[go: up one dir, main page]

WO2019035623A2 - Cancer diagnosis method using lectin-coupled nanoparticles - Google Patents

Cancer diagnosis method using lectin-coupled nanoparticles Download PDF

Info

Publication number
WO2019035623A2
WO2019035623A2 PCT/KR2018/009290 KR2018009290W WO2019035623A2 WO 2019035623 A2 WO2019035623 A2 WO 2019035623A2 KR 2018009290 W KR2018009290 W KR 2018009290W WO 2019035623 A2 WO2019035623 A2 WO 2019035623A2
Authority
WO
WIPO (PCT)
Prior art keywords
nanoparticles
lectin
cancer
janus
exosome
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KR2018/009290
Other languages
French (fr)
Korean (ko)
Other versions
WO2019035623A3 (en
Inventor
최종훈
구형준
김교범
최용현
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Industry Academic Cooperation Foundation of Chung Ang University
Original Assignee
Industry Academic Cooperation Foundation of Chung Ang University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Industry Academic Cooperation Foundation of Chung Ang University filed Critical Industry Academic Cooperation Foundation of Chung Ang University
Publication of WO2019035623A2 publication Critical patent/WO2019035623A2/en
Publication of WO2019035623A3 publication Critical patent/WO2019035623A3/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • 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
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated
    • 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
    • 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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
    • G01N2333/42—Lectins, e.g. concanavalin, phytohaemagglutinin

Definitions

  • the present invention relates to a method of diagnosing cancer by detecting exosomes using lectin-bound nanoparticles.
  • Pancreatic cancer is the 14th most common cancer in the world, with a 5-year survival rate of less than 5% and a very poor prognosis with more than half of the patients diagnosed within 6 months, making it difficult to diagnose and to have the lowest survival rate have.
  • the high mortality rate of pancreatic cancer patients is due to the fact that the diagnosis of pancreatic carcinoma is difficult due to lack of subjective symptoms and it is not easily observed by ultrasonography or CT.
  • pancreatic cancer is most resistant to conventional chemotherapeutic treatment, and it is difficult to remove it through surgical operation, so treatment is not easy unless diagnosis is made at an early stage.
  • Exosome is a small form of membrane vesicle that is secreted by most cells. It is known that exosome contains various kinds of proteins, genetic material (DNA, mRNA, miRNA) and lipids derived from cells.
  • the present inventors completed the present invention by confirming that the exosome derived from pancreatic cancer can be detected using a janus nanoparticle having lectin bound thereto and one surface of the particle surface coated with a metal.
  • It is an object of the present invention to provide a cancer diagnostic composition comprising nanoparticles to which exosome binding molecules are linked, wherein the nanoparticles are coated with a metal at a part of the particle surface.
  • Another object of the present invention is to provide a method for testing a biological sample, comprising: contacting a biological sample separated from a subject to be tested with the composition of claim 1; And identifying the exosomes bound to the nanoparticles.
  • Another object of the present invention is to provide a plasma display panel comprising a lower substrate on which electrodes are arranged; An upper substrate connected to the lower substrate to form a fluid channel through which fluid can flow; And an external power source, wherein the electrode is arranged to be orthogonal to a flow direction of the fluid moving through the channel, and the fluid channel includes nanoparticles to which exosome binding molecules are connected And a device for detecting and separating exosome.
  • One aspect of the present invention provides a composition for diagnosing cancer comprising nanoparticles to which exosome binding molecules are linked, wherein the nanoparticles are coated with a metal at a portion of the particle surface.
  • the cancer may be selected from the group consisting of breast cancer, thyroid cancer, stomach cancer, pancreatic cancer, and bile duct cancer.
  • the exosome binding molecule may be selected from the group consisting of a protein, a ligand, an aptamer, and a microRNA.
  • the protein may be lectin.
  • the nanoparticles may be polystyrene or polymethylmethacrylate.
  • the metal is selected from the group consisting of gold (Au), silver (Ag), platinum (Pt), copper (Cu), nickel (Ni), zinc (Zn) It can be selected.
  • Another aspect of the present invention provides a method for preparing a biological sample, comprising the steps of: contacting a biological sample separated from a subject to be examined with the composition of claim 1; And identifying the exosomes bound to the nanoparticles.
  • the biological sample may be selected from the group consisting of blood, serum, plasma, saliva, sputum, and urine.
  • Yet another aspect of the present invention provides a plasma display panel comprising: a lower substrate on which electrodes are disposed; An upper substrate connected to the lower substrate to form a fluid channel through which fluid can flow; And an external power source, wherein the electrode is arranged to be orthogonal to a flow direction of the fluid moving through the channel, and the fluid channel includes nanoparticles to which exosome binding molecules are connected A device for detecting and separating exosome is provided.
  • the nanoparticles may have a portion of the particle surface coated with a metal.
  • the lectin-bound nanoparticles according to an embodiment of the present invention can effectively detect and isolate exosomes secreted by cancer cells, and thus can be usefully used for cancer diagnosis.
  • FIG. 1 is a view showing a state in which a lectin and a lectin-specific glycoprotein are bound.
  • FIG. 2 is a view showing a process of separating sugar chain-specific lectin by lyophilizing mushrooms.
  • FIG. 3 is a view showing a process for producing a polystyrene nanoparticle monolayer and a process for producing a nanoparticle by forming a gold thin film on one side of the nanoparticle.
  • FIG. 4 is a schematic view of the process of synthesizing Janus nanoparticles.
  • 6 is a TEM photograph of polystyrene nanoparticles of 500 nm.
  • FIG. 7 is a TEM photograph of a gold / chromium-deposited polystyrene particle, Janus particle.
  • FIG. 8 is a schematic view showing a process of binding lectin to Janus nanoparticles.
  • FIG. 9 is a graph showing FTIR spectra of carboxyl group-bonded polystyrene nanoparticles, gold / chromium-deposited Janus nanoparticles, and lectin-bound Janus nanoparticles.
  • FIG. 10 is a graph showing the size distribution and zeta potential of carboxyl group-bonded polystyrene nanoparticles, gold / chromium-deposited Janus nanoparticles, and lectin-bound Janus nanoparticles.
  • FIG. 11 is a schematic view of a JANUS nanoparticle coupled with a TEM image and a lectin.
  • 12 is a graph showing the degree of binding of SNA lectin to Janus nanoparticles according to concentration.
  • FIG. 13 is a graph showing the degree of binding of Con A lectin to Janus nanoparticles according to concentration.
  • FIG. 14 is a graph showing the degree of binding of AAL lectin to Janus nanoparticles according to concentration.
  • CA 19-9 which is a pancreatic cancer cell marker antibody, to Janus nanoparticles according to concentration.
  • 16 is a graph showing the relative fluorescence intensities of the Janus nanoparticles coupled with the SNA lectin.
  • Fig. 17 is a graph showing the relative fluorescence intensities for the Con A lectin-bound Janus nanoparticles.
  • Fig. 18 is a graph showing the relative fluorescence intensities of the Janus nanoparticles bound with AAL lectin.
  • FIG. 19 is a graph showing the relative fluorescence intensity against pancreatic cancer cell marker antibody CA 19-9.
  • FIG. 20 is a confocal microscope photograph showing the binding affinity of lanthanum bound with Janus nanoparticles.
  • 21 is a graph showing the binding affinity between SNA lectin bound to Janus nanoparticles and each cell.
  • 22 is a graph showing the binding affinity between Con A lectin bound to Janus nanoparticles and each cell.
  • FIG. 23 is a graph showing the binding affinity of AAL lectin bound with Janus nanoparticles to each cell.
  • CA19-9 is a pancreatic cancer cell marker antibody bound to Janus nanoparticles, and each cell.
  • 25 is a graph showing binding affinity between Janus nanoparticles and each cell.
  • Fig. 26 is a confocal z-stack three-dimensional reconstruction image in PANC-1 treated with Janus nanoparticles conjugated with SNA lectin.
  • 27 is a confocal z-stack three-dimensional reconstruction image in PANC-1 treated with Con A lectin-conjugated Janus nanoparticles.
  • 29 is a graph showing cell viability analysis results for Con A lectin through MTT analysis.
  • FIG. 30 is a graph showing cell viability analysis results for AAL lectin through MTT analysis.
  • FIG. 31 is a graph showing the results of cell viability analysis of CA 19-9, which is an antibody against pancreatic cancer cell markers, by MTT assay.
  • FIG. 32 is an exemplary diagram showing the binding of exosomes with lanthanum-conjugated janus nanoparticles.
  • 33 is a schematic view showing a method of separating exosomes using a microfluid dielectrophoresis system including Janus nanoparticles.
  • FIG. 34 shows the operation principle and the simulation process of a microfluidic dielectrophoresis system including Janus nanoparticles.
  • 35 is a photograph showing pancreatic cancer exosomes isolated from Panc-1 cells.
  • Fig. 36 is a photograph showing exosomes captured in a Janus nanoparticle to which an SNA lectin protein is bound.
  • FIG. 37 is a photograph showing the expression of CD81, CD63 and EphA2 in exosome of Panc-1 cells by Western blot analysis.
  • FIG. 38 is a photograph showing the binding of exosomes derived from pancreatic cancer to Janus nanoparticles to which lectin is bound, Janus nanoparticles to which lectin is bound, and Janus-9, which is a pancreatic cancer cell marker antibody, CA 19-9.
  • 39 is a graph showing the exosome-detecting ability of Janus nanoparticles bound to each lectin.
  • One aspect of the present invention provides a composition for diagnosing cancer comprising nanoparticles to which exosome binding molecules are linked, wherein the nanoparticles are coated with a metal at a portion of the particle surface.
  • 'exosome' refers to a membrane-structured pancreas secreted from a variety of cells, which binds to other cells and tissues and functions in various roles, such as transferring membrane components, proteins, and RNA .
  • the cancer may be selected from the group consisting of breast cancer, thyroid cancer, stomach cancer, pancreatic cancer and bile duct cancer, but is not limited thereto.
  • the term " exosome binding molecule " includes biomolecules or other chemical compounds capable of specifically binding to the exosome to be detected.
  • the protein may be selected from the group consisting of aptamers, DNA, hapten, avidin, streptavidin, neutravidin, protein A, protein G and microRNA, lectin).
  • the lectin may be a lectin specific to a fucose sugar chain, and preferably a human lectin specific lectin.
  • the lectin can be isolated from mistletoe and basidiomycetes and is preferably isolated from Pholiota terrestris .
  • the nanoparticles may be made of polystyrene or polymethyl methacrylate, but the material thereof is not limited as long as the exosome binding molecule can be connected thereto.
  • the metal may be selected from the group consisting of gold (Au), silver (Ag), platinum (Pt), copper (Cu), nickel (Ni), zinc (Zn) It is preferable that it is gold.
  • the method of coating the metal on the surface of the nanoparticles may use masking, phase separation, and self assembly.
  • the cladding method uses a solid material such as wax to cover half of the particle surface and expose the other half, thereby modifying the exposed one surface to have properties different from those of the unexposed surface.
  • Phase separation is a method of making Janus particles through the mixing of two or more incompatible, incompatible components.
  • the self-assembly method is advantageous in that the surface properties of the janus particles can be controlled by using a variety of polymers to make janus particles using a block copolymer or an organic material and a polymer.
  • a nanoparticle in which a part of the surface of a particle is coated with a metal can be named as a Janus nanoparticle.
  • a god with two faces of Roman myth It refers to a structure in which two structures are included together in a particle.
  • Narrowly refers to a case where spherical particles are divided in half and each has a separate structure. In the present specification, however, the term " particle surface " .
  • Another aspect of the present invention provides a method for preparing a biological sample, comprising the steps of: contacting a biological sample separated from a subject to be examined with the composition of claim 1; And identifying the exosomes bound to the nanoparticles.
  • the biological sample may be selected from the group consisting of blood, serum, plasma, saliva, sputum, and urine, and is preferably blood, serum or plasma.
  • the method for diagnosing cancer may further include a step of analyzing DNA and protein contained in exosome by isolating exosome after identifying exosome bound to nanoparticles,
  • the type of cancer can be distinguished according to the type of exosome that is separated.
  • a lower substrate on which electrodes are disposed An upper substrate connected to the lower substrate to form a fluid channel through which fluid can flow; And an external power source,
  • the electrode is arranged to be orthogonal to a flow direction of the fluid moving through the channel, and the fluid channel includes nanoparticles to which exosome binding molecules are connected.
  • the nanoparticles are preferably those in which a part of the surface of the particles is coated with a metal, more preferably coated with gold.
  • positive / negative dielectrophoresis directions are determined according to the difference in relative dielectrophoretic force applied to the nanoparticles, and the direction of the positive / negative dielectrophoresis is determined.
  • the difference in the dielectric permittivity that is, the dielectric constant, is compared with that of the single nanoparticle, so that the exosome-bound nanoparticles can be separated by confirming the difference.
  • Pholiota terrestris and mistletoe were lyophilized to prepare mushroom powder, and extracts were obtained from the mushroom powder.
  • the extract was centrifuged to collect only supernatant, and the supernatant was filtered with gauze.
  • the gauze filtration process was repeated several times to obtain filtered mushroom extract (hereinafter referred to as 'filtered mushroom extract').
  • the haptoglobin was fixed to the sepharose column according to the manufacturer's protocol, and the obtained filter mushroom extract was contacted. After contacting, the lectin binding to the human togoglobin was eluted from the column, and the optimal lectin was selected by analyzing the lectin binding constant and carbohydrate-binding specificity.
  • other plant-derived lectins were purchased and purchased from the market as shown in Table 1 below.
  • Lectin origin Commercial Information SNA Sambucus Nigra Lectin Cherry tree L6890, Sigma Aldrich Cone Concanavaline Soybean 61760, Sigma Aldrich AAL Aleuria Aurantia Lectin Sole mushroom L-1390, Vector laboratory
  • Fig. 1 shows the binding of lectin to a lectin-specific glycoprotein.
  • FIG. 2 shows the process of preparing an extract by lyophilizing mushrooms and separating lectin from the extract.
  • a monolayer of polystyrene particles was prepared using a convective colloid assembly method and an air-water interface self-assembly method. Specifically, a high-density polystyrene particle monolayer was prepared on a glass substrate or a silicon wafer by immersing a glass substrate or a silicon wafer in a suspension in which polystyrene particles having a size of 5 to 100 nm were dispersed, and the solution components were evaporated.
  • Polystyrene particles (hereinafter, referred to as 'Janus nanoparticles') coated with a half of the surface of the particles by depositing an anisotropic metal (chromium to 20 nm and gold to several tens nm) on the monolayer of polystyrene particles, ) was prepared.
  • anisotropic metal chromium to 20 nm and gold to several tens nm
  • gold deposition was performed before the gold deposition.
  • a thermal or e-beam evaporator was used for the deposition process. Janus nanoparticles formed on a glass substrate or a silicon wafer were redispersed in a water / ethanol mixed solvent through spraying and sonication, and a small amount of surfactant was added to prevent agglomeration.
  • FIG. 3 is a view showing a process of manufacturing a Janus nanoparticle in which gold is deposited on a monolayer of polystyrene nanoparticles to form a gold thin film on only one side of the particle.
  • FIG. 4 is a schematic view of the process of synthesizing Janus nanoparticles.
  • TEM transmission electron microscopy
  • the Janus nanoparticles which were confirmed through TEM analysis, showed a dark color in the region where the electron density was increased due to the metal deposition and a relatively soft color in the polymer portion which was not deposited. .
  • 6 is a TEM photograph of polystyrene nanoparticles of 500 nm.
  • FIG. 7 is a TEM photograph of a gold / chromium-deposited polystyrene particle, Janus particle.
  • the Janus nanoparticles prepared in the above 2-1 were treated with an amine to link primary amines to the surface of the particles.
  • N-Succinimidyl 3- (2-pyridyldithio) propionate hereinafter referred to as SPDP
  • DTT dithiothreitol
  • SPDP contains an amine-reactive NHS ester ( N- Hydroxysuccinimide ester) and a thiol-reactive pyridyl disulfide group.
  • the Janus nanoparticles to which primary amine is attached are activated by reaction with SPDP to form a thiol reactive derivative . Since lectins partially contain thiols, they can be combined with activated Janus nanoparticles to produce lanthanum-bound Janus nanoparticles.
  • sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate was added to activated Janus nanoparticles by reacting with SPDP to induce the reaction. Since lectins are rich in amine groups, they can induce the binding of lectins to Janus nanoparticles.
  • Figure 8 shows the process of activating Janus nanoparticles and binding them to lectins.
  • FTIR measurements were made on ALPHA-T (Bruker optics) equipment using pulverized samples and measured in the 300-4000 cm -1 wavenumber area.
  • DLS and ELS were measured using a Malvern Zetasizer nano ZS90 instrument.
  • the polystyrene was characteristic peak (peak) observed at 700cm -1, 755cm -1 wave number through FTIR analysis, the characteristics of the polystyrene particle carboxyl groups was also observed at 1600cm -1.
  • CNH complex peaks and NH bond peaks due to protein binding were observed at 1653 cm -1 and 3310 cm -1 , respectively.
  • the size of particles determined by DLS analysis varied depending on the metal deposition and protein binding. The size was observed from 500 nm to 633 nm, and the zeta potential from the ELS analysis also changed with the treatment of the particles.
  • FIG. 9 is a graph showing FTIR spectra of carboxyl group-bonded polystyrene nanoparticles, gold / chromium-deposited Janus nanoparticles, and lectin-bound Janus nanoparticles.
  • FIG. 10 is a graph showing the size distribution and zeta potential of carboxyl group-bonded polystyrene nanoparticles, gold / chromium-deposited Janus nanoparticles, and lectin-bound Janus nanoparticles.
  • FIG. 11 is a schematic view of a JANUS nanoparticle coupled with a TEM image and a lectin.
  • Lectins bound to the polystyrene surface are identified by the Wolff-Kishner reaction between the hydrazide-functionalized gold nanoparticles and the carbohydrate aldehyde group of lectins.
  • BCA analysis was performed to confirm the degree of lectin binding to Janus nanoparticles. Specifically, quantitative analysis of BCA protein was carried out using commercially available BCA protein analysis kit (23225, thermo scientific), and analysis was performed according to the standard protocol of the present product.
  • each lectin showed a protein concentration of 250 ⁇ g / ml to 563 ⁇ g / ml. Through this, it was confirmed that lectin binds well to Janus nanoparticles.
  • 12 to 15 are graphs showing the degree of binding of SNA, Con A, AAL lectin, and CA 19-9, which is a pancreatic cancer cell marker antibody, to Janus nanoparticles according to their concentrations.
  • the affinity of lanthanum - bound Janus nanoparticles was analyzed by cell fluorescence analysis of Janus nanoparticles.
  • the affinity of lectins for each cell line was evaluated by binding each lectin (SNA, Con A, AAL) or antibody (CA 19-9) selected in Example 1 to the Janus nano-particles, The relative fluorescence intensities of the nanoparticles were measured according to time after cell line treatment. Fluorescence analysis was performed using a Synergy H1 (BioTeK) instrument and lysine-bound janus particles were treated and compared to cell samples cultured on 96-well plates. Measurement conditions are as follows: Excitation: 485, Emission: 528, Optics: Top, Gain: 125, ead Speed: Normal, Delay: 100 msec, Measurements / Data Point: 10, Read Height: 5 mm.
  • SNA lectin showed a specific affinity for pancreatic cancer cell line (PANC-1) and the affinity of the remaining lectin group for cancer cell line was weak or not.
  • CA19-9 also showed affinity to PANC-1, but showed affinity for SNA lectin rather than affinity for pancreatic cancer cell line, confirming excellent pancreatic cancer cell line affinity of SNA lectin.
  • FIGS. 16 to 19 are graphs showing relative fluorescence intensities relative to Janus nanoparticles bound with SNA, Con A, AAL lectin or CA19-9 antibody, respectively.
  • the values at each time (2h, 6h, 12h, 24h) are normalized to the fluorescence values at 24 hours and Panc-1 in FIG.
  • Confocal microscopy analysis was performed to confirm the fluorescence properties of lanthanum-bound Janus nanoparticles.
  • Cells cultured for 24 hours were treated with a certain concentration of the sample. After 24 hours of sample immobilization, confocal microscopy analysis was carried out. Cells were treated with lanthanum-bound Janus nanoparticles for 24 hours.
  • the blue fluorescence is from DAPI (ex: 358 nm, em: 461 nm) and the red fluorescence is from polystyrene of Janus nanoparticles (ex: 520 nm, em: 540 nm).
  • cell analysis was performed using a confocal microscope LSM710 (Carl Zeiss, Germany), and a 405 nm laser for DAPI staining and a 514 nm laser for Janus particle observation were used.
  • Cell samples for confocal microscopy analysis were cultured in an incubator for one day and then treated with 5 ⁇ g / ml of lanthanum-bound Janus nanoparticles on a protein concentration basis. After 24 hours of particle treatment, 2% paraformaldehyde conditions The immobilization process was performed together with the washing process.
  • SNA lectin showed a specific affinity for pancreatic cancer cell line, and fluorescence of janus particles was well observed around the cells, but the remaining lectin group showed weak affinity for cancer cell lines, or showed no affinity.
  • CA19-9 also showed affinity to PANC-1, and fluorescence of the particles was observed.
  • SNA lectin showed weak affinity to pancreatic cancer cell line, indicating that SNA lectin had excellent affinity for pancreatic cancer cell line Respectively.
  • FIG. 20 is a confocal microscope photograph showing the binding affinity of lanthanum bound with Janus nanoparticles.
  • an image to be analyzed for fluorescence intensity was loaded through a program, an analysis area was set using an area selection tool in the program, and an entire image was selected.
  • an entire image was selected.
  • FIGS. 21 to 25 are graphs showing binding affinities of each lectin, CA19-9 or lectin and antibody bound to Janus nanoparticles when they are not bound to each cell.
  • FIG. 21 is graphs showing binding affinities of each lectin, CA19-9 or lectin and antibody bound to Janus nanoparticles when they are not bound to each cell.
  • Figures 26 and 27 are confocal z-stack three-dimensional reconstructed images in PANC-1 treated with Janus nanoparticles conjugated with SNA or Con A lectin.
  • Exosomes were isolated to confirm the ability of the lectins selected in Example 1 to detect exosome.
  • human pancreatic ductal carcinoma cell line Panc-1
  • human cervical cancer cell HeLa
  • human breast adenocarcinoma MDA-MB-231
  • human skin fibroblast dermal fibroblast HDFB
  • human embryonic kidney cell HEK293T
  • the toxicity of lanthanum-bound Janus nanoparticles to the cell line of Example 4-1 was evaluated through MTT analysis. Toxicity was evaluated at a protein concentration of 50 ⁇ g / ml to 2.5 ⁇ g / ml and after 24 hours of treatment with lanthanum or antibody-conjugated Janus nanoparticles, the viability of the cell line was checked for toxicity.
  • MTT assay was performed by preparing 2 mg / ml of MTT reagent powder from AMRESCO Inc., culturing the cells on a 96-well plate for each concentration, culturing on a 96-well plate for one day, culturing on an incubator for one more day, Lt; / RTI > The cytotoxicity of the MTT-treated sample was measured by measuring the absorbance in the wavelength region of 570 nm using the above-described fluorescence analyzer.
  • FIGS. 28 to 31 are graphs showing cell viability analysis results for each lectin or CA19-9 antibody through MTT analysis.
  • a mask with the desired electrode design was placed on a glass substrate or silicon wafer and chromium and gold were continuously deposited. Then, a chamber capable of holding the particle suspension on the electrode was fabricated to fabricate a non-fluidized dielectrophoretic device with no fluid flow.
  • an AC voltage is applied to a dielectrophoretic system
  • the direction of positive / negative dielectrophoresis is determined by the difference in relative dielectrophoretic forces applied to the particles and the medium.
  • Exopeomes can be separated by precisely analyzing the dielectrophoretic behavior according to the frequency of AC voltage, particle size and dielectric properties of media, and applying it to particle separation.
  • exosome-specific marker CD63
  • ELISA and western analysis of exosome-specific marker (CD63) for exosome-bound Janus nanoparticles separated by each dielectrophoretic system can be used to identify pancreatic cancer-specific exosomes.
  • the DNA present in the separated exosome can be analyzed by PCR to confirm a K-Ras point mutation specifically expressed in pancreatic cancer cells.
  • FIG. 32 is an illustration showing an example of binding of lectin-binding Janus nanoparticles to exosome in which the lectin-specific glycoprotein is expressed.
  • 33 is a view showing a method of separating exosomes using a microfluid dielectrophoresis system including Janus nanoparticles.
  • Figure 34 shows the operating principle and simulation process of a microfluidic dielectrophoresis system containing Janus nanoparticles.
  • the diophoretic behavior of the Janus particles was confirmed in a nonflow system without fluid flow, and the system was applied to a microfluid system to implement a continuous exosome detection system.
  • Parameters that can affect the diurnal flow of Janus nanoparticles include AC frequency and voltage magnitude, electrode design, particle size, electrolyte media type, and gold film coverage in Janus particles.
  • the middle panel shows the electrode pattern to be applied to the microfluidic dielectrophoresis system and the right panel shows the simulation process to theoretically predict / analyze the results of the dielectrophoresis experiment.
  • exosomal markers and pancreatic cancer cell line markers were identified by western blotting analysis to determine whether or not exosomes of the pancreatic cancer cell line were discriminated.
  • the exosomes isolated from each cancer cell line and normal cells were lysed with RIPA buffer containing protease inhibitor cocktail & Phosphatase inhibitor cocktail (Invitrogen). From each sample, proteins were quantitated by BCA analysis (Invitrogen), and the same amount of normalized protein was electrophoretically graded through polyacrylamide gel. The separated proteins were then transferred to a nitrocellulose membrane (Merck Millipore), and protein blotting was blocked for 1 hour at room temperature with 5% Skim milk in TBST. Respectively.
  • the cells were then incubated with primary antibody (1: 1000 anti-CD63, 1: 000 anti-CD81 and 1: 1000 anti-EphA2 (Santa-Cruz)) overnight at 4 ° C.
  • the HRP-conjugated secondary antibody (Cell signaling) was then reacted for 1 hour at room temperature. Then, it was washed three times with TBST for 10 minutes, and blotting was confirmed using Chemidoc (biorad).
  • the exosomes extracted from each cell were subjected to centrifugation at 500 g for 10 minutes to obtain an supernatant. After centrifugation at 12,000 g for 20 minutes, supernatant was obtained. The exosomes were then extracted according to the manufacturer's protocol using Exoquick (System biosciences).
  • exosome-type micro vesicle As a result, an exosome-type micro vesicle was observed.
  • exosomal markers CD81, CD63
  • pancreatic cancer cell line marker EphA2
  • 35 is a photograph showing pancreatic cancer exosomes isolated from Panc-1 cells.
  • Fig. 36 is a photograph showing exosomes captured in a Janus nanoparticle to which an SNA lectin protein is bound.
  • FIG. 37 is a photograph showing the expression of CD81, CD63 and EphA2 in exosome of Panc-1 cells by Western blot analysis.
  • the number of particles was determined by measuring the residual exosomes in the supernatant after separation of the janus nanoparticles bound to the exosomes by centrifugation after the reaction between the exosomes and the janus particles.
  • the energy-filtering transmission electron microscope (LIBRA 120, Carl Zeiss) equipment The sample to be measured was prepared by loading on Cu gird.
  • 2% uranyl acetate was applied to each sample for negative staining.
  • the presence of particles or exosomes in the solvent was confirmed by nanoparticle tracking analysis and was performed using a Malvern Panalytical NS300 instrument.
  • pancreatic cancer cell line produces a fucosylated protein.
  • intracellular budding to the intracellular multivescular body (MVB) results in the formation of exosomes, .
  • FIG. 38 is a photograph showing the binding of exosomes derived from pancreatic cancer to Janus nanoparticles to which lectin is bound, Janus nanoparticles to which lectin is bound, and Janus-9, which is a pancreatic cancer cell marker antibody, CA 19-9.
  • 39 is a graph showing the exosome-detecting ability of Janus nanoparticles bound to each lectin.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Inorganic Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Nanotechnology (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

본 발명은 렉틴이 결합된 나노입자를 이용하여 엑소좀을 검출함으로써 암을 진단하는 방법에 관한 것으로, 상기 나노입자는 암세포가 분비하는 엑소좀을 효과적으로 검출 및 분리할 수 있으므로 암 진단 용도로 유용하게 사용될 수 있다.The present invention relates to a method for diagnosing cancer by detecting exosomes using lectin-bound nanoparticles. The nanoparticles are useful for cancer diagnosis because they can effectively detect and isolate exosomes secreted by cancer cells. Can be used.

Description

렉틴이 결합된 나노입자를 이용한 암 진단 방법Diagnostic method using lectin-bound nanoparticles

본 발명은 렉틴이 결합된 나노입자를 이용하여 엑소좀을 검출함으로써 암을 진단하는 방법에 관한 것이다.The present invention relates to a method of diagnosing cancer by detecting exosomes using lectin-bound nanoparticles.

췌장암은 세계적으로 14번째로 다발하는 암으로 5년 생존율이 5% 미만이고, 6개월 이내에 진단된 환자의 반 이상이 사망하는 매우 불량한 예후를 가지고 있어, 진단이 어렵고 가장 낮은 생존율을 나타내는 질환으로 알려져 있다. 췌장암 환자의 사망률이 높은 것은 췌장암의 경우 자각 증상이 없어 초기 진단이 어려울 뿐만 아니라, 초음파나 CT 촬영 등을 이용하여도 쉽게 관찰되지 않기 때문이다. 또한 췌장암은 기존의 화학요법적 치료에 대하여 강한 내성을 가지고 있는 경우가 대부분이고, 외과적인 수술을 통하여 제거하기가 쉽지 않아 초반에 진단이 되지 않으면 치료가 쉽지 않기 때문이다.Pancreatic cancer is the 14th most common cancer in the world, with a 5-year survival rate of less than 5% and a very poor prognosis with more than half of the patients diagnosed within 6 months, making it difficult to diagnose and to have the lowest survival rate have. The high mortality rate of pancreatic cancer patients is due to the fact that the diagnosis of pancreatic carcinoma is difficult due to lack of subjective symptoms and it is not easily observed by ultrasonography or CT. In addition, pancreatic cancer is most resistant to conventional chemotherapeutic treatment, and it is difficult to remove it through surgical operation, so treatment is not easy unless diagnosis is made at an early stage.

따라서 췌장암에 대한 표적 치료 및 진단을 위하여 분자적 특징을 규명하려는 연구가 시도되고 있으나, 암세포는 이형성(heterogeneity)으로 자라기 때문에 췌장암을 비롯한 암의 분자적 특징을 명확하게 규명하기는 어려운 실정이다. 췌장암의 조기 진단 필요성이 꾸준히 제기됨에 따라 최근에는 암 특이적인 바이오마커를 이용하여 췌장암을 진단하려는 연구가 계속되고 있다.Therefore, studies have been made to investigate molecular features for target treatment and diagnosis of pancreatic cancer. However, since cancer cells grow to heterogeneity, it is difficult to clearly identify the molecular characteristics of cancer including pancreatic cancer. As the need for early diagnosis of pancreatic cancer has been steadily raised, studies are currently underway to diagnose pancreatic cancer using cancer - specific biomarkers.

엑소좀은 대부분의 세포에서 분비되는 작은 형태의 소포체(membrane vesicle)로 엑소좀 안에는 세포에서 유래된 다양한 종류의 단백질, 유전물질(DNA, mRNA, miRNA), 지질 등이 포함되어 있는 것으로 알려져 있다.Exosome is a small form of membrane vesicle that is secreted by most cells. It is known that exosome contains various kinds of proteins, genetic material (DNA, mRNA, miRNA) and lipids derived from cells.

본 연구자들은 렉틴이 결합되어 있고, 입자 표면의 한쪽면이 금속으로 코팅된 야누스 나노입자(janus nanoparticle)를 이용하여 췌장암 유래 엑소좀을 검출할 수 있음을 확인하여 본 발명을 완성하였다.The present inventors completed the present invention by confirming that the exosome derived from pancreatic cancer can be detected using a janus nanoparticle having lectin bound thereto and one surface of the particle surface coated with a metal.

본 발명의 일 목적은 엑소좀 결합 분자가 연결된 나노입자를 포함하는 암 진단용 조성물로서, 상기 나노입자는 입자 표면의 일부분이 금속으로 코팅된 것인 암 진단용 조성물을 제공하는 것이다.It is an object of the present invention to provide a cancer diagnostic composition comprising nanoparticles to which exosome binding molecules are linked, wherein the nanoparticles are coated with a metal at a part of the particle surface.

본 발명의 다른 목적은 검사하고자 하는 대상체로부터 분리한 생물학적 시료와 제1항의 조성물을 접촉시키는 단계; 및 나노입자와 결합한 엑소좀을 확인하는 단계를 포함하는 암 진단 방법을 제공하는 것이다.Another object of the present invention is to provide a method for testing a biological sample, comprising: contacting a biological sample separated from a subject to be tested with the composition of claim 1; And identifying the exosomes bound to the nanoparticles.

본 발명의 다른 목적은 전극이 배치된 하부 기판; 상기 하부 기판과 연결되어 유체가 유동할 수 있는 유체 채널을 형성하는 상부 기판; 및 외부 전원을 포함하는 엑소좀 검출 및 분리 장치에 있어서, 상기 전극은 채널을 통하여 이동하는 유체의 흐름 방향과 직교하도록 배치되고, 상기 유체 채널은 엑소좀 결합 분자가 연결된 나노입자를 포함하는 것인 엑소좀 검출 및 분리 장치를 제공하는 것이다.Another object of the present invention is to provide a plasma display panel comprising a lower substrate on which electrodes are arranged; An upper substrate connected to the lower substrate to form a fluid channel through which fluid can flow; And an external power source, wherein the electrode is arranged to be orthogonal to a flow direction of the fluid moving through the channel, and the fluid channel includes nanoparticles to which exosome binding molecules are connected And a device for detecting and separating exosome.

본 발명의 일 양상은 엑소좀 결합 분자가 연결된 나노입자를 포함하는 암 진단용 조성물로서, 상기 나노입자는 입자 표면의 일부분이 금속으로 코팅된 것인 암 진단용 조성물을 제공한다.One aspect of the present invention provides a composition for diagnosing cancer comprising nanoparticles to which exosome binding molecules are linked, wherein the nanoparticles are coated with a metal at a portion of the particle surface.

본 발명의 일 구체예에 따르면, 상기 암은 유방암, 갑상선암, 위암, 췌장암 및 담도암으로 이루어진 군에서 선택되는 것일 수 있다.According to one embodiment of the present invention, the cancer may be selected from the group consisting of breast cancer, thyroid cancer, stomach cancer, pancreatic cancer, and bile duct cancer.

본 발명의 일 구체예에 따르면, 상기 엑소좀 결합 분자는 단백질, 리간드, 앱타머 및 마이크로 RNA로 이루어진 군에서 선택되는 것일 수 있다.According to one embodiment of the present invention, the exosome binding molecule may be selected from the group consisting of a protein, a ligand, an aptamer, and a microRNA.

본 발명의 일 구체예에 따르면, 상기 단백질은 렉틴일 수 있다.According to one embodiment of the present invention, the protein may be lectin.

본 발명의 일 구체예에 따르면, 상기 나노입자는 폴리스티렌 또는 폴리메틸메타크릴레이트일 수 있다.According to one embodiment of the present invention, the nanoparticles may be polystyrene or polymethylmethacrylate.

본 발명의 일 구체예에 따르면, 상기 금속은 금(Au), 은(Ag), 백금(Pt), 구리(Cu), 니켈(Ni), 아연(Zn) 및 크롬(Cr)으로 이루어진 군에서 선택되는 것일 수 있다.According to one embodiment of the present invention, the metal is selected from the group consisting of gold (Au), silver (Ag), platinum (Pt), copper (Cu), nickel (Ni), zinc (Zn) It can be selected.

본 발명의 다른 양상은 검사하고자 하는 대상체로부터 분리한 생물학적 시료와 제1항의 조성물을 접촉시키는 단계; 및 나노입자와 결합한 엑소좀을 확인하는 단계를 포함하는 암 진단 방법을 제공한다.Another aspect of the present invention provides a method for preparing a biological sample, comprising the steps of: contacting a biological sample separated from a subject to be examined with the composition of claim 1; And identifying the exosomes bound to the nanoparticles.

본 발명의 일 구체예에 따르면, 상기 생물학적 시료는 혈액, 혈청, 혈장, 타액, 객담, 및 뇨로 이루어진 군으로부터 선택되는 것일 수 있다.According to one embodiment of the present invention, the biological sample may be selected from the group consisting of blood, serum, plasma, saliva, sputum, and urine.

본 발명의 또 다른 양상은 전극이 배치된 하부 기판; 상기 하부 기판과 연결되어 유체가 유동할 수 있는 유체 채널을 형성하는 상부 기판; 및 외부 전원을 포함하는 엑소좀 검출 및 분리 장치에 있어서, 상기 전극은 채널을 통하여 이동하는 유체의 흐름 방향과 직교하도록 배치되고, 상기 유체 채널은 엑소좀 결합 분자가 연결된 나노입자를 포함하는 것인 엑소좀 검출 및 분리 장치를 제공한다.Yet another aspect of the present invention provides a plasma display panel comprising: a lower substrate on which electrodes are disposed; An upper substrate connected to the lower substrate to form a fluid channel through which fluid can flow; And an external power source, wherein the electrode is arranged to be orthogonal to a flow direction of the fluid moving through the channel, and the fluid channel includes nanoparticles to which exosome binding molecules are connected A device for detecting and separating exosome is provided.

본 발명의 일 구체예에 따르면, 상기 나노입자는 입자 표면의 일부분이 금속으로 코팅된 것일 수 있다.According to one embodiment of the present invention, the nanoparticles may have a portion of the particle surface coated with a metal.

본 발명의 일 실시예에 따른 렉틴이 결합된 나노입자는 암세포가 분비하는 엑소좀을 효과적으로 검출 및 분리할 수 있으므로 암 진단 용도로 유용하게 사용될 수 있다.The lectin-bound nanoparticles according to an embodiment of the present invention can effectively detect and isolate exosomes secreted by cancer cells, and thus can be usefully used for cancer diagnosis.

도 1은 렉틴과 렉틴 특이적 당단백질이 결합하는 모습을 보여주는 도이다.FIG. 1 is a view showing a state in which a lectin and a lectin-specific glycoprotein are bound.

도 2는 버섯류를 동결건조하여 당쇄 특이적 렉틴을 분리하는 과정을 보여주는 도이다.FIG. 2 is a view showing a process of separating sugar chain-specific lectin by lyophilizing mushrooms.

도 3은 폴리스티렌 나노입자 단층 제조과정 및 나노입자의 한쪽면에 금 박막을 형성시켜 야누스 나노입자를 제조하는 과정을 보여주는 도이다.FIG. 3 is a view showing a process for producing a polystyrene nanoparticle monolayer and a process for producing a nanoparticle by forming a gold thin film on one side of the nanoparticle.

도 4는 야누스 나노입자 합성 과정의 개략도이다.4 is a schematic view of the process of synthesizing Janus nanoparticles.

도 5는 실리콘 웨이퍼상에서 단층 정렬화된(monocrystallized) 폴리스티렌 나노입자의 SEM 이미지이다.5 is a SEM image of monocrystallized polystyrene nanoparticles on a silicon wafer.

도 6은 500nm의 폴리스티렌 나노 입자의 TEM 사진이다.6 is a TEM photograph of polystyrene nanoparticles of 500 nm.

도 7은 금/크롬 증착된 폴리스티렌 입자인 야누스 입자의 TEM 사진이다.FIG. 7 is a TEM photograph of a gold / chromium-deposited polystyrene particle, Janus particle.

도 8은 야누스 나노입자에 렉틴을 결합시키는 과정을 개략적으로 보여주는 도이다.8 is a schematic view showing a process of binding lectin to Janus nanoparticles.

도 9는 카르복실기 결합된 폴리스티렌 나노입자, 금/크롬 증착된 야누스 나노 입자 및 렉틴이 결합된 야누스 나노 입자의 FTIR 스펙트럼을 나타낸 그래프이다.9 is a graph showing FTIR spectra of carboxyl group-bonded polystyrene nanoparticles, gold / chromium-deposited Janus nanoparticles, and lectin-bound Janus nanoparticles.

도 10은 카르복실기 결합된 폴리스티렌 나노입자, 금/크롬 증착된 야누스 나노입자 및 렉틴이 결합된 야누스 나노입자의 크기 분포 및 제타 전위를 나타낸 그래프이다.10 is a graph showing the size distribution and zeta potential of carboxyl group-bonded polystyrene nanoparticles, gold / chromium-deposited Janus nanoparticles, and lectin-bound Janus nanoparticles.

도 11은 TEM 이미지 및 렉틴이 결합된 야누스 나노입자의 개략도이다.11 is a schematic view of a JANUS nanoparticle coupled with a TEM image and a lectin.

도 12는 SNA 렉틴의 야누스 나노입자에 대한 결합 정도를 농도에 따라 나타낸 그래프이다.12 is a graph showing the degree of binding of SNA lectin to Janus nanoparticles according to concentration.

도 13은 Con A 렉틴의 야누스 나노입자에 대한 결합 정도를 농도에 따라 나타낸 그래프이다.13 is a graph showing the degree of binding of Con A lectin to Janus nanoparticles according to concentration.

도 14는 AAL 렉틴의 야누스 나노입자에 대한 결합 정도를 농도에 따라 나타낸 그래프이다.14 is a graph showing the degree of binding of AAL lectin to Janus nanoparticles according to concentration.

도 15는 췌장암세포 마커 항체인 CA 19-9의 야누스 나노입자에 대한 결합 정도를 농도에 따라 나타낸 그래프이다.15 is a graph showing the degree of binding of CA 19-9, which is a pancreatic cancer cell marker antibody, to Janus nanoparticles according to concentration.

도 16은 SNA 렉틴이 결합된 야누스 나노입자에 대한 상대 형광 강도를 나타낸 그래프이다.16 is a graph showing the relative fluorescence intensities of the Janus nanoparticles coupled with the SNA lectin.

도 17은 Con A 렉틴이 결합된 야누스 나노입자에 대한 상대 형광 강도를 나타낸 그래프이다.Fig. 17 is a graph showing the relative fluorescence intensities for the Con A lectin-bound Janus nanoparticles.

도 18은 AAL 렉틴이 결합된 야누스 나노입자에 대한 상대 형광 강도를 나타낸 그래프이다.Fig. 18 is a graph showing the relative fluorescence intensities of the Janus nanoparticles bound with AAL lectin.

도 19는 췌장암세포 마커 항체인 CA 19-9에 대한 상대 형광 강도를 나타낸 그래프이다.FIG. 19 is a graph showing the relative fluorescence intensity against pancreatic cancer cell marker antibody CA 19-9. FIG.

도 20은 야누스 나노입자와 결합된 렉틴의 결합 친화성을 조사한 공초점 현미경 사진이다.FIG. 20 is a confocal microscope photograph showing the binding affinity of lanthanum bound with Janus nanoparticles.

도 21은 야누스 나노입자와 결합된 SNA 렉틴과 각 세포와의 결합 친화성을 나타낸 그래프이다.21 is a graph showing the binding affinity between SNA lectin bound to Janus nanoparticles and each cell.

도 22는 야누스 나노입자와 결합된 Con A 렉틴과 각 세포와의 결합 친화성을 나타낸 그래프이다.22 is a graph showing the binding affinity between Con A lectin bound to Janus nanoparticles and each cell.

도 23은 야누스 나노입자와 결합된 AAL 렉틴과 각 세포와의 결합 친화성을 나타낸 그래프이다.23 is a graph showing the binding affinity of AAL lectin bound with Janus nanoparticles to each cell.

도 24는 야누스 나노입자와 결합된 췌장암세포 마커 항체인 CA 19-9과 각 세포와의 결합 친화성을 나타낸 그래프이다.24 is a graph showing binding affinity between CA19-9, which is a pancreatic cancer cell marker antibody bound to Janus nanoparticles, and each cell.

도 25는 야누스 나노입자와 각 세포와의 결합 친화성을 나타낸 그래프이다.25 is a graph showing binding affinity between Janus nanoparticles and each cell.

도 26은 SNA 렉틴이 결합된 야누스 나노입자가 처리된 PANC-1에서의 공초점 z-stack 3차원 재구성 이미지이다.Fig. 26 is a confocal z-stack three-dimensional reconstruction image in PANC-1 treated with Janus nanoparticles conjugated with SNA lectin.

도 27은 Con A 렉틴이 결합된 야누스 나노입자가 처리된 PANC-1에서의 공초점 z-stack 3차원 재구성 이미지이다.27 is a confocal z-stack three-dimensional reconstruction image in PANC-1 treated with Con A lectin-conjugated Janus nanoparticles.

도 28은 MTT 분석을 통한 SNA 렉틴에 대한 세포 생존력 분석 결과를 나타낸 그래프이다.28 is a graph showing cell viability analysis results for SNA lectin through MTT analysis.

도 29는 MTT 분석을 통한 Con A 렉틴에 대한 세포 생존력 분석 결과를 나타낸 그래프이다.29 is a graph showing cell viability analysis results for Con A lectin through MTT analysis.

도 30은 MTT 분석을 통한 AAL 렉틴에 대한 세포 생존력 분석 결과를 나타낸 그래프이다.30 is a graph showing cell viability analysis results for AAL lectin through MTT analysis.

도 31은 MTT 분석을 통한 췌장암세포 마커 항체인 CA 19-9에 대한 세포 생존력 분석 결과를 나타낸 그래프이다.FIG. 31 is a graph showing the results of cell viability analysis of CA 19-9, which is an antibody against pancreatic cancer cell markers, by MTT assay.

도 32는 렉틴이 결합된 야누스 나노입자(janus nanoparticle)와 엑소좀의 결합 모습을 예시적으로 보여주는 도이다.FIG. 32 is an exemplary diagram showing the binding of exosomes with lanthanum-conjugated janus nanoparticles.

도 33은 야누스 나노입자를 포함하는 미세유체 유전영동 시스템(microfluid dielectrophoresis system)을 이용하여 엑소좀을 분리하는 방법을 개략적으로 보여주는 도이다.33 is a schematic view showing a method of separating exosomes using a microfluid dielectrophoresis system including Janus nanoparticles.

도 34은 야누스 나노입자를 포함하는 미세유체 유전영동 시스템의 작동 원리 및 시뮬레이션 과정을 보여주는 도이다.FIG. 34 shows the operation principle and the simulation process of a microfluidic dielectrophoresis system including Janus nanoparticles. FIG.

도 35은 Panc-1 세포에서 분리된 췌장암 엑소좀을 나타낸 사진이다.35 is a photograph showing pancreatic cancer exosomes isolated from Panc-1 cells.

도 36는 SNA 렉딘 단백질이 결합된 야누스 나노입자에 포획된 엑소좀을 나타낸 사진이다.Fig. 36 is a photograph showing exosomes captured in a Janus nanoparticle to which an SNA lectin protein is bound.

도 37은 Panc-1 세포의 엑소좀에서의 CD81, CD63 및 EphA2 발현을 웨스턴 블롯팅 분석에 의하여 확인한 사진이다.FIG. 37 is a photograph showing the expression of CD81, CD63 and EphA2 in exosome of Panc-1 cells by Western blot analysis.

도 38은 렉틴이 결합되지 않은 야누스 나노입자, 렉틴이 결합된 야누스 나노입자 및 췌장암세포 마커 항체인 CA 19-9가 결합된 야누스 나노입자와 췌장암 유래 엑소좀의 결합을 확인한 사진이다.FIG. 38 is a photograph showing the binding of exosomes derived from pancreatic cancer to Janus nanoparticles to which lectin is bound, Janus nanoparticles to which lectin is bound, and Janus-9, which is a pancreatic cancer cell marker antibody, CA 19-9.

도 39는 각 렉틴에 결합된 야누스 나노입자의 엑소좀 검출능을 나타낸 그래프이다.39 is a graph showing the exosome-detecting ability of Janus nanoparticles bound to each lectin.

본 발명의 일 양상은 엑소좀 결합 분자가 연결된 나노입자를 포함하는 암 진단용 조성물로서, 상기 나노입자는 입자 표면의 일부분이 금속으로 코팅된 것인 암 진단용 조성물을 제공한다.One aspect of the present invention provides a composition for diagnosing cancer comprising nanoparticles to which exosome binding molecules are linked, wherein the nanoparticles are coated with a metal at a portion of the particle surface.

본 명세서에서 사용되는 용어, '엑소좀(exosome)'은 여러 종류의 세포들로부터 분비되는 막 구조의 소낭체로, 다른 세포 및 조직에 결합하여 막 구성요소, 단백질, RNA를 전달하는 등 다양한 역할을 하는 것으로 알려져 있다.As used herein, the term 'exosome' refers to a membrane-structured pancreas secreted from a variety of cells, which binds to other cells and tissues and functions in various roles, such as transferring membrane components, proteins, and RNA .

본 발명의 일 실시예에 따르면 상기 암은 유방암, 갑상선암, 위암, 췌장암 및 담도암으로 이루어진 군에서 선택될 수 있으나 이에 제한되지 아니한다.According to an embodiment of the present invention, the cancer may be selected from the group consisting of breast cancer, thyroid cancer, stomach cancer, pancreatic cancer and bile duct cancer, but is not limited thereto.

본 명세서에서 사용되는 용어, '엑소좀 결합 분자'란 검출하고자 하는 엑소좀과 특이적으로 결합할 수 있는 바이오분자 또는 기타 화합물질 등을 포함하는 것으로 본 발명의 일 실시예에 따르면 단백질, 리간드, 앱타머, DNA, 합텐 (hapten), 아비딘 (avidin), 스트렙타비딘 (streptavidin), 뉴트라비딘 (neutravidin), 프로테인 A, 프로테인 G 및 마이크로 RNA로 이루어진 군에서 선택될 수 있으며, 상기 단백질은 렉틴(lectin)인 것이 바람직하다.As used herein, the term " exosome binding molecule " includes biomolecules or other chemical compounds capable of specifically binding to the exosome to be detected. According to one embodiment of the present invention, The protein may be selected from the group consisting of aptamers, DNA, hapten, avidin, streptavidin, neutravidin, protein A, protein G and microRNA, lectin).

본 발명의 일 실시예에 따르면 상기 렉틴은 푸코오스 당쇄 특이적인 렉틴일 수 있으며, 햅토글로빈 특이적 렉틴인 것이 바람직하다. 상기 렉틴은 겨우살이 및 버섯류 (basidiomycetes)로부터 분리할 수 있으며, 땅비늘버섯(Pholiota terrestris)에서 분리한 것이 바람직하다.According to one embodiment of the present invention, the lectin may be a lectin specific to a fucose sugar chain, and preferably a human lectin specific lectin. The lectin can be isolated from mistletoe and basidiomycetes and is preferably isolated from Pholiota terrestris .

본 발명의 일 실시예에 따르면 상기 나노입자는 폴리스티렌(polystyrene) 또는 폴리메틸메타크릴레이트(polymethyl methacrylate) 재질로 이루어진 것일 수 있으나, 엑소좀 결합 분자가 연결될 수 있는 한 그 재질은 제한되지 아니한다.According to one embodiment of the present invention, the nanoparticles may be made of polystyrene or polymethyl methacrylate, but the material thereof is not limited as long as the exosome binding molecule can be connected thereto.

본 발명의 일 실시예에 따르면 상기 금속은 금(Au), 은(Ag), 백금(Pt), 구리(Cu), 니켈(Ni), 아연(Zn) 및 크롬(Cr)으로 이루어진 군에서 선택될 수 있으며, 금인 것이 바람직하다. According to an embodiment of the present invention, the metal may be selected from the group consisting of gold (Au), silver (Ag), platinum (Pt), copper (Cu), nickel (Ni), zinc (Zn) It is preferable that it is gold.

상기 금속을 나노입자의 표면에 코팅하는 방법은 가리움법(masking), 상분리법(phase separation), 및 자가조립법(self assembly)을 이용할 수 있다. 가리움법은 왁스 등의 고체상 물질을 이용하여 입자표면의 절반을 가리고 나머지 절반을 노출 시킴으로써, 노출된 한쪽 표면을 개질시켜 노출되지 않은 면과 다른 성질을 가지게 하는 방법이다. 상 분리법은 둘 혹은 그 이상의 비호환적인, 섞이지않는(incompatible) 구성물들의 혼합을 통해 야누스 입자를 만드는 방법이다. 자가조립법은 블록 공중합체(block copolymer) 또는 유기물과 고분자를 이용하여 야누스 입자를 만드는 방법으로 다양한 종류의 폴리머들을 이용하여 야누스 입자의 표면특성을 조절할 수 있는 점이 장점이다.The method of coating the metal on the surface of the nanoparticles may use masking, phase separation, and self assembly. The cladding method uses a solid material such as wax to cover half of the particle surface and expose the other half, thereby modifying the exposed one surface to have properties different from those of the unexposed surface. Phase separation is a method of making Janus particles through the mixing of two or more incompatible, incompatible components. The self-assembly method is advantageous in that the surface properties of the janus particles can be controlled by using a variety of polymers to make janus particles using a block copolymer or an organic material and a polymer.

본 발명의 일 실시예에 따르면 입자 표면의 일부분이 금속으로 코팅된 나노입자를 야누스 나노입자(Janus nanoparticle)로 명명할 수 있으며, 로마신화의 두 얼굴을 가진 신의 이름을 따서 지어진 이름으로, 하나의 입자에 2개의 구조가 함께 포함된 구조를 말한다. 좁게는 구형 입자를 반으로 나누어 각각이 별도의 구조를 갖는 경우를 의미하지만, 본 명세서에서는 소재 입자 표면을 둘로 나누어 각각의 부분에 서로 다른 특성의 나노입자들이 부착된 경우를 포함하는 의미로 사용하였다.According to one embodiment of the present invention, a nanoparticle in which a part of the surface of a particle is coated with a metal can be named as a Janus nanoparticle. In the name of a god with two faces of Roman myth, It refers to a structure in which two structures are included together in a particle. Narrowly refers to a case where spherical particles are divided in half and each has a separate structure. In the present specification, however, the term " particle surface " .

본 발명의 다른 양상은 검사하고자 하는 대상체로부터 분리한 생물학적 시료와 제1항의 조성물을 접촉시키는 단계; 및 나노입자와 결합한 엑소좀을 확인하는 단계를 포함하는 암 진단 방법을 제공한다.Another aspect of the present invention provides a method for preparing a biological sample, comprising the steps of: contacting a biological sample separated from a subject to be examined with the composition of claim 1; And identifying the exosomes bound to the nanoparticles.

본 발명의 일 실시예에 따르면 생물학적 시료는 혈액, 혈청, 혈장, 타액, 객담, 및 뇨로 이루어진 군으로부터 선택될 수 있으며, 혈액, 혈청 또는 혈장인 것이 바람직하다.According to one embodiment of the present invention, the biological sample may be selected from the group consisting of blood, serum, plasma, saliva, sputum, and urine, and is preferably blood, serum or plasma.

본 발명의 일 실시예에 따르면 상기 암 진단 방법은 나노입자와 결합한 엑소좀을 확인하는 단계 이후, 엑소좀을 분리하여 엑소좀에 포함된 DNA, 단백질을 분석하는 단계를 추가로 포함할 수 있으며, 분리된 엑소좀의 종류에 따라 암의 종류를 판별할 수 있다.According to an embodiment of the present invention, the method for diagnosing cancer may further include a step of analyzing DNA and protein contained in exosome by isolating exosome after identifying exosome bound to nanoparticles, The type of cancer can be distinguished according to the type of exosome that is separated.

본 발명의 또 다른 양상은Yet another aspect of the present invention is

전극이 배치된 하부 기판; 상기 하부 기판과 연결되어 유체가 유동할 수 있는 유체 채널을 형성하는 상부 기판; 및 외부 전원을 포함하는 엑소좀 검출 및 분리 장치에 있어서, A lower substrate on which electrodes are disposed; An upper substrate connected to the lower substrate to form a fluid channel through which fluid can flow; And an external power source,

상기 전극은 채널을 통하여 이동하는 유체의 흐름 방향과 직교하도록 배치되고, 상기 유체 채널은 엑소좀 결합 분자가 연결된 나노입자를 포함하는 것인 엑소좀 검출 및 분리 장치를 제공한다.Wherein the electrode is arranged to be orthogonal to a flow direction of the fluid moving through the channel, and the fluid channel includes nanoparticles to which exosome binding molecules are connected.

본 발명의 일 실시예에 따르면 상기 나노입자는 입자 표면의 일부분이 금속으로 코팅된 것인 바람직하며, 금으로 코팅된 것이 더욱 바람직하다.According to an embodiment of the present invention, the nanoparticles are preferably those in which a part of the surface of the particles is coated with a metal, more preferably coated with gold.

상기 엑소좀 검출 및 분리 장치에 교류 전원이 공급되면 나노입자와 가해지는 상대적인 유전영동 힘의 차이에 따라 양/음의 유전영동(positive/negative dielectrophoresis) 방향이 결정되고, 엑소좀과 결합한 나노입자의 경우 단독의 나노입자와 비교하여 유전영동 방향, 즉 유전율에 차이가 발생하므로 이 차이를 확인함으로써 엑소좀과 결합한 나노입자를 분리할 수 있다.When the AC power is supplied to the exosome detecting and separating device, positive / negative dielectrophoresis directions are determined according to the difference in relative dielectrophoretic force applied to the nanoparticles, and the direction of the positive / negative dielectrophoresis is determined. The difference in the dielectric permittivity, that is, the dielectric constant, is compared with that of the single nanoparticle, so that the exosome-bound nanoparticles can be separated by confirming the difference.

이하 하나 이상의 구체예를 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.

실시예 1: 당 특이적 렉틴 스크리닝Example 1: Sugar specific lectin screening

땅비늘버섯(Pholiota terrestris) 및 겨우살이를 동결건조하여 버섯 분말을 제조하고, 상기 버섯 분말로부터 추출물을 수득하였다. 상기 추출물을 원심분리하여 상층액만을 회수하고, 상층액을 거즈로 여과하였다. 거즈 여과 과정을 수차례 반복하여 여과된 버섯 추출물(이하 '여과 버섯 추출물'로 기재함)을 수득하였다. Pholiota terrestris and mistletoe were lyophilized to prepare mushroom powder, and extracts were obtained from the mushroom powder. The extract was centrifuged to collect only supernatant, and the supernatant was filtered with gauze. The gauze filtration process was repeated several times to obtain filtered mushroom extract (hereinafter referred to as 'filtered mushroom extract').

상기 여과 버섯 추출물에서 푸코오스(fucose) 당쇄 특이적인 렉틴을 분리하기 위하여 하기와 같이 실험하였다. 먼저 췌장암 세포에서 분비되는 엑소좀에서 예상되는 당류가 햅토글로빈(haptoglobin)이기 때문에 세파로스(sepharose) 컬럼에 제조사의 프로토콜에 따라 햅토글로빈을 고정시키고, 수득한 여과 버섯 추출물을 접촉시켰다. 접촉시킨 후 햅토글로빈과의 결합하는 렉틴을 컬럼에서 용출시키고, 렉틴의 결합 상수, 탄수화물-결합 특이성을 분석하여 최적의 렉틴을 선별하였다. 땅비늘버섯 이외에, 다른 식물류 유래 렉틴은 하기 표 1과 같은 시중에서 판매되는 렉틴을 구매하여 사용하였다.In order to separate the fucose sugar chain specific lectin from the filter mushroom extract, the following experiment was conducted. First, since the saccharide expected in the exosome secreted from pancreatic cancer cells is haptoglobin, the haptoglobin was fixed to the sepharose column according to the manufacturer's protocol, and the obtained filter mushroom extract was contacted. After contacting, the lectin binding to the human togoglobin was eluted from the column, and the optimal lectin was selected by analyzing the lectin binding constant and carbohydrate-binding specificity. In addition to the scaly mushroom, other plant-derived lectins were purchased and purchased from the market as shown in Table 1 below.

명칭designation 렉틴Lectin 유래origin 시판 정보Commercial Information SNASNA Sambucus Nigra LectinSambucus Nigra Lectin 딱총나무Cherry tree L6890, Sigma AldrichL6890, Sigma Aldrich Con ACone Concanavalin AConcanavaline 작두콩Soybean 61760, Sigma Aldrich61760, Sigma Aldrich AALAAL Aleuria Aurantia LectinAleuria Aurantia Lectin 들주발버섯Sole mushroom L-1390, Vector laboratoryL-1390, Vector laboratory

도 1은 렉틴과 렉틴 특이적 당단백질이 결합하는 모습을 보여주는 것이다.Fig. 1 shows the binding of lectin to a lectin-specific glycoprotein.

도 2에 버섯을 동결건조하여 추출물을 제조하고, 추출물로부터 렉틴을 분리하는 과정을 보여주는 것이다.FIG. 2 shows the process of preparing an extract by lyophilizing mushrooms and separating lectin from the extract.

실시예 2: 렉틴이 결합된 야누스 나노입자(Janus nanoparticle) 제조Example 2: Preparation of Janus nanoparticle conjugated with lectin

2-1. 금속이 코팅된 폴리스티렌 입자 제조2-1. Manufacture of metal-coated polystyrene particles

대류콜로이드조립(convective colloid assembly) 방법 및 공기-물 계면(air-water interface) 자가 조립 방법을 이용하여 폴리스티렌 입자 단층을 제조하였다. 구체적으로 5 ㎛ 내지 100 ㎚ 크기의 폴리스티렌(polystyrene) 입자가 분산된 현탁액에 유리 기판 또는 실리콘 웨이퍼를 담그고, 용액 성분을 증발시켜 유리 기판 또는 실리콘 웨이퍼 위에 고밀도 폴리스티렌 입자 단층(monolayer)을 제조하였다. 상기 폴리스티렌 입자 단층에 이방성 금속(크롬(chromium)은 ~20 ㎚; 금(gold)은 수십 ㎚)을 증착(deposition)시켜 입자 표면의 절반이 금속으로 코팅된 폴리스티렌 입자(이하, '야누스 나노입자'로 기재함)를 제조하였다. 이때 금 박막의 안정적인 형성을 위해 금 증착 전 크롬 증착을 먼저 진행하였다. 증착 과정은 열 또는 전자빔 증발기(thermal or e-beam evaporator)를 이용하였다. 유리 기판 또는 실리콘 웨이퍼 위에 형성된 야누스 나노입자는 스프레이 및 소니케이션(sonication) 과정을 통해 물/에탄올 혼합용매에 재분산시키고, 응집을 방지하기 위해 미량의 계면활성제를 첨가하였다.A monolayer of polystyrene particles was prepared using a convective colloid assembly method and an air-water interface self-assembly method. Specifically, a high-density polystyrene particle monolayer was prepared on a glass substrate or a silicon wafer by immersing a glass substrate or a silicon wafer in a suspension in which polystyrene particles having a size of 5 to 100 nm were dispersed, and the solution components were evaporated. Polystyrene particles (hereinafter, referred to as 'Janus nanoparticles') coated with a half of the surface of the particles by depositing an anisotropic metal (chromium to 20 nm and gold to several tens nm) on the monolayer of polystyrene particles, ) Was prepared. In order to stabilize the formation of the gold thin film, chromium deposition was performed before the gold deposition. A thermal or e-beam evaporator was used for the deposition process. Janus nanoparticles formed on a glass substrate or a silicon wafer were redispersed in a water / ethanol mixed solvent through spraying and sonication, and a small amount of surfactant was added to prevent agglomeration.

도 3은 폴리스티렌 나노입자 단층에 금을 증착시켜 입자의 한쪽면에만 금 박막이 형성된 야누스 나노입자 제조 과정을 보여주는 것이다.FIG. 3 is a view showing a process of manufacturing a Janus nanoparticle in which gold is deposited on a monolayer of polystyrene nanoparticles to form a gold thin film on only one side of the particle.

도 4는 야누스 나노입자 합성 과정의 개략도이다.4 is a schematic view of the process of synthesizing Janus nanoparticles.

도 5는 실리콘 웨이퍼상에서 단층 정렬화된(monocrystallized) 폴리스티렌 나노입자의 SEM 이미지이다.5 is a SEM image of monocrystallized polystyrene nanoparticles on a silicon wafer.

한편, 합성된 야누스 나노입자의 형상분석을 위해 투과전자현미경(TEM) 분석을 진행하였다. 구체적으로, Energy-Filtering Transmission Electron Microscope(LIBRA 120, Carl Zeiss) 장비를 이용해 투과전자형미경 분석을 수행하였으며, 측정하고자 하는 샘플은 Cu gird 위에 로딩하여 준비하였다.On the other hand, transmission electron microscopy (TEM) analysis was carried out to analyze the shape of synthesized Janus nanoparticles. Specifically, transmission electron microscope analysis was performed using an Energy-Filtering Transmission Electron Microscope (LIBRA 120, Carl Zeiss). The sample to be measured was prepared by loading on a Cu gird.

그 결과, TEM 분석을 통해 확인된 야누스 나노입자는 금속증착으로 인하여 전자 밀도가 높아진 영역은 진하게, 증착이 되지 않은 폴리머 부분은 상대적으로 연하게 색이 대조되어 나타남을 확인하여, 야누스 나노입자가 성공적으로 제조되었음을 확인하였다.As a result, it was confirmed that the Janus nanoparticles, which were confirmed through TEM analysis, showed a dark color in the region where the electron density was increased due to the metal deposition and a relatively soft color in the polymer portion which was not deposited. .

도 6은 500nm의 폴리스티렌 나노 입자의 TEM 사진이다.6 is a TEM photograph of polystyrene nanoparticles of 500 nm.

도 7은 금/크롬 증착된 폴리스티렌 입자인 야누스 입자의 TEM 사진이다.FIG. 7 is a TEM photograph of a gold / chromium-deposited polystyrene particle, Janus particle.

2-2. 렉틴이 결합된 야누스 나노입자 제작2-2. Production of Janus nanoparticles coupled with lectin

상기 2-1에서 제조한 야누스 나노입자에 아민(amine)을 처리하여 입자 표면에 1차 아민(primary amine)이 연결되도록 하였다. 1차 아민이 연결된 야누스 나노입자에 N-Succinimidyl 3-(2-pyridyldithio)propionate (이하, SPDP로 기재함) 및 DTT(dithiothreitol)를 첨가하여 나노입자와 SPDP의 반응을 유도하였다. SPDP는 아민 반응성 NHS ester(N-Hydroxysuccinimide ester)와 티올(thiol) 반응성 피리딜 디설파이드(pyridyl disulfide) 그룹을 함유하고 있으며, 1차 아민이 연결된 야누스 나노입자는 SPDP와의 반응에 의해 활성화되어 티올 반응성 유도체를 형성하였다. 렉틴은 부분적으로 티올을 함유하고 있기 때문에 활성화된 야누스 나노입자와 결합하여 렉틴이 결합된 야누스 나노입자를 제작할 수 있다.The Janus nanoparticles prepared in the above 2-1 were treated with an amine to link primary amines to the surface of the particles. N-Succinimidyl 3- (2-pyridyldithio) propionate (hereinafter referred to as SPDP) and DTT (dithiothreitol) were added to the Janus nanoparticles to which the primary amine was connected to induce the reaction of nanoparticles with SPDP. SPDP contains an amine-reactive NHS ester ( N- Hydroxysuccinimide ester) and a thiol-reactive pyridyl disulfide group. The Janus nanoparticles to which primary amine is attached are activated by reaction with SPDP to form a thiol reactive derivative . Since lectins partially contain thiols, they can be combined with activated Janus nanoparticles to produce lanthanum-bound Janus nanoparticles.

또한, 렉틴에 티올기가 충분하지 않을 경우를 대비하기 위하여 SPDP와 반응하여 활성화된 야누스 나노입자에 sulfo SMCC(sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate)를 첨가하고, 반응을 유도하였다. 렉틴에는 아민기가 풍부하기 때문에 야누스 나노입자와 렉틴의 결합을 유도할 수 있다.In order to prepare for lack of sufficient thiol groups in the lectin, sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate was added to activated Janus nanoparticles by reacting with SPDP to induce the reaction. Since lectins are rich in amine groups, they can induce the binding of lectins to Janus nanoparticles.

도 8는 야누스 나노입자를 활성화시켜 렉틴과 결합시키는 과정을 보여주는 것이다.Figure 8 shows the process of activating Janus nanoparticles and binding them to lectins.

2-3. 야누스 나노입자의 특성 및 단백질 결합 가능 여부 평가2-3. Characterization of Janus nanoparticles and assessment of protein binding potential

상기 2-1 및 2-2에서 제조한 야누스 나노입자에 대한 특성 분석을 위하여, FTIR, DLS 및 ELS 측정을 진행하였다. 또한, 야누스 표면 위 렉틴 수식을 위한 입자의 단백질 결합능 평가를 위하여, BSA를 결합하여 단백질 결합 가능 여부를 평가하였다.For characterization of the Janus nanoparticles prepared in 2-1 and 2-2 above, FTIR, DLS and ELS measurements were carried out. In order to evaluate the protein binding ability of particles for Janus surface wectin expression, BSA was bound to evaluate the protein binding ability.

구체적으로, FTIR 은 분말화된 샘플을 이용하여 ALPHA-T(Bruker optics) 장비에서 측정이 이뤄졌으며, 300-4000cm-1 파수 영역에서 측정하였다. 한편, DLS 및 ELS는 Malvern 사의 zetasizer nano ZS90 장비를 이용하여 측정하였다.Specifically, FTIR measurements were made on ALPHA-T (Bruker optics) equipment using pulverized samples and measured in the 300-4000 cm -1 wavenumber area. On the other hand, DLS and ELS were measured using a Malvern Zetasizer nano ZS90 instrument.

그 결과, FTIR 분석을 통해 700cm-1, 755cm-1 파수에서 폴리스티렌 특성피크(peak)가 관측 되었으며, 폴리스티렌 입자의 카르복실기의 특성 또한 1600cm-1 에서 관측되었다. 또한, 단백질 결합으로 인한 CNH 복합 피크와 NH 결합 피크가 각각 1653cm-1 및 3310cm-1 에서 관측되었다. DLS 분석을 통하여 확인한 입자의 크기는 금속 증착과 단백질 결합 여부에 따라 변하였는데, 그 크기는 500nm에서 633nm까지 관측되었으며, ELS 분석을 통한 제타전위 또한 입자의 처리에 따라 변화하였다.As a result, the polystyrene was characteristic peak (peak) observed at 700cm -1, 755cm -1 wave number through FTIR analysis, the characteristics of the polystyrene particle carboxyl groups was also observed at 1600cm -1. In addition, CNH complex peaks and NH bond peaks due to protein binding were observed at 1653 cm -1 and 3310 cm -1 , respectively. The size of particles determined by DLS analysis varied depending on the metal deposition and protein binding. The size was observed from 500 nm to 633 nm, and the zeta potential from the ELS analysis also changed with the treatment of the particles.

도 9는 카르복실기 결합된 폴리스티렌 나노입자, 금/크롬 증착된 야누스 나노 입자 및 렉틴이 결합된 야누스 나노 입자의 FTIR 스펙트럼을 나타낸 그래프이다.9 is a graph showing FTIR spectra of carboxyl group-bonded polystyrene nanoparticles, gold / chromium-deposited Janus nanoparticles, and lectin-bound Janus nanoparticles.

도 10은 카르복실기 결합된 폴리스티렌 나노입자, 금/크롬 증착된 야누스 나노입자 및 렉틴이 결합된 야누스 나노입자의 크기 분포 및 제타 전위를 나타낸 그래프이다.10 is a graph showing the size distribution and zeta potential of carboxyl group-bonded polystyrene nanoparticles, gold / chromium-deposited Janus nanoparticles, and lectin-bound Janus nanoparticles.

도 11은 TEM 이미지 및 렉틴이 결합된 야누스 나노입자의 개략도이다. 폴리스티렌 표면에 결합된 렉틴은 히드라지드(hydrazide) 작용화된 금 나노 입자와 렉틴의 탄수화물 알데히드 그룹 사이의 Wolff-Kishner 반응을 통해 확인된다.11 is a schematic view of a JANUS nanoparticle coupled with a TEM image and a lectin. Lectins bound to the polystyrene surface are identified by the Wolff-Kishner reaction between the hydrazide-functionalized gold nanoparticles and the carbohydrate aldehyde group of lectins.

2-4. 야누스 나노입자에 결합된 렉틴의 정량 분석2-4. Quantitative analysis of lectins bound to Janus nanoparticles

야누스 나노입자에 렉틴의 결합 정도를 확인하기 위하여, BCA 분석을 수행하였다. 구체적으로, BCA 단백질 정량분석은 시중에 판매되는 BCA 단백질 분석 키트(23225, thermo scientific)를 사용하여 진행하였으며, 본 제품의 스탠다드 프로토콜에 따라 분석을 수행하였다.BCA analysis was performed to confirm the degree of lectin binding to Janus nanoparticles. Specifically, quantitative analysis of BCA protein was carried out using commercially available BCA protein analysis kit (23225, thermo scientific), and analysis was performed according to the standard protocol of the present product.

그 결과, 각 렉틴은 250㎍/㎖ 내지 563㎍/㎖의 단백질 농도를 나타내었다. 이를 통하여, 야누스 나노입자에 렉틴이 결합이 잘 되었음을 확인하였다. As a result, each lectin showed a protein concentration of 250 μg / ml to 563 μg / ml. Through this, it was confirmed that lectin binds well to Janus nanoparticles.

도 12 내지 도 15는 각각 SNA, Con A, AAL 렉틴 및 췌장암세포 마커 항체인 CA 19-9의 야누스 나노입자에 대한 결합 정도를 농도에 따라 나타낸 그래프이다.12 to 15 are graphs showing the degree of binding of SNA, Con A, AAL lectin, and CA 19-9, which is a pancreatic cancer cell marker antibody, to Janus nanoparticles according to their concentrations.

실시예 3: 렉틴이 결합된 야누스 나노입자의 친화도 확인Example 3 Confirmation of affinity of lanthanum-bound Janus nanoparticles

3-1. 렉틴이 결합된 야누스 나노입자의 세포에 따른 친화도 분석3-1. Cell affinity analysis of lanthanum-bound Janus nanoparticles

야누스 나노입자의 세포 표면 위 형광분석을 통하여 렉틴이 결합된 야누스 나노입자의 세포에 따른 친화도를 분석하였다.The affinity of lanthanum - bound Janus nanoparticles was analyzed by cell fluorescence analysis of Janus nanoparticles.

구체적으로, 야누스 나노입자에 실시예 1에서 선별한 각 렉틴(SNA, Con A, AAL) 또는 항체(CA 19-9)를 결합하여 각 세포주별 렉틴의 친화도를 평가하였으며, 렉틴이 결합된 야누스 나노입자의 상대 형광 강도는 세포주 처리 후 시간에 따라 측정하였다. 형광분석은 Synergy H1(BioTeK) 장비를 이용하여 진행하였으며, 96-웰 플레이트에 배양된 세포 샘플에 렉틴이 결합된 야누스 입자를 처리하여 비교하였다. 측정 조건은 다음과 같다: Excitation: 485, Emission: 528, Optics: Top, Gain: 125, ead Speed: Normal, Delay: 100msec, Measurements/Data Point: 10, Read Height: 5mm.Specifically, the affinity of lectins for each cell line was evaluated by binding each lectin (SNA, Con A, AAL) or antibody (CA 19-9) selected in Example 1 to the Janus nano-particles, The relative fluorescence intensities of the nanoparticles were measured according to time after cell line treatment. Fluorescence analysis was performed using a Synergy H1 (BioTeK) instrument and lysine-bound janus particles were treated and compared to cell samples cultured on 96-well plates. Measurement conditions are as follows: Excitation: 485, Emission: 528, Optics: Top, Gain: 125, ead Speed: Normal, Delay: 100 msec, Measurements / Data Point: 10, Read Height: 5 mm.

그 결과, SNA 렉틴이 췌장암 세포주(PANC-1)에 특이적인 친화도를 보이며 나머지 렉틴군의 암세포주에 대한 친화도는 약하거나 나타나지 않았다. CA19-9 또한 PANC-1 에 친화도를 보였으나 SNA 렉틴의 췌장암 세포주에 대한 친화도 보다는 약한 친화도를 보였다는 점에서, SNA 렉틴의 우수한 췌장암 세포주 친화도를 확인하였다.As a result, SNA lectin showed a specific affinity for pancreatic cancer cell line (PANC-1) and the affinity of the remaining lectin group for cancer cell line was weak or not. CA19-9 also showed affinity to PANC-1, but showed affinity for SNA lectin rather than affinity for pancreatic cancer cell line, confirming excellent pancreatic cancer cell line affinity of SNA lectin.

도 16 내지 도 19는 각각 SNA, Con A, AAL 렉틴 또는 CA19-9 항체가 결합된 야누스 나노입자에 대한 상대 형광 강도를 나타낸 그래프이다. 각 시간(2h, 6h, 12h, 24h)에서의 값은 그림 도 16의 24시간 및 Panc-1에서의 형광 값으로 정규화된 것이다.FIGS. 16 to 19 are graphs showing relative fluorescence intensities relative to Janus nanoparticles bound with SNA, Con A, AAL lectin or CA19-9 antibody, respectively. The values at each time (2h, 6h, 12h, 24h) are normalized to the fluorescence values at 24 hours and Panc-1 in FIG.

3-2. 렉틴이 결합된 야누스 나노입자의 공초점 현미경 분석3-2. Confocal microscopy analysis of lanthanum-conjugated Janus nanoparticles

렉틴이 결합된 야누스 나노입자의 형광특성을 확인하기 위하여 공초점 현미경분석을 수행하였다. 24시간 배양된 세포에 일정농도의 샘플을 처리하고, 24시간 후 샘플 고정화 처리한 다음 공초점 현미경 분석을 진행하였으며, 24시간 동안 렉틴이 결합된 야누스 나노입자를 처리한 후 세포주를 모니터링 하였다. 청색 형광은 DAPI(ex: 358nm, em: 461nm)에서 나온 것이며, 적색 형광은 야누스 나노입자(ex: 520nm, em: 540nm)의 폴리스티렌에서 나온 것이다.Confocal microscopy analysis was performed to confirm the fluorescence properties of lanthanum-bound Janus nanoparticles. Cells cultured for 24 hours were treated with a certain concentration of the sample. After 24 hours of sample immobilization, confocal microscopy analysis was carried out. Cells were treated with lanthanum-bound Janus nanoparticles for 24 hours. The blue fluorescence is from DAPI (ex: 358 nm, em: 461 nm) and the red fluorescence is from polystyrene of Janus nanoparticles (ex: 520 nm, em: 540 nm).

구체적으로, LSM710(Carl Zeiss, Germany) 공초점 현미경을 이용하여 세포분석을 진행하였으며, 세포핵 DAPI 염색 관찰을 위한 405nm 레이저와 야누스 입자 관측을 위한 514nm 레이저를 사용하였다. 공초점 현미경 분석을 위한 세포 샘플은 인큐베이터에서 하루 배양 후, 각 렉틴이 결합된 야누스 나노입자를 단백질 농도 기준으로 5㎍/㎖ 처리해주었으며, 입자 처리 후 24시간 후에 2% 파라포름알데하이드(paraformaldehyde) 조건에서 워싱 과정과 함께 고정화 처리하였다.Specifically, cell analysis was performed using a confocal microscope LSM710 (Carl Zeiss, Germany), and a 405 nm laser for DAPI staining and a 514 nm laser for Janus particle observation were used. Cell samples for confocal microscopy analysis were cultured in an incubator for one day and then treated with 5 μg / ml of lanthanum-bound Janus nanoparticles on a protein concentration basis. After 24 hours of particle treatment, 2% paraformaldehyde conditions The immobilization process was performed together with the washing process.

그 결과, SNA 렉틴이 췌장암 세포주에 특이적인 친화도를 보여 세포 주변에 야누스 입자의 형광이 잘 관측되었으나, 나머지 렉틴군은 암세포주에 대해약한 친화도를 나타내거나, 또는 친화도를 나타내지 않았다. CA19-9 또한 PANC-1 에 친화도를 보이며, 이에 따른 입자의 형광이 관측되었으나, SNA 렉틴의 췌장암 세포주에 대한 친화도 보다는 약한 친화도를 보였다는 점에서, SNA 렉틴의 우수한 췌장암 세포주 친화도를 확인하였다.As a result, SNA lectin showed a specific affinity for pancreatic cancer cell line, and fluorescence of janus particles was well observed around the cells, but the remaining lectin group showed weak affinity for cancer cell lines, or showed no affinity. CA19-9 also showed affinity to PANC-1, and fluorescence of the particles was observed. However, SNA lectin showed weak affinity to pancreatic cancer cell line, indicating that SNA lectin had excellent affinity for pancreatic cancer cell line Respectively.

도 20은 야누스 나노입자와 결합된 렉틴의 결합 친화성을 조사한 공초점 현미경 사진이다.FIG. 20 is a confocal microscope photograph showing the binding affinity of lanthanum bound with Janus nanoparticles.

또한, Image J 프로그램을 이용하여, 렉틴이 결합된 야누스 나노입자의 형광이미지를 분석하였다.In addition, fluorescence images of lanthanum-bound Janus nanoparticles were analyzed using the Image J program.

구체적으로, 형광세기를 분석하고자 하는 이미지를 프로그램을 통해 불러온 후, 프로그램 내 영역 선택 Tool을 이용하여 분석 영역을 설정하였으며, 이미지 전체를 선택하였다. Analyze 탭의 Measure를 통해 형광감도를 분석한 후, intergrated density 수치를 비교하여 형광이미지의 분석을 수행하였다.Specifically, an image to be analyzed for fluorescence intensity was loaded through a program, an analysis area was set using an area selection tool in the program, and an entire image was selected. After analyzing the fluorescence sensitivity through Measure in the Analyze tab, we analyzed fluorescence images by comparing intergrated density values.

그 결과, SNA 렉틴이 결합된 야누스 나노입자의 PANC-1에 대한 강한 형광 수치를 확인하였으며, CA19-9 에서도 PANC-1 세포주에 대한 강한 형광수치를 확인하였다. 이를 통하여 췌장암 세포주 표면단백질에 시알산(sialic acid)이 많이 당화 되었음을 확인하였고, ALL 렉틴로 인하여 약한 Fucosylation이 나타났음을 확인하였다.As a result, strong fluorescence values for PANC-1 of SNA lectin-conjugated Janus nanoparticles were confirmed, and strong fluorescence values for PANC-1 cell line were also confirmed in CA19-9. Through this, it was confirmed that sialic acid was highly glycosylated on the surface protein of pancreatic cancer cell line, and weak fucosylation was shown due to ALL lectin.

도 21 내지 도 25는 야누스 나노입자와 결합된 각 렉틴, CA19-9 또는 렉틴 및 항체가 결합되지 않은 경우, 각 세포와의 결합 친화성을 나타낸 그래프이다.FIGS. 21 to 25 are graphs showing binding affinities of each lectin, CA19-9 or lectin and antibody bound to Janus nanoparticles when they are not bound to each cell. FIG.

한편, 렉틴이 결합된 야누스 나노입자의 형광이미지를 3D z-stack 분석하였다. On the other hand, fluorescence images of lanthanum-bound Janus nanoparticles were analyzed by 3D z-stack.

구체적으로, LSM710 공초점 장비를 이용하여, 장비 프로그램 내 있는 Stack 옵션을 선택하여 2분동안 총 10장의 이미지를 공초점 위치에 따라 획득하였으며, 프로그램 내에서 분석되어 3차원으로 표시된 이미지를 최종 획득하였다.Specifically, using the LSM710 confocal equipment, a total of 10 images were acquired according to the confocal position for 2 minutes by selecting the Stack option in the instrument program, .

그 결과, SNA 렉틴이 결합된 야누스 나노입자가 처리된 PANC-1에서는 세포주변에 야누스 나노입자의 붉은 형광이 잘 관측되었으나, Con A 렉틴이 결합된 야누스 나노입자가 처리된 PANC-1에서는 야누스 나노입자의 형광을 관측하기 어려웠다. 이를 통하여, SNA 렉틴의 PANC-1 세포주에 대한 특이적 친화성을 확인하였다.As a result, in PANC-1 treated with Janus nanoparticles conjugated with SNA lectin, the red fluorescence of the Janus nanoparticles was well observed around the cells, whereas in PANC-1 treated with Con A lectin-conjugated Janus nanoparticles, It was difficult to observe the fluorescence of the particles. Through this, the specific affinity of SNA lectin to the PANC-1 cell line was confirmed.

도 26 및 도 27은 SNA 또는 Con A 렉틴이 결합된 야누스 나노입자가 처리된 PANC-1에서의 공초점 z-stack 3차원 재구성 이미지이다.Figures 26 and 27 are confocal z-stack three-dimensional reconstructed images in PANC-1 treated with Janus nanoparticles conjugated with SNA or Con A lectin.

실시예 4: 렉틴이 결합된 야누스 나노입자를 이용한 엑소좀 확인Example 4: Examination of exosomes using lanthanum-conjugated Janus nanoparticles

4-1. 엑소좀 분리4-1. Isolation of exosome

상기 실시예 1에서 선별한 렉틴의 엑소좀 검출 능력을 확인하기 위하여 엑소좀을 분리하였다.Exosomes were isolated to confirm the ability of the lectins selected in Example 1 to detect exosome.

구체적으로, 췌장암 세포주(Human pancreatic ductal carcinoma cell line: Panc-1), 자궁경부암 세포주(human cervical cancer cell: HeLa), 유방암 세포주(human breast adenocarcinoma: MDA-MB-231), 인간 피부 섬유아세포(human dermal fibro blast: HDFB), 인간 배아 신장세포(human embryonic kidney cell: HEK293T) 및 정상 표피세포를 각각 48시간 동안 배양한 후 배양 배지를 수거하여 원심분리로 엑소좀(exosome)을 분리하였다.Specifically, human pancreatic ductal carcinoma cell line (Panc-1), human cervical cancer cell (HeLa), human breast adenocarcinoma (MDA-MB-231), human skin fibroblast dermal fibroblast (HDFB), human embryonic kidney cell (HEK293T) and normal epidermal cells were cultured for 48 hours, respectively. The culture medium was collected and the exosome was separated by centrifugation.

4-2. 세포 독성 확인4-2. Cytotoxicity check

렉틴이 결합된 야누스 나노입자의 세포주에 대한 독성을 나타내는지 확인하였다.It was confirmed that the lectin exhibited toxicity to the cell line of the Janus nanoparticles bound thereto.

구체적으로, MTT 분석을 통하여 실시예 4-1의 세포주에 대한 렉틴이 결합된 야누스 나노입자의 독성을 평가하였다. 50㎍/㎖ 내지 2.5㎍/㎖의 단백질 농도에서 독성을 평가하였으며, 렉틴 또는 항체가 결합된 야누스 나노 입자로 24시간 동안 처리한 후 세포주의 생존율을 확인하여 독성을 평가하였다. MTT 분석은 AMRESCO 사의 MTT 시약분말을 2㎎/㎖ 준비하고, 96-웰 플레이트 상에 하루 배양된 세포에 각 농도별로 샘플을 처리해준 후 하루 더 인큐베이터 상에 배양한 다음, 마지막으로 MTT solution을 각 웰에 100㎕ 씩 적용하여 수행하였다. MTT 처리가 완료된 샘플에 대하여, 전술한 형광 분석장비를 이용하여 570nm 파장 영역 에서의 흡광도를 측정함으로써, 세포 독성을 확인하였다.Specifically, the toxicity of lanthanum-bound Janus nanoparticles to the cell line of Example 4-1 was evaluated through MTT analysis. Toxicity was evaluated at a protein concentration of 50 μg / ml to 2.5 μg / ml and after 24 hours of treatment with lanthanum or antibody-conjugated Janus nanoparticles, the viability of the cell line was checked for toxicity. MTT assay was performed by preparing 2 mg / ml of MTT reagent powder from AMRESCO Inc., culturing the cells on a 96-well plate for each concentration, culturing on a 96-well plate for one day, culturing on an incubator for one more day, Lt; / RTI > The cytotoxicity of the MTT-treated sample was measured by measuring the absorbance in the wavelength region of 570 nm using the above-described fluorescence analyzer.

그 결과, HeLa 세포주에서 SNA 및 Con A 렉틴은 5㎍/㎖ 이상의 농도에서 70% 이하의 생존을 보였고, AAL 렉틴 및 CA19-9 항체는 HEK293 세포주에서 다소 낮은 생존율을 보였으나, 대부분의 경우, SNA, Con A 및 AAL 렉틴은 5㎍/㎖ 농도 이하에서 70% 이상의 생존률을 나타내었다.As a result, in the HeLa cell line, SNA and Con A lectin survived less than 70% at a concentration of 5 μg / ml or more, while AAL lectin and CA19-9 antibody showed a somewhat lower survival rate in the HEK293 cell line, , Con A and AAL lectin showed a survival rate of 70% or more at a concentration of 5 μg / ml or less.

도 28 내지 도 31은 MTT 분석을 통한 각 렉틴 또는 CA19-9 항체에 대한 세포 생존력 분석 결과를 나타낸 그래프이다.FIGS. 28 to 31 are graphs showing cell viability analysis results for each lectin or CA19-9 antibody through MTT analysis. FIG.

4-2. 렉틴이 결합된 야누스 나노입자를 이용한 췌장암 유래 엑소좀 확인4-2. Identify exosome from pancreatic cancer using lanthanum-conjugated Janus nanoparticles

엑소좀을 분리하기 위한 비유동 유전영동 시스템(non-current dielectrophoresis system)을 제작하기 위하여 유리 기판 또는 실리콘 웨이퍼 위에 원하는 전극 디자인을 가지는 마스크를 올리고, 크롬과 금을 연속적으로 증착시켰다. 이후 전극 위에 입자 현탁액을 가둘 수 있는 챔버를 제작하여 유체의 흐름이 없는 비유동 유전영동 소자를 제작하였다. 유전영동 시스템에 교류전압을 가할 경우 입자와 미디어에 가해지는 상대적인 유전영동 힘의 차이에 따라 양/음의 유전영동(positive/negative dielectrophoresis) 방향이 결정된다. 교류전압의 진동수, 입자의 크기 및 미디어의 유전특성 등에 따른 유전영동 거동을 정확히 분석하고 이를 입자 분리에 적용하면 엑소좀을 분리할 수 있다.To fabricate a non-current dielectrophoresis system for separating exosomes, a mask with the desired electrode design was placed on a glass substrate or silicon wafer and chromium and gold were continuously deposited. Then, a chamber capable of holding the particle suspension on the electrode was fabricated to fabricate a non-fluidized dielectrophoretic device with no fluid flow. When an AC voltage is applied to a dielectrophoretic system, the direction of positive / negative dielectrophoresis is determined by the difference in relative dielectrophoretic forces applied to the particles and the medium. Exopeomes can be separated by precisely analyzing the dielectrophoretic behavior according to the frequency of AC voltage, particle size and dielectric properties of media, and applying it to particle separation.

비유동 유전영동 시스템을 이용하여 야누스 입자와 엑소좀의 결합 유무에 따른 유전영동 거동을 1차적으로 확인하고, 연속적인 미세유체 유전영동(microfluid dielectrophoresis) 소자를 구현하여 시간당 엑소좀 분리 수율을 향상시켰다. 미세유체 시스템에서 분리된 엑소좀 결합 야누스 입자를 분리하여 PCR 또는 광학장비로 확인하여 엑소좀 분리 효율을 판단하였다.By using a non - ferroelectric dielectrophoresis system, we first confirmed the dielectrophoretic behavior according to the presence or absence of janus particles and exosomes, and implemented a continuous microfluid dielectrophoresis device to improve the isolation yield of exosome per hour. The exosome - bound janus particles isolated from the microfluidic system were separated and identified by PCR or optical equipment to determine the exosome isolation efficiency.

각 유전영동 시스템으로 분리한, 엑소좀이 결합된 야누스 나노입자에 대하여 엑소좀 특이마커(CD63)에 대한 ELISA 및 웨스턴 분석을 수행하면 췌장암 특이적인 엑소좀을 확인할 수 있다. 또한, 분리된 엑소좀 내에 존재하는 DNA를 PCR로 분석하여 췌장암 세포에서 특이적으로 발현되는 K-Ras 점 돌연변이(point mutation)를 확인할 수 있다.ELISA and western analysis of exosome-specific marker (CD63) for exosome-bound Janus nanoparticles separated by each dielectrophoretic system can be used to identify pancreatic cancer-specific exosomes. In addition, the DNA present in the separated exosome can be analyzed by PCR to confirm a K-Ras point mutation specifically expressed in pancreatic cancer cells.

도 32는 렉틴이 결합된 야누스 나노입자와 상기 렉틴 특이적 당단백질이 발현된 엑소좀의 결합 모습을 예시적으로 보여주는 것이다.FIG. 32 is an illustration showing an example of binding of lectin-binding Janus nanoparticles to exosome in which the lectin-specific glycoprotein is expressed.

도 33은 야누스 나노입자를 포함하는 미세유체 유전영동 시스템(microfluid dielectrophoresis system)을 이용하여 엑소좀을 분리하는 방법을 보여주는 도이다.33 is a view showing a method of separating exosomes using a microfluid dielectrophoresis system including Janus nanoparticles.

도 34은 야누스 나노입자를 포함하는 미세유체 유전영동 시스템의 작동 원리 및 시뮬레이션 과정을 보여주는 것이다. 왼쪽 패널은 유체의 흐름이 없는 비유동 시스템에서 야누스 입자의 유전영동 거동을 확인한 후 이를 미세유체 시스템에 적용하여 연속적인 엑소좀 검출 시스템을 구현한 것이다. 야누스 나노입자의 유전영동에 영향을 미칠 수 있는 파라미터에는 AC 주파수 및 전압의 크기, 전극 디자인, 입자의 크기, 전해질 매질의 종류 및 야누스 입자에서 금 박막의 커버리지 등이 있다. 중간 패널은 미세유체 유전영동 시스템에 적용할 전극 패턴을 보여주는 것이고, 오른쪽 패널은 유전영동 실험 결과를 이론적으로 예측/분석할 수 있는 시뮬레이션 프로세스를 보여주는 것이다.Figure 34 shows the operating principle and simulation process of a microfluidic dielectrophoresis system containing Janus nanoparticles. In the left panel, the diophoretic behavior of the Janus particles was confirmed in a nonflow system without fluid flow, and the system was applied to a microfluid system to implement a continuous exosome detection system. Parameters that can affect the diurnal flow of Janus nanoparticles include AC frequency and voltage magnitude, electrode design, particle size, electrolyte media type, and gold film coverage in Janus particles. The middle panel shows the electrode pattern to be applied to the microfluidic dielectrophoresis system and the right panel shows the simulation process to theoretically predict / analyze the results of the dielectrophoresis experiment.

한편, PANC-1 췌장암 세포주로부터 추출한 엑소좀과 각 렉틴이 결합된 야누스 나노입자의 결합능을 비교하였다.On the other hand, the binding ability of exosomes extracted from PANC-1 pancreatic cancer cell line to Janus nanoparticles bound with each lectin was compared.

구체적으로, 췌장암 세포주의 엑소좀 판별 여부를 위하여 엑소좀 마커와 췌장암 세포주 마커를 웨스턴 블롯팅(western blotting) 분석을 통해 확인하였다. 각 암세포주 및 일반 세포로부터 분리된 엑소좀을 RIPA buffer containing protease inhibitor cocktail & Phosphatase inhibitor cocktail(Invitrogen)로 용해(lysis)하였다. 각 샘플로부터, BCA 분석(Invitrogen)을 통하여 단백질을 정량(quantification) 하였으며, 이를 통하여 정규화된(normalized) 동량의 단백질을 폴리아크릴아마이드 젤(polyacrylamide gel)을 통하여 전기영동 구배(electrophoretic gradient)를 주었다. 그 후, 분리된 단백질을 니트로셀룰로오스(nitrocellulose) 멤브레인(Merck millipore)으로 트랜스퍼(transfer) 하였으며, 단백질 블롯(blot)을 1시간 동안 실온(room temperature)에서 5% Skim milk in TBST로 블로킹(blocking) 하였다. 그리고 나서 일차 항체(primary antibody)(1:1000 anti-CD63, 1:000 anti-CD81 및 1:1000 anti-EphA2(Santa-Cruz))와 함께 4℃에서 밤새 인큐베이션하였다. 그 후, HRP-컨쥬게이트된 이차 항체(conjugated secondary antibody)(Cell signaling)를 1시간 실온에서 반응시켰다. 그 다음, TBST로 3회 10분씩 세척 하였으며, Chemidoc(biorad)를 이용하여 블롯팅(blotting)을 확인하였다. 각 세포로부터 추출된 엑소좀은 미디어를 추출하여, 500g에서 10분간 원심분리 후 상층액을 수득한 다음, 12,000g에서 20분간 원심분리 후 상층액을 수득하였다. 그 후, Exoquick(System biosciences)을 이용하여 엑소좀을 제조사의 프로토콜에 따라 추출하였다.Specifically, exosomal markers and pancreatic cancer cell line markers were identified by western blotting analysis to determine whether or not exosomes of the pancreatic cancer cell line were discriminated. The exosomes isolated from each cancer cell line and normal cells were lysed with RIPA buffer containing protease inhibitor cocktail & Phosphatase inhibitor cocktail (Invitrogen). From each sample, proteins were quantitated by BCA analysis (Invitrogen), and the same amount of normalized protein was electrophoretically graded through polyacrylamide gel. The separated proteins were then transferred to a nitrocellulose membrane (Merck Millipore), and protein blotting was blocked for 1 hour at room temperature with 5% Skim milk in TBST. Respectively. The cells were then incubated with primary antibody (1: 1000 anti-CD63, 1: 000 anti-CD81 and 1: 1000 anti-EphA2 (Santa-Cruz)) overnight at 4 ° C. The HRP-conjugated secondary antibody (Cell signaling) was then reacted for 1 hour at room temperature. Then, it was washed three times with TBST for 10 minutes, and blotting was confirmed using Chemidoc (biorad). The exosomes extracted from each cell were subjected to centrifugation at 500 g for 10 minutes to obtain an supernatant. After centrifugation at 12,000 g for 20 minutes, supernatant was obtained. The exosomes were then extracted according to the manufacturer's protocol using Exoquick (System biosciences).

그 결과, 엑소좀 형태의 미세소낭이 관측되었다. 또한, 웨스턴 블롯팅을 통하여 엑소좀 마커(CD81, CD63)의 여부를 확인하였으며, 동시에 췌장암 세포주 마커 (EphA2)를 확인하여, 췌장암 유래 엑소좀임을 확인하였다. 이를 통하여, SNA 렉틴이 결합된 야누스 나노입자의 췌장암 엑소좀 검출능을 확인하였다.As a result, an exosome-type micro vesicle was observed. In addition, the presence of exosomal markers (CD81, CD63) was confirmed by Western blotting, and pancreatic cancer cell line marker (EphA2) was confirmed to confirm exocrine origin of pancreatic cancer. Through this, the ability to detect pancreatic cancer exosome of Janus nanoparticles coupled with SNA lectin was confirmed.

도 35은 Panc-1 세포에서 분리된 췌장암 엑소좀을 나타낸 사진이다.35 is a photograph showing pancreatic cancer exosomes isolated from Panc-1 cells.

도 36는 SNA 렉딘 단백질이 결합된 야누스 나노입자에 포획된 엑소좀을 나타낸 사진이다.Fig. 36 is a photograph showing exosomes captured in a Janus nanoparticle to which an SNA lectin protein is bound.

도 37은 Panc-1 세포의 엑소좀에서의 CD81, CD63 및 EphA2 발현을 웨스턴 블롯팅 분석에 의하여 확인한 사진이다.FIG. 37 is a photograph showing the expression of CD81, CD63 and EphA2 in exosome of Panc-1 cells by Western blot analysis.

한편, TEM 분석 및 입자 개수를 통하여 각 렉틴이 결합된 야누스 나노입자와 추출된 엑소좀의 반응 후 엑소좀 결합능을 비교하였다.On the other hand, through the TEM analysis and the number of particles, the exosome binding ability was compared after the reaction between the lanthanum-bound Janus nanoparticles and the extracted exosomes.

구체적으로, 입자 개수는 엑소좀과 야누스 입자의 반응 후 원심분리를 이용하여 엑소좀과 결합된 야누스 나노입자의 분리 후, 상등액에 존재하는 잉여 엑소좀을 검측하여 수행하였으며, Energy-Filtering Transmission Electron Microscope(LIBRA 120, Carl Zeiss) 장비를 이용해 투과전자형미경 분석을 진행하였다. 측정하고자 하는 샘플은 Cu gird 위에 로딩하여 준비하였으며, 엑소좀 분석에는 negative staining을 위해 각 샘플마다 2% uranyl acetate 를 적용하여 염색하였다. 용매 내 존재하는 입자 또는 엑소좀은 Nanoparticle tracking 분석을 통하여 확인하였으며, Malvern Panalytical 사의 NS300 장비를 이용하였다.Specifically, the number of particles was determined by measuring the residual exosomes in the supernatant after separation of the janus nanoparticles bound to the exosomes by centrifugation after the reaction between the exosomes and the janus particles. The energy-filtering transmission electron microscope (LIBRA 120, Carl Zeiss) equipment. The sample to be measured was prepared by loading on Cu gird. For exosomal analysis, 2% uranyl acetate was applied to each sample for negative staining. The presence of particles or exosomes in the solvent was confirmed by nanoparticle tracking analysis and was performed using a Malvern Panalytical NS300 instrument.

그 결과, AAL 렉틴이 엑소좀 검출능이 가장 우수한 것으로 확인되었다. 이는췌장암 세포주는 Fucosylated 단백질을 만들어 내는 것으로 알려져 있는데, 세포의 엑소좀 생성과정중, 세포내 Multivescular body(MVB)로의 intracellular budding 과정을 통해 엑소좀이 형성 되면서 유입된 새포내 단백질 및 엑소좀 단백질의 영향으로 확인된다.As a result, it was confirmed that AAL lectin had the best ability to detect exosome. It is known that the pancreatic cancer cell line produces a fucosylated protein. During exocytic production of cells, intracellular budding to the intracellular multivescular body (MVB) results in the formation of exosomes, .

선별된 췌장암 유래 엑소좀과 각 렉틴이 수식된 야누스 나노입자의 반응을 통하여, 렉틴이 결합된 야누스 나노입자의 엑소좀 검출능을 확인하였다.Through the reaction of the selected pancreatic cancer-derived exosomes with the respective lectin-modified Janus nanoparticles, the ability to detect exosome of lanthanum-bound Janus nanoparticles was confirmed.

도 38은 렉틴이 결합되지 않은 야누스 나노입자, 렉틴이 결합된 야누스 나노입자 및 췌장암세포 마커 항체인 CA 19-9가 결합된 야누스 나노입자와 췌장암 유래 엑소좀의 결합을 확인한 사진이다.FIG. 38 is a photograph showing the binding of exosomes derived from pancreatic cancer to Janus nanoparticles to which lectin is bound, Janus nanoparticles to which lectin is bound, and Janus-9, which is a pancreatic cancer cell marker antibody, CA 19-9.

도 39는 각 렉틴에 결합된 야누스 나노입자의 엑소좀 검출능을 나타낸 그래프이다.39 is a graph showing the exosome-detecting ability of Janus nanoparticles bound to each lectin.

이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (10)

엑소좀 결합 분자가 연결된 나노입자를 포함하는 암 진단용 조성물로서,A composition for diagnosing cancer comprising nanoparticles to which an exosome binding molecule is linked, 상기 나노입자는 입자 표면의 일부분이 금속으로 코팅된 것인 암 진단용 조성물.Wherein the nanoparticles are coated with a metal at a portion of the particle surface. 제1항에 있어서, 상기 암은 유방암, 갑상선암, 위암, 췌장암 및 담도암으로 이루어진 군에서 선택되는 것인 암 진단용 조성물.The cancer diagnostic composition according to claim 1, wherein the cancer is selected from the group consisting of breast cancer, thyroid cancer, gastric cancer, pancreatic cancer, and bile duct cancer. 제1항에 있어서, 상기 엑소좀 결합 분자는 단백질, 리간드, 앱타머 및 마이크로 RNA로 이루어진 군에서 선택되는 것인 암 진단용 조성물.The cancer diagnostic composition according to claim 1, wherein the exosome binding molecule is selected from the group consisting of a protein, a ligand, an aptamer, and a microRNA. 제3항에 있어서, 상기 단백질은 렉틴인 것인 암 진단용 조성물.4. The composition according to claim 3, wherein the protein is lectin. 제1항에 있어서, 상기 나노입자는 폴리스티렌 또는 폴리메틸메타크릴레이트인 것인 암 진단용 조성물.The cancer diagnostic composition according to claim 1, wherein the nanoparticles are polystyrene or polymethyl methacrylate. 제1항에 있어서, 상기 금속은 금(Au), 은(Ag), 백금(Pt), 구리(Cu), 니켈(Ni), 아연(Zn) 및 크롬(Cr)으로 이루어진 군에서 선택되는 것인 암 진단용 조성물.The method of claim 1, wherein the metal is selected from the group consisting of Au, Ag, Pt, Cu, Ni, Zn, and Cr / RTI > 검사하고자 하는 대상체로부터 분리한 생물학적 시료와 제1항의 조성물을 접촉시키는 단계; 및Contacting the biological sample separated from the subject to be examined with the composition of claim 1; And 나노입자와 결합한 엑소좀을 확인하는 단계를 포함하는 암 진단 방법.And identifying the exosome associated with the nanoparticle. 제7항에 있어서, 상기 생물학적 시료는 혈액, 혈청, 혈장, 타액, 객담, 및 뇨로 이루어진 군으로부터 선택되는 것인 암 진단 방법.8. The method according to claim 7, wherein the biological sample is selected from the group consisting of blood, serum, plasma, saliva, sputum, and urine. 전극이 배치된 하부 기판;A lower substrate on which electrodes are disposed; 상기 하부 기판과 연결되어 유체가 유동할 수 있는 유체 채널을 형성하는 상부 기판; 및An upper substrate connected to the lower substrate to form a fluid channel through which fluid can flow; And 외부 전원을 포함하는 엑소좀 검출 및 분리 장치에 있어서, An exosome detection and separation device comprising an external power supply, 상기 전극은 채널을 통하여 이동하는 유체의 흐름 방향과 직교하도록 배치되고,Wherein the electrode is arranged to be orthogonal to the flow direction of the fluid moving through the channel, 상기 유체 채널은 엑소좀 결합 분자가 연결된 나노입자를 포함하는 것인 엑소좀 검출 및 분리 장치.Wherein the fluid channel comprises nanoparticles to which exosomal binding molecules are linked. 제9항에 있어서, 상기 나노입자는 입자 표면의 일부분이 금속으로 코팅된 것인 엑소좀 검출 및 분리 장치.The apparatus of claim 9, wherein the nanoparticles are coated with a metal at a portion of the particle surface.
PCT/KR2018/009290 2017-08-14 2018-08-13 Cancer diagnosis method using lectin-coupled nanoparticles Ceased WO2019035623A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2017-0103250 2017-08-14
KR20170103250 2017-08-14

Publications (2)

Publication Number Publication Date
WO2019035623A2 true WO2019035623A2 (en) 2019-02-21
WO2019035623A3 WO2019035623A3 (en) 2019-05-16

Family

ID=65362700

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2018/009290 Ceased WO2019035623A2 (en) 2017-08-14 2018-08-13 Cancer diagnosis method using lectin-coupled nanoparticles

Country Status (2)

Country Link
KR (1) KR102095018B1 (en)
WO (1) WO2019035623A2 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114720683A (en) * 2022-05-20 2022-07-08 南京鼓楼医院 Preparation method and application of magnetic Janus microcarrier for bladder cancer exosome multivariate analysis

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102179001B1 (en) * 2019-04-22 2020-11-16 중앙대학교 산학협력단 Device for detecting the glycan and method of the same
KR102865711B1 (en) * 2022-09-27 2025-09-26 중앙대학교 산학협력단 Marker capturing cancer diagnosis device and method for diagnosing cancer by market capturing

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100931303B1 (en) * 2008-12-12 2009-12-11 한국과학기술원 Microfluidic Chips for Fine Particle Alignment and Separation Using Inclined Substrates
EP2498094B1 (en) 2010-01-21 2016-02-24 J-Oil Mills, Inc. Method for detecting pancreatic cancer
KR101546076B1 (en) 2013-05-29 2015-08-20 전남대학교산학협력단 Rapid Methods for Detecting Hepatitis A Virus or Norovirus in a Sample Using a Magnetic Bead and Plant-Derived Lectins
US20150110882A1 (en) * 2013-10-18 2015-04-23 Japanese Foundation For Cancer Research Multifunctional metallic nanostructure and method for manufacturing the same

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114720683A (en) * 2022-05-20 2022-07-08 南京鼓楼医院 Preparation method and application of magnetic Janus microcarrier for bladder cancer exosome multivariate analysis

Also Published As

Publication number Publication date
KR20190018395A (en) 2019-02-22
KR102095018B1 (en) 2020-03-30
WO2019035623A3 (en) 2019-05-16

Similar Documents

Publication Publication Date Title
WO2009136741A1 (en) Novel au / ag core-shell composite useful for biosensor
Fan et al. High-sensitive and multiplex biosensing assay of NSCLC-derived exosomes via different recognition sites based on SPRi array
Cooper et al. Tubulovesicular processes emerge from trans-Golgi cisternae, extend along microtubules, and interlink adjacent trans-Golgi elements into a reticulum
WO2019035623A2 (en) Cancer diagnosis method using lectin-coupled nanoparticles
Reza et al. Parallel profiling of cancer cells and proteins using a graphene oxide functionalized ac-EHD SERS immunoassay
O’Brien et al. Monitoring metabolic responses to chemotherapy in single cells and tumors using nanostructure-initiator mass spectrometry (NIMS) imaging
Ortega et al. EGFR detection in extracellular vesicles of breast cancer patients through immunosensor based on silica-chitosan nanoplatform
Kalmykov et al. Bioelectrical interfaces with cortical spheroids in three-dimensions
US20250334579A1 (en) Nanoplasmonic quantification of tumor-derived extracellular vesicles in plasma microsamples for detection and treatment monitoring
Li et al. SERS analysis of carcinoma-associated fibroblasts in a tumor microenvironment based on targeted 2D nanosheets
KR20160128213A (en) Method for searching candidate material for anti-cancer drug using localized surface plasmon resonance
WO2020080833A1 (en) Biomaterial-detecting microparticle and biomaterial detection method using same
Lee et al. Dual-function nanostructured platform for isolation of nasopharyngeal carcinoma circulating tumor cells and EBV DNA detection
WO2017039356A1 (en) Allergen detection apparatus using electrochemical detection method
Chen et al. Comparison of the variability of small extracellular vesicles derived from human liver cancer tissues and cultured from the tissue explants based on a simple enrichment method
WO2020141893A1 (en) Antibody-bound nanowire for exosome separation, and exosome separation method using same
EP2823302B1 (en) Process and device for the determination of alterations in neuronal connectivity and/or morphology
WO2012015125A1 (en) Stimulus sensitive magnetic nanocomposite using pyrene polymer, and contrast medium composition containing the nanocomposite
Li et al. SERS based Y-shaped aptasensor for early diagnosis of acute kidney injury
Wang et al. Study of NSCLC cell migration promoted by NSCLC-derived extracellular vesicle using atomic force microscopy
WO2023113556A1 (en) Two-dimensional or three-dimensional nanocube self-assembly structure, and preparation method thereof
Wakakura et al. Rapid increase in cytosolic calcium ion concentration mediated by acetylcholine receptors in cultured retinal neurons and Müller cells
CN116930480A (en) A portable multi-channel nanochip for detecting biomarkers and its preparation method and application
Chakraborty et al. Acriflavine-induced surface changes in three tumor cell types and differential sensitivity to lectins
Cárdenas et al. Visualization of inositol 1, 4, 5-trisphosphate receptors on the nuclear envelope outer membrane by freeze-drying and rotary shadowing for electron microscopy

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18846092

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18846092

Country of ref document: EP

Kind code of ref document: A2