WO2020233572A1 - 多肽磁性纳米颗粒、其制备方法及应用 - Google Patents

多肽磁性纳米颗粒、其制备方法及应用 Download PDF

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WO2020233572A1
WO2020233572A1 PCT/CN2020/091063 CN2020091063W WO2020233572A1 WO 2020233572 A1 WO2020233572 A1 WO 2020233572A1 CN 2020091063 W CN2020091063 W CN 2020091063W WO 2020233572 A1 WO2020233572 A1 WO 2020233572A1
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polypeptide
cancer
magnetic
add
pbs
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PCT/CN2020/091063
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English (en)
French (fr)
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江涛
李男男
薛建
白雪皎
于骞
李妲
蔡桢
蔡倩
徐安
仇海燕
韩宇
王冉
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北京中科纳泰生物科技有限公司
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Priority to JP2021569454A priority Critical patent/JP2022533445A/ja
Priority to EP20810007.3A priority patent/EP3974830A4/en
Priority to US17/611,657 priority patent/US20220196648A1/en
Priority to CN202080029253.5A priority patent/CN113826013B/zh
Publication of WO2020233572A1 publication Critical patent/WO2020233572A1/zh

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    • G—PHYSICS
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    • 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
    • G01N33/57492—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 compounds localized on the membrane of tumor or cancer cells
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/5434—Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • 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
    • G01N33/574—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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • 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
    • G01N33/57423—Specifically defined cancers of lung
    • G—PHYSICS
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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6818—Sequencing of polypeptides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y25/00—Nanomagnetism, e.g. magnetoimpedance, anisotropic magnetoresistance, giant magnetoresistance or tunneling magnetoresistance
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • the invention belongs to the field of medical detection, and specifically relates to a polypeptide magnetic nano particle, a preparation method and application thereof.
  • CTC Circulating tumor cells
  • Tumors are highly heterogeneous. Even tumors with the same histology and morphology have different molecular biological changes. Different biological changes have different biological behaviors and treatment sensitivities.
  • clinical staging is commonly used Traditional pathological classifications such as grading and classification have limited ability to predict tumors.
  • tumor targeted therapy and immunotherapy have attracted more and more attention.
  • molecular diagnosis and precise classification of tumor types are the key to treatment. Therefore, molecular classification of tumors is an inevitable requirement for individualized tumor treatment.
  • breast cancer can be used as an example.
  • Breast cancer is one of the common malignant tumors in women, and its incidence is increasing year by year, which seriously threatens women's physical and mental health.
  • HER2 human epidermal growth factor receptor-2
  • ER estrogen receptor
  • PR progesterone receptor
  • AR androgen receptor Body
  • the clinical efficacy shows that the drug can improve the treatment efficiency of patients before and after the adjuvant treatment of advanced breast cancer, and prolong the survival time of patients. Therefore, molecular classification of breast cancer is of great significance in tumor therapy, especially targeted drugs. Because the CTC in the peripheral blood carries almost all the genetic and protein information of the tumor tissue in situ, the molecular classification of the CTC in the peripheral blood of the tested patient with HER2, ER, PR, etc. is of great significance for guiding the clinical treatment of the patient . Similarly, for almost all other malignant tumors, molecular typing and companion diagnosis of targeted drugs have important clinical value.
  • tumor immunotherapy has also made a series of progress in recent years, which has changed the treatment pattern of many cancers.
  • the PD-1/PD-L1 antibody drug for immune checkpoints is currently the most anticipated and fastest-growing tumor immunotherapy. Therefore, the expression of PD-L1 in tumor cells is very important for the pre-evaluation of the effect of this immunotherapy.
  • the accompanying diagnosis of PD-L1 expression at the CTC level has important clinical guiding significance for the immunotherapy of PD-1/PD-L1 antibody drugs.
  • the purpose of the present invention is to overcome the defects in the prior art and provide a polypeptide magnetic nanoparticle for circulating tumor cell detection and tumor marker molecular typing, and a preparation method and application thereof.
  • PBS phosphate buffered saline
  • HEPES 4-hydroxyethylpiperazine ethanesulfonic acid buffer.
  • P-L1 refers to: programmed death receptor ligand-1.
  • HER2 refers to: human epidermal growth factor receptor 2.
  • ER refers to: estrogen receptor.
  • PR refers to: progesterone receptor.
  • AR refers to: Androgen Receptor.
  • EGFR epidermal growth factor receptor
  • CXCR4 refers to: chemokine receptor 4.
  • VEGFR refers to: vascular endothelial cell growth factor receptor.
  • the first aspect of the present invention provides a polypeptide nanomagnetic nanoparticle.
  • the polypeptide magnetic nanoparticle includes: a specific targeting polypeptide and a magnetic nanoparticle; wherein the amino acid sequence of the specific targeting polypeptide is VRRDAPRFSMQGLDA-X ,
  • the C-terminal X is 5-20, preferably 5-15, more preferably 9-12 amino acid sequence, and X is not CGGNCC, CGGNCN, CGGNNC, CGGNNN, CGGNCCN, CGGNCCNN, CGGNCNN, CGGNCNNN, CGGNNCN , CGGNNCNN, CGGNNNN, CGGNNNNN, CGGNNNNN;
  • amino acids in the X amino acid sequence are selected from one or more of the following: C, G, and N.
  • the polypeptide is a specific recognition polypeptide targeting an adhesion molecule of epithelial cells
  • the amino acid sequence of the specific targeting polypeptide is: SEQ ID NO: 1 to SEQ ID NO: 9; most preferably, the amino acid sequence of the specific targeting polypeptide is: SEQ ID NO: 1.
  • the magnetic nanoparticles are magnetic nanoparticles with streptavidin; preferably, the magnetic nanoparticles have a particle size of 100-900 nm; more preferably, the magnetic nanoparticles The particle size of the magnetic nanoparticles is 300 nm to 800 nm.
  • the second aspect of the present invention provides a method for preparing the polypeptide magnetic nanoparticles described in the first aspect, and the method includes the following steps:
  • step (2) The polypeptide prepared in step (1) and the magnetic nanoparticle solution are mixed and reacted to obtain the polypeptide magnetic nanoparticle.
  • the solvent for preparing the polypeptide solution is selected from one or more of the following: water, physiological saline, PBS, HEPES; and/or
  • the solvent for preparing the magnetic nanoparticle solution is selected from one or more of the following: water, PBS, HEPES.
  • the final concentration of the polypeptide solution is 1-1000 ⁇ g/mL, preferably 100-500 ⁇ g/mL; and/or the concentration of the magnetic nanoparticle solution
  • the final concentration is 1-10000 ⁇ g/mL, preferably 1000-5000 ⁇ g/mL.
  • the mass ratio of the polypeptide and the magnetic nanoparticles is 1:10-5:1, preferably 2:5.
  • the third aspect of the present invention provides that the polypeptide magnetic nanoparticles of the first aspect or the polypeptide magnetic nanoparticles prepared according to the preparation method of the second aspect are used in the preparation of drugs and/or medical products for the diagnosis or treatment of cancer. In the application.
  • the fourth aspect of the present invention provides a method for diagnosing or treating cancer, the method comprising: administering the polypeptide magnetic nanoparticles according to the first aspect or the polypeptide magnetic nanoparticles according to the second aspect to a subject in need Preparation method of polypeptide magnetic nanoparticles.
  • the cancer is selected from one or more of the following: esophageal cancer, liver cancer, lung cancer, gastric cancer, breast cancer, colorectal cancer, cervical cancer, thyroid Cancer, prostate cancer, pancreatic cancer, kidney cancer, bladder cancer, skin cancer, melanoma, etc.; preferably breast cancer, esophageal cancer, stomach cancer, liver cancer, lung cancer, colorectal cancer, cervical cancer, and/or prostate cancer.
  • the fifth aspect of the present invention provides that the polypeptide magnetic nanoparticles of the first aspect or the polypeptide magnetic nanoparticles prepared according to the preparation method of the second aspect are used for circulating tumor cell detection and/or molecular typing. Application in medicines and/or medical products.
  • the sixth aspect of the present invention provides a polypeptide magnetic nanoparticle for diagnosing or treating cancer and/or a polypeptide magnetic nanoparticle for circulating tumor cell detection and/or molecular typing.
  • the polypeptide magnetic nanoparticle includes the first The polypeptide magnetic nanoparticle described in one aspect or the polypeptide magnetic nanoparticle prepared according to the preparation method described in the second aspect.
  • the seventh aspect of the present invention provides a method for circulating tumor cell detection and/or molecular typing, the method comprising: administering the polypeptide magnetic nanoparticles of the first aspect to a subject in need or according to The polypeptide magnetic nanoparticles prepared by the preparation method described in the second aspect.
  • the biomarker for circulating tumor cell detection and/or molecular typing is selected from one of the following or Multiple: PD-L1, HER2, ER, PR, AR, EGFR, CXCR4, VEGFR, etc.
  • the present invention provides a polypeptide magnetic nanoparticle for CTC detection, including:
  • EpCAM epithelial cell adhesion molecule
  • the method includes the following steps:
  • the solvent is selected from the good solvent of the polypeptide is water, physiological saline, PBS, HEPES.
  • the solvent is magnetic bead dispersant water, PBS, HEPES.
  • the invention also provides the detection of CTC by the polypeptide nano detection device.
  • the CTC is SK-BR-3, MCF-7, MDA-MB-231, H1975, H1650 and A549 tumor cells.
  • the present invention also provides the polypeptide nano-detection device for detecting CTC in peripheral blood of tumor patients and performing molecular typing of tumor markers on the detected CTC.
  • it is suitable for the detection and molecular typing of CTC in the peripheral blood of cancer patients such as breast cancer, esophageal cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, cervical cancer, and prostate cancer.
  • the steps of the polypeptide nanotechnology to detect CTC include incubation, washing, centrifugation, fixation, blocking, immunofluorescence staining, CTC identification and the like.
  • the present invention also provides related molecular typing of the detected CTC.
  • the identification of CTC-related molecular typing includes statistical analysis of the fluorescence intensity of CTC corresponding molecules, and the definition of expression intensity according to a certain threshold.
  • Molecular typing includes positive expression and negative expression, where positive expression includes high, medium, and low expression.
  • the biomarkers of CTC molecular typing include PD-L1, HER2, ER, PR, AR, EGFR, CXCR4 and VEGFR and other solid tumor cell biomarkers.
  • Polypeptide nano magnetic bead technology can be used for almost all solid tumors except brain tumors, osteosarcoma, and lymphoma, including esophageal cancer, liver cancer, lung cancer, stomach cancer, breast cancer, colorectal cancer, cervical cancer, thyroid cancer, and prostate cancer. Cancer, pancreatic cancer, kidney cancer, bladder cancer, skin cancer, melanoma, etc.
  • One of the objectives of the present invention is to provide a polypeptide magnetic nanoparticle for circulating tumor cell detection and tumor marker molecular typing and its application.
  • This method can realize in vitro diagnosis and molecular typing of breast cancer.
  • the method is simple to operate, low in cost, rapid in detection process, and non-invasive, avoiding the pain caused by conventional pathological detection to patients.
  • this method is expected to track the condition in real time, adjust the treatment plan in time according to the development of the condition, and provide guidance for the realization of personalized medicine.
  • the present invention discloses the application of HER2, ER, PR, AR, EGFR, VEGFR, PD-L1 and other proteins of circulating tumor cells as cancer diagnosis and molecular typing markers.
  • the present invention uses an inverted fluorescence microscope to detect the expression of protein markers as an application for molecular typing of tumor patients.
  • the experiment of the present invention confirmed the feasibility of the method for diagnosis and molecular typing in the detection of clinical blood samples.
  • the tumor includes one or more of breast cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, prostate cancer and cervical cancer.
  • the polypeptide nanotechnology of the present invention has high sensitivity and specificity for the detection of CTC, and can detect CTC in the peripheral blood of clinical patients with various tumors, including breast cancer, liver cancer, lung cancer, gastric cancer, and esophageal cancer , Colorectal cancer, prostate cancer and cervical cancer.
  • the present invention relates to a method for molecular typing of tumor-related markers for detected CTCs.
  • this method is expected to achieve early disease screening and real-time tracking of the disease, providing a new method for assisting tumor detection and tracking treatment effects
  • it can also become an important means of prognostic evaluation, providing guidance for the realization of personalized medicine, and has a good application prospect in improving the quality of life of patients and extending the survival period.
  • Figure 1 shows the results of test example 1 NO:1 polypeptide nanomagnetic beads enriching breast cancer cells and performing HER2 molecular typing, where Figure 1A shows the experimental example 1 SEQ ID NO:1 polypeptide nanomagnetic beads pair SK-BR-3 , MCF-7, MDA-MB-231 breast cancer cell capture. Figure 1B shows typical breast cancer cells with different HER2 expression levels detected by the SEQ ID NO:1 polypeptide nanomagnetic beads of Test Example 1.
  • Figure 2 shows the results of experiment 2 SEQ ID NO:1 polypeptide nanomagnetic beads enriching lung cancer cells and performing PD-L1 molecular typing
  • Figure 2A shows the experimental example 2 SEQ ID NO:1 polypeptide nanomagnetic beads H1975, Capture of H1650 and A549 lung cancer cells.
  • Figure 2B shows typical lung cancer cells with different PD-L1 expression levels detected by the SEQ ID NO:1 polypeptide nanomagnetic beads of Test Example 2.
  • Figure 3 shows typical CTCs in the peripheral blood of breast cancer patients with different HER2 expression levels detected in Test Example 3.
  • Figure 4 shows the typical CTCs in the peripheral blood of breast cancer patients with different ER expression levels detected in Test Example 4.
  • Figure 5 shows the CTCs in the peripheral blood of breast cancer patients with different expression levels of typical PR molecules detected in Test Example 5.
  • Figure 6 shows the CTCs in the peripheral blood of breast cancer patients with different expression levels of typical AR molecules detected in Test Example 6.
  • Figure 7 shows the CTCs in the peripheral blood of esophageal cancer patients with different expression levels of typical PD-L1 molecules detected in Test Example 7.
  • Figure 8 shows the CTCs in the peripheral blood of lung cancer patients with different expression levels of typical PD-L1 molecules detected in Test Example 8.
  • Figure 9 shows the CTCs in the peripheral blood of lung cancer patients with different expression levels of typical EGFR molecules detected in Test Example 9.
  • Figure 10 shows the CTCs in the peripheral blood of liver cancer patients with different expression levels of typical PD-L1 molecules detected in Test Example 10.
  • Figure 11 shows the CTCs in the peripheral blood of cervical cancer patients with different expression levels of typical PD-L1 molecules detected in Test Example 11.
  • Figure 12 shows the CTCs in the peripheral blood of gastric cancer patients with different expression levels of typical PD-L1 molecules detected in Test Example 12.
  • FIG. 13 shows the CTCs in the peripheral blood of breast cancer patients with different expression levels of typical CXCR4 molecules detected in Test Example 13.
  • Figure 14 shows the CTCs in the peripheral blood of gastric cancer patients with different expression levels of typical HER2 molecules detected in Test Example 14.
  • Figure 15 shows the CTCs in the peripheral blood of colon cancer patients with different expression levels of typical HER2 molecules detected in Test Example 15.
  • Fig. 16 shows the CTCs in the peripheral blood of colorectal cancer patients with different expression levels of typical PD-L1 molecules detected in Test Example 16.
  • Figure 17 shows the CTCs in the peripheral blood of colorectal cancer patients with different expression levels of typical VEGFR molecules detected in Test Example 17.
  • human tumor cell lines SK-BR-3, MCF-7, MDA-MB-231, H1975, H1650, and A549 used in the following examples were all purchased from the cell bank of the Institute of Basic Research, Chinese Academy of Medical Sciences.
  • the purity of the polypeptides used in the following examples is 98% or more.
  • the solvents of the aqueous solutions used in the following examples are all sterile ultrapure aqueous solutions with a resistivity of 18.2 M ⁇ cm.
  • reagents used in the following examples are all analytical reagents.
  • the scanning microscopes used in the following examples are all Olympus microscope IX73.
  • Magnetic beads purchased from Thermo Fisher.
  • the peptide was synthesized by Beijing Zhongkenate Biotechnology Co., Ltd., with a purity of 98%.
  • the magnetic stand is made by Beijing Zhongkenatai Biotechnology Co., Ltd. It can hold 15ml centrifuge tube.
  • ZEISS Axio Vert A1 and ZEISS Z2 were purchased from ZEISS Far East Co., Ltd.
  • Thermo Fisher CX5 purchased from Thermo Fisher.
  • ZEISS Z2 is recommended first, Olympus IX73 and Thermo Fisher CX5 second.
  • Test Example 1-2 uses the polypeptide magnetic nanoparticle assembly of SEQ ID NO:1-9
  • Test Examples 3-17 all use the polypeptide magnetic nanoparticle assembly of SEQ ID NO:1.
  • Test Example 1 Polypeptide magnetic nanoparticles enrich breast cancer cells and perform HER2 molecular typing
  • complete medium containing 10% fetal bovine serum, 100U/mL penicillin, 100 ⁇ g/mL chain Count the cell concentration, add about 1000 of each cell to 2ml healthy human blood, add 10 ⁇ L peptide nanomagnetic beads to mix
  • CTCs DAPI+/CK+/CD45- and cells conforming to cell morphology
  • CTCs expressing HER2 DAPI+/CK+/HER2+/CD45- cells
  • the HER2 expression level of CTCs is judged according to the fluorescence intensity of the HER2 channel.
  • SEQ ID NO: 1 polypeptide nanomagnetic bead captures SK-BR-3, MCF-7 and MDA-MB-231 with good stability and all reach more than 90%, indicating that the polypeptide nanomagnetic bead It has very high enrichment and detection efficiency for breast cancer cells.
  • Figure 1B shows breast cancer cells with different HER2 expression levels enriched by SEQ ID NO: 1 polypeptide nanomagnetic beads.
  • Table 1 shows SEQ ID NO: 1-9 for SK-BR-3, MCF-7 and MDA-MB- The detection rate of 231 three types of breast cancer cells.
  • Table 1 The detection rate of SK-BR-3, MCF-7 and MDA-MB-231 breast cancer cells
  • Test Example 2 Polypeptide magnetic nanoparticles enrich lung cancer cells and perform PD-L1 molecular typing
  • Collect lung cancer H1975, H1650 and A549 cells in the logarithmic growth phase resuspend the cells in their respective complete medium (containing 10% fetal bovine serum, 100U/mL penicillin, 100 ⁇ g/mL streptomycin), and count the cell concentration. Take about 1000 of each cell and add it to 2ml of healthy human blood, add 10 ⁇ L of peptide nanomagnetic beads to mix, and incubate for 1 hour at room temperature. Remove the centrifuge tube, add 5mL of PBS and mix gently, and place it on the magnetic rack. Then put the magnetic stand on a horizontal shaker for 30 min.
  • complete medium containing 10% fetal bovine serum, 100U/mL penicillin, 100 ⁇ g/mL streptomycin
  • CTCs DAPI+/CK+/CD45- and cells conforming to cell morphology
  • PD-L1+/CD45- cells are referred to as PD-L1 expressing CTCs
  • CTCs are judged based on the fluorescence intensity of the PD-L1 channel The expression level of PD-L1.
  • the polypeptide nanomagnetic beads have a capture rate of over 60% for H1975, H1650 and A549, indicating that the polypeptide nanomagnetic beads have very high enrichment and detection efficiency for breast cancer cells.
  • Figure 2B shows lung cancer cells with different expression levels of PD-L1 molecules enriched by SEQ ID NO:1 polypeptide nanomagnetic beads.
  • Table 2 shows the detection rate of SEQ ID NO: 1-9 on lung cancer H1975, H1650 and A549 cells.
  • Test Example 3 Polypeptide magnetic nanoparticles detect CTC in peripheral blood of breast cancer patients and perform HER2 molecular typing
  • CTC identification and HER2 fluorescence intensity analysis were performed on the detected cells.
  • CTCs DAPI+/CK+/CD45- and cells conforming to cell morphology
  • CTCs expressing HER2 DAPI+/CK+/HER2+/CD45- cells
  • the HER2 expression level of CTCs is judged according to the fluorescence intensity of the HER2 channel.
  • Figure 3 shows the typical detected CTCs in the peripheral blood of breast cancer patients with different expression levels of HRE2.
  • Test Example 4 Polypeptide magnetic nanoparticles detect CTC in peripheral blood of breast cancer patients and perform ER molecular typing
  • CTC identification and ER fluorescence intensity analysis were performed on the detected cells.
  • CTCs DAPI+/CK+/CD45- and cells conforming to the cell morphology
  • CTCs expressing ER DAPI+/CK+/ER+/CD45- cells
  • the ER expression of CTC is judged based on the fluorescence intensity of the ER channel.
  • Figure 4 shows the detected CTCs in the peripheral blood of breast cancer patients with different expression levels of typical ER molecules.
  • Test Example 5 Polypeptide magnetic nanoparticles detect CTC in peripheral blood of breast cancer patients and perform PR molecular typing
  • CTC identification and PR fluorescence intensity analysis were performed on the detected cells.
  • CTCs DAPI+/CK+/CD45- and cells conforming to cell morphology
  • CTCs expressing PR DAPI+/CK+/PR+/CD45- cells
  • the PR expression of CTCs is judged according to the fluorescence intensity of the PR channel.
  • Figure 5 shows the detected CTCs in the peripheral blood of breast cancer patients with different expression levels of typical PR molecules.
  • Test Example 6 Detection of CTC in peripheral blood of breast cancer patients and AR molecular typing by polypeptide magnetic nanoparticles
  • CTC identification and AR fluorescence intensity analysis were performed on the detected cells.
  • CTCs DAPI+/CK+/CD45- and cells conforming to cell morphology
  • CTCs expressing AR DAPI+/CK+/AR+/CD45- cells
  • the AR expression level of CTCs is judged according to the fluorescence intensity of the AR channel.
  • Figure 6 shows the detected CTCs in the peripheral blood of breast cancer patients with different expression levels of typical AR molecules.
  • Test Example 7 Detection of CTC in peripheral blood of patients with esophageal cancer and PD-L1 molecular typing by polypeptide magnetic nanoparticles
  • CTCs DAPI+/CK+/CD45- and cells conforming to cell morphology
  • PD-L1 expressing CTCs DAPI+/CK+/PD-L1+/CD45- cells
  • CTCs are judged based on the fluorescence intensity of the PD-L1 channel The expression level of PD-L1.
  • Figure 7 shows the detected CTCs in the peripheral blood of esophageal cancer patients with different expression levels of typical PD-L1 molecules.
  • Test Example 8 Polypeptide magnetic nanoparticles were used to detect CTC in peripheral blood of patients with lung cancer and perform PD-L1 molecular typing
  • CTCs DAPI+/CK+/CD45- and cells conforming to cell morphology
  • PD-L1 expressing CTCs DAPI+/CK+/PD-L1+/CD45- cells
  • CTCs are judged based on the fluorescence intensity of the PD-L1 channel The expression level of PD-L1.
  • Figure 8 shows the detected CTCs in the peripheral blood of lung cancer patients with different expression levels of typical PD-L1 molecules.
  • Test Example 9 Detecting CTC in peripheral blood of patients with lung cancer and EGFR molecular typing by polypeptide magnetic nanoparticles
  • CTCs DAPI+/CK+/CD45- and cells conforming to cell morphology
  • CTCs expressing EGFR DAPI+/CK+/EGFR+/CD45- cells
  • the EGFR expression level of CTCs is determined according to the fluorescence intensity of the EGFR channel.
  • Figure 9 shows the detected CTCs in the peripheral blood of lung cancer patients with different expression levels of typical EGFR molecules.
  • Test Example 10 Detection of CTC in peripheral blood of patients with liver cancer and PD-L1 molecular typing by polypeptide magnetic nanoparticles
  • CTCs DAPI+/CK+/CD45- and cells conforming to cell morphology
  • PD-L1 expressing CTCs DAPI+/CK+/PD-L1+/CD45- cells
  • CTCs are judged based on the fluorescence intensity of the PD-L1 channel The expression level of PD-L1.
  • Figure 10 shows the detected CTCs in the peripheral blood of liver cancer patients with different expression levels of typical PD-L1 molecules.
  • Test Example 11 Polypeptide magnetic nanoparticles detect CTC in peripheral blood of patients with cervical cancer and perform PD-L1 molecular analysis type
  • CTCs DAPI+/CK+/CD45- and cells conforming to cell morphology
  • PD-L1 expressing CTCs DAPI+/CK+/PD-L1+/CD45- cells
  • CTCs are judged based on the fluorescence intensity of the PD-L1 channel The expression level of PD-L1.
  • Figure 11 shows the detected CTCs in the peripheral blood of cervical cancer patients with different expression levels of typical PD-L1 molecules.
  • Test Example 12 Polypeptide magnetic nanoparticles were used to detect CTC in peripheral blood of patients with gastric cancer and perform PD-L1 molecular typing
  • CTCs DAPI+/CK+/CD45- and cells conforming to cell morphology
  • PD-L1 expressing CTCs DAPI+/CK+/PD-L1+/CD45- cells
  • CTCs are judged based on the fluorescence intensity of the PD-L1 channel The expression level of PD-L1.
  • Figure 12 shows the detected CTCs in the peripheral blood of gastric cancer patients with different expression levels of typical PD-L1 molecules.
  • Test Example 13 Polypeptide magnetic nanoparticles detect CTC in peripheral blood of breast cancer patients and perform CXCR4 molecules Type
  • CTC identification and CXCR4 fluorescence intensity analysis were performed on the detected cells.
  • CTCs DAPI+/CK+/CD45- and cells conforming to cell morphology
  • CTCs expressing CXCR4 DAPI+/CK+/CXCR4+/CD45- cells
  • the CXCR4 expression level of CTCs is judged according to the fluorescence intensity of the CXCR4 channel.
  • Figure 13 shows the detected CTCs in the peripheral blood of breast cancer patients with different expression levels of typical CXCR4 molecules.
  • Test Example 14 Polypeptide magnetic nanoparticles detect CTC in peripheral blood of patients with gastric cancer and perform HER2 molecular typing
  • CTCs DAPI+/CK+/CD45- and cells conforming to cell morphology
  • CTCs expressing HER2 DAPI+/CK+/HER2+/CD45- cells
  • the HER2 expression level of CTCs is judged according to the fluorescence intensity of the HER2 channel.
  • Figure 14 shows the detected CTCs in the peripheral blood of gastric cancer patients with different expression levels of typical HER2 molecules.
  • Test Example 15 Polypeptide magnetic nanoparticles were used to detect CTC in peripheral blood of patients with colorectal cancer and perform HER2 molecular typing
  • CTCs DAPI+/CK+/CD45- and cells conforming to cell morphology
  • CTCs expressing HER2 DAPI+/CK+/HER2+/CD45- cells
  • the HER2 expression level of CTCs is judged according to the fluorescence intensity of the HER2 channel.
  • Figure 15 shows the detected CTCs in the peripheral blood of colon cancer patients with different expression levels of typical HER2 molecules.
  • Test Example 16 Polypeptide magnetic nanoparticles to detect CTC in peripheral blood of patients with colorectal cancer and perform PD-L1 molecular typing
  • CTCs DAPI+/CK+/CD45- and cells conforming to cell morphology
  • PD-L1 expressing CTCs DAPI+/CK+/PD-L1+/CD45- cells
  • CTCs are judged based on the fluorescence intensity of the PD-L1 channel The expression level of PD-L1.
  • Figure 16 shows the detected CTCs in the peripheral blood of colon cancer patients with different expression levels of typical PD-L1 molecules.
  • Test Example 17 Polypeptide magnetic nanoparticles detect CTC in peripheral blood of patients with colorectal cancer and perform VEGFR molecular analysis type
  • CTCs DAPI+/CK+/CD45- and cells conforming to cell morphology
  • CTCs expressing VEGFR DAPI+/CK+/VEGFR+/CD45- cells
  • the VEGFR expression level of CTC is judged based on the fluorescence intensity of the VEGFR channel.
  • Figure 17 shows the detected CTCs in the peripheral blood of colorectal cancer patients with different expression levels of typical VEGFR molecules.

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Abstract

一种多肽磁性纳米颗粒，包括：特异性靶向多肽和磁性纳米颗粒；其中，特异性靶向多肽的氨基酸序列为VRRDAPRFSMQGLDA-X，其C末端X为5～20个氨基酸的序列；C末端X的氨基酸序列中的氨基酸选自以下一种或多种：C，G，N。多肽磁性纳米颗粒可用于多种癌症类型的CTC检测和分子分型，包括食管癌、肝癌、肺癌、胃癌、膀胱癌、皮肤癌、黑色素瘤、乳腺癌、结直肠癌、宫颈癌等；CTC分子分型的生物标志物包括PD-L1、HER2、ER、PR、AR、EGFR、VEGFR和CXCR4等。多肽磁性纳米颗粒有助于对CTC检测并在CTC水平进行生物标志物表达水平的定性和半定量分析，从而对患者进行有针对性的个体化靶向治疗或免疫治疗等精准治疗。

Description

多肽磁性纳米颗粒、其制备方法及应用
相关申请的交叉引用
本申请要求2019年05月21日提交的第CN201910424124.5号中国发明专利申请的优先权，所述申请以引用的方式整体并入本文。
技术领域
本发明属于医学检测领域，具体涉及一种多肽磁性纳米颗粒、其制备方法和应用。
背景技术
癌症已经成为当今严重危害人类健康和生命的一类疾病，全球每年800多万人死于癌症。临床上肿瘤常规检测方法多为影像学和组织活检，受限于影像学的分辨率，5mm以下的肿瘤难以发现。而组织活检又很难实现多次取样，并且给病人带来痛苦和风险。从肿瘤原发灶脱落进入血液循环的循环肿瘤细胞(CTC)，携带原位肿瘤组织几乎所有的遗传和蛋白质信息，CTC检测作为目前液体活检的一种形式，可以动态反映肿瘤进展情况，为肿瘤的疗效预测、预后评估和复发监测等提供依据。
肿瘤是高度异质性的，即使组织学和形态相同的肿瘤，其分子生物学改变也不尽相同，不同的生物学改变具有不同的生物学行为以及治疗的敏感性，目前临床上常用的分期、分级等传统的病理分型对肿瘤的预测能力有限。近年来，随着科学技术的发展，肿瘤的靶向治疗和免疫治疗越来越受到关注，为了达到最大疗效和最小毒性，肿瘤类型的分子诊断和精确分型是治疗的关键。因此对肿瘤进行分子分型是肿瘤个体化治疗的必然要求。对于肿瘤靶向药的分子诊断和精确分型以及靶向用药的伴随诊断，可以以乳腺癌为例进行阐述。乳腺癌是女性常见的恶性肿瘤之一，其发病率逐年上升，严重威胁着女性的身心健康。目前已发现与乳腺癌发生发展有关的基因多达40多个，最重要的有人表皮生长因子受体-2(HER2)、ER(雌激素受体)、PR(孕激素受体)和雄激素受体(AR)等，并研究和开发了各种靶向治疗和激素治疗。美国食品药品管理局(FDA)在2002年已批准了靶向HER2阳性乳腺癌的药物—单克隆抗体赫赛汀用于治疗乳腺癌。临床疗效显示该药在晚期乳腺癌的术前和术后辅助治疗中均能提高患者的治疗效率，延长患者的生存期。因此，乳腺癌分子分型在肿瘤治疗，特别是靶向用药方面有着非常重要的意义。因外周血中的CTC携带原位肿瘤组织几乎所有的遗传和蛋白质信息，所以对检测的患者外周血中的CTC进行HER2、ER、PR等进行分子分型对指导患者临床治疗具有非常重要的意义。同样的，对于其他几乎所有的恶性肿瘤，靶向药的分子分型和伴随诊断都有重要的临床价值。
除肿瘤的靶向治疗外，肿瘤的免疫疗法在近年来也取得了一系列进展，改变了许多癌症的治疗格局。针对免疫检查点的PD-1/PD-L1抗体药物是当前备受瞩目同时发展最快的肿瘤免疫疗法，因此肿瘤细胞的PD-L1表达量对于该免疫治疗效果的预评估至关重要， 所以对CTC水平上PD-L1表达量的伴随诊断对PD-1/PD-L1抗体药物的免疫治疗具有重要的临床指导意义。
发明内容
因此，本发明的目的在于克服现有技术中的缺陷，提供一种用于循环肿瘤细胞检测以及肿瘤标志物分子分型的多肽磁性纳米颗粒及其制备方法和应用。
在阐述本发明内容之前，定义本文中所使用的术语如下：
术语“PBS”是指：磷酸盐缓冲液。
术语“HEPES”是指：4-羟乙基哌嗪乙磺酸缓冲液。
术语“PD-L1”是指：程序性死亡受体配体-1。
术语“HER2”是指：人表皮生长因子受体2。
术语“ER”是指：雌激素受体。
术语“PR”是指：孕激素受体。
术语“AR”是指：雄激素受体。
术语“EGFR”是指：表皮生长因子受体。
术语“CXCR4”是指：趋化因子受体4。
术语“VEGFR”是指：血管内皮细胞生长因子受体。
为实现上述目的，本发明的技术方案如下：
本发明的第一方面提供了一种多肽纳米磁性纳米颗粒，所述多肽磁性纳米颗粒包括：特异性靶向多肽和磁性纳米颗粒；其中，所述特异性靶向多肽的氨基酸序列为VRRDAPRFSMQGLDA-X，其C末端X为5～20个、优选为5～15个、更优选为9～12个氨基酸的序列，且X不为CGGNCC、CGGNCN、CGGNNC、CGGNNN、CGGNCCN、CGGNCCNN、CGGNCNN、CGGNCNNN、CGGNNCN、CGGNNCNN、CGGNNNN、CGGNNNNN；
优选地，所述X氨基酸序列中的氨基酸选自以下一种或多种：C，G，N。
根据本发明第一方面多肽磁性纳米颗粒，其中，所述多肽为靶向上皮细胞粘附分子的特异性识别多肽；
优选地，所述特异性靶向多肽的氨基酸序列为：SEQ ID NO:1～SEQ ID NO:9；最优选地，所述特异性靶向多肽的氨基酸序列为：SEQ ID NO：1。
根据第一方面多肽磁性纳米颗粒，其中，所述磁性纳米颗粒为带有链霉亲和素的磁性纳米颗粒；优选地，所述磁性纳米颗粒粒径为100～900nm；更优选地，所述磁性纳米颗粒粒径为300nm～800nm。
本发明的第二方面提供了第一方面所述的多肽磁性纳米颗粒的制备方法，所述方法包括以下步骤：
(1)制备多肽和磁性纳米颗粒溶液；
(2)将步骤(1)制得的多肽和磁性纳米颗粒溶液混合反应得到所述多肽磁性纳米 颗粒。
根据本发明第二方面方法，其中，所述步骤(1)中，制备所述多肽溶液的溶剂选自以下一种或多种：水、生理盐水、PBS、HEPES；和/或
制备所述磁性纳米颗粒溶液的溶剂选自以下一种或多种：水、PBS、HEPES。
根据本发明第二方面方法，其中，所述步骤(1)中，所述多肽溶液的终浓度为1-1000μg/mL，优选为100-500μg/mL；和/或所述磁性纳米颗粒溶液的终浓度为1-10000μg/mL，优选为1000-5000μg/mL。
根据本发明第二方面方法，其中，所述步骤(2)中，所述多肽和磁性纳米颗粒的质量比为1:10-5:1，优选为2:5。
本发明的第三方面提供了第一方面所述的多肽磁性纳米颗粒或按照第二方面所述的制备方法制得的多肽磁性纳米颗粒在制备用于诊断或治疗癌症的药物和/或医疗产品中的应用。
本发明的第四方面提供了一种用于诊断或治疗癌症的方法，所述方法包括：对有需要的受试者给予第一方面所述的多肽磁性纳米颗粒或按照第二方面所述的制备方法制得的多肽磁性纳米颗粒。
根据本发明第三方面的应用或第四方面所述的方法，其中所述癌症选自以下一种或多种：食管癌、肝癌、肺癌、胃癌、乳腺癌、结直肠癌、宫颈癌、甲状腺癌、前列腺癌、胰腺癌、肾癌、膀胱癌、皮肤癌、黑色素瘤等；优选为乳腺癌、食管癌、胃癌、肝癌、肺癌、结直肠癌、宫颈癌和/或前列腺癌等。
本发明的第五方面提供了第一方面所述的多肽磁性纳米颗粒或按照第二方面所述的制备方法制得的多肽磁性纳米颗粒在制备用于循环肿瘤细胞检测和/或分子分型的药物和/或医疗产品中的应用。
本发明的第六方面提供了一种用于诊断或治疗癌症的多肽磁性纳米颗粒和/或用于循环肿瘤细胞检测和/或分子分型的多肽磁性纳米颗粒，所述多肽磁性纳米颗粒包括第一方面所述的多肽磁性纳米颗粒或按照第二方面所述的制备方法制得的多肽磁性纳米颗粒。
本发明的第七方面提供了一种用于循环肿瘤细胞检测和/或分子分型的方法，所述方法包括：对有需要的受试者给予第一方面所述的多肽磁性纳米颗粒或按照第二方面所述的制备方法制得的多肽磁性纳米颗粒。
根据本发明第五方面的应用或第六方面所述的多肽磁性纳米颗粒或第七方面的方法，其中，所述循环肿瘤细胞检测和/或分子分型的生物标志物选自以下一种或多种：PD-L1、HER2、ER、PR、AR、EGFR、CXCR4、VEGFR等。
本发明提供了一种用于CTC检测的多肽磁性纳米颗粒包括：
1)靶向上皮细胞粘附分子(EpCAM)的特异性识别多肽，对应的优选序列为VRRDAPRFSMQGLDACGGNNCNNNNN及其可能的突变体。。
2)带有链霉亲和素的磁性纳米颗粒，粒径为100～900nm，优选地，粒径为300nm～800nm)将上述1)和2)中的结合。
所述方法包括以下步骤：
a)将多肽粉末溶解于一定量的溶剂中，得到浓度为1-1000μg/mL的多肽溶液；
优选地，所述溶剂选自多肽的良溶剂为水、生理盐水、PBS、HEPES。
b)将磁性纳米颗粒用一定量的溶剂稀释，得到浓度为1-10000μg/mL的磁性纳米颗粒溶液；
优选地，所述溶剂为磁珠分散剂水、PBS、HEPES。
c)将上述多肽溶液和磁性纳米颗粒溶液按一定比例混合，置于25-37℃温度，100-160rpm转速的摇床上发生反应0.5-2h，将得到多肽磁性纳米颗粒组装体离心洗涤，优选地，离心转速为5000-10000rpm，将得到的多肽磁性纳米颗粒悬液置于4℃保存。
本发明还提供了所述的多肽纳米检测装置对CTC的检测。
优选地，所述CTC为SK-BR-3、MCF-7、MDA-MB-231、H1975、H1650和A549肿瘤细胞。
本发明还提供了所述的多肽纳米检测装置对肿瘤患者外周血的CTC检测和检出的CTC进行肿瘤标志物的分子分型。优选地，适用于乳腺癌、食管癌、胃癌、肝癌、肺癌、结直肠癌、宫颈癌、前列腺癌等癌症患者外周血中的CTC检测和分子分型。所述的多肽纳米技术检测CTC的步骤包括孵育、洗涤、离心、固定、封闭、免疫荧光染色、CTC鉴定等。
本发明还提供了对于检测出的CTC进行相关的分子分型。CTC相关分子分型的鉴定包括对CTC相应分子的荧光强度统计分析，并按照一定的阈值进行表达强度的界定。分子分型包括阳性表达和阴性表达，其中阳性表达包括高、中、低表达。
CTC分子分型的生物标志物包括PD-L1、HER2、ER、PR、AR、EGFR、CXCR4和VEGFR等多种实体肿瘤细胞的生物标志物。
多肽纳米磁珠技术可以用于除脑部肿瘤、骨肉瘤、淋巴瘤之外的几乎所有实体肿瘤，包括食管癌、肝癌、肺癌、胃癌、乳腺癌、结直肠癌、宫颈癌、甲状腺癌、前列腺癌、胰腺癌、肾癌、膀胱癌、皮肤癌、黑色素瘤等。
本发明的目的之一是提供一种利用一种用于循环肿瘤细胞检测以及肿瘤标志物分子分型的多肽磁性纳米颗粒和应用。该方法可对乳腺癌实现体外诊断及分子分型。该方法操作简便、成本低，检测过程迅速，非侵入的特性避免了常规的病理检测给患者带来的痛苦。除此之外，该方法有望对病情进行实时追踪，根据病情发展及时调节治疗方案，为实现个性化医疗提供了指导思路。
本发明公开了循环肿瘤细胞的HER2、ER、PR、AR、EGFR、VEGFR、PD-L1等蛋白作为癌症诊断及分子分型标志物的应用。本发明借助倒置荧光显微镜检测蛋白标志物 表达量作为肿瘤患者分子分型的应用。本发明的实验，在临床血液样本的检测中证实了该方法用于诊断和分子分型的可行性。
根据本发明的具体实施方案，本发明的应用中，所述肿瘤包括乳腺癌、肝癌、肺癌、胃癌、食管癌、结直肠癌、前列腺癌和宫颈癌中的一种或多种。
本发明的用于循环肿瘤细胞检测及分子分型的多肽磁性纳米颗粒可以具有但不限于以下有益效果：
1.本发明所述的多肽纳米技术对CTC的检测具有较高的灵敏度和特异性，能对临床多种肿瘤患者的外周血进行CTC的检测，包括乳腺癌、肝癌、肺癌、胃癌、食管癌、结直肠癌、前列腺癌和宫颈癌等多种肿瘤。
2.本发明涉及对检测到的CTC进行肿瘤相关标志物的分子分型的方法。利用循环肿瘤细胞中目标蛋白标志物的表达量对受试者进行体外诊断及分子分型，该方法有望实现疾病的早筛及病情的实时追踪，为辅助肿瘤检测及跟踪治疗效果提供了新方法，同时还可成为预后评价的重要手段，为实现个性化医疗提供了指导思路，在提高患者的生存质量，延长生存期方面有着很好的应用前景。
附图的简要说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1示出了试验例1NO:1多肽纳米磁珠富集乳腺癌细胞并进行HER2分子分型的结果，其中，图1A为试验例1SEQ ID NO:1多肽纳米磁珠对SK-BR-3、MCF-7、MDA-MB-231乳腺癌细胞的捕获。图1B为试验例1SEQ ID NO:1多肽纳米磁珠检测到的HER2表达量不同的典型乳腺癌细胞。
图2示出了试验例2SEQ ID NO:1多肽纳米磁珠富集肺癌细胞并进行PD-L1分子分型的结果，其中，图2A为试验例2SEQ ID NO:1多肽纳米磁珠对H1975、H1650和A549细胞肺癌细胞的捕获。图2B为试验例2SEQ ID NO:1多肽纳米磁珠检测到的PD-L1表达量不同的典型肺癌细胞。
图3为试验例3检测到的HER2表达量不同的乳腺癌患者外周血中典型的CTC。
图4为试验例4检测到的ER表达量不同的乳腺癌患者外周血中典型的CTC。
图5为试验例5检测到的典型PR分子表达量不同的乳腺癌患者外周血的CTC。
图6为试验例6检测到的典型AR分子表达量不同的乳腺癌患者外周血的CTC。
图7为试验例7检测到的典型PD-L1分子表达量不同的食管癌患者外周血的CTC。
图8为试验例8检测到的典型PD-L1分子表达量不同的肺癌患者外周血的CTC。
图9为试验例9检测到的典型EGFR分子表达量不同的肺癌患者外周血的CTC。
图10为试验例10检测到的典型PD-L1分子表达量不同的肝癌患者外周血的CTC。
图11为试验例11检测到的典型PD-L1分子表达量不同的宫颈癌患者外周血的CTC。
图12为试验例12检测到的典型PD-L1分子表达量不同的胃癌患者外周血的CTC。
图13为试验例13检测到的典型CXCR4分子表达量不同的乳腺癌癌患者外周血的CTC。
图14为试验例14检测到的典型HER2分子表达量不同的胃癌癌患者外周血的CTC。
图15为试验例15检测到的典型HER2分子表达量不同的肠癌癌患者外周血的CTC。
图16为试验例16检测到的典型PD-L1分子表达量不同的肠癌癌患者外周血的CTC。
图17为试验例17检测到的典型VEGFR分子表达量不同的肠癌癌患者外周血的CTC。
实施发明的最佳方式
下面通过具体的实施例进一步说明本发明，但是，应当理解为，这些实施例仅仅是用于更详细具体地说明之用，而不应理解为用于以任何形式限制本发明。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
本部分对本发明试验中所使用到的材料以及试验方法进行一般性的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的，但是本发明仍然在此作尽可能详细描述。本领域技术人员清楚，在上下文中，如果未特别说明，本发明所用材料和操作方法是本领域公知的。
除非特别指明，以下实施例中所用的人源肿瘤细胞系SK-BR-3、MCF-7、MDA-MB-231、H1975、H1650、A549均购自中国医学科学院基础研究所的细胞库。
除非特别指明，以下实施例中所用的多肽纯度为98％以上。
除非特别指明，以下实施例中所用水溶液的溶剂均为无菌超纯水溶液，电阻率18.2MΩ.cm。
除非特别指明，以下实施例中所用的试剂均为分析纯试剂。
除非特别指明，以下实施例中所用的扫描显微镜均为奥林巴斯显微镜IX73。
以下实施例中使用的试剂和仪器如下：
试剂：
磁珠，购自Thermo Fisher。
多肽，由北京中科纳泰生物科技有限公司自行合成，纯度为98％。
PBS，多聚甲醛，完全培养基，DAPI工作液，免疫荧光染色封闭液，均购自Hyclone。
仪器：
磁力架，北京中科纳泰生物科技有限公司自制，可放置15毫升离心管的磁力架。
荧光显微镜：
Olympus IX73，购自北京冷泉科技有限公司。
ZEISS Axio Vert A1和ZEISS Z2，购自蔡司遠東有限公司。
Thermo Fisher CX5，购自Thermo Fisher。
Nikon Ti-S，购自北京恒三江仪器销售有限公司。
优先推荐ZEISS Z2，其次Olympus IX73和Thermo Fisher CX5。
实施例1：多肽磁性纳米颗粒组装体配制
1)取400μL500nm的磁珠于2毫升Ep管中，加入1毫升PBS洗涤，然后把管放于磁力架上富集磁珠10分钟，弃上清。
2)加入2毫升PBS洗涤，然后把管放于磁力架上富集磁珠10分钟，弃上清。
3)加入1毫升PBS溶解多肽粉末，震荡涡旋，将多肽溶液加入到盛有磁珠的Ep管中，涡旋仪涡旋1分钟，将多肽磁珠混合液放置在脱色摇床上，转速调为60rpm，室温孵育1小时。
4)将Ep管放于磁力架上富集多肽磁珠10分钟，弃上清。再加1.5毫升PBS洗3遍。
5)加入400μLPBS，涡旋1分钟，将配制好的多肽磁珠存放于4℃冰箱。
以下试验例中，试验例1-2采用SEQ ID NO:1-9的多肽磁性纳米颗粒组装体，试验例3-17均采用SEQ ID NO:1的多肽磁性纳米颗粒组装体。
试验例1：多肽磁性纳米颗粒富集乳腺癌细胞并进行HER2分子分型
收集对数生长期的SK-BR-3、MCF-7和MDA-MB-231细胞，将细胞重悬至各自的完全培养基(含10％胎牛血清、100U/mL青霉素、100μg/mL链霉素)中，计数细胞浓度，每种细胞各取约1000个加入到2毫升健康人血中，加入10μL多肽纳米磁珠混匀，室温摇床孵育1小时，取下离心管，加5mL PBS轻轻混匀，放到磁力架上，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，加5mL PBS，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，将离心管从磁力架上取下，用多聚甲醛将管壁上的磁珠吹下，室温固定30分钟，加5mL PBS离心洗涤；滴加DAPI工作液染细胞核，染核结束后加加5mL PBS离心洗涤；加200μL免疫荧光染色封闭液，室温封闭30分钟，加5mL PBS离心洗涤；富集到的细胞分别用FITC-CK、PE-CD45和Alexa Fluor647-HER2(Abcam)抗体染色1h；加5mL PBS离心洗涤封片，20倍物镜下观察找到细胞界面，设置DAPI、FITC、PE和Alexa Fluor 647各荧光通道相应的曝光时间并对样品区域进行荧光扫描，对检测到的细胞进行CTC鉴定和HER2荧光强度分析。其中DAPI+/CK+/CD45-并且符合细胞形态的细胞指认为CTC，DAPI+/CK+/HER2+/CD45-的细胞指认为表达HER2的CTC，并根据HER2通道的荧光强度来判读CTC的HER2表达量。如图1A所示，SEQ ID NO:1多肽纳米磁珠对SK-BR-3、MCF-7和MDA-MB-231的捕获率稳定性好且都达到90％以上，表明该多肽纳米磁珠对乳腺癌细胞具有非常高的富集和检出效率。图1B为SEQ ID NO:1多肽纳米磁珠富集到的HER2表达量不同的乳腺癌细胞，表1为SEQ ID NO:1-9对SK-BR-3、MCF-7和MDA-MB-231三种乳腺癌细胞的检出率。
表1对SK-BR-3、MCF-7和MDA-MB-231三种乳腺癌细胞的检出率
Figure PCTCN2020091063-appb-000001
Figure PCTCN2020091063-appb-000002
试验例2：多肽磁性纳米颗粒富集肺癌细胞并进行PD-L1分子分型
收集对数生长期的肺癌H1975、H1650和A549细胞，将细胞重悬至各自的完全培养基(含10％胎牛血清、100U/mL青霉素、100μg/mL链霉素)中，计数细胞浓度，每种细胞各取约1000个加入到2毫升健康人血中，加入10μL多肽纳米磁珠混匀，室温摇床孵育1小时，取下离心管，加5mL PBS轻轻混匀，放到磁力架上，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，加5mL PBS，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，将离心管从磁力架上取下，用多聚甲醛将管壁上的磁珠吹下，室温固定30分钟，加5mL PBS离心洗涤；滴加DAPI工作液染细胞核，染核结束后加5mL PBS离心洗涤；加200μL免疫荧光染色封闭液，室温封闭30分钟，加5mL PBS离心洗涤；富集到的CTC分别用FITC-CK、PE-CD45和Alexa Fluor 647-PD-L1抗体染色1h；加5mL PBS离心洗涤封片，20倍物镜下观察，设置DAPI、FITC、PE和Alexa Fluor 647各荧光通道相应的曝光时间并对样品区域进行荧光扫描和荧光强度分析。其中DAPI+/CK+/CD45-并且符合细胞形态的细胞指认为CTC，DAPI+/CK+/PD-L1+/CD45-的细胞指认为表达PD-L1的CTC，并根据PD-L1通道的荧光强度来判读CTC的PD-L1表达量。如图2A所示，该多肽纳米磁珠对H1975、H1650和A549的捕获率都达到60％以上，表明该多肽纳米磁珠对乳腺癌细胞具有非常高的富集和检出效率。图2B为SEQ ID NO:1多肽纳米磁珠富集到的PD-L1分子表达量不同的肺癌细胞。表2为SEQ ID NO:1-9对肺癌H1975、H1650和A549细胞的检出率。
表2对肺癌H1975、H1650和A549细胞的检出率
Figure PCTCN2020091063-appb-000003
试验例3：多肽磁性纳米颗粒检测乳腺癌患者外周血的CTC并进行HER2分子分型
取2mL乳腺癌患者外周血到15mL离心管中，加入10μL多肽纳米磁珠混匀，室温摇床孵育1小时，取下离心管，加5mL PBS轻轻混匀，放到磁力架上，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，加5mL PBS，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，将离心管从磁力架上取下，用多聚甲醛 将管壁上的磁珠吹下，室温固定30分钟，加5mL PBS离心洗涤；滴加DAPI工作液染细胞核，染核结束后加5mL PBS离心洗涤；加200μL免疫荧光染色封闭液，室温封闭30分钟，加5mL PBS离心洗涤；富集到的细胞分别用FITC-CK、PE-CD45和Alexa Fluor647-HER2(Abcam)抗体染色1h；加5mL PBS离心洗涤封片，20倍物镜下观察找到细胞界面，设置DAPI、FITC、PE和Alexa Fluor 647各荧光通道相应的曝光时间并对样品区域进行荧光扫描，对检测到的细胞进行CTC鉴定和HER2荧光强度分析。其中DAPI+/CK+/CD45-并且符合细胞形态的细胞指认为CTC，DAPI+/CK+/HER2+/CD45-的细胞指认为表达HER2的CTC，并根据HER2通道的荧光强度来判读CTC的HER2表达量。图3为检测到的典型HRE2表达量不同的乳腺癌患者外周血的CTC。
试验例4：多肽磁性纳米颗粒检测乳腺癌患者外周血的CTC并进行ER分子分型
取2mL乳腺癌患者外周血到15mL离心管中，加入10μL多肽纳米磁珠混匀，室温摇床孵育1小时，取下离心管，加5mL PBS轻轻混匀，放到磁力架上，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，加5mL PBS，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，将离心管从磁力架上取下，用多聚甲醛将管壁上的磁珠吹下，室温固定30分钟，加5mL PBS离心洗涤；滴加DAPI工作液染细胞核，染核结束后加5mL PBS离心洗涤；加200μL免疫荧光染色封闭液，室温封闭30分钟，加5mL PBS离心洗涤；富集到的细胞分别用FITC-CK、PE-CD45和Alexa Fluor647-ER(Abcam)抗体染色1h；加5mL PBS离心洗涤封片，20倍物镜下观察找到细胞界面，设置DAPI、FITC、PE和Alexa Fluor 647各荧光通道相应的曝光时间并对样品区域进行荧光扫描，对检测到的细胞进行CTC鉴定和ER荧光强度分析。其中DAPI+/CK+/CD45-并且符合细胞形态的细胞指认为CTC，DAPI+/CK+/ER+/CD45-的细胞指认为表达ER的CTC，并根据ER通道的荧光强度来判读CTC的ER表达量。图4为检测到的典型ER分子表达量不同的乳腺癌患者外周血的CTC。
试验例5：多肽磁性纳米颗粒检测乳腺癌患者外周血的CTC并进行PR分子分型
取2mL乳腺癌患者外周血到15mL离心管中，加入10μL多肽纳米磁珠混匀，室温摇床孵育1小时，取下离心管，加5mL PBS轻轻混匀，放到磁力架上，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，加5mL PBS，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，将离心管从磁力架上取下，用多聚甲醛将管壁上的磁珠吹下，室温固定30分钟，加5mL PBS离心洗涤；滴加DAPI工作液染细胞核，染核结束后加5mL PBS离心洗涤；加200μL免疫荧光染色封闭液，室温封闭30分钟，加5mL PBS离心洗涤；富集到的细胞分别用FITC-CK、PE-CD45和Alexa Fluor647-PR(Abcam)抗体染色1h；加5mL PBS离心洗涤封片，20倍物镜下观察找到细胞界面，设置DAPI、FITC、PE和Alexa Fluor 647各荧光通道相应的曝光时间并对样品区域进行荧光扫描，对检测到的细胞进行CTC鉴定和PR荧光强度分析。其中DAPI+/CK+/CD45-并且符合细胞形态的细胞指认为CTC，DAPI+/CK+/PR+/CD45-的细胞指认为表达PR的CTC，并根据PR通道的荧光强度来判读CTC的PR表达量。图5为检测到的典型PR分子表达量不同的乳腺癌患者外周血的CTC。
试验例6：多肽磁性纳米颗粒检测乳腺癌患者外周血的CTC并进行AR分子分型
取2mL乳腺癌患者外周血到15mL离心管中，加入10μL多肽纳米磁珠混匀，室温摇床孵育1小时，取下离心管，加5mL PBS轻轻混匀，放到磁力架上，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，加5mL PBS，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，将离心管从磁力架上取下，用多聚甲醛将管壁上的磁珠吹下，室温固定30分钟，加5mL PBS离心洗涤；滴加DAPI工作液染细胞核，染核结束后加5mL PBS离心洗涤；加200μL免疫荧光染色封闭液，室温封闭30分钟，加5mL PBS离心洗涤；富集到的细胞分别用FITC-CK、PE-CD45和Alexa Fluor647-AR(Abcam)抗体染色1h；加5mL PBS离心洗涤封片，20倍物镜下观察找到细胞界面，设置DAPI、FITC、PE和Alexa Fluor 647各荧光通道相应的曝光时间并对样品区域进行荧光扫描，对检测到的细胞进行CTC鉴定和AR荧光强度分析。其中DAPI+/CK+/CD45-并且符合细胞形态的细胞指认为CTC，DAPI+/CK+/AR+/CD45-的细胞指认为表达AR的CTC，并根据AR通道的荧光强度来判读CTC的AR表达量。图6为检测到的典型AR分子表达量不同的乳腺癌患者外周血的CTC。
试验例7：多肽磁性纳米颗粒检测食管癌患者外周血的CTC并进行PD-L1分子分型
取2mL食管癌患者外周血到15mL离心管中，加入10μL多肽纳米磁珠混匀，室温摇床孵育1小时，取下离心管，加5mL PBS轻轻混匀，放到磁力架上，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，加5mL PBS，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，将离心管从磁力架上取下，用多聚甲醛将管壁上的磁珠吹下，室温固定30分钟，加5mL PBS离心洗涤；滴加DAPI工作液染细胞核，染核结束后加5mL PBS离心洗涤；加200μL免疫荧光染色封闭液，室温封闭30分钟，加5mL PBS离心洗涤；富集到的细胞分别用FITC-CK、PE-CD45和Alexa Fluor647-PD-L1(Abcam)抗体染色1h；加5mL PBS离心洗涤封片，20倍物镜下观察找到细胞界面，设置DAPI、FITC、PE和Alexa Fluor 647各荧光通道相应的曝光时间并对样品区域进行荧光扫描，对检测到的细胞进行CTC鉴定和PD-L1荧光强度分析。其中DAPI+/CK+/CD45-并且符合细胞形态的细胞指认为CTC，DAPI+/CK+/PD-L1+/CD45-的细胞指认为表达PD-L1的CTC，并根据PD-L1通道的荧光强度来判读CTC的PD-L1表达量。图7为检测到的典型PD-L1分子表达量不同的食管癌患者外周血的CTC。
试验例8：多肽磁性纳米颗粒检测肺癌患者外周血的CTC并进行PD-L1分子分型
取2mL肺癌患者外周血到15mL离心管中，加入10μL多肽纳米磁珠混匀，室温摇床孵育1小时，取下离心管，加5mL PBS轻轻混匀，放到磁力架上，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，加5mL PBS，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，将离心管从磁力架上取下，用多聚甲醛将管壁上的磁珠吹下，室温固定30分钟，加5mL PBS离心洗涤；滴加DAPI工作液染细胞核，染核结束后加5mL PBS离心洗涤；加200μL免疫荧光染色封闭液，室温封闭30分钟，加5mL PBS离心洗涤；富集到的细胞分别用FITC-CK、PE-CD45和Alexa Fluor  647-PD-L1(Abcam)抗体染色1h；加5mL PBS离心洗涤封片，20倍物镜下观察找到细胞界面，设置DAPI、FITC、PE和Alexa Fluor 647各荧光通道相应的曝光时间并对样品区域进行荧光扫描，对检测到的细胞进行CTC鉴定和PD-L1荧光强度分析。其中DAPI+/CK+/CD45-并且符合细胞形态的细胞指认为CTC，DAPI+/CK+/PD-L1+/CD45-的细胞指认为表达PD-L1的CTC，并根据PD-L1通道的荧光强度来判读CTC的PD-L1表达量。图8为检测到的典型PD-L1分子表达量不同的肺癌患者外周血的CTC。
试验例9：多肽磁性纳米颗粒检测肺癌患者外周血的CTC并进行EGFR分子分型
取2mL肺癌患者外周血到15mL离心管中，加入10μL多肽纳米磁珠混匀，室温摇床孵育1小时，取下离心管，加5mL PBS轻轻混匀，放到磁力架上，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，加5mL PBS，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，将离心管从磁力架上取下，用多聚甲醛将管壁上的磁珠吹下，室温固定30分钟，加5mL PBS离心洗涤；滴加DAPI工作液染细胞核，染核结束后加5mL PBS离心洗涤；加200μL免疫荧光染色封闭液，室温封闭30分钟，加5mL PBS离心洗涤；富集到的细胞分别用FITC-CK、PE-CD45和Alexa Fluor 647-EGFR(Abcam)抗体染色1h；加5mL PBS离心洗涤封片，20倍物镜下观察找到细胞界面，设置DAPI、FITC、PE和Alexa Fluor 647各荧光通道相应的曝光时间并对样品区域进行荧光扫描，对检测到的细胞进行CTC鉴定和EGFR荧光强度分析。其中DAPI+/CK+/CD45-并且符合细胞形态的细胞指认为CTC，DAPI+/CK+/EGFR+/CD45-的细胞指认为表达EGFR的CTC，并根据EGFR通道的荧光强度来判读CTC的EGFR表达量。图9为检测到的典型EGFR分子表达量不同的肺癌患者外周血的CTC。
试验例10：多肽磁性纳米颗粒检测肝癌患者外周血的CTC并进行PD-L1分子分型
取2mL肝癌患者外周血到15mL离心管中，加入10μL多肽纳米磁珠混匀，室温摇床孵育0.5-1小时，取下离心管，加5mL PBS轻轻混匀，放到磁力架上，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，加5mL PBS，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，将离心管从磁力架上取下，用多聚甲醛将管壁上的磁珠吹下，室温固定30分钟，加5mL PBS离心洗涤；滴加DAPI工作液染细胞核，染核结束后加5mL PBS离心洗涤；加200μL免疫荧光染色封闭液，室温封闭30分钟，加5mL PBS离心洗涤；富集到的细胞分别用FITC-CK、PE-CD45和Alexa Fluor647-PD-L1(Abcam)抗体染色1h；加5mL PBS离心洗涤封片，20倍物镜下观察找到细胞界面，设置DAPI、FITC、PE和Alexa Fluor 647各荧光通道相应的曝光时间并对样品区域进行荧光扫描，对检测到的细胞进行CTC鉴定和PD-L1荧光强度分析。其中DAPI+/CK+/CD45-并且符合细胞形态的细胞指认为CTC，DAPI+/CK+/PD-L1+/CD45-的细胞指认为表达PD-L1的CTC，并根据PD-L1通道的荧光强度来判读CTC的PD-L1表达量。图10为检测到的典型PD-L1分子表达量不同的肝癌患者外周血的CTC。
试验例11：多肽磁性纳米颗粒检测宫颈癌患者外周血的CTC并进行PD-L1分子分 型
取2mL宫颈癌患者外周血到15mL离心管中，加入10μL多肽纳米磁珠混匀，室温摇床孵育1小时，取下离心管，加5mL PBS轻轻混匀，放到磁力架上，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，加5mL PBS，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，将离心管从磁力架上取下，用多聚甲醛将管壁上的磁珠吹下，室温固定30分钟，加5mL PBS离心洗涤；滴加DAPI工作液染细胞核，染核结束后加5mL PBS离心洗涤；加200μL免疫荧光染色封闭液，室温封闭30分钟，加5mL PBS离心洗涤；富集到的细胞分别用FITC-CK、PE-CD45和Alexa Fluor647-PD-L1(Abcam)抗体染色1h；加5mL PBS离心洗涤封片，20倍物镜下观察找到细胞界面，设置DAPI、FITC、PE和Alexa Fluor 647各荧光通道相应的曝光时间并对样品区域进行荧光扫描，对检测到的细胞进行CTC鉴定和PD-L1荧光强度分析。其中DAPI+/CK+/CD45-并且符合细胞形态的细胞指认为CTC，DAPI+/CK+/PD-L1+/CD45-的细胞指认为表达PD-L1的CTC，并根据PD-L1通道的荧光强度来判读CTC的PD-L1表达量。图11为检测到的典型PD-L1分子表达量不同的宫颈癌患者外周血的CTC。
试验例12：多肽磁性纳米颗粒检测胃癌患者外周血的CTC并进行PD-L1分子分型
取2mL胃癌患者外周血到15mL离心管中，加入10μL多肽纳米磁珠混匀，室温摇床孵育1小时，取下离心管，加5mL PBS轻轻混匀，放到磁力架上，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，加5mL PBS，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，将离心管从磁力架上取下，用多聚甲醛将管壁上的磁珠吹下，室温固定30分钟，加5mL PBS离心洗涤；滴加DAPI工作液染细胞核，染核结束后加5mL PBS离心洗涤；加200μL免疫荧光染色封闭液，室温封闭30分钟，加5mL PBS离心洗涤；富集到的细胞分别用FITC-CK、PE-CD45和Alexa Fluor 647-PD-L1(Abcam)抗体染色1h；加5mL PBS离心洗涤封片，20倍物镜下观察找到细胞界面，设置DAPI、FITC、PE和Alexa Fluor 647各荧光通道相应的曝光时间并对样品区域进行荧光扫描，对检测到的细胞进行CTC鉴定和PD-L1荧光强度分析。其中DAPI+/CK+/CD45-并且符合细胞形态的细胞指认为CTC，DAPI+/CK+/PD-L1+/CD45-的细胞指认为表达PD-L1的CTC，并根据PD-L1通道的荧光强度来判读CTC的PD-L1表达量。图12为检测到的典型PD-L1分子表达量不同的胃癌患者外周血的CTC。
试验例13：多肽磁性纳米颗粒检测乳腺癌患者外周血的CTC并进行CXCR4分子 分型
取2mL乳腺癌患者外周血到15mL离心管中，加入10μL多肽纳米磁珠混匀，室温摇床孵育1小时，取下离心管，加5mL PBS轻轻混匀，放到磁力架上，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，加5mL PBS，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，将离心管从磁力架上取下，用多聚甲醛将管壁上的磁珠吹下，室温固定30分钟，加5mL PBS离心洗涤；滴加DAPI工作液染细胞核，染核结束后加5mL PBS离心洗涤；加200μL免疫荧光染色封闭液，室温封闭30分钟，加5mL PBS离心洗涤；富集到的细胞分别用FITC-CK、PE-CD45和Alexa Fluor647-CXCR4(Abcam)抗体染色1h；加5mL PBS离心洗涤封片，20倍物镜下观察找到 细胞界面，设置DAPI、FITC、PE和Alexa Fluor 647各荧光通道相应的曝光时间并对样品区域进行荧光扫描，对检测到的细胞进行CTC鉴定和CXCR4荧光强度分析。其中DAPI+/CK+/CD45-并且符合细胞形态的细胞指认为CTC，DAPI+/CK+/CXCR4+/CD45-的细胞指认为表达CXCR4的CTC，并根据CXCR4通道的荧光强度来判读CTC的CXCR4表达量。图13为检测到的典型CXCR4分子表达量不同的乳腺癌患者外周血的CTC。
试验例14：多肽磁性纳米颗粒检测胃癌患者外周血的CTC并进行HER2分子分型
取2mL胃癌患者外周血到15mL离心管中，加入10μL多肽纳米磁珠混匀，室温摇床孵育1小时，取下离心管，加5mL PBS轻轻混匀，放到磁力架上，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，加5mL PBS，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，将离心管从磁力架上取下，用多聚甲醛将管壁上的磁珠吹下，室温固定30分钟，加5mL PBS离心洗涤；滴加DAPI工作液染细胞核，染核结束后加5mL PBS离心洗涤；加200μL免疫荧光染色封闭液，室温封闭30分钟，加5mL PBS离心洗涤；富集到的细胞分别用FITC-CK、PE-CD45和Alexa Fluor 647-HER2(Abcam)抗体染色1h；加5mL PBS离心洗涤封片，20倍物镜下观察找到细胞界面，设置DAPI、FITC、PE和Alexa Fluor 647各荧光通道相应的曝光时间并对样品区域进行荧光扫描，对检测到的细胞进行CTC鉴定和HER2荧光强度分析。其中DAPI+/CK+/CD45-并且符合细胞形态的细胞指认为CTC，DAPI+/CK+/HER2+/CD45-的细胞指认为表达HER2的CTC，并根据HER2通道的荧光强度来判读CTC的HER2表达量。图14为检测到的典型HER2分子表达量不同的胃癌患者外周血的CTC。
试验例15：多肽磁性纳米颗粒检测肠癌患者外周血的CTC并进行HER2分子分型
取2mL肠癌患者外周血到15mL离心管中，加入10μL多肽纳米磁珠混匀，室温摇床孵育1小时，取下离心管，加5mL PBS轻轻混匀，放到磁力架上，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，加5mL PBS，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，将离心管从磁力架上取下，用多聚甲醛将管壁上的磁珠吹下，室温固定30分钟，加5mL PBS离心洗涤；滴加DAPI工作液染细胞核，染核结束后加5mL PBS离心洗涤；加200μL免疫荧光染色封闭液，室温封闭30分钟，加5mL PBS离心洗涤；富集到的细胞分别用FITC-CK、PE-CD45和Alexa Fluor 647-HER2(Abcam)抗体染色1h；加5mL PBS离心洗涤封片，20倍物镜下观察找到细胞界面，设置DAPI、FITC、PE和Alexa Fluor 647各荧光通道相应的曝光时间并对样品区域进行荧光扫描，对检测到的细胞进行CTC鉴定和HER2荧光强度分析。其中DAPI+/CK+/CD45-并且符合细胞形态的细胞指认为CTC，DAPI+/CK+/HER2+/CD45-的细胞指认为表达HER2的CTC，并根据HER2通道的荧光强度来判读CTC的HER2表达量。图15为检测到的典型HER2分子表达量不同的肠癌患者外周血的CTC。
试验例16：多肽磁性纳米颗粒检测肠癌患者外周血的CTC并进行PD-L1分子分型
取2mL肠癌患者外周血到15mL离心管中，加入10μL多肽纳米磁珠混匀，室温摇床孵育1小时，取下离心管，加5mL PBS轻轻混匀，放到磁力架上，再将磁力架置于水 平摇床上，富集30min。取下磁力架，弃去上清，加5mL PBS，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，将离心管从磁力架上取下，用多聚甲醛将管壁上的磁珠吹下，室温固定30分钟，加5mL PBS离心洗涤；滴加DAPI工作液染细胞核，染核结束后加5mL PBS离心洗涤；加200μL免疫荧光染色封闭液，室温封闭30分钟，加5mL PBS离心洗涤；富集到的细胞分别用FITC-CK、PE-CD45和Alexa Fluor 647-PD-L1(Abcam)抗体染色1h；加5mL PBS离心洗涤封片，20倍物镜下观察找到细胞界面，设置DAPI、FITC、PE和Alexa Fluor 647各荧光通道相应的曝光时间并对样品区域进行荧光扫描，对检测到的细胞进行CTC鉴定和PD-L1荧光强度分析。其中DAPI+/CK+/CD45-并且符合细胞形态的细胞指认为CTC，DAPI+/CK+/PD-L1+/CD45-的细胞指认为表达PD-L1的CTC，并根据PD-L1通道的荧光强度来判读CTC的PD-L1表达量。图16为检测到的典型PD-L1分子表达量不同的肠癌患者外周血的CTC。
试验例17：多肽磁性纳米颗粒检测肠癌患者外周血的CTC并进行VEGFR分子分 型
取2mL肠癌患者外周血到15mL离心管中，加入10μL多肽纳米磁珠混匀，室温摇床孵育1小时，取下离心管，加5mL PBS轻轻混匀，放到磁力架上，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，加5mL PBS，再将磁力架置于水平摇床上，富集30min。取下磁力架，弃去上清，将离心管从磁力架上取下，用多聚甲醛将管壁上的磁珠吹下，室温固定30分钟，加5mL PBS离心洗涤；滴加DAPI工作液染细胞核，染核结束后加5mL PBS离心洗涤；加200μL免疫荧光染色封闭液，室温封闭30分钟，加5mL PBS离心洗涤；富集到的细胞分别用FITC-CK、PE-CD45和Alexa Fluor 647-VEGFR(Abcam)抗体染色1h；加5mL PBS离心洗涤封片，20倍物镜下观察找到细胞界面，设置DAPI、FITC、PE和Alexa Fluor 647各荧光通道相应的曝光时间并对样品区域进行荧光扫描，对检测到的细胞进行CTC鉴定和VEGFR荧光强度分析。其中DAPI+/CK+/CD45-并且符合细胞形态的细胞指认为CTC，DAPI+/CK+/VEGFR+/CD45-的细胞指认为表达VEGFR的CTC，并根据VEGFR通道的荧光强度来判读CTC的VEGFR表达量。图17为检测到的典型VEGFR分子表达量不同的肠癌患者外周血的CTC。
尽管本发明已进行了一定程度的描述，明显地，在不脱离本发明的精神和范围的条件下，可进行各个条件的适当变化。可以理解，本发明不限于所述实施方案，而归于权利要求的范围，其包括所述每个因素的等同替换。

Claims (14)

  1. 一种多肽纳米磁性纳米颗粒，其特征在于，所述多肽磁性纳米颗粒包括：特异性靶向多肽和磁性纳米颗粒；其中，所述特异性靶向多肽的氨基酸序列为VRRDAPRFSMQGLDA-X，其C末端X为5～20个、优选为5～15个、更优选为9～12个氨基酸的序列，且X不为CGGNCC、CGGNCN、CGGNNC、CGGNNN、CGGNCCN、CGGNCCNN、CGGNCNN、CGGNCNNN、CGGNNCN、CGGNNCNN、CGGNNNN、CGGNNNNN；
    优选地，所述X氨基酸序列中的氨基酸选自以下一种或多种：C，G，N。
  2. 根据权利要求1所述的多肽磁性纳米颗粒，其特征在于，所述多肽为靶向上皮细胞粘附分子的特异性识别多肽；
    优选地，所述特异性靶向多肽的氨基酸序列为SEQ ID NO：1～9；
    最优选地，所述特异性靶向多肽的氨基酸序列为SEQ ID NO：1。
  3. 根据权利要求1或2所述的多肽磁性纳米颗粒，其特征在于，所述磁性纳米颗粒为带有链霉亲和素的磁性纳米颗粒；优选地，所述磁性纳米颗粒粒径为100～900nm；更优选地，所述磁性纳米颗粒粒径为300nm～800nm。
  4. 根据权利要求1至3中任一项所述的多肽磁性纳米颗粒的制备方法，其特征在于，所述方法包括以下步骤：
    (1)制备多肽和磁性纳米颗粒溶液；
    (2)将步骤(1)制得的多肽和磁性纳米颗粒溶液混合反应得到所述多肽磁性纳米颗粒。
  5. 根据权利要求4所述的方法，其特征在于，所述步骤(1)中，制备所述多肽溶液的溶剂选自以下一种或多种：水、生理盐水、PBS、HEPES；和/或
    制备所述磁性纳米颗粒溶液的溶剂选自以下一种或多种：水、PBS、HEPES。
  6. 根据权利要求4或5所述的方法，其特征在于，所述步骤(1)中，所述多肽溶液的终浓度为1-1000μg/mL，优选为100-500μg/mL；和/或所述磁性纳米颗粒溶液的终浓度为1-10000μg/mL，优选为1000-5000μg/mL。
  7. 根据权利要求4至6中任一项所述的方法，其特征在于，所述步骤(2)中，所述多肽和磁性纳米颗粒的质量比为1:10-5:1，优选为2:5。
  8. 根据权利要求1至3中任一项所述的多肽磁性纳米颗粒或按照权利要求4至7中任一项所述的制备方法制得的多肽磁性纳米颗粒在制备用于诊断或治疗癌症的药物和/或医疗产品中的应用。
  9. 一种用于诊断或治疗癌症的方法，其特征在于，所述方法包括：对有需要的受试者给予权利要求1至3中任一项所述的多肽磁性纳米颗粒或按照权利要求4至7中任一项所述的制备方法制得的多肽磁性纳米颗粒。
  10. 根据权利要求8所述的应用或权利要求9所述的方法，其特征在于，所述癌症选自以下一种或多种：食管癌、肝癌、肺癌、胃癌、乳腺癌、结直肠癌、宫颈癌、甲状腺癌、前列腺癌、胰腺癌、肾癌、膀胱癌、皮肤癌、黑色素瘤；优选为乳腺癌、食管癌、胃癌、肝癌、肺癌、结直肠癌、宫颈癌和/或前列腺癌。
  11. 根据权利要求1至3中任一项所述的多肽磁性纳米颗粒或按照权利要求4至7中任一项所述的制备方法制得的多肽磁性纳米颗粒在制备用于循环肿瘤细胞检测和/或分子分型的药物和/或医疗产品中的应用。
  12. 一种用于诊断或治疗癌症的多肽磁性纳米颗粒和/或用于循环肿瘤细胞检测和/或分子分型的多肽磁性纳米颗粒，其特征在于，所述多肽磁性纳米颗粒包括根据权利要求1至3中任一项所述的多肽磁性纳米颗粒或按照权利要求4至7中任一项所述的制备方法制得的多肽磁性纳米颗粒。
  13. 一种用于循环肿瘤细胞检测和/或分子分型的方法，其特征在于，所述方法包括： 对有需要的受试者给予权利要求1至3中任一项所述的多肽磁性纳米颗粒或按照权利要求4至7中任一项所述的制备方法制得的多肽磁性纳米颗粒。
  14. 根据权利要求11所述的应用或权利要求12所述的多肽磁性纳米颗粒或权利要求13所述的方法，其特征在于，所述循环肿瘤细胞检测和/或分子分型的生物标志物选自以下一种或多种：PD-L1、HER2、ER、PR、AR、EGFR、VEGFR和CXCR4。
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