KR101467938B1 - Bio imaging probe for detecting intracellular target molecules and curing a disease - Google Patents

Abstract

The present invention provides a bioimaging probe for detection and treatment of a target molecule in a cell, a method for producing the same, a composition for detecting a target molecule containing the probe, a method for obtaining an image of a living body or a sample using the probe, and a pharmaceutical composition containing the same. The bioimaging probe according to the present invention adopts a method in which the on / off of the fluorescence signal is controlled according to the presence of target molecules in the cell. Therefore, identification of a specific target biomarker, evaluation of intracellular gene, cell development, It can be useful for therapeutic evaluation. In particular, the bioimaging probe according to the present invention can provide a magnetic image as well as an optical image upon detection of target molecules in in vitro and in vivo. In addition, when the bioimaging probe according to the present invention is used, non-invasive and repetitive analysis in a living body is possible, and when a substance involved in disease induction is selected as a target molecule, Hybridization of a molecular beacon and a target molecule can inhibit disease induction.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a bioimaging probe for detecting intracellular target molecules and treating diseases,

The present invention relates to a bioimaging probe that can be used for the detection of intracellular target molecules and for the treatment of diseases.

MicroRNAs (miRNAs, miRs), a non-coding small RNA molecule composed of 22 nucleotides, are involved in a variety of biological processes, including cell proliferation and differentiation. Various miRNAs associated with neurodevelopment have been identified using microarray and Northern blot analysis [ L. Smirnova, et al ., Eur . J. Neurosci . 2005, 21, 1469; AM Krichevsky , et al ., Stem Cells 2006, 24, 85.]. Recently, we first reported in vivo imaging of neuro- and cancer-specific miogenesis using an optical gene imaging system ( HJ Kim , et & lt ; RTI ID = 0.0 & gt; al ., J. Nucl. Med . 2008, 49, 1686 .; HJ Kim , et al ., Mol . Imaging Biol . 2008, 11, 71 .; JY Lee , et al ., J. Nucl. Med . 2008, 49, 285 .; MH Ko , et al ., FEBS J. 2008, 275, 2605.]. To monitor the intrinsic miRNA expression patterns in living cells, a completely complementary sequence of mature miRNAs located in the 30 untranslated regions (UTRs) of the bioluminescent reporter gene tracks the individual miRNAs to investigate the expression patterns of mature miRNAs Has been widely used to [ HJ Kim, et al ., J. Nucl . Med . 2008, 49, 1686 .; X. Cao , et al ., Genes Dev . 2007, 21, 531 .; AJ Giraldez, et al ., Science 2005, 308, 833.]. However, miRNA detection based on such a reporter represents a signal-off system when the miRNA binds to and destabilizes the target mRNA. In this case, it is difficult to determine whether the observed signal-off data is indicative of miRNA expression or apoptosis in in vivo. Therefore, there is a need to develop a signal-on system for the detection of intracellular target molecules such as miRNAs. Imaging signals of fluorescently labeled ligands targeting cell membrane proteins or receptors are solely dependent on the binding affinity of the ligand to their targets at the outer boundary of the cell. However, the signals of fluorescent probes used for intracellular targets are very vague due to the presence of probes in the cells, whether or not there is interaction with their targets. To overcome this problem of imaging intracellular targets, an application comprising an amorphous dye black hole-quenching (BHQ) molecule or 4- (dimethylaminoazo) benzene-4-carboxylic acid (DABCYL) Molecular beacons with single-stranded or double-stranded DNA, recently developed imaging probes, including tamer, are designed to be conjugated after fluorescent nanoparticles. This is used to eliminate the fluorescence of the fluorescent material by fluorescence resonance energy transfer (FRET) in the absence of the target, and the fluorescence signal is recovered by separation from the quencher in the presence of the sequence-specific binding target [ Y. Li , et al., Biochem . Biophys . Res . Commun . 2008, 373, 457 .; W. Tan , et al ., Curr . Opin . Chem . Biol . 2004, 8, 547 .; X. Mao , et al ., Chem . Commun . 2009, 3065.]. A quencher-based molecular-targeting system developed on the basis of molecular beacons provides a good method for the detection of intracellular target proteins and for an improved understanding of the distribution of living mRNA in cells [ N. Nitin , et al ., Nucleic Acids Res. 2004, 32, e 58.1; MM Mhlanga , et al ., Nucleic Acids Res . 2005, 33, 1902.]. Peng et al. Reported intracellular molecular imaging using molecular beacon probes by targeting various cancer-associated genes at the mRNA level [ XH Peng , et al ., Cancer Res . 2005, 65, 1909.].

Recent advances in nanotechnology have led to a wide range of applications in relation to both therapeutic and diagnostic processes in biomedical and nanoimaging molecules [ Y. Liu , et al ., Int . J. Cancer 2007, 120, 2527; SP Leary , CY Liu , ML Yu , C. Apuzzo , Neurosurgery 2006, 58, 1009 .; P. Sharma, et al ., Adv . Colloid Interface Sci . 2006, 16, 123.]. Many multimodal molecular imaging probes with optical and radionuclide probes have emerged as powerful tools for noninvasive bioimaging in living subjects [W. Cai , X. Chen, Small 2007, 3, 1840.]. Nanoscale materials such as quantum dots and magnetic nanoparticles provide useful imaging information for detecting the extent of cancer spread in vivo using cancer-specific peptides [ W. Cai , et al ., Nano Lett . 2006, 6, 669 .; PC Wu , et al ., Bioconjugate Chem . 2008, 19, 1972.]. Recently, rhodamine-coated cobalt ferrite magnetic fluorescence (MF) particles have been used for multi-modal image acquisition; This technique is well suited for in-vivo tracking as well as in-vitro tracking of cells as bioimaging probes [ TJ Yoon , et al ., Small 2006, 2, 209.]. This dual MF imaging probe consists of a fluorescent dye coated with cobalt ferrite and silica shell in the center core, due to improved fluorescence due to the caging effect, light stability to photobleaching and low toxicity Have improved biocompatibility properties [ TJ Yoon , et al ., Small 2006, 2, 209 .; JS Kim , et al ., Toxicol . Sci . 2006, 89, 338.].

The present invention provides a new bioimaging probe that can be usefully used for the detection of target molecules and for the treatment of diseases.

The present invention provides, as means for solving the above problems, a nanoparticle capable of binding with an intracellular delivery ligand; And a molecular beacon capable of recognizing the target molecule by being hybridized with the target molecule is bound through a cleavable linkage in the cell where the bond can be cleaved.

Hereinafter, the bioimaging probe of the present invention will be described in detail.

The bioimaging probe of the present invention comprises nanoparticles that serve to transport molecular beacons to target cells; A molecular beacon capable of recognizing a target molecule by hybridizing with a target molecule in a cell; And a region capable of cleaving the bond in the cell, which binds the nanoparticle with the molecular beacon.

In the present invention, the shape of the molecular beacons is not particularly limited, and may be, for example, a hairpin structure or a linear partial double-stranded structure.

In the present invention, when the molecular beacon is in the form of a hairpin structure, a fluorescent substance is bonded near one end of the molecular beacon, a region in the center portion of the hairpin structure is hybridizable with the target molecule, The fluorescence suppressing substance may be designed to be in a position close to the fluorescent substance. In addition, one end of the molecular beacon to which the fluorescent substance is bound can be bound to the nanoparticle through a region where the bond can be cleaved in the cell.

In the present invention, when the molecular beacon is in the form of a linear partial double-stranded structure, a fluorescent substance is bonded at one end thereof, and a region including a region capable of hybridizing with the target molecule, A nucleic acid probe; And a region hybridizable with the nucleic acid probe, wherein an oligomer having a fluorescence control substance bound to the end of the nucleic acid probe, which is adjacent to one end of the nucleic acid probe, is hybridized.

In the present invention, the " region capable of hybridizing with the target molecule " contained in the nucleic acid probe means a sequence having a sequence complementary to a part of the target molecule and capable of hybridizing with the target molecule. For example, the " region capable of hybridizing with the target molecule " may have a sequence complementary to at least 90%, preferably 95% or more, of a portion of the target molecule. When the target molecule is a protein, the above-mentioned " region capable of hybridization with the target molecule " means a region capable of hybridizing with a protein as a target molecule in an aptamer serving as a nucleic acid probe. The " region capable of hybridization with the target molecule " can appropriately adjust its length according to the target molecule, and is not limited to a specific length. For example, when the target molecule is miRNA, the " region capable of hybridizing with the target molecule " may be composed of about 22 or more nucleotides, but when the target molecule is mRNA or protein, Region " may be shorter or longer.

In the present invention, the fluorescent substance bound to one end of the nucleic acid probe may be any fluorescent substance that can be used for bio (bio) imaging. In the present invention, the specific kind of the fluorescent substance is not particularly limited, and examples thereof include rhodamine and its derivatives, fluorescein and its derivatives, coumarin and its derivatives, acridine and its derivatives, pyrene and its derivatives , Erythrosine and its derivatives, eosin and its derivatives, and 4-acetamido-4'-isothiocyanatostilbene-2,2 'disulfonic acid. More specifically, the fluorescent material that can be used in the present invention is as follows.

Rhodamine and its derivatives as 6-carboxy -X- rhodamine (ROX), 6-carboxy rhodamine (R6G), Lisa Min rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, N, N, N ' -tetramethyl-6-carboxyhodamine < RTI ID = 0.0 > (R) < / RTI & (TAMRA), tetramethylrhodamine, tetramethylrhodamine isothiocyanate (TRITC), riboflavin, rosol acid, terbium chelate derivatives, Alexa derivatives, Alexa-350, Alexa-488, Alexa-547 and Alexa- Can be heard;

Fluorescein and its derivatives as 5-carboxy-fluorescein (FAM), 5- (4,6- dichloro-triazin-2-yl) amino fluorescein (DTAF), 2'7'--dimethoxy-4 ' (JOE), fluorescein, fluorescein isothiocyanate, QFITC (XRITC), fluorescamine, IR144, IR1446, malachite green isothiocyanate, 4-methylumbelliferone, orthocresolphthalein, nitrotyrosine, pararosaniline, phenol red, B-picoerythrin and o-phthalaldehyde;

Coumarin and derivatives thereof as coumarin, 7-amino-4-methyl coumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethyl coumarin (coumarin 151), didanosine cyano, 4'-6-dia mini dino- 2-phenylindole (DAPI), 5 ', 5 "-dibromophile, Bromopyrogallol Red, 7-diethylamino-3- (4'-isothiocyanatophenyl) Coumarin diethylenetriamine pentaacetate, 4- (4'-diisothiocyanato dihydro-stilbene-2,2'-disulfonic acid, 4,4'-diisothiocyanatostilbene- (DNS), 4- (4'-dimethylaminophenylazo) benzoic acid (DABCYL) and 4-dimethylaminophenyl azophenyl-4'-disulfonic acid, 5- [dimethylamino] naphthalene-1-sulfonyl chloride, -Isothiocyanate (DABITC); and the like;

Acridine and its derivatives include acridine, acridine isothiocyanate, 5- (2'-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS), 4-amino- (4-anilino-1-naphthyl) maleimide, anthranylamide and Brilliant Yellow, and the like;

Pyrene and its derivatives include pyrene, pyrene butyrate, succinimidyl 1-pyrene butyrate, Reactive Red 4 (Cibacron Brilliant Red 3B-A) and the like;

Erythrosine and its derivatives such as erythrosine B, erythrosine isothiocyanate, and ethidium;

Eosin and its derivatives include eosin and eosin isothiocyanate, and the like;

4- acetamido- 4 & apos ; -isothiocyanato stilbene- 2,2 ' disulfonic acid .

In the present invention, the nucleic acid probe may further include a linker region bound between the fluorescent substance and an area capable of hybridizing with the target molecule. In the present invention, the linker region can be used for facilitating the introduction of the above-mentioned fluorescent substance into one end of the nucleic acid probe. The specific kind of the linker region is not particularly limited, but, for example, And may be a nucleotide having 4 to 10 short nucleic acid sequences.

In the present invention, the oligomer having a region capable of hybridizing with the nucleic acid probe and having a fluorescence control substance bound thereto is bound to one end of a region where the fluorescence control substance can hybridize with the nucleic acid probe, It can be designed to be positioned in close proximity to the fluorescent material bound to the probe.

In the present invention, as described above, when the nucleic acid probe further comprises a linker region, the oligomer may further include a linker region capable of hybridizing with the linker region of the nucleic acid probe, in addition to a region capable of hybridizing with the nucleic acid probe As shown in FIG.

In the present invention, a cleavable linkage in the cell is bound to one end of the nucleic acid probe to which the fluorescent substance is bound, The other end of the region that can be bonded to the nanoparticle serves to connect the nanoparticle with the molecular beacon. After the bioimaging probe is introduced into the cell, the bond is cleaved within the cleavable region, Beacons can be separated from the nanoparticles.

The disruptable region of the present invention includes a disulfide linkage in the region. After the bioimaging probe is introduced into the cell, the disulfide bond is cleaved under an acidic cytoplasmic environment, To be separated from the particles. It is well known in the art that disulfide bonds can easily be cleaved under acidic cytoplasmic conditions. In particular, in the case of cells that cause diseases such as cancer cells, molecular beacons can easily be separated from nanoparticles in disease-causing cells, since they induce an acidic cytoplasmic environment by inducing acid in metabolism.

In the present invention, a nucleic acid probe of a molecular beacon separated from nanoparticles can be hybridized with a target molecule in the presence of the target molecule to achieve target molecule detection and disease treatment.

Hereinafter, the mechanism by which the nucleic acid probe is hybridized with the target molecule will be described in detail.

The molecular beacon of the present invention can be designed so that the hybridization of the nucleic acid probe and the target molecule is performed more effectively than the hybridization of the nucleic acid probe and the oligomer with the fluorescence control substance. The hybridization between the nucleic acid probe and the target molecule must be well performed so that the oligomer having the fluorescence control substance bound thereto may be separated from the nucleic acid probe.

For this purpose, in the present invention, the region where the target molecule can be hybridized with the nucleic acid probe is designed to be larger than the region where the oligomer to which the fluorescence control substance is bonded is hybridizable with the nucleic acid probe, The oligomer hybridized with the nucleic acid probe can be easily separated. More specifically, the nucleic acid sequence length of the region contained in the oligomer that can hybridize with the nucleic acid probe may be 20% to 25% of the length of the nucleic acid sequence of the region contained in the nucleic acid probe and capable of hybridizing with the target molecule have.

Also, in order to prevent the oligomer hybridized with the nucleic acid probe from falling off when the target molecule is not present, the hybridization between the nucleic acid probe and the oligomer can be designed to be stabilized at at least 40 ° C.

In the present invention, the specific kind of the fluorescence control substance that can be bound to the oligomer is not particularly limited and any fluorescence control substance commonly known in the art can be used without limitation. Specific examples of the fluorescent control material usable in the present invention include 4- (dimethylamino) azobenzene-4-carboxylic acid (DABCYL), 4- (dimethylamino) azobenzenesulfonic acid (DABSYL), BHQ- BHQ (Biosearch Technologies, Inc) or Eclipse (Amersham Bioscience) such as BHQ-2 or BHQ-3.

The specific kind of the target molecule that can be detected by the molecular beacon of the present invention is not particularly limited and may be, for example, microRNA, mRNA or protein. In the following examples of the present invention to be described later, microRNAs are targeted as target molecules, but the present invention is not limited thereto. For example, when the target molecule is a protein, a specific protein may be detected by synthesizing a suitable aptamer as a nucleic acid probe capable of detecting it and introducing it into a molecular beacon.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention, (13) are combined with each other. 1, the molecular beacon 10 includes a linker region 11a, a fluorescent substance 11c bonded to the linker region 11a, and a region 11b capable of hybridizing with the target molecule A nucleic acid probe 11; And an oligomer 12 including a region 12b that can be hybridized with the nucleic acid probe and to which a fluorescence control material 12a is bound. In addition, the region 13 in which the bond can be cleaved in the cell can be bound to the end of the nucleic acid probe 11. As shown in FIG. 1, since the fluorescent control material 12a is bonded to the oligomer at a position close to the fluorescent material 11c, the fluorescent material 11c is quenched.

That is, since the oligomer to which the fluorescence control substance is bound is hybridized with the nucleic acid probe when the target molecule is not present, fluorescence is not generated by the fluorescence control substance.

2 is a schematic diagram of a state 20 in which a nucleic acid probe 11 to which a fluorescent material is bound and a target molecule 21 are hybridized. 2, when the target molecule 21 is present, the nucleic acid probe 11 of the molecular beacon 10 is immobilized on the target molecule (not shown) instead of the oligomer 12 to which the fluorescence control material 12a is bonded 21, the oligomer 12 to which the fluorescence control substance 12a having the quenching (suppressing) the fluorescent material is bound is separated, and fluorescence can be emitted again. FIG. 2 is a graph showing a relationship between a region (13) in which a bond can be cleaved in the cell after the bioimaging probe including nanoparticles and a molecular beacon is introduced into the cell, 11b. As shown in Fig. That is, when the molecular beacon 10 bound to the nanoparticles through the region 13 in which the bond is cleaved in the cell is introduced into a disease-causing cell such as a cancer cell, the cell is subjected to an acidic cytoplasmic environment , The region 13 in which the bond can be cleaved in the cell is separated from the molecular beacon 10 and can be a molecular beacon 40 separated from the nanoparticles as described above. The molecular beacon 40 separated from the nanoparticles is separated from the region 11b capable of hybridizing with the target molecule of the nucleic acid probe 11 in a state where the region 13 in which the bond can be cleaved is separated The oligomer 12 is separated from the nucleic acid probe 11 in the presence of the target molecule 21 and the nucleic acid probe 11 is hybridized with the target molecule 21 so that the fluorescent material 11c can again fluoresce have.

FIG. 3 is a flow chart illustrating a process of turning on a fluorescent signal by hybridizing with a target molecule in a cell after the bioimaging probe according to the present invention is introduced into a cell. 3 attached herewith shows a bioimaging probe 30 according to one embodiment of the present invention in which an intracellular delivery ligand 31 and a molecular beacon 10 are bonded to the nanoparticles 23, The molecular beacon 10 is separated from the nanoparticles 32 under an acidic cytoplasmic environment and the nucleic acid probe 11 of the separated molecular beacon 40 is detected in the presence of the target molecule 21 FIG. 5 is a schematic view illustrating a process of separating the oligomer 12 to which the fluorescence control material 12a is bound and hybridizing the target molecule 21. As shown in FIG. 3, when the target molecule is not present, it is signal-off, and when the target molecule is bound to the molecular beacon according to the present invention, the signal- A molecular detection system can be provided.

The intracellular delivery ligand 31 of the bioimaging probe 30 may serve to allow the bioimaging probe 30 to be introduced into cells through endocytosis and the nanoparticles 32 may serve as a target A fluorescence image or a magnetic resonance (MR) image or the like can be provided so that it can be confirmed whether or not a cell to be present is present.

Therefore, the use of the bioimaging probe 30 of the present invention can confirm the presence or absence of a target cell. After the bioimaging probe 30 is introduced into a targeted cell, The presence or absence of the target molecule can be confirmed by on-off of the fluorescence signal.

3, as the target molecule 21 involved in disease induction is hybridized to the molecular beacon 40, the disease inducing process 50 is affected, and the disease inducing cells 51a, 51b , 51c may be killed.

Although the linker region 11a is used for easily introducing the fluorescent material 11c to one end of the nucleic acid probe in the above-described FIG. 1 to FIG. 3, in the structure of the molecular beacon according to the present invention, The region 11a is not necessarily required. Even if the linker region 11a of the nucleic acid probe 11 does not exist, it can be modified to have a fluorescent substance at one end of the region 11b that can be hybridized with the target molecule.

In the present invention, the nanoparticles can be used without limitation as long as the particles have a diameter of 1 nm to 1000 nm, preferably 2 nm to 100 nm. In the present invention, the specific type of the nanoparticles is not particularly limited, and examples thereof include magnetic nanoparticles, biocompatible materials, quantum dots, carbon nanotubes, gold, and SERS dot (Surface Enhanced Raman Spectroscopic dot) ≪ / RTI >

When the magnetic nanoparticles are used as the nanoparticles of the present invention, the magnetic nanoparticles may be a metal material, a magnetic material, or a magnetic alloy.

In the present invention, the metal material may be at least one selected from the group consisting of Pt, Pd, Ag, Cu and Au though not particularly limited.

The magnetic material in the present invention is also not particularly limited, Co, Mn, Fe, Ni , Gd, Mo, MM '2 O 4, And M _p O _q (Where M and M 'each independently represent Co, Fe, Ni, Mn, Zn, Gd or Cr, and p and q satisfy the expressions "0 <p? 3" and "0 <q? ). &Lt; / RTI &gt;

In the present invention, the magnetic alloy is not particularly limited, but may be at least one selected from the group consisting of CoCu, CoPt, FePt, CoSm, NiFe and NiFeCo.

In the present invention, the magnetic nanoparticles may have a coating layer containing a fluorescent material and a biocompatible material. In the present invention, the fluorescent material contained in the coating layer is not limited as long as it can be used for living body imaging. In the present invention, the specific kind of the fluorescent substance is not particularly limited, and for example, the same components as the fluorescent substance bound to one end of the above-mentioned nucleic acid probe can be used.

However, in the present invention, the fluorescent substance contained in the magnetic nanoparticles is used for the purpose of confirming the existence of a target cell, and the fluorescent substance bound to one end of the nucleic acid probe is a target molecule Is used for the purpose of confirming the presence or absence of fluorescence.

When the biocompatible material is used as the nanoparticle of the present invention, the biocompatible material may include a material selected from the group consisting of silica, polymer, lipid, and dextran.

In the present invention, the polymer may be at least one selected from the group consisting of a polyalkylene glycol, a polyetherimide, a polyvinylpyrrolidone, a hydrophilic vinyl polymer, and a copolymer of two or more of the above, have.

When a quantum dot is used as the nanoparticle of the present invention, the quantum dot may have a structure of a core body, a cell portion surrounding the core body, and a polymer coating layer coating the cell portion. In the present invention, the specific kind of the quantum dot is not particularly limited, and any quantum dot can be used without limitation as long as it has biocompatibility so that it can be used for living body imaging. In the present invention, the quantum dots having a particle diameter of less than 5.5 nm can be used. Since a heavy metal such as CdSe (cadmium-cesium), CdTe (cadmium-telluride) or CdS (cadmium sulfide) is mainly used as a constituent of the central body of the quantum dots, the use of the quantum dots may be harmful to the human body. However, in the present invention, by controlling the diameter of the quantum dots to be less than 5.5 nm, it is possible to relatively shorten the time to stay in the body through the rapid in vitro release of the quantum dots, thereby relatively reducing the toxicity ( Natuer biotechnology 2007 25 (10), 1165-1170 ).

When the SERS dot is used as the nanoparticle of the present invention, the SERS dot is a silica nanoparticle having a size of about 100 nm having a raman signal and capable of performing multiple analysis using Raman scattering.

Meanwhile, when another diagnostic probe is introduced into the bioimaging probe according to the present invention, it can be used as multiple diagnostic probes. For example, when a T1 or T2 magnetic resonance imaging probe is combined with a bioimaging probe according to the present invention, T1 magnetic resonance imaging or T2 magnetic resonance imaging can be performed at the same time, and an additional optical diagnostic probe In addition to optical imaging using magnetic resonance imaging and fluorescence imaging, other types of optical imaging can be performed simultaneously. Combining a CT diagnostic probe can simultaneously perform magnetic resonance imaging and CT diagnosis. Also, when combined with radioisotope, magnetic resonance imaging, PET and SPECT diagnosis can be performed at the same time.

Accordingly, the present invention can provide a bioimaging probe further comprising one or more probes selected from the group consisting of T1 or T2 magnetic resonance imaging probes, optical diagnostic probes, CT diagnostic probes, and radioactive isotopes. In the present invention, the probe may be used in combination with the nanoparticles of the bioimaging probe according to the functional group of the probe used or the hydrophobic / hydrophilic characteristic thereof.

For example, in the T1 by magnetic resonance imaging probe of the present invention comprises a compound, such as Gd and Mn compound, the T2 MRI diagnostic probe Fe ₃ O _4, and MnFe ₂ O ₄ The optical diagnostic probe includes a quantum dot, and the CT diagnostic probe includes an I (iodine) compound and gold nanoparticles. The radioisotope includes In (indium), Tc (technetium ) And F (fluorine).

Meanwhile, the bioimaging probe of the present invention can be introduced into cells by endocytosis or transfection.

The bioimaging probe according to the present invention may further include intracellular delivery ligands bound to the nanoparticles. Quot; intracellular delivery ligand &quot; refers to a substance that facilitates the passage of the bioimaging probe of the present invention through a cell membrane or an additional intracellular organ membrane. For example, specific examples of the &quot; intracellular delivery ligand &quot; include a protein transduction domain, a transmembrane domain, a cell penetrating peptide, an aptamer, an amphipathic polymer amphiphilic polymers or biocompatible copolymers. The intracellular delivery ligand of the present invention and the method of introducing it into an imaging probe are well known in the art.

In addition, the bioimaging probe according to the present invention may further include a cell or tissue targeting ligand bound to the nanoparticle. A &quot; cell or tissue targeting ligand &quot; can serve to induce the fluorescent imaging probe of the present invention to be introduced into a tissue or cell in which a desired target nucleic acid molecule is present. In the present invention, the &quot; cell or tissue targeting ligand &quot; may be an antibody, an aptamer, or a peptide, and may include a specific marker depending on the type of cell or tissue, a targeting ligand capable of binding thereto, Methods for incorporation into imaging probes are well known in the art.

Another object of the present invention is to provide a method for binding a molecular beacon capable of recognizing a target molecule by hybridizing with a target molecule to one end of a region in which a bond can be cleaved in a cell, And binding the other end of the region to nanoparticles capable of binding to an intracellular delivery ligand.

In the present invention, in order to allow the bioimaging probe to separate from the nanoparticles and to perform an independent function after the bioimaging probe is introduced into the cell, a nucleic acid probe To the opposite side of the one end of the strand), one end of the region where the bond can be cleaved in the cell. Such conjugation is by methods well known in the art (see Bioconjugate Chem ., 2009, 20 (1), 5-14).

In the present invention, the step of binding the other end of the cleavable region on the nanoparticles may include a step of immobilizing functional groups introduced onto the nanoparticles through surface modification, functional groups introduced into the other end of the cleavable region Lt; / RTI &gt;

In the present invention, functional groups capable of mutually covalent bonding exist on the surface of the nanoparticle and the other end of the region that can be cleaved, which is possible through appropriate modification. That is, the surface of the nanoparticle and the functional groups introduced at the other end of the cleavable region can be selected from known functional group binding examples so as to form a bond with each other. Representative examples of the known functional group binding examples are shown in Table 1 below.

I II III R-NH ₂ R'-COOH R-NHCO-R ' R-SH R'-SH R-SS-R R-OH R '- (epoxy group) R-OCH ₂ C (OH) CH ₂ -R ' RH-NH ₂ R '- (epoxy group) _{R-NHCH 2 C (OH)} CH 2 -R ' R-SH R '- (epoxy group) R-SCH ₂ C (OH) CH ₂ -R ' R-NH ₂ R'-COH R-N = CH-R ' R-NH ₂ R'-NCO R-NHCONH-R ' R-NH ₂ R'-NCS R-NHCSNH-R ' R-SH R'-COCH ₂ R'-COCH ₂ SR R-SH R ' -O (C = O) X _{R-OCH 2 (C = O} ) O-R ' R- (aziridine group) R'-SH R-CH ₂ CH (NH ₂ ) CH ₂ S-R ' R-CH = CH ₂ R'-SH R-CH ₂ CHS-R ' R-OH R'-NCO R'-NHCOO-R R-SH R'-COCH ₂ X R-SCH ₂ CO-R ' R-NH ₂ R'-CON ₃ R-NHCO-R ' R-COOH R'-COOH R- (C = O) O (C = O) -R '+ H ₂ O R-SH R'-X R-S-R ' R-NH _{2 ^{R'CH 2 C (NH 2 +)}} OCH 3 ^{R-NHC (NH 2 +)} CH 2 -R ' ^{R-OP (O 2 -)} OH R'-NH ₂ ^{R-OP (O 2 -)} -NH-R ' R-CONHNH ₂ R'-COH R-CONHN = CH-R &apos; R-NH ₂ R'-SH R-NHCO (CH ₂ ) ₂ SS-R ' I or II: functional group introduced at the nanoparticle surface or at the other end of the region that can be cleaved
III: Examples of bonding by reactions of I and II

In the present invention, the functional groups introduced on the surfaces of the nanoparticles may include -COOH, -CHO, -NH ₂ , -SH, -CONH ₂ , -PO ₃ H, -PO ₄ H, -SO ₃ H, -SO ₄ H , -OH, -sulfonate, -nitrate, -phosphonate, -succinimidyl, -maleimide and -alkyl groups. Also, in the present invention, the functional groups introduced at the other end of the cleavable region include -NH ₂ , -COOH, -COH, -NCO, -NCS, -COCH ₂ , -SH, -CON ₃ and -CH ₂ C (NH ²⁺⁾ may be selected from the group consisting of OCH _3.

In particular, in the present invention, the functional group introduced into the nanoparticle surface is -COOH, and the functional group introduced at the other end of the cleavable region may be -NH ₂ . That is, by binding nanoparticles having -COOH in the cleavable region having -NH ₂ at the 5 'end, the nanoparticles can be bound to the molecular beacon through a region that can be cleaved, but the present invention is not limited thereto .

In the present invention, the binding between the functional group introduced on the nanoparticle surface and the functional group introduced to the other end of the cleavable region can be carried out using a known crosslinking agent. In the present invention, the specific kind of the cross-linking agent is not particularly limited and includes, for example, N-ethyl-N'-dimethylaminopropyl carbodiimide, 1,4-Diisothiocyanatobenzene, 1,4-Phenylene diisocyanate, 1,6-Diisocyanatohexane, 4- (4-Diisocyanatohexane) (4-maleimidophenyl) butyric acid N-hydroxysuccinimide ester, Phosgene solution, 4- (maleimido) phenyl isocyanate (4- ( Maleinimido) phenyl isocyanate, 1,6-hexanediamine, p-Nitrophenyl chloroformate, N-Hydroxysuccinimide, 1,3 1,3-Dicyclohexylcarbodiimide, 1,1'-Carbonyldiimidazole, 3- 3-maleimidobenzoic acid N-hydroxysuccinimide ester, ethylenediamine, bis (4-nitrophenyl) carbonate, and succinic anhydride. Succinyl chloride, N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide Hydrochloride, N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride, N- N-succinimidyl 3- (2-pyridyldithio) propionate, and succinic anhydride (N, N'-disuccinimidyl carbonate) But are not limited to, those selected from the group consisting of sucroic anhydride. In the present invention, the coupling reaction may be carried out in a suitable solvent known in the art.

The method for preparing a bioimaging probe according to the present invention may further comprise the step of binding an intracellular delivery ligand onto the nanoparticles.

Intracellular transduction ligands introduced on the nanoparticles may be required to introduce the bioimaging probe prepared in the present invention into cells.

Methods for binding intracellular delivery ligands to nanoparticles in the present invention are well known in the art. In the present invention, intracellular delivery ligands can be bound onto surface-modified nanoparticles using the same components as the known crosslinking agents described above.

The present invention provides, as yet another means for solving the above problems, a composition for detecting a target molecule comprising a bioimaging probe according to the present invention.

As described above, the bioimaging probe of the present invention can be used to detect and image intracellular target molecules in in vivo or in vitro. The composition for detecting a target molecule according to the present invention may comprise a carrier and a vehicle commonly used in the medical field. The composition for detecting a target molecule of the present invention may be, for example, an ion exchange resin, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., various phosphates, glycine, sor Salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal silica, magnesium triacetate, magnesium triacetate, But are not limited to, silicates, polyvinyl pyrrolidone, cellulose based substrates, polyethylene glycols, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene glycols or wool. The composition of the present invention may further include, in addition to the above components, a lubricant, a wetting agent, an emulsifying agent, a suspending agent, or a preservative.

The composition for detecting a target molecule according to the present invention may be prepared as an aqueous solution for parenteral administration, preferably a buffer solution such as Hank's solution, Ringer's solution or physically buffered saline solution Etc. may be used. Water-soluble injection suspensions may contain a substrate capable of increasing the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

Another preferred embodiment of the target molecule detection composition of the present invention may be in the form of a sterile injectable preparation of a sterile injectable aqueous or oleaginous suspension. In the present invention, the suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents (ex. Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (e.g., a solution in 1,3-butanediol). Vehicles and solvents that may be used in the present invention include mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, nonvolatile oils are conventionally used as a solvent or suspending medium. For this purpose, any non-volatile oil including synthetic mono or diglycerides and less irritant may be used.

In another aspect of the present invention, there is provided a method for detecting a target molecule, comprising the steps of: administering a composition for detecting a target molecule according to the present invention to a living body or a sample; And detecting a signal emitted from the living body or the sample by the bioimaging probe to obtain an image.

As used herein, the term &quot; sample &quot; means a tissue or cell isolated from a subject to be diagnosed. In addition, the step of administering the composition for detecting a target molecule of the present invention to a living body or a sample may be administered through a route commonly used in the field of medicine, but parenteral administration is preferable. For example, intravenous, Intravenous, intramuscular, subcutaneous, or topical routes. In the step of obtaining the image of the present invention, it is preferable to use magnetic resonance imaging (MRI) and optical imaging to sense the signal emitted by the fluorescence imaging probe.

In the present invention, the &quot; magnetic resonance imaging apparatus &quot; described above is a device that puts a living body in a strong magnetic field and irradiates a radio wave of a specific frequency to absorb energy into atomic nuclei such as hydrogen in the living body tissue, The energy of the atomic nucleus such as hydrogen is released, and this energy is converted into a signal and processed by a computer for imaging. In the present invention, the type of the MRI apparatus is not particularly limited and may be, for example, a T2 spin-spin relaxation MRI apparatus, but is not limited thereto. On the other hand, in the present invention, a confocal microscope, a fluorescent microscope, a living optical apparatus, or the like can be used for the optical imaging, but the present invention is not limited thereto.

The present invention provides, as yet another means for solving the above-mentioned problems, a pharmaceutical composition comprising the bioimaging probe according to the present invention.

In the present invention, by designing the molecular beacon contained in the bioimaging probe to be hybridizable with a target molecule as a disease-causing substance, the bioimaging probe of the present invention can be used not only for the detection of target molecules, May be used to inhibit the activity of the target molecule, which is a triggering substance, so as to be able to treat the disease.

In the present invention, the type of disease that can be treated using the bioimaging probe is not particularly limited, and the molecular beacon contained in the bioimaging probe of the present invention may be appropriately selected depending on the type of disease to which the target molecule is hybridized Can be selected. In the present invention, various cancer diseases such as gastric cancer, blood cancer, pancreatic cancer, prostate cancer, lung cancer, breast cancer and ovarian cancer; Parkinson's disease, neurological diseases such as Alzheimer's disease; Cardiovascular disease; And metabolic diseases such as diabetes mellitus ( REBECCA T. MARQUEZ and ANTON P. MCCAFFREY , &quot; Advances in MicroRNAs : Implications for Gene Therapists ", HUMAN GENE THERAPY , 19 , 2008, p. 27-37) can be designed.

When the target molecule to be hybridized with the molecular beacon contained in the bioimaging probe according to the present invention is selected as a substance involved in cancer induction, as well as the detection of target molecules, cancer treatment is simultaneously achieved . This is because, when a substance involved in cancer induction, which is the target molecule, is present, the fluorescence inhibition substance contained in the molecular beacon is separated from the molecular beacon, and as the target molecule hybridizes with the molecular beacon, the target molecule involved in cancer induction And the cancer-induced cells may gradually be killed.

In the present invention, the pharmaceutical composition for treating diseases may include a carrier and a vehicle commonly used in the field of medicine, like the above-described composition for detecting a target molecule. Specifically, the carrier and the vehicle specifically include the above- May be the same as the detection composition.

In the present invention, the use of the pharmaceutical composition for treating diseases may also be the same as the above-described composition for detecting target molecules.

The bioimaging probe according to the present invention adopts a method in which the on / off of the fluorescence signal is controlled according to the presence of target molecules in the cell. Therefore, identification of a specific target biomarker, evaluation of intracellular gene, It can be useful for therapeutic evaluation. In particular, the bioimaging probe according to the present invention can provide an optical image as well as a magnetic resonance image upon detection of target molecules in in vitro and in vivo. In addition, when the bioimaging probe according to the present invention is used, non-invasive and repetitive analysis in a living body is possible, and when a substance involved in disease induction is selected as a target molecule, Hybridization of a molecular beacon and a target molecule can inhibit disease induction.

FIG. 1 is a view showing a state in which a molecular beacon according to an embodiment of the present invention, in which a nucleic acid probe having a fluorescent substance bonded thereto and an oligomer having a fluorescence control substance bonded thereto, It is a drawing
FIG. 2 is a diagram schematically showing a state in which a nucleic acid probe to which a fluorescent material is bound and a target molecule are hybridized.
FIG. 3 is a graph showing the results of a method in which a molecular beacon is separated from a nanoparticle after a bioimaging probe according to an embodiment of the present invention including nanoparticles, an intracellular delivery ligand and a molecular beacon is introduced into a target cell, The nucleic acid probe of the present invention is separated from the oligomer to which the fluorescence control substance is bound in the presence of the target molecule and is hybridized with the target molecule, and as the target molecule involved in the disease induction is hybridized with the molecular beacon, And a process of killing disease-causing cells by influencing the induction process.
4 is a TEM photograph of magnetic fluorescent nanoparticles (MF) (left figure) and magnetic fluorescent nanoparticles (likelihood) into which miR221 molecular beacons are introduced.
FIG. 5 is a graph showing the fluorescence activity of miR221 molecular beacon in a test tube according to the concentration of miR221 which is a target molecule.
FIG. 6 is a photograph of a confocal laser scanning microscope in which a NuMF substance having Nuclear Aptamer (Nu) bound to magnetic fluorescent nanoparticles (MF) was bound to various cancer cells to confirm cancer affinity.
FIG. 7 is a photograph of a confocal laser scanning microscope in which NuMF substance bound with nucleolin aptamer to magnetic fluorescent nanoparticles is bound to various normal cells and compared with cancer affinity.
8 is a graph showing fluorescence activity in C6 (glioma) cells according to the concentration of NuMF.
FIG. 9 is a photograph of a live cell image showing the process of NuMF being transferred into cancer cells in cancer cells.
10 is a photograph showing a T2 magnetic resonance image signal according to the concentration of NuMF and NuMFmt (mutant type of NuMF) in cancer cells.
11 is a graph showing real time PCR results of miR221 showing that miR221 is almost not expressed in astrocyte but overexpressed in thyroid cancer cells (NPA, TPC) and glioma cells (C6 glioma).
12 is a graph showing the cytotoxic effect of C6 cancer cells according to the concentration of miR221 antagomir using only a nucleic acid probe (miR221 antagomir) of miR221 molecular beacon as MTT assay effect.
13 is a graph showing the cytotoxic effect of C6 cancer cells using the bioimaging probe (miR221MB-NuMF) according to an embodiment of the present invention in which miR221 molecular beacon is bound to NuMF, by MTT analysis effect.
FIG. 14 is a photograph of a confocal laser scanning microscope image showing that miR221 molecular beacon is separated from NuMF after the bioimaging probe (miR221MB-NuMF) according to an embodiment of the present invention is introduced into C6 cancer cells.
FIG. 15 is a photograph of a bio-optical imaging result showing that a bioimaging probe (miR221MB-NuMF) according to an embodiment of the present invention is effective for the treatment of C6 cancer cells.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

[ Example ]

All data presented in the following examples are expressed as means ± standard error and are calculated using Student's t-test. * And ** shown in the following figures indicate statistical significance of P < 0.05 and P &lt; 0.005, respectively.

Example  1: Magnetic fluorescent nanoparticles and miR221  Reactive / Treatability  molecule Beacon Conjugated  Bio Imaging Of the probe  Produce

(One) miR221  Reactive / Treatability  molecule Beacon  Produce

A linear partial double stranded miR221 molecular beacon (miR221MB) was constructed which can detect miR221 specifically expressed in glioma and thyroid cancer cells as a target molecule and simultaneously treat cancer cells.

First, a nucleic acid probe including a region capable of hybridizing with miR221 was prepared. To this end, several random linker sequences (TTC GCT GT) were linked to the 3 'end of the complementary nucleotide with mature miR221 and a Cy5.0 fluorescent probe was coupled to the random linker sequence at the 3' end. In order to allow the miR221 molecular beacon to separate from the magnetic fluorescent nanoparticle after the introduction of the bioimaging probe (the conjugate of the magnetic fluorescent nanoparticle and the miR221 molecular beacon) into the cancer cell, Disulfide bond (-SS bond) was added, and -NH ₂ was again bound to the 5 'end so that the miR221 molecular beacon could be bound to the magnetic fluorescent nanoparticle.

An oligomer was synthesized which hybridized with the nucleic acid probe and to which Black Hole Quencher 2 (BHQ2; Bionics, Inc, Korea) was coupled as a fluorescence control substance. The overall miR221 molecular beacon is as follows.

5'- NH ₂ -SS- tcg atg taa cag acg acc caa ag TGT CGC TT-Cy5.0-3 '

3'-gtt tc ACA GCG-BHQ2-5 '

500 pmol of an oligomer containing a fluorescent control substance (BHQ2) and 500 pmol of a nucleic acid probe containing Cy5.0 were mixed with 1 ml of an annealing buffer (1x TE buffer, 100 mM NaCl, pH 7.8) For 15 minutes to synthesize a miR221 molecular beacon.

(2) Fluorescent material ( fluorophore ) &Lt; / RTI &gt; with a biocompatible coating layer &lt; RTI ID = 0.0 & MF ) Nanoparticles (MNP @ SiO2 RITC ) - PEG / COOH )

MNP @ SiO2 (RITC) -PEG / COOH was prepared as described in T. J. Yoon, et al., Small 2006, 2, 209 by purchasing nanoparticles (Biterials, Korea).

In summary, the cobalt ferrite magnetic core was incubated with polyvinylpyrrolidone (PVP; Sigma, St. Louis, Mo., USA) and then centrifuged at 4000 rpm for 10 minutes to obtain PVP-stabilized particles. The trimethoxysilane modified with RITC (rhodamine B isothiocyanate, excitation / emission = 555/578 nm) was injected into PVP-stabilized cobalt ferrite particles. The polymerization was induced by adding an ammonia solvent to produce silica-coated magnetic nanoparticles (size: about 50 nm, hydrodynamic diameter: 58.1 nm) into which RITC (4 x 10 ³ per nanoparticle) was introduced. (4.4 x 10 ⁴ per nanoparticle) and polyethylene glycol (PEG) (1 x 10 ⁴ per nanoparticle, 500 g mol ^-1 ) were introduced into the surface of MNP @ SiO 2 (RITC) ) -PEG / COOH. &Lt; / RTI &gt;

(3) magnetic fluorescence ( MF ) Nanoparticles and miR221  Reactive / Treatability  molecule Beacon Conjugation

Covalent conjugation of the carboxyl MF nanoparticles to the miR221 molecular beacon (concentration: 20 mM) was carried out by adding N-ethyl-N'-dimethylaminopropyl carbodiimide (EDC) thereto and incubating at room temperature for 1 hour, And the coupling efficiency between the carboxyl groups [MK So, et al., Nat . Protoc . 2006, 1, 1160]. The MF particle: miR221 molecular beacon: EDC reaction ratio was 1: 3: 200. The reaction product was centrifuged at 15000 rpm for 10 minutes to remove unconjugated free miR221 molecular beacon and EDC, and then washed several times with a Tris buffer solution (10 mM Tris-HCl, pH 7.4). After brief sonication, the final conjugate product was mixed with a borate buffer solution (10 mM sodium borate, pH 7.4; Sigma, St. Louis, Mo., USA). The spherical MF nanoparticles were homogeneously dispersed in the aqueous solution, and the hydrodynamic diameter measured by TEM was about 58.1 nm. Figure 4 shows a TEM photograph of MF nanoparticles (left) and MF nanoparticles (right) into which miR221 molecular beacons were introduced. In the miR221 molecular beacon, the oligomer is stained with 2% uranyl acetate and appears as a dark frame as indicated by the black arrow.

Mature miR221 consisting of 22 nucleotides from the PicTar database ( http://pictar.bio.nyu.edu ) was synthesized to determine if it could hybridize to the miR221 binding region of the miR221 molecular beacon and Cy5 0.0 &gt; miR221 &lt; / RTI &gt; in the presence of mature miR221. As a result of testing the fluorescence intensity of miR221 molecular beacon in microtube with different concentrations of mature miR221 (0, 25, 50, 100, 200, 400 pmol), fluorescence intensity of miR221 Strength increased. These results confirmed the specificity of the miR221 molecular beacon for miR221.

Example  2: in various cancer cells Necleolin In the nucleolin aptamer (Nu)  Confirmation of cancer affinity effect by

(1) magnetic fluorescence ( MF ) Nanoparticles and Necleolin Aptamer (Nu) Conjugation

Nucleolin Aptamer: NH ₂ -C ₆ -5'-TTGGTGGTGGTGGTTGTGGTGGTGG TGG-3

Nucleolytic aptamer mutation: NH ₂ -C ₆ -5'-TTCCTCCTCCTCCTTCTCCTCCTCCTCC-3

Covalent conjugation of carboxyl MF nanoparticles with nucleolin aptamer (concentration: 400 pM) was carried out by adding N-ethyl-N'-dimethylaminopropyl carbodiimide (EDC) thereto and incubating at room temperature for 1 hour By increasing the coupling efficiency between the amine and the carboxyl group [MK So, et al., Nat . Protoc . 2006, 1, 1160]. The reaction ratio of the MF nanoparticle: nucleolin aptamer: EDC was 1: 3: 200. The reaction was centrifuged at 15000 rpm for 10 minutes to remove unconjugated free nucleolin aptamer and EDC, and then washed several times with a Tris buffer solution (10 mM Tris-HCl, pH 7.4). After sonication, the final conjugate product was mixed with a borate buffer solution (10 mM sodium borate, pH 7.4; Sigma, St. Louis, Mo., USA) The preparation of TAMMER-MF nanoparticles (NuMF) was completed.

(2) Culture of cells

To observe the specific target force for cancer cells using NuMF, a total of 16 cancer cell lines and 7 normal cell lines were cultured based on the cell culture standard of ATCC. (Human glioblastoma), CRT (human astrocytoma), PC-12 (adrenalgland / pheochormycytoma), F11 (mouse neuroblastoma), C6 (mouse glioma), HepG-2 (human hepatocellular carcinoma) hepatocellular carcinoma, Caco-2, human colon adenocarcinoma, papillary thyroid carcinoma, NPA (papillary thyroid carcinoma), U-2OS (osteogenic sarcoma) HeLa (human cervical cancer cell), A-549 (lung carcinoma), PC-3 (prostate cancer cell line) and F9 (testis teratoma cell line) were used as normal cell lines. MSE (mesenchymal stem cells) neuronal stem cell, CHO (Chinese hamster ovary cell), 293 (kidney epithelial cell), L-132 lung normal cell, CCD-986sk (skin fibroblast) and TM-4 (testis cell). As can be seen from the results of FIGS. 6 and 7, NuMF targets were confirmed in all cancer cell lines except for the CRT, F11, and U-2OS cancer cell lines, and even infiltration into the cells was observed. On the other hand, NuMFmt (mutant type) failed to target cancer cell lines at all. In addition, NuMF-induced target was not observed in all cell lines except CCD-986sk in normal cell line. Thus, the selective specificity of necleolin aptamer to cancer cells could be confirmed by confocal microscopy.

(3) NuMF of C6  (Glioma) cell target affinity observation

After C6 cells were treated with various concentrations of NuMF, the cancer target affinity was analyzed by a fluorescence analyzer.

C6 cells were dispensed into 6-well plates pre-coated with gelatin and covered with sterile cover slips. The cells were then washed twice with phosphate-buffered saline (PBS). NuMF was then exposed to C6 cells and incubated at 37.8 [deg.] C for 4 hours. Nuclease Aptamer (Nu) can be introduced into cells by endocytosis, and NuMF particles have been shown to be suitable for intracellular transfer [Ko et al., Small , 2009, 5 (10), 1207 ; Hwang, et al., J. Nucl . Med. 2010, 51 (1), 98]. In order to confirm intracellular entry of NuMF, NuMFmt, in which Nu mutant was conjugated to MF nanoparticles, was used as a comparative example, in which the normal function of the nucleolin aptamer was not exhibited. For fluorescence analysis in vitro, C6 cells treated with different concentrations of NuMF and NuMFmt (0, 50, 100, 200, 400, 800 pmol) were rinsed with PBS and harvested by trypsinization. The collected cells were transferred to dark 96-well microplates and their fluorescence intensity was measured with a Varioskan Flash microplate reader (Thermo Fisher Scientific, Vantaa, Finland).

As a result, as shown in FIG. 8, it was confirmed that the C6 cell target power with increasing NuMF concentration was increased and the rhodamine fluorescence signal of NuMF was increased. On the other hand, NuMFmt, a mutant, is shown to be consistently low regardless of its concentration, so the specificity of NuMF for C6 cells can be confirmed.

Live cell imaging was performed using a real-time optical microscope to confirm the affinity of the C6 cell carcinoma target of NuMF by imaging.

As a result, as shown in FIG. 9, NuMF was adsorbed on the cell membrane after 5 minutes of exposure to the C6 cell, and 15 minutes and 30 minutes later, it was confirmed that the cell enters the C6 cell. This is a result of showing the specificity of NuMF to be delivered into the cell by the nucleolin aptamer.

An in vitro T2 weight image analysis was performed to identify the C6 cell carcinoma target affinity of NuMF by magnetic resonance signals. The gelatin is a line in 24-well plates coated with ⁵ x 10 5 / well of a frequency divider, and C6 cells, different concentrations of NuMF and NuMFmt (0, 50, 100, 200, 400, 800pmol) 1 day after the treatment C6 cells were rinsed with PBS and harvested by trypsinization. T2 MR signals were analyzed using a 1.5 T MRI scanner (GE Healthcare, Inc.).

As a result, as shown in FIG. 10, the T2 weighted Magnetic Resonance signal decreased in C6 cancer cells as the concentration of NuMF increased. On the other hand, the mutant NuMFmt was unable to target C6 cells, so that the T2 magnetic resonance signal remained constant regardless of the concentration.

Example  3: miR221  molecule Beacon  Nucleic acid In the probe  Observation of cancer cell therapy by

(1) mature miR221 To detect qRT - PCR  analysis

To evaluate the expression pattern of miR221 in normal cells and cancer cells, small RNA extracts of normal cells astrocyte, cancer cells (NPA, TPC) and C6 glioma cells were used for quantitative real-time PCR Chain Reaction (qRT-PCR) analysis was performed.

First, the entire small RNA extracted with nuclease-free water was isolated from each cell using the mirVana miRNA isolation kit (Ambion, Austin, TX, USA). To investigate the copy number of miR221 in cancer cells, qRT-PCR was performed using the mirVana qRT-PCR primer set (Ambion) and the mirVana qRT-PCR miRNA detection kit (Ambion). Mature miR221 levels were verified by PCR amplification using iCycer (Bio-Rad) with SYBR Premix Ex Taq (2 ×) (Takara, Japan) (95 ° C for 3 min, 95 ° C for 15 sec, Sec, 40 cycles of amplification). U6 snRNA primer set (Ambion, Austin, TX, USA) was used as an internal control.

As a result, as can be seen from FIG. 11, it was confirmed that miR221 was hardly expressed in astrocyte and overexpressed in thyroid cancer cells (NPA, TPC) and C6 glioma cells.

(2) miR221  molecule Beacon  Nucleic acid In the probe  Observation of cancer cell therapy by

C6 cell MTT analysis was performed by nucleic acid probe using miR221 as a target molecule in order to analyze that the nucleic acid probe of miR221 molecular beacon exerts a therapeutic effect on cancer cells by inhibiting the function of mature miR221 in C6 cells. A nucleic acid probe having miR221 as a target molecule was introduced into C6 cells, and 3 x 10 &lt; ⁴ &gt; / well C6 cells were dispensed into a 24-well plate. Thereafter, the cells were cultured in a 5% CO ₂ incubator at 37 ° C for 48 hours. After the medium was removed the following day, 250 쨉 l of MTT (2 mg / ml) reagent was added, followed by blocking of light using a foil and incubation in an incubator at 37 캜 for 4 hours. The supernatant was removed, treated with 1 mL of DMSO, and left at 37 째 C for 15 minutes. Subsequently, 200 μl of the solution was transferred to 96 wells and absorbance was measured at 540 nm using an ELISA spectrophotometer recorder.

As a result, as can be seen from FIG. 12, after 2 days, the MTT assay showed that the C6 cell killing effect was statistically significant from 200 pmol concentration. This confirms that the nucleic acid probe of the miR221 molecular beacon can act as an antagomir of miR221 to inhibit the function of miR221, ultimately killing cancer cells.

Example  4: Bio Imaging The probe (miR221MB-NuMF)  Cancer diagnosis / treatment observation

(1) magnetic fluorescence ( MF ) For nanoparticles miR221  Reactive / Treatability  molecule Beacon (miR221MB) and  Necleolin Aptamer ( Nu ) Each Conjugation

Covalent conjugation of the miR221 molecular beacon (concentration: 20 mM) and the nucleolin aptamer (concentration: 400 pM) to the carboxyl MF nanoparticles resulted in the addition of N-ethyl-N'-dimethylaminopropyl carbodiimide EDC) and incubated at room temperature for 1 hour to enhance the coupling efficiency between amine and carboxyl groups [MK So, et al., Nat . Protoc. 2006, 1, 1160]. The MF nanoparticle: miR221 molecular beacon: nucleolin aptamer: EDC reaction ratio was 1: 3: 3: 200. The reaction product was centrifuged at 15000 rpm for 10 minutes to remove unconjugated free miR221 molecular beacon and EDC, and then washed several times with a Tris buffer solution (10 mM Tris-HCl, pH 7.4). Thereafter, sonication was performed briefly and the final conjugate product was mixed with a borate buffer solution (10 mM sodium borate, pH 7.4; Sigma, St. Louis, Mo., USA) -NuMF).

(2) Bio Imaging The probe (miR221MB-NuMF)  Observation of cancer cell therapy by

C6 cell MTT assay was performed by miR221MB-NuMF in order to analyze the therapeutic effect of cancer cells on inhibition of miR221 function by biomimaging probe (miR221MB-NuMF) in C6 cells. In order to perform a comparative analysis MTT, it was dispensed a MF, NuMF, and after the transfection in NuMF miR221MB-C6 cells, 3X 10 ⁴ / well of C6 cells in a 24-well plate. Thereafter, the cells were cultured in a 5% CO ₂ incubator at 37 ° C for up to 48 hours. After incubation (1 hour, 3 hours, 5 hours, 24 hours, 48 hours) at various times, media was removed. After addition of 250 쨉 l of MTT (2 mg / ml) reagent, the light was blocked using a foil and incubated in an incubator at 37 째 C for 4 hours. Thereafter, the supernatant was removed, treated with 1 mL of DMSO, and left at 37 DEG C for 15 minutes. 200 [mu] l of the solution was transferred into 96 wells and absorbance was measured at 540 nm using an ELISA spectrophotometer recorder.

As a result, as shown in FIG. 13, almost no cell death was observed when NuMF conjugated with pure magnetic (MF) nanoparticles and Nucleolin aptamer (Nu) was introduced into C6 cells However, when miR221MB-NuMF, to which miR221 molecular beacon was additionally bound, was introduced into C6 cells, it was confirmed that cell death was rapidly induced 3 hours after the transfer of the above substance.

As described above, miR221MB-NuMF is induced by endocytosis in the cell membrane of C6 cell by the nucleolin aptamer, and is transferred into the inside of the cell. Then, the miR221MB-NuMF is present in the nucleic acid probe under the environment of the acidic cytoplasm The binding of the miR221 molecular beacon to NuMF is released, and mature miR221 overexpressed in the C6 cell binds to the nucleic acid probe of the free miR221 molecular beacon to inhibit miR221 function, thereby inducing the death of the C6 cell .

(3) Internalized miR221MB - NuMF of Confocal  Microscopic observation

Confocal microscopy of miR221MB-NuMF was performed during the incubation period of C6 cells to obtain in-vitro fluorescent images of miR221MB-NuMF in C6 cells. First, C6 cells were fixed with 3.7% formaldehyde solution (Sigma, St. Louis, Mo., USA) and rinsed with PBS for 3 minutes for 10 minutes. (Vector Laboratories, Inc., CA, USA) containing 4 ', 6'-diamidino-2-phenylindole dihydrochloride (DAPI) solution was added and the cover slip was removed from the 6- And they were observed with a confocal microscope.

As a result, as shown in FIG. 14, the blue image is a DAPI nucleus image, the green color is a Rhodamine fluorescence signal contained in the MF nanoparticles, and the red color is a fluorescence signal in the miR221 molecular beacon It is the fluorescence signal of Cy5.0. miR221MB-NuMF targets C6 cells by nucleolin aptamer. In the cell membrane, green fluorescence signal and red fluorescence signal are present at the same position. However, in the cell, -SS- As the binding is broken, the miR221 molecular beacon is released from the NuMF and the mature miR221 overexpressed in the C6 cell binds to the nucleic acid probe of the miR221 molecular beacon, so that the fluorescence control substance is separated from the molecular beacon and the red fluorescence signal is increased there was. In particular, it was found that the miR221 molecular beacon was separated from the NuMF by confirming that the green fluorescence signal and the red fluorescence signal were independently located at different positions within the cell.

(4) miR221MB - NuMF Biomechanical imaging of cancer using

In vivo in order to perform the cancer treatment effect studies, harvesting the C6 cells not treated with the C6 cells treated for miR221MB-NuMF 5 × 10 ⁶ / well one each from the PBS, and nude mice (male BALB / c, 7 weeks old). In the subcutaneous tissues of both thighs. At this time, in order to confirm the number of living cells to be transplanted, a plasmid construct containing a firefly luciferase gene induced by the cytomegalovirus (CMV) promoter was introduced into the C6 cell together with the miR221MB-NuMF.

As a result, as shown in FIG. 15, it was confirmed that the same number of cells in the left and right thighs were transferred into the living body and survived by the first day luciferase reporter gene after transplantation into C6 cells. However, two days after transplantation, it was confirmed that the image signal dropped sharply on the right femur treated with miR221MB-NuMF than on the left femur without treatment of miR221MB-NuMF. This means that C6 cell death was caused by miR221MB-NuMF.

10: molecular beacon 11: nucleic acid probe
11a: Linker area
11b: a region capable of hybridizing with the target molecule
11c: Fluorescent material
12: oligomer 12a: fluorescent control substance
12b: region capable of hybridization with nucleic acid probe
13: A cleavable linkage in the cell where the bond can be cleaved
20: A state in which a nucleic acid probe of a molecular beacon hybridizes with a target molecule
21: target molecule
30: Bioimaging probe
31: intracellular delivery ligand 32: nanoparticles
40: molecular beacon in which a region that can be cleaved in the cell is removed
50: Disease inducing process
51a, 51b, 51c: disease-causing cells
60: inside the cell

Claims (23)

Nanoparticles capable of binding to an intracellular delivery ligand; And
Molecular beacons capable of recognizing the target molecule by hybridizing with the target molecule are bound through a cleavable linkage within the cell,
Wherein the molecular beacon comprises a nucleic acid probe having a fluorophore at one end and a region capable of hybridizing with the target molecule, the probe being bound to a region where the other end is capable of cleavage; And a region hybridizable with the nucleic acid probe, wherein an oligomer having a fluorescence control substance bound to the end of the nucleic acid probe adjacent to the one end of the nucleic acid probe to which the fluorescent material is bound is hybridized
Bioimaging probe. delete The method according to claim 1,
Fluorescent materials include, but are not limited to, rhodamine and its derivatives, fluorescein and its derivatives, coumarin and its derivatives, acridine and its derivatives, pyrene and its derivatives, erythrosine and its derivatives, eosin and its derivatives, 4'-isothiocyanato-stilbene-2,2'-disulfonic acid,
The rhodamine derivatives include 6-carboxy-X-rhodamine (ROX), 6-carboxyhodamine (R6G), lysaminrodamine B sulfonyl chloride, rhodamine, rhodamine B, rhodamine 123, N, N, N ', N'-tetramethyl-6-carboxyindanamine (Texas Red), sulfonyl chloride derivatives of sulfolodamine 101 TAMRA), tetramethylrhodamine, tetramethylrhodamine isothiocyanate (TRITC), riboflavin, rosol acid, Alexa-350, Alexa-488, Alexa-547 or Alexa-647;
The fluorescein derivative may be selected from the group consisting of 5-carboxyfluorescein (FAM), 5- (4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF), 2'7'-dimethoxy- (JOE), fluororesin, fluororesin isothiocyanate, QFITC (XRITC), fluorescamine, IR144, IR1446, malachite green isothiocyanate, 4-dichloro-6-carboxyfluorescein - methylumbelliferone, orthocresolphthalein, nitrotyrosine, pararosaniline, phenol red, B-picoerythrine or o-phthalaldehyde;
The coumarin derivatives include 7-amino-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (coumarin 151), cyanosine, 4'- Indole (DAPI), 5 ', 5''- dibromopirogol Bromopyrogallol Red, 7-diethylamino-3- (4'-isothiocyanatophenyl) Ethylene triamine pentaacetate, 4- (4'-diisothiocyanatodi-stilbene-2,2'-disulfonic acid, 4,4'-diisothiocyanatostilbene- Dimethylamino] naphthalene-1-sulfonyl chloride or 4- (4'-dimethylaminophenylazo) benzoic acid (DABCYL) 4-Dimethylaminophenyl azophenyl-4'-isothio Cyanate (DABITC);
The acridine derivatives include acridine isothiocyanate, 5- (2'-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N- [3- vinylsulfonyl) phenyl] (Lucifer Yellow VS), N- (4-anilino-1-naphthyl) maleimide, anthranylamide or Brilliant Yellow;
The pyrene derivative is pyrene butyrate, succinimidyl 1-pyrene butyrate or ^{Reactive Red 4 (Cibacron ® Brilliant Red} 3B-A) , and;
Wherein said erythrosine derivative is erythrosine B, erythrosine isothiocyanate or ethidium;
The eosin derivative is eosine isothiocyanate
Bioimaging probe. The method according to claim 1,
The nucleic acid probe may be a fluorescent material; A linker region coupled to the fluorescent material; And a region coupled to the linker region and capable of hybridizing with the target molecule. 5. The method of claim 4,
Oligomers include fluorescent control materials; A linker region coupled to the fluorescent control material and capable of hybridizing with a linker region of a nucleic acid probe; And a region capable of hybridizing with a portion of the hybridization region of the nucleic acid probe. The method according to claim 1,
Wherein the region where the target molecule can hybridize with the nucleic acid probe is larger than the region where the oligomer to which the fluorescence control substance is bound is capable of hybridizing with the nucleic acid probe. The method according to claim 1,
The fluorescent control material is selected from the group consisting of 4- (dimethylamino) azobenzene-4-carboxylic acid (DABCYL), 4- (dimethylamino) azobenzene sulfonic acid (DABSYL), BHQ-1, BHQ-2, BHQ-3 and ECLIPSE Wherein the bioimaging probe is a bioimaging probe. The method according to claim 1,
The target molecule is a microRNA, mRNA or protein bioimaging probe. The method according to claim 1,
Wherein the region in the cell where the bond can be cleaved comprises a disulfide linkage. The method according to claim 1,
Wherein the nanoparticles are selected from the group consisting of magnetic nanoparticles, biocompatible materials, quantum dots, carbon nanotubes, gold, and SERS dots. 11. The method of claim 10,
The magnetic nanoparticles are metal materials, magnetic materials, or magnetic alloys. 12. The method of claim 11,
Wherein the magnetic nanoparticles have a coating layer comprising a fluorescent substance and a biocompatible substance. 13. The method of claim 12,
Wherein the fluorescent substance contained in the coating layer of the magnetic nanoparticles and the fluorescent substance bound to one end of the nucleic acid probe are different from each other. 11. The method of claim 10,
Wherein the biocompatible material comprises a material selected from the group consisting of silica, polymers, lipids, and dextran. 11. The method of claim 10,
The quantum dot has a diameter of less than 5.5 nm. The method according to claim 1,
A bioimaging probe in which nanoparticles are further bound with an intracellular delivery ligand. 17. The method of claim 16,
Wherein the intracellular delivery ligand is selected from the group consisting of a protein delivery domain, a transmembrane domain, a cell penetration peptide, an aptamer, an amphipathic polymer, and a biocompatible copolymer. A molecular beacon capable of recognizing a target molecule by hybridizing with a target molecule can be bound to one end of a region where a bond can be cleaved in the cell and the other end of the cleavable region can be coupled with an intracellular delivery ligand &Lt; / RTI &gt; wherein the nanoparticles are nanoparticles. 19. The method of claim 18,
Wherein the bond between the nanoparticles and the end of the cleavable region is through covalent bonding between the functional group introduced on the nanoparticle and the functional group introduced into the other end of the cleavable region. A composition for detecting a target molecule comprising a bioimaging probe according to any one of claims 1 to 17. Administering the composition according to claim 20 to a living body or a sample; And
Detecting a signal emitted by the bioimaging probe from the living body or the sample to obtain an image,
A method for obtaining an image of a living body or a sample. Nanoparticles capable of binding to an intracellular delivery ligand; And
Molecular beacons capable of recognizing microRNA 221 by hybridization with microRNA 221 are bound through a cleavable linkage within the cell,
Wherein the molecular beacon comprises a nucleic acid probe having a fluorophore at one end and a region capable of hybridizing with the microRNA 221, the other end of the nucleic acid probe being bound to the cleavable region; And a region hybridizable with the nucleic acid probe, wherein an oligomer having a fluorescence control substance bound to the end of the nucleic acid probe adjacent to the one end of the nucleic acid probe to which the fluorescent material is bound is hybridized
A pharmaceutical composition for treating glioma comprising a bioimaging probe.

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