WO2022092352A1 - Method for isolating disease-specific exosome - Google Patents

Method for isolating disease-specific exosome Download PDF

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WO2022092352A1
WO2022092352A1 PCT/KR2020/014890 KR2020014890W WO2022092352A1 WO 2022092352 A1 WO2022092352 A1 WO 2022092352A1 KR 2020014890 W KR2020014890 W KR 2020014890W WO 2022092352 A1 WO2022092352 A1 WO 2022092352A1
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exosomes
magnetic nanoparticles
disease
exosome
bound
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Korean (ko)
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함승주
문병걸
정혜인
김륜형
강병훈
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연세대학교 산학협력단
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    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/06—Tissue, human, animal or plant cell, or virus culture apparatus with filtration, ultrafiltration, inverse osmosis or dialysis means
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention relates to a method for isolating disease-specific exosomes from body fluids, and more particularly, to a method for multiple isolation of exosomes overexpressing disease-specific markers from all exosomes at once.
  • Liquid biopsy is an in vitro diagnostic method for diagnosing diseases using circulating tumor cells, cell free circulating tumor DNA, and exosomes present in blood. It is emerging as a next-generation strategy for cancer diagnosis and treatment because it can be used for precise-targeted anti-cancer treatment and can be used for detailed observation of cancer occurrence and metastasis with only the test.
  • Exosomes are spherical vesicles (30-100 nm) that are discharged from cells and contain various proteins, lipids, and genomes that are indicators of cancer progression, metastasis, and drug response. , pleural fluid, saliva, cerebrospinal fluid, breast milk, semen, amniotic fluid, ascites, etc.). In addition, it is attracting attention as the most promising biomarker because it exists at a higher concentration in body fluids than in circulating tumor cells.
  • exosomes derived from disease-related cells such as cancer cells and analyze the genes in the exosomes and disease-specific markers on the surface of the exosomes together.
  • disease-related cells such as cancer cells
  • the number of exosomes overexpressing disease-specific markers is very few, about 1000. Therefore, it is necessary to separate the two types of exosomes so that the signals of exosomes overexpressing disease-specific markers are not obscured by signals of exosomes discharged from normal cells in disease diagnosis and prognosis discrimination.
  • a representative method for isolating exosomes is a method using centrifugation. It takes a long time to separate exosomes by co-precipitating them with PEG or dextran polymers, and each time the exosomes are separated, the sample is lost. .
  • exosomes overexpressing disease-specific markers and non-expressing exosomes cannot be distinguished, in order to separate normal cell-derived exosomes from exosomes overexpressing disease-specific markers, after separating the entire exosomes from body fluids, specific markers should be used to further isolate exosomes overexpressing disease-specific markers. This method has a disadvantage in that the sample loss rate is high and a considerable amount of time (6 hours or more) is required.
  • exosomes to which the magnetic nanoparticles are bound are injected into the microfluidic channel and separated into different recovery paths in the microfluidic channel,
  • the magnetic nanoparticles have a different magnetization from the first magnetic nanoparticles and the first magnetic nanoparticles to which a probe for detecting an exosome common expression marker is bound, and a probe for detecting a disease-specific exosome overexpression marker is bound It provides a method for separating disease-specific exosomes containing two magnetic nanoparticles.
  • the present inventors studied a method for separating disease-specific exosomes present in a small number in body fluids from normal cell-derived exosomes at once. Based on the presence of a large number of , magnetic nanoparticles labeled with a probe for detecting a disease-specific exosome overexpression marker or a probe for detecting a common exosome expression marker were prepared. Relatively few disease-specific markers exist in normal cell-derived exosomes. Therefore, when a sample containing exosomes is brought into contact with the two types of magnetic nanoparticles, both types of magnetic nanoparticles bind to the disease-specific exosomes, or a probe for detecting disease-specific exosome overexpression markers is labeled. A lot of magnetic nanoparticles bind. On the other hand, a lot of magnetic nanoparticles labeled with exosome common expression markers bind to normal cell-derived exosomes.
  • first magnetic nanoparticles and “second magnetic nanoparticles” are for distinguishing nanoparticles having different sizes and labeled probes, and do not limit the scope of magnetic nanoparticles, and may be used interchangeably.
  • the size of the first magnetic nanoparticles may be 100 to 800 nm, and the size of the second magnetic nanoparticles may be 200 to 1000 nm.
  • the size of the first magnetic nanoparticles is 100 to 600 nm
  • the size of the second magnetic nanoparticles may be 200 to 800 nm
  • more preferably the size of the first magnetic nanoparticles is 100 to 500 nm
  • the size of the second magnetic nanoparticles may be 200 to 600 nm.
  • the size of the magnetic nanoparticles is less than 100 nm in diameter, the exosome size is less than 100 nm and the separation ability is lowered, and the magnetic nanoparticles of 1000 nm or more have remarkably reduced dispersibility, making separation impossible.
  • the magnetic nanoparticles may be in the form of magnetic nanocluster.
  • Magnetic nanocluster is a particle synthesized by aggregation of several single magnetic nanoparticles, and can be synthesized in various sizes by controlling the amount of single magnetic nanoparticles. Since the magnetization of the cluster can be increased by the sum of the magnetizations of a single magnetic nanoparticle, the magnetization increases as the size of the magnetic nanocluster increases.
  • the first magnetic nanoparticles and the second magnetic nanoparticles have the same composition so that factors other than the size of the nanoparticles do not affect the magnetic sensitivity or the strength of magnetization.
  • the exosome common expression marker refers to a marker found in all exosomes regardless of the parent cell from which the exosomes are derived, and may be selected from the group consisting of CD9, CD63, CD81 and CD82.
  • CD81 was used as an exosome common expression marker.
  • disease-specific exosome refers to an exosome derived from a disease-associated cell, and a representative disease-associated cell includes cancer cells.
  • the disease-specific exosome expression marker refers to a marker specifically or commonly found in exosomes discharged from disease-associated cells, and may have a specific marker depending on the derived disease-associated cell.
  • the disease-specific exosome expression marker is HER2 (human epidermal growth factor receptor 2), PSA (prostate specific antigen), CEA (carcinoembryonic antigen), AFP (alpha fetoprotein), CA125 (cancer antigen 125), CA 15-3 ( It can be selected from the group consisting of cancer antigen 15-3) and CA 19-9 (cancer antigen 19-9), and HER2 was used in the present invention.
  • the term "probe” refers to a substance capable of specifically binding to a marker expressed in exosomes, monoclonal antibody, polyclonal antibody, chimeric antibody, Fab, F(ab') 2 , Fab' , scFv (single chain fragment variable), single-domain antibody (single-domain antibody), aptamer, it may be selected from the group consisting of a peptide and a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • a HER2 antibody was used as a probe for detecting a disease-specific exosome expression marker
  • a CD9 antibody was used as a probe for detecting a common exosome expression marker.
  • a probe for detecting a common exosome expression marker was coupled to magnetic nanoparticles with a small magnetization (size), and magnetic nanoparticles with a large magnetization (size)
  • a disease-specific exosome expression marker detection probe was bound to the particle.
  • the binding between the magnetic nanoparticles and the probe may be achieved by amide bond, but any binding method may be used as long as it can induce stable binding between the magnetic nanoparticles and the probe.
  • the disease-specific exosome separation method starts with the step of obtaining exosomes to which magnetic nanoparticles are bound by contacting a sample containing exosomes with two or more types of magnetic nanoparticles having different degrees of magnetization.
  • a method of binding exosomes to magnetic nanoparticles may use an antigen-antibody reaction.
  • the exosome and buffer to which the magnetic nanoparticles are bound are injected into the microfluidic channel to which a magnetic field is applied, and the injection rate of the exosome to which the magnetic nanoparticles are bound may vary depending on the size of the microfluidic channel, but 2 ml/hr It is preferable to use from 10 ml/hr. When it is in the above range, the exosomes treated with magnetic nanoparticles can be effectively affected by the magnetic field.
  • the buffer used for a constant laminar flow is preferably injected at a rate of 8 ml/hr to 40 ml/hr according to the injection rate of the sample injection unit and a ratio of 1:4.
  • the injection volume per unit length of the sample injection unit is 1, the injection volume of the buffer injection unit is 4 times larger.
  • the buffer It is desirable to quadruple the injection rate of the injection unit.
  • the injection rate of the buffer injection unit compared to the injection rate of the sample injection unit may be changed according to the design of the microfluidic channel.
  • the flow of the sample and the buffer in the microfluidic channel structure can be controlled using a method well known in the art. These include electroosmotic flow that electrically moves a small amount of liquid sample, a method using a membrane pump, a syringe pump, and the like.
  • the exosomes bound with magnetic nanoparticles are injected into the microfluidic channel through the sample injection unit and pass through the main channel to which an external magnetic field of a certain size is applied. .
  • the first magnetic nanoparticles to which the probe for detecting the common exosome expression marker is bound to the normal cell-derived exosomes, but the first magnetic nanoparticles as well as the first magnetic nanoparticles to the disease-specific exosomes for detecting the disease-specific exosome expression marker Since the second magnetic nanoparticles to which the probe is bound are also bound, there is a large difference in the intensity of magnetic sensitivity or magnetization between the two exosomes.
  • the strength of the magnetic field is preferably set to 500 G to 3000 G (0.05 T to 0.3 T). If the intensity is too weak, the exosomes do not separate from each other, so only disease-specific exosomes cannot be separated.
  • the magnetic device may include applying an external magnetic field from a permanent magnet or an electromagnet.
  • the permanent magnets are produced from nickel, cobalt, iron, alloys thereof and alloys of non-ferromagnetic materials that become ferromagnetic by alloying, ie, alloys known as Heusler alloys (eg, alloys of copper, tin and manganese).
  • Heusler alloys eg, alloys of copper, tin and manganese.
  • suitable alloys for permanent magnets are known and commercially available for making magnets usable in one embodiment of the present invention.
  • a typical such material is a transition metal-semimetal (metalloid) alloy, with a transition metal (usually Fe, Co, or Ni) about 80% and a melting point lowering metalloid component (boron, carbon, silicon, phosphorus or aluminum).
  • Permanent magnets may be crystalline or amorphous.
  • An example of an amorphous alloy is Fe80B20 (Metglas 2605).
  • the external magnetic field is provided by a rectangular (2.5 cm x 2.5 cm x 4.0 cm) neodymium (NdFeB) magnet (K&J Magnetics, Jamison, PA) attached to the upper and lower surfaces of the main channel of the microfluidic channel. do.
  • the method may further include the step of inhaling the exosomes combined with magnetic nanoparticles separated by different recovery paths after step (b) (c), wherein the suction is performed by using a pump connected to the outlet can be done through
  • the inhalation may be made at a rate ranging from 10 ml/hr to 50 ml/hr.
  • disease-specific exosomes can be separated from normal cell-derived exosomes at once, and this method is different from the existing exosome separation method of isolating the disease-related exosomes again after separating the entire exosomes.
  • the process is simple and only disease-related exosomes can be concentrated and isolated, and the isolated disease-specific exosomes can be utilized for disease diagnosis and prognosis prediction.
  • FIG. 1 is a schematic diagram of the principle of multiple separation of exosomes using a microfluidic channel, according to an example of the present invention.
  • Figure 2 shows a schematic diagram of a microfluidic channel for multiple separation of exosomes, according to an example of the present invention.
  • FIG. 3 shows a microfluidic channel structure for multiple separation of exosomes manufactured according to an example of the present invention.
  • Figure 4 is the result of confirming the magnetic nanoparticles of triiron tetraoxide (Fe 3 O 4 ) of various sizes synthesized according to a preparation example of the present invention by a transmission electron microscope: (A) is 100 nm, (B) is 200 nm, (C) is 300 nm and (D) is a 400 nm size of magnetic nanoparticles.
  • VSM vibrating sample magnetometer
  • (A) is a transmission electron microscope image of exosomes not treated with magnetic nanoparticles
  • B is CD9- bound to exosomes derived from HCC_1143 cells Transmission electron microscope image of magnetic nanoparticles
  • C is a scanning electron microscope image of CD9-magnetic nanoparticles bound to HCC_1143 cell-derived exosomes
  • D is a transmission electron microscope image of HER2-magnetic nanoparticles bound to HCC_1954 cell-derived exosomes
  • E is a scanning electron microscope image of HER2-magnetic nanoparticles bound to HCC_1954 cell-derived exosomes.
  • HCC_1954 is an image obtained by confocal microscopy after binding each of magnetic nanoparticles of different sizes (labeled with HER2 antibody or CD9 antibody) with exosomes derived from a HER2-overexpressing cell line (HCC_1954).
  • Figure 10 is the result of confirming the trajectory of the exosomes by optical microscopy after binding each of magnetic nanoparticles (labeled with HER2 antibody or CD9 antibody) of different sizes with exosomes derived from the HER2-overexpressing cell line (HCC_1954) and injecting them into microfluidic channels: (A-C) Exosome behavior by location in microfluidic channels in the absence of an external magnetic field; (D-F) Exosome behavior by location in microfluidic channels under an external magnetic field; and (G-J) exosome behavior by outlet under an external magnetic field.
  • A-C Exosome behavior by location in microfluidic channels in the absence of an external magnetic field
  • D-F Exosome behavior by location in microfluidic channels under an external magnetic field
  • G-J exosome behavior by outlet under an external magnetic field.
  • each of magnetic nanoparticles (labeled with HER2 antibody or CD9 antibody) of different sizes is combined with exosomes derived from a HER2-overexpressing cell line (HCC_1954), and these are injected into microfluidic channels at different total flow rates, and then outlet 2 compared to the total flow rate. And it is a result of confirming the ratio of the exosomes recovered in 4.
  • FIG. 12 is a diagram showing each of magnetic nanoparticles (labeled with HER2 antibody or CD9 antibody) of different sizes is combined with exosomes derived from a HER2-overexpressing cell line (HCC_1954), and after injecting them into microfluidic channels, exosomes are recovered from each outlet to indicate disease markers. This is the result of confirming the expression rate of the gene.
  • a pattern using SU-8 photosensitive resin on a silicon wafer was fabricated as shown in FIG. After that, liquid polydimethylsiloxane (PDMS) was solidified in a casting mold to make a chip, and the chip was attached to a slide glass to make a microfluidic channel device.
  • PDMS liquid polydimethylsiloxane
  • the injection hole is divided into a sample inlet (one channel, 1 in FIG. 2), one channel with a buffer inlet (divided into 8 channels after injection, 2 in FIG. 2), and a magnetic field is applied from the outside. It includes an outlet composed of a main channel (4 in FIG. 2) and a total of 4 channels (grouped by 2, 3 in FIG. 2). 3 shows a photograph of the actually fabricated microfluidic channel.
  • the sample inlet was arranged on the side to confirm the behavior of the magnetic nanoparticle-coated exosome sample, and the buffer inlet was increased to eight channels in order to reduce the parabolic fluid flow due to the laminar flow effect. composed.
  • a phosphate buffered saline (PBS) solution mixed with 10% of bovine serum albumin (BSA) was quickly flowed after production. was produced.
  • magnetic nanoclusters having diameters of 200 nm and 400 nm, respectively, were used.
  • ferric chloride FeCl 3
  • sodium acetate sodium citric acid
  • the composition of the magnetic nanoparticles was triiron tetraoxide (Fe 3 O 4 ), and had a surface and various sizes as shown in FIG. 4 .
  • VSM vibrating sample magnetometer
  • an antibody binding to the disease-specific marker and the magnetic nanoparticles synthesized in 2-1 were combined.
  • HER2 antibody EPR19547-12 (ab214275), purchased from Abcam
  • CD9 antibody EPR23105-121 (ab236630), Abcam
  • CD9 antibody binds to CD81 on the surface of exosomes.
  • the antibodies were combined with magnetic nanoparticles of different sizes.
  • amide bonds were induced with 1-ethyl 3-dimethylaminopropyl carbodiimide and hydroxysuccinimide to prepare antibody-bound magnetic nanoparticles: 400 nm magnetic nanoparticles 10 mg + anti-HER2 antibody 10 ⁇ g; and 10 mg of 200 nm magnetic particles + 10 ⁇ g of anti-CD9 antibody.
  • HER2-magnetic nanoparticles 400 nm
  • CD9-magnetic nanoparticles 200 nm
  • exosomes were collected and obtained in a culture medium according to a method known in the art. After culturing HCC_1143 cells, which are HER2 non-expressing cells, exosomes were isolated in the same manner. Each of the isolated exosomes was mixed with 2 mg of HER2-magnetic nanoparticles (400 nm) or 2 mg of CD9-magnetic nanoparticles (200 nm) to prepare a total of four exosome-magnetic nanoparticle complexes.
  • A is a transmission electron microscope image of exosomes not treated with magnetic nanoparticles
  • B is a transmission electron microscope image of CD9-magnetic nanoparticles bound to HCC_1143 cell-derived exosomes
  • C is CD9 bound to HCC_1143 cell-derived exosomes.
  • D is a transmission electron microscope image of HER2-magnetic nanoparticles bound to HCC_1954 cell-derived exosomes
  • E is a scanning electron microscope image of HER2-magnetic nanoparticles bound to HCC_1954 cell-derived exosomes.
  • HCC_1954 cells which are HER2-overexpressing cells.
  • FIG. 10 shows an image that appears when a magnet is placed in the lower part of the main channel in the microfluidic channel shown in FIG. 2 and exosomes having magnetic nanoparticles attached thereto are flowed through the microfluidic channel.
  • FIG. 11 shows the fraction of exosomes obtained from outlets 2 and 4 compared to the amount of exosomes injected after performing an exosome separation experiment by changing the total flow rate to 5, 15, 25, 35, and 50 ml/hr shown in a circular table. It was confirmed that the sample could be obtained with high efficiency when the separation experiment was performed at a total flow rate of 20 ml/hr (sample inlet 4 ml/hr).

Abstract

The present invention relates to a method whereby multiple exosomes overexpressing a disease-specific marker are isolated at the same time from the total population of exosomes. Compared to existing exosome isolation methods, in which exosomes are isolated from the total population of exosomes and then disease-related exosomes are isolated from the exosomes isolated from the total population, the method is simple and has the advantage of being able to concentrate and isolate only the disease-related exosomes, and the isolated disease-related exosome can be used for diagnosing and predicting the prognosis of diseases.

Description

질병 특이적 엑소좀의 분리 방법Isolation method for disease-specific exosomes
본 발명은 체액으로부터 질병 특이적 엑소좀을 분리하는 방법에 관한 것으로, 구체적으로 질병 특이적 마커를 과발현하는 엑소좀을 전체 엑소좀과 한번에 다중 분리하는 방법에 관한 것이다.The present invention relates to a method for isolating disease-specific exosomes from body fluids, and more particularly, to a method for multiple isolation of exosomes overexpressing disease-specific markers from all exosomes at once.
액체 생체검사는 혈액 내 존재하는 순환종양세포(circulating tumor cells), 세포유리순환종양 DNA (cell free circulating tumor DNA), 엑소좀(exosome) 등을 이용하여 질병을 진단하는 체외 진단법으로 혈액 등의 체액 검사만으로 암 발생 및 전이 등에 대한 상세관찰이 가능하고, 정밀-표적 항암 치료에 사용될 수 있기 때문에 암 진단 및 치료를 위한 차세대 전략으로 떠오르고 있다.Liquid biopsy is an in vitro diagnostic method for diagnosing diseases using circulating tumor cells, cell free circulating tumor DNA, and exosomes present in blood. It is emerging as a next-generation strategy for cancer diagnosis and treatment because it can be used for precise-targeted anti-cancer treatment and can be used for detailed observation of cancer occurrence and metastasis with only the test.
엑소좀은 세포에서 배출되는 구형의 소낭(30~100 ㎚)으로 암의 진행, 전이, 약제 반응의 지표가 되는 다양한 단백질, 지방질, 유전체를 담고 있으며, 매우 안정한 물질이므로 거의 모든 체액 (혈액, 오줌, 흉수, 침, 뇌척수, 모유, 정액, 양수, 복수 등)에서 발견된다. 또한, 순환종양세포보다 체액에 높은 농도로 존재하기 때문에 가장 유망한 바이오마커로 주목을 받고 있다.Exosomes are spherical vesicles (30-100 nm) that are discharged from cells and contain various proteins, lipids, and genomes that are indicators of cancer progression, metastasis, and drug response. , pleural fluid, saliva, cerebrospinal fluid, breast milk, semen, amniotic fluid, ascites, etc.). In addition, it is attracting attention as the most promising biomarker because it exists at a higher concentration in body fluids than in circulating tumor cells.
질병 진단 및 예후 판별에 있어 암세포와 같은 질병 관련 세포에서 유래된 엑소좀을 분리하여 엑소좀 안에 있는 유전자와 엑소좀 표면에 있는 질병 특이적 마커를 함께 분석하면 도움이 된다. 그러나 체액 1 ㎖ 내에 10억 개 이상으로 존재하는 엑소좀은 대부분 정상 세포에서 유래된 엑소좀이며, 질병 특이적 마커를 과발현하는 엑소좀은 1000개 정도로 매우 소수이다. 따라서 질병 진단 및 예후 판별에 있어 질병 특이적 마커를 과발현하는 엑소좀의 신호가 정상 세포에서 배출된 엑소좀의 신호에 가려지지 않도록 상기 두 종류의 엑소좀을 분리할 필요가 있다.In disease diagnosis and prognosis discrimination, it is helpful to isolate exosomes derived from disease-related cells such as cancer cells and analyze the genes in the exosomes and disease-specific markers on the surface of the exosomes together. However, most of the exosomes present in 1 ml of body fluid are exosomes derived from normal cells, and the number of exosomes overexpressing disease-specific markers is very few, about 1000. Therefore, it is necessary to separate the two types of exosomes so that the signals of exosomes overexpressing disease-specific markers are not obscured by signals of exosomes discharged from normal cells in disease diagnosis and prognosis discrimination.
현재 엑소좀을 분리하는 대표적인 방법은 원심분리를 이용한 방법으로 PEG나 덱스트란류의 고분자로 엑소좀을 공동침전시켜 분리하는데 시간이 오래 걸리고, 엑소좀을 분리할 때마다 시료가 소실되는 단점이 있다. 또한, 질병 특이적 마커를 과발현하는 엑소좀과 비발현 엑소좀을 구별할 수 없어 정상 세포 유래 엑소좀과 질병 특이적 마커를 과발현하는 엑소좀을 분리하려면 체액으로부터 전체 엑소좀을 분리한 후 특정 마커를 사용하여 질병 특이적 마커를 과발현하는 엑소좀을 추가적으로 분리해야 한다. 이러한 방법은 시료의 소실률이 높고 상당한 시간(6시간 이상)이 소요되는 단점이 있다.Currently, a representative method for isolating exosomes is a method using centrifugation. It takes a long time to separate exosomes by co-precipitating them with PEG or dextran polymers, and each time the exosomes are separated, the sample is lost. . In addition, since exosomes overexpressing disease-specific markers and non-expressing exosomes cannot be distinguished, in order to separate normal cell-derived exosomes from exosomes overexpressing disease-specific markers, after separating the entire exosomes from body fluids, specific markers should be used to further isolate exosomes overexpressing disease-specific markers. This method has a disadvantage in that the sample loss rate is high and a considerable amount of time (6 hours or more) is required.
따라서, 현재 질병 마커 과발현 엑소좀을 전체 엑소좀과 한번에 다중 분리하는 액체 생검 기술이 전무한 실정이다. 이러한 상황에서 본 발명자들은 자화도 및 크기가 상이한 2종 이상의 자성 나노입자로 정상 세포 유래 엑소좀과 질병 관련 세포에서 유래하여 질병 특이적 마커를 과발현하는 엑소좀을 한번에 분리하는 방법을 개발하여 본 발명을 완성하였다.Therefore, there is currently no liquid biopsy technology for multiple separation of exosomes overexpressing disease markers from all exosomes at once. In this situation, the present inventors developed a method for separating normal cell-derived exosomes and exosomes derived from disease-related cells and overexpressing disease-specific markers at once with two or more types of magnetic nanoparticles having different degrees of magnetization and size, and the present invention was completed.
본 발명의 목적은 자성 나노입자의 자화도 차이를 이용하여 질병 특이적 엑소좀을 정상 세포 유래 엑소좀과 한번에 분리하는 방법을 제공하는 것이다.It is an object of the present invention to provide a method for separating disease-specific exosomes from normal cell-derived exosomes at once by using the difference in magnetization degree of magnetic nanoparticles.
상기 목적을 달성하기 위하여, 본 발명의 일 양상은 In order to achieve the above object, an aspect of the present invention is
(a) 엑소좀이 포함된 시료와 자화도가 서로 상이한 2종 이상의 자성 나노입자를 접촉시켜 자성 나노입자가 결합된 엑소좀을 얻는 단계; 및(a) contacting a sample containing exosomes with two or more types of magnetic nanoparticles having different degrees of magnetization to obtain exosomes to which magnetic nanoparticles are bound; and
(b) 자성 나노입자가 결합된 엑소좀 및 버퍼를 자기장이 인가된 미세유체채널에 주입하는 단계를 포함하되,(b) injecting the exosomes and the buffer to which the magnetic nanoparticles are bound into the microfluidic channel to which the magnetic field is applied,
상기 자성 나노입자가 결합된 엑소좀은 미세유체채널에 주입되어 미세유체채널 내의 서로 다른 회수 경로로 분리되고,The exosomes to which the magnetic nanoparticles are bound are injected into the microfluidic channel and separated into different recovery paths in the microfluidic channel,
상기 자성 나노입자는 엑소좀 공통 발현 마커를 탐지하는 프로브가 결합된 제1 자성 나노입자 및 제1 자성 나노입자와 자화도가 상이하고, 질병 특이적 엑소좀 과발현 마커를 탐지하는 프로브가 결합된 제2 자성 나노입자를 포함하는 질병 특이적 엑소좀의 분리방법을 제공한다.The magnetic nanoparticles have a different magnetization from the first magnetic nanoparticles and the first magnetic nanoparticles to which a probe for detecting an exosome common expression marker is bound, and a probe for detecting a disease-specific exosome overexpression marker is bound It provides a method for separating disease-specific exosomes containing two magnetic nanoparticles.
본 발명자들은 체액 내에 소수로 존재하는 질병 특이적 엑소좀을 정상 세포 유래 엑소좀과 한번에 분리할 수 있는 방법을 연구한 결과, 질병 특이적 엑소좀에는 엑소좀 공통 발현 마커뿐만 아니라 질병 특이적 발현 마커가 많이 존재하는 점에 근거하여 질병 특이적 엑소좀 과발현 마커 탐지용 프로브, 또는 엑소좀 공통 발현 마커 탐지용 프로브가 표지된 자성 나노입자를 제조하였다. 정상 세포 유래 엑소좀에는 질병 특이적 마커가 상대적으로 적게 존재한다. 따라서, 엑소좀이 포함된 시료와 상기 2종의 자성 나노입자를 접촉시키면 질병 특이적 엑소좀에는 2종의 자성 나노입자 모두가 결합하거나, 질병 특이적 엑소좀 과발현 마커를 탐지하는 프로브가 표지된 자성 나노입자가 많이 결합한다. 반면, 정상 세포 유래 엑소좀에는 엑소좀 공통 발현 마커가 표지된 자성 나노입자가 많이 결합한다.The present inventors studied a method for separating disease-specific exosomes present in a small number in body fluids from normal cell-derived exosomes at once. Based on the presence of a large number of , magnetic nanoparticles labeled with a probe for detecting a disease-specific exosome overexpression marker or a probe for detecting a common exosome expression marker were prepared. Relatively few disease-specific markers exist in normal cell-derived exosomes. Therefore, when a sample containing exosomes is brought into contact with the two types of magnetic nanoparticles, both types of magnetic nanoparticles bind to the disease-specific exosomes, or a probe for detecting disease-specific exosome overexpression markers is labeled. A lot of magnetic nanoparticles bind. On the other hand, a lot of magnetic nanoparticles labeled with exosome common expression markers bind to normal cell-derived exosomes.
본 발명에서, 용어 "제1 자성 나노입자", "제2 자성 나노입자"는 크기 및 표지되는 프로브가 상이한 나노입자를 구분하기 위한 것으로 자성 나노입자의 범위를 제한하는 것은 아니며, 서로 혼용되어 사용될 수 있다. In the present invention, the terms "first magnetic nanoparticles" and "second magnetic nanoparticles" are for distinguishing nanoparticles having different sizes and labeled probes, and do not limit the scope of magnetic nanoparticles, and may be used interchangeably. can
본 발명의 일 구체예에 따르면, 상기 제1 자성 나노입자의 크기는 100 내지 800 ㎚, 상기 제2 자성 나노입자의 크기는 200 내지 1000 ㎚일 수 있다. 바람직하게는 제1 자성 나노입자의 크기는 100 내지 600 ㎚, 상기 제2 자성 나노입자의 크기는 200 내지 800 ㎚일 수 있고, 더욱 바람직하게는 제1 자성 나노입자의 크기는 100 내지 500 ㎚, 상기 제2 자성 나노입자의 크기는 200 내지 600 ㎚일 수 있다.According to an embodiment of the present invention, the size of the first magnetic nanoparticles may be 100 to 800 nm, and the size of the second magnetic nanoparticles may be 200 to 1000 nm. Preferably, the size of the first magnetic nanoparticles is 100 to 600 nm, the size of the second magnetic nanoparticles may be 200 to 800 nm, more preferably the size of the first magnetic nanoparticles is 100 to 500 nm, The size of the second magnetic nanoparticles may be 200 to 600 nm.
자성 나노입자의 크기가 지름 100 ㎚ 이하이면 엑소좀의 크기가 100 ㎚ 이하여서 분리능이 떨어지며, 1000 ㎚ 이상의 자성 나노입자는 분산성이 현저하게 감소하여 분리가 불가능해진다.If the size of the magnetic nanoparticles is less than 100 nm in diameter, the exosome size is less than 100 nm and the separation ability is lowered, and the magnetic nanoparticles of 1000 nm or more have remarkably reduced dispersibility, making separation impossible.
본 발명의 일 구체예에 따르면, 상기 자성 나노입자는 자성 나노 클러스터 형태일 수 있다. 자성 나노 클러스터는 여러 개의 단일 자성 나노입자가 뭉쳐져서 합성되는 입자로 단일 자성 나노입자의 양을 조절하여 다양한 크기로 합성이 가능하다. 단일 자성 나노입자의 자화도의 합으로 클러스터의 자화도를 높일 수 있기 때문에 자성 나노 클러스터의 크기가 커질수록 자화도가 커지게 된다. According to one embodiment of the present invention, the magnetic nanoparticles may be in the form of magnetic nanocluster. Magnetic nanocluster is a particle synthesized by aggregation of several single magnetic nanoparticles, and can be synthesized in various sizes by controlling the amount of single magnetic nanoparticles. Since the magnetization of the cluster can be increased by the sum of the magnetizations of a single magnetic nanoparticle, the magnetization increases as the size of the magnetic nanocluster increases.
본 발명에서는 자성 나노입자의 크기에 따른 자화도 차이를 이용하여 암세포 유래 엑소좀, 정상 엑소좀을 동시에 분리하였다. 따라서, 상기 제1 자성 나노입자 및 제2 자성 나노입자는 나노입자의 크기 이외에 다른 요인이 자기 민감도 또는 자화도의 세기에 영향을 미치지 않도록 동일한 조성을 사용하는 것이 바람직하다. In the present invention, cancer cell-derived exosomes and normal exosomes were simultaneously separated using the difference in magnetization according to the size of magnetic nanoparticles. Therefore, it is preferable that the first magnetic nanoparticles and the second magnetic nanoparticles have the same composition so that factors other than the size of the nanoparticles do not affect the magnetic sensitivity or the strength of magnetization.
본 발명에서, 상기 엑소좀 공통 발현 마커는 엑소좀이 유래한 부모세포와 무관하게 모든 엑소좀에서 발견되는 마커를 말하며, CD9, CD63, CD81 및 CD82로 이루어진 군에서 선택될 수 있다. 본 발명에서는 엑소좀 공통 발현 마커로 CD81을 사용하였다.In the present invention, the exosome common expression marker refers to a marker found in all exosomes regardless of the parent cell from which the exosomes are derived, and may be selected from the group consisting of CD9, CD63, CD81 and CD82. In the present invention, CD81 was used as an exosome common expression marker.
본 발명에서, 용어 "질병 특이적 엑소좀"은 질병 연관 세포에서 유래한 엑소좀을 말하며, 대표적인 질병 연관 세포에는 암세포가 있다. 상기 질병 특이적 엑소좀 발현 마커는 질병 연관 세포에서 배출된 엑소좀에서 특이적 또는 공통적으로 발견되는 마커를 말하며, 유래한 질병 연관 세포에 따라 특이적 마커를 가질 수 있다. 상기 질병 특이적 엑소좀 발현 마커는 HER2 (human epidermal growth factor receptor 2), PSA (prostate specific antigen), CEA (carcinoembryonic antigen), AFP (alpha fetoprotein), CA125 (cancer antigen 125), CA 15-3 (cancer antigen 15-3) 및 CA 19-9 (cancer antigen 19-9)로 이루어진 군에서 선택될 수 있으며, 본 발명에서는 HER2를 사용하였다.In the present invention, the term “disease-specific exosome” refers to an exosome derived from a disease-associated cell, and a representative disease-associated cell includes cancer cells. The disease-specific exosome expression marker refers to a marker specifically or commonly found in exosomes discharged from disease-associated cells, and may have a specific marker depending on the derived disease-associated cell. The disease-specific exosome expression marker is HER2 (human epidermal growth factor receptor 2), PSA (prostate specific antigen), CEA (carcinoembryonic antigen), AFP (alpha fetoprotein), CA125 (cancer antigen 125), CA 15-3 ( It can be selected from the group consisting of cancer antigen 15-3) and CA 19-9 (cancer antigen 19-9), and HER2 was used in the present invention.
본 발명에서, 용어 "프로브"는 엑소좀에 발현된 마커에 특이적으로 결합할 수 있는 물질을 말하며, 단일클론항체, 폴리클론항체, 키메릭 항체, Fab, F(ab') 2, Fab', scFv(single chain fragment variable), 단일-도메인 항체(single-domain antibody), 압타머, 펩타이드 및 PNA(Peptide nucleic acid)로 이루어진 군에서 선택될 수 있다.In the present invention, the term "probe" refers to a substance capable of specifically binding to a marker expressed in exosomes, monoclonal antibody, polyclonal antibody, chimeric antibody, Fab, F(ab') 2 , Fab' , scFv (single chain fragment variable), single-domain antibody (single-domain antibody), aptamer, it may be selected from the group consisting of a peptide and a peptide nucleic acid (PNA).
본 발명에서는 질병 특이적 엑소좀 발현 마커를 탐지하는 프로브로 HER2 항체, 엑소좀 공통 발현 마커 탐지 프로브로는 CD9 항체를 사용하였다. 이때 자성 나노입자의 자화도 차이를 이용하여 상기 두 엑소좀을 분리하기 위해 자화도(크기)가 작은 자성 나노입자에는 엑소좀 공통 발현 마커 탐지 프로브를 결합시켰고, 자화도(크기)가 큰 자성 나노입자에는 질병 특이적 엑소좀 발현 마커 탐지 프로브를 결합시켰다. In the present invention, a HER2 antibody was used as a probe for detecting a disease-specific exosome expression marker, and a CD9 antibody was used as a probe for detecting a common exosome expression marker. At this time, in order to separate the two exosomes using the difference in magnetization of the magnetic nanoparticles, a probe for detecting a common exosome expression marker was coupled to magnetic nanoparticles with a small magnetization (size), and magnetic nanoparticles with a large magnetization (size) A disease-specific exosome expression marker detection probe was bound to the particle.
본 발명의 일 구체예에 따르면, 상기 자성 나노입자와 프로브의 결합은 아마이드 결합에 의해 이루어질 수 있으나, 자성 나노입자와 프로브 사이에 안정적인 결합을 유도할 수 있다면 어떠한 결합 방법도 사용할 수 있다.According to one embodiment of the present invention, the binding between the magnetic nanoparticles and the probe may be achieved by amide bond, but any binding method may be used as long as it can induce stable binding between the magnetic nanoparticles and the probe.
본 발명의 일 예에 따른 질병 특이적 엑소좀 분리 방법은 엑소좀을 포함하는 시료와 자화도가 서로 상이한 2종 이상의 자성 나노입자를 접촉시켜 자성 나노입자가 결합된 엑소좀을 얻는 단계에서 시작된다. 엑소좀과 자성 나노입자를 결합시키는 방법은 항원-항체 반응을 이용할 수 있다. The disease-specific exosome separation method according to an embodiment of the present invention starts with the step of obtaining exosomes to which magnetic nanoparticles are bound by contacting a sample containing exosomes with two or more types of magnetic nanoparticles having different degrees of magnetization. . A method of binding exosomes to magnetic nanoparticles may use an antigen-antibody reaction.
이후 자성 나노입자가 결합된 엑소좀 및 버퍼를 자기장이 인가된 미세유체채널에 주입하며, 자성 나노입자가 결합된 엑소좀의 주입 속도는 미세유체채널의 크기에 따라 달라질 수 있으나, 2 ㎖/hr 내지 10 ㎖/hr으로 사용하는 것이 바람직하다. 상기 범위일 때 자성 나노입자를 처리(treat)한 엑소좀이 효과적으로 자기장의 영향을 받을 수 있다. 또한, 일정한 층류를 위해 사용되는 버퍼는 시료 주입부의 주입 속도와 1:4의 비율로 맞추어 8 ㎖/hr 내지 40 ㎖/hr으로 주입하는 것이 바람직하다. 도 2에 제시된 미세유체채널 구조에서 시료 주입부의 단위 길이당 주입 부피를 1로 보면 버퍼 주입부의 주입 부피가 4배 크므로 시료와 버퍼가 만나는 곳(분리 부분)의 유속을 일정하게 유지하기 위해서는 버퍼 주입부의 주입 속도를 4배로 하는 것이 바람직하다. 그러나 시료 주입부의 주입 속도 대비 버퍼 주입부의 주입 속도는 미세유체채널의 디자인에 따라 변화될 수 있다.Afterwards, the exosome and buffer to which the magnetic nanoparticles are bound are injected into the microfluidic channel to which a magnetic field is applied, and the injection rate of the exosome to which the magnetic nanoparticles are bound may vary depending on the size of the microfluidic channel, but 2 ml/hr It is preferable to use from 10 ml/hr. When it is in the above range, the exosomes treated with magnetic nanoparticles can be effectively affected by the magnetic field. In addition, the buffer used for a constant laminar flow is preferably injected at a rate of 8 ml/hr to 40 ml/hr according to the injection rate of the sample injection unit and a ratio of 1:4. In the microfluidic channel structure shown in FIG. 2, if the injection volume per unit length of the sample injection unit is 1, the injection volume of the buffer injection unit is 4 times larger. In order to maintain a constant flow rate at the place where the sample and the buffer meet (separation part), the buffer It is desirable to quadruple the injection rate of the injection unit. However, the injection rate of the buffer injection unit compared to the injection rate of the sample injection unit may be changed according to the design of the microfluidic channel.
상기 미세유체채널 구조물에서 시료 및 버퍼의 흐름은 이 분야에 널리 알려진 방법을 사용하여 조절할 수 있다. 여기에는 전기적으로 적은 양의 액체시료를 이동시키는 전기삼투유체흐름(electroosmotic flow), 박막펌프(membrane pump), 시린지펌프(syringe pump)를 사용하는 방법 등이 포함된다.The flow of the sample and the buffer in the microfluidic channel structure can be controlled using a method well known in the art. These include electroosmotic flow that electrically moves a small amount of liquid sample, a method using a membrane pump, a syringe pump, and the like.
자성 나노입자가 결합된 엑소좀은 시료 주입부를 통해 미세유체채널에 주입되어 일정 크기의 외부 자기장이 인가된 메인 채널을 거치며, 이 과정에서 자성 나노입자의 자화도 차이에 따라 엑소좀의 경로가 달라진다. 구체적으로 정상 세포 유래 엑소좀에는 엑소좀 공통 발현 마커 탐지용 프로브가 결합된 제1 자성 나노입자만 결합하나, 질병 특이적 엑소좀에는 제1 자성 나노입자뿐만 아니라 질병 특이적 엑소좀 발현 마커 탐지용 프로브가 결합된 제2 자성 나노입자도 결합하기 때문에 두 엑소좀 사이에 자기 민감도 또는 자화도의 세기가 차이가 크게 발생한다. 이러한 엑소좀들이 일정한 크기의 자기장이 인가된 메인 채널을 거치면서 이동 경로가 상이해지고, 이동 경로에 따라 서로 다른 배출구를 사용하면 크기가 다른 자성 입자들이 결합된 엑소좀을 한번에 분리할 수 있다. The exosomes bound with magnetic nanoparticles are injected into the microfluidic channel through the sample injection unit and pass through the main channel to which an external magnetic field of a certain size is applied. . Specifically, only the first magnetic nanoparticles to which the probe for detecting the common exosome expression marker is bound to the normal cell-derived exosomes, but the first magnetic nanoparticles as well as the first magnetic nanoparticles to the disease-specific exosomes for detecting the disease-specific exosome expression marker Since the second magnetic nanoparticles to which the probe is bound are also bound, there is a large difference in the intensity of magnetic sensitivity or magnetization between the two exosomes. As these exosomes pass through the main channel to which a magnetic field of a certain size is applied, the movement paths are different, and if different outlets are used according to the movement paths, the exosomes to which magnetic particles of different sizes are bound can be separated at once.
이때, 외부 자기장은 미세유체채널 내 유체 흐름 방향과 수직 방향으로 걸어 주는 것이 이동경로 차이의 극대화를 유도할 수 있어 효과적인 분리에 유리하다. 또한, 자기장의 세기는 500 G 내지 3000 G(0.05 T 내지 0.3 T)로 걸어주는 것이 바람직하다. 세기가 너무 약하면 엑소좀의 서로 분리되지 않아 질병 특이적 엑소좀만 분리할 수 없고, 세기가 너무 강하면 이동 경로가 너무 급격하게 변하여 질병 특이적 엑소좀을 특정 배출구를 통해 농축하여 분리할 수 없는 문제가 있다.At this time, applying the external magnetic field in the direction perpendicular to the flow direction of the fluid in the microfluidic channel can induce the maximization of the difference in the movement path, which is advantageous for effective separation. In addition, the strength of the magnetic field is preferably set to 500 G to 3000 G (0.05 T to 0.3 T). If the intensity is too weak, the exosomes do not separate from each other, so only disease-specific exosomes cannot be separated. there is
상기 자기장치로는 영구 자석 또는 전자석으로부터 외부 자기장을 인가하는 것을 포함할 수 있다. 상기 영구 자석은 니켈, 코발트, 철, 이들의 합금 및 합금됨으로써 강자성이 되는 비 강자성 물질들의 합금, 즉 Heusler 합금으로 알려진 합금(예를 들어, 구리, 주석 및 망간의 합금)으로부터 생성된다. 영구 자석을 위한 많은 적절한 합금들은 알려져 있고, 본 발명의 일 실시예에서 사용할 수 있는 자석 제작을 위하여 상업적으로 이용할 수 있다. 전형적인 그러한 물질은 전이금속-반금속(메탈로이드) 합금으로서, 전이금속(대개 Fe, Co, 또는 Ni) 약 80%와 녹는점을 낮춰주는 반금속 성분(보론, 탄소, 실리콘, 인 또는 알루미늄)으로부터 만들어진다. 영구 자석은 결정형 또는 무정형일 수 있다. 무정형 합금의 일례로는 Fe80B20 (Metglas 2605)이다. 본 발명의 실시예에서 외부 자기장은 미세유체채널의 메인 채널의 윗면 및 아래면에 부착된 직사각형(2.5 ㎝ x 2.5 ㎝ x 4.0 ㎝) 네오디뮴 (NdFeB) 자석(K&J Magnetics, Jamison, PA)에 의하여 제공된다.The magnetic device may include applying an external magnetic field from a permanent magnet or an electromagnet. The permanent magnets are produced from nickel, cobalt, iron, alloys thereof and alloys of non-ferromagnetic materials that become ferromagnetic by alloying, ie, alloys known as Heusler alloys (eg, alloys of copper, tin and manganese). Many suitable alloys for permanent magnets are known and commercially available for making magnets usable in one embodiment of the present invention. A typical such material is a transition metal-semimetal (metalloid) alloy, with a transition metal (usually Fe, Co, or Ni) about 80% and a melting point lowering metalloid component (boron, carbon, silicon, phosphorus or aluminum). is made from Permanent magnets may be crystalline or amorphous. An example of an amorphous alloy is Fe80B20 (Metglas 2605). In an embodiment of the present invention, the external magnetic field is provided by a rectangular (2.5 cm x 2.5 cm x 4.0 cm) neodymium (NdFeB) magnet (K&J Magnetics, Jamison, PA) attached to the upper and lower surfaces of the main channel of the microfluidic channel. do.
본 발명에서, 상기 방법은 단계 (b) 이후 (c) 서로 다른 회수 경로로 분리된 자성 나노입자와 결합된 엑소좀을 흡입하는 단계를 추가로 포함할 수 있으며, 상기 흡입은 배출구에 연결된 펌프를 통해 이루어질 수 있다. 또한, 상기 흡입은 10 ㎖/hr 내지 50 ㎖/hr 속도 범위에서 이루어질 수 있다.In the present invention, the method may further include the step of inhaling the exosomes combined with magnetic nanoparticles separated by different recovery paths after step (b) (c), wherein the suction is performed by using a pump connected to the outlet can be done through In addition, the inhalation may be made at a rate ranging from 10 ml/hr to 50 ml/hr.
본 발명의 일 예에 따르면 질병 특이적 엑소좀을 정상 세포 유래 엑소좀과 한번에 분리할 수 있고, 이 방법은 전체 엑소좀을 분리한 후 질병 관련 엑소좀을 다시 분리하는 기존의 엑소좀 분리 방법과 비교하여 공정이 간단하고 질병 관련 엑소좀만 농축 분리할 수 있는 장점이 있으며, 분리된 질병 특이적 엑소좀은 질병의 진단 및 예후 예측에 활용될 수 있다.According to an example of the present invention, disease-specific exosomes can be separated from normal cell-derived exosomes at once, and this method is different from the existing exosome separation method of isolating the disease-related exosomes again after separating the entire exosomes. In comparison, there is an advantage in that the process is simple and only disease-related exosomes can be concentrated and isolated, and the isolated disease-specific exosomes can be utilized for disease diagnosis and prognosis prediction.
도 1은 본 발명의 일 예에 따른, 미세유체채널을 이용한 엑소좀 다중 분리 원리를 도식화한 것이다.1 is a schematic diagram of the principle of multiple separation of exosomes using a microfluidic channel, according to an example of the present invention.
도 2는 본 발명의 일 예에 따른, 엑소좀 다중 분리용 미세유체채널의 모식도를 나타낸 것이다.Figure 2 shows a schematic diagram of a microfluidic channel for multiple separation of exosomes, according to an example of the present invention.
도 3은 본 발명의 일 예에 따라 제작한 엑소좀 다중 분리용 미세유체채널 구조물을 나타낸 것이다.3 shows a microfluidic channel structure for multiple separation of exosomes manufactured according to an example of the present invention.
도 4는 본 발명의 일 제조예에 따라 합성한 다양한 크기의 사산화삼철(Fe 3O 4) 자성 나노입자를 투과전자현미경으로 확인한 결과이다: (A)는 100 ㎚, (B)는 200 ㎚, (C)는 300 ㎚ 및 (D)는 400 ㎚ 크기의 자성 나노입자이다.Figure 4 is the result of confirming the magnetic nanoparticles of triiron tetraoxide (Fe 3 O 4 ) of various sizes synthesized according to a preparation example of the present invention by a transmission electron microscope: (A) is 100 nm, (B) is 200 nm, (C) is 300 nm and (D) is a 400 nm size of magnetic nanoparticles.
도 5는 다양한 크기를 갖는 자성 입자의 자화도를 진동시료 자화율측정기(VSM, vibrating sample magnetometer)로 측정한 결과이다.5 is a result of measuring the magnetization of magnetic particles having various sizes with a vibrating sample magnetometer (VSM).
도 6은 다양한 크기를 갖는 자성 나노입자의 결정구조를 엑스레이 회절분석기(XRD, X-ray Diffraction)으로 확인한 결과이다.6 is a result of confirming the crystal structure of magnetic nanoparticles having various sizes with an X-ray diffraction analyzer (XRD, X-ray Diffraction).
도 7은 엑소좀과 자성 나노입자의 결합 여부를 전자현미경으로 확인한 결과이다: (A)는 자성 나노입자를 처리하지 않은 엑소좀의 투과전자현미경 이미지, B는 HCC_1143 세포 유래 엑소좀과 결합한 CD9-자성 나노입자의 투과전자현미경 이미지, C는 HCC_1143 세포 유래 엑소좀과 결합한 CD9-자성 나노입자의 주사전자현미경 이미지, D는 HCC_1954 세포 유래 엑소좀과 결합한 HER2-자성 나노입자의 투과전자현미경 이미지 및 E는 HCC_1954 세포 유래 엑소좀과 결합한 HER2-자성 나노입자의 주사전자현미경 이미지이다.7 is a result of confirming the binding of exosomes and magnetic nanoparticles with an electron microscope: (A) is a transmission electron microscope image of exosomes not treated with magnetic nanoparticles, B is CD9- bound to exosomes derived from HCC_1143 cells Transmission electron microscope image of magnetic nanoparticles, C is a scanning electron microscope image of CD9-magnetic nanoparticles bound to HCC_1143 cell-derived exosomes, D is a transmission electron microscope image of HER2-magnetic nanoparticles bound to HCC_1954 cell-derived exosomes, and E is a scanning electron microscope image of HER2-magnetic nanoparticles bound to HCC_1954 cell-derived exosomes.
도 8은 크기가 상이한 자성 나노입자 (HER2 항체 또는 CD9 항체 표지) 각각을 HER2 과발현 세포주(HCC_1954) 유래 엑소좀과 결합시킨 후 공초점 현미경으로 확인한 이미지이다.8 is an image obtained by confocal microscopy after binding each of magnetic nanoparticles of different sizes (labeled with HER2 antibody or CD9 antibody) with exosomes derived from a HER2-overexpressing cell line (HCC_1954).
도 9는 크기가 상이한 자성 나노입자 (HER2 항체 또는 CD9 항체 표지) 각각을 HER2 비발현 세포주 (HCC_1143) 유래 엑소좀과 결합시킨 후 공초점 현미경으로 확인한 이미지이다.9 is an image confirmed by confocal microscopy after binding magnetic nanoparticles of different sizes (labeled with HER2 antibody or CD9 antibody) with exosomes derived from a HER2 non-expressing cell line (HCC_1143), respectively.
도 10은 크기가 상이한 자성 나노입자 (HER2 항체 또는 CD9 항체 표지) 각각을 HER2 과발현 세포주(HCC_1954) 유래 엑소좀과 결합시킨 후 미세유체채널에 주입하여 광학현미경으로 엑소좀의 궤적을 확인한 결과이다: (A-C) 외부 자기장이 없을 때 미체유체채널 내 위치별 엑소좀 거동; (D-F) 외부 자기장 하에서 미체유체채널 내 위치별 엑소좀 거동; 및 (G-J) 외부 자기장 하에서 배출구별 엑소좀 거동.Figure 10 is the result of confirming the trajectory of the exosomes by optical microscopy after binding each of magnetic nanoparticles (labeled with HER2 antibody or CD9 antibody) of different sizes with exosomes derived from the HER2-overexpressing cell line (HCC_1954) and injecting them into microfluidic channels: (A-C) Exosome behavior by location in microfluidic channels in the absence of an external magnetic field; (D-F) Exosome behavior by location in microfluidic channels under an external magnetic field; and (G-J) exosome behavior by outlet under an external magnetic field.
도 11은 크기가 상이한 자성 나노입자 (HER2 항체 또는 CD9 항체 표지) 각각을 HER2 과발현 세포주(HCC_1954) 유래 엑소좀과 결합시키고, 이를 미세유체채널에 서로 다른 총 유량으로 주입한 후 총 유량 대비 배출구 2 및 4에서 회수되는 엑소좀의 비율을 확인한 결과이다.11 shows each of magnetic nanoparticles (labeled with HER2 antibody or CD9 antibody) of different sizes is combined with exosomes derived from a HER2-overexpressing cell line (HCC_1954), and these are injected into microfluidic channels at different total flow rates, and then outlet 2 compared to the total flow rate. And it is a result of confirming the ratio of the exosomes recovered in 4.
도 12는 크기가 상이한 자성 나노입자 (HER2 항체 또는 CD9 항체 표지) 각각을 HER2 과발현 세포주(HCC_1954) 유래 엑소좀과 결합시키고, 이를 미세유체채널에 주입한 후 각 배출구에서 엑소좀을 회수하여 질병 마커 유전자의 발현율을 확인한 결과이다.12 is a diagram showing each of magnetic nanoparticles (labeled with HER2 antibody or CD9 antibody) of different sizes is combined with exosomes derived from a HER2-overexpressing cell line (HCC_1954), and after injecting them into microfluidic channels, exosomes are recovered from each outlet to indicate disease markers. This is the result of confirming the expression rate of the gene.
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 specific examples will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.
제조예 1: 미세유체채널 장치 제조Preparation Example 1: Preparation of microfluidic channel device
실리콘 웨이퍼에 SU-8 감광 수지를 이용한 무늬를 도 2의 A에 제시된 대로 제작하여 주조틀(높이 100 ㎛)을 만들었다. 이후 액상의 폴리디메틸실록산(PDMS)을 주조틀에 고형화시켜 칩을 만들고, 칩을 슬라이드 글라스에 붙여 미세유체채널 장치를 만들었다.A pattern using SU-8 photosensitive resin on a silicon wafer was fabricated as shown in FIG. After that, liquid polydimethylsiloxane (PDMS) was solidified in a casting mold to make a chip, and the chip was attached to a slide glass to make a microfluidic channel device.
상기 미세유체채널은 주입구가 시료 주입구(1개 채널, 도 2에서 ①), 버퍼 주입구 통합 1개 채널 (주입 후 8개 채널로 나뉨, 도 2에서 ②)로 나뉘어 있고, 외부에서 자기장이 인가되는 메인 채널(도 2에서 ④)과 총 4개 채널 (2개씩 묶음, 도 2에서 ③)로 구성된 배출구를 포함한다. 도 3에 실제 제작한 미세유체채널의 사진을 나타내었다.In the microfluidic channel, the injection hole is divided into a sample inlet (one channel, ① in FIG. 2), one channel with a buffer inlet (divided into 8 channels after injection, ② in FIG. 2), and a magnetic field is applied from the outside. It includes an outlet composed of a main channel (④ in FIG. 2) and a total of 4 channels (grouped by 2, ③ in FIG. 2). 3 shows a photograph of the actually fabricated microfluidic channel.
상기 미세유체채널은 자성 나노입자가 코팅된 엑소좀 시료의 거동을 확인하기 위하여 시료 주입구를 옆면에 배치하였고, 버퍼 주입구는 층류 효과로 인한 포물선 모양의 유체 흐름을 줄이기 위하여 채널의 수를 8개로 늘려 구성하였다. 또한, 세포가 미세유체채널 표면에 붙는 현상과 유체 방울 형성을 줄이기 위하여 제작 후 신속하게 소혈청 알부민(Bovine serum albumin, BSA)이 10% 섞인 인산완충생리식염수 (Phosphate buffered saline, PBS) 용액을 흐르게 하여 제작하였다.In the microfluidic channel, the sample inlet was arranged on the side to confirm the behavior of the magnetic nanoparticle-coated exosome sample, and the buffer inlet was increased to eight channels in order to reduce the parabolic fluid flow due to the laminar flow effect. composed. In addition, in order to reduce the adhesion of cells to the surface of the microfluidic channel and the formation of fluid droplets, a phosphate buffered saline (PBS) solution mixed with 10% of bovine serum albumin (BSA) was quickly flowed after production. was produced.
제조예 2: HER2 결합용 프로브가 결합된 자성 나노입자 제조Preparation Example 2: Preparation of magnetic nanoparticles bound to HER2 binding probe
2-1. 자성 나노입자 합성2-1. Magnetic Nanoparticle Synthesis
자성 나노입자는 각각 지름이 200 ㎚, 400 ㎚인 자성 나노클러스터를 사용하였다. 자성 나노클러스터는 염화제이철 (FeCl 3), 소듐아세테이트 및 소듐시트릭산을 디에틸렌글라이콜과 에틸렌글라이콜의 혼합용액에 섞은 후, 용매열합성법 (Solvothermal) 방식으로 전기로에서 12시간 이상 반응시켜 얻었다. 자성 나노입자의 조성은 사산화삼철(Fe 3O 4)이었고, 도 4와 같은 표면과 다양한 크기를 가진다. 상기 염화제이철의 양을 조절하면 다양한 크기의 자성 입자를 합성할 수 있다.As the magnetic nanoparticles, magnetic nanoclusters having diameters of 200 nm and 400 nm, respectively, were used. For magnetic nanoclusters, ferric chloride (FeCl 3 ), sodium acetate, and sodium citric acid are mixed with a mixed solution of diethylene glycol and ethylene glycol, and then reacted in an electric furnace using the solvothermal method for more than 12 hours. got it done The composition of the magnetic nanoparticles was triiron tetraoxide (Fe 3 O 4 ), and had a surface and various sizes as shown in FIG. 4 . By controlling the amount of the ferric chloride, magnetic particles of various sizes can be synthesized.
도 5는 진동시료 자화율측정기(VSM, vibrating sample magnetometer)로 크기가 상이한 각 자성 나노입자의 자성 민감도를 측정한 결과이다. 자성 나노입자의 크기에 따라 자성 민감도가 상이한 것을 알 수 있으며, 이러한 차이를 이용하여 엑소좀의 분리가 가능하다.5 is a result of measuring the magnetic sensitivity of each magnetic nanoparticles having different sizes with a vibrating sample magnetometer (VSM). It can be seen that the magnetic sensitivity is different depending on the size of the magnetic nanoparticles, and it is possible to separate the exosomes using this difference.
2-2. HER2 결합용 항체가 표지된 자성 나노입자 제조2-2. Preparation of magnetic nanoparticles labeled with HER2 binding antibody
질병 특이적 마커를 과발현하는 엑소좀을 분리하기 위해 상기 질병 특이적 마커에 결합하는 항체와 2-1에서 합성한 자성 나노입자를 결합시켰다. 본 제조예에서는 HER2 항체 (EPR19547-12 (ab214275), Abcam에서 구입) 및 CD9 항체 (EPR23105-121 (ab236630), Abcam)를 사용하였다. CD9 항체는 엑소좀 표면의 CD81에 결합한다. 또한, 자화도 차이를 이용하여 질병 특이적 마커를 과발현하는 엑소좀과 정상 세포 유래 엑소좀을 분리하기 위해 상기 항체는 서로 크기가 상이한 자성 나노입자와 결합시켰다.In order to isolate an exosome overexpressing a disease-specific marker, an antibody binding to the disease-specific marker and the magnetic nanoparticles synthesized in 2-1 were combined. In this preparation, HER2 antibody (EPR19547-12 (ab214275), purchased from Abcam) and CD9 antibody (EPR23105-121 (ab236630), Abcam) were used. CD9 antibody binds to CD81 on the surface of exosomes. In addition, in order to separate the exosomes overexpressing disease-specific markers from the exosomes derived from normal cells using the difference in magnetization, the antibodies were combined with magnetic nanoparticles of different sizes.
구체적으로 1-에틸 3-디메틸아미노프로필 카르보디이미드와 하이드록시숙시니미드 (hydroxysuccinimide)로 아마이드 결합을 유도하여 항체가 결합된 자성 나노입자를 제조하였다: 400 ㎚ 자성 나노입자 10 ㎎ + anti-HER2 antibody 10 ㎍; 및 200 ㎚ 자성입자 10 ㎎ + anti-CD9 antibody 10 ㎍.Specifically, amide bonds were induced with 1-ethyl 3-dimethylaminopropyl carbodiimide and hydroxysuccinimide to prepare antibody-bound magnetic nanoparticles: 400 nm magnetic nanoparticles 10 mg + anti-HER2 antibody 10 μg; and 10 mg of 200 nm magnetic particles + 10 μg of anti-CD9 antibody.
이하에서는 HER2 항체 또는 CD9 항체로 표지된 자성 나노입자를 각각 HER2-자성 나노입자(400 ㎚) 및 CD9-자성 나노입자(200 ㎚)로 기재한다.Hereinafter, magnetic nanoparticles labeled with HER2 antibody or CD9 antibody will be referred to as HER2-magnetic nanoparticles (400 nm) and CD9-magnetic nanoparticles (200 nm), respectively.
제조예 3: 자성 나노입자가 결합된 엑소좀 제조Preparation Example 3: Preparation of exosomes bound to magnetic nanoparticles
HER2 과발현 세포인 HCC_1954 세포를 배양한 후 배양 배지에서 당업계에 알려진 방법에 따라 엑소좀을 포집하고, 획득하였다. HER2 비발현 세포인 HCC_1143 세포를 배양한 후 동일한 방법으로 엑소좀을 분리하였다. 분리한 엑소좀 각각을 HER2-자성 나노입자(400 ㎚) 2 ㎎ 또는 CD9-자성 나노입자(200 ㎚) 2 ㎎과 혼합하여 총 4종의 엑소좀-자성 나노입자 복합체를 제조하였다.After culturing HCC_1954 cells, which are HER2 overexpressing cells, exosomes were collected and obtained in a culture medium according to a method known in the art. After culturing HCC_1143 cells, which are HER2 non-expressing cells, exosomes were isolated in the same manner. Each of the isolated exosomes was mixed with 2 mg of HER2-magnetic nanoparticles (400 nm) or 2 mg of CD9-magnetic nanoparticles (200 nm) to prepare a total of four exosome-magnetic nanoparticle complexes.
도 7은 위와 같이 처리한 후 엑소좀과 자성 나노입자의 결합 여부를 전자현미경으로 확인한 사진이다. 도 6에서 A는 자성 나노입자를 처리하지 않은 엑소좀의 투과전자현미경 이미지, B는 HCC_1143 세포 유래 엑소좀과 결합한 CD9-자성 나노입자의 투과전자현미경 이미지, C는 HCC_1143 세포 유래 엑소좀과 결합한 CD9-자성 나노입자의 주사전자현미경 이미지, D는 HCC_1954 세포 유래 엑소좀과 결합한 HER2-자성 나노입자의 투과전자현미경 이미지, E는 HCC_1954 세포 유래 엑소좀과 결합한 HER2-자성 나노입자의 주사전자현미경 이미지이다.7 is a photograph confirming with an electron microscope whether the exosomes and magnetic nanoparticles are bound after the above treatment. In FIG. 6, A is a transmission electron microscope image of exosomes not treated with magnetic nanoparticles, B is a transmission electron microscope image of CD9-magnetic nanoparticles bound to HCC_1143 cell-derived exosomes, and C is CD9 bound to HCC_1143 cell-derived exosomes. -Scanning electron microscope image of magnetic nanoparticles, D is a transmission electron microscope image of HER2-magnetic nanoparticles bound to HCC_1954 cell-derived exosomes, and E is a scanning electron microscope image of HER2-magnetic nanoparticles bound to HCC_1954 cell-derived exosomes. .
도 8은 HER2 과발현 세포인 HCC_1954 세포에서 획득한 엑소좀에 HER2 항체 또는 CD9 항체로 표지된 자성 나노입자를 각각 처리한 후 공초점 현미경으로 획득한 이미지이다.8 is an image obtained by confocal microscopy after each treatment with HER2 antibody or CD9 antibody-labeled magnetic nanoparticles on exosomes obtained from HCC_1954 cells, which are HER2-overexpressing cells.
도 9는 HER2 비발현 세포인 HCC_1143 세포에서 획득한 엑소좀에 HER2 항체 또는 CD9 항체로 표지된 자성 나노입자를 각각 처리한 후 공초점 현미경으로 획득한 이미지이다.9 is an image obtained by confocal microscopy after each treatment with HER2 antibody or CD9 antibody-labeled magnetic nanoparticles on exosomes obtained from HCC_1143 cells, which are HER2 non-expressing cells.
실시예 1: 엑소좀 분리 실험Example 1: Exosome isolation experiment
상기 제조예 3에서 얻은 자성 나노입자가 결합된 엑소좀을 미체유체채널에 주입하여 자화도의 차이에 따라 분리할 수 있는지 확인하였다. 미세유체채널 내 유량은 총 20 ㎖/hr (시료 주입구 4 ㎖/hr, 버퍼 주입구 16 ㎖/hr)로 고정하고, 외부 자기장의 크기는 평균적으로 0.3T를 사용하였다.It was confirmed whether the exosomes to which the magnetic nanoparticles obtained in Preparation Example 3 were bound could be separated according to the difference in magnetization by injecting them into the microfluidic channel. The flow rate in the microfluidic channel was fixed at a total of 20 ml/hr (sample inlet 4 ml/hr, buffer inlet 16 ml/hr), and the size of the external magnetic field was 0.3T on average.
도 10은 도 2에 제시된 미세유체채널에서 메인 채널의 아래 부분에 자석을 두고 미세유체채널에 각 자성 나노입자를 붙인 엑소좀을 흘려 보냈을 때 나타나는 영상을 나타낸 것이다.10 shows an image that appears when a magnet is placed in the lower part of the main channel in the microfluidic channel shown in FIG. 2 and exosomes having magnetic nanoparticles attached thereto are flowed through the microfluidic channel.
도 11은 총 유량을 5, 15, 25, 35, 50 ㎖/hr로 변화시켜 엑소좀 분리 실험을 진행한 후 주입한 엑소좀의 양 대비 2번 및 4번 배출구에서 획득한 엑소좀의 분율을 원형 표로 나타낸 것이다. 총 유량을 20 ㎖/hr (시료 주입구 4 ㎖/hr)로 분리 실험 진행 시 높은 효율로 시료 획득이 가능하다는 것을 확인하였다.11 shows the fraction of exosomes obtained from outlets 2 and 4 compared to the amount of exosomes injected after performing an exosome separation experiment by changing the total flow rate to 5, 15, 25, 35, and 50 ml/hr shown in a circular table. It was confirmed that the sample could be obtained with high efficiency when the separation experiment was performed at a total flow rate of 20 ml/hr (sample inlet 4 ml/hr).
도 12는 총 유량을 20 ㎖/hr (시료 주입구 4 ㎖/hr)로 고정하여 엑소좀 분리 실험을 진행하고, 각 배출구에서 획득한 엑소좀에서 핵산을 분리한 후 중합효소연쇄반응(PCR)으로 질병 특이적 마커 유전자의 발현율을 확인한 결과이다. 확인 결과, 4번 배출구에서 획득한 엑소좀에서 HER2 유전자의 발현율이 가장 높은 것을 확인하였다.12 shows an exosome separation experiment by fixing the total flow rate to 20 ml/hr (sample inlet 4 ml/hr), and after separating nucleic acids from the exosomes obtained from each outlet, polymerase chain reaction (PCR) This is the result of confirming the expression rate of the disease-specific marker gene. As a result, it was confirmed that the expression rate of the HER2 gene was the highest in the exosomes obtained from outlet #4.

Claims (11)

  1. (a) 엑소좀이 포함된 시료와 자화도가 서로 상이한 2종 이상의 자성 나노입자를 접촉시켜 자성 나노입자가 결합된 엑소좀을 얻는 단계; 및(a) contacting a sample containing exosomes with two or more types of magnetic nanoparticles having different degrees of magnetization to obtain exosomes to which magnetic nanoparticles are bound; and
    (b) 자성 나노입자가 결합된 엑소좀 및 버퍼를 자기장이 인가된 미세유체채널에 주입하는 단계를 포함하되,(b) injecting the exosomes and the buffer to which the magnetic nanoparticles are bound into the microfluidic channel to which the magnetic field is applied,
    상기 자성 나노입자가 결합된 엑소좀은 미세유체채널에 주입되어 미세유체채널 내의 서로 다른 회수 경로로 분리되고,The exosomes to which the magnetic nanoparticles are bound are injected into the microfluidic channel and separated into different recovery paths in the microfluidic channel,
    상기 자성 나노입자는 엑소좀 공통 발현 마커를 탐지하는 프로브가 결합된 제1 자성 나노입자 및 제1 자성 나노입자와 자화도가 상이하고, 질병 특이적 엑소좀 발현 마커를 탐지하는 프로브가 결합된 제2 자성 나노입자를 포함하는 질병 특이적 엑소좀의 분리방법.The magnetic nanoparticles have a different magnetization from the first magnetic nanoparticles and the first magnetic nanoparticles to which a probe for detecting a common exosome expression marker is coupled, and a probe for detecting a disease-specific exosome expression marker is bound. 2 A method for separating disease-specific exosomes containing magnetic nanoparticles.
  2. 제1항에 있어서, 상기 방법은 단계 (b) 이후 (c) 서로 다른 회수 경로로 분리된 자성 나노입자가 결합된 엑소좀을 흡입하는 단계를 추가로 포함하는 질병 특이적 엑소좀의 분리 방법.The method of claim 1, wherein the method further comprises the step of inhaling the magnetic nanoparticles-coupled exosomes separated by different recovery routes after step (b) (c).
  3. 제2항에 있어서, 상기 흡입 속도는 10 ㎖/hr 내지 50 ㎖/hr인 질병 특이적 엑소좀의 분리방법.The method of claim 2, wherein the inhalation rate is 10 ml/hr to 50 ml/hr.
  4. 제1항에 있어서, 상기 제1 자성 나노입자와 상기 제2 자성 나노입자는 크기가 서로 상이한 질병 특이적 엑소좀의 분리 방법.The method of claim 1, wherein the first magnetic nanoparticles and the second magnetic nanoparticles are different in size from each other.
  5. 제4항에 있어서, 상기 제1 자성 나노입자의 크기는 100 내지 800 ㎚이고, 상기 제2 자성 나노입자의 크기는 200 내지 1000 ㎚인 질병 특이적 엑소좀의 분리방법.The method of claim 4, wherein the size of the first magnetic nanoparticles is 100 to 800 nm, and the size of the second magnetic nanoparticles is 200 to 1000 nm.
  6. 제1항에 있어서, 상기 엑소좀 공통 발현 마커는 CD9, CD63, CD81 및 CD82로 이루어진 군에서 선택되는 질병 특이적 엑소좀의 분리 방법.The method of claim 1, wherein the exosome common expression marker is selected from the group consisting of CD9, CD63, CD81 and CD82.
  7. 제1항에 있어서, 상기 질병 특이적 엑소좀 발현 마커는 HER2 (human epidermal growth factor receptor 2), PSA (prostate specific antigen), CEA (carcinoembryonic antigen), AFP (alpha fetoprotein), CA125 (cancer antigen 125), CA 15-3 (cancer antigen 15-3) 및 CA 19-9 (cancer antigen 19-9)로 이루어진 군에서 선택되는 질병 특이적 엑소좀의 분리 방법.According to claim 1, wherein the disease-specific exosome expression marker is HER2 (human epidermal growth factor receptor 2), PSA (prostate specific antigen), CEA (carcinoembryonic antigen), AFP (alpha fetoprotein), CA125 (cancer antigen 125) , A method for isolating a disease-specific exosome selected from the group consisting of CA 15-3 (cancer antigen 15-3) and CA 19-9 (cancer antigen 19-9).
  8. 제1항에 있어서, 상기 프로브는 단일클론항체, 폴리클론항체, 키메릭 항체, Fab, F(ab') 2, Fab', scFv(single chain fragment variable), 단일-도메인 항체(single-domain antibody), 압타머, 펩타이드 및 PNA(Peptide nucleic acid)로 이루어진 군에서 선택되는 질병 특이적 엑소좀의 분리 방법.The method of claim 1, wherein the probe is a monoclonal antibody, polyclonal antibody, chimeric antibody, Fab, F(ab') 2 , Fab', single chain fragment variable (scFv), single-domain antibody ), an aptamer, a method for separating a disease-specific exosome selected from the group consisting of peptides and PNA (Peptide nucleic acid).
  9. 제1항에 있어서, 상기 (b)에서 자성 나노입자와 결합된 엑소좀의 주입 속도는 2 ㎖/hr 내지 10 ㎖/hr인 질병 특이적 엑소좀의 분리방법.The method of claim 1, wherein the injection rate of the exosome bound to the magnetic nanoparticles in (b) is 2 ㎖/hr to 10 ㎖/hr.
  10. 제1항에 있어서, 상기 버퍼의 주입 속도는 8 ㎖/hr 내지 40 ㎖/hr인 질병 특이적 엑소좀의 분리방법.The method according to claim 1, wherein the injection rate of the buffer is 8 ml/hr to 40 ml/hr.
  11. 제1항에 있어서, 상기 자기장의 세기는 500 G 내지 3000 G인 질병 특이적 엑소좀의 분리방법.The method of claim 1, wherein the magnetic field strength is 500 G to 3000 G.
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