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WO2019035623A2 - Méthode de diagnostic d'un cancer au moyen de nanoparticules liées à la lectine - Google Patents

Méthode de diagnostic d'un cancer au moyen de nanoparticules liées à la lectine Download PDF

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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
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nanoparticles
lectin
cancer
janus
exosome
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WO2019035623A3 (fr
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최종훈
구형준
김교범
최용현
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Industry Academic Cooperation Foundation of Chung Ang University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—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.

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Abstract

La présente invention concerne une méthode de diagnostic d'un cancer à l'aide de nanoparticules liées à la lectine pour détecter des exosomes, les nanoparticules étant susceptibles de détecter et de séparer efficacement des exosomes sécrétés par les cellules cancéreuses, et pouvant ainsi être utilement appliquées au diagnostic du cancer.
PCT/KR2018/009290 2017-08-14 2018-08-13 Méthode de diagnostic d'un cancer au moyen de nanoparticules liées à la lectine Ceased WO2019035623A2 (fr)

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CN114720683A (zh) * 2022-05-20 2022-07-08 南京鼓楼医院 一种用于膀胱癌外泌体多元分析的磁性Janus微载体的制备方法及应用

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KR102179001B1 (ko) * 2019-04-22 2020-11-16 중앙대학교 산학협력단 특정 당쇄를 검출하기 위한 장치 및 검출 방법
KR102865711B1 (ko) * 2022-09-27 2025-09-26 중앙대학교 산학협력단 표지자 포획식 암 진단 장치 및 표지자 포획식 암 진단 방법

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