WO2018155880A1 - Liposome–nucleic acid nano-fusion for quantitative diagnosis of multiple ribonucleic acid markers, theoretical stability evaluation method therefor, application thereof, and production method therefor - Google Patents

Liposome–nucleic acid nano-fusion for quantitative diagnosis of multiple ribonucleic acid markers, theoretical stability evaluation method therefor, application thereof, and production method therefor Download PDF

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WO2018155880A1
WO2018155880A1 PCT/KR2018/002064 KR2018002064W WO2018155880A1 WO 2018155880 A1 WO2018155880 A1 WO 2018155880A1 KR 2018002064 W KR2018002064 W KR 2018002064W WO 2018155880 A1 WO2018155880 A1 WO 2018155880A1
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nucleic acid
liposome
fusion
quantitative diagnosis
ribonucleic acid
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French (fr)
Korean (ko)
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엄숭호
신승원
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성균관대학교산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0076—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion
    • A61K49/0084—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion liposome, i.e. bilayered vesicular structure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0089—Particulate, powder, adsorbate, bead, sphere
    • A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
    • A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
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    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/101—Temperature
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    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence
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    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/155—Particles of a defined size, e.g. nanoparticles
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    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/161—Vesicles, e.g. liposome
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/10—Detection mode being characterised by the assay principle
    • C12Q2565/101—Interaction between at least two labels
    • C—CHEMISTRY; METALLURGY
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention provides a liposome-nucleic acid nanofusion for quantitative diagnosis of multiple ribonucleic acid markers, an imaging system for confirming stability using the nanofusion molecular dynamics simulation technique, and utilizing the same for disease diagnosis, a disease diagnosis method, and a method for preparing the nanofusion. It is about.
  • Nucleic acid is a material that attracts a lot of attention and is being produced in various forms through nanobiotechnology.
  • the technology used to diagnose cancer by attaching phosphors to the nucleic acid nanostructures thus produced is drawing much attention.
  • nucleic acid is a substance constituting the living body, when inserted into the living body, it is very likely to be broken down by enzymes and lose its original function.
  • a process of binding to a target material is required.
  • cancer cells that do not express specific targets, such as triple negative breast cancer have considerable difficulty in diagnosis.
  • RNA of the target cell it has been found that the diagnosis corresponding to the entire cancer cycle, and among them, the expression of a specific miRNA is known to change rapidly in early cancer. For this reason, the importance of miRNA as a diagnostic marker is increasing day by day. In addition, precise treatment is difficult due to tumor heterogeneity in cancer tissues occurring at one site, and it is important to obtain spatial-temporal information by checking the difference between cells for precise patient-specific treatment. It is considered to be.
  • the present inventors completed the present invention by developing a liposome-nucleic acid nanofusion for quantitative diagnosis of multiple ribonucleic acid markers, which is capable of quantitative comparison between cells with high efficiency and low cost.
  • an object of the present invention is to provide a branched first nucleic acid structure having a cyclic terminal; And it is to provide a liposome-nucleic acid nano fusion for multiple ribonucleic acid marker quantitative diagnosis comprising a silica spherical body bonded to the surface of the nucleic acid nanostructures conjugated to the branched second nucleic acid structure having a cyclic terminal inside the liposome.
  • Another object of the present invention is to provide a biological diagnostic imaging system comprising the liposome-nucleic acid nanofusion for the multi-ribonucleic acid marker quantitative diagnosis.
  • Still another object of the present invention is to provide a method for preparing a liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis.
  • Still another object of the present invention is to provide an evaluation method for theoretically evaluating the stability of the liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis.
  • Still another object of the present invention is to provide a kit for diagnosing cancer comprising the liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis.
  • the present invention is a branched first nucleic acid structure having a cyclic terminal; And it provides a liposome-nucleic acid nano fusion for quantitative diagnosis of multiple ribonucleic acid markers comprising a silica spherical body bonded to the surface of the nucleic acid nanostructures conjugated to the branched second nucleic acid structure having a cyclic terminal inside the liposome.
  • the first nucleic acid structure may be a form in which linear nucleic acids selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 1 to 3 are combined in a Y-shaped branched form.
  • the second nucleic acid structure may be in a form in which linear nucleic acids selected from the group consisting of nucleotide sequences of SEQ ID NOs: 4 to 6 are combined in a Y-shaped branched form.
  • the linear nucleic acid may further include a phosphor at the 5 'end.
  • the first nucleic acid structure may comprise a nucleotide sequence of SEQ ID NO: 7 having a cyclic terminal complementary to the target RNA.
  • the second nucleic acid structure may comprise a nucleotide sequence of SEQ ID NO: 8 having a cyclic terminal complementary to the target RNA.
  • the first nucleic acid structure and the second nucleic acid structure may be further labeled with a phosphor and a quencher.
  • the nucleic acid structure may be one that is further labeled with a quencher at one end of the base sequence having a complementary sequence with the target RNA.
  • the silica sphere may be 50 to 90 nm.
  • the silica spherical surface is treated by sequentially treating aminopropyl trimethoxysilane and cyanuric chloride before the nucleic acid nanostructures are bonded to the surface. May be modified.
  • the liposome may be made of a cationic lipid.
  • the cationic lipid may be composed of a cationic lipid comprising a component of DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and cholesterol.
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • the present invention also provides a biodiagnostic imaging system comprising the liposome-nucleic acid nanofusion.
  • the system may be to measure intracellular fluorescence.
  • the present invention also provides a method for diagnosing a disease comprising administering the liposome-nucleic acid nanofusion to a subject in need thereof and measuring fluorescence.
  • the disease may be cancer.
  • the administration may be through oral administration, intravenous injection, intraperitoneal injection, intramuscular injection, arterial injection or subcutaneous injection.
  • the present invention provides a method for preparing liposome-nucleic acid nanofusions for quantitative diagnosis of multiple ribonucleic acid markers, comprising the following steps:
  • a nucleic acid nanostructure is prepared by conjugation with each other using a ligase enzyme (S1). ;
  • the solvent of the S2 step may be to include methanol, ethanol, distilled water, and ammonia water.
  • the methanol and ethanol may be mixed in a volume ratio of 6: 4 to 8: 2:
  • the present invention is an evaluation method for theoretically evaluating the stability of the liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis
  • the method provides an evaluation method comprising the step of measuring the hydrogen bonding energy generated inside the nanofusion.
  • the measurement may be performed at 35 to 40 °C.
  • the present invention also provides a kit for diagnosing cancer comprising the liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis.
  • Liposome-nucleic acid nanofusion for quantitative diagnosis of multiple ribonucleic acid markers has the effect of quantitative comparison of RNA expression patterns of various cell lines and the difference between cells in a single cell line. This enables multiple real-time diagnosis of cancer-specific RNA markers expressed in cells obtained in actual clinical practice, and has the advantage of easily and quickly obtaining information necessary for diagnosis and treatment.
  • high-sensitivity diagnosis is possible by targeting microribonucleic acid whose concentration is absolutely low in cells, and thus it is possible to predict the diagnosis and prognosis of early cancers in which relatively few target substances are expressed. Because it is possible to target a variety of target material, even difficult to diagnose carcinoma such as triple negative breast cancer can be easily diagnosed.
  • FIG. 1 is a diagram illustrating a method for preparing a liposome-nucleic acid nanofusion and a diagnostic method for diagnosing intracellular real-time multiple RNA markers of the present invention.
  • FIG. 2 shows a method for producing a nucleic acid nanostructure (afc-DNA), and a lower part shows a result of confirming the synthesis of the nucleic acid nanostructure through electrophoresis.
  • 3 is a diagram showing simulation conditions for confirming the three-dimensional structure of the nucleic acid nanostructures.
  • FIG. 4 is a view showing the results of confirming the three-dimensional structure of the nucleic acid nanostructures through the oxDNA program in accordance with the conditions of FIG.
  • 5 is a view showing a process and results of bonding the nucleic acid nanostructures to the surface of the silica nanoparticles
  • 5a is a schematic diagram of the bonding process
  • 5b is a result of confirming the binding stability (37 degrees) with temperature
  • 5c shows the particle size change according to the surface treatment
  • 5d shows the result of visualizing the nucleic acid nanostructures attached to the silica surface through a fluorescence microscope.
  • FIG. 6 is a view showing the results according to the size, the fluorescent signal amplification and the surface charge change according to the size of the liposome-nucleic acid nano fusion of the present invention.
  • FIG. 7 is a diagram showing the results obtained by treating liposome-nucleic acid nanofusions prepared in the present invention to two types of breast cancer cell lines (MCF-7 and SK-BR-3) and confirming that intracellular delivery is well performed.
  • Figure 9a shows the result of measuring the target miRNA to the nucleic acid nanostructures, resulting in the increase in the resulting fluorescent signal
  • Figure 9b is mixed with the target miRNA in various concentrations
  • Figure 9c shows the result of confirming the change of
  • Figure 9c is a view showing the result of converting the change of the fluorescent signal to color.
  • the present inventors have completed the present invention as a result of research to develop cancer cell diagnostics capable of real-time intracellular multiplex RNA marker diagnosis.
  • the present invention provides a branched first nucleic acid structure having a cyclic terminal; And a liposome-nucleic acid nano fusion for quantitative diagnosis of multiple ribonucleic acid markers, wherein the nucleic acid nanostructure to which the branched second nucleic acid structure having a cyclic terminal is conjugated is bound to the surface thereof in a liposome. It is characterized by providing a diagnostic imaging system.
  • the present invention provides a method for preparing a liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis comprising the following steps:
  • a nucleic acid nanostructure is prepared by conjugation with each other using a ligase enzyme (S1). ;
  • the order and / or configuration of the steps may be appropriately changed, but not limited to the above steps.
  • Liposome-nucleic acid nanofusions according to the present invention can be used for the diagnosis of a disease.
  • two kinds of nucleic acid structures labeled with phosphors to be implemented in a cell are attached to silica spheres.
  • the globular surface was coated with liposomes (see FIG. 1).
  • the size of the prepared nano fusion can be injected into the body If the size and shape can be appropriately adjusted, but preferably can be prepared in a sphere having a diameter of 50 nm to 90 nm.
  • the first nucleic acid structure and the second nucleic acid structure may be prepared in the form of a branch by allowing linear nucleic acids having an adhesive end to be bonded in a Y-shaped branch form or repeatedly bonded in the Y-shaped branch form.
  • Eggplant may be to label the phosphor at least three 5 'end, but is not limited thereto.
  • the nucleic acid construct has a cyclic terminus and is complementary to the target ribonucleic acid for complementary binding to intracellular target messenger ribonucleic acid (mRNA) or target microribonucleic acid (miRNA) to detect intracellular signals.
  • the nucleic acid including the sequence and the short sequence which binds to the cyclic nucleic acid structure may be attached to one or more ends of the Y-branch.
  • the cyclic nucleic acid structure binding to the target ribonucleic acid is a poster resonance energy transfer between the phosphor and the quencher (Forster Resonance Energy Transfer, FRET). That is, when the cyclic nucleic acid structure does not bind with the target ribonucleic acid, light does not occur due to the Foster resonance energy transfer effect, and when combined, light is generated from the phosphor.
  • FRET Forward Resonance Energy Transfer
  • the phosphor may be fluorescein, Texas red, rhodamine, alexa, cyanine, BODIPY or coumarin, more preferably.
  • 6-FAM Texas 615, Alexa Fluor 488, Cy5, or Cy3, preferably the quencher is TAMRA, BHQ, Iowa Black RQ or MBNQ It may be (MGBNFQ, molecular grove binding non-fluorescence quencher), more preferably Iowa Black RQ, if the phosphor or quencher can be used in vivo is not limited to this can be appropriately used by those skilled in the art.
  • Liposomes of the present invention can vary in lipid composition to control interaction with cells according to the intended use.
  • liposomes were prepared with a positively charged lipid so that the liposome-nucleic acid nanofusion according to the present invention could be introduced into the cytoplasm by non-specifically fusion with a cell membrane to a target cell, preferably the positively charged lipid.
  • the liposome-nucleic acid fluorescence fusion of the present invention is a nucleic acid nano fusion (silica sphere having a nucleic acid nanostructure conjugated to a first nucleic acid structure and a second nucleic acid structure) and liposomes added to the same solution (solvent), respectively. It can be prepared by mixing, preferably the mixing weight ratio of the silica nanoparticles and liposomes surface-treated nucleic acid nanofusions is not limited, as in one embodiment of the present invention solution and liposomes containing nucleic acid nanofusions This solution may be mixed at a weight ratio of 8: 7, but is not limited thereto.
  • the same solution should be used to prevent rupture of liposomes due to osmotic pressure.
  • the solution may include distilled water or a phosphate solution such as PBS (Phosphate buffersaline), but is not limited thereto.
  • the solvent of the step S2 of the present invention may be one containing methanol, ethanol, distilled water, and ammonia water, the methanol and ethanol may be mixed in a volume ratio of 6: 4 to 8: 2 :.
  • the liposome-nucleic acid nanofusion body according to the present invention has a feature that enables quantitative diagnosis according to the expression of the color tone by labeling phosphors having different color tones at the ends of the first and second nucleic acid structures.
  • the liposome-nucleic acid nanofusion of the present invention can be utilized as a bio-diagnostic imaging system, and the system captures multiple RNA markers to activate specific fluorescent substances, and according to fluorescence signal change and color conversion according to miRNA concentration, It has features that allow quantitative comparison between cells.
  • the present invention may provide a disease diagnosis method comprising administering the liposome-nucleic acid nanofusion to an individual in need of diagnosis of the disease and measuring intracellular fluorescence.
  • the administration may be by oral administration, intravenous injection, intraperitoneal injection, intramuscular injection, arterial injection or subcutaneous injection method, the subject means a subject in need of diagnosis of the disease, more specifically human, or non -Means human mammals such as primates, mice, rats, dogs, cats, horses, or cattle.
  • the diagnostic method is not limited to any one of the diseases because the nucleic acid sequence can be changed and liposome-nucleic acid fluorescent nanofusions can be prepared according to the type of disease requiring diagnosis. It may be to, and most preferably in accordance with an embodiment of the present invention may be for the purpose of diagnosing breast cancer.
  • the liposome-nucleic acid fluorescent nanofusion of the present invention may be prepared to include various materials because the interior thereof is empty.
  • the material that can be included therein is not limited, but preferably includes a therapeutic agent to be used as a therapeutic system at the same time as the bio-diagnostic imaging.
  • the present invention is an evaluation method for theoretically evaluating the stability of the liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis, the method comprising the steps of measuring the hydrogen bonding energy generated inside the nanofusions
  • the evaluation method can be provided.
  • oxDNA is a simulation code developed to implement a large-particle DNA model, it is possible to theoretically evaluate the physical stability of nucleic acid nanofusions through molecular dynamics.
  • the measurement of the hydrogen bond energy is characterized in that it is carried out at 35 to 40 °C, the evaluation method by measuring the hydrogen bond energy generated in the interior of the nano-fusion or nucleic acid nanostructures for a predetermined time (x10 5 ) When it is confirmed that the hydrogen bonding energy is kept constant, it is determined that the prepared nanofusion or nucleic acid nanostructures have structural stability.
  • the present invention also provides a kit for diagnosing cancer comprising the liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis.
  • the cancer may include breast cancer, bile duct cancer, bladder cancer, brain tumor, cervical cancer, chorionic cancer, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, multiple myeloma, AIDS-related leukemia and adult T-cell lymphoma / leukemia, epithelial cancer, Liver cancer, lung cancer, lymphoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer or renal cell cancer.
  • kit may include one or more other component compositions, solutions or devices suitable for analytical methods.
  • the kit may include all biological or chemical reagents necessary for diagnosing cancer, differences between multiple cell lines of cancer, or between cells within a single cell line, essential elements necessary for performing PCR, and a guide.
  • PCR kits include test tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), enzymes such as deoxynucleotides (dNTPs), Taq-polymerases and reverse transcriptases, DNases, RNAse inhibitors, DEPC-water, sterile water and the like.
  • the guide is a printed document which explains how to use the kit, e.g. the reaction conditions presented.
  • the instructions may include brochures in the form of pamphlets or leaflets, labels affixed to the kit, and instructions on the surface of the package containing the kit.
  • the guide may include information disclosed or provided through an electronic medium such as the Internet.
  • liposome-nucleic acid nanofusions of the present invention may have been delivered intracellularly with a molecular beacon structure and nanostructures capable of specific binding to intercellular miRNAs within a single cell line or between different cell lines of a target cancer. At this time, since the nanostructure itself is composed of a labeled fluorescent factor capable of quantification, cancer can be diagnosed by confirming it through fluorescence signal increase and color conversion.
  • the target miRNA was selected and used as miR-21 and miR-22, miRNAs that are known to be closely related to cancer in the previous studies.
  • Simple intracellular delivery of diagnostic probes has limitations that are difficult to quantitatively analyze due to cell-specific intracellular migration rates.For example, if the diagnostic probe is simply delivered in large quantities due to cellular characteristics, background noise of the diagnostic probe itself Because of this, even if there is no actual target miRNA, it is possible to cause positive false, and even if there is a target miRNA, it may cause negative false.
  • the nanotechnology-based diagnostic probes developed to date have mostly the characteristics of relative quantification, and the present inventors attempt to overcome the above limitations, for the efficient diagnosis of intracellular miRNA, as shown in FIG. 1. Specific liposome-nucleic acid nanofusions have been developed.
  • the sequence having the nucleotide sequence of the nucleic acid oligomer constituting the first nucleic acid structure (fc1-DNA) and the second nucleic acid structure (fc2-DNA) and the complementary sequence with the target RNA is shown in Table 1.
  • Table 1 After dissolving in TE buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA), each nucleic acid oligomer was mixed in the same molar ratio (0.1 mM). Thereafter, the first nucleic acid structure and the second nucleic acid structure were synthesized by gradually lowering the temperature of the solution at 60 ° C. to 20 ° C. by 1 ° C. per minute.
  • nucleic acid nanostructures (afc-DNA) was performed by conjugation through ligase enzymes between the first and second nucleic acid constructs as shown above in FIG. 2.
  • the same mole number unit nucleic acid nanostructure mixture (0.1 mM) was added to 3 Weiss units of T4 ligase enzyme and buffer (4 ° C., 16 hours).
  • nucleic acid nanostructure synthesis was confirmed through electrophoresis.
  • the oxDNA program was used to confirm the three-dimensional structure of the synthesized nucleic acid nanostructure, specific simulation conditions are shown in FIG. 3, and the results are shown in FIG. 4. As can be seen in Figure 4, the nucleic acid structure and nucleic acid nanostructures synthesized in the present invention was confirmed that the hydrogen bond energy generated inside is kept constant, having stability.
  • methanol and ethanol were mixed in various volume ratios so that the final volume was 46 mL, and then 1 mL of distilled water and 3 mL of ammonia water (28-30 w / w%) were added and stirred for 10 minutes. Afterwards, 0.6 ml of Tetraethyl orthosilicate (TEOS) solution was slowly treated to prepare silica nanoparticles. The prepared silica nanoparticles were washed with ethanol three times or more through centrifugation (15,000 G, 30 minutes).
  • TEOS Tetraethyl orthosilicate
  • silica nanoparticles were treated with 0.8 mL of APTMS (aminopropyltrimethoxysilane, (3-Aminopropyl) trimethoxysilane), 38 ml of ethanol, 2 ml of distilled water, and 0.25 ml of acetic acid. Added. After rinsing with ethanol and acetonitrile through three or more centrifugations, 115.2 mg of cyanuric chloride was treated and stirred for 2 hours. Washed three times or more with acetonitrile, ethanol, distilled water, borate buffer (Borate buffer, pH 8.5).
  • APTMS aminopropyltrimethoxysilane, (3-Aminopropyl) trimethoxysilane
  • nucleic acid nanofusions were prepared by treating nucleic acid nanostructures in which 25 pmole amine groups per 4 mg of silica nanoparticles were substituted at the ends, and reacting for 16 hours or more. Unreacted nucleic acid nanostructures were removed by washing with distilled water through centrifugation.
  • Silica nanoparticles to which the nucleic acid nanoparticles were bound were treated with cationic lipid DOTAP (1,2-dioleoyl-3-trimethylammonium-propane).
  • DOTAP cationic lipid
  • 3.5 mg of DOTAP phospholipid was dispersed by sonication in 1.4 ml of distilled water at 10% amplitude (amplitude, Qsonica Q500) (dispersed at 4 ° C. and stored at low temperature until reaction).
  • the DOTAP solution and the nanofusion solution were mixed, reacted with stirring for 1 hour, and then washed with distilled water and physiological saline through centrifugation.
  • Example 2 various sizes of silica nanoparticle bodies were prepared for signal amplification and efficient intracellular delivery of nucleic acid nanostructures, thereby deriving an optimal size of silica nanoparticle bodies. Based on the synthesis method using a mixture of methanol and ethanol, silica nanoparticles having a size of 20 ⁇ 270 nm were synthesized.
  • aminopropyl trimethoxysilane and cyanuric chloride are sequentially treated on silica nanoparticles to bond the nucleic acid nanostructures to the surface, as shown in FIG. 5A. It was allowed to react with the amine group.
  • Figure 5b shows the result of checking how much the fluorescent factor attached to the surface of the silica drops over time at 37 °C, the intensity of the fluorescence signal generated in the nucleic acid nanostructure is different depending on the size of the silica nanoparticles It confirmed that there was an optimum point.
  • silica nanoparticles coated with nucleic acid nanostructures are treated with DOTAP (Dioleoyl-trimethylammonium-propane), a cationic phospholipid.
  • DOTAP Dioleoyl-trimethylammonium-propane
  • a liposome-nucleic acid nanofusion and use it for diagnosis.
  • the size and surface charge change of the particle body according to the DOTAP treatment was confirmed.
  • nucleic acid nanostructure attached to the surface of the micro-sized silica beads through the same aminopropyl trimethoxysilane and cyanuric chloride chemical reaction fluorescence microscope (Zeiss Axiovert 200M, Carl Zeiss) Confirmed through to verify the adhesion of the nucleic acid nano fusion on the surface of the silica nanoparticles in the present invention.
  • FIG. 6 shows the results of confirming the size according to the preparation step of the liposome-nucleic acid nanofusion of the present invention, the fluorescence signal amplification and the surface charge change according to the size. Fluorescence intensity was measured using a fluorescence photometer (SpectraMax M5, Molecular Devices), and the change of surface charge was measured by dynamic light scattering technique (Zetasizer Nano ZS, Malvern Instruments).
  • Figure 6a schematically shows the manufacturing step of the present invention, attaching the nucleic acid nanostructures to the surface of the silica nanoparticles produced using various fractions of methanol / ethanol mixture, and finally treated with DOTAP to coat the surface It was.
  • FIG. 6b and 6c show the images taken using the size of the silica spheres and the scanning electron microscope (JEM-3010, JEOL) according to the mixing volume ratio of methanol and ethanol
  • Figure 6d is bound according to the sphere size It shows the amount of the nucleic acid nanostructure and the fluorescent signal. According to the result, it can be seen that the amount of aggregates on the surface of the nucleic acid nanostructure is changed according to the size of the spherical body, thereby changing the size of the fluorescent signal.
  • Figure 6e shows the surface charge change before and after the DOTAP treatment, it can be seen that the surface is coated with DOTAP (positive charge) through the result of the surface charge is changed from negative charge to positive charge when the DOTAP treatment.
  • DOTAP positive charge
  • the prepared liposome-nucleic acid nanofusions were treated with two types of breast cancer cell lines (MCF-7 and SK-BR-3) to confirm that intracellular delivery was well achieved. As shown in FIG. 7A, it was confirmed that the DOTAP was better delivered into the cells than the presence of DOTAP, and that the liposome-nucleic acid nanofusion having a final size of about 70 nm was best delivered into the cells.
  • liposomes of the present invention in five breast cancer cell lines (HCC-1937, MCF-7, MDA-MB-453, MDA-MB-231, and SK-BR-3)
  • RT-PCR results obtained through flow cytometry were compared with the results of RT-PCR, which is currently standard. All cancer cells were cultured in 24-well plates and treated with 0.5 mg of liposome-nucleic acid nanofusions on 1 x 10 5 cells. After treatment, the cells were extracted after incubation for 2 hours at 37 ° C. and 5% CO 2 concentration, and measured by flow cytometry.
  • RT-PCR In the case of RT-PCR, it was measured using TaqMan Fast Universal PCR Master Mix (Applied Biosystems), and a miRNA specific primer kit was purchased from the company (miR-21: Hs04231424_s1, miR-22: Hs00993773_g1). .
  • the expression level of the target miRNA was compared with the result obtained by the method proposed in the present invention and the result confirmed by RT-PCR in FIG. 8A.
  • the primer sequences used are shown in Table 2 below. As a result, it was confirmed that the relative ratios of the results obtained in RT-PCR and the results obtained in the present invention are consistent with all five types of cancer cell tumors used.
  • the liposome-nucleic acid nanofusion of the present invention is composed of a fluorescence beacon structure capable of specific binding to a target miRNA and a label fluorescent factor capable of quantifying the nanostructure itself when the nanostructure is delivered into a cell. It confirmed through.
  • the structural stability of the synthesized liposome-nucleic acid nanofusions was verified by electrophoresis technique and molecular dynamic simulation.In the case of specific detection ability of the target miRNA, a comparison of the mixture of different concentrations of the miRNA with different concentrations of the miRNA was performed. Experiments confirmed that miRNA specificity and detectability were effective.
  • the molecular dynamics simulation program used to confirm the structural stability was the freeware oxDNA.
  • the nucleic acid strand having the same nucleotide sequence used in the present invention was input, and it was confirmed in FIG. 4 that the internal energy was stably maintained over time.
  • various concentrations of target miRNAs were treated to nucleic acid nanostructures in order to confirm the detectability, and the increase in fluorescence signal generated therefrom was measured using a fluorescence spectrometer (SpectraMax M5, Molecular Devices), and is shown in FIG. 9A.

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Abstract

The present invention relates to: a liposome–nucleic acid nano-fusion for the quantitative diagnosis of multiple ribonucleic acid markers, the nano-fusion containing, in a liposome, a silica sphere having bound to the surface thereof a nucleic acid nano-structure having a first nucleic acid structure, which is branched and has a cyclic end, bonded with a second nucleic acid structure, which is branched and has a cyclic end; a theoretical stability evaluation method for the nano-fusion; an imaging system using the nano-fusion in disease diagnosis; and a production method for the nano-fusion. By using the liposome–nucleic acid nano-fusion according to the present invention, an advantage may be achieved whereby the difference in aspects of RNA expression among various cell lines may be quantitatively compared, and the difference among cells in a single cell line may be checked, and thus multiple intracellularly-expressed cancer-specific RNA markers acquired in an actual clinical setting may be diagnosed in real-time, and on the basis thereof, information required for diagnosis and treatment may be easily acquired.

Description

다중 리보핵산 마커 정량진단용 리포솜-핵산 나노융합체, 이의 이론적 안정성 평가 방법, 이의 응용, 및 이의 제조 방법Liposome-Nucleic Acid Nanofusions for Quantitative Diagnosis of Multiple Ribonucleic Acid Markers, Methods for Evaluating Theoretical Stability, Applications thereof, and Methods for Preparing the Same
본 발명은 다중 리보핵산 마커 정량진단용 리포솜-핵산 나노융합체, 상기 나노융합체 분자동력학적 시뮬레이션 기법을 이용하여 안정성을 확인하고 이를 질병 진단에 활용하는 이미징 시스템, 질병 진단 방법, 및 상기 나노융합체의 제조방법에 관한 것이다.The present invention provides a liposome-nucleic acid nanofusion for quantitative diagnosis of multiple ribonucleic acid markers, an imaging system for confirming stability using the nanofusion molecular dynamics simulation technique, and utilizing the same for disease diagnosis, a disease diagnosis method, and a method for preparing the nanofusion. It is about.
현재 대부분의 암 진단은 금속, 고분자 중합체 등 인체에 유해한 물질을 사용하고 정확한 진단을 위해서 적게는 3일, 많게는 한 달 이상이 소요된다. 그리고 대부분의 진단체가 가시광선 파장대의 빛을 발생하지 않기 때문에 암이 상당히 진행되어 종양을 형성하기 전까지는 눈으로만 암을 진단하는 데에 한계가 있다. 또한 같은 이유로 진단이 생체 내에서 실시간으로 진행되는 데에 한계가 발생한다.Most cancer diagnosis currently uses materials harmful to the human body, such as metals and polymers, and it takes at least three days and a month or more for accurate diagnosis. And since most diagnostic agents do not generate light in the visible wavelength range, there is a limit in diagnosing cancer by eye only until the cancer progresses considerably and forms a tumor. For the same reason, there is a limit to the diagnosis being made in real time in vivo.
이에 따라 최근에는 인체 유해성을 최소화하기 위해 생체 친화적 물질로 진단체를 제작하는 연구가 상당수 진행되고 있다. 핵산은 이들 중 많은 주목을 받고 있는 물질로 나노생명공학기술을 통해 다양한 형태로 제작되고 있다. 이렇게 만들어진 핵산 나노구조체에 형광체를 붙여 암 진단에 사용하는 기술이 크게 이목을 끌고 있다.Accordingly, in recent years, a lot of research has been conducted to manufacture a diagnostic material using biocompatible materials in order to minimize human hazards. Nucleic acid is a material that attracts a lot of attention and is being produced in various forms through nanobiotechnology. The technology used to diagnose cancer by attaching phosphors to the nucleic acid nanostructures thus produced is drawing much attention.
그러나 핵산이 생체를 구성하는 물질인 만큼 생체 내에 삽입되었을 시 효소에 의해 부서지게 되어 본래 기능을 상실하게 될 가능성이 매우 높다. 또한 진단체가 암 세포 뿐 아니라 일반 세포에까지 비 특이적으로 결합하는 것을 최소화하기 위해 표적물질과 결합시키는 과정이 요구된다. 하지만 현재까지 상기 요구사항들을 동시에 만족시키는 연구는 아직까지 많이 진행되고 있지 않은 실정이다. 게다가 삼중음성유방암 등과 같이 특이적인 표적체가 발현되지 않는 암세포는 진단에 상당한 어려움을 겪고 있다.However, since nucleic acid is a substance constituting the living body, when inserted into the living body, it is very likely to be broken down by enzymes and lose its original function. In addition, in order to minimize the non-specific binding of the diagnostic material to cancer cells as well as general cells, a process of binding to a target material is required. However, until now, studies that satisfy the above requirements at the same time have not been much progressed yet. In addition, cancer cells that do not express specific targets, such as triple negative breast cancer, have considerable difficulty in diagnosis.
한편, 최근 대상 세포의 RNA의 발현 양상에 따라 암 전주기에 해당하는 진단이 가능한 것으로 밝혀지고 있으며, 그중에서 특정 miRNA의 발현 양상은 초기암에서 빠르게 변화하는 것이 알려져있다. 이로 인해 진단 마커로서 miRNA의 중요성이 나날이 부각되고 있다. 또한 한 부위에서 발생한 암조직 내부에 존재하는 종양 이질성 (Tumor heterogeneity) 으로 인해 정밀한 치료가 어려운 실정으로, 정밀한 환자 맞춤형 치료를 위해서 세포 간 차이를 확인하여 시공적인 (Spatio-temporal) 정보를 얻는 것이 중요하게 여겨지고 있다. On the other hand, according to the expression of the RNA of the target cell, it has been found that the diagnosis corresponding to the entire cancer cycle, and among them, the expression of a specific miRNA is known to change rapidly in early cancer. For this reason, the importance of miRNA as a diagnostic marker is increasing day by day. In addition, precise treatment is difficult due to tumor heterogeneity in cancer tissues occurring at one site, and it is important to obtain spatial-temporal information by checking the difference between cells for precise patient-specific treatment. It is considered to be.
이러한 필요성에 근거하여 단일 세포 실시간 중합효소 연쇄반응 (Single cell real time polymerase chain reaction), 고효율 시퀀싱 (High-throughput sequencing) 등 기술들이 개발되고 있으나, 이 역시 분석에 필요한 시간 및 비용이 크다는 뚜렷한 한계를 가지고 있다. 또한 위의 문제점을 극복하기 위하여 나노기술을 이용한 세포내 실시간 분석을 위해 다양한 진단 시스템들이 보고되고 있으나(한국등록특허 제1464100호), 진단 시스템의 세포내 전달이 세포간 특성으로 크게 좌우되기 때문에 세포 종류에 따른 정량적 비교가 불가능하며, 이로 인해 실질적인 임상 적용에 문제가 있다.Based on these needs, technologies such as single cell real time polymerase chain reaction and high-throughput sequencing have been developed, but they also have significant limitations in the time and cost required for analysis. Have. In addition, various diagnostic systems have been reported for real-time intracellular analysis using nanotechnology in order to overcome the above problems (Korea Patent No. 1464100), but because the intracellular delivery of the diagnostic system is largely dependent on intercellular characteristics, Quantitative comparisons by type are not possible, which causes problems in practical clinical applications.
이에, 다종의 세포주 간 RNA 발현 양상 차이를 비교하여, 빠르게 암을 진단할 수 있는 고성능 암 진단체가 요구되고 있는 실정이다.Accordingly, there is a need for a high-performance cancer diagnostic agent capable of rapidly diagnosing cancer by comparing RNA expression patterns among various cell lines.
본 발명자들은 종래기술의 문제점을 극복하기 위하여, 고효율, 저비용인 동시에 세포 간의 정량 비교가 가능한 다중 리보핵산 마커 정량진단용 리포솜-핵산 나노융합체를 개발함으로써 본 발명을 완성하였다.The present inventors completed the present invention by developing a liposome-nucleic acid nanofusion for quantitative diagnosis of multiple ribonucleic acid markers, which is capable of quantitative comparison between cells with high efficiency and low cost.
따라서 본 발명의 목적은 고리형 말단을 가지는 가지형태의 제1핵산구조체; 및 고리형 말단을 가지는 가지형태의 제2핵산구조체가 접합된 핵산 나노 구조체가 표면에 결합된 실리카 구형체를 리포솜 내부에 포함하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체를 제공하는 것이다.Accordingly, an object of the present invention is to provide a branched first nucleic acid structure having a cyclic terminal; And it is to provide a liposome-nucleic acid nano fusion for multiple ribonucleic acid marker quantitative diagnosis comprising a silica spherical body bonded to the surface of the nucleic acid nanostructures conjugated to the branched second nucleic acid structure having a cyclic terminal inside the liposome.
본 발명의 다른 목적은 상기 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체를 포함하는 생체 진단 이미징 시스템을 제공하는 것이다.Another object of the present invention is to provide a biological diagnostic imaging system comprising the liposome-nucleic acid nanofusion for the multi-ribonucleic acid marker quantitative diagnosis.
본 발명의 또 다른 목적은 상기 다중 리보핵산 마커 정량진단용 리포솜-핵산 나노융합체의 제조방법을 제공하는 것이다.Still another object of the present invention is to provide a method for preparing a liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis.
본 발명의 또 다른 목적은 상기 다중 리보핵산 마커 정량진단용 리포솜-핵산 나노융합체의 안정성을 이론적으로 평가하는 평가방법을 제공하는 것이다.Still another object of the present invention is to provide an evaluation method for theoretically evaluating the stability of the liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis.
본 발명의 또 다른 목적은 상기 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체를 포함하는 암 진단용 키트를 제공하는 것이다. Still another object of the present invention is to provide a kit for diagnosing cancer comprising the liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis.
그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.
상기와 같은 본 발명의 목적을 달성하기 위해서, 본 발명은 고리형 말단을 가지는 가지형태의 제1핵산구조체; 및 고리형 말단을 가지는 가지형태의 제2핵산구조체가 접합된 핵산 나노 구조체가 표면에 결합된 실리카 구형체를 리포솜 내부에 포함하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체를 제공한다.In order to achieve the object of the present invention as described above, the present invention is a branched first nucleic acid structure having a cyclic terminal; And it provides a liposome-nucleic acid nano fusion for quantitative diagnosis of multiple ribonucleic acid markers comprising a silica spherical body bonded to the surface of the nucleic acid nanostructures conjugated to the branched second nucleic acid structure having a cyclic terminal inside the liposome.
본 발명의 일실시예에 있어서, 상기 제1핵산구조체는 서열번호 1 내지 3의 염기서열로 이루어진 군으로부터 선택되는 선형핵산들이 Y자 가지형태로 결합한 형태인 것일 수 있다.In one embodiment of the present invention, the first nucleic acid structure may be a form in which linear nucleic acids selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 1 to 3 are combined in a Y-shaped branched form.
본 발명의 다른 실시예에 있어서, 상기 제2핵산구조체는 서열번호 4 내지 6의 염기서열로 이루어진 군으로부터 선택되는 선형핵산들이 Y자 가지형태로 결합한 형태인 것일 수 있다.In another embodiment of the present invention, the second nucleic acid structure may be in a form in which linear nucleic acids selected from the group consisting of nucleotide sequences of SEQ ID NOs: 4 to 6 are combined in a Y-shaped branched form.
본 발명의 또 다른 실시예에 있어서, 상기 선형핵산은 5' 말단에 형광체를 더 포함하는 것일 수 있다.In another embodiment of the present invention, the linear nucleic acid may further include a phosphor at the 5 'end.
본 발명의 또 다른 실시예에 있어서, 상기 제1핵산구조체는 고리형 말단이 표적 RNA와 상보적 서열을 가지는 서열번호 7의 염기서열을 포함하는 것일 수 있다.In another embodiment of the present invention, the first nucleic acid structure may comprise a nucleotide sequence of SEQ ID NO: 7 having a cyclic terminal complementary to the target RNA.
본 발명의 또 다른 실시예에 있어서, 상기 제2핵산구조체는 고리형 말단이 표적 RNA와 상보적 서열을 가지는 서열번호 8의 염기서열을 포함하는 것일 수 있다.In another embodiment of the present invention, the second nucleic acid structure may comprise a nucleotide sequence of SEQ ID NO: 8 having a cyclic terminal complementary to the target RNA.
본 발명의 또 다른 실시예에 있어서, 상기 제1핵산구조체 및 제2핵산구조체는 형광체 및 소광제가 추가로 표지된 것일 수 있다.In another embodiment of the present invention, the first nucleic acid structure and the second nucleic acid structure may be further labeled with a phosphor and a quencher.
본 발명의 또 다른 실시예에 있어서, 상기 핵산구조체는 표적 RNA와 상보적서열을 가지는 염기서열의 한 쪽 말단에 소광제가 추가로 표지된 것일 수 있다.In another embodiment of the present invention, the nucleic acid structure may be one that is further labeled with a quencher at one end of the base sequence having a complementary sequence with the target RNA.
본 발명의 또 다른 실시예에 있어서, 상기 실리카 구형체는 50 내지 90 nm인 것일 수 있다.In another embodiment of the present invention, the silica sphere may be 50 to 90 nm.
본 발명의 또 다른 실시예에 있어서, 상기 실리카 구형체는 핵산 나노 구조체가 표면에 결합되기 전에, 아미노프로필 트리메톡시실란(Aminopropyl trimethoxysilane) 및 사이아누릭 클로라이드 (Cyanuric chloride)를 순차적으로 처리하여 표면개질된 것일 수 있다.In another embodiment of the present invention, the silica spherical surface is treated by sequentially treating aminopropyl trimethoxysilane and cyanuric chloride before the nucleic acid nanostructures are bonded to the surface. May be modified.
본 발명의 또다른 실시예에 있어서, 상기 리포솜은 양이온성 지질로 이루어진 것일 수 있다.In another embodiment of the present invention, the liposome may be made of a cationic lipid.
본 발명의 또 다른 실시예에 있어서, 상기 양이온성 지질은 DOTAP(1,2-dioleoyl-3-trimethylammonium-propane) 및 콜레스테롤을 구성 성분으로 하는 양이온성 지질로 이루어진 것일 수 있다.In another embodiment of the present invention, the cationic lipid may be composed of a cationic lipid comprising a component of DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and cholesterol.
또한, 본 발명은 상기 리포솜-핵산 나노융합체를 포함하는 생체 진단 이미징 시스템을 제공한다.The present invention also provides a biodiagnostic imaging system comprising the liposome-nucleic acid nanofusion.
본 발명의 일실시예에 있어서, 상기 시스템은 세포 내 형광을 측정하는 것일 수 있다.In one embodiment of the present invention, the system may be to measure intracellular fluorescence.
또한, 본 발명은 질병의 진단을 필요로 하는 개체에 상기 리포솜-핵산 나노융합체를 투여하고 형광을 측정하는 단계를 포함하는 질병 진단 방법을 제공한다.The present invention also provides a method for diagnosing a disease comprising administering the liposome-nucleic acid nanofusion to a subject in need thereof and measuring fluorescence.
본 발명의 일실시예에 있어서, 상기 질병은 암일 수 있다.In one embodiment of the invention, the disease may be cancer.
본 발명의 다른 실시예에 있어서, 상기 투여는 경구 투여, 정맥 주사, 복강 주사, 근육 주사, 동맥 주사 또는 피하 주사를 통해 이루어지는 것일 수 있다.In another embodiment of the present invention, the administration may be through oral administration, intravenous injection, intraperitoneal injection, intramuscular injection, arterial injection or subcutaneous injection.
이에 더하여, 본 발명은 하기 단계를 포함하는 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체의 제조방법을 제공한다:In addition, the present invention provides a method for preparing liposome-nucleic acid nanofusions for quantitative diagnosis of multiple ribonucleic acid markers, comprising the following steps:
고리형 말단을 가지는 가지형태의 제1핵산구조체, 및 고리형 말단을 가지는 가지형태의 제2핵산구조체를 제조한 후, 리가아제 효소를 이용하여 서로 접합시켜 핵산 나노 구조체를 제조하는 단계(S1);After preparing a branched first nucleic acid structure having a cyclic terminal and a branched second nucleic acid structure having a cyclic terminal, a nucleic acid nanostructure is prepared by conjugation with each other using a ligase enzyme (S1). ;
용매에 실리케이트 용액을 첨가하여 실리카 나노입자체를 합성하는 단계(S2);Adding a silicate solution to a solvent to synthesize silica nanoparticles (S2);
상기 S2 단계의 나노입자체에 아미노프로필 트리메톡시실란(Aminopropyl trimethoxysilane) 및 사이아누릭 클로라이드 (Cyanuric chloride)를 순차처리하는 단계(S3);Sequentially treating aminopropyl trimethoxysilane and cyanuric chloride on the nanoparticles of step S2 (S3);
상기 S3 단계의 나노입자체와 S1 단계의 핵산 나노 구조체를 반응시켜 핵산 나노구조체를 제조하는 단계(S4);Preparing a nucleic acid nanostructure by reacting the nanoparticle body of step S3 with the nucleic acid nanostructure of step S1 (S4);
상기 S4 단계의 핵산 나노구조체에 양이온성 지질을 첨가한 후 초음파 처리하여 리포좀-핵산 나노융합체를 제조하는 단계(S5).Adding a cationic lipid to the nucleic acid nanostructure of step S4, followed by ultrasonication to prepare a liposome-nucleic acid nanofusion (S5).
본 발명의 일실시예에 있어서, 상기 S2 단계의 용매는 메탄올, 에탄올, 증류수, 및 암모니아수를 포함하는 것일 수 있다.In one embodiment of the present invention, the solvent of the S2 step may be to include methanol, ethanol, distilled water, and ammonia water.
본 발명의 다른 실시예에 있어서, 상기 메탄올 및 에탄올은 6 : 4 내지 8 : 2의 부피비율로 혼합되는 것일 수 있다.In another embodiment of the present invention, the methanol and ethanol may be mixed in a volume ratio of 6: 4 to 8: 2:
아울러, 본 발명은 상기 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체의 안정성을 이론적으로 평가하는 평가방법으로,In addition, the present invention is an evaluation method for theoretically evaluating the stability of the liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis,
상기 방법은 나노융합체 내부에서 발생하는 수소결합 에너지를 측정하는 단계를 포함하는 것을 특징으로 하는, 평가방법을 제공한다.The method provides an evaluation method comprising the step of measuring the hydrogen bonding energy generated inside the nanofusion.
본 발명의 일실시예에 있어서, 상기 측정은 35 내지 40℃에서 수행되는 것일 수 있다.In one embodiment of the present invention, the measurement may be performed at 35 to 40 ℃.
또한, 본 발명은 상기 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체를 포함하는 암 진단용 키트를 제공한다. The present invention also provides a kit for diagnosing cancer comprising the liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis.
본 발명에 따른 다중 리보핵산 마커 정량진단용 리포솜-핵산 나노융합체는 다종의 세포주간 RNA 발현 양상 차이의 정량적 비교 및 단일 세포주내 세포간 차이를 확인할 수 있는 효과를 가진다. 이를 통해 실제 임상에서 얻어진 세포내에 발현되고 있는 암특이적 RNA 마커의 다중 실시간 진단이 가능해지고, 이를 토대로 하여 진단 및 치료에 필요한 정보를 쉽고 빠르게 얻을 수 있는 장점이 있다. Liposome-nucleic acid nanofusion for quantitative diagnosis of multiple ribonucleic acid markers according to the present invention has the effect of quantitative comparison of RNA expression patterns of various cell lines and the difference between cells in a single cell line. This enables multiple real-time diagnosis of cancer-specific RNA markers expressed in cells obtained in actual clinical practice, and has the advantage of easily and quickly obtaining information necessary for diagnosis and treatment.
특히 세포 내에서 그 농도가 절대적으로 적은 마이크로 리보핵산을 표적으로 하여 고감도 진단이 가능하므로, 상대적으로 표적물질이 많이 발현되지 않는 초기 암의 진단 및 예후를 예측하는 것이 가능하며, 세포막 내외부에 발현되는 다양한 표적물질을 표적화할 수 있기 때문에 삼중음성유방암 등과 같이 진단이 어려운 암종 역시 용이하게 진단이 가능하다.In particular, high-sensitivity diagnosis is possible by targeting microribonucleic acid whose concentration is absolutely low in cells, and thus it is possible to predict the diagnosis and prognosis of early cancers in which relatively few target substances are expressed. Because it is possible to target a variety of target material, even difficult to diagnose carcinoma such as triple negative breast cancer can be easily diagnosed.
이에 더하여, 다양한 형광체를 사용하여 다양한 종류의 miRNA 농도에 따른 다중 비색(Multi-colorimetric) 진단 평가가 가능하고, 단일 세포 내에서 다양한 종류의 miRNA 진단을 통해 세포 간 이종성(Heterogeneity)을 평가할 수 있는 특징이 있으며, 융합체 표면의 리포솜 이중막이 생체 내에서 진단체가 분해되는 것을 늦춰주기 때문에 생체 내 적용이 용이한 장점이 있다.In addition, multi-colorimetric diagnostic evaluation according to various miRNA concentrations using various phosphors is possible, and heterogeneity between cells can be evaluated through diagnosis of various miRNAs in a single cell. In addition, since the liposome bilayer on the surface of the fusion slows down the degradation of the diagnostic body in vivo, there is an advantage that it is easy to apply in vivo.
도 1은 본 발명의 세포내 실시간 다중 RNA 마커 진단을 위한 리포좀-핵산 나노융합체의 제조방법 및 진단 방법을 도시적으로 나타낸 도면이다.1 is a diagram illustrating a method for preparing a liposome-nucleic acid nanofusion and a diagnostic method for diagnosing intracellular real-time multiple RNA markers of the present invention.
도 2의 위쪽은 핵산 나노 구조체(afc-DNA)의 제조방법을 나타낸 것이고, 아래쪽은 핵산 나노 구조체의 합성을 전기영동기법을 통해 확인한 결과를 나타낸 도면이다.2 shows a method for producing a nucleic acid nanostructure (afc-DNA), and a lower part shows a result of confirming the synthesis of the nucleic acid nanostructure through electrophoresis.
도 3은 핵산 나노구조체의 삼차원적 구조를 확인하기 위한 시뮬레이션 조건을 나타낸 도면이다.3 is a diagram showing simulation conditions for confirming the three-dimensional structure of the nucleic acid nanostructures.
도 4는 핵산 나노구조체의 삼차원적 구조를 도 3의 조건에 따라 oxDNA 프로그램을 통해 확인한 결과를 나타낸 도면이다.4 is a view showing the results of confirming the three-dimensional structure of the nucleic acid nanostructures through the oxDNA program in accordance with the conditions of FIG.
도 5는 실리카 나노입자체의 표면에 핵산 나노구조체를 접합하는 과정과 결과를 나타낸 도면으로, 5a는 접합 과정의 모식도를 나타낸 것이고, 5b는 온도에 따른 결합 안정성(37도)을 확인한 결과이며, 5c는 표면 처리 단계별 입자체 크기 변화를 나타낸 것이고, 5d는 실리카 표면에 붙은 핵산 나노구조체를 형광현미경을 통해 시각화한 결과를 나타낸 도면이다.5 is a view showing a process and results of bonding the nucleic acid nanostructures to the surface of the silica nanoparticles, 5a is a schematic diagram of the bonding process, 5b is a result of confirming the binding stability (37 degrees) with temperature, 5c shows the particle size change according to the surface treatment, and 5d shows the result of visualizing the nucleic acid nanostructures attached to the silica surface through a fluorescence microscope.
도 6은 본 발명의 리포좀-핵산 나노융합체의 제조 단계에 따른 크기와, 크기에 따른 형광 시그널 증폭 및 표면 전하 변화를 확인한 결과를 나타낸 도면이다.6 is a view showing the results according to the size, the fluorescent signal amplification and the surface charge change according to the size of the liposome-nucleic acid nano fusion of the present invention.
도 7은 본 발명에서 제조된 리포좀-핵산 나노융합체를 두가지 종류(MCF-7 및 SK-BR-3)의 유방암 세포주에 처리하여, 세포 내 전달이 잘 이루어지는지를 확인한 결과를 나타낸 도면이다.FIG. 7 is a diagram showing the results obtained by treating liposome-nucleic acid nanofusions prepared in the present invention to two types of breast cancer cell lines (MCF-7 and SK-BR-3) and confirming that intracellular delivery is well performed.
도 8은 본 발명의 나노융합체가 다중 진단능을 가지는지 확인한 결과로, 5종의 유방암 세포주(HCC-1937, MCF-7, MDA-MB-453, MDA-MB-231 및 SK-BR-3)를 대상으로 본 발명의 리포좀-핵산 나노융합체를 처리한 후, 유세포 분석을 통해 얻어지는 결과를 현재 표준적으로 사용되고 있는 RT-PCR의 결과와 비교한 결과를 나타낸 도면이다.8 is a result confirming that the nanofusion of the present invention has a multi-diagnostic ability, five breast cancer cell lines (HCC-1937, MCF-7, MDA-MB-453, MDA-MB-231 and SK-BR-3 After treating the liposome-nucleic acid nanofusion of the present invention, the result obtained by flow cytometry is compared with the results of RT-PCR which is currently used as a standard.
도 9a는 대상 miRNA를 핵산 나노구조체에 처리하고, 그로 인해 발생하는 형광시그널의 증대를 측정한 결과를 나타낸 것이고, 도 9b는 대상 miRNA를 다양한 농도로 혼합하고, 상기 혼합액에의 농도에 따른 형광시그널의 변화를 확인한 결과를 나타낸 것이며, 도 9c는 상기 형광시그널의 변화를 색깔로 변환한 결과를 나타낸 도면이다.Figure 9a shows the result of measuring the target miRNA to the nucleic acid nanostructures, resulting in the increase in the resulting fluorescent signal, Figure 9b is mixed with the target miRNA in various concentrations, the fluorescent signal according to the concentration in the mixed solution Figure 9c shows the result of confirming the change of, Figure 9c is a view showing the result of converting the change of the fluorescent signal to color.
본 발명자들은 실시간 세포내 다중 RNA 마커 진단이 가능한 암세포 진단체를 개발하기 위해 연구한 결과 본 발명을 완성하게 되었다.The present inventors have completed the present invention as a result of research to develop cancer cell diagnostics capable of real-time intracellular multiplex RNA marker diagnosis.
따라서 본 발명은 고리형 말단을 가지는 가지형태의 제1핵산구조체; 및 고리형 말단을 가지는 가지형태의 제2핵산구조체가 접합된 핵산 나노 구조체가 표면에 결합된 실리카 구형체를 리포솜 내부에 포함하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체 및 이를 포함하는 생체 진단 이미징 시스템을 제공하는 것을 그 특징으로 한다.Accordingly, the present invention provides a branched first nucleic acid structure having a cyclic terminal; And a liposome-nucleic acid nano fusion for quantitative diagnosis of multiple ribonucleic acid markers, wherein the nucleic acid nanostructure to which the branched second nucleic acid structure having a cyclic terminal is conjugated is bound to the surface thereof in a liposome. It is characterized by providing a diagnostic imaging system.
또한, 본 발명은 하기 단계를 포함하는 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체의 제조방법을 제공한다:In addition, the present invention provides a method for preparing a liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis comprising the following steps:
고리형 말단을 가지는 가지형태의 제1핵산구조체, 및 고리형 말단을 가지는 가지형태의 제2핵산구조체를 제조한 후, 리가아제 효소를 이용하여 서로 접합시켜 핵산 나노 구조체를 제조하는 단계(S1);After preparing a branched first nucleic acid structure having a cyclic terminal and a branched second nucleic acid structure having a cyclic terminal, a nucleic acid nanostructure is prepared by conjugation with each other using a ligase enzyme (S1). ;
용매에 실리케이트 용액을 첨가하여 실리카 나노입자체를 합성하는 단계(S2);Adding a silicate solution to a solvent to synthesize silica nanoparticles (S2);
상기 S2 단계의 나노입자체에 아미노프로필 트리메톡시실란(Aminopropyl trimethoxysilane) 및 사이아누릭 클로라이드 (Cyanuric chloride)를 순차처리하는 단계(S3);Sequentially treating aminopropyl trimethoxysilane and cyanuric chloride on the nanoparticles of step S2 (S3);
상기 S3 단계의 나노입자체와 S1 단계의 핵산 나노 구조체를 반응시켜 핵산 나노구조체를 제조하는 단계(S4);Preparing a nucleic acid nanostructure by reacting the nanoparticle body of step S3 with the nucleic acid nanostructure of step S1 (S4);
상기 S4 단계의 핵산 나노구조체에 양이온성 지질을 첨가한 후 초음파 처리하여 리포좀-핵산 나노융합체를 제조하는 단계(S5).Adding a cationic lipid to the nucleic acid nanostructure of step S4, followed by ultrasonication to prepare a liposome-nucleic acid nanofusion (S5).
이때, 상기 제조방법은 본 발명에 따른 리포솜-핵산 나노융합체를 제조할 수 있다면, 적절하게 단계의 순서 및/또는 구성을 변경할 수 있으며 상기 단계에 제한되는 것은 아니다.In this case, if the preparation method can produce liposome-nucleic acid nanofusions according to the present invention, the order and / or configuration of the steps may be appropriately changed, but not limited to the above steps.
본 발명에 따른 리포솜-핵산 나노융합체는 질병의 진단을 위해 사용될 수 있으며, 이를 위해 본 발명의 일실시예에서는 세포 내에서 구현 가능하도록 형광체를 표지한 핵산구조체 2종을 실리카 구형체에 부착한 다음 리포좀으로 상기 구형체 표면을 코팅하였다(도 1 참조). 이때, 본 발명의 일실시예에서는 지지를 위한 구형체로 실리카를 이용하였으나, 생체 내 사용가능한 나노사이즈의 구형체라면 이에 제한되지 않고 사용할 수 있으며, 제조된 나노융합체의 크기는 체내에 주입할 수 있는 크기 및 모양이라면 적절하게 조절 가능 하지만, 바람직하게는 직경이 50 nm 내지 90 nm인 구형으로 제조할 수 있다.Liposome-nucleic acid nanofusions according to the present invention can be used for the diagnosis of a disease. For this purpose, in one embodiment of the present invention, two kinds of nucleic acid structures labeled with phosphors to be implemented in a cell are attached to silica spheres. The globular surface was coated with liposomes (see FIG. 1). At this time, in one embodiment of the present invention, but used as a spherical body for support, if the nano-spherical spheres that can be used in vivo can be used without limitation, the size of the prepared nano fusion can be injected into the body If the size and shape can be appropriately adjusted, but preferably can be prepared in a sphere having a diameter of 50 nm to 90 nm.
본 발명에서 제1핵산구조체 및 제2핵산구조체는 점착성 말단을 가지는 선형핵산이 Y자 가지형태로 결합하거나 상기 Y자 가지형태로 반복 결합하게 함으로써 나뭇가지 형태로 제조할 수 있으며, 상기 점착성 말단이 가지는 최소 3개의 5' 말단에 형광체를 표지하는 것일 수 있으나 이에 제한되는 것은 아니다.In the present invention, the first nucleic acid structure and the second nucleic acid structure may be prepared in the form of a branch by allowing linear nucleic acids having an adhesive end to be bonded in a Y-shaped branch form or repeatedly bonded in the Y-shaped branch form. Eggplant may be to label the phosphor at least three 5 'end, but is not limited thereto.
상기 핵산구조체는 고리형 말단을 가지는 것으로, 세포 내부 신호를 검출하기 위하여, 세포 내 표적 메신저 리보핵산(mRNA) 또는 표적 마이크로 리보핵산(miRNA)과 상보적으로 결합할 수 있도록 상기 표적 리보핵산과 상보적인 서열 및 고리형 핵산구조체로 묶이게 하는 짧은 서열을 포함하는 핵산을 Y자 가지의 어느 한 쪽 이상의 말단에 부착한 것일 수 있다. 이때, 상기 표적 리보핵산과 상보적인 서열의 한 쪽 말단에 소광제를 추가로 표지함으로써, 상기 표적 리보핵산과 결합하는 고리형 핵산구조체가 형광체와 소광제 사이의 포스터 공명에너지전달현상(Forster Resonance Energy Transfer, FRET)을 토대로 작동될 수 있도록 하였다. 즉, 상기 고리형 핵산구조체가 표적 리보핵산과 결합하지 않으면, 포스터공명에너지전달효과로 인해 빛이 발생하지 않고, 결합하면 형광체에서 빛이 발생할 수 있도록 하였다.The nucleic acid construct has a cyclic terminus and is complementary to the target ribonucleic acid for complementary binding to intracellular target messenger ribonucleic acid (mRNA) or target microribonucleic acid (miRNA) to detect intracellular signals. The nucleic acid including the sequence and the short sequence which binds to the cyclic nucleic acid structure may be attached to one or more ends of the Y-branch. At this time, by further labeling a quencher at one end of the sequence complementary to the target ribonucleic acid, the cyclic nucleic acid structure binding to the target ribonucleic acid is a poster resonance energy transfer between the phosphor and the quencher (Forster Resonance Energy Transfer, FRET). That is, when the cyclic nucleic acid structure does not bind with the target ribonucleic acid, light does not occur due to the Foster resonance energy transfer effect, and when combined, light is generated from the phosphor.
바람직하게 상기 형광체는 플루오레신(fluorescein), 텍사스레드(TexasRed), 로다민(rhodamine), 알렉사(alexa), 시아닌(cyanine), 보디피(BODIPY) 또는 쿠마린(coumarin)일 수 있고, 더욱 바람직하게는 6-FAM, Texas 615, Alexa Fluor 488, Cy5, 또는 Cy3일 수 있으며, 바람직하게 상기 소광제는 탐라(TAMRA), 비에이치큐(BHQ), 아이오와블랙 알큐(Iowa Black RQ) 또는 엠지비엔에프큐(MGBNFQ, molecular grove binding non-fluorescence quencher)일수 있고, 더욱 바람직하게는 Iowa Black RQ 일 수 있으나, 생체 내 사용 가능한 형광체 또는 소광제라면 이에 한정되지 않고 당업자가 적절하게 변경하여 사용할 수 있다.Preferably, the phosphor may be fluorescein, Texas red, rhodamine, alexa, cyanine, BODIPY or coumarin, more preferably. Preferably 6-FAM, Texas 615, Alexa Fluor 488, Cy5, or Cy3, preferably the quencher is TAMRA, BHQ, Iowa Black RQ or MBNQ It may be (MGBNFQ, molecular grove binding non-fluorescence quencher), more preferably Iowa Black RQ, if the phosphor or quencher can be used in vivo is not limited to this can be appropriately used by those skilled in the art.
본 발명의 리포솜은 사용 목적에 따라 세포와의 상호작용을 조절할 수 있도록 지질조성을 달리할 수 있다. 본 발명의 일실시예에서는, 표적 세포에 비특이적으로 세포막과 융합하여 본 발명에 따른 리포솜-핵산 나노융합체가 세포질 내로 유입될 수 있도록 양전하를 띄는 지질로 리포솜을 제조하였으며, 바람직하게 상기 양전하를 띄는 지질은 DOTAP(1,2-dioleoyl-3-trimethylammonium-propane)를 이용하여 제조할 수 있으나 이에 한정되는 것은 아니다. Liposomes of the present invention can vary in lipid composition to control interaction with cells according to the intended use. In one embodiment of the present invention, liposomes were prepared with a positively charged lipid so that the liposome-nucleic acid nanofusion according to the present invention could be introduced into the cytoplasm by non-specifically fusion with a cell membrane to a target cell, preferably the positively charged lipid. May be prepared using DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), but is not limited thereto.
또한, 본 발명의 리포솜-핵산형광 융합체는 동일한 용액(용매)에 각각 첨가한 핵산나노융합체(제1핵산구조체 및 제2핵산구조체가 접합된 핵산 나노구조체가 표면에 결합된 실리카 구형체) 및 리포솜을 혼합함으로써 제조될 수 있으며, 바람직하게는 핵산 나노융합체가 표면 처리된 실리카 나노입자체 및 리포솜의 혼합 중량비는 제한이 없으며, 본 발명의 일실시예에서와 같이 핵산 나노융합체가 포함된 용액 및 리포솜이 포함된 용액을 8:7의 무게비로 혼합할 수 있으나 이에 한정되는 것은 아니다. 이때, 동일한 용액(용매)를 사용해야 삼투압에 의한 리포솜의 파열을 방지할 수 있으며, 바람직하게 상기 용액은 증류수 또는 PBS(Phosphate buffersaline)와 같은 인산염 용액을 포함하는 것일 수 있으나 이에 한정되는 것은 아니다.In addition, the liposome-nucleic acid fluorescence fusion of the present invention is a nucleic acid nano fusion (silica sphere having a nucleic acid nanostructure conjugated to a first nucleic acid structure and a second nucleic acid structure) and liposomes added to the same solution (solvent), respectively. It can be prepared by mixing, preferably the mixing weight ratio of the silica nanoparticles and liposomes surface-treated nucleic acid nanofusions is not limited, as in one embodiment of the present invention solution and liposomes containing nucleic acid nanofusions This solution may be mixed at a weight ratio of 8: 7, but is not limited thereto. In this case, the same solution (solvent) should be used to prevent rupture of liposomes due to osmotic pressure. Preferably, the solution may include distilled water or a phosphate solution such as PBS (Phosphate buffersaline), but is not limited thereto.
또한, 본 발명의 S2 단계의 용매는 메탄올, 에탄올, 증류수, 및 암모니아수를 포함하는 것일 수 있으며, 상기 메탄올 및 에탄올은 6 : 4 내지 8 : 2의 부피비율로 혼합되는 것일 수 있다.In addition, the solvent of the step S2 of the present invention may be one containing methanol, ethanol, distilled water, and ammonia water, the methanol and ethanol may be mixed in a volume ratio of 6: 4 to 8: 2 :.
본 발명에 따른 리포솜-핵산 나노융합체는 제1핵산구조체와 제2핵산구조체의 말단에 상이한 색조의 형광체를 표지함으로써, 상기 색조의 발현에 따라 정량적 진단이 가능하도록 한 특징이 있다.The liposome-nucleic acid nanofusion body according to the present invention has a feature that enables quantitative diagnosis according to the expression of the color tone by labeling phosphors having different color tones at the ends of the first and second nucleic acid structures.
따라서 본 발명의 리포솜-핵산 나노융합체는 생체 진단 이미징 시스템으로 활용될 수 있으며, 상기 시스템은 다중 RNA 마커를 포획하여 특이적인 형광물질의 활성화시킴으로써, miRNA 농도에 따른 형광 시그널 변화 및 색상 환산에 따라, 세포간 정량 비교가 가능한 특징을 가지는 것이다.Therefore, the liposome-nucleic acid nanofusion of the present invention can be utilized as a bio-diagnostic imaging system, and the system captures multiple RNA markers to activate specific fluorescent substances, and according to fluorescence signal change and color conversion according to miRNA concentration, It has features that allow quantitative comparison between cells.
이에 더하여, 본 발명은 질병의 진단을 필요로 하는 개체에 상기 리포솜-핵산 나노융합체를 투여하고 세포 내 형광을 측정하는 단계를 포함하는 질병 진단 방법을 제공할 수 있다. 상기 투여는 경구 투여, 정맥 주사, 복강 주사, 근육 주사, 동맥 주사 또는 피하 주사 방법을 통해 이루어질 수 있으며, 상기 개체란 질병의 진단을 필요로 하는 대상을 의미하고, 보다 구체적으로는 인간, 또는 비-인간인 영장류, 생쥐(mouse), 쥐(rat), 개, 고양이, 말, 또는 소 등의 포유류를 의미한다. 또한, 상기 진단 방법은 진단을 필요로 하는 질병의 종류에 따라 핵산 서열을 변경하고 리포솜-핵산 형광 나노융합체를 제조할 수 있기 때문에 어느 하나의 질병에 한정되는 것은 아니나, 바람직하게는 암 진단을 목적으로 하는 것일 수 있고, 가장 바람직하게는 본 발명의 일실시예에 따라 유방암 진단을 목적으로 하는 것일 수 있다.In addition, the present invention may provide a disease diagnosis method comprising administering the liposome-nucleic acid nanofusion to an individual in need of diagnosis of the disease and measuring intracellular fluorescence. The administration may be by oral administration, intravenous injection, intraperitoneal injection, intramuscular injection, arterial injection or subcutaneous injection method, the subject means a subject in need of diagnosis of the disease, more specifically human, or non -Means human mammals such as primates, mice, rats, dogs, cats, horses, or cattle. In addition, the diagnostic method is not limited to any one of the diseases because the nucleic acid sequence can be changed and liposome-nucleic acid fluorescent nanofusions can be prepared according to the type of disease requiring diagnosis. It may be to, and most preferably in accordance with an embodiment of the present invention may be for the purpose of diagnosing breast cancer.
나아가 본 발명의 리포솜-핵산 형광 나노융합체는 내부가 비어 있어, 다양한 물질을 포함하도록 제조할 수 있다. 내부에 포함할 수 있는 물질에는 제한이 없으나, 바람직하게는 치료제를 포함하도록 함으로써 생체 진단 이미징과 동시에 치료용 시스템으로 활용할 수 있다.Furthermore, the liposome-nucleic acid fluorescent nanofusion of the present invention may be prepared to include various materials because the interior thereof is empty. The material that can be included therein is not limited, but preferably includes a therapeutic agent to be used as a therapeutic system at the same time as the bio-diagnostic imaging.
아울러, 본 발명은 상기 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체의 안정성을 이론적으로 평가하는 평가방법으로, 상기 방법은 나노융합체 내부에서 발생하는 수소결합 에너지를 측정하는 단계를 포함하는 것을 특징으로 하는, 평가방법을 제공할 수 있다.In addition, the present invention is an evaluation method for theoretically evaluating the stability of the liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis, the method comprising the steps of measuring the hydrogen bonding energy generated inside the nanofusions The evaluation method can be provided.
상기 평가방법은 oxDNA 시뮬레이션에 의해서 수행되는 것으로, 상기 oxDNA는 대단위-입자 DNA 모델을 구현하기 위해 개발된 시뮬레이션 코드로서, 분자동력학을 통해서 핵산 나노융합체의 물리적 안정성을 이론적으로 평가하는 것이 가능하게 한다.The evaluation method is carried out by oxDNA simulation, oxDNA is a simulation code developed to implement a large-particle DNA model, it is possible to theoretically evaluate the physical stability of nucleic acid nanofusions through molecular dynamics.
본 발명에서 상기 수소결합 에너지의 측정은 35 내지 40℃에서 수행되는 것을 특징으로 하며, 상기 평가방법은 나노융합체 또는 핵산 나노구조체의 내부에서 발생하는 수소결합 에너지를 일정시간(x105) 동안 측정하여, 상기 수소결합 에너지가 일정하게 유지되는 것이 확인될 때, 제조된 나노융합체 또는 핵산 나노구조체에 구조적 안정성이 있는 것으로 판단한다.In the present invention, the measurement of the hydrogen bond energy is characterized in that it is carried out at 35 to 40 ℃, the evaluation method by measuring the hydrogen bond energy generated in the interior of the nano-fusion or nucleic acid nanostructures for a predetermined time (x10 5 ) When it is confirmed that the hydrogen bonding energy is kept constant, it is determined that the prepared nanofusion or nucleic acid nanostructures have structural stability.
또한, 본 발명은 상기 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체를 포함하는 암 진단용 키트를 제공한다. The present invention also provides a kit for diagnosing cancer comprising the liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis.
본 발명에서 상기 암은 유방암, 담관암, 방광암, 뇌종양, 자궁경부암, 융모암, 대장암, 자궁내막암, 식도암, 위암, 다발성 골수종, AIDS-관련 백혈병 및 성인 T-세포 림프종/백혈병, 상피내암, 간암, 폐암, 림프종, 신경모세포종, 구강암, 난소암, 췌장암, 전립선암, 직장암, 육종, 피부암, 고환암, 갑상선암 또는 신세포암이고, 이에 제한되지 않는다.In the present invention, the cancer may include breast cancer, bile duct cancer, bladder cancer, brain tumor, cervical cancer, chorionic cancer, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, multiple myeloma, AIDS-related leukemia and adult T-cell lymphoma / leukemia, epithelial cancer, Liver cancer, lung cancer, lymphoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer or renal cell cancer.
본 명세서에서 용어 "키트"는 분석 방법에 적합한 한 종류 또는 그 이상의 다른 구성 성분 조성물, 용액 또는 장치가 포함될 수 있다.As used herein, the term "kit" may include one or more other component compositions, solutions or devices suitable for analytical methods.
또한, 상기 키트는 암의 진단, 암의 다종 세포주 간의 차이 또는 단일 세포주내 세포간 차이를 진단하기 위하여 필요한 모든 생물학적 또는 화학적 시약, PCR을 수행하기 위해 필요한 필수 요소, 안내서를 포함할 수 있다. PCR 키트는, 상기 프라이머 세트 외에도 테스트 튜브 또는 다른 적절한 컨테이너, 반응 완충액(pH 및 마그네슘 농도는 다양), 데옥시뉴클레오타이드 (dNTPs), Taq-폴리머라아제 및 역전사효소와 같은 효소, DNase, RNAse 억제제, DEPC-수 (DEPC-water) 및 멸균수 등을 포함할 수 있다. 상기 안내서는 키트 사용법, 예를 들면, 제시되는 반응 조건 등을 설명하는 인쇄물이다. 안내서는 팜플렛 또는 전단지 형태의 안내 책자, 키트에 부착된 라벨, 및 키트를 포함하는 패키지의 표면상에 설명을 포함할 수 있다. 또한, 안내서는 인터넷과 같이 전기 매체를 통해 공개되거나 제공되는 정보를 포함할 수 있다. 본 발명의 목적 상, 본 발명의 리포좀-핵산 나노융합체는 대상이 되는 암의 다종 세포주 간의 차이 또는 단일 세포주내 세포간 miRNA와 특이적인 결합을 할 수 있는 Molecular beacon 구조와 나노구조체가 세포내로 전달되었을 때, 나노구조체 자체적인 정량화가 가능한 표지 형광인자로 구성되어 있기 때문에, 형광시그널 증대 및 색깔의 변환을 통하여 확인하여 암을 진단할 수 있다. In addition, the kit may include all biological or chemical reagents necessary for diagnosing cancer, differences between multiple cell lines of cancer, or between cells within a single cell line, essential elements necessary for performing PCR, and a guide. In addition to the above primer sets, PCR kits include test tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), enzymes such as deoxynucleotides (dNTPs), Taq-polymerases and reverse transcriptases, DNases, RNAse inhibitors, DEPC-water, sterile water and the like. The guide is a printed document which explains how to use the kit, e.g. the reaction conditions presented. The instructions may include brochures in the form of pamphlets or leaflets, labels affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide may include information disclosed or provided through an electronic medium such as the Internet. For the purposes of the present invention, liposome-nucleic acid nanofusions of the present invention may have been delivered intracellularly with a molecular beacon structure and nanostructures capable of specific binding to intercellular miRNAs within a single cell line or between different cell lines of a target cancer. At this time, since the nanostructure itself is composed of a labeled fluorescent factor capable of quantification, cancer can be diagnosed by confirming it through fluorescence signal increase and color conversion.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.
[실시예]EXAMPLE
본 실시예에서는 효과적인 암진단 및 임상 적용을 위해, 대상 miRNA는 기존 연구에서 암과의 연관성이 깊은 것으로 알려진 miRNA들인 miR-21 및 miR-22로 선택하여 사용하였다. 진단 프로브의 단순 세포내 전달은 세포 특이적인 세포내 이입률로 인해 정량 분석이 어려운 한계가 있는 것으로, 예를 들면 진단 프로브가 세포 특성으로 인해 단순히 세포내로 다량 전달된 경우, 진단 프로브 자체의 Background noise로 인해 실제 대상 miRNA가 없을 경우에도 positive false를 야기할 가능성이 있으며, 대상 miRNA가 있을 경우에도 negative false를 야기하기도 한다. 이에 현재까지 개발된 나노기술 기반의 진단 프로브는 대부분 상대적 정량만이 가능한 특성을 가지고 있는 바, 본 발명자들은 상기 한계를 극복하고자, 세포내 효율적인 miRNA의 진단을 위해, 도 1에 도시된 방법과 같이 특이적인 리포좀-핵산 나노융합체를 개발하였다.In this example, for effective cancer diagnosis and clinical application, the target miRNA was selected and used as miR-21 and miR-22, miRNAs that are known to be closely related to cancer in the previous studies. Simple intracellular delivery of diagnostic probes has limitations that are difficult to quantitatively analyze due to cell-specific intracellular migration rates.For example, if the diagnostic probe is simply delivered in large quantities due to cellular characteristics, background noise of the diagnostic probe itself Because of this, even if there is no actual target miRNA, it is possible to cause positive false, and even if there is a target miRNA, it may cause negative false. Thus, the nanotechnology-based diagnostic probes developed to date have mostly the characteristics of relative quantification, and the present inventors attempt to overcome the above limitations, for the efficient diagnosis of intracellular miRNA, as shown in FIG. 1. Specific liposome-nucleic acid nanofusions have been developed.
실시예 1. 리포좀-핵산 나노융합체의 제조Example 1 Preparation of Liposomal-Nucleic Acid Nanofusions
1-1. 핵산 나노구조체 합성 및 분자동력학 시뮬레이션1-1. Nucleic Acid Nanostructure Synthesis and Molecular Dynamics Simulation
제1핵산구조체(fc1-DNA) 및 제2핵산구조체(fc2-DNA)를 이루는 핵산 올리고머의 염기서열과 표적 RNA와 상보적 서열을 가지는 서열은 표 1에 나타내었다. TE buffer(10 mM Tris, pH 8.0, 0.1 mM EDTA)에 녹인 후, 각 핵산 올리고머를 동일한 몰수비(0.1 mM)로 혼합하였다. 이후 용액의 온도를 60℃에서 20℃까지 분당 1℃씩 서서히 낮추는 과정을 통해 제1핵산 구조체 및 제2핵산구조체를 합성하였다. The sequence having the nucleotide sequence of the nucleic acid oligomer constituting the first nucleic acid structure (fc1-DNA) and the second nucleic acid structure (fc2-DNA) and the complementary sequence with the target RNA is shown in Table 1. After dissolving in TE buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA), each nucleic acid oligomer was mixed in the same molar ratio (0.1 mM). Thereafter, the first nucleic acid structure and the second nucleic acid structure were synthesized by gradually lowering the temperature of the solution at 60 ° C. to 20 ° C. by 1 ° C. per minute.
핵산 나노 구조체(afc-DNA)의 합성은 도 2의 위쪽에 나타낸 것과 같이 제1핵산구조체 및 제2핵산구조체 사이의 리가아제 효소를 통한 접합으로 수행되었다. 동일 몰수의 단위 핵산 나노구조체 혼합액 (0.1 mM)을 3 Weiss unit의 T4 리가아제 효소 및 버퍼에 하였다(4℃, 16시간). 도 2 아래에 나타낸 것과 같이, 핵산 나노구조체 합성은 전기영동기법을 통해 확인되었다. The synthesis of the nucleic acid nanostructures (afc-DNA) was performed by conjugation through ligase enzymes between the first and second nucleic acid constructs as shown above in FIG. 2. The same mole number unit nucleic acid nanostructure mixture (0.1 mM) was added to 3 Weiss units of T4 ligase enzyme and buffer (4 ° C., 16 hours). As shown below in Figure 2, nucleic acid nanostructure synthesis was confirmed through electrophoresis.
합성된 핵산 나노구조체의 삼차원적 구조를 확인하기 위해 oxDNA 프로그램을 사용하였으며, 구체적인 시뮬레이션 조건은 도 3에 나타내었으며, 그 결과를 도 4에 나타내었다. 도 4에서 확인할 수 있는 것과 같이, 본 발명에서 합성된 핵산구조체와 핵산 나노구조체는 내부에서 발생하는 수소결합 에너지가 일정하게 유지되어, 안정성을 가진다는 것을 확인하였다.The oxDNA program was used to confirm the three-dimensional structure of the synthesized nucleic acid nanostructure, specific simulation conditions are shown in FIG. 3, and the results are shown in FIG. 4. As can be seen in Figure 4, the nucleic acid structure and nucleic acid nanostructures synthesized in the present invention was confirmed that the hydrogen bond energy generated inside is kept constant, having stability.
[표 1]TABLE 1
1-2. 다양한 크기의 실리카 나노입자체 합성 및 핵산 나노입자체 부착1-2. Synthesis of Silica Nanoparticles of Various Sizes and Attachment of Nucleic Acid Nanoparticles
입자체 합성을 위해서, 최종 부피가 46mL가 되도록 메탄올과 에탄올을 다양한 부피 비율로 혼합한 후, 1 mL의 증류수와 3 mL의 암모니아수(28~30 w/w%)를 첨가해주었고, 10분간 교반한 뒤 Tetraethyl orthosilicate (TEOS) 용액 0.6 ml을 서서히 처리하여 실리카 나노입자체를 제조하였다. 제조된 실리카 나노입자체는 원심분리를 통해(15,000 G, 30분) 3회 이상 에탄올로 씻어내었다. For particle synthesis, methanol and ethanol were mixed in various volume ratios so that the final volume was 46 mL, and then 1 mL of distilled water and 3 mL of ammonia water (28-30 w / w%) were added and stirred for 10 minutes. Afterwards, 0.6 ml of Tetraethyl orthosilicate (TEOS) solution was slowly treated to prepare silica nanoparticles. The prepared silica nanoparticles were washed with ethanol three times or more through centrifugation (15,000 G, 30 minutes).
에탄올로 씻어낸 후, 200 mg의 실리카 나노입자체에 0.8 mL의 APTMS(아미노프로필트리메톡시실란, (3-Aminopropyl)trimethoxysilane), 38 ml의 에탄올, 2 ml의 증류수, 및 0.25 ml의 아세트산을 첨가하였다. 이후 3회 이상의 원심분리를 통해 에탄올, 아세토니트릴 (Acetonitrile)로 씻어내고, 115.2 mg의 염화시아누르(cyanuric chloride)를 처리한 후 2시간 동안 교반해주었다. 3회 이상 아세토니트릴, 에탄올, 증류수, 붕산염 버퍼용액 (Borate buffer,pH 8.5)로 씻어내었다.After rinsing with ethanol, 200 mg of silica nanoparticles were treated with 0.8 mL of APTMS (aminopropyltrimethoxysilane, (3-Aminopropyl) trimethoxysilane), 38 ml of ethanol, 2 ml of distilled water, and 0.25 ml of acetic acid. Added. After rinsing with ethanol and acetonitrile through three or more centrifugations, 115.2 mg of cyanuric chloride was treated and stirred for 2 hours. Washed three times or more with acetonitrile, ethanol, distilled water, borate buffer (Borate buffer, pH 8.5).
마지막으로 4 mg의 실리카 나노입자체 당 25 pmole의 아민기가 말단에 치환된 핵산 나노 구조체를 처리한 후 16시간 이상 반응시켜 핵산 나노융합체를 제조하였다. 미반응된 핵산 나노 구조체는 원심분리를 통해 증류수로 씻어내 제거하였다.Finally, nucleic acid nanofusions were prepared by treating nucleic acid nanostructures in which 25 pmole amine groups per 4 mg of silica nanoparticles were substituted at the ends, and reacting for 16 hours or more. Unreacted nucleic acid nanostructures were removed by washing with distilled water through centrifugation.
1-3. 양이온성 인지질막 처리1-3. Cationic Phospholipid Treatment
핵산 나노입자체가 결합된 실리카 나노입자체에 양이온성 지질인 DOTAP(1,2-dioleoyl-3-trimethylammonium-propane)을 처리하였다. 반응에 앞서 3.5 mg의 DOTAP 인지질은 1.4 ml의 증류수에 10 % 진폭 (amplitude, Qsonica Q500)으로 초음파 처리하여 분산시켰다(4℃에서 분산되어 반응 전까지 저온 보관됨). DOTAP 용액과 나노융합체 용액을 혼합하고, 1시간 동안 교반하며 반응시킨 뒤, 원심분리를 통해 증류수, 생리식염수로 씻어주었다.Silica nanoparticles to which the nucleic acid nanoparticles were bound were treated with cationic lipid DOTAP (1,2-dioleoyl-3-trimethylammonium-propane). Prior to the reaction, 3.5 mg of DOTAP phospholipid was dispersed by sonication in 1.4 ml of distilled water at 10% amplitude (amplitude, Qsonica Q500) (dispersed at 4 ° C. and stored at low temperature until reaction). The DOTAP solution and the nanofusion solution were mixed, reacted with stirring for 1 hour, and then washed with distilled water and physiological saline through centrifugation.
실시예 2. 실리카 나노입자체 합성 및 핵산 나노구조체 접합Example 2. Silica Nanoparticle Synthesis and Nucleic Acid Nanostructure Conjugation
본 실시예 2에서 핵산 나노구조체의 시그널 증폭과 효율적인 세포내 전달을 위해 다양한 크기의 실리카 나노입자체를 제조하여, 최적의 실리카 나노입자체 크기를 도출하고자하였다. 메탄올과 에탄올 혼합액을 사용한 합성법을 기반으로 하여, 20~270 nm의 크기를 갖는 실리카 나노입자체를 합성하였다. In Example 2, various sizes of silica nanoparticle bodies were prepared for signal amplification and efficient intracellular delivery of nucleic acid nanostructures, thereby deriving an optimal size of silica nanoparticle bodies. Based on the synthesis method using a mixture of methanol and ethanol, silica nanoparticles having a size of 20 ~ 270 nm were synthesized.
이후 도 5a에 나타낸 것과 같이 핵산 나노구조체를 표면에 접합하기 위해 실리카 나노입자체에 아미노프로필 트리메톡시실란(Aminopropyl trimethoxysilane) 및 사이아누릭 클로라이드 (Cyanuric chloride)를 순차적으로 처리하여 핵산 나노 구조체의 아민기 (Amine group)과 반응할 수 있도록 하였다. Subsequently, aminopropyl trimethoxysilane and cyanuric chloride are sequentially treated on silica nanoparticles to bond the nucleic acid nanostructures to the surface, as shown in FIG. 5A. It was allowed to react with the amine group.
또한 도 5b에서는 실리카 표면에 부착된 형광인자가 37℃에서 시간이 지남에 따라 어느 정도 떨어지는지 확인한 결과를 나타낸 것으로, 실리카 나노입자체의 크기에 따라 핵산 나노구조체에서 발생하는 형광 시그널의 강도가 달라지고, 최적점이 있는 것을 확인하였다.In addition, Figure 5b shows the result of checking how much the fluorescent factor attached to the surface of the silica drops over time at 37 ℃, the intensity of the fluorescence signal generated in the nucleic acid nanostructure is different depending on the size of the silica nanoparticles It confirmed that there was an optimum point.
실시예 3. 양이온성 인지질막 처리 및 세포 내 miRNA 검출능 확인Example 3 Cationic Phospholipid Treatment and Intracellular miRNA Detection
본 발명에서는, 효율적인 세포내 전달 및 엔도조말 탈출 (Endosomal escape)을 위해서, 핵산 나노 구조체가 코팅된 실리카 나노입자체(즉, 핵산 나노융합체)를 양이온성 인지질인 DOTAP (Dioleoyl-trimethylammonium-propane) 처리하여 리포좀-핵산 나노융합체로 제조하여 진단에 사용한다. 본 실시예에서는 DOTAP 처리에 따른 입자체의 크기 및 표면 전하 변화를 확인하였다. In the present invention, for efficient intracellular delivery and endosomal escape, silica nanoparticles coated with nucleic acid nanostructures (ie, nucleic acid nanofusions) are treated with DOTAP (Dioleoyl-trimethylammonium-propane), a cationic phospholipid. To prepare a liposome-nucleic acid nanofusion and use it for diagnosis. In this example, the size and surface charge change of the particle body according to the DOTAP treatment was confirmed.
그 결과, 도 5c에 나타낸 것과 같이, 다양한 크기의 실리카 나노입자체 표면에 핵산 나노구조체를 부착하고 이후 DOTAP를 처리하여 다양한 크기의 나노융합체를 제작하는 것이 가능하다는 것을 확인할 수 있으며, 메탄올 및 에탄올의 혼합 부피 비율이 8:2일 때 제작된 실리카 나노입자체를 사용하여 최종 나노융합체를 제작한 경우는 51.38 nm의 크기를, 6:4일 때는 80.56 nm의 크기를, 4:6일 때는 134.21 nm의 크기를, 2:8일 때는 225.70 nm의 크기를, 0:10일 때는 264.40 nm의 크기를 가진다는 것을 알 수 있다. 또한 도 5d에서 확인할 수 있는 것과 같이, 마이크로 크기의 실리카 비드의 표면에 동일한 아미노프로필 트리메톡시실란과 사이아누릭 클로라이드 화학반응을 통해 부착된 핵산 나노구조체를 형광현미경(Zeiss Axiovert 200M, Carl Zeiss)을 통해 확인하여 본 발명에서 실리카 나노입자체 표면의 핵산 나노융합체 부착을 검증하였다.As a result, as shown in Figure 5c, it can be seen that it is possible to attach the nucleic acid nanostructures to the surface of the silica nanoparticles of various sizes, and then to process the DOTAP to produce nano fusions of various sizes, methanol and ethanol When the final nano fusions were prepared using the silica nanoparticles produced at a mixing volume ratio of 8: 2, the size was 51.38 nm, the size was 80.56 nm at 6: 4, and the thickness was 134.21 nm. It can be seen that the size of 225.70 nm at 2: 8 and 264.40 nm at 0:10. In addition, as can be seen in Figure 5d, the nucleic acid nanostructure attached to the surface of the micro-sized silica beads through the same aminopropyl trimethoxysilane and cyanuric chloride chemical reaction fluorescence microscope (Zeiss Axiovert 200M, Carl Zeiss) Confirmed through to verify the adhesion of the nucleic acid nano fusion on the surface of the silica nanoparticles in the present invention.
도 6에서 본 발명의 리포좀-핵산 나노융합체의 제조 단계에 따른 크기와, 크기에 따른 형광 시그널 증폭 및 표면 전하 변화를 확인한 결과를 나타내었다. 형광세기는 형광광도계 (SpectraMax M5, Molecular Devices)를 이용하여 측정하였고, 표면 전하의 변화는 동적빛산란 기법으로 측정하였다 (Zetasizer Nano ZS, Malvern Instruments). 도 6a는 본 발명의 제조단계를 모식적으로 나타낸 것이며, 다양한 분율의 메탄올/에탄올 혼합액을 사용하게 제작된 실리카 나노입자체의 표면에 핵산 나노구조체를 부착하고, 최종적으로 DOTAP을 처리하여 표면을 코팅하였다. 도 6b 및 6c는 는 메탄올 및 에탄올의 혼합 부피 비율에 따른 실리카 구형체의 크기와 주사전자현미경 (JEM-3010, JEOL)을 이용하여 촬영된 이미지를 나타낸 것이고, 도 6d는 구형체 크기에 따라 결합되는 핵산 나노구조체의 양과 형광 시그널을 나타낸 것이다. 해당 결과에서 구형체의 크기에 따라 핵산 나노구조체의 표면에 응집되는 양이 달라지고 그로인해 형광시그널의 크기가 변화한다는 것을 알 수 있다. 도 6e는 DOTAP 처리 전/후의 표면 전하 변화를 나타낸 것으로, DOTAP를 처리할 때 표면 전하가 음전하에서 양전하로 바뀌는 결과를 통해 DOTAP (양전하)으로 표면이 코팅되는 것을 확인할 수 있다.6 shows the results of confirming the size according to the preparation step of the liposome-nucleic acid nanofusion of the present invention, the fluorescence signal amplification and the surface charge change according to the size. Fluorescence intensity was measured using a fluorescence photometer (SpectraMax M5, Molecular Devices), and the change of surface charge was measured by dynamic light scattering technique (Zetasizer Nano ZS, Malvern Instruments). Figure 6a schematically shows the manufacturing step of the present invention, attaching the nucleic acid nanostructures to the surface of the silica nanoparticles produced using various fractions of methanol / ethanol mixture, and finally treated with DOTAP to coat the surface It was. 6b and 6c show the images taken using the size of the silica spheres and the scanning electron microscope (JEM-3010, JEOL) according to the mixing volume ratio of methanol and ethanol, Figure 6d is bound according to the sphere size It shows the amount of the nucleic acid nanostructure and the fluorescent signal. According to the result, it can be seen that the amount of aggregates on the surface of the nucleic acid nanostructure is changed according to the size of the spherical body, thereby changing the size of the fluorescent signal. Figure 6e shows the surface charge change before and after the DOTAP treatment, it can be seen that the surface is coated with DOTAP (positive charge) through the result of the surface charge is changed from negative charge to positive charge when the DOTAP treatment.
제조된 리포좀-핵산 나노융합체를 두가지 종류(MCF-7 및 SK-BR-3)의 유방암 세포주에 처리하여, 세포 내 전달이 잘 이루어지는지를 확인하였다. 도 7a에 나타낸 것과 같이, DOTAP가 존재할 때 보다 세포 내로 잘 전달된다는 것을 확인하였고, 최종 크기 70 nm 가량의 리포좀-핵산 나노융합체가 세포내로 가장 잘 전달되는 것으로 확인하였다.The prepared liposome-nucleic acid nanofusions were treated with two types of breast cancer cell lines (MCF-7 and SK-BR-3) to confirm that intracellular delivery was well achieved. As shown in FIG. 7A, it was confirmed that the DOTAP was better delivered into the cells than the presence of DOTAP, and that the liposome-nucleic acid nanofusion having a final size of about 70 nm was best delivered into the cells.
도 7b에서 확인할 수 있는 것과 같이, 유세포 분석 (MacsQuant VYB, Miltenyi Biotec)을 통해 확인한 결과 본 발명의 핵산 나노 구조체가 세포내로 전달될 때, 형광체가 동일한 비율로 나타나 세포 내로 핵산구조체가 동일한 비율로 전달될 수 있다는 것을 확인하였다.As can be seen in Figure 7b, as confirmed by flow cytometry (MacsQuant VYB, Miltenyi Biotec), when the nucleic acid nanostructures of the present invention is delivered into the cell, the phosphors appear in the same proportion and the nucleic acid structures are delivered in the same ratio into the cell It can be confirmed.
실시간 세포내 다중 진단능을 확인하기 위해, 5종의 유방암 세포주(HCC-1937, MCF-7, MDA-MB-453, MDA-MB-231 및 SK-BR-3)를 대상으로 본 발명의 리포좀-핵산 나노융합체를 처리한 후, 유세포 분석을 통해 얻어지는 결과를 현재 표준적으로 사용되고 있는 RT-PCR의 결과와 비교하였다. 모든 암세포는 24-well plate에서 배양되었으며, 1 x 105 개의 세포를 대상으로 0.5 mg의 리포좀-핵산 나노융합체가 처리되었다. 처리 후 37℃, 5% CO2 농도에서 2시간 동안 배양 후 세포를 추출하여 유세포 분석 측정하였다. RT-PCR의 경우, TaqMan Fast Universal PCR Master Mix (Applied Biosystems)를 사용하여 측정되었으며, 동 업체에서 제공하는 miRNA 특이적 프라이머 키트를 구매하여 사용하였다(miR-21: Hs04231424_s1, miR-22: Hs00993773_g1). 대상 miRNA의 발현 정도를 본 발명에서 제시한 방식으로 얻어진 결과와 RT-PCR을 이용하여 확인한 결과를 도 8a에서 비교하였다. 사용된 프라이머 서열은 하기 표 2에 나타내었다. 그 결과 사용된 다섯가지 종류의 암세포종 모두에서 RT-PCR에서 얻어진 결과와 본 발명에서 제시된 방식으로 얻어진 결과의 상대적 비율이 일치하는 것을 확인하였다.To confirm real-time intracellular multiple diagnosis, liposomes of the present invention in five breast cancer cell lines (HCC-1937, MCF-7, MDA-MB-453, MDA-MB-231, and SK-BR-3) After treatment with the nucleic acid nanofusions, the results obtained through flow cytometry were compared with the results of RT-PCR, which is currently standard. All cancer cells were cultured in 24-well plates and treated with 0.5 mg of liposome-nucleic acid nanofusions on 1 x 10 5 cells. After treatment, the cells were extracted after incubation for 2 hours at 37 ° C. and 5% CO 2 concentration, and measured by flow cytometry. In the case of RT-PCR, it was measured using TaqMan Fast Universal PCR Master Mix (Applied Biosystems), and a miRNA specific primer kit was purchased from the company (miR-21: Hs04231424_s1, miR-22: Hs00993773_g1). . The expression level of the target miRNA was compared with the result obtained by the method proposed in the present invention and the result confirmed by RT-PCR in FIG. 8A. The primer sequences used are shown in Table 2 below. As a result, it was confirmed that the relative ratios of the results obtained in RT-PCR and the results obtained in the present invention are consistent with all five types of cancer cell tumors used.
결과적으로 세포간 miR-21과 miR-22의 발현 양상 차이를 정량적으로 비교하는 것이 가능함을 확인하였고, 동일 세포주 내 세포간 차이에 관한 데이터를 얻는 것이 가능함을 확인하였다.As a result, it was confirmed that it is possible to quantitatively compare the expression patterns of miR-21 and miR-22 between cells, and to obtain data on the difference between cells in the same cell line.
실시예 4. 핵산 나노구조체 개발 및 대상 miRNA 검출능 확인Example 4. Development of nucleic acid nanostructures and confirmation of target miRNA detection ability
본 발명의 리포좀-핵산 나노융합체는 대상 miRNA와 특이적인 결합을 할 수 있는 Molecular beacon 구조와 나노구조체가 세포내로 전달되었을 때, 나노구조체 자체적인 정량화가 가능한 표지 형광인자로 구성된다는 것을 앞선 실시예를 통해서 확인하였다. 또한 합성된 리포좀-핵산 나노융합체의 구조적 안정성은 전기영동 기법과 분자동력학적 시뮬레이션을 통해 검증되었고, 대상 miRNA에 대한 특이적 검출능의 경우, 다양한 농도의 대상 miRNA의 혼합액과 다른 종류의 miRNA와의 비교 실험을 통해 miRNA 특이성 및 검출능이 유효한 것을 확인하였다. 구조적 안정성을 확인하기 위해 사용된 분자동력학적 시뮬레이션 프로그램은 프리웨어인 oxDNA를 사용하였다. oxDNA 시뮬레이션 상에서 본 발명에 사용된 동일한 염기서열을 갖는 핵산 가닥을 입력하고, 시간이 지남에 따라 내부 에너지가 안정적으로 유지되는 것을 도 4에서 확인하였다. 또한 검출능을 확인하기 위해 다양한 농도의 대상 miRNA를 핵산 나노구조체에 처리하고, 그로 인해 발생하는 형광시그널의 증대를 형광광도계 (SpectraMax M5, Molecular Devices)를 사용하여 측정하여 도 9a에 나타내었다. 핵산 나노구조체간 조립 과정 이후에도 대상 miRNA의 검출능에 변화가 없음을 확인하였고, 대상으로하는 miR-21과 miR-22를 다양한 농도로 혼합하고, 혼합액에서 대상으로하는 miRNA의 농도에만 의존적으로 형광시그널이 증가하는 것을 도 9b에서 확인하였다. 이와 같이 얻어진 형광시그널을 색깔로 변환하여 나타낸 결과는 도 9c와 같다.According to the previous embodiment, the liposome-nucleic acid nanofusion of the present invention is composed of a fluorescence beacon structure capable of specific binding to a target miRNA and a label fluorescent factor capable of quantifying the nanostructure itself when the nanostructure is delivered into a cell. It confirmed through. In addition, the structural stability of the synthesized liposome-nucleic acid nanofusions was verified by electrophoresis technique and molecular dynamic simulation.In the case of specific detection ability of the target miRNA, a comparison of the mixture of different concentrations of the miRNA with different concentrations of the miRNA was performed. Experiments confirmed that miRNA specificity and detectability were effective. The molecular dynamics simulation program used to confirm the structural stability was the freeware oxDNA. On the oxDNA simulation, the nucleic acid strand having the same nucleotide sequence used in the present invention was input, and it was confirmed in FIG. 4 that the internal energy was stably maintained over time. In addition, various concentrations of target miRNAs were treated to nucleic acid nanostructures in order to confirm the detectability, and the increase in fluorescence signal generated therefrom was measured using a fluorescence spectrometer (SpectraMax M5, Molecular Devices), and is shown in FIG. 9A. After assembling the nucleic acid nanostructures, it was confirmed that there was no change in the detection ability of the target miRNA, and mixed the target miR-21 and miR-22 at various concentrations, and the fluorescent signal was dependent only on the concentration of the target miRNA in the mixture The increase was confirmed in FIG. 9B. The fluorescence signal thus obtained is converted to color and shown in FIG. 9C.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해되어야 한다.The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above are to be understood in all respects as illustrative and not restrictive.

Claims (27)

  1. 고리형 말단을 가지는 가지형태의 제1핵산구조체; 및 고리형 말단을 가지는 가지형태의 제2핵산구조체가 접합된 핵산 나노 구조체가 표면에 결합된 실리카 구형체를 리포솜 내부에 포함하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체.Branched first nucleic acid structure having a cyclic terminal; And Liposome-nucleic acid nano fusion for quantitative diagnosis of multiple ribonucleic acid markers comprising a silica sphere in which the nucleic acid nanostructures conjugated to the branched second nucleic acid structure having a cyclic terminal bound to the surface inside the liposome.
  2. 제1항에 있어서,The method of claim 1,
    상기 제1핵산구조체는 서열번호 1 내지 3의 염기서열로 이루어진 군으로부터 선택되는 선형핵산들이 Y자 가지형태로 결합한 형태인 것을 특징으로 하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체.The first nucleic acid structure is a liposome-nucleic acid nano fusion for quantitative diagnosis of multiple ribonucleic acid markers, characterized in that the linear nucleic acid selected from the group consisting of the base sequence of SEQ ID NO: 1 to 3 in the form of Y-shaped bound.
  3. 제1항에 있어서,The method of claim 1,
    상기 제2핵산구조체는 서열번호 4 내지 6의 염기서열로 이루어진 군으로부터 선택되는 선형핵산들이 Y자 가지형태로 결합한 형태인 것을 특징으로 하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체.The second nucleic acid structure is a liposome-nucleic acid nano fusion for quantitative diagnosis of multiple ribonucleic acid markers, characterized in that the linear nucleic acid selected from the group consisting of the base sequence of SEQ ID NO: 4 to 6 in the form of a Y-shaped bound.
  4. 제 2 항 또는 제 3 항에 있어서, The method of claim 2 or 3,
    상기 선형핵산은 5' 말단에 형광체를 더 포함하는 것을 특징으로 하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체.The linear nucleic acid is characterized in that it further comprises a phosphor at the 5 'terminal, liposome-nucleic acid nano fusion for multiple ribonucleic acid marker quantitative diagnosis.
  5. 제 2 항에 있어서, The method of claim 2,
    상기 제1핵산구조체는 고리형 말단이 표적 RNA와 상보적 서열을 가지는 서열번호 7의 염기서열을 포함하는 것을 특징으로 하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체.Wherein the first nucleic acid structure is characterized in that it comprises a nucleotide sequence of SEQ ID NO: 7 having a cyclic terminal complementary to the target RNA, liposome-nucleic acid nano fusion for multiple ribonucleic acid marker quantitative diagnosis.
  6. 제 3 항에 있어서, The method of claim 3, wherein
    상기 제2핵산구조체는 고리형 말단이 표적 RNA와 상보적 서열을 가지는 서열번호 8의 염기서열을 포함하는 것을 특징으로 하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체.The second nucleic acid structure is characterized in that it comprises a nucleotide sequence of SEQ ID NO: 8 having a cyclic terminal complementary to the target RNA, liposome-nucleic acid nano-fusion for multiple ribonucleic acid marker quantitative diagnosis.
  7. 제 1 항에 있어서, The method of claim 1,
    상기 제1핵산구조체 및 제2핵산구조체는 형광체 및 소광제가 추가로 표지된 것을 특징으로 하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체.The first nucleic acid structure and the second nucleic acid structure is characterized in that the phosphor and quencher is further labeled, liposome-nucleic acid nano fusion for quantitative diagnosis of multiple ribonucleic acid markers.
  8. 제 1 항에 있어서,The method of claim 1,
    상기 실리카 구형체는 50 nm 내지 80 nm인 것을 특징으로 하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체.The silica sphere is characterized in that 50 nm to 80 nm, liposome-nucleic acid nano fusion for multi-ribonucleic acid marker quantitative diagnosis.
  9. 제 1 항에 있어서,The method of claim 1,
    상기 실리카 구형체는 핵산 나노 구조체가 표면에 결합되기 전에, 아미노프로필 트리메톡시실란(Aminopropyl trimethoxysilane) 및 사이아누릭 클로라이드 (Cyanuric chloride)를 순차적으로 처리하여 표면개질된 것을 특징으로 하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체.The silica spherical body is surface-modified by sequentially processing aminopropyl trimethoxysilane and cyanuric chloride before the nucleic acid nanostructures are bonded to the surface. Liposome-Nucleic Acid Nanofusion for Marker Quantitative Diagnosis.
  10. 제 1 항에 있어서,The method of claim 1,
    상기 리포솜은 양이온성 지질로 이루어진 것을 특징으로 하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체.The liposome is characterized by consisting of a cationic lipid, liposome-nucleic acid nano fusion for multi-ribonucleic acid marker quantitative diagnosis.
  11. 제 10 항에 있어서,The method of claim 10,
    상기 양이온성 지질은 DOTAP(1,2-dioleoyl-3-trimethylammonium-propane) 및 콜레스테롤을 구성 성분으로 하는 양이온성 지질로 이루어진 것을 특징으로 하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체.The cationic lipid is DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and a cationic lipid comprising a cholesterol as a component, multi-ribonucleic acid marker quantitative diagnostic liposome-nucleic acid nano fusion.
  12. 제 1 항의 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체를 포함하는 생체 진단 이미징 시스템.Biological diagnostic imaging system comprising a liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis of claim 1.
  13. 제 12 항에 있어서,The method of claim 12,
    상기 시스템은 세포 내 형광을 측정하는 것을 특징으로 하는, 생체 진단 이미징 시스템.And the system measures intracellular fluorescence.
  14. 하기 단계를 포함하는 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체의 제조방법:Method for preparing a liposome-nucleic acid nanofusion for multi-ribonucleic acid marker quantitative diagnosis comprising the following steps:
    고리형 말단을 가지는 가지형태의 제1핵산구조체, 및 고리형 말단을 가지는 가지형태의 제2핵산구조체를 제조한 후, 리가아제 효소를 이용하여 서로 접합시켜 핵산 나노 구조체를 제조하는 단계(S1);After preparing a branched first nucleic acid structure having a cyclic terminal and a branched second nucleic acid structure having a cyclic terminal, a nucleic acid nanostructure is prepared by conjugation with each other using a ligase enzyme (S1). ;
    용매에 실리케이트 용액을 첨가하여 실리카 나노입자체를 합성하는 단계(S2);Adding a silicate solution to a solvent to synthesize silica nanoparticles (S2);
    상기 S2 단계의 나노입자체에 아미노프로필 트리메톡시실란(Aminopropyl trimethoxysilane) 및 사이아누릭 클로라이드 (Cyanuric chloride)를 순차처리하는 단계(S3);Sequentially treating aminopropyl trimethoxysilane and cyanuric chloride on the nanoparticles of step S2 (S3);
    상기 S3 단계의 나노입자체와 S1 단계의 핵산 나노 구조체를 반응시켜 핵산 나노구조체를 제조하는 단계(S4);Preparing a nucleic acid nanostructure by reacting the nanoparticle body of step S3 with the nucleic acid nanostructure of step S1 (S4);
    상기 S4 단계의 핵산 나노구조체에 양이온성 지질을 첨가한 후 초음파 처리하여 리포좀-핵산 나노융합체를 제조하는 단계(S5).Adding a cationic lipid to the nucleic acid nanostructure of step S4, followed by ultrasonication to prepare a liposome-nucleic acid nanofusion (S5).
  15. 제 14 항에 있어서,The method of claim 14,
    상기 제1핵산구조체는 서열번호 1 내지 3의 염기서열로 이루어진 군으로부터 선택되는 선형핵산들이 Y자 가지형태로 결합한 형태인 것을 특징으로 하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체의 제조방법.The first nucleic acid structure is characterized in that the linear nucleic acid selected from the group consisting of the base sequence of SEQ ID NO: 1 to 3 in the form of a combination of Y-shaped branched, multi-ribonucleic acid marker quantitative diagnostic liposome-nucleic acid nano fusion method .
  16. 제 14 항에 있어서,The method of claim 14,
    상기 제2핵산구조체는 서열번호 4 내지 6의 염기서열로 이루어진 군으로부터 선택되는 선형핵산들이 Y자 가지형태로 결합한 형태인 것을 특징으로 하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체의 제조방법.The second nucleic acid structure is characterized in that the linear nucleic acid selected from the group consisting of the nucleotide sequence of SEQ ID NO: 4 to 6 in the form of a combination of Y-shaped branched, multi-ribonucleic acid marker quantitative diagnostic liposome-nucleic acid nano fusion method .
  17. 제 15 항 또는 제 16 항에 있어서, The method according to claim 15 or 16,
    상기 선형핵산은 5' 말단에 형광체를 더 포함하는 것을 특징으로 하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체의 제조방법.Wherein the linear nucleic acid is characterized in that it further comprises a phosphor at the 5 'terminal, method for producing a liposome-nucleic acid nano fusion for multiple ribonucleic acid marker quantitative diagnosis.
  18. 제 15 항에 있어서, The method of claim 15,
    상기 제1핵산구조체는 고리형 말단이 표적 RNA와 상보적 서열을 가지는 서열번호 7의 염기서열을 포함하는 것을 특징으로 하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체의 제조방법.Wherein the first nucleic acid structure is characterized in that it comprises a nucleotide sequence of SEQ ID NO: 7 having a cyclic terminal complementary to the target RNA, liposome-nucleic acid nano fusion for multiple ribonucleic acid marker quantitative diagnosis.
  19. 제 16 항에 있어서, The method of claim 16,
    상기 제2핵산구조체는 고리형 말단이 표적 RNA와 상보적 서열을 가지는 서열번호 8의 염기서열을 포함하는 것을 특징으로 하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체의 제조방법.The second nucleic acid structure is characterized in that the cyclic end comprises a nucleotide sequence of SEQ ID NO: 8 having a complementary sequence to the target RNA, multiple ribonucleic acid markers quantitative diagnostic liposome-nucleic acid nano fusion method for the preparation.
  20. 제 14 항에 있어서, The method of claim 14,
    상기 제1핵산구조체 및 제2핵산구조체는 형광체 및 소광제가 추가로 표지된 것을 특징으로 하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체의 제조방법.The first nucleic acid structure and the second nucleic acid structure is characterized in that the phosphor and quencher further labeled, liposome-nucleic acid nano fusion method for quantitative diagnosis of multiple ribonucleic acid markers.
  21. 제 14 항에 있어서,The method of claim 14,
    상기 실리카 구형체는 50 nm 내지 80 nm인 것을 특징으로 하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체의 제조방법.The silica spherical body is characterized in that 50 nm to 80 nm, multi-ribonucleic acid marker quantitative diagnostic liposome-nucleic acid nano fusion method for diagnostics.
  22. 제 14 항에 있어서,The method of claim 14,
    상기 양이온성 지질은 DOTAP(1,2-dioleoyl-3-trimethylammonium-propane) 및 콜레스테롤을 구성 성분으로 하는 양이온성 지질로 이루어진 것을 특징으로 하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체의 제조방법.The cationic lipid is DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and a cationic lipid comprising cholesterol as a component, a method for producing a liposome-nucleic acid nano fusion for quantitative diagnosis of multiple ribonucleic acid markers .
  23. 제 14 항에 있어서,The method of claim 14,
    상기 S2 단계의 용매는 메탄올, 에탄올, 증류수, 및 암모니아수를 포함하는 것을 특징으로 하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체의 제조방법.The solvent of the step S2 is methanol, ethanol, distilled water, and ammonia water, characterized in that, multi-ribonucleic acid marker quantitative diagnostic liposome-nucleic acid nano fusion method for manufacturing.
  24. 제 23 항에 있어서,The method of claim 23, wherein
    상기 메탄올 및 에탄올은 6 : 4 내지 8 : 2의 부피비율로 혼합되는 것을 특징으로 하는, 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체의 제조방법.Methanol and ethanol is 6: 4 to 8: characterized in that the volume ratio is mixed, the method for producing a liposome-nucleic acid nano fusion for multi-ribonucleic acid marker quantitative diagnosis.
  25. 제 1 항의 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체의 안정성을 이론적으로 평가하는 평가방법으로,An evaluation method for theoretically evaluating the stability of liposome-nucleic acid nanofusions for quantitative diagnosis of multiple ribonucleic acid markers according to claim 1,
    상기 방법은 나노융합체 내부에서 발생하는 수소결합 에너지를 측정하는 단계를 포함하는 것을 특징으로 하는, 평가방법.The method comprises the step of measuring the hydrogen bond energy generated inside the nanofusion, evaluation method.
  26. 제 25 항에 있어서,The method of claim 25,
    상기 측정은 35 내지 40℃에서 수행되는 것을 특징으로 하는, 평가방법.The measurement method, characterized in that carried out at 35 to 40 ℃.
  27. 제1항의 다중 리보핵산 마커 정량진단용 리포좀-핵산 나노융합체를 포함하는 암 진단용 키트. A cancer diagnostic kit comprising a liposome-nucleic acid nanofusion for quantitative diagnosis of multiple ribonucleic acid markers according to claim 1.
PCT/KR2018/002064 2017-02-21 2018-02-20 Liposome–nucleic acid nano-fusion for quantitative diagnosis of multiple ribonucleic acid markers, theoretical stability evaluation method therefor, application thereof, and production method therefor WO2018155880A1 (en)

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