 수 있다. 일 구현예에서, 상기 티올기는 상기 항체 또는 이의 단편의 경쇄의 불변영역에 존재하는 것일 수 있으나, 이에 한정되지 않는다. 상기 자가항원; 및 항원제시세포 특이적 항체 또는 이의 단편은 상기 티올기를 통해 나노입자의 지질막에 결합할 수 있으나, 이에 한정되지 않는다. 바람직하게는, 상기 결합은 상기 티올기 및 지질막의 말레이미드기의 결합을 통해 이루어지는 것일 수 있으나, 이에 한정되는 것은 아니다. Preferably, the autoantigen; And the antigen-presenting cell-specific antibody or fragment thereof may have a thiol group or may be modified to have a thiol group. The thiol group includes a free thiol group. The thiol group is an autoantigen; And it may be bound to the amine group of the antigen-presenting cell-specific antibody or fragment thereof. That is, the thiol group is an autoantigen; And among the amino acids constituting the antigen-presenting cell-specific antibody or fragment thereof, it may be bound to an amino acid having a primary amine group, such as Lysine (Lys) or Arigine (Arg). In one embodiment, the thiol group may be present in the constant region of the light chain of the antibody or fragment thereof, but is not limited thereto. the autoantigen; And antigen-presenting cell-specific antibody or fragment thereof may bind to the lipid membrane of nanoparticles through the thiol group, but is not limited thereto. Preferably, the bonding may be made through bonding of the thiol group and the maleimide group of the lipid membrane, but is not limited thereto.
바람직하게는, 본 발명에 따른 나노입자는 면역조절제, 바람직하게는 면역억제제를 더 포함하는 것을 특징으로 한다. 더욱 바람직하게는, 상기 면역조절제는 나노입자의 표면을 코팅하는 지질막 상에 혹은 지질막 내부에 봉입 (결합, 포집, 또는 담지)된 것일 수 있다. 면역억제제 (Immunosuppressants)는 면역체계가 건강한 세포나 조직을 손상시키는 것을 방지하기 위해 면역 기능을 억제하는 약물을 의미한다. 주로 장기 이식 또는 줄기세포 이식을 받은 환자에서 이식거부반응을 예방하기 위해 투여되며, 자가면역질환을 치료하기 위해서도 사용된다. 즉, 상기 면역억제제는 자가항원에 대한 면역계의 면역관용을 유도할 수 있다. 본 발명에 있어서, 면역억제제는 면역기능을 억제할 수 있는 것이면 충분하고, 구체적인 종류에 한정되는 것은 아니나, 구체적인 예시로는 부신피질호르몬 (Glucocorticoids), 칼시뉴린 (Calcineurine) 억제제, 항대사제, 및 mTOR 억제제 등을 들 수 있다. Preferably, the nanoparticles according to the present invention are characterized in that they further comprise an immunomodulatory agent, preferably an immunosuppressant agent. More preferably, the immunomodulatory agent may be encapsulated (bound, captured, or supported) on the lipid membrane coating the surface of the nanoparticles or inside the lipid membrane. Immunosuppressants are drugs that suppress immune function to prevent the immune system from damaging healthy cells or tissues. It is mainly administered to prevent transplant rejection in patients undergoing organ transplantation or stem cell transplantation, and is also used to treat autoimmune diseases. That is, the immunosuppressive agent may induce immune tolerance of the immune system to the autoantigen. In the present invention, the immunosuppressive agent is sufficient as long as it can suppress immune function, and is not limited to specific types, but specific examples include corticosteroids (Glucocorticoids), calcineurin (Calcineurine) inhibitors, antimetabolites, and mTOR inhibitors; and the like.
더욱 바람직하게는, 상기 면역억제제는 덱사메타손 (Dexamethasone), 베타메타손 (Betamethasone), 프레드니손 (Prednisone), 프레드니솔론 (Prednisolone), 메틸프레드니솔론 (Methylprednisolone), 트리암시놀론 (Triamcinolone), 데플라자코트 (Deflazacort), 사이클로스포린 A (Cyclosporine A), 타크로리무스 (Tacrolimus), MPA (Mycophenolic acid), MMF (Mycophenolate mofetil), 아자티오프린 (Azathioprine), 미조리빈 (Mizoribine), 에베로리무스 (Everolimus), 라파마이신 (Rapamycin), 레티노산 (Retinoic acid), 및 비타민 D3 등으로부터 선택될 수 있으나, 이에 한정되는 것은 아니다.More preferably, the immunosuppressive agent is dexamethasone, betamethasone, prednisone, prednisolone, methylprednisolone, triamcinolone A, deflazacort. Cyclosporine A), Tacrolimus, MPA (Mycophenolic acid), MMF (Mycophenolate mofetil), Azathioprine, Mizoribine, Everolimus, Rapamycin, Retinoic acid ( Retinoic acid), and may be selected from vitamin D3, but is not limited thereto.
또한, 본 발명은 본 발명에 따른 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자를 유효성분으로 포함하는, 자가면역질환의 예방, 개선, 또는 치료용 약학적 조성물을 제공한다. 상기 치료는 자가면역질환의 질병 진행 억제를 포함한다.In addition, the present invention provides a pharmaceutical composition for preventing, improving, or treating autoimmune diseases, comprising the lipid-antioxidant nanoparticles bound to the surface of the antibody and autoantigen according to the present invention as an active ingredient. Such treatment includes inhibiting disease progression of an autoimmune disease.
상기 나노입자의 자가면역질환의 예방, 개선, 또는 치료 효과는 상기 나노입자의 자가항원에 의한 면역 억제 효과, 혹은 자가항원에 대한 면역관용 유도 효과에 의해 달성되는 것이다. The prevention, improvement, or therapeutic effect of the nanoparticles for autoimmune diseases is achieved by the immunosuppressive effect of the nanoparticles or the immune tolerance inducing effect on the autoantigens.
보다 구체적으로는, 상기 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자는 하기로 이루어진 군에서 선택된 하나 이상의 특징을 만족할 수 있다:More specifically, the lipid-antioxidant nanoparticles bound to the surface of the antibody and autoantigen may satisfy one or more characteristics selected from the group consisting of:
(a) 항원제시세포 및 T 세포의 상호작용을 억제함;(a) inhibit the interaction of antigen-presenting cells and T cells;
(b) 면역세포의 조직 침습을 억제함;(b) inhibiting tissue invasion of immune cells;
(c) 보조 T 세포, 세포독성 T 세포, 수지상세포, 및 대식세포로 이루어진 군에서 선택된 하나 이상의 수준 또는 활성을 감소시킴;(c) reducing the level or activity of one or more selected from the group consisting of helper T cells, cytotoxic T cells, dendritic cells, and macrophages;
(d) 조절 T 세포의 수준 또는 활성을 증가시킴;(d) increasing the level or activity of regulatory T cells;
(e) 염증성 사이토카인의 수준 또는 활성을 억제함; 및(e) inhibiting the level or activity of an inflammatory cytokine; and
(f) 자가항체의 생성을 억제함.(f) Inhibits the production of autoantibodies.
특히, 상기 나노입자는 개체에 투여시 림프절 또는 비장을 표적화 (targeting)하여, 즉, 림프절 또는 비장으로 이동하여 상기 효과를 발휘할 수 있다.In particular, when the nanoparticles are administered to a subject, they can target the lymph node or spleen, that is, migrate to the lymph node or spleen to exert the above effect.
상기 (a) 항원제시세포 및 T 세포의 상호작용을 억제한다는 것은, 항원제시세포에 의한 T 세포의 활성화, 바람직하게는, 세포독성 T 세포의 활성화를 억제한다는 것을 의미한다. 나아가, 이는 본 발명에 따른 나노입자에 의한 면역관용 수지상세포 (tolerogenic dendritic cells)의 유도도 포함한다.(a) Inhibiting the interaction between the antigen-presenting cell and the T cell means inhibiting the activation of the T cell by the antigen-presenting cell, preferably, the activation of the cytotoxic T cell. Furthermore, this includes induction of tolerogenic dendritic cells by the nanoparticles according to the present invention.
상기 (b) 면역세포의 조직 침습을 억제한다는 것은, 바람직하게는 수지상세포, 대식세포, 및 세포독성 T 세포의 조직 침습을 억제하는 것을 의미한다. 특히, 상기 나노입자가 뇌척수염의 예방, 개선, 또는 치료를 목적으로 하는 경우, 상기 면역세포들의 중추신경계 침습을 억제하는 것을 의미한다. The (b) suppressing the tissue invasion of immune cells, preferably means inhibiting the tissue invasion of dendritic cells, macrophages, and cytotoxic T cells. In particular, when the nanoparticles are for the purpose of preventing, improving, or treating encephalomyelitis, it means inhibiting the central nervous system invasion of the immune cells.
상기 (e) 염증성 사이토카인은 TNF-α, IL-1β, 및 IL-6로 이루어진 군에서 선택된 하나 이상일 수 있으나, 염증을 유발할 수 있는 사이토카인이라면 제한 없이 포함할 수 있다. 즉, 본 발명에 따른 나노입자는 활성화된 면역세포에 의한 상기 염증성 사이토카인들의 생성 및 분비를 억제할 수 있다.The (e) inflammatory cytokine may be one or more selected from the group consisting of TNF-α, IL-1β, and IL-6, but may include without limitation as long as it is a cytokine that can induce inflammation. That is, the nanoparticles according to the present invention can inhibit the production and secretion of the inflammatory cytokines by activated immune cells.
상기 (f)의 자가항체는 바람직하게는 상기 나노입자에 담지된 자가항원에 대한 자가항체일 수 있다. 즉, 본 발명에 따른 나노입자는 항원제시세포 특이적으로 자가항원을 전달하여 이에 대한 면역관용을 유도함으로써, 상기 자가항원에 대한 자가항체가 생성되는 것을 억제하는 기능을 수행한다. The autoantibody of (f) may preferably be an autoantibody against an autoantigen supported on the nanoparticles. That is, the nanoparticles according to the present invention perform a function of inhibiting the generation of autoantibodies against the autoantigen by inducing immune tolerance against the antigen-presenting cell-specific delivery of the autoantigen.
따라서, 본 발명은 본 발명에 따른 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자를 유효성분으로 포함하는, 면역조절용 약학적 조성물을 제공한다. 상기 면역조절은 자가항원에 대한 면역억제 용도 및 자가항원에 대한 면역관용 유도 용도를 포함한다. Accordingly, the present invention provides a pharmaceutical composition for immunomodulation, comprising, as an active ingredient, the lipid-antioxidant nanoparticles bound to the surface of the antibody and the autoantigen according to the present invention. The immunomodulation includes the use of immunosuppression against the autoantigen and the use of inducing immune tolerance against the autoantigen.
본 발명의 조성물 내의 상기 나노입자의 함량은 질환의 증상, 증상의 진행 정도, 환자의 상태 등에 따라서 적절히 조절 가능하며, 예컨대, 전체 조성물 중량을 기준으로 0.0001 내지 99.9중량%, 또는 0.001 내지 50중량%일 수 있으나, 이에 한정되는 것은 아니다. 상기 함량비는 용매를 제거한 건조량을 기준으로 한 값이다. The content of the nanoparticles in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc., for example, 0.0001 to 99.9% by weight, or 0.001 to 50% by weight based on the total weight of the composition may be, but is not limited thereto. The content ratio is a value based on the dry amount from which the solvent is removed.
본 발명에 따른 약학적 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다. 상기 부형제는 예를 들어, 희석제, 결합제, 붕해제, 활택제, 흡착제, 보습제, 필름-코팅 물질, 및 제어방출첨가제로 이루어진 군으로부터 선택된 하나 이상일 수 있다. The pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions. The excipient may be, for example, at least one selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a humectant, a film-coating material, and a controlled-release additive.
본 발명에 따른 약학적 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 서방형 과립제, 장용과립제, 액제, 점안제, 엘실릭제, 유제, 현탁액제, 주정제, 트로키제, 방향수제, 리모나아데제, 정제, 서방형정제, 장용정제, 설하정, 경질캅셀제, 연질캅셀제, 서방캅셀제, 장용캅셀제, 환제, 틴크제, 연조엑스제, 건조엑스제, 유동엑스제, 주사제, 캡슐제, 관류액, 경고제, 로션제, 파스타제, 분무제, 흡입제, 패취제, 멸균주사용액, 또는에어로졸 등의 외용제 등의 형태로 제형화하여 사용될 수 있으며, 상기 외용제는 크림, 젤, 패치, 분무제, 연고제, 경고제, 로션제, 리니멘트제, 파스타제 또는 카타플라스마제 등의 제형을 가질 수 있다. The pharmaceutical composition according to the present invention can be prepared according to a conventional method, respectively, in powders, granules, sustained-release granules, enteric granules, liquids, eye drops, elsilic, emulsions, suspensions, spirits, troches, fragrances, and limonaade. , tablets, sustained release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusates, Warnings, lotions, pasta, sprays, inhalants, patches, sterile injection solutions, or external preparations such as aerosols can be formulated and used, and the external preparations are creams, gels, patches, sprays, ointments, warning agents , lotion, liniment, pasta, or cataplasma.
본 발명에 따른 약학적 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 올리고당, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.
본 발명에 따른 정제, 산제, 과립제, 캡슐제, 환제, 트로키제의 첨가제로 옥수수전분, 감자전분, 밀전분, 유당, 백당, 포도당, 과당, 디-만니톨, 침강탄산칼슘, 합성규산알루미늄, 인산일수소칼슘, 황산칼슘, 염화나트륨, 탄산수소나트륨, 정제 라놀린, 미결정셀룰로오스, 덱스트린, 알긴산나트륨, 메칠셀룰로오스, 카르복시메칠셀룰로오스나트륨, 카올린, 요소, 콜로이드성실리카겔, 히드록시프로필스타치, 히드록시프로필메칠셀룰로오스(HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, 프로필렌글리콜, 카제인, 젖산칼슘, 프리모젤 등 부형제; 젤라틴, 아라비아고무, 에탄올, 한천가루, 초산프탈산셀룰로오스, 카르복시메칠셀룰로오스, 카르복시메칠셀룰로오스칼슘, 포도당, 정제수, 카제인나트륨, 글리세린, 스테아린산, 카르복시메칠셀룰로오스나트륨, 메칠셀룰로오스나트륨, 메칠셀룰로오스, 미결정셀룰로오스, 덱스트린, 히드록시셀룰로오스, 히드록시프로필스타치, 히드록시메칠셀룰로오스, 정제쉘락, 전분호, 히드록시프로필셀룰로오스, 히드록시프로필메칠셀룰로오스, 폴리비닐알코올, 폴리비닐피롤리돈 등의 결합제가 사용될 수 있으며, 히드록시프로필메칠셀룰로오스, 옥수수전분, 한천가루, 메칠셀룰로오스, 벤토나이트, 히드록시프로필스타치, 카르복시메칠셀룰로오스나트륨, 알긴산나트륨, 카르복시메칠셀룰로오스칼슘, 구연산칼슘, 라우릴황산나트륨, 무수규산, 1-히드록시프로필셀룰로오스, 덱스트란, 이온교환수지, 초산폴리비닐, 포름알데히드처리 카제인 및 젤라틴, 알긴산, 아밀로오스, 구아르고무(Guar gum), 중조, 폴리비닐피롤리돈, 인산칼슘, 겔화전분, 아라비아고무, 아밀로펙틴, 펙틴, 폴리인산나트륨, 에칠셀룰로오스, 백당, 규산마그네슘알루미늄, 디-소르비톨액, 경질무수규산 등 붕해제; 스테아린산칼슘, 스테아린산마그네슘, 스테아린산, 수소화식물유(Hydrogenated vegetable oil), 탈크, 석송자, 카올린, 바셀린, 스테아린산나트륨, 카카오지, 살리실산나트륨, 살리실산마그네슘, 폴리에칠렌글리콜(PEG) 4000, PEG 6000, 유동파라핀, 수소첨가대두유(Lubri wax), 스테아린산알루미늄, 스테아린산아연, 라우릴황산나트륨, 산화마그네슘, 마크로골(Macrogol), 합성규산알루미늄, 무수규산, 고급지방산, 고급알코올, 실리콘유, 파라핀유, 폴리에칠렌글리콜지방산에테르, 전분, 염화나트륨, 초산나트륨, 올레인산나트륨, dl-로이신, 경질무수규산 등의 활택제;가 사용될 수 있다.As additives for tablets, powders, granules, capsules, pills, and troches according to the present invention, corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, phosphoric acid Calcium monohydrogen, calcium sulfate, sodium chloride, sodium hydrogen carbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethyl excipients such as cellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose phthalate acetate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin , hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch powder, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. Hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, silicic anhydride, 1-hydroxy Propylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodite, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogen Added soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acid, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, A lubricant such as starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, light anhydrous silicic acid; may be used.
본 발명에 따른 액제의 첨가제로는 물, 묽은 염산, 묽은 황산, 구연산나트륨, 모노스테아린산슈크로스류, 폴리옥시에칠렌소르비톨지방산에스텔류(트윈에스텔), 폴리옥시에칠렌모노알킬에텔류, 라놀린에텔류, 라놀린에스텔류, 초산, 염산, 암모니아수, 탄산암모늄, 수산화칼륨, 수산화나트륨, 프롤아민, 폴리비닐피롤리돈, 에칠셀룰로오스, 카르복시메칠셀룰로오스나트륨 등이 사용될 수 있다.As additives for the liquid formulation according to the present invention, water, diluted hydrochloric acid, diluted sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (Twinester), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc. can be used.
본 발명에 따른 시럽제에는 백당의 용액, 다른 당류 혹은 감미제 등이 사용될 수 있으며, 필요에 따라 방향제, 착색제, 보존제, 안정제, 현탁화제, 유화제, 점조제 등이 사용될 수 있다.In the syrup according to the present invention, a sucrose solution, other sugars or sweeteners may be used, and if necessary, a fragrance, colorant, preservative, stabilizer, suspending agent, emulsifier, thickening agent, etc. may be used.
본 발명에 따른 유제에는 정제수가 사용될 수 있으며, 필요에 따라 유화제, 보존제, 안정제, 방향제 등이 사용될 수 있다.Purified water may be used in the emulsion according to the present invention, and if necessary, an emulsifier, preservative, stabilizer, fragrance, etc. may be used.
본 발명에 따른 현탁제에는 증류수, 포도당 수용액, 아카시아, 트라가칸타, 메칠셀룰로오스, 카르복시메칠셀룰로오스, 카르복시메칠셀룰로오스나트륨, 미결정셀룰로오스, 알긴산나트륨, 히드록시프로필메칠셀룰로오스(HPMC), HPMC 1828, HPMC 2906, HPMC 2910 등 현탁화제가 사용될 수 있으며, 필요에 따라 계면활성제, 보존제, 안정제, 착색제, 방향제가 사용될 수 있다.The suspending agent according to the present invention includes distilled water, aqueous glucose solution, acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906 , a suspending agent such as HPMC 2910 may be used, and if necessary, a surfactant, preservative, stabilizer, colorant, and fragrance may be used.
본 발명에 따른 주사제에는 주사용 증류수, 포도당 수용액, 0.9% 염화나트륨주사액, 링겔주사액, 덱스트로스주사액, 덱스트로스+염화나트륨주사액, 피이지(PEG), 락테이티드 링겔주사액, 에탄올, 프로필렌글리콜, 비휘발성유-참기름, 면실유, 낙화생유, 콩기름, 옥수수기름, 올레인산에칠, 미리스트산 이소프로필, 안식향산벤젠과 같은 용제; 안식향산나트륨, 살리실산나트륨, 초산나트륨, 요소, 우레탄, 모노에칠아세트아마이드, 부타졸리딘, 프로필렌글리콜, 트윈류, 니정틴산아미드, 헥사민, 디메칠아세트아마이드와 같은 용해보조제; 약산 및 그 염(초산과 초산나트륨), 약염기 및 그 염(암모니아 및 초산암모니움), 유기화합물, 단백질, 알부민, 펩 톤, 검류와 같은 완충제; 염화나트륨과 같은 등장화제; 중아황산나트륨(NaHSO3) 이산화탄소가스, 메타중아황산나트륨(Na2S2O5), 아황산나트륨(Na2SO3), 질소가스(N2), 에칠렌디아민테트라초산과 같은 안정제; 소디움비설파이드 0.1%, 소디움포름알데히드 설폭실레이트, 치오우레아, 에칠렌디아민테트라초산디나트륨, 아세톤소디움비설파이트와 같은 황산화제; 벤질알코올, 클로로부탄올, 염산프로카인, 포도당, 글루콘산칼슘과 같은 무통화제; 시엠시나트륨, 알긴산나트륨, 트윈 80, 모노스테아린산알루미늄과 같은 현탁화제를 포함할 수 있다.The injection according to the present invention includes distilled water for injection, glucose aqueous solution, 0.9% sodium chloride injection, Ringel injection, dextrose injection, dextrose + sodium chloride injection, PEG (PEG), lactated Ringel injection, ethanol, propylene glycol, non-volatile solvents such as oil-sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; Solubilizing aids such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tweens, nijeongtinamide, hexamine, and dimethylacetamide; Weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, buffers such as albumin, peptone, gum; isotonic agents such as sodium chloride; sodium bisulfite (NaHSO 3 ) carbon dioxide gas, sodium metabisulfite (Na 2 S 2 O 5 ), sodium sulfite (Na 2 SO 3 ), nitrogen gas (N 2 ), stabilizers such as ethylenediaminetetraacetic acid; sulphating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, acetone sodium bisulfite; analgesic agents such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; suspending agents such as SiMC sodium, sodium alginate, Tween 80, and aluminum monostearate.
본 발명에 따른 좌제에는 카카오지, 라놀린, 위텝솔, 폴리에틸렌글리콜, 글리세로젤라틴, 메칠셀룰로오스, 카르복시메칠셀룰로오스, 스테아린산과 올레인산의 혼합물, 수바날(Subanal), 면실유, 낙화생유, 야자유, 카카오버터+콜레스테롤, 레시틴, 라네트왁스, 모노스테아린산글리세롤, 트윈 또는 스판, 임하우젠(Imhausen), 모놀렌(모노스테아린산프로필렌글리콜), 글리세린, 아뎁스솔리두스(Adeps solidus), 부티룸 태고-G(Buytyrum Tego-G), 세베스파마 16 (Cebes Pharma 16), 헥사라이드베이스 95, 코토마(Cotomar), 히드록코테 SP, S-70-XXA, S-70-XX75(S-70-XX95), 히드록코테(Hydrokote) 25, 히드록코테 711, 이드로포스탈 (Idropostal), 마사에스트라리움(Massa estrarium, A, AS, B, C, D, E, I, T), 마사-MF, 마수폴, 마수폴-15, 네오수포스탈-엔, 파라마운드-B, 수포시로(OSI, OSIX, A, B, C, D, H, L), 좌제기제 IV 타입 (AB, B, A, BC, BBG, E, BGF, C, D, 299), 수포스탈 (N, Es), 웨코비 (W, R, S, M ,Fs), 테제스터 트리글리세라이드 기제 (TG-95, MA, 57)와 같은 기제가 사용될 수 있다.The suppository according to the present invention includes cacao fat, lanolin, witepsol, polyethylene glycol, glycerogelatin, methyl cellulose, carboxymethyl cellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, Lecithin, Lanet Wax, Glycerol Monostearate, Tween or Span, Imhausen, Monolene (Propylene Glycol Monostearate), Glycerin, Adeps Solidus, Butyrum Tego -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydroxote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydro Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium, A, AS, B, C, D, E, I, T, Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository IV type (AB, B, A, BC, BBG, E, BGF, C, D, 299), supostal (N, Es), Wecobi (W, R, S, M, Fs), tester triglyceride base (TG-95, MA, 57) and The same mechanism may be used.
경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract, for example, starch, calcium carbonate, sucrose ) or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate talc are also used.
경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜 (propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다.Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
바람직하게는, 본 발명에 따른 나노입자는 증류수 또는 등장액 (isotonic solution)에 분산되어 투여되는 것을 특징으로 한다. 상기 등장액은 구체적인 종류에 제한되지 않고, 투여 대상이 되는 개체의 체액과 등장성 (isotonic)을 보이는 용액이라면 제한 없이 포함할 수 있으나, 바람직하게는 포도당 수용액 및 생리식염수 (NaCl 용액) 등으로부터 선택될 수 있다. 더욱 바람직하게는, 상기 포도당 수용액의 농도 (w/w%)는 4 내지 6% 일 수 있고, 가장 바람직하게는 상기 농도가 5% 일 수 있으나 이에 한정되지 않는다. 또한, 상기 생리식염수 농도 (w/w%)는 0.5 내지 1% 일 수 있고, 가장 바람직하게는 상기 농도가 0.9% 일 수 있으나, 이에 한정되지 않는다.Preferably, the nanoparticles according to the present invention are dispersed in distilled water or isotonic solution and administered. The isotonic solution is not limited to a specific type, and may include without limitation as long as it is isotonic with the body fluid of the subject to be administered, but is preferably selected from aqueous glucose solution and physiological saline (NaCl solution). can More preferably, the concentration (w/w%) of the aqueous glucose solution may be 4 to 6%, and most preferably, the concentration may be 5%, but is not limited thereto. In addition, the physiological saline concentration (w/w%) may be 0.5 to 1%, and most preferably, the concentration may be 0.9%, but is not limited thereto.
본 발명에 따른 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 일 구현예에서, 본 발명에 따른 나노입자는, 항산화 나노입자의 중량을 기준으로, 개체 중량 대비 1 내지 100 mg/kg, 1 내지 90 mg/kg, 1 내지 80 mg/kg, 1 내지 70 mg/kg, 1 내지 60 mg/kg, 1 내지 50 mg/kg, 1 내지 40 mg/kg, 1 내지 30 mg/kg, 1 내지 20 mg/kg, 1 내지 10 mg/kg, 1 내지 5 mg/kg, 10 내지 100 mg/kg, 20 내지 100 mg/kg, 30 내지 100 mg/kg, 40 내지 100 mg/kg, 10 내지 80 mg/kg, 10 내지 60 mg/kg, 20 내지 60 mg/kg, 30 내지 60 mg/kg, 또는 40 내지 60 mg/kg의 용량으로 투여될 수 있으나, 이에 한정되지 않는다. 또한, 상기 나노입자는 단일 투여는 물론, 목적하는 효과를 달성할 때까지 다회 투여될 수 있다.The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and type of the patient's disease; Sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined. In one embodiment, the nanoparticles according to the present invention, based on the weight of the antioxidant nanoparticles, 1 to 100 mg / kg, 1 to 90 mg / kg, 1 to 80 mg / kg, 1 to 70 mg relative to the individual weight /kg, 1-60 mg/kg, 1-50 mg/kg, 1-40 mg/kg, 1-30 mg/kg, 1-20 mg/kg, 1-10 mg/kg, 1-5 mg/kg kg, 10-100 mg/kg, 20-100 mg/kg, 30-100 mg/kg, 40-100 mg/kg, 10-80 mg/kg, 10-60 mg/kg, 20-60 mg/kg , 30 to 60 mg / kg, or may be administered in a dose of 40 to 60 mg / kg, but is not limited thereto. In addition, the nanoparticles may be administered multiple times until the desired effect is achieved as well as a single administration.
또한, 본 발명에 따른 나노입자는 목적에 따라 투여방법이 달라질 수 있다. 예컨대, 본 발명에 따른 나노입자를 자가면역질환의 예방 목적으로 투여하는 경우, 질환의 증상이 없는 상태에서 상기 나노입자를 미리 투여할 수 있고, 본 발명에 따른 나노입자를 자가면역질환의 진행 억제 목적으로 투여하는 경우, 질환이 확인된 직후에 상기 나노입자를 투여할 수 있다. 또한, 본 발명에 따른 나노입자를 자가면역질환의 치료 목적으로 투여하는 경우, 자가면역질환의 증상이 나타난 직후부터 상기 나노입자를 투여할 수 있다.In addition, the administration method of the nanoparticles according to the present invention may vary depending on the purpose. For example, when the nanoparticles according to the present invention are administered for the purpose of preventing autoimmune diseases, the nanoparticles may be administered in advance in the absence of symptoms of the disease, and the nanoparticles according to the present invention inhibit the progression of autoimmune diseases In the case of administration for the purpose, the nanoparticles may be administered immediately after the disease is identified. In addition, when the nanoparticles according to the present invention are administered for the purpose of treating an autoimmune disease, the nanoparticles may be administered immediately after symptoms of the autoimmune disease appear.
본 발명에 따른 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 본 발명이 속하는 기술분야에 통상의 기술자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount capable of obtaining the maximum effect with a minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention pertains.
본 발명의 약학적 조성물은 개체에게 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구 복용, 피하 주사, 복강 투여, 정맥 주사, 근육 주사, 척수 주위 공간(경막내) 주사, 설하 투여, 볼점막 투여, 직장 내 삽입, 질 내 삽입, 안구 투여, 귀 투여, 비강 투여, 흡입, 입 또는 코를 통한 분무, 피부 투여, 경피 투여 등에 따라 투여될 수 있다.The pharmaceutical composition of the present invention may be administered to an individual by various routes. All modes of administration can be envisaged, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal (intrathecal) injection, sublingual administration, buccal administration, rectal insertion, vaginal It can be administered according to internal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, skin administration, transdermal administration, and the like.
본 발명의 약학적 조성물은 치료할 질환, 투여 경로, 환자의 연령, 성별, 체중 및 질환의 중등도 등의 여러 관련 인자와 함께 활성성분인 약물의 종류에 따라 결정된다.The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with several related factors such as the disease to be treated, the route of administration, the patient's age, sex, weight, and the severity of the disease.
본 발명에서 “개체”란 질병의 치료를 필요로 하는 대상을 의미하고, 보다 구체적으로는 인간 또는 비-인간인 영장류, 생쥐 (mouse), 쥐 (rat), 개, 고양이, 말, 및 소 등의 포유류를 의미한다.In the present invention, "individual" means a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses, cattle, etc. means the mammals of
본 발명에서 “투여”란 임의의 적절한 방법으로 개체에게 소정의 본 발명의 조성물을 제공하는 것을 의미한다.In the present invention, "administration" means providing a predetermined composition of the present invention to a subject by any suitable method.
본 발명에서 “예방”이란 목적하는 질환의 발병을 억제하거나 지연시키는 모든 행위를 의미하고, “치료”란 본 발명에 따른 약학적 조성물의 투여에 의해 목적하는 질환과 그에 따른 대사 이상 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미하며, “개선”이란 본 발명에 따른 조성물의 투여에 의해 목적하는 질환과 관련된 파라미터, 예를 들면 증상의 정도를 감소시키는 모든 행위를 의미한다. 특히, 상기 “개선”은 “질병의 진행 억제”를 포함한다.In the present invention, “prevention” means any action that suppresses or delays the onset of a target disease, and “treatment” means that the target disease and its metabolic abnormalities are improved or It means any action that is advantageously changed, and “improvement” means any action that reduces a parameter related to a desired disease, for example, the degree of a symptom by administration of the composition according to the present invention. In particular, the "improvement" includes "inhibiting the progression of a disease".
또한, 본 발명은 본 발명에 따른 나노입자를 유효성분으로 포함하는, 자가면역질환의 예방 또는 개선용 식품 조성물을 제공한다. 상기 식품 조성물은 건강기능식품 조성물을 포함한다. 즉, 본 발명은 상기 나노입자를 유효성분으로 포함하는 기능성 식이 및 약학 조성물을 제공하는 것을 주요 목적으로 한다.In addition, the present invention provides a food composition for preventing or improving autoimmune diseases, comprising the nanoparticles according to the present invention as an active ingredient. The food composition includes a health functional food composition. That is, the main object of the present invention is to provide a functional dietary and pharmaceutical composition comprising the nanoparticles as an active ingredient.
본 발명의 나노입자를 식품 첨가물로 사용할 경우, 상기 생체고분자 나노입자를 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용할 수 있다. 유효성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조시 본 발명의 나노입자는 원료에 대하여 15 중량% 이하, 또는 10 중량% 이하의 양으로 첨가될 수 있다. 그러나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.When the nanoparticles of the present invention are used as food additives, the biopolymer nanoparticles can be added as they are or used together with other foods or food ingredients, and can be appropriately used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, in the production of food or beverage, the nanoparticles of the present invention may be added in an amount of 15% by weight or less, or 10% by weight or less based on the raw material. However, in the case of long-term intake for health and hygiene or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.
상기 식품의 종류에는 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강기능식품을 모두 포함한다.There is no particular limitation on the type of the food. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and it includes all health functional foods in the ordinary sense.
본 발명에 따른 건강음료 조성물은 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물은 포도당 및 과당과 같은 모노사카라이드, 말토오스 및 수크로오스와 같은 디사카라이드, 덱스트린 및 시클로덱스트린과 같은 폴리사카라이드, 및 자일리톨, 소르비톨 및 에리트리톨 등의 당알콜이다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 mL당 일반적으로 약 0.01-0.20g, 또는 약 0.04-0.10g 이다.The health beverage composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, as in a conventional beverage. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetener, natural sweeteners such as taumatine and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used. The proportion of the natural carbohydrate is generally about 0.01-0.20 g, or about 0.04-0.10 g per 100 mL of the composition of the present invention.
상기 외에 본 발명의 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 조성물은 천연 과일쥬스, 과일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0.01-0.20 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, It may contain a carbonation agent used for carbonated beverages, and the like. In addition, the composition of the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The proportion of these additives is not critical, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.
본 명세서에 있어서, “건강기능식품”이란 특정보건용 식품(food for special health use, FoSHU)와 동일한 용어로, 영양 공급 외에도 생체조절기능이 효율적으로 나타나도록 가공된 의학, 의료효과가 높은 식품을 의미하는데, 상기 식품은 자가면역질환의 예방 또는 개선에 유용한 효과를 얻기 위하여 정제, 캡슐, 분말, 과립, 액상, 환 등의 다양한 형태로 제조될 수 있다.In this specification, “health functional food” is the same term as food for special health use (FoSHU), and refers to foods with high medical and medical effects processed to efficiently exhibit bioregulatory functions in addition to nutritional supply. Meaning, the food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, pills, etc. to obtain a useful effect in the prevention or improvement of autoimmune diseases.
본 발명의 건강기능식품은 당업계에서 통상적으로 사용되는 방법에 의하여 제조가능하며, 상기 제조 시에는 당업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 또한 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어날 수 있다.The health functional food of the present invention can be prepared by a method commonly used in the art, and during the manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, unlike general drugs, there are no side effects that may occur when taking the drug for a long period of time by using food as a raw material, and it can be excellent in portability.
또한, 본 발명은 본 발명에 따른 나노입자를 포함하는, 자가면역질환의 예방, 개선, 또는 치료용 키트를 제공한다.In addition, the present invention provides a kit for preventing, improving, or treating autoimmune diseases, comprising the nanoparticles according to the present invention.
본 발명에 있어서, “키트 (kit)”는 구체적인 형태나 종류에 제한이 없으며, 당업계에서 통상적으로 사용되는 형태의 키트를 사용할 수 있다. 예컨대, 본 발명에 따른 키트는 상기 나노입자 외에도 상기 나노입자의 보관 용액, 투여 도구, 투여용 현탁액 등을 추가로 포함할 수 있으며, 상기 나노입자의 특징, 제조방법 등에 관한 지시서를 추가로 포함할 수 있다.In the present invention, the “kit” is not limited to a specific form or type, and a kit of a type commonly used in the art may be used. For example, the kit according to the present invention may further include a storage solution of the nanoparticles, an administration tool, a suspension for administration, etc. in addition to the nanoparticles, and may further include instructions on the characteristics of the nanoparticles, manufacturing methods, etc. can
또한, 본 발명은 (S1) 염기성 환경에서 생체고분자의 자가조립을 유도하여 항산화 나노입자를 제조하는 단계;In addition, the present invention comprises the steps of (S1) producing antioxidant nanoparticles by inducing self-assembly of a biopolymer in a basic environment;
(S2) 상기 항산화 나노입자의 현탁액으로 지질막을 수화시켜 지질막으로 표면이 코팅된 항산화 나노입자를 제조하는 단계; 및(S2) hydrating the lipid membrane with the suspension of the antioxidant nanoparticles to prepare antioxidant nanoparticles whose surface is coated with the lipid membrane; and
(S3) 상기 지질막으로 표면이 코팅된 항산화 나노입자를 자가항원, 및 항원제시세포 특이적 항체 또는 이의 단편과 반응시키는 단계를 포함하는, 제1항의 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자의 제조방법을 제공한다.(S3) The antibody and autoantigen of claim 1 comprising the step of reacting the surface-coated antioxidant nanoparticles with an autoantigen and an antigen-presenting cell-specific antibody or fragment thereof with the surface of the lipid membrane-bound lipid-antioxidation A method for preparing nanoparticles is provided.
상기 단계 (S1)의 생체고분자의 구체적인 설명은 위에서 서술한 바와 같다. 상기 생체고분자는 폴리도파민, 탄닌, 및 세륨 산화물로 이루어진 군에서 선택된 하나 이상일 수 있으나, 이에 한정되는 것은 아니다.A detailed description of the biopolymer in step (S1) is as described above. The biopolymer may be one or more selected from the group consisting of polydopamine, tannin, and cerium oxide, but is not limited thereto.
본 발명에 있어서, 상기 단계 (S1)은 구체적으로 하기 단계를 포함할 수 있다:In the present invention, the step (S1) may specifically include the following steps:
(S1-1) 생체고분자 용액을 염기성 용액으로 적정하는 단계; 및(S1-1) titrating the biopolymer solution with a basic solution; and
(S1-2) 생체고분자의 자가조립을 유도하는 단계.(S1-2) Inducing self-assembly of the biopolymer.
상기 생체고분자 용액은, 바람직하게는 폴리도파민 용액, 탄닌 용액, 및 세륨 산화물 용액으로 이루어진 군에서 선택될 수 있으며, 바람직하게는 폴리도파민 수용액일 수 있고, 더욱 바람직하게는 도파민 하이드로클로라이드 (dopamine hydrochloride) 용액일 수 있다. 상기 생체고분자 용액의 용매는 증류수일 수 있으나, 이에 한정되는 것은 아니다.The biopolymer solution may be preferably selected from the group consisting of polydopamine solution, tannin solution, and cerium oxide solution, preferably polydopamine aqueous solution, more preferably dopamine hydrochloride. It may be a solution. The solvent of the biopolymer solution may be distilled water, but is not limited thereto.
상기 염기성 용액은 염기성 환경에서 상기 생체고분자의 자가조립을 유도하기 위한 목적으로 사용된다. 상기 염기성 용액의 구체적인 종류에는 제한이 없으나, 바람직하게는 수산화나트륨 용액, 수산화칼륨 용액, 및 수산화칼슘 용액 등으로부터 선택될 수 있다.The basic solution is used for the purpose of inducing self-assembly of the biopolymer in a basic environment. The specific type of the basic solution is not limited, but may be preferably selected from sodium hydroxide solution, potassium hydroxide solution, calcium hydroxide solution, and the like.
상기 단계 (S1-1)에서, 상기 생체고분자 용액은 염기성 용액에 의해 pH 5 내지 10, pH 6 내지 10, pH 7 내지 10, pH 8 내지 10, pH 9 내지 10, 또는 pH 9.5 내지 10으로 적정될 수 있으나, 이에 한정되는 것은 아니다.In the step (S1-1), the biopolymer solution is titrated to pH 5 to 10, pH 6 to 10, pH 7 to 10, pH 8 to 10, pH 9 to 10, or pH 9.5 to 10 with a basic solution. may be, but is not limited thereto.
상기 단계 (S1-2)의 자가조립은 생체고분자 용액을 자기교반함으로써 이루어질 수 있다. 바람직하게는, 상기 자기교반은 10 내지 35 ℃, 10 내지 30 ℃, 10 내지 27 ℃, 15 내지 30 ℃, 20 내지 30 ℃, 20 내지 27 ℃, 또는 23 내지 27 ℃에서 수행될 수 있고, 1 내지 40 시간, 1 내지 35 시간, 1 내지 30 시간, 1 내지 25 시간, 5 내지 35 시간, 10 내지 35 시간, 15 내지 35 시간, 15 내지 30 시간, 20 내지 30 시간, 또는 20 내지 25시간 동안 수행될 수 있으나, 이에 한정되는 것은 아니다.The self-assembly of the step (S1-2) may be accomplished by magnetically stirring the biopolymer solution. Preferably, the magnetic stirring may be carried out at 10 to 35 °C, 10 to 30 °C, 10 to 27 °C, 15 to 30 °C, 20 to 30 °C, 20 to 27 °C, or 23 to 27 °C, 1 to 40 hours, 1 to 35 hours, 1 to 30 hours, 1 to 25 hours, 5 to 35 hours, 10 to 35 hours, 15 to 35 hours, 15 to 30 hours, 20 to 30 hours, or 20 to 25 hours may be performed, but is not limited thereto.
또한, 상기 제조방법의 단계 (S1)은 상기 단계 (S1-2) 이후에 하기 단계들을 선택적으로 더 포함할 수 있다:In addition, the step (S1) of the manufacturing method may optionally further include the following steps after the step (S1-2):
(S1-3) 상기 단계 (S1-2)로부터 제조된 항산화 나노입자를 세척하는 단계; 및(S1-3) washing the antioxidant nanoparticles prepared in the step (S1-2); and
(S1-4) 상기 항산화 나노입자를 여과하는 단계.(S1-4) filtering the antioxidant nanoparticles.
바람직하게는, 상기 단계 (S1-3)의 세척은 증류수에 생체고분자 나노입자를 현탁한 후 원심분리함으로써 이루어질 수 있으나, 이에 제한되는 것은 아니고, 당업계에 공지된 나노입자 세척 방법을 제한 없이 사용할 수 있다.Preferably, the washing of step (S1-3) can be made by suspending the biopolymer nanoparticles in distilled water and then centrifuging, but is not limited thereto, and the nanoparticle washing method known in the art can be used without limitation. can
바람직하게는, 상기 단계 (S1-4)의 여과는 폴리카보네이트 필터를 이용하여 이루어질 수 있으나, 이에 한정되는 것은 아니다. Preferably, the filtration of step (S1-4) may be made using a polycarbonate filter, but is not limited thereto.
바람직하게는, 상기 생체고분자 나노입자는 증류수에 현탁된 상태로 보관될 수 있으나, 이에 한정되는 것은 아니다.Preferably, the biopolymer nanoparticles may be stored in a suspended state in distilled water, but is not limited thereto.
상기 제조방법에 있어서, 상기 지질막은 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), phosphorylglycerol (PG), phosphocholine (PC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide), 콜레스테롤 (cholesterol), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 및 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)로 이루어진 군에서 선택된 하나 이상을 포함할 수 있으나, 이에 한정되지 않는다. 이외에, 지질막에 대한 성분은 위에서 서술한 바와 같다.In the preparation method, the lipid membrane is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), phosphorylglycerol (PG), phosphocholine ( PC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide), cholesterol (cholesterol), 1,2-dioleoyl-sn-glycero- It may include at least one selected from the group consisting of 3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). However, the present invention is not limited thereto. In addition, the components for the lipid membrane are as described above.
바람직하게는, 상기 지질막은 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 및 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide)를 위에서 서술한 몰비율로 혼합한 후, 유기용매에 용해시키고 감압 농축함으로써 제조되는 것일 수 있다.Preferably, the lipid membrane is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), and 1,2-disteroyl-sn- It may be prepared by mixing glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide) in the molar ratio described above, dissolving it in an organic solvent and concentrating under reduced pressure.
바람직하게는, 상기 유기용매는 클로로포름, 헥산, 에틸아세테이트, 메탄올, 디클로로메탄, 사염화탄소, 벤젠, DMSO 및 DMF로 구성된 군으로부터 선택되는 어느 하나 이상일 수 있고, 바람직하게는 클로로포름 및 메탄올 용액일 수 있으나, 이에 한정되는 것은 아니다. 더욱 바람직하게는, 상기 클로로포름 및 메탄올 용액은 클로로포름 : 메탄올 (v/v)이 1 내지 10 : 1, 1 내지 8 : 1, 1 내지 6 : 1, 1 내지 5 : 1, 2 내지 10 : 1, 3 내지 10 : 1, 2 내지 8 : 1, 3 내지 7 : 1, 또는 3 내지 6 : 1의 비율로 혼합된 것일 수 있으나, 이에 한정되지 않는다.Preferably, the organic solvent may be any one or more selected from the group consisting of chloroform, hexane, ethyl acetate, methanol, dichloromethane, carbon tetrachloride, benzene, DMSO and DMF, preferably chloroform and methanol solution, The present invention is not limited thereto. More preferably, the chloroform and methanol solution is chloroform: methanol (v/v) 1 to 10: 1, 1 to 8: 1, 1 to 6: 1, 1 to 5: 1, 2 to 10: 1, 3 to 10: 1, 2 to 8: 1, 3 to 7: 1, or may be mixed in a ratio of 3 to 6: 1, but is not limited thereto.
상기 단계 (S2)에서 상기 지질막 및 항산화 나노입자는 지질막 : 항산화 나노입자의 중량비 (w/w)가 1 내지 20 : 27, 1 내지 18 : 27, 1 내지 16 : 27, 1 내지 14 : 27, 1 내지 12 : 27, 1 내지 10 : 27, 1 내지 8 : 27, 1 내지 6 : 27, 또는 1 내지 4 : 27 일 수 있으나, 이에 한정되는 것은 아니며, 상기 나노입자가 상기 지질로 코팅될 수 있는 정도면 충분하다.In the step (S2), the lipid membrane and the antioxidant nanoparticles have a weight ratio (w/w) of the lipid membrane: antioxidant nanoparticles of 1 to 20: 27, 1 to 18: 27, 1 to 16: 27, 1 to 14: 27, 1 to 12: 27, 1 to 10: 27, 1 to 8: 27, 1 to 6: 27, or 1 to 4: 27, but is not limited thereto, and the nanoparticles may be coated with the lipid. It is enough to have
바람직하게는, 상기 지질막은 면역조절제, 더욱 바람직하게는 면역억제제가 봉입된 것일 수 있다.Preferably, the lipid membrane may be an immunomodulatory agent, more preferably an immunosuppressive agent encapsulated therein.
상기 면역억제제는 상기 지질막 제조과정에서 각 지질성분을 유기용매에 용해시켜 혼합할 때 함께 첨가함으로써 지질막에 봉입될 수 있다. 이 때, 상기 면역억제제는 면역억제제 : 항산화 나노입자 (바람직하게는, 폴리도파민 나노입자)의 중량비 (w/w)가 0.0001 내지 0.01 : 1, 0.0001 내지 0.01 : 1, 0.001 내지 0.01 : 1, 또는 0.001 내지 0.005 : 1가 되도록 첨가될 수 있으나, 이에 한정되는 것은 아니다.The immunosuppressant may be encapsulated in the lipid membrane by dissolving each lipid component in an organic solvent during the preparation of the lipid membrane and adding together when mixing. In this case, the immunosuppressive agent has a weight ratio (w/w) of the immunosuppressant: antioxidant nanoparticles (preferably polydopamine nanoparticles) of 0.0001 to 0.01: 1, 0.0001 to 0.01: 1, 0.001 to 0.01: 1, or 0.001 to 0.005:1 may be added, but is not limited thereto.
상기 단계 (S3)의 자가항원; 및 항원제시세포 특이적 항체 또는 이의 단편을 상기 나노입자와 반응시키는 것은 상기 단계 (S2)로부터 수득한 나노입자의 현탁액에 자가항원; 또는 항원제시세포 특이적 항체 또는 이의 단편의 용액을 첨가하여 교반하여 이루어지는 것일 수 있다.the autoantigen of step (S3); And reacting the antigen-presenting cell-specific antibody or fragment thereof with the nanoparticles is an autoantigen in the suspension of nanoparticles obtained from step (S2); Alternatively, a solution of antigen-presenting cell-specific antibody or fragment thereof may be added and stirred.
상기 교반은 10 내지 35 ℃, 10 내지 30 ℃, 10 내지 27 ℃, 15 내지 30 ℃, 20 내지 30 ℃, 20 내지 27 ℃, 또는 23 내지 27 ℃에서 수행될 수 있고, 1 내지 30 시간, 1 내지 25 시간, 1 내지 20 시간, 1 내지 15 시간, 5 내지 30 시간, 10 내지 25 시간, 10 내지 20 시간, 또는 10 내지 15 시간 동안 수행될 수 있으나, 이에 한정되는 것은 아니다.The stirring may be performed at 10 to 35 ℃, 10 to 30 ℃, 10 to 27 ℃, 15 to 30 ℃, 20 to 30 ℃, 20 to 27 ℃, or 23 to 27 ℃, 1 to 30 hours, 1 to 25 hours, 1 to 20 hours, 1 to 15 hours, 5 to 30 hours, 10 to 25 hours, 10 to 20 hours, or 10 to 15 hours, but is not limited thereto.
일 구현예에서, 상기 항원제시세포 특이적 항체 또는 이의 단편은, 상기 항체 또는 이의 단편 : 항산화 나노입자 (바람직하게는, 폴리도파민 나노입자)의 중량비 (w/w)가 0.01 내지 1 : 1, 0.01 내지 0.9 : 1, 0.01 내지 0.8 : 1, 0.01 내지 0.7 : 1, 0.01 내지 0.6 : 1, 0.05 내지 1 : 1, 0.07 내지 1 : 1, 0.09 내지 1 : 1, 0.1 내지 1 : 1, 0.2 내지 1 : 1, 0.3 내지 1 : 1, 0.2 내지 0.8 : 1, 0.2 내지 0.6 : 1, 또는 0.4 내지 0.6 : 1가 되도록 첨가될 수 있으나, 이에 한정되지 않는다.In one embodiment, the antigen-presenting cell-specific antibody or fragment thereof has a weight ratio (w/w) of the antibody or fragment: antioxidant nanoparticles (preferably polydopamine nanoparticles) of 0.01 to 1:1, 0.01 to 0.9:1, 0.01 to 0.8:1, 0.01 to 0.7:1, 0.01 to 0.6:1, 0.05 to 1:1, 0.07 to 1:1, 0.09 to 1:1, 0.1 to 1:1, 0.2 to 1:1, 0.3 to 1:1, 0.2 to 0.8:1, 0.2 to 0.6:1, or 0.4 to 0.6:1 may be added, but is not limited thereto.
일 구현예에서, 상기 자가항원은, 상기 자가항원 : 항산화 나노입자 (바람직하게는, 폴리도파민 나노입자)의 중량비 (w/w)가 0.01 내지 1 : 1, 0.02 내지 1 : 1, 0.05 내지 1 : 1, 0.07 내지 1 : 1, 0.09 내지 1 : 1, 0.01 내지 0.8 : 1, 0.01 내지 0.6 : 1, 0.01 내지 0.5 : 1, 0.01 내지 0.3 : 1, 0.05 내지 0.3 : 1, 또는 0.09 내지 0.2 : 1가 되도록 첨가될 수 있으나, 이에 한정되지 않는다.In one embodiment, the autoantigen has a weight ratio (w/w) of the autoantigen: antioxidant nanoparticles (preferably polydopamine nanoparticles) of 0.01 to 1:1, 0.02 to 1:1, 0.05 to 1 : 1, 0.07 to 1:1, 0.09 to 1:1, 0.01 to 0.8:1, 0.01 to 0.6:1, 0.01 to 0.5:1, 0.01 to 0.3:1, 0.05 to 0.3:1, or 0.09 to 0.2: 1 may be added, but is not limited thereto.
상기 단계 (S3)에서, 상기 자가항원; 또는 항원제시세포 특이적 항체 또는 이의 단편이 티올기를 갖지 않은 것인 경우, 상기 제조방법은 상기 (S3) 단계 전에 상기 자가항원; 또는 항원제시세포 특이적 항체 또는 이의 단편이 티올기를 갖도록 변형하는 단계를 더 포함할 수 있다. 상기 티올기를 첨가하는 방법은 당업계에 공지되어 있으나, 바람직하게는 traut reagent를 상기 자가항원 또는 항체의 용액에 첨가하고 반응시켜 수행될 수 있다.In the step (S3), the autoantigen; Alternatively, when the antigen-presenting cell-specific antibody or fragment thereof does not have a thiol group, the preparation method includes the autoantigen; Alternatively, the method may further include modifying the antigen-presenting cell-specific antibody or fragment thereof to have a thiol group. The method of adding the thiol group is known in the art, but it may be preferably carried out by adding a traut reagent to a solution of the autoantigen or antibody and reacting.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.
[실시예][Example]
<실시예 1> 자가항원과 재조합 항체가 결합되어 있는 지질-항산화 나노입자 (AbaLDPN-MOG)의 제조<Example 1> Self-antigen and recombinant antibody are bound lipid-preparation of antioxidant nanoparticles (AbaLDPN-MOG)
표면이 면역관용 유도 약물을 함유한 지질층으로 코팅되어 있으며, 상기 지질층의 표면에는 자가항원 및 재조합 항체가 결합되어 있는 항산화 나노입자 (이하, “AbaLDPN-MOG”)를 제조하였다. 전체적인 제조 과정은 도 1에 나타냈다.Antioxidant nanoparticles (hereinafter, “AbaLDPN-MOG”) were prepared whose surface was coated with a lipid layer containing an immune tolerance-inducing drug, and autoantigens and recombinant antibodies were bound to the surface of the lipid layer. The overall manufacturing process is shown in FIG. 1 .
<1-1> 항산화 나노입자 (폴리도파민 나노입자, PN)의 제조<1-1> Preparation of antioxidant nanoparticles (polydopamine nanoparticles, PN)
알칼리 조건하에서 도파민의 자가 중합을 통해 폴리도파민 나노입자를 합성하였다. Polydopamine nanoparticles were synthesized through self-polymerization of dopamine under alkaline conditions.
구체적으로, 도파민 하이드로클로라이드 (dopamine hydrochloride)를 25 mL의 삼중 증류수 (TDW)에 녹여 최종 농도 2 mg/mL이 되도록 준비한 후, 1N 수산화 나트륨 용액을 이용하여 도파민 하이드로클로라이드 수용액을 pH 9.68로 적정한 후, 상온 (25 ℃)에서 24시간 동안 자기 교반 하였다. 반응이 종료되면, 13,500g에서 20분 동안 원심분리하여 흑색의 폴리도파민 나노입자 침전물을 수집하고, 상층액이 투명해질 때까지 삼중 증류수로 세척하였다. 세척을 마친 폴리도파민 나노입자는 450 nm 폴리카보네이트 필터에 여과를 하여 증류수에 현탁된 현탁액 상태로 4 ℃에 보관하였다. 이하 폴리도파민 나노입자는 PN 또는 항산화 나노입자 등으로 지칭한다.Specifically, dopamine hydrochloride was dissolved in 25 mL of triple distilled water (TDW) to prepare a final concentration of 2 mg/mL, and then the dopamine hydrochloride aqueous solution was titrated to pH 9.68 using 1N sodium hydroxide solution, Magnetic stirring was carried out at room temperature (25 °C) for 24 hours. Upon completion of the reaction, a black polydopamine nanoparticle precipitate was collected by centrifugation at 13,500 g for 20 minutes, and washed with triple distilled water until the supernatant became transparent. After washing, the polydopamine nanoparticles were filtered through a 450 nm polycarbonate filter and stored at 4° C. as a suspension in distilled water. Hereinafter, polydopamine nanoparticles are referred to as PN or antioxidant nanoparticles.
<1-2> 약물이 봉입되지 않은 지질막으로 코팅된 항산화 나노입자 (LPN)의 제조<1-2> Preparation of anti-oxidation nanoparticles (LPN) coated with a lipid membrane without drug encapsulation
상기 실시예 1-1에서 제조한 폴리도파민 나노입자를 함유한 현탁액으로, 약물을 함유하지 않은 지질층을 수화시켜, DPPC: DPPG=7:3의 지질 조성을 가진, 지질-항산화 나노입자를 제조하였다. The suspension containing the polydopamine nanoparticles prepared in Example 1-1 was hydrated with a drug-free lipid layer, and lipid-antioxidant nanoparticles having a lipid composition of DPPC: DPPG=7:3 were prepared.
구체적으로, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide)를 7 : 3 : 0.1의 몰비율로 혼합하여 클로로포름-메탄올 (4:1, v/v)에 용해시킨 후, 감압 농축하여 지질 박막을 제조하였다. 제조한 지질 박막에 상기 실시예 1-1에서 제조한 폴리도파민 수용액을 가하여 수화시켰다. 약물이 봉입 되지 않은 상태의 지질-항산화 나노입자 (LPN)는 4 ℃에 보관하였다. Specifically, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-disteroyl-sn-glycero-3-phosphoethanolamine -N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide) was mixed in a molar ratio of 7: 3: 0.1, dissolved in chloroform-methanol (4:1, v/v), and concentrated under reduced pressure. Lipid thin films were prepared. The polydopamine aqueous solution prepared in Example 1-1 was added to the prepared lipid thin film to hydrate. Lipid-antioxidant nanoparticles (LPN) in an unencapsulated state were stored at 4 °C.
<1-3> 약물이 봉입된 지질막으로 코팅된 항산화 나노입자 (LDPN)의 제조<1-3> Preparation of antioxidant nanoparticles (LDPN) coated with drug-encapsulated lipid membrane
상기 실시예 1-1에서 제조한 폴리도파민 나노입자를 함유한 현탁액으로, 약물을 함유한 지질층 (DPPC: DPPG=7:3의 지질 조성)을 수화시켜, 지질-항산화 나노입자를 제조하였다.With the suspension containing the polydopamine nanoparticles prepared in Example 1-1, a drug-containing lipid layer (DPPC: lipid composition of DPPG=7:3) was hydrated to prepare lipid-antioxidant nanoparticles.
구체적으로, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide)를 7 : 3 : 0.1의 몰비율로 혼합하여 클로로포름-메탄올 (4:1, v/v)에 용해시키고, 덱사메타손 (dexamethasone, Dexa)은 폴리도파민에 대하여 0.003 : 1 의 중량비 (w/w)로 첨가한 후 감압 농축하여 지질 박막을 제조하였다. 제조한 지질 박막은 상기 실시예 1-1에서 제조한 폴리도파민 수용액 또는 등장액인 5 % glucose 수용액을 가하여 수화시켰다. 약물이 봉입된 지질-항산화 나노입자 (LDPN)는 4 ℃에 보관하였다.Specifically, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-disteroyl-sn-glycero-3-phosphoethanolamine -N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide) was mixed in a molar ratio of 7: 3: 0.1, dissolved in chloroform-methanol (4:1, v/v), and dexamethasone (dexamethasone, Dexa) was added at a weight ratio (w/w) of 0.003: 1 with respect to polydopamine, and then concentrated under reduced pressure to prepare a lipid thin film. The prepared lipid thin film was hydrated by adding the polydopamine aqueous solution prepared in Example 1-1 or an isotonic 5% glucose aqueous solution. The drug-encapsulated lipid-antioxidant nanoparticles (LDPN) were stored at 4°C.
<1-4> 자가항원이 결합된 지질-항산화 나노입자 (LDPN-MOG)의 제조<1-4> Preparation of lipid-antioxidant nanoparticles (LDPN-MOG) bound to autoantigen
상기 실시예 1-3에서 제조한 나노입자에 자가항원 (myelin oligodendrocyte glycoprotein, MOG)을 가하여 격렬하게 교반 하였다. 먼저, thiol기를 포함하지 않은 자가항원 10 mg/mL에 traut reagent가 최종 농도 2 mg/mL가 되도록 첨가하여 25 ℃ 에서 1시간 정도 반응하여 thiolation 한 후 정제하였다. 이미 thiol기를 갖고 있는 자가항원의 경우 바로 사용이 가능하다. 정제된 자가항원은 지질-항산화 나노입자 현탁액에 처리하여 25 ℃ 에서 12 시간 동안 나노입자와의 반응을 진행하였으며, 20 분 동안 13,500g에서 원심분리하여 자가항원이 결합된 지질-항산화 나노입자 (LDPN-MOG)를 수집하고, 이를 증류수 또는 등장액인 5 % glucose 수용액에 재현탁 하여 4 ℃에 보관하였다.Autoantigen (myelin oligodendrocyte glycoprotein, MOG) was added to the nanoparticles prepared in Example 1-3 and vigorously stirred. First, traut reagent was added to a final concentration of 2 mg/mL to 10 mg/mL of an autoantigen not containing a thiol group, reacted at 25° C. for about 1 hour, followed by thiolation and purification. Autoantigens that already have a thiol group can be used immediately. The purified autoantigen was treated with a lipid-antioxidant nanoparticle suspension and reacted with nanoparticles at 25 °C for 12 hours, and centrifuged at 13,500 g for 20 minutes to bind autoantigen-bound lipid-antioxidant nanoparticles (LDPN). -MOG) was collected, resuspended in distilled water or an isotonic 5% glucose aqueous solution, and stored at 4 °C.
<1-5> 재조합 항체 및 자가항원이 결합된 지질-항산화 나노입자 (AbaLDPN-MOG)의 제조<1-5> Recombinant antibody and autoantigen conjugated lipid-preparation of antioxidant nanoparticles (AbaLDPN-MOG)
상기 실시예 1-3에서 제조한 나노입자에 재조합 항체 및 자가항원을 가하여 격렬하게 교반하였다. 재조합 항체 (10 mg/mL; CD80/86 특이적 항체) 및 자가항원 (10 mg/mL)을 traut reagent 2mg/mL 조건하에서 반응을 시켜 thiolation 한 후 정제하였다. 본 실시예에서는 상기 CD80/86 특이적 항체로서, 재조합 단백질인 Abatacept (Orencia, Bristol-Myers Squibb; BMS)를 사용하였다. Abatacept는 human cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4)을 human IgG1 의 Fc portion과 융합한 단백질로, CD80 및 CD86에 특이적으로 결합할 수 있다. Abatacept는 homodimer의 형태를 가지며, monomer는 357개의 amino acid sequence (서열번호 1)를 가지는 것으로 알려져 있다. 상기 Abatacept 대신, 또다른 상용화 약물인 Belatacept (Nulojix, BMS)를 사용할 수 있다. 이미 thiol기를 갖고 있는 자가항원 또는 항체의 경우 바로 사용이 가능하다. 정제된 자가항원 및 항체를 지질-항산화 나노입자 현탁액에 처리하여 25 ℃ 에서 12 시간 동안 나노입자와의 반응을 진행하였으며, 20 분 동안 13,500g에서 원심분리하여 자가항원 및 재조합 항체가 결합된 지질-항산화 나노입자 (AbaLDPN-MOG)를 수집하고, 이를 증류수 또는 등장액인 5 % glucose 수용액에 재현탁 하여 4 ℃에 보관하였다.Recombinant antibody and autoantigen were added to the nanoparticles prepared in Example 1-3 and vigorously stirred. Recombinant antibody (10 mg/mL; CD80/86-specific antibody) and autoantigen (10 mg/mL) were reacted under traut reagent 2 mg/mL conditions, followed by thiolation and purification. In this example, a recombinant protein Abatacept (Orencia, Bristol-Myers Squibb; BMS) was used as the CD80/86-specific antibody. Abatacept is a protein in which human cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is fused with the Fc portion of human IgG1, and can specifically bind to CD80 and CD86. Abatacept has the form of a homodimer, and the monomer is known to have a 357 amino acid sequence (SEQ ID NO: 1). Instead of the Abatacept, another commercially available drug, Belatacept (Nulojix, BMS) may be used. Autoantigens or antibodies that already have a thiol group can be used immediately. Purified autoantigens and antibodies were treated with lipid-antioxidant nanoparticle suspension to react with nanoparticles at 25° C. for 12 hours, and centrifuged at 13,500 g for 20 minutes to lipid-bound autoantigen and recombinant antibody- Antioxidant nanoparticles (AbaLDPN-MOG) were collected, resuspended in distilled water or isotonic 5% glucose aqueous solution, and stored at 4 °C.
[실험예][Experimental example]
<실험예 1> AbaLDPN-MOG의 물리화학적 특성 분석<Experimental Example 1> Analysis of physicochemical properties of AbaLDPN-MOG
<1-1> AbaLDPN-MOG의 입자 크기 및 제타전위 평가<1-1> Evaluation of particle size and zeta potential of AbaLDPN-MOG
상기 실시예 1에서 제조한 AbaLDPN-MOG의 물리화학적 특성을 파악하기 위해, 입자 크기 및 제타전위를 평가하였다. In order to understand the physicochemical properties of AbaLDPN-MOG prepared in Example 1, particle size and zeta potential were evaluated.
구체적으로, 실시예 1-1의 방법으로 제조한 폴리도파민 나노입자 (대조군) 및 실시예 1-3, 1-4, 또는 1-5의 방법으로 제조한 나노입자를 준비하였으며, 10% 소태아혈청 (fetal bovine serum), 및 100 units/㎖ 페니실린를 함유한 혈청에서 24시간 동안 보관한 후, AbaLDPN-MOG의 크기를 동적 광산란법 (ELS8000 instrument, Photal, Osaka, Japan)을 이용하여 측정하였다. 그 결과 도 2와 같이 PN, LDPN, 및 AbaLDPN-MOG간의 입자 크기에는 유의미한 차이가 없는 것을 확인 하였다. 또한 수용액 상에서의 제타전위는 레이저 도플러 전기영동법을 이용하여 측정하였으며 각 조성간의 유의미한 차이가 없음을 확인하였다.Specifically, polydopamine nanoparticles (control) prepared by the method of Example 1-1 and nanoparticles prepared by the method of Examples 1-3, 1-4, or 1-5 were prepared, and 10% fetal calf After storage for 24 hours in serum (fetal bovine serum) and serum containing 100 units/ml penicillin, the size of AbaLDPN-MOG was measured using a dynamic light scattering method (ELS8000 instrument, Photal, Osaka, Japan). As a result, as shown in FIG. 2, it was confirmed that there was no significant difference in particle size between PN, LDPN, and AbaLDPN-MOG. In addition, the zeta potential in aqueous solution was measured using laser Doppler electrophoresis, and it was confirmed that there was no significant difference between the respective compositions.
<1-2> AbaLDPN-MOG의 약물 봉입량 및 결합된 자가항원의 정량<1-2> AbaLDPN-MOG drug loading and quantification of bound autoantigen
상기 실시예 1에서 제조한 AbaLDPN-MOG에 봉입된 면역조절 약물 (덱사메타손) 및 상기 나노입자에 결합한 자가항원을 고성능 액체크로마토그래피 (HPLC, Agilent)를 이용하여 정량했다. The immunomodulatory drug (dexamethasone) encapsulated in AbaLDPN-MOG prepared in Example 1 and the autoantigen bound to the nanoparticles were quantified using high performance liquid chromatography (HPLC, Agilent).
면역조절 약물의 정량을 위하여, 나노입자를 methanol에 분산 시키고, 30 분간 초음파분산법 (ultrasonication)을 이용하여 지질층에 봉입된 약물이 모두 석출되도록 한 후, 27,000 xg에서 20분간 원심분리하여 약물이 녹아있는 상층액만을 분석하였다. 이동상은 삼차 증류수 : acetonitrile을 부피비 70:30의 조성으로 준비하였으며, 검출 파장은 254 nm이었다. 컬럼은 C18 역상 크로마토그래피 컬럼 (C18 reverse-phase HPLC column, Phenomenex)을 사용하였으며 25 ℃ 하에서 진행하였다. For quantification of immunomodulatory drugs, nanoparticles are dispersed in methanol, and all drugs encapsulated in the lipid layer are precipitated by ultrasonic dispersion for 30 minutes, followed by centrifugation at 27,000 x g for 20 minutes to dissolve the drug. Only the supernatant was analyzed. The mobile phase was prepared in a composition of tertiary distilled water: acetonitrile in a volume ratio of 70:30, and the detection wavelength was 254 nm. A C18 reverse-phase chromatography column (C18 reverse-phase HPLC column, Phenomenex) was used for the column, and was carried out at 25°C.
Thiol을 가진 자가항원 (MOG-Cys)의 경우, 나노입자와 반응시킨 후 남은 자유 항원들을 HPLC로 정량 하여 계산하였다. 이동상의 경우 gradient mobile phase를 이용하였으며, 용매 A를 0.1 % trifluoroacetic acid (TFA)/water, 용매 B를 0.1 % TFA/acetonitrile이라 할 때, 용매 B를 3%에서 시작하여 2 분간 20 %로, 10 분간 20 %에서 50 %로, 4분간 50 %에서 70 %로, 1 분간 70%에서 3%로 조성을 바꾸면서 진행하였으며 검출 파장은 562 nm에서 진행 하였다. 컬럼은 C18 역상 크로마토그래피 컬럼 (C18 reverse-phase HPLC column, Phenomenex)을 사용하였으며 25 ℃ 하에서 진행하였다.In the case of thiol-containing autoantigens (MOG-Cys), free antigens remaining after reacting with nanoparticles were quantified by HPLC and calculated. For mobile phase, gradient mobile phase was used. When solvent A was 0.1 % trifluoroacetic acid (TFA)/water and solvent B was 0.1 % TFA/acetonitrile, solvent B was started at 3% and increased to 20% for 2 minutes, 10 The composition was changed from 20% to 50% for 1 minute, from 50% to 70% for 4 minutes, and from 70% to 3% for 1 minute, and the detection wavelength was 562 nm. A C18 reverse-phase chromatography column (C18 reverse-phase HPLC column, Phenomenex) was used for the column, and was carried out at 25°C.
분석 결과는 도 3에 나타냈다. 약물이 함유된 지질박막으로 코팅되지 않은 대조군 (PN 나노입자)의 경우 약물이 검출되지 않았으나, 본 발명의 나노입자 (LDPN, LDPN-MOG, 및 AbaLDPN-MOG)는 모두 약물의 존재가 확인되었으며, 그 양은 나노입자간 유의미한 차이가 없는 것으로 나타났다. 자가항원의 경우, 자가항원이 수식되지 않은 대조군 (PN 나노입자) 및 LDPN 나노입자는 자가항원이 검출되지 않았으나, 자가항원으로 표면이 수식된 LDPN-MOG 나노입자 및 AbaLDPN-MOG 나노입자는 모두 자가항원이 높은 수준으로 존재하는 것을 확인할 수 있었다.The analysis results are shown in FIG. 3 . In the case of the control (PN nanoparticles) not coated with the drug-containing lipid film, no drug was detected, but the presence of the drug was confirmed in all of the nanoparticles (LDPN, LDPN-MOG, and AbaLDPN-MOG) of the present invention, The amount did not show any significant difference between nanoparticles. In the case of autoantigens, no autoantigens were detected in the control group (PN nanoparticles) and LDPN nanoparticles that were not modified with autoantigens, but LDPN-MOG nanoparticles and AbaLDPN-MOG nanoparticles surface-modified with autoantigens were all autologous. It was confirmed that the antigen was present at a high level.
<1-3> AbaLDPN-MOG의 분광학적 성질 평가<1-3> Evaluation of spectroscopic properties of AbaLDPN-MOG
상기 실시예 1에서 제조한 AbaLDPN-MOG의 분광학적 성질을 이용하여 화학적 작용기들을 분석하였다.Chemical functional groups were analyzed using the spectroscopic properties of AbaLDPN-MOG prepared in Example 1.
구체적으로, 실시예 1에서 제조한 나노입자들을 동결건조하고, 푸리에 변환 적외선분광법 (Fourier transformation-infrared spectroscopy, FT-IR) (FT/IR-400, JASCO)을 수행해 나노입자들의 지질 박막 코팅을 확인하였다. 적외선분광법은 attenuated total reflection (ATR)을 이용하여 측정하였다. 그 결과 2900 내지 3000 cm-1 에서 강한 피크가 나타남을 확인하였다. 이는 인지질의 지방산이 많이 갖고 있는 C-H 신축진동에 기인한 것이다. 라만 분광법 (LabRAM HR. Evolution, HORIBA)의 경우 위와 유사하게 동결건조한 나노입자들을 사용하였으며, 532 nm의 laser를 사용하여 분석하였다. 그 결과, 방향족고리들을 가진 고분자화합물의 특징인 D band와 G band가 모든 조성에서 공통적으로 나타난 것을 확인하였다 (도 4).Specifically, the nanoparticles prepared in Example 1 were freeze-dried, and Fourier transformation-infrared spectroscopy (FT-IR) (FT/IR-400, JASCO) was performed to confirm the lipid thin film coating of the nanoparticles. did. Infrared spectroscopy was measured using attenuated total reflection (ATR). As a result, it was confirmed that a strong peak appeared at 2900 to 3000 cm -1 . This is due to the CH stretching vibration that many fatty acids of phospholipids have. In the case of Raman spectroscopy (LabRAM HR. Evolution, HORIBA), freeze-dried nanoparticles were used similarly to the above and analyzed using a 532 nm laser. As a result, it was confirmed that the D band and G band, which are the characteristics of the polymer compound having aromatic rings, appeared in common in all compositions (FIG. 4).
<1-4> AbaLDPN-MOG의 형태 및 원소 분석<1-4> Morphology and elemental analysis of AbaLDPN-MOG
상기 실시예 1에서 제조한 AbaLDPN의 입자 크기 및 형태를 평가하였으며 원소분석의 경우 투과전자현미경에 탑재된 에너지 분산형 X-선 분광법 (EDS)를 통하여 분석하였다.The particle size and shape of AbaLDPN prepared in Example 1 were evaluated, and in the case of elemental analysis, energy dispersive X-ray spectroscopy (EDS) mounted on a transmission electron microscope was used.
분석 결과는 도 5에 나타냈다. 구체적으로, 투과전자현미경 관찰 결과, 입자의 형태는 동적 광산란법을 이용한 나노입자의 크기 분석 결과와 유사함을 확인할 수 있었다. 또한, EDS를 이용하여 원소분석을 수행한 결과, 항산화 나노입자의 주요 구성 원소인 탄소, 질소, 산소의 검출이 가장 지배적임을 확인하였다. 인의 검출은 인지질 층을 의미하며, 황의 검출은 표면에 결합한 단백질에 의한 것이다.The analysis results are shown in FIG. 5 . Specifically, as a result of transmission electron microscopy, it was confirmed that the shape of the particle was similar to the result of size analysis of the nanoparticles using the dynamic light scattering method. In addition, as a result of elemental analysis using EDS, it was confirmed that the detection of carbon, nitrogen, and oxygen, which are major constituent elements of antioxidant nanoparticles, was the most dominant. The detection of phosphorus refers to the phospholipid layer, and the detection of sulfur is by a protein bound to the surface.
<실험예 2> 재조합 항체의 수식에 따른 항원제시세포 특이적 결합능 확인<Experimental Example 2> Confirmation of antigen-presenting cell specific binding ability according to modification of recombinant antibody
실시예 1에서 제조한 LDPN 나노입자 및 AbaLDPN-MOG 나노입자의 CD80/86를 발현하고 있는 항원제시세포에 대한 결합능을 유세포 분석법, 공초점 현미경, 및 투과전자현미경을 통해서 확인하였으며, ex vivo 조건하에서 타겟 세포에 대한 선택성을 평가하였다.The binding ability of the LDPN nanoparticles and AbaLDPN-MOG nanoparticles prepared in Example 1 to antigen-presenting cells expressing CD80/86 was confirmed by flow cytometry, confocal microscopy, and transmission electron microscopy, and under ex vivo conditions. Selectivity for target cells was evaluated.
<2-1> 형광표지를 이용한 나노입자의 항원제시세포 결합능 평가<2-1> Evaluation of antigen-presenting cell binding ability of nanoparticles using fluorescent labeling
실시예 1에서 제조한 LDPN 및 AbaLDPN-MOG의 CD80/86-발현 수지상세포에 대한 결합능을 유세포분석법 및 공초점 현미경으로 확인 하였다. 나노입자는 실시예 1에 따라 CD80/86에 특이적으로 결합할 수 있는 재조합 항체로 표면을 수식했다. The binding ability of LDPN and AbaLDPN-MOG prepared in Example 1 to CD80/86-expressing dendritic cells was confirmed by flow cytometry and confocal microscopy. The nanoparticles were surface-modified with a recombinant antibody capable of specifically binding to CD80/86 according to Example 1.
구체적으로, C57BL/6 (8주령)으로부터 비장 유래 수지상세포를 유세포분석기를 이용하여 얻었다 (FACS Aria Sorting). 먼저 비장세포에 존재하는 세포들 중 CD11c 및 MHCII를 동시에 발현하는 세포만을 선별하여 진행하였다. 수지상세포의 경우, 대퇴골 및 경골을 적출하여 미성숙 수지상세포를 수득하고, 이를 granulocyte-macrophage colony-stimulating factor (GM-CSF) 및 interleukin-4 (IL-4)를 각각 20 ng/mL이 되도록 첨가한 배지 (10% 소태아혈청, 100 units/㎖ 페니실린, 및 100 mg/㎖ 스트렙토마이신이 보충된 Iscove's Modified Dulbecco's Medium (IMDM))에서 7일 간 분화 시킨 후 사용했다. 분화가 완료 되면 24 well plate에 well당 1×106 세포의 밀도로 파종 (seeding)한 후, 1 μg/mL의 LPS 조건하에서 24 시간 활성화 시켰다.Specifically, spleen-derived dendritic cells from C57BL/6 (8 weeks old) were obtained using flow cytometry (FACS Aria Sorting). First, among cells present in splenocytes, only cells expressing CD11c and MHCII were selected and performed. In the case of dendritic cells, immature dendritic cells were obtained by excision of the femur and tibia, and granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) were added at a concentration of 20 ng/mL, respectively. It was used after differentiation for 7 days in medium (Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin). Upon completion of differentiation, seeding was performed at a density of 1×10 6 cells per well in a 24-well plate, followed by activation for 24 hours under LPS conditions of 1 μg/mL.
이어서, 수지상세포에 Cy5로 표지된 나노입자들을 폴리도파민으로서 400 μg/mL이 되도록 처리하였다. 1시간 뒤, 나노입자들을 제거하고, 상기 수지상세포들의 Cy5 형광 세기를 유세포분석법으로 확인한다. Subsequently, dendritic cells were treated with Cy5-labeled nanoparticles as polydopamine to a concentration of 400 μg/mL. After 1 hour, the nanoparticles are removed, and the Cy5 fluorescence intensity of the dendritic cells is checked by flow cytometry.
그 결과, 도 6에 나타낸 바와 같이, 표면에 항체가 수식된 AbaLDPN-MOG 나노입자가 처리된 실험군은, LDPN-MOG가 처리된 실험군 보다 더 높은 형광 세기를 나타냈다. 상기 결과는 재조합 항체의 나노입자 수식이 수지상세포에 대한 상기 나노입자의 결합능을 높인다는 것을 보여주는 것이다.As a result, as shown in FIG. 6 , the experimental group treated with AbaLDPN-MOG nanoparticles on the surface of which the antibody was modified exhibited higher fluorescence intensity than the experimental group treated with LDPN-MOG. The above results show that the nanoparticle modification of the recombinant antibody increases the binding capacity of the nanoparticles to dendritic cells.
이어서, 공초점 현미경으로 나노입자의 세포 결합능을 확인하기 위해, 위와 동일한 방법으로 수지상세포 및 나노입자를 배양한 후, 나노입자들를 제거한 세포들을 10% formalin/PBS (v/v) 상에서 1시간 고정한 후에 2 μg/mL의 Hoechst로 15 분간 핵을 염색하여 현미경으로 관찰했다.Then, in order to confirm the cell-binding ability of nanoparticles with a confocal microscope, dendritic cells and nanoparticles were cultured in the same manner as above, and cells from which nanoparticles were removed were fixed in 10% formalin/PBS (v/v) for 1 hour. Afterwards, nuclei were stained with Hoechst at 2 μg/mL for 15 minutes and observed under a microscope.
그 결과, AbaLDPN-MOG가 처리된 실험군은 LDPN-MOG가 처리된 실험군 보다 더 높은 형광 세기를 나타내는 것을 확인했다 (도 7). 상기 결과는, 위의 실험결과와 마찬가지로, 본 발명에 따른 지질-항산화 나노입자의 표면을 재조합 항체로 수식하면 상기 나노입자의 항원제시세포에 대한 특이적 결합능이 증가한다는 것을 보여준다. As a result, it was confirmed that the experimental group treated with AbaLDPN-MOG exhibited higher fluorescence intensity than the experimental group treated with LDPN-MOG ( FIG. 7 ). The above results, like the above experimental results, show that the specific binding ability of the nanoparticles to antigen-presenting cells is increased when the surface of the lipid-antioxidant nanoparticles according to the present invention is modified with a recombinant antibody.
<2-2> 투과전자현미경을 이용한 항원제시세포에 대한 세포 결합능 평가<2-2> Evaluation of cell binding ability to antigen-presenting cells using transmission electron microscopy
실시예 1에서 제조한 LDPN 및 AbaLDPN-MOG의 CD80/86-발현 수지상세포에 대한 결합능을 투과전자현미경으로 확인 하였다. 나노입자는 실시예 1에 따라 CD80/86에 특이적으로 결합할 수 있는 재조합 항체로 표면을 수식했다.The binding ability of LDPN and AbaLDPN-MOG prepared in Example 1 to CD80/86-expressing dendritic cells was confirmed by transmission electron microscopy. The nanoparticles were surface-modified with a recombinant antibody capable of specifically binding to CD80/86 according to Example 1.
구체적으로, 활성화된 비장 유래 수지상세포에 대해 나노입자를 폴리도파민으로서 400 μg/mL이 되도록 처리하였다. 1 시간 후, 각 세포를 수집하여 Karnovsky 용액으로 2시간 동안 고정한 다음, 차가운 0.05 M 소듐 카코딜레이트 (sodium carcodylate) 완충액으로 3번 세척하고, 펠렛을 4 ℃에서 2시간 동안 1% 오스뮴 테트록시드 (osmium tetroxide)로 사후 고정시켰다. 고정시킨 펠렛을 차가운 삼중 증류수로 3회 세척한 후, 4℃에서 밤새 0.5% 우라닐 아세테이트 (uranyl acetate)로 염색하고, 에탄올 (30%, 50%, 70%, 80%, 90% 및 100% 3회)에서 탈수 시켰다. 탈수된 세포 펠렛에 50:50 프로필렌 옥사이드(propylene oxide)/스퍼(Spurr) 수지를 2시간 동안 침투시킨 후, 100% 스퍼 수지로 교체하여 이를 70 ℃ 오븐에서 24시간 동안 고체화 시켰다. 펠렛은 극미세단면 (60nm)으로 절단하여 TEM으로 관찰하였다.Specifically, for activated spleen-derived dendritic cells, nanoparticles were treated to be 400 μg/mL as polydopamine. After 1 h, each cell was collected and fixed with Karnovsky's solution for 2 h, then washed 3 times with cold 0.05 M sodium carcodylate buffer, and the pellet was washed with 1% osmium tetroxide at 4 °C for 2 h. (osmium tetroxide) was post-fixed. The fixed pellet was washed three times with cold triple distilled water, then stained with 0.5% uranyl acetate at 4°C overnight, and ethanol (30%, 50%, 70%, 80%, 90% and 100%). 3) was dehydrated. After infiltrating the dehydrated cell pellet with 50:50 propylene oxide/Spurr resin for 2 hours, it was replaced with 100% spur resin and solidified in an oven at 70° C. for 24 hours. The pellets were cut into micro-sections (60 nm) and observed by TEM.
그 결과, 도 8에 나타낸 바와 같이, LDPN-MOG 나노입자와 비교하여 훨씬 많은 수의 AbaLDPN-MOG 나노입자가 수지상세포에 결합한 것을 관찰할 수 있다. 즉, 상기 결과는 재조합 항체로 나노입자의 표면을 수식하는 것이 수지상세포에 대한 나노입자의 결합능을 높인다는 것을 입증하는 것이다.As a result, as shown in FIG. 8 , it can be observed that a much larger number of AbaLDPN-MOG nanoparticles bound to dendritic cells compared to LDPN-MOG nanoparticles. That is, the above results prove that modifying the surface of nanoparticles with a recombinant antibody increases the binding ability of nanoparticles to dendritic cells.
<실험예 3> AbaLDPN-MOG의 수지상세포 내 내포작용 (endocytosis) 확인<Experimental Example 3> Confirmation of endocytosis in dendritic cells of AbaLDPN-MOG
실시예 1에 따라 제조한, 재조합 항체 및 자가항원이 표면에 수식된 지질-항산화 나노입자가 수지상 세포 내로 효과적으로 내포작용을 일으키는 것을 공초점 현미경 및 투과전자현미경을 통하여 확인하였다. 특히, 상기 나노입자는 내포작용을 통해 수지상 세포의 리소좀 (lysosome) 내로 함입되는 것이 확인됐다. It was confirmed through confocal microscopy and transmission electron microscopy that the lipid-antioxidant nanoparticles prepared according to Example 1, on which the recombinant antibody and autoantigen were modified on the surface, effectively endocytosis into dendritic cells. In particular, it was confirmed that the nanoparticles are incorporated into the lysosome of dendritic cells through endocytosis.
구체적으로는, 실험예 2-1과 같은 조건으로, 수지상세포에 Cy5-표지된 나노입자들을 폴리도파민으로서 400 μg/mL에 해당하도록 처리하여 시간대별로 Cy5 형광의 움직임을 추적하였다. 또한 세포 내의 lysosome을 표지하기 위해서 Lysotracker (Invitrogen)을 사용했다.Specifically, under the same conditions as in Experimental Example 2-1, Cy5-labeled nanoparticles in dendritic cells were treated to correspond to 400 μg/mL as polydopamine, and the movement of Cy5 fluorescence was tracked for each time period. In addition, Lysotracker (Invitrogen) was used to label intracellular lysosomes.
그 결과, 수지상세포에 나노입자를 처리하고 1시간이 경과했을 때에는 대부분의 형광이 세포의 표면에서 검출됐으나, 4시간 뒤에는 세포 내부에서 Cy5 형광이 검출되기 시작했으며, 24시간 후에는 세포 내부의 lysotracker와 같은 위치에서 형광신호가 분산되어 존재하는 것을 확인하였다 (도 9). 상기 결과는 본 발명에 따른 나노입자가 내포작용을 통해 항원제시세포 내부로 도입될 수 있으며, 특히 리소좀 내로 이동한다는 것을 보여준다.As a result, most of the fluorescence was detected on the surface of the cell when 1 hour had elapsed after the treatment with nanoparticles on the dendritic cells, but after 4 hours, Cy5 fluorescence started to be detected inside the cell, and after 24 hours, the lysotracker inside the cell It was confirmed that the fluorescence signal was dispersed and existed at the same position as (FIG. 9). The above results show that the nanoparticles according to the present invention can be introduced into antigen-presenting cells through endocytosis, and, in particular, move into lysosomes.
<실험예 4> AbaLDPN-MOG의 수지상세포 및 T 세포간의 상호작용 억제 능력 확인<Experimental Example 4> Confirmation of the ability of AbaLDPN-MOG to inhibit the interaction between dendritic cells and T cells
실시예 1에 따라 제조한, 재조합 항체 및 자가항원이 표면에 수식된 지질-항산화 나노입자가항원제시세포 및 T 세포간 상호작용을 저해하는 것을 in vitro에서 확인하였다. 이를 위해, T 세포 활성화에 관여하는 수지상세포의 표면 단백질을 이용해 수지세상세포 및 T 세포간 상호작용을 in vitro로 구현했다. It was confirmed in vitro that the lipid-antioxidant nanoparticles prepared according to Example 1 and modified on the surface of the recombinant antibody and autoantigen inhibited the interaction between antigen-presenting cells and T cells. To this end, the interaction between dendritic cells and T cells was implemented in vitro using the surface proteins of dendritic cells involved in T cell activation.
<4-1> T 세포의 활성화 저해 작용 확인<4-1> Confirmation of T cell activation inhibitory action
본 발명에 따른 나노입자에 의한 수지상세포 및 T 세포간 상호작용 억제 효과를 도 10에 나타낸 in vitro 환경에서 T 세포의 IL-2 분비량을 통해 평가하였다.by nanoparticles according to the present invention The effect of inhibiting the interaction between dendritic cells and T cells was evaluated by the amount of IL-2 secretion of T cells in the in vitro environment shown in FIG. 10 .
구체적으로, 도 10의 시험관 조건은 수지상세포 및 T 세포 간의 상호작용을 모방한 것으로, 96 well plate에 anti-CD3 antibody (aCD3Ab) 및 CD80 Fc fusion protein (CD80 Fc)을 코팅하여 구현한 것이다. aCD3Ab 및 CD80 Fc를 각각 10 μg/mL이 되도록 PBS에 준비하여 well plate를 덮어준 후 4 ℃에서 12 시간 동안 보관했다. 12 시간 후, well plate의 코팅이 완료 되면 PBS로 1회 세척한 후 C57BL/6 (8주령)으로부터 준비한 비장 세포를 well 당 3×106 세포의 밀도로 파종하고, 나노입자는 폴리도파민으로서 400 μg/mL이 되도록 처리했다. 48 시간 후, 세포를 배양하던 배지를 수득하고, 이에 대해 제조사 protocol에 따라 ELISA (R&D systems, DY402-05)를 수행하여 IL-2를 검출했다.Specifically, the in vitro conditions of FIG. 10 mimic the interaction between dendritic cells and T cells, and are implemented by coating a 96-well plate with anti-CD3 antibody (aCD3Ab) and CD80 Fc fusion protein (CD80 Fc). ACD3Ab and CD80 Fc were each prepared in PBS at a concentration of 10 μg/mL, covered with a well plate, and stored at 4°C for 12 hours. After 12 hours, when the coating of the well plate is completed, after washing once with PBS, spleen cells prepared from C57BL/6 (8 weeks old) were seeded at a density of 3×10 6 cells per well, and the nanoparticles were 400 as polydopamine. It was processed so that it might become microgram/mL. After 48 hours, the medium in which the cells were cultured was obtained, and IL-2 was detected by performing ELISA (R&D systems, DY402-05) according to the manufacturer's protocol.
그 결과, 면역조절약물인 덱사메타손을 포함하는 나노입자 (LDPN-MOG 또는 AbaLDPN-MOG)가 처리된 그룹은 덱사메타손 및 항원을 직접 처리한 그룹 (Dexa+MOG)에 비해 T 세포에 의한 IL-2 분비가 현저하게 억제된 것을 확인할 수 있었다 (도 11). 특히, 표면이 수지상세포 특이적 항체로 수식된 AbaLDPN-MOG 나노입자는, 항체가 수식되지 않은 LDPN-MOG보다 더 효과적으로 T 세포의 IL-2 분비를 억제하는 것으로 나타났다. 상기 결과는 본 발명에 따른, 항체로 표면이 수식된 지질-항산화 나노입자가 well plate에 코팅된 수지상세포 표면 단백질과 T 세포의 상호작용을 효과적으로 차단하여, T 세포의 활성화를 억제한다는 것을 보여주는 것이다. 특히, 본 발명에 따른 나노입자는 양성대조군으로 사용한 CTLA-4 Fc와 비교하여서도 더욱 효과적으로 T 세포의 IL-2 분비를 억제하는 것으로 확인됐다. As a result, the group treated with nanoparticles (LDPN-MOG or AbaLDPN-MOG) containing the immunomodulatory drug dexamethasone had IL-2 secretion by T cells compared to the group treated with dexamethasone and antigen directly (Dexa+MOG). was remarkably suppressed (FIG. 11). In particular, it was shown that AbaLDPN-MOG nanoparticles whose surface was modified with a dendritic cell-specific antibody inhibited IL-2 secretion of T cells more effectively than LDPN-MOG on which the antibody was not modified. The above results show that the antibody-modified lipid-antioxidant nanoparticles according to the present invention effectively block the interaction between T cells and dendritic cell surface proteins coated on a well plate, thereby inhibiting T cell activation. . In particular, it was confirmed that the nanoparticles according to the present invention inhibited IL-2 secretion of T cells more effectively compared to CTLA-4 Fc used as a positive control.
<4-2> T 세포의 증식 및 분열 억제 작용 확인<4-2> Confirmation of T cell proliferation and division inhibitory action
본 발명에 따른 나노입자에 의한 수지상세포 및 T 세포간 상호작용 억제 효과를 도 10에 나타낸 in vitro 환경에서 T 세포의 증식 및 분열 수준을 통해 평가했다.by nanoparticles according to the present invention The effect of inhibiting the interaction between dendritic cells and T cells was evaluated through the proliferation and division levels of T cells in the in vitro environment shown in FIG. 10 .
구체적으로, 도 10과 동일한 조건에서 실험을 진행하였으며, 마찬가지로 96 well plate에 aCD3Ab 및 CD80 Fc를 코팅하여 구현하였다. 세포를 파종하기 전에 PBS로 1회 세척했다.Specifically, the experiment was carried out under the same conditions as in FIG. 10, and similarly, aCD3Ab and CD80 Fc were coated on a 96 well plate and implemented. Cells were washed once with PBS before seeding.
비장세포는 carboxyfluorescein succinimidyl ester (CFSE)로 염색하여 준비했다. CFSE (BioLegend, 423801)는 제조사의 protocol에 따라 준비되었으며, 형광 염색된 비장세포는 well 당 1×107 세포의 밀도로 파종하고, 나노입자는 폴리도파민으로서 400 μg/mL이 되도록 처리하였다. 72 시간 후 비장세포에 대해 유세포분석을 수행했다.Splenocytes were prepared by staining with carboxyfluorescein succinimidyl ester (CFSE). CFSE (BioLegend, 423801) was prepared according to the manufacturer's protocol, and fluorescently-stained splenocytes were seeded at a density of 1×10 7 cells per well, and the nanoparticles were treated to be 400 μg/mL as polydopamine. After 72 hours, flow cytometry was performed on splenocytes.
결과는 도 12 및 도 13에 나타냈다. 도면에서 확인 가능한 바와 같이, 본 발명에 따른 나노입자는 대조군들 (미처리대조군, MOG 처리군, 덱사메타손 및 MOG 처리군)과 비교하여 T 세포의 분열 및 증식을 현저하게 억제했다. 특히, 표면이 수지상세포 특이적 항체로 수식된 AbaLDPN-MOG 나노입자는, 항체가 수식되지 않은 LDPN-MOG보다 더 효과적으로 T 세포의 분열 및 증식을 억제한 것으로 나타났다. 형광 세기를 통한 T 세포의 확장 인덱스 (expansion index) 분석은 flowJo v10 프로그램으로 수행했다. 보조 T 세포를 의미하는 CD3 및 CD4 이중 양성 세포; 및 세포독성 T 세포를 의미하는 CD3 및 CD8 이중 양성 세포 각각에 대해서 분석했으며, 표면이 수지상세포 특이적 항체로 수식된 AbaLDPN-MOG 나노입자가 두 종류의 T 세포 모두 효과적으로 억제하는 것을 확인할 수 있었다.The results are shown in FIGS. 12 and 13 . As can be seen in the figure, the nanoparticles according to the present invention significantly inhibited the division and proliferation of T cells compared to the control groups (untreated control group, MOG-treated group, dexamethasone and MOG-treated group). In particular, it was found that AbaLDPN-MOG nanoparticles whose surface was modified with a dendritic cell-specific antibody inhibited the division and proliferation of T cells more effectively than LDPN-MOG on which the antibody was not modified. Analysis of T cell expansion index through fluorescence intensity was performed with flowJo v10 program. CD3 and CD4 double positive cells, meaning helper T cells; and CD3 and CD8 double-positive cells, which mean cytotoxic T cells, respectively, were analyzed, and it was confirmed that AbaLDPN-MOG nanoparticles whose surface was modified with a dendritic cell-specific antibody effectively inhibited both types of T cells.
<실험예 5> AbaLDPN-MOG의 면역관용 수지상세포 유도 능력 평가<Experimental Example 5> Evaluation of immune-tolerance dendritic cell induction ability of AbaLDPN-MOG
<5-1> 면역관용 수지상세포 유도 능력 확인<5-1> Confirmation of immune tolerance dendritic cell induction ability
실시예 1의 방법으로 제조한, 재조합 항체 및 자가항원이 표면에 수식된 지질-항산화 나노입자가 효과적으로 면역관용 수지상세포를 유도할 수 있음을 유세포분석법을 통해 확인 하였다. It was confirmed through flow cytometry that the lipid-antioxidant nanoparticles prepared by the method of Example 1 and modified on the surface of recombinant antibodies and autoantigens could effectively induce immune-tolerant dendritic cells.
구체적으로, 비장세포에 폴리도파민으로서 400 μg/mL에 해당하는 나노입자를 1시간 동안 처리하고, 나노입자를 제거한 상태로 세포를 72 시간 동안 배양 했다. 배양을 마친 비장세포에 1 μg/mL LPS를 처리하여 세포를 활성화 시킨 후 6 시간 뒤에 유세포분석법으로 세포를 확인했다. 세포질에 존재하는 단백질을 분석하기 위해서는 LPS와 함께 brefeldin A를 5 μg/mL이 되도록 처리하여 세포질 내 단백질의 유출을 막았다. 세포의 형광 항체 염색은 해당 제품 (BioLegend 424401)의 protocol을 따라서 진행하였다. 비장 세포 내에서 CD11c 및 MHCII의 이중 양성 세포만을 선택하여, 이들의 타겟 단백질에 대한 형광의 mean fluorescence intensity를 분석 하였으며, 세포막 단백질 CD86, CD80, MHCII; 및 염증성 사이토카인 TNF-α, IL-1β, IL-6의 수준을 확인했다.Specifically, splenocytes were treated with nanoparticles corresponding to 400 μg/mL as polydopamine for 1 hour, and the cells were cultured for 72 hours with the nanoparticles removed. After activating the cells by treating the cultured splenocytes with 1 μg/mL LPS, the cells were checked by flow cytometry 6 hours later. In order to analyze the protein present in the cytoplasm, it was treated with LPS at a concentration of 5 μg/mL with brefeldin A to prevent the leakage of the protein into the cytoplasm. Fluorescent antibody staining of cells was performed according to the protocol of the corresponding product (BioLegend 424401). Only double-positive cells of CD11c and MHCII were selected from spleen cells, and the mean fluorescence intensity of fluorescence for their target proteins was analyzed. Cell membrane proteins CD86, CD80, MHCII; and the levels of inflammatory cytokines TNF-α, IL-1β, and IL-6.
그 결과, 도 14에 나타낸 바와 같이, 본 발명에 따른 항산화-나노입자를 처리한 세포는 수지상세포의 활성화와 관련된 세포막 단백질인 CD80, CD86, 및 MHCII의 수준이 감소하고, 염증성 사이토카인인 TNF-α, IL-1β, 및 IL-6의 분비도 억제된 것을 확인할 수 있었다. 특히, 상기 효과는 재조합 항체로 표면이 수식된 지질-항산화 나노입자 (AbaLDPN-MOG)를 처리한 세포에서 더욱 현저하게 나타났다. 상기 결과는 본 발명에 따른 나노입자가 수지상세포에서 CD80, CD86, 및 MHCII에 의한 T 세포 활성화를 효과적으로 차단함으로써, 면역관용 수지상세포를 유도할 수 있음을 보여준다 (도 15). As a result, as shown in FIG. 14, the cells treated with the antioxidant-nanoparticles according to the present invention decreased the levels of CD80, CD86, and MHCII, which are cell membrane proteins related to the activation of dendritic cells, and the inflammatory cytokine TNF- It was confirmed that the secretion of α, IL-1β, and IL-6 was also suppressed. In particular, the effect was more pronounced in cells treated with lipid-antioxidant nanoparticles (AbaLDPN-MOG), the surface of which was modified with a recombinant antibody. The above results show that the nanoparticles according to the present invention can induce immune-tolerance dendritic cells by effectively blocking T cell activation by CD80, CD86, and MHCII in dendritic cells (FIG. 15).
<실험예 6> 동물모델에서의 AbaLDPN-MOG의 면역조절 효과 확인<Experimental Example 6> Confirmation of immunomodulatory effect of AbaLDPN-MOG in animal model
2차 면역기관인 림프절과 비장은 수지상세포를 비롯한 항원제시세포가 많이 분포하는 곳으로, 본 발명에 따른 면역조절을 위한 나노입자의 주요 타겟 장기이다. 본 발명자들은 동물모델에 상기 나노입자를 피하주사하였을 때, 상기 나노입자가 동물모델의 2차 면역기관을 표적화하는 것을 전임상 단층광학 이미징, 유세포분석법 및 면역형광조직법을 통하여 확인하였다.Lymph nodes and spleen, which are secondary immune organs, are the places where antigen-presenting cells, including dendritic cells, are abundantly distributed, and are major target organs for the nanoparticles for immunomodulation according to the present invention. The present inventors confirmed through preclinical tomographic imaging, flow cytometry and immunofluorescence histology that the nanoparticles were targeted to the secondary immune organs of the animal model when the nanoparticles were subcutaneously injected into an animal model.
<6-1> 단층광학 이미징을 이용한 AbaLDPN-MOG의 림프절 표적화 능력 평가<6-1> Evaluation of lymph node targeting ability of AbaLDPN-MOG using tomographic imaging
실시예 1에 따라 제조한, 재조합 항체 (CD80/86-특이적 재조합 항체) 및 자가항원이 표면에 수식된 지질-항산화 나노입자의 림프절 표적화 능력을 전임상 단층광학 이미징을 통하여 확인하였다.The lymph node targeting ability of the recombinant antibody (CD80/86-specific recombinant antibody) and the lipid-antioxidant nanoparticles on the surface of which the recombinant antibody (CD80/86-specific recombinant antibody) and autoantigen were modified, prepared according to Example 1, was confirmed through preclinical tomographic optical imaging.
구체적으로는, C57BL/6 (8주령)의 요추에 해당하는 부분의 피하에 Cy5로 표지 된 나노입자를 주사했다. 나노입자는 폴리도파민으로서 50 mg/kg으로 투여 하였으며, 투여 후 6, 24, 48, 및 72시간이 경과했을 때 림프절에서의 Cy5의 형광 세기를 측정했다.Specifically, Cy5-labeled nanoparticles were injected subcutaneously in the lumbar spine of C57BL/6 (8 weeks old). Nanoparticles were administered as polydopamine at 50 mg/kg, and when 6, 24, 48, and 72 hours had elapsed after administration, Cy5 fluorescence intensity in lymph nodes was measured.
그 결과, 도 16 및 도 17에 나타낸 바와 같이, 재조합 항체가 표면에 수식된 나노입자는 항체가 수식되지 않은 나노입자에 비해 림프절에 더 많은 양이 축적되었으며, 더 오랜 시간 동안 축적되어 있는 것을 확인하였다. 이와 같은 결과는 양쪽 림프절 모두에서 유사하게 나타났다. As a result, as shown in FIGS. 16 and 17 , it was confirmed that the nanoparticles on the surface of which the recombinant antibody was modified were accumulated in a larger amount in the lymph nodes compared to the nanoparticles in which the antibody was not modified, and accumulated for a longer time. did. These results were similar in both lymph nodes.
<6-2> AbaLDPN-MOG의 림프절 내 수지상세포의 표적화 능력 평가<6-2> Evaluation of AbaLDPN-MOG targeting ability of dendritic cells in lymph nodes
실시예 1에 따라 제조한, 재조합 항체 (CD80/86-특이적 재조합 항체) 및 자가항원이 표면에 수식된 지질-항산화 나노입자의 비장 표적화 능력을 림프절 내 수지상세포를 분석하여 확인하였다.The spleen-targeting ability of the recombinant antibody (CD80/86-specific recombinant antibody) and autoantigen-modified lipid-antioxidant nanoparticles prepared according to Example 1 on the surface was confirmed by analyzing dendritic cells in the lymph nodes.
이를 위해, 실험예 6-2와 동일한 방법으로 본 발명에 따른 나노입자를 마우스에 투여한 후, 마우스의 림프절을 분리하여 유세포분석법으로 확인하였다. To this end, the nanoparticles according to the present invention were administered to mice in the same manner as in Experimental Example 6-2, and then lymph nodes of the mice were isolated and confirmed by flow cytometry.
림프절에 존재하는 세포집단 중 수지상세포 (CD11c+MHCII+)에 대하여 Cy5형광을 나타내는 세포의 분포를 분석한 결과, 재조합 항체로 표면이 수식된 지질-항산화 나노입자를 처리한 그룹에서 Cy5 양성인 수지상세포의 비율이 대조군 (재조합 항체로 수식되지 않은 지질-항산화 나노입자를 처리한 그룹)에 비해 현저하게 증가한 것으로 나타났다 (도 18). 상기 결과는 재조합 항체로 표면이 수식된 지질-항산화 나노입자는 항체로 수식되지 않은 나노입자와 비교해 더욱 효과적으로 수지상세포에 결합하며, 림프절 타겟화 능력 역시 증가한다는 것을 보여준다. As a result of analyzing the distribution of cells exhibiting Cy5 fluorescence with respect to dendritic cells (CD11c + MHCII + ) among the cell populations present in the lymph nodes, lipid-modified lipid-antioxidant nanoparticles were treated with a recombinant antibody, and Cy5-positive dendritic cells It was shown that the ratio of the control group (lipid not modified with recombinant antibody-treated with antioxidant nanoparticles) was significantly increased compared to (FIG. 18). The above results show that lipid-antioxidant nanoparticles surface-modified with recombinant antibody bind to dendritic cells more effectively compared to nanoparticles not modified with antibody, and their ability to target lymph nodes is also increased.
<6-3> 면역형광조직법을 이용한 나노입자의 림프절 분포 능력 평가<6-3> Evaluation of lymph node distribution ability of nanoparticles using immunofluorescence histology
재조합 항체 (CD80/86-특이적 재조합 항체) 및 자가항원이 표면에 수식된 지질-항산화 나노입자의 림프절 표적화 능력을 면역형광조직법을 이용하여 확인하였다.The lymph node targeting ability of the recombinant antibody (CD80/86-specific recombinant antibody) and the lipid-antioxidant nanoparticles modified on the surface of the autoantigen was confirmed by immunofluorescence histology.
실험예 6-1과 동일한 방법으로 마우스에 Cy5로 표지된 나노입자를 투여한 후, 마우스의 림프절을 분리하여 공초점현미경 및 Automated Multimodal Tissue Analysis System (PerkinElmer, Vectra)을 이용하여 조직학적 분석을 실시하였다. 분리된 림프절은 10 % formalin/PBS (v/v)에서 24시간 고정한 후, 30 % sucrose 용액에서 cryoprotection process를 24시간 진행 하였다. 조직의 embedding medium은 OCT compound tissue-TEK을 이용했으며, 액화 질소를 이용하여 진행 후 -80 ℃에 보관하였다. Cryosection slide는 동결절편기 (Leica)를 이용하여 준비하였으며 조직 절편의 두께는 5 μm로 하였다. 형광 항체로 염색하기 전에, 10 % FBS/PBS로 2 시간 동안 blocking을 진행 하였고, PBS로 washing 후, 항체를 4 ℃에서 12시간 이상 처리하였다. 그 후, 샘플을 PBST를 이용하여 washing 하였으며, 2 μg/mL의 Hoechst로 1시간 동안 염색을 진행 하였다. 염색이 완료된 샘플은 washing한 후 fluoromount (Sigma-aldrich)를 처리하고 slide를 완성했다. Slide는 공초점 현미경과 Automated Multimodal Tissue Analysis System을 이용하여 분석하였다.After administering Cy5-labeled nanoparticles to the mouse in the same manner as in Experimental Example 6-1, the lymph nodes of the mouse were isolated and histological analysis was performed using a confocal microscope and an Automated Multimodal Tissue Analysis System (PerkinElmer, Vectra). did. The isolated lymph nodes were fixed in 10% formalin/PBS (v/v) for 24 hours, and then the cryoprotection process was performed in 30% sucrose solution for 24 hours. The tissue embedding medium was OCT compound tissue-TEK, and was stored at -80 °C after processing using liquid nitrogen. The cryosection slide was prepared using a cryosection (Leica), and the thickness of the tissue section was 5 μm. Before staining with a fluorescent antibody, blocking was performed with 10% FBS/PBS for 2 hours, and after washing with PBS, the antibody was treated at 4°C for more than 12 hours. After that, the samples were washed with PBST and stained with 2 μg/mL Hoechst for 1 hour. The stained samples were washed and treated with fluoromount (Sigma-aldrich) to complete the slide. Slides were analyzed using a confocal microscope and an Automated Multimodal Tissue Analysis System.
관찰 결과는 도 19에 나타냈다. 이미지에서 명확히 확인할 수 있듯이, 재조합 항체로 표면이 수식된 지질-항산화 나노입자를 처리한 마우스의 림프절에서는 대조군 (항체로 표면이 수식되지 않은 나노입자)에 비해 매우 높은 수준의 Cy5 형광이 관측됐다. 즉, 이는 항체로 표면이 수식된 지질-항산화 나노입자가 림프절에 높은 수준으로 축적되었음을 의미한다. 상기 결과는 본 발명에 따른 재조합 항체로 표면이 수식된 지질-항산화 나노입자의 림프절 표적화 기능이 우수하며, 따라서 림프절 내에 효과적으로 분포될 수 있음을 보여준다.The observation results are shown in FIG. 19 . As can be clearly seen from the image, a very high level of Cy5 fluorescence was observed in the lymph nodes of mice treated with lipid-antioxidant nanoparticles surface-modified with the recombinant antibody compared to the control group (nanoparticles surface-modified with an antibody). That is, this means that the lipid-antioxidant nanoparticles surface-modified with the antibody were accumulated at a high level in the lymph nodes. The above results show that the surface-modified lipid-antioxidant nanoparticles with the recombinant antibody according to the present invention have an excellent lymph node targeting function, and thus can be effectively distributed in lymph nodes.
<실험예 7> 자가면역질환 모델에서의 AbaLDPN-MOG 효능 평가<Experimental Example 7> AbaLDPN-MOG efficacy evaluation in autoimmune disease model
본 발명에 따른 나노입자의 효능을 오브알부민 (Ovalbumin, OVA)을 이용한 자가면역질환 동물모델에서 확인하였다. 나노입자의 체액성 면역반응 조절 효과는 혈청 내 자가항체의 농도 분석을 통해 평가하였고, 세포성 면역반응의 조절 효과는 ELISPOT을 통해 평가하였다.The efficacy of the nanoparticles according to the present invention was confirmed in an animal model of autoimmune disease using ovalbumin (OVA). The effects of nanoparticles on the humoral immune response were evaluated by analyzing the concentration of autoantibodies in serum, and the modulating effect of the cellular immune response was evaluated by ELISPOT.
<7-1> Ovalbumin을 이용한 자가면역질환 모델의 구축<7-1> Construction of Autoimmune Disease Model Using Ovalbumin
병원성 항원이 아닌 자가항원 (오브알부민, OVA)에 대하여 면역반응이 일어나도록 C57BL/6를 이용한 (12 주령) 자가면역질환 동물 모델을 구축하였다.An autoimmune disease animal model (12 weeks old) using C57BL/6 was constructed so that an immune response to an autoantigen (ovalbumin, OVA), not a pathogenic antigen, occurs.
구체적으로는, 2 mg/mL의 OVA와 동일한 부피에 해당하는 complete freund’s adjuvant (CFA; invivogen)을 균일한 유제 (emulsion)가 될 때까지 교반하였으며, 제조된 자가항원 용액 200 μL를 마우스에 피하주사하여 자가면역질환을 유도했다. 2 주 후, OVA 만을 추가로 100 μL 피하 주사하여 재활성화 시켰다. 실험의 전체적인 계획표는 도 20a과 같다.Specifically, complete freund's adjuvant (CFA; invivogen) corresponding to the same volume as 2 mg/mL OVA was stirred until it became a uniform emulsion, and 200 μL of the prepared autoantigen solution was subcutaneously injected into the mouse. This led to autoimmune disease. After 2 weeks, only OVA was reactivated by subcutaneous injection of an additional 100 μL. The overall schedule of the experiment is shown in FIG. 20A.
<7-2> 나노입자의 체액성 면역 반응 조절 능력 평가<7-2> Evaluation of the ability of nanoparticles to modulate humoral immune response
재조합 항체 및 자가항원이 표면에 수식된 지질-항산화 나노입자의 체액성 면역 반응 조절 능력 확인하기 위해, 동물모델의 체내 자가항체 농도를 측정했다.In order to confirm the ability of the lipid-antioxidant nanoparticles to modulate the humoral immune response of the recombinant antibody and the autoantigen-modified surface, the concentration of the autoantibody in the body of the animal model was measured.
구체적으로는, 자가항원으로 OVA가 담지된 본 발명의 나노입자 (AbaLDPN-OVA)를 7일 간격으로 3회 피하 주사하였으며, 자가면역질환 모델 유도시작일로부터 14일이 경과한 시점부터 매주 개체의 꼬리 정맥으로부터 혈액을 10 μL 수득했다. 혈액을 10% FBS를 함유한 5 mM EDTA/PBS 수용액에 잘 희석한 후, 2,000g 에서 10분간 원심 분리를 하여 적혈구를 분리해냈다. 분리된 혈청 샘플은 ELISA의 감도에 맞게 희석을 하여 OVA가 coating 된 well plate를 이용하여 ELISA를 진행했다.Specifically, the nanoparticles of the present invention (AbaLDPN-OVA) loaded with an autoantigen (AbaLDPN-OVA) were subcutaneously injected three times with an interval of 7 days, and the tail of the individual every week from the time point 14 days elapsed from the start date of induction of the autoimmune disease model. 10 μL of blood was obtained from the vein. Blood was well diluted in 5 mM EDTA/PBS aqueous solution containing 10% FBS, and then centrifuged at 2,000 g for 10 minutes to separate red blood cells. The separated serum samples were diluted according to the sensitivity of the ELISA, and ELISA was performed using an OVA-coated well plate.
그 결과, 도 20b에 나타낸 바와 같이, 본 발명의 나노입자가 투여된 마우스의 체내 자가항체 (anti-OVA IgG) 수준은 기타 대조군들에 비해 유의하게 감소하였으며, 특히 재조합 항체가 표면에 수식된 지질-항산화 나노입자를 투여 받은 마우스는 항체가 수식되지 않은 나노입자를 투여 받은 마우스보다도 더욱 현저하게 자가항체의 수준이 감소한 것을 확인하였다. 상기 결과는 본 발명에 따른 나노입자가 지속적인 항원 노출에도 불구하고 체액성 면역반응을 장기간 효과적으로 억제할 수 있으며, 항체 및 자가항원으로 표면이 수식된 나노입자의 체액성 면역 억제 효과가 특히 우수하다는 것을 뒷받침 한다.As a result, as shown in Figure 20b, the level of autoantibody (anti-OVA IgG) in the mouse administered with the nanoparticles of the present invention was significantly reduced compared to other controls, in particular, the recombinant antibody was modified on the surface of the lipid - It was confirmed that the level of autoantibodies in the mice receiving the antioxidant nanoparticles decreased more significantly than the mice receiving the nanoparticles in which the antibody was not modified. The above results show that the nanoparticles according to the present invention can effectively suppress the humoral immune response for a long period of time despite continuous antigen exposure, and that the humoral immunosuppressive effect of the nanoparticles surface-modified with antibodies and autoantigens is particularly excellent. back it up
<실험예 8> 자가면역 뇌척수염 모델에서의 AbaLDPN-MOG 효능 평가<Experimental Example 8> Efficacy evaluation of AbaLDPN-MOG in autoimmune encephalomyelitis model
본 발명에 따른 나노입자의 효능을 실험적 자가면역 뇌척수염 (experimental autoimmune encephalomyelitis; EAE) 모델을 이용하여 평가하였다. 효능 평가는 개체의 임상적 증상 (clinical score) 및 체중 변화를 비롯하여, 면역세포의 중추신경계 침습 정도 및 탈수초화 정도를 확인하여 이루어졌다.The efficacy of the nanoparticles according to the present invention was evaluated using an experimental autoimmune encephalomyelitis (EAE) model. Efficacy evaluation was made by confirming the degree of central nervous system invasion and demyelination of immune cells, including changes in the subject's clinical symptoms (clinical score) and body weight.
<8-1> 실험적 자가면역 뇌척수염 (EAE) 동물 모델의 구축 및 나노입자 투여에 따른 임상증상 평가<8-1> Construction of experimental autoimmune encephalomyelitis (EAE) animal model and evaluation of clinical symptoms according to nanoparticle administration
실험적 자가면역 뇌척수염 (EAE) 모델은 인간의 다발성 경화증 (multiple sclerosis) 연구에 널리 사용되는 실험적 동물 모델로, 건강한 개체의 신경계에 존재하는 myelin oligodendrocyte glycoprotein (MOG)의 35에서 55번까지의 fragment peptide (MOG35-55)에 대한 자가 면역반응을 유도하여 구축한 모델이다.The experimental autoimmune encephalomyelitis (EAE) model is an experimental animal model widely used in the study of multiple sclerosis in humans. Fragment peptides 35 to 55 of myelin oligodendrocyte glycoprotein (MOG) present in the nervous system of healthy individuals ( It is a model constructed by inducing an autoimmune response to MOG 35-55 ).
구체적으로는, MOG35-55 (MOG peptide) (Prospec Bio, PRO-371) 2 mg/mL과 complete freund’s adjuvant (CFA; Heat inactivated M. tuberculosis, 4 mg/mL) (Chondrex)를 동일한 부피로 혼합하여 유제 (emulsion) 형태로 준비했다. 균일한 유제를 C57BL/6 (12주령, female)에 200 μL씩, 피하투여한 후 2 시간 이내로 pertussis toxin (PTX) (List Biological Lab) 400 ng을 복강투여 하였다. 유제 피하 주사 48 시간 뒤에 PTX를 추가로 400 ng 복강투여 하였다.Specifically, MOG 35-55 (MOG peptide) (Prospec Bio, PRO-371) 2 mg/mL and complete freund's adjuvant (CFA; Heat inactivated M. tuberculosis, 4 mg/mL) (Chondrex) were mixed in the same volume. Thus, it was prepared in the form of an emulsion. 200 μL of a uniform emulsion was subcutaneously administered to C57BL/6 (12 weeks old, female), and 400 ng of pertussis toxin (PTX) (List Biological Lab) was intraperitoneally administered within 2 hours. An additional 400 ng of PTX was administered intraperitoneally 48 hours after the subcutaneous injection of the emulsion.
이후 개체의 임상적 증상 (임상 스코어 (clinical score))과 체중을 추적관찰했다. 임상적 증상 (clinical score)에 대한 criteria는 선행 문헌 Nature Protocols 1 (2006) 1810-1819를 기반으로 평가하였으며 매일 체중과 함께 평가하였다.Afterwards, the subjects' clinical symptoms (clinical score) and body weight were followed up. Criteria for clinical score were evaluated based on the prior literature Nature Protocols 1 (2006) 1810-1819, and daily weight was evaluated.
투여 스케쥴은 실험 목적 (자가면역질환의 예방, 진행 억제, 및 치료)에 따라 다르게 진행했다. 자가면역질환의 예방을 목적으로 할 경우, 질환모델을 유도하기 2주 전부터 1주일 간격으로 2회 나노입자 투여를 진행하였으며 (도 21a), 자가면역질환의 진행 억제를 목적으로 할 경우, 자가면역질환 유도 직후 1주일 간격으로 2회 나노입자 투여를 진행하였고 (도 21b), 자가면역질환의 치료가 목적인 경우 증상이 나타나기 시작하였을 때, clinical score가 0.5인 개체들을 무작위적으로 선별하여 대조군 및 실험군으로 나누고, 나노입자를 폴리도파민으로서 50 mg/kg이 되도록 3일 마다 4회 피하 주사로 투여했다 (도 21c).The administration schedule differed depending on the experimental purpose (prevention, progression inhibition, and treatment of autoimmune diseases). For the purpose of preventing autoimmune diseases, the nanoparticles were administered twice at intervals of 1 week from 2 weeks before inducing the disease model (FIG. 21a). Immediately after disease induction, the nanoparticles were administered twice at intervals of 1 week (FIG. 21b), and when symptoms began to appear when the purpose of the treatment of autoimmune disease was to start, the subjects with a clinical score of 0.5 were randomly selected, and the control group and the experimental group , and the nanoparticles were administered by subcutaneous injection 4 times every 3 days to 50 mg/kg as polydopamine ( FIG. 21c ).
총 28일동안 개체들의 임상 스코어와 체중 변화를 추적관찰 하였다. 그 결과, 미처리 대조군에 비해 본 발명의 지질-항산화 나노입자가 처리된 마우스는 효과적으로 임상 증상을 개선하고 체중을 정상적으로 조절하며, 특히 재조합 항체가 표면에 수식된 AbaLDPN-MOG가 그렇지 않은 LDPN-MOG에 비해서 더 고무적인 결과를 보여주는 것을 확인했다 (도 22a 내지 22c).Clinical scores and weight changes of the subjects were followed up for a total of 28 days. As a result, compared to the untreated control group, the mice treated with the lipid-antioxidant nanoparticles effectively improved clinical symptoms and normally controlled body weight, and in particular, AbaLDPN-MOG on which the recombinant antibody was modified on the surface of LDPN-MOG was not treated. It was confirmed that it showed more encouraging results compared to that ( FIGS. 22a to 22c ).
<8-2> 나노입자의 면역세포의 중추신경계 침습 개선 및 조절 T 세포 증가 효과 확인<8-2> Confirmation of the effect of nanoparticles to improve the central nervous system invasion of immune cells and increase regulatory T cells
본 발명에 따른 항산화-나노입자에 의한 면역세포의 중추신경계 침습 개선 능력을 유세포분석법으로 평가하였다. Antioxidant according to the present invention - The ability to improve the central nervous system invasion of immune cells by nanoparticles was evaluated by flow cytometry.
구체적으로는, 실험예 8-1과 동일한 방법으로 자가면역질환 모델에 나노입자를 투여하였으며, 실험 개시일로부터 28일이 경과한 후 각 개체로부터 척수를 적출하였다. 척수는 1mg/mL collagenase (Sigma-Aldrich) 및 cell strainer (40 μm) (SPL)을 이용하여 single cell로 분리 되었으며, hypotonic buffer내에서 적혈구를 용혈한 후 사용하였다. 각 면역세포는 기본적으로 CD45를 발현하는 것을 선별하였고, 수지상세포의 경우 CD11c+ MHCII+를, 대식세포의 경우 CD11b+ F4/80+를, 조력 T 세포의 경우 CD3+ CD4+를, 세포독성 T 세포의 경우 CD3+ CD8+를, 조절 T 세포의 경우 조력 T 세포 내에서 CD25+ FoxP3+를 마커로 하여 분석하였다. 병인성 보조 T 세포의 경우, CD3+ CD4+를 공통으로 표지하며, IFN-γ 또는 IL-17A를 발현하는 것으로 분석하였다. 표지 방법은 상기 유세포분석법과 동일하게 진행하였다.Specifically, nanoparticles were administered to the autoimmune disease model in the same manner as in Experimental Example 8-1, and after 28 days from the start date of the experiment, the spinal cord was extracted from each individual. The spinal cord was isolated into single cells using 1 mg/mL collagenase (Sigma-Aldrich) and a cell strainer (40 μm) (SPL), and was used after hemolysis of red blood cells in hypotonic buffer. Each immune cell was basically selected to express CD45, CD11c + MHCII + for dendritic cells, CD11b + F4/80 + for macrophages, CD3 + CD4 + for helper T cells, and cytotoxic T In the case of cells, CD3 + CD8 + , and in the case of regulatory T cells, CD25 + FoxP3 + in helper T cells were analyzed as markers. For pathogenic helper T cells, they were analyzed to commonly label CD3 + CD4 + and express either IFN-γ or IL-17A. The labeling method was performed in the same manner as the flow cytometry method.
결과는 도 23a 내지 23c에 나타냈다. 본 발명에 따른 항산화 나노입자를 자가면역질환의 예방, 진행 억제, 또는 치료 목적으로 투여한 경우 모두 미처리 대조군에 비해 면역세포들의 중추신경계로의 침습이 감소한 것을 확인할 수 있었다. 특히, 상기 효과는 보다 LDPN-MOG 투여군보다는 AbaLDPN-MOG 투여군에서 더욱 확연하게 나타났다. 보다 자세하게는, 수지상세포, 대식세포, 및 세포독성 T 세포 모두 AbaLDPN-MOG가 가장 효과적으로 중추신경계로의 침습을 억제하였으며, 조력 T 세포에 대해서는 LDPN-MOG 및 AbaLDPN-MOG가 유사한 정도의 효과를 보였으나, 면역관용 효과가 있는 조절 T 세포의 경우, 후술하는 바와 같이 AbaLDPN-MOG를 투여한 실험군에서 가장 높은 수준을 나타내는 것을 확인하였다 (도 24a 내지 24c). 즉, 이는 본 발명에 따른 항산화 나노입자가 과잉의 면역반응을 조절할 수 있는 조절 T 세포의 비율을 증가시키는 효과가 있음을 보여주는 것이다. 뿐만 아니라, 조직을 손상시킬 수 있는 IFN-γ 또는 IL-17A를 발현하는 보조 T 세포의 비율은 AbaLDPN-MOG를 투여한 실험군에서 가장 확연하게 감소했다.The results are shown in FIGS. 23A-23C. When the antioxidant nanoparticles according to the present invention were administered for the purpose of prevention, progression inhibition, or treatment of autoimmune diseases, it was confirmed that the invasion of immune cells into the central nervous system was reduced compared to the untreated control group. In particular, the effect was more pronounced in the AbaLDPN-MOG administration group than in the LDPN-MOG administration group. More specifically, AbaLDPN-MOG inhibited invasion into the central nervous system most effectively for dendritic cells, macrophages, and cytotoxic T cells, and LDPN-MOG and AbaLDPN-MOG showed similar effects on helper T cells. B, in the case of regulatory T cells having an immune tolerance effect, it was confirmed that the highest level was exhibited in the experimental group administered with AbaLDPN-MOG as described below ( FIGS. 24a to 24c ). That is, this shows that the antioxidant nanoparticles according to the present invention have an effect of increasing the ratio of regulatory T cells capable of regulating an excessive immune response. In addition, the ratio of helper T cells expressing IFN-γ or IL-17A, which can damage tissues, was most significantly decreased in the experimental group administered with AbaLDPN-MOG.
<8-3> 나노입자의 자가항원 특이적 세포성 면역반응 조절 능력 평가<8-3> Evaluation of the ability of nanoparticles to modulate autoantigen-specific cellular immune response
본 발명에 따른 항산화-나노입자의 면역세포의 항원특이적 세포성 면역 반응 조절 능력을 ELISPOT을 이용하여 평가하였다.Antioxidant according to the present invention - The ability of nanoparticles to modulate antigen-specific cellular immune response of immune cells was evaluated using ELISPOT.
구체적으로는, 실험예 8-1과 동일한 방법으로 자가면역질환 모델에 나노입자를 투여하였으며, 실험 개시일로부터 28일이 지난 뒤 개체들로부터 비장세포를 적출하여, 5 μg/mL의 MOG peptide 처리 하에서 ELISPOT (BD, Mouse IFN-γ ELISPOT Set, 551083)을 진행 하였다. 구체적인 실험은 제품의 protocol을 따라 진행 하였다Specifically, nanoparticles were administered to the autoimmune disease model in the same manner as in Experimental Example 8-1, and splenocytes were extracted from the subjects after 28 days from the start date of the experiment, under the treatment of 5 μg/mL MOG peptide. ELISPOT (BD, Mouse IFN-γ ELISPOT Set, 551083) was performed. Specific experiments were carried out according to the protocol of the product.
결과는 도 25a 내지 25c에 나타냈다. 본 발명에 따른 항산화 나노입자를 자가면역질환의 예방, 진행 억제, 또는 치료 목적으로 투여한 경우, 상기 나노입자가 각 동물모델의 비장세포가 자가항원 (MOG peptide)에 의해 활성화되어 IFN-γ를 분비하는 것을 감소시키는 것을 확인했다. 즉, 상기 나노입자는 MOG peptide에 의해 활성화 되는 면역세포들의 세포성 면역반응을 효과적으로 억제할 수 있으며 특히, 재조합 항체로 수식된 지질-항산화 나노입자의 경우 재조합 항체가 수식 되지 않은 지질-항산화 나노입자보다 효과적으로 면역반응을 조절하는 것으로 나타났다.The results are shown in Figures 25a to 25c. When the antioxidant nanoparticles according to the present invention are administered for the purpose of preventing, inhibiting progression, or treating autoimmune diseases, the nanoparticles activate the splenocytes of each animal model by autoantigen (MOG peptide) to produce IFN-γ. It was confirmed that secretion was reduced. That is, the nanoparticles can effectively inhibit the cellular immune response of immune cells activated by the MOG peptide, and in particular, in the case of lipid-antioxidant nanoparticles modified with recombinant antibody, lipid-antioxidant nanoparticles that are not modified with recombinant antibody It has been shown to more effectively modulate the immune response.
<8-4> 나노입자의 면역세포의 중추신경계 침습 개선 능력 및 탈수초화 억제 효과 평가<8-4> Evaluation of the ability of nanoparticles to improve the central nervous system invasion and the inhibitory effect of demyelination of immune cells
본 발명에 따른 항산화-나노입자의 면역세포의 중추신경계 침습 개선 능력 탈수초화 억제 효과를 면역형광조직법을 이용하여 분석하였다.Antioxidant according to the present invention - The ability of the nanoparticles to improve the central nervous system invasion of immune cells and the inhibitory effect of demyelination was analyzed using immunofluorescence histology.
구체적으로는, 실험예 8-1과 동일한 방법으로 자가면역질환 모델에 나노입자를 투여하였으며, 실험 개시일로부터 28일이 지난 뒤, 각 개체로부터 척수를 적출하였다. 적출한 척수는 실험예 6-3과 동일한 방법으로 처리하여 면역형광조직법을 수행하였다. CD45는 중추신경계를 침습한 면역세포의 마커이며, myelin basic protein (MBP)은 수초의 마커로서, 탈수초화 정도를 확인 할 수 있다.Specifically, nanoparticles were administered to the autoimmune disease model in the same manner as in Experimental Example 8-1, and after 28 days from the start date of the experiment, the spinal cord was extracted from each individual. The extracted spinal cord was treated in the same manner as in Experimental Example 6-3, and immunofluorescence histology was performed. CD45 is a marker of immune cells that invaded the central nervous system, and myelin basic protein (MBP) is a myelin marker, which can confirm the degree of demyelination.
결과는 도 26에 나타냈다. 본 발명에 따른 항산화 나노입자를 투여 받은 마우스는 미처리 대조군과 비교하여 면역세포들의 중추신경계 침습이 두드러지게 감소한 것으로 나타났으며, 특히 면역세포 침습 억제 효과는 AbaLDPN-MOG를 투여한 실험군에서 가장 뛰어난 것으로 나타났다. 상기 결과는 본 발명에 따른 항산화 나노입자가 뇌척수염 동물모델에서 마이엘린 수초 (myelin sheath)를 보호하며, 면역세포의 중추신경계 침습을 차단하여 면역반응을 조절할 수 있음을 보여준다.The results are shown in FIG. 26 . The mice receiving the antioxidant nanoparticles according to the present invention showed a remarkably reduced central nervous system invasion of immune cells compared to the untreated control group. appear. The above results show that the antioxidant nanoparticles according to the present invention can protect the myelin sheath in an animal model of encephalomyelitis and regulate the immune response by blocking the invasion of the central nervous system by immune cells.
이상에서 살펴본 바와 같이, 본 발명에 따른 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자는 개체에 투여되었을 때 비장 또는 림프절을 효과적으로 타겟팅하고, 항원제시세포에 특이적으로 결합하여 지질막 내에 봉입된 면역억제제를 효과적으로 전달할 뿐만 아니라, 자가항원에 대한 면역관용을 유도하는 것이 확인되었다 (도 27). 즉, 본 발명에 따른 나노입자는 자가항원에 대한 자가항체 생성을 억제하고, 면역세포들의 과할성화를 억제하며, 면역세포에 의한 정상조직 침습을 억제할 수 있으므로, 다양한 자가면역질환의 예방 및 치료 목적으로 활용될 것으로 기대된다.As described above, the lipid-antioxidant nanoparticles to which the antibody and the autoantigen according to the present invention are bound to the surface effectively target the spleen or lymph node when administered to a subject, bind specifically to antigen-presenting cells, and are encapsulated in the lipid membrane. It was confirmed that not only effectively delivered the immunosuppressive agent, but also induces immune tolerance to autoantigens ( FIG. 27 ). That is, the nanoparticles according to the present invention can inhibit the generation of autoantibodies against autoantigens, suppress hyperactivity of immune cells, and suppress invasion of normal tissues by immune cells, thus preventing and treating various autoimmune diseases. It is expected to be used for this purpose.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야 한다. The foregoing description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.
본 발명에 따른 지질-항산화 나노입자는 자가항원 및 항원제시세포 특이적 항체가 표면에 수식되어 있어 항원제시세포 특이적으로 자가항원을 전달할 수 있고, 표면은 면역조절제가 봉입된 지질막으로 코팅되어 있어 자가항원에 대해 면역관용을 유도할 수 있다. 특히 본 발명에 따른 나노입자는 자가면역질환 동물모델에 투여하였을 때 면역기관인 림프절 및 비장을 효과적으로 타겟팅하여 항원제시세포들에 의한 과잉의 면역 활성화를 효과적으로 억제함으로써, 자가면역질환을 예방, 지연, 및 치료할 수 있음이 확인되었다. 따라서, 본 발명에 따른 나노입자는 뇌척수염 등을 포함한 다양한 자가면역질환의 예방 내지 치료를 위해 유용히 활용될 것으로 기대된다.Lipid-antioxidant nanoparticles according to the present invention have autoantigens and antigen-presenting cell-specific antibodies modified on the surface to deliver the autoantigen specifically to antigen-presenting cells, and the surface is coated with a lipid membrane encapsulated with an immunomodulatory agent. Immune tolerance to autoantigens can be induced. In particular, when the nanoparticles according to the present invention are administered to an animal model of autoimmune disease, they effectively target lymph nodes and spleen, which are immune organs, and effectively inhibit excessive immune activation by antigen-presenting cells, thereby preventing, delaying, and It has been shown to be curable. Therefore, the nanoparticles according to the present invention are expected to be usefully utilized for the prevention or treatment of various autoimmune diseases, including encephalomyelitis.

Claims (29)

  1. 항산화 나노입자; 상기 나노입자 표면을 코팅하는 지질막; 상기 지질막 표면에 결합된 자가항원; 및 상기 지질막 표면에 결합된 항원제시세포 특이적 항체 또는 이의 단편을 포함하는, 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자.antioxidant nanoparticles; a lipid film coating the surface of the nanoparticles; an autoantigen bound to the surface of the lipid membrane; And An antigen-presenting cell-specific antibody or fragment thereof bound to the surface of the lipid membrane, wherein the antibody and the autoantigen are bound to the surface of the lipid-antioxidant nanoparticles.
  2. 제1항에 있어서,According to claim 1,
    상기 항산화 나노입자는 폴리도파민 나노입자, 탄닌 나노입자, 및 세륨 산화물 나노입자로 이루어진 군에서 선택된 하나 이상인, 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자.The antioxidant nanoparticles are polydopamine nanoparticles, tannin nanoparticles, and at least one selected from the group consisting of cerium oxide nanoparticles, wherein the antibody and autoantigen are bound to the surface of the lipid-antioxidant nanoparticles.
  3. 제1항에 있어서,According to claim 1,
    상기 지질막은 페길화된 것을 특징으로 하는, 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자.The lipid membrane is characterized in that the pegylated, antibody and autoantigen bound to the surface of the lipid-antioxidant nanoparticles.
  4. 제1항에 있어서,According to claim 1,
    상기 지질막은 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), phosphorylglycerol (PG), phosphocholine (PC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide), 콜레스테롤 (cholesterol), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 및 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)로 이루어진 군에서 선택된 하나 이상을 포함하는 것을 특징으로 하는, 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자.The lipid membrane is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), phosphorylglycerol (PG), phosphocholine (PC), 1,2 -distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide), cholesterol (cholesterol), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) , 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) comprising at least one selected from the group consisting of, an antibody and Lipid-antioxidant nanoparticles with autoantigen bound to the surface.
  5. 제4항에 있어서5. The method of claim 4
    상기 DPPC : DPPG의 몰비율은 1 내지 10 : 1인 것을 특징으로 하는, 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자.The DPPC: DPPG molar ratio is 1 to 10: characterized in that 1 to 1, the antibody and the autoantigen bound to the surface of the lipid-antioxidant nanoparticles.
  6. 제4항에 있어서5. The method of claim 4
    상기 DPPC : DPPG : DSPE-PEG2000-maleimide의 몰비율은 100 내지 1000 : 10 내지 500 : 1인 것을 특징으로 하는, 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자.The molar ratio of the DPPC: DPPG: DSPE-PEG2000-maleimide is 100 to 1000: 10 to 500: 1, characterized in that the antibody and autoantigen are bound to the surface of the lipid-antioxidant nanoparticles.
  7. 제1항에 있어서,According to claim 1,
    상기 항원제시세포 특이적 항체 또는 이의 단편은 수지상세포 또는 대식세포에 특이적으로 결합하는 항체 또는 이의 단편인 것을 특징으로 하는, 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자.The antigen-presenting cell-specific antibody or fragment thereof is an antibody or fragment thereof that specifically binds to dendritic cells or macrophages, wherein the antibody and autoantigen are surface-bound lipid-antioxidant nanoparticles.
  8. 제7항에 있어서,8. The method of claim 7,
    상기 항원제시세포 특이적 항체 또는 이의 단편은 CD80, CD86, CD123, CD303, CD304, CD68, CD11b, CD11c, BDCA-1, DC-SIGN, MHCII, F4/80, CD206, 및 CSF1-R로 이루어진 군에서 선택된 하나 이상의 단백질에 결합하는 것을 특징으로 하는, 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자.The antigen-presenting cell-specific antibody or fragment thereof is from the group consisting of CD80, CD86, CD123, CD303, CD304, CD68, CD11b, CD11c, BDCA-1, DC-SIGN, MHCII, F4/80, CD206, and CSF1-R Characterized in binding to one or more proteins selected from, antibodies and autoantigens bound to the surface of the lipid-antioxidant nanoparticles.
  9. 제1항에 있어서,According to claim 1,
    상기 항원제시세포 특이적 항체 또는 이의 단편은 IgG, Fab', F(ab')2, Fab, Fv, 재조합 IgG (rIgG), 단일쇄 Fv (scFv), 및 디아바디 (diabody)로 이루어진 군에서 선택된 하나 이상인 것인, 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자.The antigen-presenting cell-specific antibody or fragment thereof is from the group consisting of IgG, Fab', F(ab') 2 , Fab, Fv, recombinant IgG (rIgG), single chain Fv (scFv), and diabody. One or more selected, antibody and autoantigen bound to the surface of the lipid-antioxidant nanoparticles.
  10. 제1항에 있어서,According to claim 1,
    상기 자가항원은 개체에서 자가면역질환을 유도할 수 있는 단백질, 이의 단편, 또는 이의 변이체인, 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자.The autoantigen is a protein capable of inducing an autoimmune disease in an individual, a fragment thereof, or a variant thereof, wherein the antibody and the autoantigen are bound to the surface of the lipid-antioxidant nanoparticles.
  11. 제1항에 있어서,According to claim 1,
    상기 자가항원은 콜라겐, 인슐린, 인슐린 B 사슬, 프로인슐린, 미엘린 단백질, 미엘린 염기성 단백질 (myelin basic protein), 미엘린 프로테오리피드 단백질 (myelin proteolipid protein), 미엘린 희소돌기아교세포 당단백질 (myelin oligodendrocyte glycoprotein), Hsp60, 및 Hsp65로 이루어진 군에서 선택된 하나 이상으로부터 유래된 단백질, 이의 단편, 또는 이의 변이체인 것을 특징으로 하는, 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자. The autoantigen is collagen, insulin, insulin B chain, proinsulin, myelin protein, myelin basic protein, myelin proteolipid protein, myelin oligodendrocyte glycoprotein , Hsp60, and Hsp65, characterized in that the protein derived from one or more selected from the group consisting of, a fragment thereof, or a variant thereof, an antibody and an autoantigen bound to the surface-antioxidant nanoparticles.
  12. 제1항에 있어서,According to claim 1,
    상기 자가항원; 및 항원제시세포 특이적 항체 또는 이의 단편은 티올기를 갖는 것이거나, 티올기를 갖도록 변형된 것을 특징으로 하는, 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자.the autoantigen; And antigen-presenting cell-specific antibody or fragment thereof is characterized in that it has a thiol group or is modified to have a thiol group, wherein the antibody and the autoantigen are bound to the surface of the lipid-antioxidant nanoparticles.
  13. 제12항에 있어서,13. The method of claim 12,
    상기 지질막은 말레이미드기를 갖는 지질을 포함하고, 상기 자가항원; 및 항원제시세포 특이적 항체 또는 이의 단편은 티올기 및 말레이미드기의 결합을 통해 지질막에 결합되는 것을 특징으로 하는, 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자.The lipid membrane comprises a lipid having a maleimide group, and the autoantigen; And Antigen-presenting cell-specific antibody or fragment thereof is characterized in that the binding to the lipid membrane through the binding of a thiol group and a maleimide group, the antibody and the autoantigen are bound to the surface of the lipid-antioxidant nanoparticles.
  14. 제1항에 있어서,According to claim 1,
    상기 나노입자는 면역억제제를 더 포함하는 것을 특징으로 하는, 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자.The nanoparticles, characterized in that it further comprises an immunosuppressant, antibodies and autoantigens bound to the surface of the lipid-antioxidant nanoparticles.
  15. 제14항에 있어서,15. The method of claim 14,
    상기 면역억제제는 상기 지질막에 봉입되어 있는 것을 특징으로 하는, 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자.The immunosuppressant is characterized in that it is encapsulated in the lipid membrane, the antibody and the autoantigen bound to the surface of the lipid-antioxidant nanoparticles.
  16. 제14항에 있어서,15. The method of claim 14,
    상기 면역억제제는 부신피질호르몬 (Glucocorticoids), 칼시뉴린 (Calcineurine) 억제제, 항대사제, mTOR 억제제, 및 비타민 D3로 이루어진 군에서 선택된 하나 이상인, 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자.The immunosuppressant is at least one selected from the group consisting of corticosteroids (Glucocorticoids), calcineurin (Calcineurine) inhibitors, antimetabolites, mTOR inhibitors, and vitamin D3, antibodies and autoantigens bound to the surface of lipid-antioxidant nanoparticles.
  17. 제1항에 있어서,According to claim 1,
    상기 나노입자는 직경이 50 내지 200 nm인, 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자.The nanoparticles have a diameter of 50 to 200 nm, wherein the antibody and the autoantigen are bound to the surface of the lipid-antioxidant nanoparticles.
  18. 제1항 내지 제17항 중 어느 한 항의 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자를 유효성분으로 포함하는, 자가면역질환의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for the prevention or treatment of autoimmune diseases, comprising as an active ingredient the lipid-antioxidant nanoparticles bound to the surface of the antibody and the autoantigen of any one of claims 1 to 17.
  19. 제18항에 있어서,19. The method of claim 18,
    상기 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자는 하기로 이루어진 군에서 선택된 하나 이상의 특징을 만족하는 것인, 약학적 조성물:The antibody and the autoantigen bound to the surface of the lipid-antioxidant nanoparticles satisfy one or more characteristics selected from the group consisting of, a pharmaceutical composition:
    (a) 항원제시세포 및 T 세포의 상호작용을 억제함;(a) inhibit the interaction of antigen-presenting cells and T cells;
    (b) 면역세포의 조직 침습을 억제함;(b) inhibiting tissue invasion of immune cells;
    (c) 보조 T 세포, 세포독성 T 세포, 수지상세포, 및 대식세포로 이루어진 군에서 선택된 하나 이상의 수준 또는 활성을 감소시킴;(c) reducing the level or activity of one or more selected from the group consisting of helper T cells, cytotoxic T cells, dendritic cells, and macrophages;
    (d) 조절 T 세포의 수준 또는 활성을 증가시킴;(d) increasing the level or activity of regulatory T cells;
    (e) 염증성 사이토카인의 수준 또는 활성을 억제함; 및(e) inhibiting the level or activity of an inflammatory cytokine; and
    (f) 자가항체의 생성을 억제함.(f) Inhibits the production of autoantibodies.
  20. 제18항에 있어서,19. The method of claim 18,
    상기 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자는 림프절 또는 비장을 표적화 (targeting)하는 것을 특징으로 하는, 약학적 조성물.The antibody and the autoantigen bound to the surface of the lipid-antioxidant nanoparticles are characterized in that the lymph node or the spleen is targeted (targeting), a pharmaceutical composition.
  21. 제18항에 있어서,19. The method of claim 18,
    상기 자가면역질환은 류마티스 관절염, 소아 류마티스 관절염, 전신성 경피증, 성인 스틸씨병, 전신 홍반성 루푸스, 아토피 피부염, 베체트병 (Behcet's disease), 다발성 경화증, 전신성 경화증, 쇼그렌 증후군, 원발성 담즙성 경변증, 셀리악병, 염증성 장질환, 제1형 당뇨병,　자가면역성 용혈성 빈혈증, 굿파스쳐 증후군, 그레이브스 병, 하시모토 갑상선염, 감상선 항진증, 중증근무력증, 천포창, 혈관염, 뇌척수염, 뇌하수체염, 백반증, 천식, 원발성 담즙성 간경변, 시신경 척수염, 심상성 천포창 (pemphigus vulgaris), 과민성 장질환, 크론병, 대장염, 궤양성 대장염, 건선, 심근병증, 중증 근무력증 (myasthenia gravis), 결절성 다발동맥염, 길랑바레 증후군, 및 강직성 척추염 (ankylosing spondylitis)으로 이루어진 군에서 선택된 하나 이상인 것을 특징으로 하는, 자가면역질환의 예방 또는 치료용 약학적 조성물.The autoimmune diseases include rheumatoid arthritis, juvenile rheumatoid arthritis, systemic scleroderma, adult Still's disease, systemic lupus erythematosus, atopic dermatitis, Behcet's disease, multiple sclerosis, systemic sclerosis, Sjogren's syndrome, primary biliary cirrhosis, celiac disease , inflammatory bowel disease, type 1 diabetes, autoimmune hemolytic anemia, Goodpasture's syndrome, Graves' disease, Hashimoto's thyroiditis, hyperthyroidism, myasthenia gravis, pemphigus, vasculitis, encephalomyelitis, pituitary, vitiligo, asthma, primary biliary cirrhosis, Neuromyelitis optica, pemphigus vulgaris, irritable bowel disease, Crohn's disease, colitis, ulcerative colitis, psoriasis, cardiomyopathy, myasthenia gravis, polyarteritis nodosa, Guillain-Barré syndrome, and ankylosing spondylitis ), characterized in that at least one selected from the group consisting of, a pharmaceutical composition for the prevention or treatment of autoimmune diseases.
  22. 제18항의 약학적 조성물을 포함하는, 자가면역질환의 예방 또는 치료용 키트.A kit for preventing or treating autoimmune diseases, comprising the pharmaceutical composition of claim 18.
  23. 제1항 내지 제17항 중 어느 한 항의 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자를 유효성분으로 포함하는, 자가면역질환의 예방 또는 개선용 식품 조성물.A food composition for preventing or improving autoimmune diseases, comprising as an active ingredient the lipid-antioxidant nanoparticles bound to the surface of the antibody and the autoantigen of any one of claims 1 to 17.
  24. (S1) 염기성 환경에서 생체고분자의 자가조립을 유도하여 항산화 나노입자를 제조하는 단계;(S1) inducing self-assembly of biopolymers in a basic environment to prepare antioxidant nanoparticles;
    (S2) 상기 항산화 나노입자의 현탁액으로 지질막을 수화시켜 지질막으로 표면이 코팅된 항산화 나노입자를 제조하는 단계; 및(S2) hydrating the lipid membrane with the suspension of the antioxidant nanoparticles to prepare antioxidant nanoparticles whose surface is coated with the lipid membrane; and
    (S3) 상기 지질막으로 표면이 코팅된 항산화 나노입자를 자가항원, 및 항원제시세포 특이적 항체 또는 이의 단편과 반응시키는 단계를 포함하는, 제1항의 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자의 제조방법.(S3) The antibody and autoantigen of claim 1 comprising the step of reacting the surface-coated antioxidant nanoparticles with an autoantigen and an antigen-presenting cell-specific antibody or fragment thereof with the surface of the lipid membrane-bound lipid-antioxidation A method for preparing nanoparticles.
  25. 제24항에 있어서,25. The method of claim 24,
    상기 생체고분자는 폴리도파민, 탄닌, 및 세륨 산화물로 이루어진 군에서 선택된 하나 이상인, 제조방법.The biopolymer is polydopamine, tannin, and at least one selected from the group consisting of cerium oxide, a manufacturing method.
  26. 제24항에 있어서,25. The method of claim 24,
    상기 지질막은 면역억제제가 봉입되어 있는 것을 특징으로 하는, 제조방법.The lipid membrane is characterized in that the immunosuppressant is encapsulated, the manufacturing method.
  27. 제1항의 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자를 이를 필요로 하는 개체에 투여하는 단계를 포함하는, 자가면역질환의 예방 또는 치료방법.The method of preventing or treating an autoimmune disease, comprising administering the antibody and the autoantigen of claim 1 to the surface-bound lipid-antioxidant nanoparticles to an individual in need thereof.
  28. 자가면역질환의 치료용 약제의 제조를 위한 제1항의 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자의 용도.The use of lipid-antioxidant nanoparticles bound to the surface of the antibody and autoantigen of claim 1 for the manufacture of a medicament for the treatment of autoimmune diseases.
  29. 제1항의 항체 및 자가항원이 표면에 결합된 지질-항산화 나노입자의 자가면역질환의 예방 또는 치료 용도.The antibody and autoantigen of claim 1, wherein the surface is bound to the lipid-antioxidant nanoparticles for the prevention or treatment of autoimmune diseases.
PCT/KR2022/006518 2021-05-06 2022-05-06 Lipid-biopolymer nanoparticles having antibody and autoantigen bound to surface thereof, and use thereof WO2022235125A1 (en)

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