WO2020141893A1 - Antibody-bound nanowire for exosome separation, and exosome separation method using same - Google Patents

Antibody-bound nanowire for exosome separation, and exosome separation method using same Download PDF

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WO2020141893A1
WO2020141893A1 PCT/KR2020/000038 KR2020000038W WO2020141893A1 WO 2020141893 A1 WO2020141893 A1 WO 2020141893A1 KR 2020000038 W KR2020000038 W KR 2020000038W WO 2020141893 A1 WO2020141893 A1 WO 2020141893A1
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antibody
exosomes
circulating
exosome
nanowires
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PCT/KR2020/000038
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French (fr)
Korean (ko)
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조영남
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주식회사 제놉시
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to a nanowire for separating exosomes bound to an antibody and a method for separating exosomes using the same.
  • the cancer diagnosis method mainly consists of invasive methods such as tissue sample collection and endoscopic examination, and liquid biopsy is receiving attention as an alternative to the existing invasive diagnosis and examination method.
  • Liquid biopsy allows detailed diagnosis of cancer development and metastasis by analyzing circulating tumor cells or cfDNA derived from tumor cells or circulating exosomes present in the body from urine, saliva, blood, etc. And, since it uses a non-invasive method, it is actively studied in the field of cancer diagnosis.
  • circulating exosomes have a diameter of 30 nm to 150 nm, unlike extracellular microvesicles (EMV) having a diameter of 500 nm to 5000 nm, which are secreted directly from the cell membrane, and proteins derived from the lysosome pathway in cells, It contains the main molecules of cells such as mRNA and microRNA.
  • EMV extracellular microvesicles
  • circulating exosomes have been reported to promote cancer cell progression, invasion and metastasis. Accordingly, attempts have been made to predict cancer diagnosis and prognosis by isolating circulating exosomes and analyzing proteins, mRNA, and microRNAs in circulating exosomes.
  • exosome separation methods include ultracentrifugation, density gradient centrifugation, size exclusion chromatography, exosome precipitation, and immunoaffinity capture. ) Etc. are used.
  • this method has disadvantages in that the purity and separation efficiency of exosomes that are relatively separated are low, and labor intensive, resulting in a lot of time and cost.
  • the present inventors completed the present invention by confirming that the nanowires are capable of specifically binding exosomes, thereby efficiently separating circulating exosomes from a small sample.
  • an aspect of the present invention as an antibody-coupled nanowire for exosome separation, wherein the nanowire is composed of a conductive polymer, provides a nanowire for exosome separation.
  • Another aspect of the present invention as a method for separating circulating exosomes from a sample, the method comprising: (a) mixing the nanowire and a sample; (b) separating the nanowires; (c) treating the separated nanowires with a reducing agent; And (d) obtaining exosomes.
  • Another aspect of the present invention is a method for providing information for diagnosing cancer or predicting prognosis by detecting circulating exosomes in a sample isolated from a subject suspected of developing a cancer disease, the method comprising: (a ) Mixing the nanowire and a sample; (b) separating the nanowires; (c) processing a marker on the separated nanowire; (d) removing unbound markers; And (e) detecting the marker, provides a method for providing information for diagnosing cancer or predicting prognosis.
  • Circulating exosomes with a diameter of 40 nm to 150 nm were separated within 1 hour from a small sample using a nanowire for separating the exosomes bound with the antibody according to the present invention.
  • the amount of circulating exosomes detected in the sample obtained from the cancer patient was three times higher than that of the normal patient. Therefore, the nanowire for separating the exosomes to which the antibody of the present invention is bound and the method for separating the exosomes using the same can be useful for cancer diagnosis or prognosis prediction.
  • FIG. 1 is a diagram schematically illustrating a method for separating circulating exosomes using nanowires containing a conductive polymer bound with an antibody.
  • FIG. 2 is a view taken by a scanning electron microscope (scale bar: 500 nm) of a conductive polymer bound with an antibody, which is an embodiment of the present invention.
  • FIG. 3 is a view taken by a transmission electron microscope (left scale bar: 500 nm, right scale bar: 100 nm) of a conductive polymer to which an antibody, which is an embodiment of the present invention, is bound.
  • MNW magnetic nanowires
  • NW nanowires
  • FIG. 5 shows the amount of exosomes isolated from concentrated culture medium of MDA-MB-231, HeLa, HCT116 or MCF7 cancer cell lines using magnetic beads (Dyna Beads_CD9, Dyna Beads_CD81) or magnetic nanowires (CD9_MNWs, CD81_MNWs, Abs_MNWs).
  • CD9_MNWs are magnetic nanowires to which the anti-CD9 antibody is bound
  • the CD81_MNWs are magnetic nanowires to which the anti-CD81 antibody is bound.
  • Abs_MNWs are nanowires to which anti-CD9 antibody, anti-CD63 antibody and anti-CD81 antibody are bound.
  • FIG. 6 shows ELISA of exosomes isolated from concentrated culture medium of MDA-MB-231, HeLa, HCT116 or MCF7 cancer cell lines using magnetic beads (Dyna Beads_CD9, Dyna Beads_CD81) or magnetic nanowires (CD9_MNWs, CD81_MNWs, Abs_MNWs). It is a figure quantified through.
  • FIG. 7 is a total of exosomes isolated from concentrated culture medium of MDA-MB-231, HeLa, HCT116 or MCF7 cancer cell lines using magnetic beads (Dyna Beads_CD9, Dyna Beads_CD81) or magnetic nanowires (CD9_MNWs, CD81_MNWs, Abs_MNWs). Protein is quantified through BCA analysis.
  • 9 to 11 are fluorescence images of Abs_MNWs without DTT treatment after exosomes were collected from plasma of lung cancer patients (scale bar: 10 ⁇ m; insertion scale bar: 5 ⁇ m).
  • FIG. 15 is a diagram illustrating RNA size distribution by extracting RNA in exosomes isolated from plasma of lung cancer patients using Abs_MNWs.
  • 16 is a diagram showing the expression level of miR-21 by extracting RNA in exosomes isolated from plasma of healthy donor and lung cancer patients using Abs_MNWs.
  • 17 is a diagram comparing the amount of exosomes isolated using Abs_MNWs from plasma of healthy donors and cancer patients.
  • 18 is a diagram quantifying total protein in exosomes isolated from plasma of healthy donors and cancer patients using Abs_MNWs through BCA analysis.
  • FIG. 19 is a diagram confirming Western blotting using antibodies against HSP70, TSG101, CD81, CD9, CD63, and GAPDH from exosomes isolated using plasma Abs_MNWs from healthy donor and lung cancer patients.
  • FIG. 20 is a diagram comparing the amount of exosomes isolated from plasma of healthy donors, breast cancer patients and lung cancer patients using Abs_MNWs, ExoQuick and Invitrogen total exosome separation kits.
  • FIG. 21 is a diagram comparing diameters of exosomes isolated from plasma of healthy donors, breast cancer patients, and lung cancer patients using the Abs_MNWs, ExoQuick, and Invitrogen total exosome separation kits.
  • FIG. 22 is a diagram quantifying exosomes isolated from plasma of healthy donors, breast cancer patients and lung cancer patients using Abs_MNWs, ExoQuick and Invitrogen total exosome separation kits by ELISA.
  • FIG. 23 is a diagram showing the process, processing time, cost, and minimum sample amount of a method for separating circulating exosomes using Abs_MNWs, which is an embodiment of the present invention.
  • the nanowire is composed of a conductive polymer, and provides a nanowire for exosome separation.
  • the exosome may be characterized by being a circulating exosome.
  • the circulating exosomes are derived from intracellular lysosomal pathways and include major molecules of cells such as proteins, mRNA and microRNA, and have a diameter of 40 nm to 150 nm.
  • the antibody may be an antibody that specifically binds to exosomes.
  • the type is not limited as long as it can specifically bind to exosomes.
  • the antibody is an anti-CD9 antibody, anti-CD24 antibody, anti-CD41 antibody, anti-CD44 antibody, anti-CD63 antibody, anti-CD81 antibody, anti-CD82 antibody, anti-Flotillin antibody, anti-Caveolin- 1 antibody, anti-Rab5 antibody, anti-TSG101 antibody, anti-Alix antibody, anti-CXCR4 antibody, anti-FLOT-1 antibody, anti-TM9SF4 antibody, anti-TM9SF3 antibody, anti-HSPA8 antibody, anti-HSC70 antibody, It may be any one antibody selected from the group consisting of anti-TSTA3 antibody, anti-Thr-181 antibody, anti-HSP70 antibody and combinations thereof.
  • the nanowire may be a combination of two or more antibodies.
  • the nanowire may be a combination of three or more antibodies.
  • combinations of two or more antibodies include the anti-CD9 antibody and anti-CD24 antibody; Anti-CD9 antibody and anti-CD41 antibody; Anti-CD9 antibody and anti-CD44 antibody; Anti-CD9 antibody and anti-CD63 antibody; Anti-CD9 antibody and anti-CD81 antibody; Anti-CD9 antibody and anti-CD82 antibody; Anti-CD9 antibody and anti-Flotillin antibody; Anti-CD9 antibody and anti-Caveolin-1 antibody; Anti-CD9 antibody and anti-Rab5 antibody; Anti-CD9 antibody and anti-TSG101 antibody; Anti-CD9 antibody and anti-Alix antibody; Anti-CD9 antibody and anti-CXCR4 antibody; Anti-CD9 antibody and anti-FLOT-1 antibody; Anti-CD9 antibody and anti-TM9SF4 antibody; Anti-CD9 antibody and anti-TM9SF3 antibody; Anti-CD9 antibody and anti-HSPA8 antibody; Anti-CD9 antibody and anti-HSC70 antibody; Anti-CD9 antibody and anti-TSTA3 antibody; Anti-CD9 antibody and anti-Thr-181
  • Examples of combinations of three or more antibodies include the anti-CD9 antibody, anti-CD63 antibody and anti-CD24 antibody; Anti-CD9 antibodies, anti-CD63 antibodies and anti-CD41 antibodies; Anti-CD9 antibodies, anti-CD63 antibodies and anti-CD44 antibodies; Anti-CD9 antibody, anti-CD63 antibody and anti-CD81 antibody; Anti-CD9 antibody, anti-CD63 antibody and anti-CD82 antibody; Anti-CD9 antibodies, anti-CD63 antibodies and anti-Flotillin antibodies; Anti-CD9 antibody, anti-CD63 antibody and anti-Caveolin-1 antibody; Anti-CD9 antibody, anti-CD63 antibody and anti-Rab5 antibody; Anti-CD9 antibody, anti-CD63 antibody and anti-TSG101 antibody; Anti-CD9 antibodies, anti-CD63 antibodies and anti-Alix antibodies; Anti-CD9 antibody, anti-CD63 antibody and anti-CXCR4 antibody; Anti-CD9 antibody, anti-CD63 antibody and anti-FLOT-1 antibody; Anti-CD9 antibody, anti-CD63 antibody and anti-TM9SF
  • anti-CD9 antibody, anti-CD63 antibody and/or anti-CD81 antibody were used.
  • an anti-CD9 antibody or an anti-CD81 antibody was used as a single antibody, and an antibody mixture of anti-CD9 antibody, anti-CD63 antibody and anti-CD81 antibody was used as multiple antibodies.
  • the antibody may be a biotinylated antibody.
  • biotin-linked anti-CD9 antibody, biotin-linked anti-CD63 antibody, and biotin-linked anti-CD81 antibody were used.
  • the conductive polymer may be polyacetylene, polypyrrole, polythiophene, polydot (PEDOT), poly(3,4-ethylenedioxythiophene), polyaniline, or a derivative thereof.
  • the conductive polymer may be polypyrrole
  • nanowires are prepared using polypyrrole as the conductive polymer.
  • the nanowire may be a combination of biotin.
  • Biotin-coupled nanowires may be doped with a conductive polymer and biotin together during an electrochemical deposition process.
  • the nanowires may be prepared in the form of bio-doped free-standing Ppy NWs.
  • Antibodies and nanowires may be bound via streptavidin, traptavidin or neutravidin.
  • traptavidin refers to a variant of streptavidin, indicating a dissociation rate for biotin that is about 10 times slower, increased mechanical strength, and improved thermal stability. It is a protein. Treptavidin also specifically binds biotin.
  • the term "neutravidin” of the present invention is also referred to as "deglycosylated avidin” and is prepared to avoid the main disadvantages of native avidin and streptavidin.
  • the nanowire may have a diameter of 100 nm to 300 nm, may be 5 ⁇ m to 30 ⁇ m in length, and may be characterized by having an average length of 18 ⁇ m.
  • anti-CD9 antibody-coupled nanowires CD9_NWs
  • anti-CD81 antibody-coupled nanowires CD81_NWs
  • antibody mixture-coupled nanowires Abs_NWs
  • the nanowires may further include magnetic nanoparticles, which are termed "magnetic nanowires".
  • the magnetic nanowires may be doped with conductive polymers, magnetic nanoparticles, and biotin together during an electrochemical deposition process.
  • the magnetic nanowire is equipped with a large amount of magnetic nanoparticles, and has a greater transverse relaxation rate (R2) than the nanoparticles at the same iron (Fe) concentration, and the transverse relaxation of 20 mMFeS-1 to 60 mMFeS-1 It may have a value, and may have a saturation magnetization value of -90 to 90 emu/g, but is not limited thereto.
  • anti-CD9 antibody-coupled magnetic nanowires CD9_MNWs
  • anti-CD81 antibody-coupled magnetic nanowires CD81_MNWs
  • antibody mixture-coupled nanowires Abs_MNWs
  • Another aspect of the present invention as a method for separating circulating exosomes from a sample, the method comprising: (a) mixing the nanowire and a sample; (b) separating the nanowires; (c) treating the separated nanowires with a reducing agent; And (d) obtaining exosomes.
  • the nanowire refers to a nanowire for separating exosomes from which the antibody is bound, and is the same as described above for the nanowire for separating exosomes to which the antibody is bound.
  • the magnetic nanowires also mean the magnetic nanowires to which the antibody is bound, and are the same as described above in the nanowires for separating the exosomes to which the antibody is bound.
  • the circulating exosomes are the same as described above in nanowires for exosome separation.
  • the circulating exosomes are anti-CD9 antibody, anti-CD24 antibody, anti-CD41 antibody, anti-CD44 antibody, anti-CD63 antibody, anti-CD81 antibody, anti-CD82 antibody, anti-Flotillin antibody, anti-Caveolin -1 antibody, anti-Rab5 antibody, anti-TSG101 antibody, anti-Alix antibody, anti-CXCR4 antibody, anti-FLOT-1 antibody, anti-TM9SF4 antibody, anti-TM9SF3 antibody, anti-HSPA8 antibody, anti-HSC70 antibody , Anti-TSTA3 antibody, anti-Thr-181 antibody, anti-HSP70 antibody, and may be specifically binding to any one antibody selected from the group consisting of combinations thereof.
  • circulating exosomes were isolated using anti-CD9 antibody, anti-CD63 antibody and/or anti-CD81 antibody that specifically binds circulating exosomes. Specifically, in one embodiment of the present invention, circulating exosomes were isolated using an anti-CD9 antibody or an anti-CD81 antibody as a single antibody, and an antibody mixture of anti-CD9 antibody, anti-CD63 antibody and anti-CD81 antibody was used. Circulating exosomes were isolated.
  • sample refers to cerebrospinal fluid, pleural fluid, ascites, plasma, or body fluid samples isolated from the human body.
  • the sample may be a liquid sample separated from the human body.
  • plasma can be obtained from the blood.
  • the reducing agent may be DTT (Dithiothreitol), glutathione or TCEP (tris(2-carboxyethyl)phosphine).
  • the sample may be subjected to a pre-treatment process.
  • the pre-treatment process may include a centrifugation step and a filtration step.
  • the centrifugation step may be performed by primary centrifugation and/or secondary centrifugation.
  • the sample may be subjected to a first centrifugation step to remove cells and cell debris by centrifugation at 3,000 ⁇ g for 10 minutes.
  • a sample (blood) collected from an individual was centrifuged at 3,000 ⁇ g for 10 minutes.
  • the sample may be subjected to the first centrifugation to remove cells and cell debris by centrifugation at 300 ⁇ g for 10 minutes, followed by a second centrifugation at 2,000 ⁇ g for 20 minutes.
  • the sample was centrifuged at 300 ⁇ g for 10 minutes, and centrifuged again at 2,000 ⁇ g for 20 minutes.
  • the sample may be filtered through a sterile 0.22 ⁇ m filter.
  • the centrifuged sample was filtered through a sterile 0.22 ⁇ m filter (Merck Millipore, USA).
  • step (a) after the nanowire is treated with a sample that has undergone a pre-treatment process, in order to promote exosome adhesion, it can be reacted for 30 minutes at room temperature with gentle shaking.
  • the nanowire attached to the exosome and the remaining sample may be separated through centrifugation.
  • the magnetic nanowire to which the exosome is attached and the remaining sample can be separated using a magnet.
  • magnetic nanowires attached with exosomes and the remaining sample were separated using MagneSphere® Technology Magnetic Separation Stands (Promega, USA).
  • the reducing agent glutathione (glutathione) or DTT (Dithiothreitol) solution may be added to break the disulfide bond to separate the exosomes trapped in the nanowires or the magnetic nanowires.
  • the exosomes captured in the nanowires or the magnetic nanowires were separated by treating the DTT solution at a concentration of 50 mM.
  • Another aspect of the present invention provides a method for providing information for diagnosing cancer or predicting prognosis by detecting circulating exosomes in a sample isolated from a subject suspected of developing cancer disease.
  • the method comprises: (a) mixing the nanowire and a sample; (b) separating the nanowires; (c) processing a marker on the separated nanowire; (d) removing unbound markers; And (e) detecting the marker, provides a method for providing information for diagnosing cancer or predicting prognosis.
  • the nanowire refers to a nanowire for separating exosomes from which the antibody is bound, and is the same as described above for the nanowire for separating exosomes to which the antibody is bound.
  • the magnetic nanowires also mean the magnetic nanowires to which the antibody is bound, and are the same as described above in the nanowires for separating the exosomes to which the antibody is bound.
  • the circulating exosomes are the same as described above in nanowires for exosome separation.
  • the circulating exosomes are anti-CD9 antibody, anti-CD24 antibody, anti-CD41 antibody, anti-CD44 antibody, anti-CD63 antibody, anti-CD81 antibody, anti-CD82 antibody, anti-Flotillin antibody, anti-Caveolin -1 antibody, anti-Rab5 antibody, anti-TSG101 antibody, anti-Alix antibody, anti-CXCR4 antibody, anti-FLOT-1 antibody, anti-TM9SF4 antibody, anti-TM9SF3 antibody, anti-HSPA8 antibody, anti-HSC70 antibody , Anti-TSTA3 antibody, anti-Thr-181 antibody, anti-HSP70 antibody, and may be specifically binding to any one antibody selected from the group consisting of combinations thereof.
  • circulating exosomes were isolated using anti-CD9 antibody, anti-CD63 antibody and/or anti-CD81 antibody that specifically binds circulating exosomes. Specifically, in one embodiment of the present invention, circulating exosomes were isolated using an anti-CD9 antibody or an anti-CD81 antibody as a single antibody, and an antibody mixture of anti-CD9 antibody, anti-CD63 antibody and anti-CD81 antibody was used. Circulating exosomes were isolated.
  • Steps (a) and (b) are the same as described above in the circulating exosome separation method.
  • the isolated nanowires can be treated with a marker, wherein the marker can be a preparation capable of detecting circulating exosomes.
  • the agent capable of detecting the circulating exosome may be an exosome staining reagent or an antibody specifically binding to the exosome, but is not limited thereto.
  • a Vybrant TM DiO dye solution capable of staining an exosome membrane as a marker and an anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody specifically binding to exosome, Anti-TSG101 antibody and anti-HSP70 antibody were used.
  • the marker can be detected by a suitable method according to the marker.
  • a suitable method for example, in one embodiment of the present invention, after treating the Vybrant TM DiO dye solution (Life Technologies) as a marker, fluorescence intensity was detected and measured using a suitable fluorescence microscope.
  • an anti-CD9 antibody, an anti-CD63 antibody, an anti-CD81 antibody, an anti-TSG101 antibody and an anti-HSP70 antibody that specifically binds exosomes as a marker Western blot Exosomes were detected through.
  • suitable analysis methods include Western blot, ELISA (enzyme linked immunosorbent assay, ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, flowescence activated cell sorter (FACS), Protein chips, and the like, but is not limited thereto.
  • the method for providing information may include (f) comparing the detected amount of circulating exosomes of the individual with the detected amount of circulating exosomes of a normal control group; And (g) when the circulating exosome detection amount of the individual is higher than the circulating exosome detection amount of the normal control group, further comprising determining that a cancer disease has occurred.
  • step (g) when the circulating exosome detection amount of the individual is more than 2 times higher than the circulating exosome detection amount of the normal control group, it can be determined that a cancer disease has occurred. Specifically, in the step (g), when the circulating exosome detection amount of the individual is 3 times or more higher than the circulating exosome detection amount of the normal control group, it can be determined that cancer disease has occurred. In one embodiment of the present invention, compared to a healthy donor, it was confirmed that circulating exosomes in cancer patients are more than 3 times higher (FIG. 17 ).
  • Example 1 Preparation of nanowires comprising a conductive polymer bound with an antibody
  • Polypyrrole nanowires (CD9_NWs, CD81_NWs, and Abs_NWs) with anti-CD9-antibody, anti-CD81-antibody, and antibody mixtures were prepared.
  • a gold (Au) layer having a thickness of about 150 nm was deposited on one side of a porous alumina mold (AAO template, Whatman, pore diameter, 200 nm) by thermal evaporation. All electrochemical experiments were measured using a potentiostat/galvanostat (BioLogic SP-150) equipped with a platinum wire counter electrode and an Ag/AgCl (3.0 M NaCl type) comparison electrode in a gold (Au) coated AAO template.
  • the AAO template was washed several times with ultrapure water and immersed in a 2 M sodium hydroxide (NaOH, Sigma Aldrich) solution for 2 hours to remove the AAO template.
  • polypyrrole nanowires (NWs Ppy) of carboxylic acid (-COOH; carboxylic acid) in order to activate group 30 mM of EDC (N- (3-Dimethylaminopropyl the polypyrrole nanowires (NWs Ppy)) -N '-ethylcarbodiimide hydrochloride, Sigma Aldrich) and 6 mM N-hydroxy succinimide (NHS) were added and reacted for 45 minutes.
  • EDC N- (3-Dimethylaminopropyl the polypyrrole nanowires (NWs Ppy)) -N '-ethylcarbodiimide hydrochloride, Sigma Aldrich
  • NHS N-hydroxy succinimide
  • the resulting polypyrrole nanowires (Ppy NWs) were reacted with streptavidin (10 ⁇ g/ml) for an additional 45 minutes, and then washed with water. Subsequently, the anti-CD9 antibody, anti-CD81 antibody or antibody mixture (anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody) at a concentration of 10 ⁇ l/ml was introduced into the nanowire end of streptavidin nano-polypyrrole.
  • Polypyrrole nanowires (CD9_NWs, CD81_NWs and Abs_NWs) with anti-CD9 antibody, anti-CD81 antibody or antibody mixture were prepared by reacting with wire (Ppy NWs) overnight at 4°C.
  • biotinylated anti-CD63 antibody and the biotinylated anti-CD81 antibody were purchased from AnCell (Oak Park, Minnesota, USA).
  • anti-CD9 antibody with biotin attached was purchased from Abcam (Cambridge, UK).
  • Anti-CD9-antibody, anti-CD81-antibody and antibody mixture bound polypyrrole magnetic nanowires were prepared.
  • a gold (Au) layer having a thickness of about 150 nm was deposited on one side of a porous alumina mold (AAO template, Whatman, pore diameter, 200 nm) by thermal evaporation. All electrochemical experiments were measured using a potentiostat/galvanostat (BioLogic SP-150) equipped with a platinum wire counter electrode and an Ag/AgCl (3.0 M NaCl type) comparison electrode in a gold (Au) coated AAO template. At room temperature, 30 ⁇ l of magnetic nanoparticles (average diameter, 10 nm, Sigma Aldrich) were attached onto a gold (Au) coated AAO disk and introduced into the pores of AAO.
  • AAO template Whatman, pore diameter, 200 nm
  • the AAO template was washed several times with ultrapure water and immersed in a 2 M sodium hydroxide (NaOH, Sigma Aldrich) solution for 2 hours to remove the AAO template.
  • the carboxylic acid of polypyrrole magnetic nanowires (Ppy MNWs) (-COOH; carboxylic acid) in order to activate group
  • polypyrrole magnetic nanowires (Ppy MNWs) 30 mM of EDC (N- (3-Dimethylaminopropyl a) - N '-ethylcarbodiimide Hydrochloride, Sigma Aldrich) and 6 mM NHS (N-hydroxy succinimide, Sigma Aldrich) were added and reacted for 45 minutes.
  • the resulting polypyrrole magnetic nanowires (Ppy MNWs) were reacted with streptavidin (10 ⁇ g/ml) for an additional 45 minutes, and then washed with water. Subsequently, the anti-CD9 antibody, anti-CD81 antibody or antibody mixture (anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody) at a concentration of 10 ⁇ l/ml was introduced into the nanowire end of streptavidin and introduced into the polypyrrole magnetism.
  • CD9_MNWs Polypyrrole magnetic nanowires (CD9_MNWs, CD81_MNWs, combined with anti-CD9 antibodies, anti-CD81 antibodies or antibody mixtures having a final antibody concentration of 0.4 ⁇ g/ml) by reacting with nanowires (Ppy MNWs) overnight at a temperature of 4° C. And Abs_MNWs).
  • the CD9_MNWs are magnetic nanowires to which anti-CD9 antibodies are bound
  • CD81_MNWs are magnetic nanowires to which anti-CD81 antibodies are bound
  • Abs_MNWs are nanowires to which anti-CD9 antibody, anti-CD63 antibody and anti-CD81 antibody are bound.
  • biotinylated anti-CD63 antibody and the biotinylated anti-CD81 antibody were purchased from AnCell (Oak Park, Minnesota, USA).
  • anti-CD9 antibody with biotin attached was purchased from Abcam (Cambridge, UK).
  • CD9_MNWs, CD81_MNWs, and Abs_MNWs were treated with HRP (Horseradish peroxidase) labeled anti-mouse IgG antibody and reacted for 1 hour, followed by 3% concentration of BSA solution to prevent non-specific binding.
  • HRP Haseradish peroxidase
  • the antibody conjugated on 1.26 ⁇ 10 6 MNWs/ml was detected and its quantity was quantified, and the results were read using a spectrophotometer at a wavelength of 650 nm. .
  • CD9_MNWs, CD81_MNWs, and Abs_MNWs prepared in Example 2 were analyzed and magnetic force was measured using a scanning electron microscope and a transmission electron microscope of CD9_MNWs, CD81_MNWs, and Abs_MNWs.
  • CD9_MNWs, CD81_MNWs, and Abs_MNWs was observed using a scanning electron microscope (G2F30, Tecnai) with an acceleration voltage of 15 kV and a transmission electron microscope with 300 kV.
  • a scanning electron microscope (SEM) image (scale bar 500 nm) of a diameter of about 200 nm and a comparatively long nanowire having an average length of about 18 ⁇ m was confirmed to be synthesized (Fig. 2).
  • SEM scanning electron microscope
  • FIG. 3 magnetic nanoparticles ( ⁇ 10 nm diameter) are irregular in the polypyrrole magnetic nanowire (Ab mixture_mPpyNW) to which the antibody mixture is bound through an electron transmission microscope (TEM) image (scale bar 50 nm). It was confirmed that it was randomly distributed and buried in a densely arranged state.
  • the magnetism of the nanowires is more synergistic, and the spatial constraints of the nanowires make the nanowires more sensitive to the magnetic field, It was confirmed that by oriented the magnetic moments of each nanoparticle, precise control and selective manipulation in the separation of circulating exosomes is possible.
  • Example 4 Analysis of exosome separation and separation efficiency using magnetic nanowires containing an antibody-conductive conductive polymer
  • Example 4.1 Separation of exosomes using CD9_MNWs, CD81_MNWs, Abs_MNWs and magnetic beads
  • a concentrated culture medium was prepared.
  • cancer cell lines (MDA-MB-231 and MCF7 breast cancer cells, HCT116 colon cancer cells or HeLa cervical cancer cells) were treated with 5% CO 2 and 10% FBS and 1% penicillin-streptomycin at a temperature of 37° C., respectively.
  • Cultured in containing RPMI-1640 medium (Invitrogen, Carlsbad, CA). Cancer cells of about 2 ⁇ 10 9 cell numbers were pelletized, washed three times with RPMI-1640 medium, and the medium was replaced with serum-free RPMI medium. Each cancer cell was cultured in serum-free RPMI medium for 2 days to obtain a culture medium.
  • Dyna Beads_CD9 (5.0 ⁇ 10 5 beads/ ⁇ l), Dyna Beads_CD81 (5.0 ⁇ 10 5 beads/ ⁇ l), CD9_MNWs (1.0 ⁇ 10 3 MNWs/ ⁇ l), CD81_MNWs (1.0 ⁇ 10 3 MNWs/ ⁇ L), and Abs_MNWs (1.0 ⁇ 10 3 MNWs/ ⁇ L) were treated in 250 ⁇ L to 3 mL of concentrated culture medium, and then reacted for 30 minutes at room temperature with gentle shaking to promote exosome adhesion. Ordered.
  • NanoSight NS300 (Malvern Instruments, Malvern, UK) and Malvern Zetasizer Nano-Z (Malvern Instruments, Malvern, UK) to evaluate the concentration and size of the isolated exosomes.
  • ELISA analysis was performed by sandwich ELISA analysis using anti-CD9 antibody and anti-CD81 antibody.
  • 100 ⁇ l of an anti-CD9 antibody at a concentration of 1 ⁇ g/100 ⁇ l was treated in each well of a 96-well-plate, and 4° C. The mixture was allowed to react overnight. Thereafter, the 96-well-plate was treated with PBS containing 1% BSA at a temperature of 37° C. to perform a blocking process for 1 hour. After washing three times with PBS added with 0.1% BSA, each well of a 96-well-plate was treated with 100 ⁇ l of PBS and the exosomes isolated in Example 3.1, and then incubated at 37°C.
  • the Abs_MNWs to which the three types of anti-CD9 antibody, anti-CD63 antibody and anti-CD81 antibody are bound are significantly larger compared to the magnetic beads, CD9_MNWs and CD81_MNWs to which the anti-CD81 antibody or anti-CD9 antibody is folded. Exosomes were isolated.
  • the diameter of the exosomes separated by ultracentrifugation is mainly in the range of 100 nm to 300 nm, while the diameter of the exosomes separated by Abs_MNWs is mainly 40 nm. To 150 nm. Through this, it was confirmed that Abs_MNWs had a uniform size distribution of the separated exosomes (FIG. 8).
  • Exosomes were isolated from plasma of lung cancer patients using Abs_MNWs, and their separation efficiency was analyzed. At this time, the exosome isolated to Abs_MNWs was labeled with a membrane-specific fluorescent dye.
  • blood was collected in a vacuum blood drawer containing an anticoagulant ethlenediaminetetracetic acid (EDTA) according to a procedure approved by the National Cancer Center Institutional Review Board.
  • EDTA anticoagulant ethlenediaminetetracetic acid
  • the collected blood was centrifuged at 3,000 ⁇ g for 10 minutes and stored at a temperature of -80° C. until analysis.
  • the plasma and Abs_MNWs were separated from the exosomes in the same manner as in Example 3.1, and Abs_MNWs in a state where exosomes not treated with DTT were collected were also prepared. After culturing the Abs_MNWs collected with DTT-treated exosomes and Abs_MNWs treated with DTT for 8 minutes at a temperature of 37°C, a 5 ⁇ l/ml Vybrant TM DiO dye solution (Life Technologies) staining the exosome membrane was prepared. Treatment. The exosomes were then washed with phosphate-buffered saline (PBS) and DiO-labeled exosomes were analyzed by Zeiss fluorescence microscopy.
  • PBS phosphate-buffered saline
  • Example 5.2 RNA analysis in exosomes isolated from plasma of lung cancer patients using Abs_MNWs
  • exosomes were isolated from the plasma of lung cancer patients using Abs_MNWs, RNA was extracted from the exosomes, and the Bioanalyzer profile was examined to analyze its robustness, purity, and size distribution.
  • RNA in exosomes was extracted using a TRIzol kit (Invitrogen, Paisley, UK). At this time, RNA was extracted according to the manufacturer's manual. Additionally, the extracted RNA was treated with chloroform (Merck, Darmstadt, Germany) and centrifuged at 12,000 x g for 15 minutes at 4°C to separate the mixture into an aqueous phase and an organic phase. At this time, isopropanol was used to precipitate the supernatant.
  • chloroform Merck, Darmstadt, Germany
  • miR21-cDNA was synthesized using 10 ng of RNA eluent as a random nuclear atom using a TaqMan MicroRNA reverse transcription kit (Applied Biosystems, Foster city, CA, USA).
  • quantitative reverse transcription polymerase chain reaction qRT-PCR was performed using an LC480 real-time PCR system (Roche, Basel, Switzerland) for 5 minutes at 25°C, 20 minutes at 46°C, and 1 minute at 95°C. Was performed. After completion of the reaction, it was stored at 4°C.
  • RNA sizes mainly less than 400 nucleotides
  • RNA extracted from exosomes was further amplified using the cDNA synthesis kit, and the expression of miR-21 was evaluated.
  • a unique miR-21 expression pattern was observed in lung cancer patients (FIG. 16 ).
  • Exosomes were isolated from plasma of healthy donors, breast cancer patients and lung cancer patients using Abs_MNWs, and their total amount was measured by NTA and BCA analysis. NTA and BCA analysis was performed in the same manner as in Example 4.2.
  • cancer patients showed circulating exosomes three times or more (FIG. 17).
  • BCA analysis also showed that the exosome protein in cancer patients was 3.9 times higher than in healthy donors (FIG. 18).
  • Exosomes isolated from plasma of healthy donor and lung cancer patients using Abs_MNWs were confirmed by Western blot using antibodies against HSP70, TSG101, CD81, CD9, CD63, and GAPDH.
  • exosomes isolated using Abs_MNWs were dissolved in M-PER reagent (Thermo Fisher Scientific, Massachusetts, Waltham, USA). Thereafter, 20 ⁇ g of the dissolved sample was electrophoresed on a 10% sodium dodecyl sulfate polyacrylamide gel and transferred to a poly-vinylidene fluoride (PVDF) membrane (0.45 ⁇ m, Millipore).
  • PVDF poly-vinylidene fluoride
  • the PVDF membrane was blocked for 1 hour with 3% skim milk powder at room temperature, and as a primary antibody, mouse anti-TSG101 antibody (1:1,000), rabbit anti-HSP70 antibody (1:1,000), rabbit anti -GAPDH antibody (1:1,000), rabbit anti-CD9 antibody (1:1,000), rabbit anti-CD63 antibody (1:1,000), rabbit anti-CD81 antibody (1:1,000), and rabbit monoclonal anti-GAPDH antibody ( 1:1,000) and reacted overnight.
  • ExoQuick EXOQ5TM-1, System Biosciences, Palo Alto, CA, USA
  • Invitrogen total exosome separation kit 4484451, Thermo Fisher Scientific, Massachusetts, Waltham, USA
  • exosome-human CD81 flow detection reagent 10622D, Thermo Fisher Scientific, USA
  • Quantification of purified exosomes was performed using the BCA assay kit (Thermo Scientific, Waltham, MA, USA) in the same manner as in Example 4.2 to determine protein concentration. To improve reproducibility, all analyzes were performed under the same experimental conditions.
  • the size distribution of most of the exosomes separated by Abs_MNWs was analyzed through a nanoparticle tracking analysis. As a result, the size distribution of most of the exosomes separated by Abs_MNWs was uniform and ranged from 40 nm to 150 nm (FIG. 21).
  • exosome proteins were analyzed through Western blot.
  • anti-CD63 antibody, anti-CD81 antibody, anti-TSG101 antibody and HSP70 antibody were used.
  • Abs_MNWs was used compared to the two conventional methods, a higher level of exosome protein was confirmed (FIG. 22 ).

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Abstract

The present invention relates to an antibody-bound nanowire for exosome separation, and an exosome separation method using same. By using an antibody-bound nanowire for exosome separation, according to the present invention, circulating exosomes having a diameter of 40 nm to 150 nm have been separated from a small amount of a sample within 1 hour in a high yield. In addition, it has been confirmed that the amount of circulating exosomes detected in a sample obtained from a cancer patient is 3 times greater than that of a normal person. Therefore, an antibody-bound nanowire for exosome separation, and the exosome separation method using same, of the present invention, can be effectively used in cancer diagnosis or prognosis prediction.

Description

항체가 결합된 엑소좀 분리용 나노와이어 및 이를 이용한 엑소좀 분리 방법Nanowire for separating antibody-bound exosomes and method for separating exosomes using the same
본 발명은 항체가 결합된 엑소좀 분리용 나노와이어 및 이를 이용한 엑소좀 분리 방법에 관한 것이다.The present invention relates to a nanowire for separating exosomes bound to an antibody and a method for separating exosomes using the same.
최근 전 세계적으로 암 질환의 조기 진단 중요성이 크게 부각되고 있으며, 이에 따라 암 조기 진단 방법에 대한 연구 비중이 증가하고 있는 추세이다. 그러나 현재까지 암 진단 방법은 조직 샘플의 채취 및 내시경 검사 등의 침습적인 방법이 주를 이루고 있어, 기존의 침습적인 진단 및 검사 방법의 대안으로 액체 생체검사가 주목 받고 있다. 액체 생체검사는 소변, 타액, 혈액 등으로부터 체내에 존재하는 순환 종양 세포(Circulating Tumor Cell)나 종양 세포 유래의 cfDNA 또는 순환 엑소좀(circulating exosome)을 분석하여 암 발생 및 전이 등에 대한 상세한 진단이 가능하고, 비침습적인(non-invasive) 방법을 사용하기 때문에, 암 진단 분야에서 활발히 연구되고 있다.Recently, the importance of early diagnosis of cancer diseases has been highlighted worldwide, and accordingly, the proportion of research on methods for early diagnosis of cancer is increasing. However, until now, the cancer diagnosis method mainly consists of invasive methods such as tissue sample collection and endoscopic examination, and liquid biopsy is receiving attention as an alternative to the existing invasive diagnosis and examination method. Liquid biopsy allows detailed diagnosis of cancer development and metastasis by analyzing circulating tumor cells or cfDNA derived from tumor cells or circulating exosomes present in the body from urine, saliva, blood, etc. And, since it uses a non-invasive method, it is actively studied in the field of cancer diagnosis.
한편, 순환 엑소좀은 세포막으로부터 직접 분비되는 500 nm 내지 5000 nm 직경의 세포외 미세소낭(extracellular microvesicles, EMV)과는 달리, 30 nm 내지 150 nm 직경을 가지며, 세포 내 리소좀 경로로부터 유래되어 단백질, mRNA 및 microRNA 등과 같은 세포의 주요 분자를 포함하고 있다. 특히, 순환 엑소좀은 암 세포의 진행, 침습 및 전이를 촉진하는 것으로 보고된 바 있다. 이에, 순환 엑소좀을 분리하여, 순환 엑소좀 내 단백질, mRNA, microRNA 등의 분석을 통해 암 진단 및 예후를 예측하고자는 시도가 이루어지고 있다.On the other hand, circulating exosomes have a diameter of 30 nm to 150 nm, unlike extracellular microvesicles (EMV) having a diameter of 500 nm to 5000 nm, which are secreted directly from the cell membrane, and proteins derived from the lysosome pathway in cells, It contains the main molecules of cells such as mRNA and microRNA. In particular, circulating exosomes have been reported to promote cancer cell progression, invasion and metastasis. Accordingly, attempts have been made to predict cancer diagnosis and prognosis by isolating circulating exosomes and analyzing proteins, mRNA, and microRNAs in circulating exosomes.
현재, 엑소좀 분리 방법으로는 초원심분리(ultracentrifugation), 밀도 구배 원심분리(density gradient centrifugation), 크기 배제 크로마토그래피(size exclusion chromatography), 엑소좀 침전(exosome precipitation) 및 면역친화성 포집(immunoaffinity capture) 등이 이용되고 있다. 그러나, 이러한 방법은 상대적으로 분리되는 엑소좀의 순도 및 분리 효율이 낮고, 노동 집약적이여서 많은 시간 및 비용이 들어간다는 단점이 있다.Currently, exosome separation methods include ultracentrifugation, density gradient centrifugation, size exclusion chromatography, exosome precipitation, and immunoaffinity capture. ) Etc. are used. However, this method has disadvantages in that the purity and separation efficiency of exosomes that are relatively separated are low, and labor intensive, resulting in a lot of time and cost.
따라서, 단시간 내에 고가의 장비나 복잡한 단계 없이 엑소좀을 고효율로 분리할 수 있는 기술의 개발이 요구되고 있다.Therefore, there is a need to develop a technology capable of separating exosomes with high efficiency without expensive equipment or complicated steps in a short time.
이에 본 발명자는 엑소좀에 특이적으로 결합할 수 있는 항체가 결합된 나노와이어를 제조하여, 상기 나노와이어가 소량의 시료로부터 순환 엑소좀을 효율적으로 분리하는 것을 확인함으로써 본 발명을 완성하였다.Accordingly, the present inventors completed the present invention by confirming that the nanowires are capable of specifically binding exosomes, thereby efficiently separating circulating exosomes from a small sample.
상기 목적을 달성하기 위해, 본 발명의 일 측면은, 항체가 결합된 엑소좀 분리용 나노와이어로서, 상기 나노와이어는 전도성 고분자로 구성된 것인, 엑소좀 분리용 나노와이어를 제공한다.To achieve the above object, an aspect of the present invention, as an antibody-coupled nanowire for exosome separation, wherein the nanowire is composed of a conductive polymer, provides a nanowire for exosome separation.
본 발명의 다른 측면은, 시료로부터, 순환 엑소좀을 분리하기 위한 방법으로서, 상기 방법은 (a) 상기 나노와이어와 시료를 혼합하는 단계; (b) 상기 나노와이어를 분리하는 단계; (c) 상기 분리한 나노와이어에 환원제를 처리하는 단계; 및 (d) 엑소좀을 수득하는 단계를 포함하는 순환 엑소좀을 분리하는 방법을 제공한다.Another aspect of the present invention, as a method for separating circulating exosomes from a sample, the method comprising: (a) mixing the nanowire and a sample; (b) separating the nanowires; (c) treating the separated nanowires with a reducing agent; And (d) obtaining exosomes.
본 발명의 또 다른 측면은, 암 질환의 발병이 의심되는 개체로부터 분리된 시료에서 순환 엑소좀을 검출함으로써, 암을 진단 또는 예후를 예측하기 위한 정보를 제공하기 위한 방법으로서, 상기 방법은 (a) 상기 나노와이어와 시료를 혼합하는 단계; (b) 상기 나노와이어를 분리하는 단계; (c) 상기 분리한 나노와이어에 표지자를 처리하는 단계; (d) 결합하지 않은 표지자를 제거하는 단계; 및 (e) 상기 표지자를 검출하는 단계를 포함하는, 암을 진단 또는 예후를 예측하기 위한 정보 제공 방법을 제공한다.Another aspect of the present invention is a method for providing information for diagnosing cancer or predicting prognosis by detecting circulating exosomes in a sample isolated from a subject suspected of developing a cancer disease, the method comprising: (a ) Mixing the nanowire and a sample; (b) separating the nanowires; (c) processing a marker on the separated nanowire; (d) removing unbound markers; And (e) detecting the marker, provides a method for providing information for diagnosing cancer or predicting prognosis.
본 발명에 따른 항체가 결합된 엑소좀 분리용 나노와이어를 이용하여 소량의 시료로부터 40 nm 내지 150 nm 직경의 순환 엑소좀을 높은 수율로 1시간 내에 분리하였다. 또한, 암 환자로부터 수득한 시료에서 검출되는 순환 엑소좀의 양이 암 환자가 정상인보다 3배 이상 많은 것을 확인하였다. 따라서, 본 발명의 항체가 결합된 엑소좀 분리용 나노와이어 및 이를 이용한 엑소좀 분리방법은 암 진단 또는 예후 예측에 유용하게 사용될 수 있다.Circulating exosomes with a diameter of 40 nm to 150 nm were separated within 1 hour from a small sample using a nanowire for separating the exosomes bound with the antibody according to the present invention. In addition, it was confirmed that the amount of circulating exosomes detected in the sample obtained from the cancer patient was three times higher than that of the normal patient. Therefore, the nanowire for separating the exosomes to which the antibody of the present invention is bound and the method for separating the exosomes using the same can be useful for cancer diagnosis or prognosis prediction.
도 1은 항체가 결합된 전도성 고분자를 포함하는 나노와이어를 이용해 순환 엑소좀을 분리하는 방법을 도식화한 도면이다.1 is a diagram schematically illustrating a method for separating circulating exosomes using nanowires containing a conductive polymer bound with an antibody.
도 2는 본 발명의 일 실시예인 항체가 결합된 전도성 고분자를 주사 전자 현미경(스케일 바: 500 nm)으로 촬영한 도면이다.2 is a view taken by a scanning electron microscope (scale bar: 500 nm) of a conductive polymer bound with an antibody, which is an embodiment of the present invention.
도 3은 본 발명의 일 실시예인 항체가 결합된 전도성 고분자를 투과 전자 현미경(왼쪽 스케일 바: 500 nm, 오른쪽 스케일 바: 100 nm)으로 촬영한 도면이다.3 is a view taken by a transmission electron microscope (left scale bar: 500 nm, right scale bar: 100 nm) of a conductive polymer to which an antibody, which is an embodiment of the present invention, is bound.
도 4는 본 발명의 일 실시예인 자성입자가 포함된 자성 나노와이어(MNW) 및 나노와이어(NW)의 자기화 정도를 측정한 도면이다.4 is a view measuring the degree of magnetization of magnetic nanowires (MNW) and nanowires (NW) containing magnetic particles, which is an embodiment of the present invention.
도 5는 MDA-MB-231, HeLa, HCT116 또는 MCF7 암세포주의 농축된 배양 배지로부터 자성 비드(Dyna Beads_CD9, Dyna Beads_CD81) 또는 자성 나노와이어(CD9_MNWs, CD81_MNWs, Abs_MNWs)를 이용하여 분리한 엑소좀 양을 비교한 도면이다. 이때, 상기 CD9_MNWs는 항-CD9 항체가 결합된 자성 나노와이어이며, CD81_MNWs는 항-CD81 항체가 결합된 자성 나노와이어이다. 또한, Abs_MNWs는 항-CD9 항체, 항-CD63 항체 및 항-CD81 항체가 결합된 나노와이어이다.FIG. 5 shows the amount of exosomes isolated from concentrated culture medium of MDA-MB-231, HeLa, HCT116 or MCF7 cancer cell lines using magnetic beads (Dyna Beads_CD9, Dyna Beads_CD81) or magnetic nanowires (CD9_MNWs, CD81_MNWs, Abs_MNWs). It is a comparison drawing. In this case, the CD9_MNWs are magnetic nanowires to which the anti-CD9 antibody is bound, and the CD81_MNWs are magnetic nanowires to which the anti-CD81 antibody is bound. In addition, Abs_MNWs are nanowires to which anti-CD9 antibody, anti-CD63 antibody and anti-CD81 antibody are bound.
도 6은 MDA-MB-231, HeLa, HCT116 또는 MCF7 암세포주의 농축된 배양 배지로부터 자성 비드(Dyna Beads_CD9, Dyna Beads_CD81) 또는 자성 나노와이어(CD9_MNWs, CD81_MNWs, Abs_MNWs)를 이용하여 분리한 엑소좀을 ELISA 통해 정량한 도면이다.FIG. 6 shows ELISA of exosomes isolated from concentrated culture medium of MDA-MB-231, HeLa, HCT116 or MCF7 cancer cell lines using magnetic beads (Dyna Beads_CD9, Dyna Beads_CD81) or magnetic nanowires (CD9_MNWs, CD81_MNWs, Abs_MNWs). It is a figure quantified through.
도 7은 MDA-MB-231, HeLa, HCT116 또는 MCF7 암세포주의 농축된 배양 배지로부터 자성 비드(Dyna Beads_CD9, Dyna Beads_CD81) 또는 자성 나노와이어(CD9_MNWs, CD81_MNWs, Abs_MNWs)를 이용하여 분리한 엑소좀의 총 단백질을 BCA 분석을 통해 정량한 도면이다.FIG. 7 is a total of exosomes isolated from concentrated culture medium of MDA-MB-231, HeLa, HCT116 or MCF7 cancer cell lines using magnetic beads (Dyna Beads_CD9, Dyna Beads_CD81) or magnetic nanowires (CD9_MNWs, CD81_MNWs, Abs_MNWs). Protein is quantified through BCA analysis.
도 8은 MDA-MB-231, HeLa, HCT116 또는 MCF7 암세포주의 농축된 배양 배지로부터 초원심분리 또는 Abs_MNWs를 이용하여 분리한 엑소좀의 직경을 측정한 도면이다.8 is a view measuring the diameter of exosomes isolated using ultracentrifugation or Abs_MNWs from the concentrated culture medium of MDA-MB-231, HeLa, HCT116 or MCF7 cancer cell lines.
도 9 내지 도 11은 폐암 환자의 혈장으로부터 엑소좀이 포집한 후, DTT를 처리하지 않은 Abs_MNWs의 형광 이미지를 촬영한 도면이다(스케일 바: 10 ㎛; 삽입 스케일 바: 5 ㎛).9 to 11 are fluorescence images of Abs_MNWs without DTT treatment after exosomes were collected from plasma of lung cancer patients (scale bar: 10 μm; insertion scale bar: 5 μm).
도 12 내지 도 14는 폐암 환자의 혈장으로부터 엑소좀이 포집한 후, DTT를 처리한 Abs_MNWs의 형광 이미지를 촬영한 도면이다(스케일 바: 10 ㎛; 삽입 스케일 바: 5 ㎛).12 to 14 are fluorescence images of Abs_MNWs treated with DTT after exosomes were collected from plasma of lung cancer patients (scale bar: 10 μm; insertion scale bar: 5 μm).
도 15는 폐암 환자의 혈장으로부터 Abs_MNWs를 이용하여 분리한 엑소좀 내 RNA를 추출하여 RNA의 크기 분포를 측정한 도면이다.FIG. 15 is a diagram illustrating RNA size distribution by extracting RNA in exosomes isolated from plasma of lung cancer patients using Abs_MNWs.
도 16은 건강한 공여자 및 폐암 환자의 혈장으로부터 Abs_MNWs를 이용하여 분리한 엑소좀 내 RNA를 추출하여 miR-21의 발현 정도를 측정한 도면이다.16 is a diagram showing the expression level of miR-21 by extracting RNA in exosomes isolated from plasma of healthy donor and lung cancer patients using Abs_MNWs.
도 17은 건강한 공여자 및 암 환자의 혈장으로부터 Abs_MNWs를 이용하여 분리한 엑소좀 양을 비교한 도면이다.17 is a diagram comparing the amount of exosomes isolated using Abs_MNWs from plasma of healthy donors and cancer patients.
도 18은 건강한 공여자 및 암 환자의 혈장으로부터 Abs_MNWs를 이용하여 분리한 엑소좀 내 총 단백질을 BCA 분석을 통해 정량한 도면이다.18 is a diagram quantifying total protein in exosomes isolated from plasma of healthy donors and cancer patients using Abs_MNWs through BCA analysis.
도 19는 건강한 공여자 및 폐암 환자의 혈장으로부터 Abs_MNWs를 이용하여 분리한 엑소좀을 HSP70, TSG101, CD81, CD9, CD63, 및 GAPDH에 대한 항체를 이용한 웨스턴 블롯을 통해 확인한 도면이다.FIG. 19 is a diagram confirming Western blotting using antibodies against HSP70, TSG101, CD81, CD9, CD63, and GAPDH from exosomes isolated using plasma Abs_MNWs from healthy donor and lung cancer patients.
도 20은 건강한 공여자, 유방암 환자 및 폐암 환자의 혈장으로부터 Abs_MNWs, ExoQuick 및 Invitrogen 총 엑소좀 분리 키트를 이용하여 분리한 엑소좀의 양을 비교한 도면이다.FIG. 20 is a diagram comparing the amount of exosomes isolated from plasma of healthy donors, breast cancer patients and lung cancer patients using Abs_MNWs, ExoQuick and Invitrogen total exosome separation kits.
도 21은 건강한 공여자, 유방암 환자 및 폐암 환자의 혈장으로부터 Abs_MNWs, ExoQuick 및 Invitrogen 총 엑소좀 분리 키트를 이용하여 분리한 엑소좀의 직경을 비교한 도면이다.FIG. 21 is a diagram comparing diameters of exosomes isolated from plasma of healthy donors, breast cancer patients, and lung cancer patients using the Abs_MNWs, ExoQuick, and Invitrogen total exosome separation kits.
도 22는 건강한 공여자, 유방암 환자 및 폐암 환자의 혈장으로부터 Abs_MNWs, ExoQuick 및 Invitrogen 총 엑소좀 분리 키트를 이용하여 분리한 엑소좀을 ELISA 통해 정량한 도면이다.FIG. 22 is a diagram quantifying exosomes isolated from plasma of healthy donors, breast cancer patients and lung cancer patients using Abs_MNWs, ExoQuick and Invitrogen total exosome separation kits by ELISA.
도 23은 본 발명의 일 실시예인 Abs_MNWs를 이용하여 순환 엑소좀을 분리하는 방법의 과정, 처리 시간, 비용 및 최소 시료량을 정리한 도면이다.23 is a diagram showing the process, processing time, cost, and minimum sample amount of a method for separating circulating exosomes using Abs_MNWs, which is an embodiment of the present invention.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
항체가 결합된 엑소좀 분리용 나노와이어Nanowire for separating antibody-bound exosomes
본 발명의 일 측면은, 항체가 결합된 엑소좀 분리용 나노와이어로서, 상기 나노와이어는 전도성 고분자로 구성된 것인, 엑소좀 분리용 나노와이어를 제공한다.One aspect of the present invention, as an antibody-coupled nanowire for exosome separation, the nanowire is composed of a conductive polymer, and provides a nanowire for exosome separation.
상기 엑소좀은 순환 엑소좀(circulating exosome)인 것을 특징으로 하는 것일 수 있다. 상기 순환 엑소좀은 세포 내 리소좀 경로로부터 유래되어 단백질, mRNA 및 microRNA 등과 같은 세포의 주요 분자를 포함하며, 40 nm 내지 150 nm 직경을 갖는다.The exosome may be characterized by being a circulating exosome. The circulating exosomes are derived from intracellular lysosomal pathways and include major molecules of cells such as proteins, mRNA and microRNA, and have a diameter of 40 nm to 150 nm.
상기 항체는 엑소좀에 특이적으로 결합하는 항체일 수 있다. 엑소좀에 특이적으로 결합할 수 있는 한, 종류를 제한하지 않는다. 구체적으로, 상기 항체는 항-CD9 항체, 항-CD24 항체, 항-CD41 항체, 항-CD44 항체, 항-CD63 항체, 항-CD81 항체, 항-CD82 항체, 항-Flotillin 항체, 항-Caveolin-1 항체, 항-Rab5 항체, 항-TSG101 항체, 항-Alix 항체, 항-CXCR4 항체, 항-FLOT-1 항체, 항-TM9SF4 항체, 항-TM9SF3 항체, 항-HSPA8 항체, 항-HSC70 항체, 항-TSTA3 항체, 항-Thr-181 항체, 항-HSP70 항체 및 이들의 조합으로 이루어진 군으로부터 선택되는 어느 하나의 항체일 수 있다.The antibody may be an antibody that specifically binds to exosomes. The type is not limited as long as it can specifically bind to exosomes. Specifically, the antibody is an anti-CD9 antibody, anti-CD24 antibody, anti-CD41 antibody, anti-CD44 antibody, anti-CD63 antibody, anti-CD81 antibody, anti-CD82 antibody, anti-Flotillin antibody, anti-Caveolin- 1 antibody, anti-Rab5 antibody, anti-TSG101 antibody, anti-Alix antibody, anti-CXCR4 antibody, anti-FLOT-1 antibody, anti-TM9SF4 antibody, anti-TM9SF3 antibody, anti-HSPA8 antibody, anti-HSC70 antibody, It may be any one antibody selected from the group consisting of anti-TSTA3 antibody, anti-Thr-181 antibody, anti-HSP70 antibody and combinations thereof.
상기 나노와이어는 2개 이상의 항체가 결합된 것일 수 있다. 바람직하게는 상기 나노와이어는 3개 이상의 항체가 결합된 것일 수 있다.The nanowire may be a combination of two or more antibodies. Preferably, the nanowire may be a combination of three or more antibodies.
2개 이상의 항체 조합의 예로는, 상기 항체는 항-CD9 항체 및 항-CD24 항체; 항-CD9 항체 및 항-CD41 항체; 항-CD9 항체 및 항-CD44 항체; 항-CD9 항체 및 항-CD63 항체; 항-CD9 항체 및 항-CD81 항체; 항-CD9 항체 및 항-CD82 항체; 항-CD9 항체 및 항-Flotillin 항체; 항-CD9 항체 및 항-Caveolin-1 항체; 항-CD9 항체 및 항-Rab5 항체; 항-CD9 항체 및 항-TSG101 항체; 항-CD9 항체 및 항-Alix 항체; 항-CD9 항체 및 항-CXCR4 항체; 항-CD9 항체 및 항-FLOT-1 항체; 항-CD9 항체 및 항-TM9SF4 항체; 항-CD9 항체 및 항-TM9SF3 항체; 항-CD9 항체 및 항-HSPA8 항체; 항-CD9 항체 및 항-HSC70 항체; 항-CD9 항체 및 항-TSTA3 항체; 항-CD9 항체 및 항-Thr-181 항체; 항-CD63 항체 및 항-CD81 항체; 또는 항-CD9 항체 및 항-HSP70 항체의 조합일 수 있으나, 이에 제한되는 것은 아니다.Examples of combinations of two or more antibodies include the anti-CD9 antibody and anti-CD24 antibody; Anti-CD9 antibody and anti-CD41 antibody; Anti-CD9 antibody and anti-CD44 antibody; Anti-CD9 antibody and anti-CD63 antibody; Anti-CD9 antibody and anti-CD81 antibody; Anti-CD9 antibody and anti-CD82 antibody; Anti-CD9 antibody and anti-Flotillin antibody; Anti-CD9 antibody and anti-Caveolin-1 antibody; Anti-CD9 antibody and anti-Rab5 antibody; Anti-CD9 antibody and anti-TSG101 antibody; Anti-CD9 antibody and anti-Alix antibody; Anti-CD9 antibody and anti-CXCR4 antibody; Anti-CD9 antibody and anti-FLOT-1 antibody; Anti-CD9 antibody and anti-TM9SF4 antibody; Anti-CD9 antibody and anti-TM9SF3 antibody; Anti-CD9 antibody and anti-HSPA8 antibody; Anti-CD9 antibody and anti-HSC70 antibody; Anti-CD9 antibody and anti-TSTA3 antibody; Anti-CD9 antibody and anti-Thr-181 antibody; Anti-CD63 antibody and anti-CD81 antibody; Or it may be a combination of anti-CD9 antibody and anti-HSP70 antibody, but is not limited thereto.
3개 이상의 항체 조합의 예로는, 상기 항체는 항-CD9 항체, 항-CD63 항체 및 항-CD24 항체; 항-CD9 항체, 항-CD63 항체 및 항-CD41 항체; 항-CD9 항체, 항-CD63 항체 및 항-CD44 항체; 항-CD9 항체, 항-CD63 항체 및 항-CD81 항체; 항-CD9 항체, 항-CD63 항체 및 항-CD82 항체; 항-CD9 항체, 항-CD63 항체 및 항-Flotillin 항체; 항-CD9 항체, 항-CD63 항체 및 항-Caveolin-1 항체; 항-CD9 항체, 항-CD63 항체 및 항-Rab5 항체; 항-CD9 항체, 항-CD63 항체 및 항-TSG101 항체; 항-CD9 항체, 항-CD63 항체 및 항-Alix 항체; 항-CD9 항체, 항-CD63 항체 및 항-CXCR4 항체; 항-CD9 항체, 항-CD63 항체 및 항-FLOT-1 항체; 항-CD9 항체, 항-CD63 항체 및 항-TM9SF4 항체; 항-CD9 항체, 항-CD63 항체 및 항-TM9SF3 항체; 항-CD9 항체, 항-CD63 항체 및 항-HSPA8 항체; 항-CD9 항체, 항-CD63 항체 및 항-HSC70 항체; 항-CD9 항체, 항-CD63 항체 및 항-TSTA3 항체; 항-CD9 항체, 항-CD63 항체 및 항-Thr-181 항체; 또는 항-CD9 항체, 항-CD63 항체 및 항-HSP70 항체의 조합일 수 있으나, 이에 제한되는 것은 아니다.Examples of combinations of three or more antibodies include the anti-CD9 antibody, anti-CD63 antibody and anti-CD24 antibody; Anti-CD9 antibodies, anti-CD63 antibodies and anti-CD41 antibodies; Anti-CD9 antibodies, anti-CD63 antibodies and anti-CD44 antibodies; Anti-CD9 antibody, anti-CD63 antibody and anti-CD81 antibody; Anti-CD9 antibody, anti-CD63 antibody and anti-CD82 antibody; Anti-CD9 antibodies, anti-CD63 antibodies and anti-Flotillin antibodies; Anti-CD9 antibody, anti-CD63 antibody and anti-Caveolin-1 antibody; Anti-CD9 antibody, anti-CD63 antibody and anti-Rab5 antibody; Anti-CD9 antibody, anti-CD63 antibody and anti-TSG101 antibody; Anti-CD9 antibodies, anti-CD63 antibodies and anti-Alix antibodies; Anti-CD9 antibody, anti-CD63 antibody and anti-CXCR4 antibody; Anti-CD9 antibody, anti-CD63 antibody and anti-FLOT-1 antibody; Anti-CD9 antibody, anti-CD63 antibody and anti-TM9SF4 antibody; Anti-CD9 antibody, anti-CD63 antibody and anti-TM9SF3 antibody; Anti-CD9 antibody, anti-CD63 antibody and anti-HSPA8 antibody; Anti-CD9 antibody, anti-CD63 antibody and anti-HSC70 antibody; Anti-CD9 antibody, anti-CD63 antibody and anti-TSTA3 antibody; Anti-CD9 antibody, anti-CD63 antibody and anti-Thr-181 antibody; Or it may be a combination of anti-CD9 antibody, anti-CD63 antibody and anti-HSP70 antibody, but is not limited thereto.
본 발명의 일 실시예에서는, 항-CD9 항체, 항-CD63 항체 및/또는 항-CD81 항체를 사용하였다. 구체적으로, 본 발명의 일 실시예에서는 단일 항체로서 항-CD9 항체 또는 항-CD81 항체를 사용하였으며, 다중 항체로서 항-CD9 항체, 항-CD63 항체 및 항-CD81 항체의 항체 혼합물을 사용하였다.In one embodiment of the present invention, anti-CD9 antibody, anti-CD63 antibody and/or anti-CD81 antibody were used. Specifically, in one embodiment of the present invention, an anti-CD9 antibody or an anti-CD81 antibody was used as a single antibody, and an antibody mixture of anti-CD9 antibody, anti-CD63 antibody and anti-CD81 antibody was used as multiple antibodies.
또한, 상기 항체는 비오틴이 결합된 항체(biotinylated antibody)일 수 있다. 본 발명의 일 실시예에서는, 비오틴이 결합된 항-CD9 항체, 비오틴이 결합된 항-CD63 항체 및 비오틴이 결합된 항-CD81 항체를 사용하였다.In addition, the antibody may be a biotinylated antibody. In one embodiment of the present invention, biotin-linked anti-CD9 antibody, biotin-linked anti-CD63 antibody, and biotin-linked anti-CD81 antibody were used.
상기 전도성 고분자는 폴리아세틸렌(polyacetylene), 폴리피롤(polypyrrole), 폴리티오펜(polythiophene), 폴리피닷(PEDOT: poly(3,4-ethylenedioxythiophene), 폴리아닐린(polyaniline) 또는 이들의 유도체일 수 있다. 바람직하게는, 상기 전도성 고분자는 폴리피롤일 수 있다. 본 발명의 일 실시예에서는, 전도성 고분자로서 폴리피롤을 사용하여 나노와이어를 제조하였다.The conductive polymer may be polyacetylene, polypyrrole, polythiophene, polydot (PEDOT), poly(3,4-ethylenedioxythiophene), polyaniline, or a derivative thereof. The conductive polymer may be polypyrrole In one embodiment of the present invention, nanowires are prepared using polypyrrole as the conductive polymer.
상기 나노와이어는 비오틴이 결합된 것일 수 있다. 비오틴이 결합된 나노와이어는 전기화학적 증착 과정 동안 전도성 고분자 및 비오틴이 함께 도핑(doping)된 것일 수 있다. 구체적으로, 상기 나노와이어는 비오틴이 도핑된 프리-스탠딩 폴리피롤 나노와이어(free-standing Ppy NWs) 형태로 제조될 수 있다. 항체와 나노와이어는 스트렙타비딘(straptavidin), 트랩타비딘(traptavidin) 또는 뉴트라비딘(neutravidin)을 통해 결합된 것일 수 있다.The nanowire may be a combination of biotin. Biotin-coupled nanowires may be doped with a conductive polymer and biotin together during an electrochemical deposition process. Specifically, the nanowires may be prepared in the form of bio-doped free-standing Ppy NWs. Antibodies and nanowires may be bound via streptavidin, traptavidin or neutravidin.
본 발명에서 사용된 용어, "스트렙타비딘"은 스트렙토마이세스 아비디니(Streptomyces avidinii)로부터 분리되는 분자량 60 kDa의 4량체 비오틴 결합 단백질을 의미한다. 아비딘과는 매우 낮은 상동성을 가지나 그 구조는 매우 유사하다. 스트렙타비딘은 아비딘과 마찬가지로 항균활성을 가지며 비오틴에 대해 매우 높은 결합력을 갖는다. 한편, 스트렙타비딘은 산성 등전점(pI=5)을 나타내고 아비딘에 비해 현저히 낮은 용해도를 갖는다. 상업적으로 사용가능한 스트렙타비딘은 예컨대, Thermo Scientific Pierce Streptavidin은 53 kDa의 분자량을 갖는 재조합 형태의 스트렙타비딘으로 중성에 가까운 등전점(pI=6.8 내지 7.5)를 갖는다.As used herein, the term “streptavidin” refers to a tetrameric biotin-binding protein having a molecular weight of 60 kDa isolated from Streptomyces avidinii. It has very low homology to avidin, but its structure is very similar. Streptavidin, like avidin, has antibacterial activity and very high binding capacity to biotin. On the other hand, streptavidin has an acidic isoelectric point (pI=5) and has a significantly lower solubility than avidin. Commercially available streptavidin is, for example, Thermo Scientific Pierce Streptavidin is a recombinant form of streptavidin having a molecular weight of 53 kDa and has a near-neutral isoelectric point (pI=6.8 to 7.5).
본 발명에서 사용된 용어, "트렙타비딘(traptavidin)"은 스트렙타비딘의 변이체를 지칭하는 용어로, 약 10배 가량 느린 비오틴에 대한 해리속도를 나타내며, 기계적 강도가 증가되고, 열적 안정성이 향상된 단백질이다. 트렙타비딘 역시 비오틴에 특이적으로 결합한다.As used in the present invention, the term "traptavidin (traptavidin)" refers to a variant of streptavidin, indicating a dissociation rate for biotin that is about 10 times slower, increased mechanical strength, and improved thermal stability. It is a protein. Treptavidin also specifically binds biotin.
본 발명의 용어 "뉴트라비딘(neutravidin)"은 "탈당화아비딘"라고도 하며 천연형의 아비딘과 스트렙타비딘의 주된 단점을 피하고자 제조된 것으로 이름에 나타난 바와 같이 아비딘을 탈당화하여 생성되는 것으로 아비딘에 비해 감소된 분자량(60 kDa)을 가지면서도 높은 비오틴 결합력을 유지하는 단백질을 의미한다. 상기 아비딘의 탈당화는 등전점을 낮추어(pI=6.3) 아비딘에 대한 비특이적인 결합의 주된 원인을 효과적으로 제거한다. 라이신 잔기는 사용가능한 채로 유지되므로 스트렙타비딘과 같이 용이하게 유도체화하거나 복합체화할 수 있다. 또한 높은 비오틴 결합력 및 낮은 비특이적 결합을 나타나므로 이상적인 비오틴 결합 단백질로 다양하게 이용될 수 있다.The term "neutravidin" of the present invention is also referred to as "deglycosylated avidin" and is prepared to avoid the main disadvantages of native avidin and streptavidin. As shown in the name, avidin is produced by deglycosylating avidin. It means a protein that has a reduced molecular weight (60 kDa) and maintains high biotin binding power as compared to. Deglycosylation of avidin lowers the isoelectric point (pI=6.3) to effectively eliminate the main cause of nonspecific binding to avidin. Lysine residues remain usable, so they can be easily derivatized or complexed, such as streptavidin. In addition, since it exhibits high biotin binding power and low non-specific binding, it can be used in various ways as an ideal biotin-binding protein.
상기 나노와이어는 100 ㎚ 내지 300 ㎚의 직경을 가질 수 있고, 5 ㎛ 내지 30 ㎛의 길이일 수 있으며, 평균 18 ㎛의 길이를 갖는 것을 특징으로 하는 것일 수 있다.The nanowire may have a diameter of 100 nm to 300 nm, may be 5 μm to 30 μm in length, and may be characterized by having an average length of 18 μm.
본 발명의 일 실시예로서, 항-CD9 항체가 결합된 나노와이어(CD9_NWs), 항-CD81 항체가 결합된 나노와이어(CD81_NWs) 및 항체 혼합물이 결합된 나노와이어(Abs_NWs)를 제조하였다. 이때, 상기 항체 혼합물은 항-CD9 항체, 항-CD63 항체 및 항-CD81 항체의 혼합물이다.As an embodiment of the present invention, anti-CD9 antibody-coupled nanowires (CD9_NWs), anti-CD81 antibody-coupled nanowires (CD81_NWs), and antibody mixture-coupled nanowires (Abs_NWs) were prepared. At this time, the antibody mixture is a mixture of anti-CD9 antibody, anti-CD63 antibody and anti-CD81 antibody.
상기 나노와이어는 자성 나노 입자를 더 포함할 수 있으며, 이를 "자성 나노와이어"로 명명하였다. 상기 자성 나노와이어는 전기화학적 증착 과정 동안 전도성 고분자, 자성 나노 입자 및 비오틴이 함께 도핑(doping)된 것일 수 있다. 상기 자성 나노와이어는 다량의 자성 나노 입자를 탑재하여, 같은 철(Fe) 농도에서 나노 입자보다 큰 횡축이완 값(transverse relaxation rate; R2)을 가지며, 20 mMFeS-1 내지 60 mMFeS-1의 횡축이완 값을 가질 수 있으며, -90 내지 90 emu/g의 포화자화 값을 가질 수 있으나, 이에 제한되는 것은 아니다.The nanowires may further include magnetic nanoparticles, which are termed "magnetic nanowires". The magnetic nanowires may be doped with conductive polymers, magnetic nanoparticles, and biotin together during an electrochemical deposition process. The magnetic nanowire is equipped with a large amount of magnetic nanoparticles, and has a greater transverse relaxation rate (R2) than the nanoparticles at the same iron (Fe) concentration, and the transverse relaxation of 20 mMFeS-1 to 60 mMFeS-1 It may have a value, and may have a saturation magnetization value of -90 to 90 emu/g, but is not limited thereto.
본 발명의 일 실시예로서, 항-CD9 항체가 결합된 자성 나노와이어(CD9_MNWs), 항-CD81 항체가 결합된 자성 나노와이어(CD81_MNWs) 및 항체 혼합물이 결합된 나노와이어(Abs_MNWs)를 제조하였다. 이때, 상기 항체 혼합물은 항-CD9 항체, 항-CD63 항체 및 항-CD81 항체의 혼합물이다.As an embodiment of the present invention, anti-CD9 antibody-coupled magnetic nanowires (CD9_MNWs), anti-CD81 antibody-coupled magnetic nanowires (CD81_MNWs), and antibody mixture-coupled nanowires (Abs_MNWs) were prepared. At this time, the antibody mixture is a mixture of anti-CD9 antibody, anti-CD63 antibody and anti-CD81 antibody.
순환 엑소좀 분리방법Circulating exosome separation method
본 발명의 다른 측면은, 시료로부터, 순환 엑소좀을 분리하기 위한 방법으로서, 상기 방법은 (a) 상기 나노와이어와 시료를 혼합하는 단계; (b) 상기 나노와이어를 분리하는 단계; (c) 상기 분리한 나노와이어에 환원제(reducing agent)를 처리하는 단계; 및 (d) 엑소좀을 수득하는 단계를 포함하는 순환 엑소좀을 분리하는 방법을 제공한다.Another aspect of the present invention, as a method for separating circulating exosomes from a sample, the method comprising: (a) mixing the nanowire and a sample; (b) separating the nanowires; (c) treating the separated nanowires with a reducing agent; And (d) obtaining exosomes.
이때, 상기 나노와이어는 항체가 결홥된 엑소좀 분리용 나노와이어를 의미하며, 항체가 결합된 엑소좀 분리용 나노와이어에서 상술한 바와 동일하다. 또한, 자성 나노와이어도 항체가 결합된 자성 나노와이어를 의미하며, 항체가 결합된 엑소좀 분리용 나노와이어에서 상술한 바와 동일하다.At this time, the nanowire refers to a nanowire for separating exosomes from which the antibody is bound, and is the same as described above for the nanowire for separating exosomes to which the antibody is bound. In addition, the magnetic nanowires also mean the magnetic nanowires to which the antibody is bound, and are the same as described above in the nanowires for separating the exosomes to which the antibody is bound.
상기 순환 엑소좀은 엑소좀 분리용 나노와이어에서 상술한 바와 동일하다.The circulating exosomes are the same as described above in nanowires for exosome separation.
또한, 상기 순환 엑소좀은 항-CD9 항체, 항-CD24 항체, 항-CD41 항체, 항-CD44 항체, 항-CD63 항체, 항-CD81 항체, 항-CD82 항체, 항-Flotillin 항체, 항-Caveolin-1항체, 항-Rab5 항체, 항-TSG101 항체, 항-Alix 항체, 항-CXCR4 항체, 항-FLOT-1 항체, 항-TM9SF4 항체, 항-TM9SF3 항체, 항-HSPA8 항체, 항-HSC70 항체, 항-TSTA3 항체, 항-Thr-181 항체, 항-HSP70 항체 및 이들의 조합으로 이루어진 군으로부터 선택되는 어느 하나의 항체와 특이적으로 결합하는 것일 수 있다.In addition, the circulating exosomes are anti-CD9 antibody, anti-CD24 antibody, anti-CD41 antibody, anti-CD44 antibody, anti-CD63 antibody, anti-CD81 antibody, anti-CD82 antibody, anti-Flotillin antibody, anti-Caveolin -1 antibody, anti-Rab5 antibody, anti-TSG101 antibody, anti-Alix antibody, anti-CXCR4 antibody, anti-FLOT-1 antibody, anti-TM9SF4 antibody, anti-TM9SF3 antibody, anti-HSPA8 antibody, anti-HSC70 antibody , Anti-TSTA3 antibody, anti-Thr-181 antibody, anti-HSP70 antibody, and may be specifically binding to any one antibody selected from the group consisting of combinations thereof.
본 발명의 일 실시예에서는, 순환 엑소좀에 특이적으로 결합하는 항-CD9 항체, 항-CD63 항체 및/또는 항-CD81 항체를 사용하여 순환 엑소좀을 분리하였다. 구체적으로, 본 발명의 일 실시예에서는 단일 항체로서 항-CD9 항체 또는 항-CD81 항체를 이용하여 순환 엑소좀을 분리하였으며, 항-CD9 항체, 항-CD63 항체 및 항-CD81 항체의 항체 혼합물을 이용하여 순환 엑소좀을 분리하였다.In one embodiment of the present invention, circulating exosomes were isolated using anti-CD9 antibody, anti-CD63 antibody and/or anti-CD81 antibody that specifically binds circulating exosomes. Specifically, in one embodiment of the present invention, circulating exosomes were isolated using an anti-CD9 antibody or an anti-CD81 antibody as a single antibody, and an antibody mixture of anti-CD9 antibody, anti-CD63 antibody and anti-CD81 antibody was used. Circulating exosomes were isolated.
본 명세서에서 사용된 용어, "시료"는 인체로부터 분리된 뇌척수액, 흉수, 복수, 혈장, 또는 체액 시료를 의미한다. 상기 시료는 인체에서 분리된 액상 시료일 수 있다. 이때, 혈장은 혈액에서 수득될 수 있다.As used herein, the term "sample" refers to cerebrospinal fluid, pleural fluid, ascites, plasma, or body fluid samples isolated from the human body. The sample may be a liquid sample separated from the human body. At this time, plasma can be obtained from the blood.
상기 환원제는 DTT(Dithiothreitol), 글루타치온 또는 TCEP(tris(2-carboxyethyl)phosphine)일 수 있다.The reducing agent may be DTT (Dithiothreitol), glutathione or TCEP (tris(2-carboxyethyl)phosphine).
상기 (a) 단계에서, 상기 시료는 전처리 과정을 거친 것일 수 있다. 상기 전처리 과정은 원심분리 단계 및 여과 단계로 이루어질 수 있다. 또한, 상기 원심분리 단계는 1차 원심분리 및/또는 2차 원심분리로 진행될 수 있다.In step (a), the sample may be subjected to a pre-treatment process. The pre-treatment process may include a centrifugation step and a filtration step. Also, the centrifugation step may be performed by primary centrifugation and/or secondary centrifugation.
구체적으로, 상기 시료는 10분 동안 3,000×g에서 원심분리하여 세포 및 세포 파편을 제거하는 1차 원심분리 단계를 거친 것일 수 있다. 본 발명의 일 실시예에서는 개체로부터 채취한 시료(혈액)를 10분 동안 3,000×g에서 원심분리하였다. 또한, 상기 시료는 10분 동안 동안 300×g에서 원심분리하여 세포 및 세포 파편을 제거하는 1차 원심분리한 후, 다시 20분 동안 2,000×g에서 2차 원심분리를 거친 것일 수 있다. 본 발명의 일 실시예에서는 시료를 10분 동안 300×g에서 원심분리하고, 다시 20분 동안 2,000×g에서 원심분리하였다.Specifically, the sample may be subjected to a first centrifugation step to remove cells and cell debris by centrifugation at 3,000×g for 10 minutes. In one embodiment of the present invention, a sample (blood) collected from an individual was centrifuged at 3,000×g for 10 minutes. In addition, the sample may be subjected to the first centrifugation to remove cells and cell debris by centrifugation at 300 × g for 10 minutes, followed by a second centrifugation at 2,000 × g for 20 minutes. In one embodiment of the present invention, the sample was centrifuged at 300×g for 10 minutes, and centrifuged again at 2,000×g for 20 minutes.
상기 시료는 원심분리 단계 후, 무균 0.22 ㎛ 필터로 여과시킨 것일 수 있다. 본 발명의 일 실시예에서는 원심분리한 시료를 무균 0.22 ㎛ 필터(Merck Millipore, USA)를 통해 여과하였다.After the centrifugation step, the sample may be filtered through a sterile 0.22 μm filter. In one embodiment of the present invention, the centrifuged sample was filtered through a sterile 0.22 μm filter (Merck Millipore, USA).
또한, 상기 (a) 단계에서, 상기 나노와이어를 전처리 과정을 거친 시료에 처리한 후, 엑소좀 부착을 촉진하기 위해, 부드럽게 진탕하면서 실온에서 30분 동안 반응시킬 수 있다.In addition, in the step (a), after the nanowire is treated with a sample that has undergone a pre-treatment process, in order to promote exosome adhesion, it can be reacted for 30 minutes at room temperature with gentle shaking.
상기 (b) 단계에서, 원심분리를 통해 엑소좀이 부착된 나노와이어와 남은 시료를 분리할 수 있다. 이때, 자성 나노와이어의 경우, 자석을 이용하여 엑소좀이 부착된 자성 나노와이어와 남은 시료를 분리할 수 있다. 본 발명의 일 실시예에서는, MagneSphere® Technology Magnetic Separation Stands(Promega, USA)를 이용하여 엑소좀이 부착된 자성 나노와이어와 남은 시료를 분리하였다.In step (b), the nanowire attached to the exosome and the remaining sample may be separated through centrifugation. In this case, in the case of the magnetic nanowire, the magnetic nanowire to which the exosome is attached and the remaining sample can be separated using a magnet. In one embodiment of the present invention, magnetic nanowires attached with exosomes and the remaining sample were separated using MagneSphere® Technology Magnetic Separation Stands (Promega, USA).
상기 (c) 단계에서, 환원제인 글루타치온(glutathione) 또는 DTT(Dithiothreitol) 용액을 첨가하여 이화항 결합(disulfide bond)을 끊어 나노와이어 또는 자성 나노와이어에 포획된 엑소좀을 분리시킬 수 있다. 본 발명의 일 실시예에서는 50 mM 농도의 DTT 용액을 처리하여 나노와이어 또는 자성 나노와이어에 포획된 엑소좀을 분리하였다.In the step (c), the reducing agent glutathione (glutathione) or DTT (Dithiothreitol) solution may be added to break the disulfide bond to separate the exosomes trapped in the nanowires or the magnetic nanowires. In one embodiment of the present invention, the exosomes captured in the nanowires or the magnetic nanowires were separated by treating the DTT solution at a concentration of 50 mM.
암 진단 또는 예후 예측을 위한 정보 제공 방법How to provide information for cancer diagnosis or prognosis
본 발명의 또 다른 측면은, 암 질환의 발병이 의심되는 개체로부터 분리된 시료에서 순환 엑소좀을 검출함으로써, 암을 진단 또는 예후를 예측하기 위한 정보를 제공하기 위한 방법을 제공한다. 구체적으로, 상기 방법은 (a) 상기 나노와이어와 시료를 혼합하는 단계; (b) 상기 나노와이어를 분리하는 단계; (c) 상기 분리한 나노와이어에 표지자를 처리하는 단계; (d) 결합하지 않은 표지자를 제거하는 단계; 및 (e) 상기 표지자를 검출하는 단계를 포함하는, 암을 진단 또는 예후를 예측하기 위한 정보 제공 방법을 제공한다.Another aspect of the present invention provides a method for providing information for diagnosing cancer or predicting prognosis by detecting circulating exosomes in a sample isolated from a subject suspected of developing cancer disease. Specifically, the method comprises: (a) mixing the nanowire and a sample; (b) separating the nanowires; (c) processing a marker on the separated nanowire; (d) removing unbound markers; And (e) detecting the marker, provides a method for providing information for diagnosing cancer or predicting prognosis.
이때, 상기 나노와이어는 항체가 결홥된 엑소좀 분리용 나노와이어를 의미하며, 항체가 결합된 엑소좀 분리용 나노와이어에서 상술한 바와 동일하다. 또한, 자성 나노와이어도 항체가 결합된 자성 나노와이어를 의미하며, 항체가 결합된 엑소좀 분리용 나노와이어에서 상술한 바와 동일하다.At this time, the nanowire refers to a nanowire for separating exosomes from which the antibody is bound, and is the same as described above for the nanowire for separating exosomes to which the antibody is bound. In addition, the magnetic nanowires also mean the magnetic nanowires to which the antibody is bound, and are the same as described above in the nanowires for separating the exosomes to which the antibody is bound.
상기 순환 엑소좀은 엑소좀 분리용 나노와이어에서 상술한 바와 동일하다.The circulating exosomes are the same as described above in nanowires for exosome separation.
또한, 상기 순환 엑소좀은 항-CD9 항체, 항-CD24 항체, 항-CD41 항체, 항-CD44 항체, 항-CD63 항체, 항-CD81 항체, 항-CD82 항체, 항-Flotillin 항체, 항-Caveolin-1항체, 항-Rab5 항체, 항-TSG101 항체, 항-Alix 항체, 항-CXCR4 항체, 항-FLOT-1 항체, 항-TM9SF4 항체, 항-TM9SF3 항체, 항-HSPA8 항체, 항-HSC70 항체, 항-TSTA3 항체, 항-Thr-181 항체, 항-HSP70 항체 및 이들의 조합으로 이루어진 군으로부터 선택되는 어느 하나의 항체와 특이적으로 결합하는 것일 수 있다.In addition, the circulating exosomes are anti-CD9 antibody, anti-CD24 antibody, anti-CD41 antibody, anti-CD44 antibody, anti-CD63 antibody, anti-CD81 antibody, anti-CD82 antibody, anti-Flotillin antibody, anti-Caveolin -1 antibody, anti-Rab5 antibody, anti-TSG101 antibody, anti-Alix antibody, anti-CXCR4 antibody, anti-FLOT-1 antibody, anti-TM9SF4 antibody, anti-TM9SF3 antibody, anti-HSPA8 antibody, anti-HSC70 antibody , Anti-TSTA3 antibody, anti-Thr-181 antibody, anti-HSP70 antibody, and may be specifically binding to any one antibody selected from the group consisting of combinations thereof.
본 발명의 일 실시예에서는, 순환 엑소좀에 특이적으로 결합하는 항-CD9 항체, 항-CD63 항체 및/또는 항-CD81 항체를 사용하여 순환 엑소좀을 분리하였다. 구체적으로, 본 발명의 일 실시예에서는 단일 항체로서 항-CD9 항체 또는 항-CD81 항체를 이용하여 순환 엑소좀을 분리하였으며, 항-CD9 항체, 항-CD63 항체 및 항-CD81 항체의 항체 혼합물을 이용하여 순환 엑소좀을 분리하였다.In one embodiment of the present invention, circulating exosomes were isolated using anti-CD9 antibody, anti-CD63 antibody and/or anti-CD81 antibody that specifically binds circulating exosomes. Specifically, in one embodiment of the present invention, circulating exosomes were isolated using an anti-CD9 antibody or an anti-CD81 antibody as a single antibody, and an antibody mixture of anti-CD9 antibody, anti-CD63 antibody and anti-CD81 antibody was used. Circulating exosomes were isolated.
상기 (a) 단계 및 (b) 단계는 순환 엑소좀 분리 방법에서 상술한 바와 동일하다.Steps (a) and (b) are the same as described above in the circulating exosome separation method.
상기 (c) 단계에서, 상기 분리한 나노와이어에 표지자를 처리할 수 있으며, 이때, 상기 표지자는 순환 엑소좀을 검출할 수 있는 제제일 수 있다. 구체적으로, 상기 순환 엑소좀을 검출할 수 있는 제제는 엑소좀 염색 시약 또는 엑소좀에 특이적으로 결합하는 항체일 수 있으며, 이에 제한되는 것은 아니다. 본 발명의 일 실시예에서는, 표지자로써 엑소좀 막을 염색시킬 수 있는 VybrantTM DiO 염료 용액(Life Technologies)과 엑소좀에 특이적으로 결합하는 항-CD9 항체, 항-CD63 항체, 항-CD81 항체, 항-TSG101 항체 및 항-HSP70 항체를 사용하였다. 상기 (d) 단계에서 결합하지 않은 표지자를 제거함으로써, 노이즈를 제거할 수 있다.In step (c), the isolated nanowires can be treated with a marker, wherein the marker can be a preparation capable of detecting circulating exosomes. Specifically, the agent capable of detecting the circulating exosome may be an exosome staining reagent or an antibody specifically binding to the exosome, but is not limited thereto. In one embodiment of the present invention, a Vybrant TM DiO dye solution (Life Technologies) capable of staining an exosome membrane as a marker and an anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody specifically binding to exosome, Anti-TSG101 antibody and anti-HSP70 antibody were used. By removing the unbound marker in the step (d), noise can be removed.
상기 (e) 단계에서 표지자에 따라 적합한 방법으로 표지자를 검출할 수 있다. 예를 들면, 본 발명의 일 실시예에서는 표지자로서 VybrantTM DiO 염료 용액(Life Technologies)을 처리한 후, 이에 적합한 형광 현미경을 사용하여 형광 세기를 검출 및 측정하였다. 또한, 본 발명의 일 실시예에서는 표지자로서 엑소좀에 특이적으로 결합하는 항-CD9 항체, 항-CD63 항체, 항-CD81 항체, 항-TSG101 항체 및 항-HSP70 항체를 처리한 후, 웨스턴 블랏을 통해 엑소좀을 검출하였다. 또한, 엑소좀에 특이적으로 결합하는 항체를 이용할 경우, 적합한 분석 방법으로는 웨스턴 블랏, 엘라이자(enzyme linked immunosorbent assay, ELISA), 방사선면역분석(Radioimmunoassay. RIA), 방사면역확산법(radioimmunodiffusion), 오우크테로니(Ouchterlony) 면역확산법, 로케트(rocket) 면역전기영동, 조직면역염색, 면역침전분석법(Immunoprecipitation Assay), 보체 고정 분석법(Complement Fixation Assay), 유세포분석(Fluorescence Activated Cell Sorter, FACS), 단백질 칩(protein chip) 등이 있으나, 이로 제한되는 것은 아니다.In step (e), the marker can be detected by a suitable method according to the marker. For example, in one embodiment of the present invention, after treating the Vybrant ™ DiO dye solution (Life Technologies) as a marker, fluorescence intensity was detected and measured using a suitable fluorescence microscope. In addition, in one embodiment of the present invention, after treatment with an anti-CD9 antibody, an anti-CD63 antibody, an anti-CD81 antibody, an anti-TSG101 antibody and an anti-HSP70 antibody that specifically binds exosomes as a marker, Western blot Exosomes were detected through. In addition, when using an antibody that specifically binds to exosomes, suitable analysis methods include Western blot, ELISA (enzyme linked immunosorbent assay, ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, flowescence activated cell sorter (FACS), Protein chips, and the like, but is not limited thereto.
상기 정보 제공 방법은 (f) 상기 개체의 순환 엑소좀 검출량과 정상 대조군의 순환 엑소좀 검출량을 비교하는 단계; 및 (g) 상기 개체의 순환 엑소좀 검출량이 정상 대조군의 순환 엑소좀 검출량에 비해 높은 경우, 암 질환이 발병 되었다고 판정하는 단계를 추가적으로 더 포함할 수 있다.The method for providing information may include (f) comparing the detected amount of circulating exosomes of the individual with the detected amount of circulating exosomes of a normal control group; And (g) when the circulating exosome detection amount of the individual is higher than the circulating exosome detection amount of the normal control group, further comprising determining that a cancer disease has occurred.
상기 (g) 단계에서, 상기 개체의 순환 엑소좀 검출량이 정상 대조군의 순환 엑소좀 검출량에 비해 2배 이상 높은 경우 암 질환이 발병 되었다고 판정할 수 있다. 구체적으로, 상기 (g) 단계에서, 상기 개체의 순환 엑소좀 검출량이 정상 대조군의 순환 엑소좀 검출량에 비해 3배 이상 높은 경우 암 질환이 발병 되었다고 판정할 수 있다. 본 발명의 일 실시예에서는, 건강한 공여자와 비교하여, 암 환자가 순환 엑소좀이 3배 이상 많은 것을 확인하였다(도 17).In the step (g), when the circulating exosome detection amount of the individual is more than 2 times higher than the circulating exosome detection amount of the normal control group, it can be determined that a cancer disease has occurred. Specifically, in the step (g), when the circulating exosome detection amount of the individual is 3 times or more higher than the circulating exosome detection amount of the normal control group, it can be determined that cancer disease has occurred. In one embodiment of the present invention, compared to a healthy donor, it was confirmed that circulating exosomes in cancer patients are more than 3 times higher (FIG. 17 ).
이하, 본 발명을 하기 실시예에 의하여 더욱 상세하게 설명한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐, 본 발명의 범위가 이들만으로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail by examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited to them.
실시예 1. 항체가 결합된 전도성 고분자를 포함하는 나노와이어 제조Example 1. Preparation of nanowires comprising a conductive polymer bound with an antibody
항-CD9-항체, 항-CD81-항체, 및 항체 혼합물이 결합된 폴리피롤 나노와이어(CD9_NWs, CD81_NWs 및 Abs_NWs)를 제조하였다.Polypyrrole nanowires (CD9_NWs, CD81_NWs, and Abs_NWs) with anti-CD9-antibody, anti-CD81-antibody, and antibody mixtures were prepared.
먼저, 다공성 알루미나 주형(AAO template, Whatman, pore diameter, 200 ㎚)의 한쪽 면에 약 150 ㎚ 두께의 금(Au)층을 가열증발(thermal evaporation)시켜 증착시켰다. 모든 전기화학적 실험은 금(Au) 코팅된 AAO 주형에서 백금 와이어 상대전극과 Ag/AgCl(3.0 M NaCl type) 비교전극을 구비한 potentiostat/galvanostat(BioLogic SP-150)를 사용하여 측정하였다.First, a gold (Au) layer having a thickness of about 150 nm was deposited on one side of a porous alumina mold (AAO template, Whatman, pore diameter, 200 nm) by thermal evaporation. All electrochemical experiments were measured using a potentiostat/galvanostat (BioLogic SP-150) equipped with a platinum wire counter electrode and an Ag/AgCl (3.0 M NaCl type) comparison electrode in a gold (Au) coated AAO template.
AAO 주형의 기공에 0.01M 폴리(4-스티렌설폰산)(poly(4-styrene sulfonic acid), Sigma Aldrich) 및 1 mg/㎖ NHS-SS-biotin(Succinimidyl-2-(biotinamido)-ethyl-1,3'-dithiopropionate, CovaChem)을 함유하는 0.01 M 피롤(pyrrole, Sigma Aldrich) 용액과 함께 1.5 V(vs. Ag/AgCl)에서 7분 동안 크로노암페로메트리(chronoamperometry)를 적용하여 전기 화학적 증착을 수행하였다.0.01M poly(4-styrene sulfonic acid), Sigma Aldrich) and 1 mg/ml NHS-SS-biotin (Succinimidyl-2-(biotinamido)-ethyl-1) in the pores of the AAO template Electrochemical deposition by applying chronoamperometry at 1.5 V (vs. Ag/AgCl) for 7 min with a 0.01 M pyrrole (Sigma Aldrich) solution containing 3'-dithiopropionate, CovaChem Was performed.
그 후, AAO 주형을 초순수(ultrapure water)로 여러 번 세척하고, 2 M의 수산화나트륨(NaOH, Sigma Aldrich) 용액에 2시간 동안 담궈 AAO 주형을 제거하였다. 이어서, 폴리피롤 나노와이어(Ppy NWs)의 카복실산(-COOH; carboxylic acid)기를 활성화하기 위해, 폴리피롤 나노와이어(Ppy NWs)에 30 mM의 EDC(N-(3-Dimethylaminopropyl)-N' -ethylcarbodiimide hydrochloride, Sigma Aldrich) 및 6 mM의 NHS(N-hydroxy succinimide, Sigma Aldrich)를 첨가하고 45분 동안 반응시켰다.Thereafter, the AAO template was washed several times with ultrapure water and immersed in a 2 M sodium hydroxide (NaOH, Sigma Aldrich) solution for 2 hours to remove the AAO template. Then, polypyrrole nanowires (NWs Ppy) of carboxylic acid (-COOH; carboxylic acid) in order to activate group, 30 mM of EDC (N- (3-Dimethylaminopropyl the polypyrrole nanowires (NWs Ppy)) -N '-ethylcarbodiimide hydrochloride, Sigma Aldrich) and 6 mM N-hydroxy succinimide (NHS) were added and reacted for 45 minutes.
생성된 폴리피롤 나노와이어(Ppy NWs)를 스트렙타비딘(10 ㎍/㎖)과 함께 추가로 45분 동안 반응시킨 후 물로 세척하였다. 이어서, 10 ㎕/㎖의 농도의 항-CD9 항체, 항-CD81 항체 또는 항체 혼합물(항-CD9 항체, 항-CD63 항체, 항-CD81 항체)을 나노와이어 말단에 스트렙타비딘이 도입된 폴리피롤 나노와이어(Ppy NWs)와 함께 4℃ 온도에서 하룻밤 동안 반응시켜, 항-CD9 항체, 항-CD81 항체 또는 항체 혼합물이 결합된 폴리피롤 나노와이어(CD9_NWs, CD81_NWs 및 Abs_NWs)를 제조하였다.The resulting polypyrrole nanowires (Ppy NWs) were reacted with streptavidin (10 μg/ml) for an additional 45 minutes, and then washed with water. Subsequently, the anti-CD9 antibody, anti-CD81 antibody or antibody mixture (anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody) at a concentration of 10 μl/ml was introduced into the nanowire end of streptavidin nano-polypyrrole. Polypyrrole nanowires (CD9_NWs, CD81_NWs and Abs_NWs) with anti-CD9 antibody, anti-CD81 antibody or antibody mixture were prepared by reacting with wire (Ppy NWs) overnight at 4°C.
이때, 비오틴이 부착된 항-CD63 항체 및 비오틴이 부착된 항-CD81 항체는 AnCell(Oak Park, Minnesota, USA)로부터 구매하였다. 또한, 비오틴이 부착된 항-CD9 항체는 Abcam(Cambridge, UK)로부터 구매하였다.At this time, the biotinylated anti-CD63 antibody and the biotinylated anti-CD81 antibody were purchased from AnCell (Oak Park, Minnesota, USA). In addition, anti-CD9 antibody with biotin attached was purchased from Abcam (Cambridge, UK).
실시예 2. 항체가 결합된 전도성 고분자를 포함하는 자성 나노와이어 제조Example 2. Preparation of magnetic nanowires containing a conductive polymer bound with an antibody
항-CD9-항체, 항-CD81-항체 및 항체 혼합물이 결합된 폴리피롤 자성 나노와이어(CD9_MNWs, CD81_MNWs 및 Abs_MNWs)를 제조하였다.Anti-CD9-antibody, anti-CD81-antibody and antibody mixture bound polypyrrole magnetic nanowires (CD9_MNWs, CD81_MNWs and Abs_MNWs) were prepared.
먼저, 다공성 알루미나 주형(AAO template, Whatman, pore diameter, 200 ㎚)의 한쪽 면에 약 150 ㎚ 두께의 금(Au)층을 가열증발(thermal evaporation)시켜 증착시켰다. 모든 전기화학적 실험은 금(Au) 코팅된 AAO 주형에서 백금 와이어 상대전극과 Ag/AgCl(3.0 M NaCl type) 비교전극을 구비한 potentiostat/galvanostat(BioLogic SP-150)를 사용하여 측정하였다. 상온에서 30 ㎕의 자성 나노 입자(평균 직경, 10 ㎚, Sigma Aldrich)를 금(Au) 코팅된 AAO 디스크 위에 부착시켜 AAO의 기공(pore)에 도입하였다.First, a gold (Au) layer having a thickness of about 150 nm was deposited on one side of a porous alumina mold (AAO template, Whatman, pore diameter, 200 nm) by thermal evaporation. All electrochemical experiments were measured using a potentiostat/galvanostat (BioLogic SP-150) equipped with a platinum wire counter electrode and an Ag/AgCl (3.0 M NaCl type) comparison electrode in a gold (Au) coated AAO template. At room temperature, 30 μl of magnetic nanoparticles (average diameter, 10 nm, Sigma Aldrich) were attached onto a gold (Au) coated AAO disk and introduced into the pores of AAO.
AAO 주형의 기공에 0.01M 폴리(4-스티렌설폰산)(poly(4-styrene sulfonic acid), Sigma Aldrich) 및 1 mg/㎖ NHS-SS-biotin(Succinimidyl-2-(biotinamido)-ethyl-1,3'-dithiopropionate, CovaChem)을 함유하는 0.01 M 피롤(pyrrole, Sigma Aldrich) 용액과 함께 1.5 V(vs. Ag/AgCl)에서 7분 동안 크로노암페로메트리(chronoamperometry)를 적용하여 전기 화학적 증착을 수행하였다.0.01M poly(4-styrene sulfonic acid), Sigma Aldrich) and 1 mg/ml NHS-SS-biotin (Succinimidyl-2-(biotinamido)-ethyl-1) in the pores of the AAO template Electrochemical deposition by applying chronoamperometry at 1.5 V (vs. Ag/AgCl) for 7 min with a 0.01 M pyrrole (Sigma Aldrich) solution containing 3'-dithiopropionate, CovaChem Was performed.
그 후, AAO 주형을 초순수(ultrapure water)로 여러 번 세척하고, 2 M의 수산화나트륨(NaOH, Sigma Aldrich) 용액에 2시간 동안 담궈 AAO 주형을 제거하였다. 이어서, 폴리피롤 자성 나노와이어(Ppy MNWs)의 카복실산(-COOH; carboxylic acid)기를 활성화하기 위해, 폴리피롤 자성 나노와이어(Ppy MNWs)에 30 mM의 EDC(N-(3-Dimethylaminopropyl)-N' -ethylcarbodiimide hydrochloride, Sigma Aldrich) 및 6 mM의 NHS(N-hydroxy succinimide, Sigma Aldrich)를 첨가하고 45분 동안 반응시켰다.Thereafter, the AAO template was washed several times with ultrapure water and immersed in a 2 M sodium hydroxide (NaOH, Sigma Aldrich) solution for 2 hours to remove the AAO template. Subsequently, the carboxylic acid of polypyrrole magnetic nanowires (Ppy MNWs) (-COOH; carboxylic acid) in order to activate group, polypyrrole magnetic nanowires (Ppy MNWs) 30 mM of EDC (N- (3-Dimethylaminopropyl a) - N '-ethylcarbodiimide Hydrochloride, Sigma Aldrich) and 6 mM NHS (N-hydroxy succinimide, Sigma Aldrich) were added and reacted for 45 minutes.
생성된 폴리피롤 자성 나노와이어(Ppy MNWs)를 스트렙타비딘(10 ㎍/㎖)과 함께 추가로 45분 동안 반응시킨 후 물로 세척하였다. 이어서, 10 ㎕/㎖의 농도의 항-CD9 항체, 항-CD81 항체 또는 항체 혼합물(항-CD9 항체, 항-CD63 항체, 항-CD81 항체)을 나노와이어 말단에 스트렙타비딘이 도입된 폴리피롤 자성 나노와이어(Ppy MNWs)와 함께 4℃ 온도에서 하룻밤 동안 반응시켜, 0.4 ㎍/㎖의 최종 항체 농도를 갖는 항-CD9 항체, 항-CD81 항체 또는 항체 혼합물이 결합된 폴리피롤 자성 나노와이어(CD9_MNWs, CD81_MNWs 및 Abs_MNWs)를 제조하였다.The resulting polypyrrole magnetic nanowires (Ppy MNWs) were reacted with streptavidin (10 μg/ml) for an additional 45 minutes, and then washed with water. Subsequently, the anti-CD9 antibody, anti-CD81 antibody or antibody mixture (anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody) at a concentration of 10 μl/ml was introduced into the nanowire end of streptavidin and introduced into the polypyrrole magnetism. Polypyrrole magnetic nanowires (CD9_MNWs, CD81_MNWs, combined with anti-CD9 antibodies, anti-CD81 antibodies or antibody mixtures having a final antibody concentration of 0.4 μg/ml) by reacting with nanowires (Ppy MNWs) overnight at a temperature of 4° C. And Abs_MNWs).
상기 CD9_MNWs는 항-CD9 항체가 결합된 자성 나노와이어이며, CD81_MNWs는 항-CD81 항체가 결합된 자성 나노와이어이다. 또한, Abs_MNWs는 항-CD9 항체, 항-CD63 항체 및 항-CD81 항체가 결합된 나노와이어이다.The CD9_MNWs are magnetic nanowires to which anti-CD9 antibodies are bound, and CD81_MNWs are magnetic nanowires to which anti-CD81 antibodies are bound. In addition, Abs_MNWs are nanowires to which anti-CD9 antibody, anti-CD63 antibody and anti-CD81 antibody are bound.
이때, 비오틴이 부착된 항-CD63 항체 및 비오틴이 부착된 항-CD81 항체는 AnCell(Oak Park, Minnesota, USA)로부터 구매하였다. 또한, 비오틴이 부착된 항-CD9 항체는 Abcam(Cambridge, UK)로부터 구매하였다.At this time, the biotinylated anti-CD63 antibody and the biotinylated anti-CD81 antibody were purchased from AnCell (Oak Park, Minnesota, USA). In addition, anti-CD9 antibody with biotin attached was purchased from Abcam (Cambridge, UK).
또한, CD9_MNWs, CD81_MNWs 및 Abs_MNWs에 결합된 각 항체의 농도는 HRP(Horseradish peroxidase)가 표지된 항-마우스 IgG 항체를 처리하여 1시간 동안 반응시킨 후, 비특이적 결합을 방지하기 위해 3% 농도의 BSA 용액을 처리하였다. CD9_MNWs, CD81_MNWs 및 Abs_MNWs를 조심스럽게 세척하여 결합하지 않은 항-마우스 IgG 항체를 제거한 후, HRP의 발색 기질인 TMB(tetramethylbenzidine)를 처리하였다. 항체의 상대적인 양을 HRP 항-마우스 IgG 표준 곡선과 비교하여, 1.26×106 MNWs/㎖ 상에 접합된 항체를 검출하고 이의 양을 정량화하였으며, 650 nm 파장에서 분광 광도계를 사용하여 결과를 판독하였다.In addition, the concentration of each antibody bound to CD9_MNWs, CD81_MNWs, and Abs_MNWs was treated with HRP (Horseradish peroxidase) labeled anti-mouse IgG antibody and reacted for 1 hour, followed by 3% concentration of BSA solution to prevent non-specific binding. Was treated. CD9_MNWs, CD81_MNWs and Abs_MNWs were carefully washed to remove unbound anti-mouse IgG antibody, followed by treatment with HRP's chromogenic substrate, tetramethylbenzidine (TMB). By comparing the relative amount of antibody to the HRP anti-mouse IgG standard curve, the antibody conjugated on 1.26×10 6 MNWs/ml was detected and its quantity was quantified, and the results were read using a spectrophotometer at a wavelength of 650 nm. .
실시예 3. 항-CD9-항체, 항-CD81-항체 또는 항체 혼합물이 결합된 폴리피롤 자성 나노와이어(CD9_MNWs, CD81_MNWs 및 Abs_MNWs)의 특성 분석Example 3. Characterization of anti-CD9-antibody, anti-CD81-antibody or antibody mixture bound polypyrrole magnetic nanowires (CD9_MNWs, CD81_MNWs and Abs_MNWs)
상기 실시예 2에서 제조한 CD9_MNWs, CD81_MNWs 및 Abs_MNWs의 특성을 분석하기 위해, CD9_MNWs, CD81_MNWs 및 Abs_MNWs의 주사전자현미경 및 투과전자현미경을 이용해 형태를 분석하고 자기력을 측정하였다.To analyze the properties of CD9_MNWs, CD81_MNWs, and Abs_MNWs prepared in Example 2, the morphology was analyzed and magnetic force was measured using a scanning electron microscope and a transmission electron microscope of CD9_MNWs, CD81_MNWs, and Abs_MNWs.
구체적으로, CD9_MNWs, CD81_MNWs 및 Abs_MNWs의 형태(morphology)는 가속 전압 15 kV의 주사전자현미경(G2F30, Tecnai) 및 300 kV의 투과전자현미경을 이용하여 관찰하였다. 자기력 측정은 SQUID-VSM magnetometer(MPMS-VSM, Quantum design)를 이용하여 상온에서 수행하였다. 자기장은 70 Oe 내지 70 kOe의 세기로 변화시켜 인가하였고, 횡축이완시간(transverse relaxation time, T2)은 7 테슬라(Tesla) MRI(Bruker BioSpin MRI GmbH; echo time[TE]=6.5 ㎳ 및 repetition time[TR]=1,600 ㎳)를 이용하여 측정하였다.Specifically, the morphology of CD9_MNWs, CD81_MNWs, and Abs_MNWs was observed using a scanning electron microscope (G2F30, Tecnai) with an acceleration voltage of 15 kV and a transmission electron microscope with 300 kV. Magnetic force measurement was performed at room temperature using a SQUID-VSM magnetometer (MPMS-VSM, Quantum design). The magnetic field was applied by varying the intensity between 70 Oe and 70 kOe, and the transverse relaxation time (T 2 ) was 7 Tesla MRI (Bruker BioSpin MRI GmbH; echo time [TE]=6.5 ㎳ and repetition time) It was measured using [TR]=1,600 kPa).
그 결과, 도 2에 나타난 바와 같이, 주사전자현미경(SEM) 이미지(스케일바 500 nm)를 통해 약 200 ㎚의 직경 및 약 18 ㎛의 평균 길이를 갖는 비교적 긴 길이의 나노와이어가 합성된 것을 확인하였다(도 2). 또한, 도 3에 나타난 바와 같이, 전자투과현미경(TEM) 이미지(스케일바 50 ㎚)를 통해 항체 혼합물이 결합된 폴리피롤 자성 나노와이어(Ab mixture_mPpyNW) 안에 자성 나노 입자(<10 ㎚의 직경)가 불규칙하게 분포(randomly distributed)되고, 밀도 있게 배열된 상태로 매립되어 있음을 확인하였다. 나아가, 도 4에 나타낸 바와 같이, 항체 혼합물이 결합된 폴리피롤 자성 나노와이어(Ab mixture_mPpyNW) 및 자성 나노 입자(MNPs)의 포화자화 값을 측정한 결과, 제조된 나노와이어(Ms = 57 emu/g)는 큰 포화자화 값(saturation magnetization value)을 가지는 것을 확인하였다.As a result, as shown in Figure 2, a scanning electron microscope (SEM) image (scale bar 500 nm) of a diameter of about 200 nm and a comparatively long nanowire having an average length of about 18 μm was confirmed to be synthesized (Fig. 2). In addition, as shown in FIG. 3, magnetic nanoparticles (<10 nm diameter) are irregular in the polypyrrole magnetic nanowire (Ab mixture_mPpyNW) to which the antibody mixture is bound through an electron transmission microscope (TEM) image (scale bar 50 nm). It was confirmed that it was randomly distributed and buried in a densely arranged state. Furthermore, as shown in Figure 4, as a result of measuring the saturation magnetization values of the polypyrrole magnetic nanowires (Ab mixture_mPpyNW) and magnetic nanoparticles (MNPs) to which the antibody mixture is bound, the prepared nanowires (Ms = 57 emu/g) Was confirmed to have a large saturation magnetization value.
이를 통해, 산화철 자성 나노 입자의 조립(assembly)으로 인하여, 나노와이어의 자성(magnetism)이 더욱 상승(synergistic)하였으며, 나노와이어의 공간적 제약(confined geometry)은 나노와이어가 자기장에 더욱 민감하도록 하여, 각각의 나노 입자가 자기 모멘트를 배향함으로써, 순환 엑소좀의 분리에 있어서 정확한 제어 및 선택적인 조작이 가능하도록 하는 것을 확인하였다.Through this, due to the assembly of the iron oxide magnetic nanoparticles, the magnetism of the nanowires is more synergistic, and the spatial constraints of the nanowires make the nanowires more sensitive to the magnetic field, It was confirmed that by oriented the magnetic moments of each nanoparticle, precise control and selective manipulation in the separation of circulating exosomes is possible.
실시예 4. 항체가 결합된 전도성 고분자를 포함하는 자성 나노와이어를 이용한 엑소좀 분리 및 분리 효율 분석Example 4. Analysis of exosome separation and separation efficiency using magnetic nanowires containing an antibody-conductive conductive polymer
실시예 4.1. CD9_MNWs, CD81_MNWs, Abs_MNWs 및 자성비드를 이용한 엑소좀 분리Example 4.1. Separation of exosomes using CD9_MNWs, CD81_MNWs, Abs_MNWs and magnetic beads
비특이적으로 결합된 단백질 응집체 및 막 소포체를 최소화하면서 항체가 결합된 전도성 고분자를 포함하는 자성 나노와이어를 이용해 배양 배지(CCM)로부터 엑소좀을 분리 효율을 확인하기 위해, 농축된 배양 배지를 제조하였다.To minimize the efficiency of separating exosomes from the culture medium (CCM) using magnetic nanowires containing a conductive polymer bound with an antibody while minimizing non-specifically bound protein aggregates and membrane vesicles, a concentrated culture medium was prepared.
구체적으로, 4가지의 암 세포주(MDA-MB-231 및 MCF7 유방암 세포, HCT116 결장암 세포 또는 HeLa 자궁경부암 세포)를 각각 5% CO2, 37℃ 온도에서 10% FBS 및 1% 페니실린-스트렙토마이신을 함유하는 RPMI-1640 배지(Invitrogen, Carlsbad, CA)에서 배양하였다. 약 2×109 세포수의 암세포를 각각 펠릿화하고, RPMI-1640 배지로 3회 세척한 후, 무혈청 RPMI 배지로 배지를 교체하였다. 각각의 암세포를 무혈청 RPMI 배지에서 2일 동안 배양하고 배양액을 수득하였다. 이때, 10분 동안 300×g에서 원심분리하고, 다시 20분 동안 2,000×g에서 원심분리하여 온전한 세포 및 세포 파편을 제거하였다. 농축된 배양 배지를 수집하고 무균 0.22 ㎛ 필터(Merck Millipore, USA)를 통해 여과하였다.Specifically, four cancer cell lines (MDA-MB-231 and MCF7 breast cancer cells, HCT116 colon cancer cells or HeLa cervical cancer cells) were treated with 5% CO 2 and 10% FBS and 1% penicillin-streptomycin at a temperature of 37° C., respectively. Cultured in containing RPMI-1640 medium (Invitrogen, Carlsbad, CA). Cancer cells of about 2×10 9 cell numbers were pelletized, washed three times with RPMI-1640 medium, and the medium was replaced with serum-free RPMI medium. Each cancer cell was cultured in serum-free RPMI medium for 2 days to obtain a culture medium. At this time, centrifugation at 300 × g for 10 minutes, and centrifugation at 2,000 × g for 20 minutes again to remove intact cells and cell debris. The concentrated culture medium was collected and filtered through a sterile 0.22 μm filter (Merck Millipore, USA).
그 후, 상기 제조한 4가지의 농축된 배양 배지로부터 항-CD81(Dyna Beads® _CD81, ThermoFisher Scientific Inc.)와 접합된 자성 비드, 항-CD9(Dyna Beads® _CD9, ThermoFisher Scientific Inc.)와 접합된 자성 비드, CD9_MNWs, CD81_MNWs 및 Abs_MNWs를 이용하여 엑소좀을 분리하였다.Then, magnetic beads, anti-CD9 (Dyna Beads® _CD9, ThermoFisher Scientific Inc.) conjugated with anti-CD81 (Dyna Beads® _CD81, ThermoFisher Scientific Inc.) from the four concentrated culture medium prepared above Exosomes were isolated using magnetic beads, CD9_MNWs, CD81_MNWs and Abs_MNWs.
구체적으로, 엑소좀을 분리하기 위해, Dyna Beads_CD9(5.0×105 비드/㎕), Dyna Beads_CD81(5.0×105 비드/㎕), CD9_MNWs(1.0×103 MNWs/㎕), CD81_MNWs(1.0×103 MNWs/㎕), 및 Abs_MNWs(1.0×103 MNWs/㎕)를 250 ㎕ 내지 3 ㎖의 농축된 배양 배지에 처리한 후, 엑소좀 부착을 촉진하기 위해, 부드럽게 진탕하면서 실온에서 30분 동안 반응시켰다. 그 후, MagneSphere® Technology Magnetic Separation Stands(Promega, USA) 및 1.5 ㎖ 튜브를 이용해 상등액을 제거하고, 50 mM 농도의 DTT(Dithiothreitol) 용액을 첨가하여 이화항 결합(disulfide bond)을 끊어 자성 비드 및 자성 나노와이어에 포획된 엑소좀을 분리하였다.Specifically, to isolate exosomes, Dyna Beads_CD9 (5.0×10 5 beads/μl), Dyna Beads_CD81 (5.0×10 5 beads/μl), CD9_MNWs (1.0×10 3 MNWs/μl), CD81_MNWs (1.0×10 3 MNWs/μL), and Abs_MNWs (1.0×10 3 MNWs/μL) were treated in 250 μL to 3 mL of concentrated culture medium, and then reacted for 30 minutes at room temperature with gentle shaking to promote exosome adhesion. Ordered. Then, the supernatant was removed using MagneSphere® Technology Magnetic Separation Stands (Promega, USA) and a 1.5 mL tube, and a 50 mM concentration of DTT (Dithiothreitol) solution was added to break the disulfide bond to break the magnetic beads and magnetism. The exosomes captured on the nanowires were isolated.
실시예 4.2. CD9_MNWs, CD81_MNWs, Abs_MNWs 및 자성비드의 엑소좀 분리 효율 분석Example 4.2. Analysis of exosome separation efficiency of CD9_MNWs, CD81_MNWs, Abs_MNWs and magnetic beads
상기 실시예 4.1에서 항-CD81(Dyna Beads® _CD81)와 접합된 자성 비드, 항-CD9(Dyna Beads® _CD9)와 접합된 자성 비드, CD9_MNWs, CD81_MNWs 또는 Abs_MNWs를 이용하여 분리한 엑소좀을 나노입자 추적 분석(NTA), ELISA 및 BCA 분석을 통해 분리 효율을 비교하였다.Nanoparticles of exosomes separated using anti-CD81 (Dyna Beads® _CD81) and magnetic beads conjugated in Example 4.1, magnetic beads conjugated with anti-CD9 (Dyna Beads® _CD9), CD9_MNWs, CD81_MNWs or Abs_MNWs Separation efficiency was compared by follow-up analysis (NTA), ELISA and BCA analysis.
먼저, 나노입자 추적 분석은 NanoSight NS300(Malvern Instruments, Malvern, UK) 및 Malvern Zetasizer Nano-Z(Malvern Instruments, Malvern, UK)를 사용하여 분리된 엑소좀의 농도 및 크기를 평가하였다.First, nanoparticle tracking analysis was performed using NanoSight NS300 (Malvern Instruments, Malvern, UK) and Malvern Zetasizer Nano-Z (Malvern Instruments, Malvern, UK) to evaluate the concentration and size of the isolated exosomes.
또한, BCA 분석은 BCA 분석 키트(Thermo Scientific, Waltham, MA, USA)를 사용하여 제조사의 매뉴얼에 따라 총 단백질 농도를 측정하였다. 구체적으로, 1 ㎕의 분리된 엑소좀을 19 ㎕의 M-PER 시약(Thermo Fisher Scientific, Massachusetts, Waltham, USA)에서 희석하고, 200 ㎕의 BCA 시약 A 및 B 혼합물(A:B = 50:1)을 첨가하여 37℃ 온도에서 30분 동안 반응시켰다. 그 후, UV/VIS 분광광도계를 이용하여 562 nm의 파장에서 O.D값을 측정하였다. 단백질 농도는 표준 BCA 곡선(r2 = 99.8 %)으로부터 산출하였다. 모든 측정은 일정한 실험 조건에서 수행되었다. In addition, BCA analysis was performed using the BCA analysis kit (Thermo Scientific, Waltham, MA, USA) to measure the total protein concentration according to the manufacturer's manual. Specifically, 1 μl of isolated exosomes was diluted in 19 μl of M-PER reagent (Thermo Fisher Scientific, Massachusetts, Waltham, USA), and 200 μl of BCA reagent A and B mixture (A:B = 50:1 ) Was added and reacted at a temperature of 37° C. for 30 minutes. Then, the O.D value was measured at a wavelength of 562 nm using a UV/VIS spectrophotometer. Protein concentration was calculated from a standard BCA curve (r2 = 99.8%). All measurements were performed under constant experimental conditions.
나아가, ELISA 분석은 항-CD9 항체 및 항-CD81 항체를 이용한 샌드위치 ELISA 분석을 진행하였다. 먼저, 항-CD9 항체를 96-웰-플레이트(Thermo Fischer Scientific)에 코팅하기 위해, 1 ㎍/100 ㎕ 농도의 항-CD9 항체 100 ㎕을 96-웰-플레이트의 각 웰에 처리하고, 4℃ 온도에서 하룻밤 동안 반응시켰다. 그 후, 96-웰-플레이트를 37℃ 온도에서 1% BSA가 첨가된 PBS를 처리하여 1시간 동안 블로킹 과정을 진행하였다. 0.1% BSA가 첨가된 PBS로 3회 세척한 후, 96-웰-플레이트의 각 웰에 100 ㎕의 PBS 및 실시예 3.1에서 분리한 엑소좀을 처리한 후, 37℃ 온도에서 배양하였다.Furthermore, ELISA analysis was performed by sandwich ELISA analysis using anti-CD9 antibody and anti-CD81 antibody. First, to coat an anti-CD9 antibody on a 96-well-plate (Thermo Fischer Scientific), 100 μl of an anti-CD9 antibody at a concentration of 1 μg/100 μl was treated in each well of a 96-well-plate, and 4° C. The mixture was allowed to react overnight. Thereafter, the 96-well-plate was treated with PBS containing 1% BSA at a temperature of 37° C. to perform a blocking process for 1 hour. After washing three times with PBS added with 0.1% BSA, each well of a 96-well-plate was treated with 100 μl of PBS and the exosomes isolated in Example 3.1, and then incubated at 37°C.
그 후, 남아있는 용액을 제거하고, 각 웰을 0.1% BSA가 첨가된 PBS로 2회 세척하고, 비오틴이 접합된 항-CD81 항체(LifeSpan Biosciences, Inc., Seattle, WA, USA)에 첨가한 후, 실온에서 1시간 동안 배양하였다. 0.1% BSA가 첨가된 PBS로 3회 세척한 후, 96-웰-플레이트의 각 웰에 1:1,000 비율로 HRP가 접합된 스트렙타비딘이 첨가된 100 ㎕의 PBS를 처리하고, 실온에서 30분 동안 반응시켰다. 0.1% BSA가 첨가된 PBS로 3회 세척한 후, TMB 용액(Thermo Fisher Scientific)을 각 웰에 첨가하고 실온에서 15분 동안 반응시켰다. 그 후, 각 웰에 50 ㎕의 정지 용액을 첨가한 후, UV/VIS 분광광도계를 이용하여 450 nm 파장에서 흡광도를 측정하였다.Thereafter, the remaining solution was removed, and each well was washed twice with PBS added with 0.1% BSA, and added to biotin-conjugated anti-CD81 antibody (LifeSpan Biosciences, Inc., Seattle, WA, USA). Then, the cells were incubated at room temperature for 1 hour. After washing three times with PBS with 0.1% BSA, 100 µl of PBS with HRP-conjugated streptavidin added in a 1:1,000 ratio to each well of a 96-well-plate was treated, and 30 minutes at room temperature. During the reaction. After washing three times with PBS with 0.1% BSA, TMB solution (Thermo Fisher Scientific) was added to each well and reacted at room temperature for 15 minutes. Thereafter, 50 µl of the stop solution was added to each well, and absorbance was measured at a wavelength of 450 nm using a UV/VIS spectrophotometer.
상기 5가지의 상이한 방법으로 분리한 엑소좀을 나노입자 추적 분석, ELISA 및 BCA 분석을 통해 확인한 결과, 4가지의 농축된 배양 배지로부터 Abs_MNWs를 이용하여 엑소좀을 분리한 경우가 가장 높은 수율 및 순도로 엑소좀을 분리하였다. 특히, ELISA 결과에서 CD9_MNWs, CD81_MNWs 또는 자성 비드를 이용하여 분리한 엑소좀과 비교하여 Abs_MNWs를 이용해 분리한 엑소좀에 대해 가장 높은 O.D값이 측정되었다(도 5 내지 도 7).As a result of confirming exosomes separated by the above 5 different methods through nanoparticle tracking analysis, ELISA and BCA analysis, the highest yield and purity are obtained when exosomes are separated using Abs_MNWs from 4 concentrated culture media. Exosomes were isolated. In particular, in the ELISA results, the highest O.D value was measured for exosomes isolated using Abs_MNWs compared to CD9_MNWs, CD81_MNWs, or exosomes isolated using magnetic beads (FIGS. 5 to 7 ).
즉, 3가지 유형의 항-CD9 항체, 항-CD63 항체 및 항-CD81 항체가 결합된 Abs_MNWs는 항-CD81 항체 또는 항-CD9 항체가 접찹된 자성 비드, CD9_MNWs 및 CD81_MNWs와 비교하여 상당히 많은 양의 엑소좀을 분리하였다.That is, the Abs_MNWs to which the three types of anti-CD9 antibody, anti-CD63 antibody and anti-CD81 antibody are bound are significantly larger compared to the magnetic beads, CD9_MNWs and CD81_MNWs to which the anti-CD81 antibody or anti-CD9 antibody is folded. Exosomes were isolated.
이를 통해, 단일 항체보다 다중 항체가 결합된 나노와이어가 엑소좀 분리 효율이 높은 것을 확인하였다. 이는 소량의 시료로부터 많은 양의 엑소좀을 분리가 가능해짐으로써, 더 민감하고 1시간 미만으로 엑소좀 분리 시간을 크게 단축시키는 것을 확인하였다.Through this, it was confirmed that the nanowires to which the multiple antibodies were bound than the single antibody had higher exosome separation efficiency. This confirmed that it is possible to separate a large amount of exosomes from a small amount of samples, which makes it more sensitive and significantly shortens the exosome separation time to less than 1 hour.
한편, 전자현미경을 통해 엑소좀의 직경을 분석한 결과, 초원심분리로 분리한 엑소좀의 직경은 주로 100 nm 내지 300 nm의 범위인 반면, Abs_MNWs에 의해 분리된 엑소좀의 직경은 주로 40 nm 내지 150 nm의 범위에 있었다. 이를 통해, Abs_MNWs를 사용하여 분리된 엑소좀의 균일한 크기의 분포를 갖는 것을 확인하였다(도 8).On the other hand, as a result of analyzing the diameter of the exosomes through an electron microscope, the diameter of the exosomes separated by ultracentrifugation is mainly in the range of 100 nm to 300 nm, while the diameter of the exosomes separated by Abs_MNWs is mainly 40 nm. To 150 nm. Through this, it was confirmed that Abs_MNWs had a uniform size distribution of the separated exosomes (FIG. 8).
실시예 5. 암 환자의 생물학적 시료로부터의 엑소좀 분리 및 분석Example 5. Isolation and analysis of exosomes from biological samples of cancer patients
실시예 5.1. Abs_MNWs를 이용한 폐암 환자의 혈장으로부터의 엑소좀 분리 여부 확인Example 5.1. Confirmation of separation of exosomes from plasma of lung cancer patients using Abs_MNWs
폐암 환자의 혈장으로부터 Abs_MNWs를 이용하여 엑소좀을 분리하고, 이의 분리 효율을 분석하였다. 이때, Abs_MNWs에 분리된 엑소좀은 막-특이적 형광 염료가 표지되도록 하였다.Exosomes were isolated from plasma of lung cancer patients using Abs_MNWs, and their separation efficiency was analyzed. At this time, the exosome isolated to Abs_MNWs was labeled with a membrane-specific fluorescent dye.
구체적으로, 먼저, 국립 암센터 기관 생명 윤리 위원회(National Cancer Center Institutional Review Board)에 의해 승인된 절차에 따라 항응고제인 EDTA(ethlenediaminetetracetic acid)를 포함하는 진공 채혈기에 혈액을 수집하였다. 혈장 분리를 위해, 수집된 혈액을 10분 동안 3,000×g에서 원심분리하고, 이를 분석 전까지 -80℃ 온도에 보관하였다.Specifically, first, blood was collected in a vacuum blood drawer containing an anticoagulant ethlenediaminetetracetic acid (EDTA) according to a procedure approved by the National Cancer Center Institutional Review Board. For plasma separation, the collected blood was centrifuged at 3,000×g for 10 minutes and stored at a temperature of -80° C. until analysis.
상기 혈장과 Abs_MNWs를 실시예 3.1과 동일한 방법으로 엑소좀을 분리하였으며, DTT를 처리하지 않은 엑소좀이 포집된 상태의 Abs_MNWs도 준비하였다. DTT를 처리하지 않은 엑소좀이 포집된 Abs_MNWs 및 DTT를 처리한 Abs_MNWs를 37℃ 온도에서 8분 동안 각각 배양한 후, 엑소좀 막을 염색시키는 5 ㎕/㎖의 VybrantTM DiO 염료 용액(Life Technologies)을 처리하였다. 그 후, 엑소좀을 인산염-완충된 식염수(PBS)로 세척하고, DiO-표지된 엑소좀을 Zeiss 형광 현미경에 의해 분석하였다.The plasma and Abs_MNWs were separated from the exosomes in the same manner as in Example 3.1, and Abs_MNWs in a state where exosomes not treated with DTT were collected were also prepared. After culturing the Abs_MNWs collected with DTT-treated exosomes and Abs_MNWs treated with DTT for 8 minutes at a temperature of 37°C, a 5 µl/ml Vybrant TM DiO dye solution (Life Technologies) staining the exosome membrane was prepared. Treatment. The exosomes were then washed with phosphate-buffered saline (PBS) and DiO-labeled exosomes were analyzed by Zeiss fluorescence microscopy.
그 결과, DTT를 처리하지 않은 엑소좀이 포집된 Abs_MNWs의 표면에서 강한 형광 신호가 검출되었으며, 이를 통해 엑소좀이 Abs_MNWs에 직접적으로 포집된 것을 확인하였다(도 9 내지 도 11). 또한, DTT를 처리한 Abs_MNWs에서는 형광 신호가 검출되지 않았다(도 12 내지 도 14).As a result, a strong fluorescence signal was detected on the surface of Abs_MNWs where DTT-treated exosomes were collected, and it was confirmed that exosomes were directly captured by Abs_MNWs (FIGS. 9 to 11 ). In addition, a fluorescent signal was not detected in Abs_MNWs treated with DTT (FIGS. 12 to 14 ).
실시예 5.2. Abs_MNWs를 이용한 폐암 환자의 혈장으로부터 분리한 엑소좀 내 RNA 분석Example 5.2. RNA analysis in exosomes isolated from plasma of lung cancer patients using Abs_MNWs
실시예 5.1과 동일한 방법으로, 폐암 환자의 혈장으로부터 Abs_MNWs를 이용하여 엑소좀을 분리하고, 엑소좀에서 RNA를 추출하여 Bioanalyzer 프로파일을 조사하여 이의 견고성, 순도, 및 크기 분포를 분석하였다.In the same manner as in Example 5.1, exosomes were isolated from the plasma of lung cancer patients using Abs_MNWs, RNA was extracted from the exosomes, and the Bioanalyzer profile was examined to analyze its robustness, purity, and size distribution.
구체적으로, 엑소좀 내 RNA를 TRIzol 키트(Invitrogen, Paisley, UK)를 이용하여 추출하였다. 이때, 제조사의 매뉴얼에 따라 RNA를 추출하였다. 추가로, 추출한 RNA에 클로로폼(Merck, Darmstadt, Germany)을 처리하고 4℃ 온도에서 12,000 ×g에서 15분 동안 원심분리하여 혼합물을 수성상 및 유기상으로 분리하였다. 이때, 상등액을 침전시키기 위해 이소프로판올을 사용하였다.Specifically, RNA in exosomes was extracted using a TRIzol kit (Invitrogen, Paisley, UK). At this time, RNA was extracted according to the manufacturer's manual. Additionally, the extracted RNA was treated with chloroform (Merck, Darmstadt, Germany) and centrifuged at 12,000 x g for 15 minutes at 4°C to separate the mixture into an aqueous phase and an organic phase. At this time, isopropanol was used to precipitate the supernatant.
그 후, miR21-cDNA를 TaqMan MicroRNA 역전사 키트(Applied Biosystems, Foster city, CA, USA)를 사용하여 무작위 핵사머로 10 ng의 RNA 용리액을 이용하여 합성하였다. 이때, 25℃ 온도에서 5분, 46℃ 온도에서 20분, 및 95℃ 온도에서 1분 동안 LC480 실시간 PCR 시스템(Roche, Basel, Switzerland)을 이용하여 정량적 역전사 중합효소연쇄반응(qRT-PCR)을 수행하였다. 반응 종료 후, 4℃ 온도에 보관하였다. 이때, 제조사에 매뉴얼에 따라 miR-21(has-miR-21-3p, 분석 ID 477973_mir)에 대해 사전 설계된 프라이머 및 프로브(TaqManTM Advanced miRNA Assay, Thermo Fisher Scientific)를 사용하였다.Thereafter, miR21-cDNA was synthesized using 10 ng of RNA eluent as a random nuclear atom using a TaqMan MicroRNA reverse transcription kit (Applied Biosystems, Foster city, CA, USA). At this time, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using an LC480 real-time PCR system (Roche, Basel, Switzerland) for 5 minutes at 25°C, 20 minutes at 46°C, and 1 minute at 95°C. Was performed. After completion of the reaction, it was stored at 4°C. At this time, pre-designed primers and probes (TaqMan TM Advanced miRNA Assay, Thermo Fisher Scientific) for miR-21 (has-miR-21-3p, analysis ID 477973_mir) were used according to the manufacturer's manual.
그 결과, 광범위한 RNA 크기(주로 400개 미만의 뉴클레오티드)가 검출되었으며, 이들 대부분은 전기영동(electropherogram)에서 대략 170개의 뉴클레오티드 크기를 나타냈다(도 15).As a result, a wide range of RNA sizes (mainly less than 400 nucleotides) were detected, most of which showed approximately 170 nucleotide sizes in the electropherogram (FIG. 15).
또한, cDNA 합성 키트를 사용하여 엑소좀에서 추출한 RNA를 추가로 증폭시키고, miR-21의 발현에 대해 평가하였다. Abs_MNWs를 이용하여 건강한 공여자 및 폐암 환자의 혈장으로부터 추출한 엑소좀의 miRNA 발현 수준을 측정한 결과, 폐암 환자에서 독특한 miR-21 발현 양상이 관찰되었다(도 16).In addition, RNA extracted from exosomes was further amplified using the cDNA synthesis kit, and the expression of miR-21 was evaluated. As a result of measuring the level of miRNA expression of exosomes extracted from plasma of healthy donor and lung cancer patients using Abs_MNWs, a unique miR-21 expression pattern was observed in lung cancer patients (FIG. 16 ).
실시예 5.3. Abs_MNWs를 이용한 유방암 환자 및 폐암 환자의 혈장으로부터 분리한 엑소좀 분석Example 5.3. Analysis of exosomes isolated from plasma of breast and lung cancer patients using Abs_MNWs
Abs_MNWs를 이용하여 건강한 공여자, 유방암 환자 및 폐암 환자의 혈장으로부터 엑소좀을 분리하고, NTA 및 BCA 분석을 통해 이들의 총량을 측정하였다. NTA 및 BCA 분석은 실시예 4.2와 동일한 방법으로 수행하였다.Exosomes were isolated from plasma of healthy donors, breast cancer patients and lung cancer patients using Abs_MNWs, and their total amount was measured by NTA and BCA analysis. NTA and BCA analysis was performed in the same manner as in Example 4.2.
그 결과, 건강한 공여자와 비교하여, 암 환자가 순환 엑소좀이 3배 이상 많은 것으로 나타났다(도 17). 또한, BCA 분석에서도 건강한 공여자와 비교하여 암 환자의 엑소좀 단백질이 3.9배 높은 것으로 나타났다(도 18).As a result, compared to a healthy donor, cancer patients showed circulating exosomes three times or more (FIG. 17). In addition, BCA analysis also showed that the exosome protein in cancer patients was 3.9 times higher than in healthy donors (FIG. 18).
실시예 5.3. Abs_MNWs를 이용한 폐암 환자의 혈장으로부터 분리한 엑소좀 확인Example 5.3. Identification of exosomes isolated from plasma of lung cancer patients using Abs_MNWs
건강한 공여자 및 폐암 환자의 혈장으로부터 Abs_MNWs를 이용하여 분리한 엑소좀을 HSP70, TSG101, CD81, CD9, CD63, 및 GAPDH에 대한 항체를 이용하여 웨스턴 블롯으로 엑소좀을 확인하였다.Exosomes isolated from plasma of healthy donor and lung cancer patients using Abs_MNWs were confirmed by Western blot using antibodies against HSP70, TSG101, CD81, CD9, CD63, and GAPDH.
구체적으로, Abs_MNWs를 이용하여 분리한 엑소좀을 M-PER 시약(Thermo Fisher Scientific, Massachusetts, Waltham, USA)에 용해시켰다. 그 후, 용해시킨 샘플 20 ㎍을 10% 소듐 도데실 설페이트 폴리아크릴아미드 겔(sodium Dodecyl Sulfate Polyacrylamide Gel)에서 전기영동하고 PVDF(poly-vinylidene fluoride) 막(0.45 ㎛, Millipore)에 옮겼다. 그 후, PVDF 막을 실온에서 3% 탈지 분유로 1시간 동안 블로킹 과정을 진행하고, 1차 항체로써, 마우스 항-TSG101 항체(1:1,000), 토끼 항-HSP70 항체(1:1,000), 토끼 항-GAPDH 항체(1:1,000), 토끼 항-CD9 항체(1:1,000), 토끼 항-CD63 항체(1:1,000), 토끼 항-CD81항체(1:1,000), 및 토끼 단클론 항-GAPDH 항체(1:1,000)를 처리하고 하룻밤 동안 반응시켰다. 다음 날, 막을 TBST로 3회 세척한 후, 2차 항체로써, 염소 항-마우스 IgG 항체(1:3,000) 또는 염소 항-토끼 IgG 항체(1:3,000)를 처리하고 1시간 동안 반응시켰다. 그 후, TBST 완충액으로 3회 세척하고, SuperSignal® West Pico 화학발광 기질 시약(34077, Thermo Scientific)을 처리한 후 형광 분석하였다.Specifically, exosomes isolated using Abs_MNWs were dissolved in M-PER reagent (Thermo Fisher Scientific, Massachusetts, Waltham, USA). Thereafter, 20 μg of the dissolved sample was electrophoresed on a 10% sodium dodecyl sulfate polyacrylamide gel and transferred to a poly-vinylidene fluoride (PVDF) membrane (0.45 μm, Millipore). Subsequently, the PVDF membrane was blocked for 1 hour with 3% skim milk powder at room temperature, and as a primary antibody, mouse anti-TSG101 antibody (1:1,000), rabbit anti-HSP70 antibody (1:1,000), rabbit anti -GAPDH antibody (1:1,000), rabbit anti-CD9 antibody (1:1,000), rabbit anti-CD63 antibody (1:1,000), rabbit anti-CD81 antibody (1:1,000), and rabbit monoclonal anti-GAPDH antibody ( 1:1,000) and reacted overnight. The next day, after washing the membrane three times with TBST, as a secondary antibody, goat anti-mouse IgG antibody (1:3,000) or goat anti-rabbit IgG antibody (1:3,000) was treated and reacted for 1 hour. Then, washed three times with TBST buffer, treated with SuperSignal® West Pico chemiluminescent substrate reagent (34077, Thermo Scientific), followed by fluorescence analysis.
그 결과, Abs_MNWs로 분리된 엑소좀에는 다양한 엑소좀 단백질이 포함되어 있어, 분리된 물질이 엑소좀임을 다시 한 번 확인하였다(도 19).As a result, the exosomes isolated with Abs_MNWs contain various exosome proteins, and it was confirmed once again that the separated substances were exosomes (FIG. 19).
실시예 5.4. Abs_MNWs의 엑소좀 분리 효율 비교Example 5.4. Comparison of Abs_MNWs exosome separation efficiency
유방암 환자 및 폐암 환자의 혈장으로부터 Abs_MNWs, ExoQuick, Invitrogen 총 엑소좀 분리 키트, 및 엑소좀-인간 CD81 유동 검출 시약(10622D, Thermo Fisher Scientific, USA)을 사용하여 제조사의 매뉴얼에 따라 엑소좀을 분리하고 정제하였다.Isolate the exosomes according to the manufacturer's manual using Abs_MNWs, ExoQuick, Invitrogen total exosome separation kit, and exosome-human CD81 flow detection reagent (10622D, Thermo Fisher Scientific, USA) from plasma of breast cancer patients and lung cancer patients. Purified.
구체적으로, ExoQuick(EXOQ5TM-1, System Biosciences, Palo Alto, CA, USA), Invitrogen 총 엑소좀 분리 키트(4484451, Thermo Fisher Scientific, Massachusetts, Waltham, USA), 및 엑소좀-인간 CD81 유동 검출 시약(10622D, Thermo Fisher Scientific, USA)을 사용하여 제조사의 매뉴얼에 따라 엑소좀을 분리하고 정제하였다. 정제한 엑소좀의 정량은 실시예 4.2와 동일한 방법으로 BCA 분석 키트(Thermo Scientific, Waltham, MA, USA)를 사용하여 단백질 농도를 결정하였다. 재현성을 향상시키기 위해, 모든 분석을 동일한 실험 조건 하에 수행하였다.Specifically, ExoQuick (EXOQ5TM-1, System Biosciences, Palo Alto, CA, USA), Invitrogen total exosome separation kit (4484451, Thermo Fisher Scientific, Massachusetts, Waltham, USA), and exosome-human CD81 flow detection reagent ( 10622D, Thermo Fisher Scientific, USA) to isolate and purify the exosomes according to the manufacturer's manual. Quantification of purified exosomes was performed using the BCA assay kit (Thermo Scientific, Waltham, MA, USA) in the same manner as in Example 4.2 to determine protein concentration. To improve reproducibility, all analyzes were performed under the same experimental conditions.
그 결과, 도 20에 나타난 바와 같이, Abs_MNWs를 이용한 경우, 암 환자의 혈장으로부터 높은 수율 및 순도로 엑소좀을 분리하였으며, 평균 NTA 값은 6.3±0.15×109 입자/㎖ 이었다. 한편, Exoquick 및 Invitrogen 키트를 이용한 경우, 암 환자의 혈장으로부터 분리된 엑소좀의 평균 NTA 값은 각각 2.4±0.12×109 입자/㎖ 및 1.73±0.26×109 입자/㎖이었다. 이때, NTA 분석을 실시예 3.2와 동일한 방법으로 수행하였다. 즉, Abs_MNWs는 종래 2가지 방법에 비해 약 3배 더 높은 수율로 엑소좀을 분리하였다.As a result, as shown in FIG. 20, when Abs_MNWs was used, exosomes were separated from plasma of cancer patients with high yield and purity, and the average NTA value was 6.3±0.15×10 9 particles/ml. On the other hand, when using the Exoquick and Invitrogen kits, the mean NTA values of exosomes isolated from the plasma of cancer patients were 2.4±0.12×10 9 particles/ml and 1.73±0.26×10 9 particles/ml, respectively. At this time, NTA analysis was performed in the same manner as in Example 3.2. That is, Abs_MNWs isolated exosomes in a yield approximately 3 times higher than the two conventional methods.
또한, Abs_MNWs에 의해 분리된 대부분의 엑소좀의 크기 분포를 나노입자추적분석기 (nanoparticle tracking analysis)를 통하여 분석하였다. 그 결과, Abs_MNWs에 의해 분리된 대부분의 엑소좀의 크기 분포는 균일하고 40 nm 내지 150 nm 범위였다(도 21).In addition, the size distribution of most of the exosomes separated by Abs_MNWs was analyzed through a nanoparticle tracking analysis. As a result, the size distribution of most of the exosomes separated by Abs_MNWs was uniform and ranged from 40 nm to 150 nm (FIG. 21).
나아가, 분리된 엑소좀을 용출(lysis)한 후, 웨스턴 블랏을 통해 엑소좀 단백질을 분석하였다. 이때, 항-CD63 항체, 항-CD81 항체, 항-TSG101 항체 및 HSP70 항체를 사용하였다. 그 결과, 종래 2가지 방법에 비해 Abs_MNWs를 사용한 경우, 더 높은 수준의 엑소좀 단백질이 확인되었다(도 22).Furthermore, after lysis of the separated exosomes, exosome proteins were analyzed through Western blot. At this time, anti-CD63 antibody, anti-CD81 antibody, anti-TSG101 antibody and HSP70 antibody were used. As a result, when Abs_MNWs was used compared to the two conventional methods, a higher level of exosome protein was confirmed (FIG. 22 ).
상기 실시예 1 내지 실시예 5.4를 통해, Abs_MNWs를 사용하여 소량의 시료로부터 간단하고, 빠르며, 높은 민감도를 갖는 엑소좀을 분리하는 방법을 설명하였다. Abs_MNWs에 의한 엑소좀 분리에 필요한 절차, 처리 시간, 비용 및 최소 시료량을 도 23에 나타내었다.Through Examples 1 to 5.4, a method of separating exosomes having a simple, fast, and high sensitivity from a small amount of samples using Abs_MNWs was described. The procedure, processing time, cost and minimum sample amount required for exosome separation by Abs_MNWs are shown in FIG. 23.

Claims (21)

  1. 항체가 결합된 엑소좀 분리용 나노와이어로서,As an antibody-coupled nanowire for exosome separation,
    상기 나노와이어는 전도성 고분자로 구성된 것인, 엑소좀 분리용 나노와이어.The nanowire is composed of a conductive polymer, nanowire for exosome separation.
  2. 제1항에 있어서,According to claim 1,
    상기 항체는 항-CD9 항체, 항-CD24 항체, 항-CD41 항체, 항-CD44 항체, 항-CD63 항체, 항-CD81 항체, 항-CD82 항체, 항-Flotillin 항체, 항-Caveolin-1항체, 항-Rab5 항체, 항-TSG101 항체, 항-Alix 항체, 항-CXCR4 항체, 항-FLOT-1 항체, 항-TM9SF4 항체, 항-TM9SF3 항체, 항-HSPA8 항체, 항-HSC70 항체, 항-TSTA3 항체, 항-Thr-181 항체, 항-HSP70 항체 및 이들의 조합으로 이루어진 군으로부터 선택되는 어느 하나인 것인, 엑소좀 분리용 나노와이어.The antibody is an anti-CD9 antibody, anti-CD24 antibody, anti-CD41 antibody, anti-CD44 antibody, anti-CD63 antibody, anti-CD81 antibody, anti-CD82 antibody, anti-Flotillin antibody, anti-Caveolin-1 antibody, Anti-Rab5 antibody, anti-TSG101 antibody, anti-Alix antibody, anti-CXCR4 antibody, anti-FLOT-1 antibody, anti-TM9SF4 antibody, anti-TM9SF3 antibody, anti-HSPA8 antibody, anti-HSC70 antibody, anti-TSTA3 Nanowire for exosome separation, which is any one selected from the group consisting of antibodies, anti-Thr-181 antibodies, anti-HSP70 antibodies and combinations thereof.
  3. 제1항에 있어서,According to claim 1,
    상기 나노와이어는 2개 이상의 항체가 결합된 것인, 엑소좀 분리용 나노와이어.The nanowires are two or more antibodies are bound, nanowires for exosome separation.
  4. 제1항에 있어서,According to claim 1,
    상기 항체는 비오틴(biotin)이 결합된 것인, 엑소좀 분리용 나노와이어.The antibody is biotin (biotin) is bound, nanowires for exosome separation.
  5. 제1항에 있어서,According to claim 1,
    상기 전도성 고분자는 폴리아세틸렌(polyacetylene), 폴리피롤(polypyrrole), 폴리티오펜(polythiophene), 폴리피닷(PEDOT: poly(3,4-ethylenedioxythiophene), 폴리아닐린(polyaniline) 또는 이들의 유도체인 것인, 엑소좀 분리용 나노와이어.The conductive polymer is polyacetylene (polyacetylene), polypyrrole (polypyrrole), polythiophene (polythiophene), polypidot (PEDOT: poly(3,4-ethylenedioxythiophene), polyaniline (polyaniline) or a derivative thereof, which is an exosome Separation nanowires.
  6. 제1항에 있어서,According to claim 1,
    상기 나노와이어는 ss-비오틴이 결합된 것인, 엑소좀 분리용 나노와이어.The nanowire is ss-biotin is bound, nanowires for exosome separation.
  7. 제1항에 있어서,According to claim 1,
    상기 항체와 나노와이어는 스트렙타비딘(straptavidin), 트랩타비딘(traptavidin) 또는 뉴트라비딘(neutravidin)을 통해 결합된 것인, 엑소좀 분리용 나노와이어.The antibody and the nanowires are streptavidin (straptavidin), traptavidin (traptavidin) or neutravidin (neutravidin) is a nanowire for exosome separation.
  8. 제1항에 있어서,According to claim 1,
    상기 엑소좀은 순환 엑소좀(circulating exosome)인 것을 특징으로 하는, 엑소좀 분리용 나노와이어.The exosome is characterized in that the circulating exosome (circulating exosome), nanowires for exosome separation.
  9. 시료로부터 순환 엑소좀을 분리하기 위한 방법으로서,As a method for isolating circulating exosomes from a sample,
    상기 방법은The above method
    (a) 제1항의 나노와이어와 시료를 혼합하는 단계;(a) mixing the nanowire of claim 1 with a sample;
    (b) 상기 나노와이어를 분리하는 단계;(b) separating the nanowires;
    (c) 상기 분리한 나노와이어에 환원제(reducing agent)를 처리하는 단계; 및 (c) treating the separated nanowires with a reducing agent; And
    (d) 엑소좀을 수득하는 단계를 포함하는 순환 엑소좀 분리방법.(d) Circulating exosome separation method comprising the step of obtaining an exosome.
  10. 제9항에 있어서,The method of claim 9,
    상기 시료는 뇌척수액, 혈장, 혈액, 흉수, 복수 및 체액으로 이루어진 군에서 선택되는 어느 하나인 것인, 순환 엑소좀 분리방법.The sample is any one selected from the group consisting of cerebrospinal fluid, plasma, blood, pleural fluid, ascites and body fluids, circulating exosome separation method.
  11. 제9항에 있어서,The method of claim 9,
    상기 환원제는 DTT(Dithiothreitol), 글루타치온 또는 TCEP(tris(2-carboxyethyl)phosphine)인 것인, 순환 엑소좀 분리방법.The reducing agent is DTT (Dithiothreitol), glutathione or TCEP (tris(2-carboxyethyl)phosphine), circulating exosome separation method.
  12. 제9항에 있어서,The method of claim 9,
    상기 시료는 10분 동안 3,000×g에서 원심분리 처리한 것인, 순환 엑소좀 분리방법.The sample is centrifuged at 3,000 × g for 10 minutes, circulating exosome separation method.
  13. 제9항에 있어서, The method of claim 9,
    상기 순환 엑소좀은 직경이 40 nm 내지 150 nm인 것인, 순환 엑소좀 분리방법. The circulating exosomes will have a diameter of 40 nm to 150 nm, circulating exosome separation method.
  14. 제9항에 있어서,The method of claim 9,
    상기 순환 엑소좀은 항-CD9 항체, 항-CD24 항체, 항-CD41 항체, 항-CD44 항체, 항-CD63 항체, 항-CD81 항체, 항-CD82 항체, 항-Flotillin 항체, 항-Caveolin-1항체, 항-Rab5 항체, 항-TSG101 항체, 항-Alix 항체, 항-CXCR4 항체, 항-FLOT-1 항체, 항-TM9SF4 항체, 항-TM9SF3 항체, 항-HSPA8 항체, 항-HSC70 항체, 항-TSTA3 항체, 항-Thr-181 항체, 항-HSP70 항체 및 이들의 조합으로 이루어진 군으로부터 선택되는 어느 하나의 항체와 결합하는 것인, 순환 엑소좀 분리 방법.The circulating exosomes are anti-CD9 antibody, anti-CD24 antibody, anti-CD41 antibody, anti-CD44 antibody, anti-CD63 antibody, anti-CD81 antibody, anti-CD82 antibody, anti-Flotillin antibody, anti-Caveolin-1 Antibody, anti-Rab5 antibody, anti-TSG101 antibody, anti-Alix antibody, anti-CXCR4 antibody, anti-FLOT-1 antibody, anti-TM9SF4 antibody, anti-TM9SF3 antibody, anti-HSPA8 antibody, anti-HSC70 antibody, anti -TSTA3 antibody, anti-Thr-181 antibody, anti-HSP70 antibody and any one selected from the group consisting of a combination of antibodies, the method of circulating exosome separation.
  15. 암 질환의 발병이 의심되는 개체로부터 분리된 시료에서 순환 엑소좀을 검출함으로써, 암을 진단 또는 예후를 예측하기 위한 정보를 제공하기 위한 방법으로서, 상기 방법은A method for providing information for diagnosing cancer or predicting prognosis by detecting circulating exosomes in a sample isolated from a subject suspected of developing a cancer disease, the method comprising
    (a) 제1항의 나노와이어와 시료를 혼합하는 단계;(a) mixing the nanowire of claim 1 with a sample;
    (b) 상기 나노와이어를 분리하는 단계;(b) separating the nanowires;
    (c) 상기 분리한 나노와이어에 표지자를 처리하는 단계;(c) processing a marker on the separated nanowire;
    (d) 결합하지 않은 표지자를 제거하는 단계; 및 (d) removing unbound markers; And
    (e) 상기 표지자를 검출하는 단계를 포함하는, 암을 진단 또는 예후를 예측하기 위한 정보 제공 방법.(e) detecting the marker, the method of providing information for predicting a cancer diagnosis or prognosis.
  16. 제15항에 있어서,The method of claim 15,
    상기 정보 제공 방법은 (f) 상기 개체의 순환 엑소좀 검출량과 정상 대조군의 순환 엑소좀 검출량을 비교하는 단계; 및 (g) 상기 개체의 순환 엑소좀 검출량이 정상 대조군의 순환 엑소좀 검출량에 비해 높은 경우, 암 질환이 발병 되었다고 판정하는 단계를 추가적으로 더 포함하는 것인, 암을 진단 또는 예후를 예측하기 위한 정보 제공 방법.The method for providing information may include (f) comparing the detected amount of circulating exosomes of the individual with the detected amount of circulating exosomes of a normal control group; And (g) when the circulating exosome detection amount of the individual is higher than the circulating exosome detection amount of the normal control group, further comprising determining that a cancer disease has occurred, information for diagnosing cancer or predicting prognosis. How to provide.
  17. 제16항에 있어서,The method of claim 16,
    상기 (g) 단계에서 상기 개체의 순환 엑소좀 검출량이 정상 대조군의 순환 엑소좀 검출량에 비해 2배 이상 높은 경우 암 질환이 발병 되었다고 판정하는 것을 특징으로 하는, 암을 진단 또는 예후를 예측하기 위한 정보 제공 방법.In the step (g), when the detection amount of the circulating exosomes of the individual is more than 2 times higher than the detection amount of the circulating exosomes of the normal control group, it is determined that a cancer disease has occurred, information for predicting a cancer diagnosis or prognosis How to provide.
  18. 제15항에 있어서,The method of claim 15,
    상기 시료는 뇌척수액, 혈장, 혈액, 흉수, 복수 및 체액으로 이루어진 군에서 선택되는 어느 하나인 것인, 암을 진단 또는 예후를 예측하기 위한 정보 제공 방법.The sample is any one selected from the group consisting of cerebrospinal fluid, plasma, blood, pleural fluid, ascites, and body fluids.
  19. 제15항에 있어서,The method of claim 15,
    상기 시료는 10분 동안 3,000×g에서 원심분리한 것인, 암을 진단 또는 예후를 예측하기 위한 정보 제공 방법.The sample is centrifuged at 3,000 × g for 10 minutes, a method for providing information for diagnosing cancer or predicting prognosis.
  20. 제15항에 있어서,The method of claim 15,
    상기 순환 엑소좀은 직경이 40 nm 내지 150 nm인 것인, 암을 진단 또는 예후를 예측하기 위한 정보 제공 방법.The circulating exosomes having a diameter of 40 nm to 150 nm, a method for providing information for diagnosing cancer or predicting prognosis.
  21. 제15항에 있어서,The method of claim 15,
    상기 표지자는 순환 엑소좀을 검출할 수 있는 제제인 것인, 암을 진단 또는 예후를 예측하기 위한 정보 제공 방법.The marker is an agent capable of detecting circulating exosomes, a method for providing information for diagnosing cancer or predicting prognosis.
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