KR20110099591A - Magnetic fluorescent nanoparticle imaging probes for detecting intracellular target molecules - Google Patents

Abstract

The present invention provides a magnetic fluorescent nanoparticle imaging probe that can be used for intracellular target molecule imaging, a method for preparing the same, a composition for detecting the target molecule including the same, and a method for obtaining an image of a living body or a sample using the same. The magnetic fluorescence nanoparticle imaging probe according to the present invention has a mode in which on-off of the fluorescence signal is regulated in accordance with the presence of the target molecule in the cell. It can be usefully used for disease diagnosis / treatment evaluation. In particular, the magnetic fluorescent nanoparticle imaging probe according to the present invention based on magnetic nanoparticles can provide magnetic resonance images as well as optical images according to detection of target molecules in vitro and in vivo. In addition, the magnetic fluorescent nanoparticle imaging probe according to the present invention enables non-invasive and repeated analysis in a living subject.

Description

Magnetic fluorescent nanoparticle imaging probes for detecting intracellular target molecules

The present invention relates to magnetic fluorescent nanoparticle imaging probes that can be used for intracellular target molecule imaging.

MicroRNAs (miRNAs, miRs), a non-coding small RNA molecule consisting of 22 nucleotides, are involved in various biological processes, including cell proliferation and differentiation. Various miRNAs related to neural development have been identified using microarray and northern blot analysis [L. Smirnova, et al., Eur. J. Neurosci. 2005, 21, 1469; A. M. Krichevsky, et al., Stem Cells 2006, 24, 85.]. Recently, we have reported for the first time in vivo imaging of neuro- and cancer-specific miRNA production using an optical gene imaging system [H. J. Kim, et al., J. Nucl. Med. 2008, 49, 1686 .; H. J. Kim, et al., Mol. Imaging Biol. 2008, 11, 71; J. Y. Lee, et al., J. Nucl. Med. 2008, 49, 285 .; M. H. Ko, et al., FEBS J. 2008, 275, 2605.]. To monitor the intrinsic miRNA expression patterns in living cells, fully complementary sequences of mature miRNAs located in 30 untranslated regions (UTRs) of the bioluminescent reporter genes track individual miRNAs to investigate expression patterns of mature miRNAs. It has been widely used for the purpose [H. J. Kim, et al., J. Nucl. Med. 2008, 49, 1686 .; X. Cao, et al., Genes Dev. 2007, 21, 531; A. J. Giraldez, et al., Science 2005, 308, 833.]. However, miRNA detection based on such reporters 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 means miRNA expression or cell death in vivo. Therefore, there is a need to develop a signal-on system for detection of intracellular target molecules such as miRNAs.

Imaging signals of fluorescently labeled ligands targeting cell membrane proteins or receptors are only dependent on the binding affinity of ligands at their outer boundaries and their targets. However, the signals of fluorescent probes used for intracellular targets are very vague due to the presence of probes in the cells, with or without interaction to their targets. To overcome this problem of imaging intracellular targets, an app comprising a black hole-quenching (BHQ) molecule or 4- (dimethylaminoazo) benzene-4-carboxylic acid (DABCYL), a non-fluorescent dye Molecular beacons with single- or double-stranded DNA, including recently developed imaging probes, are designed to be conjugated after fluorescent nanoparticles. It is used to fluoresce the fluorescence 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 bound 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.] The developed quencher-based molecular-targeting system based on molecular beacons provides a good method for the detection of intracellular target proteins and an improved understanding of the distribution of live intracellular mRNAs [N. Nitin, et al., Nucleic Acids Res. 2004, 32, e58.1 .; M. M. Mhlanga, et al., Nucleic Acids Res. 2005, 33, 1902.]. Peng et al report on intracellular molecular imaging with molecular beacon probes by targeting various cancer-associated genes at the mRNA level [X. H. Peng, et al., Cancer Res. 2005, 65, 1909.].

Recent advances in nanotechnology have led to wide applications for both therapeutic and diagnostic processes in biopharmaceutical and nanoimaging molecules [Y. Liu, et al., Int. J. Cancer 2007, 120, 2527 .; S. P. Leary, C. Y. Liu, M. L. 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 non-invasive 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 spread of cancer in vivo using cancer-specific peptides [W. Cai, et al., Nano Lett. 2006, 6, 669 .; P. C. Wu, et al., Bioconjugate Chem. 2008, 19, 1972.]. Recently, rhodamine-coated cobalt ferrite magnetic fluorescence (MF) particles have been used for multimodal image acquisition; This technique is well suited for in vivo tracking as well as in vitro tracking of cells as a bioimaging probe [T. J. Yoon, et al., Small 2006, 2, 209.], this dual MF imaging probe consists of a fluorescent dye coated with a silica shell and cobalt ferrite in the central core, which is caused by the casing effect. Have improved biocompatibility properties due to improved fluorescence, photostability to photobleaching and low toxicity [T. J. Yoon, et al., Small 2006, 2, 209 .; J. S. Kim, et al., Toxicol. Sci. 2006, 89, 338.].

The present invention seeks to provide new magnetic fluorescent nanoparticle imaging probes that can be usefully used for detection of target molecules.

The present invention provides a magnetic nanoparticle having a coating layer comprising a fluorophore and a biocompatible material; And a molecular beacon coupled to the coating layer and including a nucleic acid probe comprising a region capable of hybridizing with a target molecule and a region capable of hybridizing with the nucleic acid probe and having an oligomer to which a fluorescent control material is bound. It provides a magnetic fluorescent nanoparticle imaging probe for the detection of a target molecule comprising a.

In the present invention, it refers to a sequence capable of hybridizing with a target molecule by having a sequence complementary to a portion of the “region capable of hybridizing with a target molecule” contained in the nucleic acid probe. For example, the “region that can hybridize with a target molecule” may be one having a sequence that is at least 90%, preferably at least 95%, complementary to a portion of the target molecule. If the target molecule is a protein, the “region that can hybridize with the target molecule” means a region that can hybridize with a protein that is a target molecule in an aptamer serving as a nucleic acid probe. The "region that can hybridize with the target molecule" may be appropriately adjusted depending on the target molecule, and need not be limited to a specific length. For example, if the target molecule is miRNA, the region that can hybridize with the target molecule may be composed of about 22 nucleotides, but if the target molecule is mRNA or protein, the region that can hybridize with the target molecule may be shorter or It can be longer.

In an oligomer comprising a region capable of hybridizing with the nucleic acid probe and having a fluorescent control material bound thereto, the fluorescent control material is bound to one end of the region capable of hybridizing with the nucleic acid probe, wherein the nucleic acid probe is a magnetic fluorescent nanoparticle. It is designed so that the fluorescent control material can be located in close proximity to one end that is bonded to the coating layer.

On the other hand, the magnetic fluorescent nanoparticle imaging probe of the present invention can be designed such that hybridization of the nucleic acid probe and the target molecule is better than hybridization of the oligomer to which the nucleic acid probe and the fluorescent control material are bound. This is because the oligomer to which the fluorescent control material is bound can be separated from the nucleic acid probe with the target molecule.

To this end, in one embodiment, the region where the target molecule can hybridize with the nucleic acid probe is designed to be larger than the region where the target molecule can hybridize with the oligomer to which the fluorescent control material is bound, so that in the presence of the target molecule, The oligomers that have been hybridized with the nucleic acid probe can be easily separated. In addition, hybridization between the nucleic acid probe and the oligomer may be designed to be stable at least at 40 ° C. in order to prevent the oligomer that has been hybridized with the nucleic acid probe from falling off when the target molecule is not present.

In one embodiment, there is provided a magnetic fluorescent nanoparticle imaging probe further comprises a linker at one end of the nucleic acid probe. The linker may be used to facilitate the introduction of a functional group capable of binding the nucleic acid probe to the coating layer of the magnetic fluorescent nanoparticles. In one embodiment, the linker may be a nucleotide having 4 to 10 short nucleic acid sequences. In this case, the oligomer may be modified to include a region capable of hybridizing with the linker in addition to the region capable of hybridizing with the nucleic acid probe.

In one embodiment of the present invention, in order to synthesize fluorescent-nanoparticle-based molecular beacons, a partially double stranded oligonucleotide with a functional group capable of binding to a magnetic nanoparticle coating layer at one end was designed. Referring to FIG. 1, the molecular beacon 30 includes a linker 42 including a functional group (not shown in the drawing, located in front of the linker 42) that can be bonded to a coating layer of magnetic fluorescent nanoparticles. An oligomer 50 comprising a nucleic acid probe 40 comprising a region 44 capable of hybridizing with a target molecule, and a region 54 capable of hybridizing with the nucleic acid probe and having a fluorescent control material 52 bound thereto It includes. In the following example, a linker 42 was used to easily introduce a functional group (not shown in the drawing, present in the front of the linker 42) that can be bonded to the coating layer of the magnetic fluorescent nanoparticles. Such molecular beacons are conjugated to the magnetic fluorescent nanoparticles through a bond formed by a functional group present on the coating layer and a functional group present on the nucleic acid probe. In this case, since the fluorescent control material is located close to the magnetic fluorescent nanoparticles, the fluorescence represented by the magnetic fluorescent nanoparticles is quenched.

That is, since the oligomer to which the fluorescent control material is bound is hybridized with the nucleic acid probe bound to the magnetic fluorescent nanoparticles in the absence of the target molecule, fluorescence does not appear by the fluorescent control material. On the other hand, as shown in Figure 2, when the target molecule 60, the nucleic acid probe 40 of the molecular beacon 30 is instead of the oligomer 50 to which the fluorescent control material 52 is bound to the target molecule ( 60), the oligomer 50 to which the fluorescent control material 52, which was quenched fluorescence of the magnetic fluorescent nanoparticles, is separated, thereby fluorescing again.

In summary, the magnetic fluorescent nanoparticle imaging probe according to the present invention is signal-off when the target molecule is not present, as shown in FIG. 3, and the target molecule is separated from the magnetic fluorescent nanoparticle imaging probe of the present invention. The combination provides a molecular detection system that is signaled on.

Therefore, using the magnetic fluorescent nanoparticle imaging probe of the present invention, it is possible to confirm the presence or absence of a target molecule to be detected through on-off of a fluorescence signal, and to display it as an MR image at the same time as displaying a fluorescent image.

Although, in the following embodiment, the linker 42 was used to easily introduce a functional group (not shown in the figure, present in the front of the linker 42) that can be bonded to the coating layer of the magnetic fluorescent nanoparticles. The linker 42 is not necessarily required in the configuration of the molecular beacon of the present invention. Even if the linker 42 is not present, it is possible to modify it to have a functional group capable of bonding with the coating layer at one end of the region 44 that can hybridize with the target molecule.

In the present invention, the magnetic nanoparticles (magnetic nanoparticles) are magnetic and can be used without limitation as long as the particle diameter is 1nm to 1000nm, preferably 2nm to 100nm. Meanwhile, in consideration of the presence of a biocompatible coating layer on the magnetic nanoparticles and the biological immune system, the diameter of the magnetic fluorescent nanoparticle imaging probe may be 10 nm to 200 nm.

In one embodiment, the magnetic nanoparticles may be a metal material, a magnetic material, or a magnetic alloy.

The metal material is not particularly limited but may be one or more selected from the group consisting of Pt, Pd, Ag, Cu, and Au.

The magnetic material is also not particularly limited, but Co, Mn, Fe, Ni, Gd, Mo, MM ' ₂ O ₄ , And M _p O _q (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 = 5", respectively). It may be one or more selected from the group consisting of.

In addition, 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 addition, the fluorescent material included in the magnetic fluorescent nanoparticles of the present invention may be any fluorescent material that can be used for biological imaging. In one embodiment, the fluorescent material is, but is not limited to, rhodamine and its derivatives, fluorescein and its derivatives, coumarin and its derivatives, acridine and its derivatives, pyrene and its derivatives, erythrocin and its derivatives, eosin And derivatives thereof, and 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid. More specifically illustrating the fluorescent material is as follows:

Rhodamine and its derivatives : 6-carboxy-X-rhodamine (ROX), 6-carboxyrodamine (R6G), lysamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, Rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivatives of sulforhodamine 101 (Texas Red), N, N, N ', N'-tetramethyl-6-carboxyrodamine (TAMRA), tetramethyl rhodamine, tetramethyl rhodamine isothiocyanate (TRITC), riboflavin, rosolic acid, terbium chelate derivatives, Alexa derivatives, Alexa-350, Alexa-488, Alexa-547, Alexa-647;

Fluorescein and its derivatives : 5-carboxyfluorescein (FAM), 5- (4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF), 2'7'-dimethoxy-4 '5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate, QFITC (XRITC), fluorescamine, IR144, IR1446, malachite green isothiocyanate, 4-methylumbeliferon, orthocresol phthalein, nitrotyrosine, pararoaniline, phenol red, B-phycoerythrin, o-phthaldialdehyde;

Coumarin and its derivatives : coumarin, 7-amino-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (coumarin 151), cyanosine, 4'-6-diaminidino- 2-phenylindole (DAPI), 5 ', 5''-dibromopyrogallol-sulfonphthalein (Bromopyrogallol Red), 7-diethylamino-3- (4'-isothiocyanatophenyl) -4- Methylcoumarin diethylenetriamine pentaacetate, 4- (4'-diisothiocyanatodihydro-stilben-2,2'-disulfonic acid, 4,4'-diisothiocyanatostilben-2,2 '-Disulfonic acid, 5- [dimethylamino] naphthalene-1-sulfonyl chloride (DNS, dansyl chloride), 4- (4'-dimethylaminophenylazo) benzoic acid (DABCYL) 4-dimethylaminophenylazophenyl-4' Isothiocyanate (DABITC);

Acridine and its derivatives : acridine, acridine isothiocyanate, 5- (2'-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N- [3-vinylsulfur Ponyl) phenyl] naphthalimide-3,5 disulfonate (LuciferYellow VS), N- (4-anilino-1-naphthyl) maleimide, anthranilamide, Brilliant Yellow;

Pyrene and its derivatives : pyrene, pyrene butyrate, succinimidyl 1-pyrene butyrate, Reactive Red 4 (Cibacron ^® Brilliant Red 3B-A);

Erythrosine and its derivatives : erythrosin B, erythrosin isothiocyanate, ethidium;

Eosin and its derivatives : eosin, eosin isothiocyanate;

4- Acetamido -4'- Isothiocyanatostilbene -2,2 ' Disulfonic acid .

Meanwhile, the biocompatible material used for the coating layer including the fluorophore and the biocompatible material may be used as long as it is known in the art as a biocompatible material. In one embodiment, the biocompatible material may be one comprising a material selected from the group consisting of silica, polymers, lipids and dextran. The biocompatible materials can be modified to provide better biocompatibility. For example, it is possible to introduce and use a polymer in silica or to use a lipid in combination with a polymer. Specific examples of biocompatible polymers include biopolymers such as chitosan, elastin, hyaluronic acid, alginate, gelatin, collagen, cellulose, polyethylene glycol (PEG), polyethylene oxide (PEO), polycaprolactone (PCL), and polylactic acid ( PLA), polyglycolic acid (PGA), poly [(lactic-co- (glycolic acid)) (PLGA), poly [(3-hydroxybutyrate) -co- (3-hydroxybalarate) (PHBV), Polydioxanone (PDO), poly [(L-lactide) -co- (caprolactone)], poly (esterurethane) (PEUU), poly [(L-lactide) -co- (D-lactide) ], Poly [ethylene-co- (vinyl alcohol)] (PVOH), polyacrylic acid (PAA), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polystyrene (PS), polyaniline (PAN), etc. Chemical polymers can be used. In one embodiment, the biocompatible material may be silica. In other embodiments, the biocompatible material may be a polymer such as polyethylene glycol. In another embodiment, the biocompatible coating layer comprises, for example, a cationic surfactant comprising an alkyl trimethylammonium halide; Saturated or unsaturated fatty acids such as oleic acid, lauric acid, or dodecylic acid, trioctylphosphine oxide (TOPO), trioctylphosphine (TOP), Or alkyl amines such as trialkylphosphine or trialkylphosphine oxides such as tributylphosphine, oleic amine, trioctylamine, or octylamine Or neutral surfactants including alkyl thiols; And anionic surfactants including sodium alkyl sulfate, or sodium alkyl phosphate.

Fluorescent control materials that can be used in the magnetic fluorescent nanoparticle imaging probe of the present invention can be used as long as they are known in the art as fluorescent control materials. Such fluorescence control material may be used as, but not limited to, 4- (dimethylamino) azobenzene-4-carboxylic acid (DABCYL) or 4- (dimethylamino) azobenzene sulfonic acid (DABSYL). It is also possible to use Eclipse available from Amersham Biosciences or BHQ-1, BHQ-2, BHQ-3, etc., sold under the trademark Black Hole Quencher (BHQ) available from Biosearch Technologies, Inc.

On the other hand, the kind of target molecule detectable with the magnetic fluorescent nanoparticle imaging probe of the present invention is not particularly limited. In one embodiment, the target molecule can be a microRNA, mRNA or protein. In the following examples of the present invention, a magnetic fluorescent nanoparticle imaging probe that detects microRNA as a target molecule and detects the microRNA is detected, and shows the result of detecting the microRNA, but the present invention is not limited thereto. no. For example, if a target molecule is a protein, a suitable aptamer may be synthesized as a nucleic acid probe capable of detecting it, and then introduced into a magnetic fluorescent nanoparticle imaging probe to detect a specific protein.

Meanwhile, when introducing another diagnostic probe into the magnetic fluorescent nanoparticle imaging probe according to the present invention, it may be used as a multiple diagnostic probe. For example, combining T1 magnetic resonance imaging probes with magnetic fluorescence nanoparticle imaging probes allows simultaneous diagnosis of T2 magnetic resonance imaging and T1 magnetic resonance imaging, and by combining additional optical diagnostic probes in addition to fluorescent materials, magnetic resonance imaging In addition to optical imaging by the fluorescent material and other optical imaging can be performed at the same time, combined with the CT diagnostic probe can be used for magnetic resonance imaging and CT diagnosis at the same time. In addition, when combined with radioisotopes, magnetic resonance imaging, PET, and SPECT can be simultaneously diagnosed.

Accordingly, the present invention also provides a magnetic fluorescent nanoparticle imaging probe further comprising one or more probes selected from the group consisting of T1 magnetic resonance imaging diagnostic probes, optical diagnostic probes, CT diagnostic probes and radioisotopes. These probes can be used by binding to magnetic fluorescent nanoparticle imaging probes or by encapsulating in a coating layer, depending on the functional groups and hydrophobic / hydrophilic characteristics of the probes used.

For example, the T1 magnetic resonance imaging probe includes a Gd compound, a Mn compound, and the like, the optical diagnostic probe includes a quantum dot, the CT diagnostic probe includes an I (iodine) compound, gold nanoparticles, Isotopes include In, Tc, F, and the like.

Magnetic fluorescent nanoparticle imaging probes of the invention can be introduced into cells by endocytosis or transfection.

In one embodiment, the magnetic fluorescent nanoparticle imaging probe may be further coupled to an intracellular delivery ligand. Intracellular delivery ligands refer to materials that facilitate the magnetic fluorescent nanoparticle imaging probe of the present invention to easily cross cell membranes or additional intracellular organ membranes. For example, a protein transduction domain, a transmembrane domain, or a cell penetrating peptide may be used as the intracellular delivery ligand. These intracellular delivery ligands and methods for introducing them into imaging probes are well known in the art.

In addition, the fluorescent nanoparticle imaging probe may be further bound to a cell or tissue targeting ligand. The cell or tissue targeting ligand may serve to guide the fluorescent nanoparticle imaging probe of the present invention to be introduced into the tissue or cell in which the desired target nucleic acid molecule is present. As the cell or tissue targeting ligand, antibodies, aptamers, peptides, and the like may be used. Specific markers according to cell or tissue types or targeting ligands capable of binding thereto, and the targeting ligands may be introduced into the imaging probe. Methods are well known in the art.

The present invention also forms a coating layer comprising a fluorophore and a biocompatible material on the magnetic nanoparticles; On the coating layer, a molecular beacon comprising a nucleic acid probe including a region capable of hybridizing with a target molecule and an oligomer including a region capable of hybridizing with the nucleic acid probe and having a fluorescence control agent bound thereto is hybridized. It provides a method of producing a magnetic fluorescent nanoparticle imaging probe for the detection of a target molecule comprising a.

The binding of the molecular beacon and the coating layer may be made through a covalent bond between a functional group present on the coating layer of the magnetic nanoparticles and a functional group introduced on the nucleic acid probe of the molecular beacon.

The coating layer of the magnetic nanoparticles has a functional group capable of binding to a functional group introduced on the nucleic acid probe. Such functional groups may be inherently present on the coating layer or the coating layer may be appropriately modified to have a functional group to which the nucleic acid probe may be bound. The type of functional groups present on the coating layer may vary depending on which one is selected. That is, the functional group present on the coating layer and the functional group introduced on the nucleic acid probe may be selected from known functional group binding examples so as to form a bond with each other. Representative examples of such 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) R-OCH ₂ C (OH) CH ₂ -R ' RH-NH ₂ R '-(epoxy) R-NHCH ₂ C (OH) CH ₂ -R ' R-SH R '-(epoxy) 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 ' R-NH ₂ R'-SH R-NHCO (CH ₂ ) ₂ SS-R ' I or II: functional group present on the coating layer or functional group introduced on the nucleic acid probe
III: binding example according to reaction of I and II

In one embodiment, the functional groups present on the coating layer are -COOH, -CHO, -NH ₂ , -SH, -CONH ₂ , -PO ₃ H, -PO ₄ H, -SO ₃ H, -SO ₄ H, And -OH, -sulfonate, -nitrate, -phosphonate, -succinimidyl group, -maleimide group, and -alkylli.

In one embodiment, the functional group introduced to the nucleic acid probe is -NH ₂ , -COOH, -COH, -NCO, -NCS, -COCH ₂ , -SH, -CON ₃ and -CH ₂ C (NH ^{2 +} ) OCH It may be selected from the group consisting of _three .

In one embodiment, the functional group present on the coating layer is -COOH, the functional group introduced to the nucleic acid probe may be -NH ₂ . That is, by binding the nucleic acid probe having -NH ₂ at the 5 'end to the coating layer having -COOH on the surface, one end of the nucleic acid probe can be connected to the magnetic fluorescent nanoparticles.

The binding between the functional group present on the coating layer and the functional group introduced into the nucleic acid probe may be performed using a known crosslinking agent. Such crosslinkers include, but are not limited to, N-ethyl-N'dimethylaminopropyl carbodiimide, 1,4-diisothiocyanatobenzene, 1,4-Diisothiocyanatobenzene, 1,4-Phenylene diisocyanate, 1,6-Diisocyanatohexane, 4- (4-maleimidophenyl) butyric acid normal-hydroxysuccinate Imide ester (4- (4-Maleimidophenyl) butyric acid N-hydroxysuccinimide ester), phosgene solution, 4- (maleimido) phenyl isocyanate, 1,6-hexanediamine 1,6-Hexanediamine, p-Nitrophenyl chloroformate, N-Hydroxysuccinimide, 1,3-dicyclohexylcarbodiimide (1,3-Dicyclohexylcarbodiimide ), 1,1'-carbonyldiimidazole, 3-maleimidobenzoic acid normal-hydroxysuccinate 3-Maleimidobenzoic acid N-hydroxysuccinimide ester, Ethylenediamine, Bis (4-nitrophenyl) carbonate, Succinyl chloride, N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (N- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide Hydrochloride), N, N'-disuccinimidyl carbonate (N, N ' Group consisting of -Disuccinimidyl carbonate, N-succinimidyl 3- (2-pyridyldithio) propionate, and succinic anhydride Can be selected from. Such binding reaction may be carried out in a suitable solvent known in the art.

In addition, the present invention provides a composition for detecting a target molecule comprising the magnetic fluorescent nanoparticle imaging probe. As described above, the magnetic fluorescent nanoparticle imaging probe of the present invention can be used to detect and image intracellular target molecules in vivo or in vitro. The composition for detecting a target molecule according to the present invention may include a carrier and a vehicle commonly used in the medical field. Particularly glyceride mixtures of ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (e.g. human serum albumin), buffer substances (e.g. various phosphates, glycine, sorbic acid, potassium sorbate, saturated vegetable fatty acids ), Water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, hydrogen carbonate, sodium chloride and zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose based substrates, polyethylene glycols, sodium carboxy Methylcellulose, polyarylate, wax, polyethylene glycol or wool, and the like. The compositions of the present invention may also further comprise lubricants, wetting agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components.

In one embodiment, the composition for detecting a target molecule according to the present invention may be prepared as an aqueous solution for parenteral administration, preferably Hank's solution, Ringer's solution or physically buffered saline. Buffer solutions such as can be used. Aqueous injection suspensions can be added with a substrate that can increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

Another preferred embodiment of the composition for detecting target molecules of the present invention may be in the form of sterile injectable preparations of sterile injectable aqueous or oily suspensions. Such suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents (eg Tween 80) and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions (eg solutions in 1,3-butanediol) in nontoxic parenterally acceptable diluents or solvents. Vehicles and solvents that may be used include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, nonvolatile oils are conventionally employed as a solvent or suspending medium. For this purpose any non-irritating non-volatile oil can be used including synthetic mono or diglycerides.

The present invention also administers the target molecule detection composition to a living body or a sample;

The present invention provides a method of obtaining an image of a living body or a sample, comprising detecting a signal emitted by a magnetic fluorescent nanoparticle imaging probe from the living body or a sample to obtain an image.

The term "sample" as used above refers to tissue or cells isolated from a subject to be diagnosed. In addition, the step of injecting the target molecule detection composition into a living body or a sample may be administered through a route commonly used in the medical field, parenteral administration is preferred, for example, intravenous, intraperitoneal, intramuscular, subcutaneous Or via a local route.

In the method, it is preferable to use a magnetic resonance imaging apparatus (MRI) and optical imaging to detect a signal emitted by the magnetic magnetic fluorescent nanoparticle imaging probe.

A magnetic resonance imaging apparatus puts a living body in a strong magnetic field and irradiates radio waves of a specific frequency to absorb energy into atomic nuclei such as hydrogen in biological tissues to make the energy high, and then stops propagating the atomic nuclei energy such as hydrogen. It is a device that processes the energy by converting this energy into a signal and processing it with a computer. The magnetic resonance imaging apparatus may be a T2 spin-spin relaxation magnetic resonance imaging apparatus. Meanwhile, a confocal microscope, a fluorescence microscope, or an optical optics device may be used for optical imaging.

The magnetic fluorescence nanoparticle imaging probe according to the present invention has a mode in which on-off of the fluorescence signal is regulated in accordance with the presence of the target molecule in the cell, thereby identifying specific target biomarkers or evaluating intracellular genes, cell development and It can be usefully used for disease diagnosis / treatment evaluation. In particular, the magnetic fluorescent nanoparticle imaging probe according to the present invention based on magnetic nanoparticles can provide magnetic resonance images as well as optical images according to detection of target molecules in vitro and in vivo. In addition, the magnetic fluorescent nanoparticle imaging probe according to the present invention enables non-invasive and repeated analysis in a living subject.

1 is a diagram schematically illustrating a molecular beacon of the present invention in which a nucleic acid probe and an oligomer to which the fluorescent control material is bound are hybridized.
2 is a diagram schematically illustrating a state in which a nucleic acid probe and a target molecule are hybridized.
FIG. 3 illustrates a process in which the nucleic acid probe of the magnetic fluorescent nanoparticle imaging probe conjugated to the molecular beacon of FIG. 1 is separated from the oligomer to which the fluorescent control material is bound and hybridized with the target molecule in the presence of the target molecule. One drawing.
4A is a photograph of MF particles (left) and MF particles (right) into which miR124a beacons are introduced, and FIG. 4B shows electrophoresis results of MF particles (lane 1) and MF particles (lane 2) into which miR124a beacons are introduced. 4C is a graph showing the quenching of MF particles in accordance with the concentration of miR124a beacons, and FIG. 4D is a graph showing the increase in fluorescence intensity of MF particles incorporating miR124a beacons depending on the concentration of miR124a. .
Figure 5 is a photograph showing the results of immunofluorescence staining according to the neural differentiation of P19 cells.
FIG. 6A shows qRT-PCR results showing that endogenous mature miR124a expression gradually increases after induction of neural differentiation of P19 cells, and FIG. 6B shows changes in endogenous miR124a expression following introduction of MF-miR124a beacons into P19 cells. Figure 6c is a graph showing the results observed through the change in fluorescence intensity, Figure 6c is a confocal laser scanning micrograph showing the change in fluorescence exhibited by the MF-miR124a beacon during neuronal differentiation of P19 cells.
Figure 7 shows the result of reducing the T2 Weighted MR signal in proportion to the concentration after the P19 cells into the MF-miR124a beacon of various concentrations.
Figure 8 shows the miR-124a bio-optical imaging results according to differentiation of P19 cells using MF-miR124a beacons in vivo.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the technical field to which the present invention pertains. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

[ Example ]

All data presented through the following examples were expressed as mean ± standard error, it was calculated using the Student's t-test. * And ** in the following figures indicate that they were evaluated with statistical significance of P <0.05 and P <0.005, respectively.

Example  1: magnetic fluorescence ( MF ) Nanoparticles Conjugated miR124a Reactive molecules Beacon  Produce

(One) miR124a Reactive molecules Beacon  Produce

Specific MF-miR124a beacons capable of detecting miR124a as target molecules were constructed by amine-carboxyl reaction.

First, a nucleic acid probe comprising a region capable of hybridizing with miR124a was constructed. To this end, several random linker sequences (TTC GCT GT) were linked to the 5 'end of the complementary nucleotide of the mature miR124a, and an amino group was bound to the 5' end.

An oligomer hybridized with the nucleic acid probe and having a black hole quencher 1 (BHQ1; Bionics, Inc., Korea) as a fluorescent control material was prepared. The full miR124a beacon is:

5'NH ₂ -TTC GCT GTt ggc att cac cgc gtg cct taa-3

3'BHQ1-GCG ACA ACC GT-5

(2) fluorescent material ( fluorophore Magnetic fluorescence having a biocompatible coating layer comprising MF ) Nanoparticles (MNP @ SiO2 ( RITC ) - PEG Of COOH Manufacturing

Nanoparticles were purchased from Biterials (Korea) to prepare MNP @ SiO2 (RITC) -PEG / COOH as described in TJ Yoon, et al., Small 2006, 2, 209. In summary, the cobalt ferrite magnetic cores were incubated with polyvinylpyrrolidone (PVP; Sigma, St. Louis, MO, USA) and then PVP-stabilized particles were obtained by centrifugation at 4000 rpm for 10 minutes. Trimethoxysilane modified with RITC (rhodamine B isothiocyanate, excitation / release = 555/578 nm) was injected into PVP-stabilized cobalt ferrite particles. Polymerization was induced by the addition of ammonia solvent to produce silica-coated magnetic nanoparticles incorporating RITC (4 × 10 ³ per nanoparticle) (size: approximately 50 nm, hydrodynamic diameter: 58.1 nm). . A carboxyl moiety (4.4 × 10 ⁴ per nanoparticle) and polyethylene glycol (PEG) (1 × 10 ⁴ , 500 g mol ^-1 per nanoparticle) were introduced to the surface of MNP @ SiO2 (RITC) to introduce MNP @ SiO2 (RITC). ) -PEG / COOH was formed.

(3) magnetic fluorescence ( MF ) With nanoparticles miR124a Reactive molecules Beacon Conjugation

Covalent conjugation of carboxyl MF nanoparticles with miR124a beacons (concentration: 20 mM) was performed by adding N-ethyl-N'-dimethylaminopropylcarbodiimidi (EDC) to them and incubating at room temperature for 1 hour. This was done by improving the coupling efficiency between carboxyl groups [MK So, et al., Nat . Protoc . 2006, 1, 1160. The reaction ratio of MF particles: miR124a beacon: EDC was 1: 3: 200. The reaction was centrifuged at 15000 rpm for 10 minutes to remove unconjugated free miR124a beacons and EDC, then washed several times with Tris buffer solution (10 mM Tris-HCl, pH 7.4). After brief sonication, the final conjugated product was mixed using borate buffer solution (10 mM sodium borate, pH 7.4; Sigma, St. Louis, MO, USA). The spherical MF particles were homogeneously dispersed in the aqueous solution, and the hydrodynamic diameter measured using TEM was about 58.1 nm. 4A shows TEM images of MF particles (left) and MF particles (right) into which miR124a beacons were introduced. In miR124a beacons miR124a beacon oligos are stained with 2% uranyl acetate and appear in dark frames as indicated by the black arrows.

On the other hand, in order to verify the conjugation pattern between the MF particles and the miR124a, the MF particles and the miR124a beacon-introduced MF particles were loaded on 0.5% agarose gel and subjected to gel electrophoresis. As a result, as can be seen in Figure 4b, it can be confirmed that the size of the MF particles (lane 1) and the MF particles (lane 2) into which the miR124a beacon is introduced.

To investigate the optimal concentration at which the miR124a beacons were quenched, the quenching efficiency was tested while incubating for 1 hour at room temperature with the concentration of MF particles fixed (100 pmol) and the concentration of miR124a beacons increasing. As a result, as can be seen in Figure 4c, the fluorescent signal of the MF particles was quenched depending on the concentration of miR124a beacons.

Mature miR124a consisting of 22 nucleotides from the PicTar database ( http://pictar.bio.nyu.edu ) was synthesized and hybridized to the miR124a binding region of the MF-miR124a beacon, and in the MF-miR124a beacon It was tested whether the fluorescence signal of could brighten in the presence of mature miR124a. As a result of testing the fluorescence intensity of MF-miR124a beacon in the microtube at different concentrations of mature miR124a (100, 300, 500 pmol), as shown in FIG. 4D, the fluorescence intensity increased depending on the concentration of miR124a. However, when treated with a mutant or no value compound, the fluorescent signal remained quenched continuously. These results suggested the specificity of the MF-miR124a beacons for miR124a.

Example  2: during neurogenesis miR124a  By manifestation MF - miR124a Beacon  Confirmation of fluorescence activity enhancement

(One) P19  Cell culture

P19 cells were selected to observe differentiation of neurons by neuron-specific miR124a expression during neurogenesis using MF-miR124a beacons. P19 cells, the mouse embryonic teratocarcinoma cell line used in the experiment, were 7.5% bovine calf serum (Gibco) and 2.5% FBS (Cellgro, Herndon, VA, USA), penicillin in a 5% CO2-wetting chamber. (10 UmL_1; Invitrogen, Grand Island, NY, USA), and streptomycin (10 mgmL_1; Invitrogen) were grown in a-modified minimum essential medium (a-MEM) (Gibco, Grand Island, NY, USA).

(2) P19  Induction of neuronal differentiation of cells and Immunofluorescence  Differentiation confirmation by dyeing

Monolayer culture method for inducing neuronal differentiation of P19 cells [J. Pachernik, et al., Physiol . Res. 2005, 54 , 115]. P19 cells on gelatin-coated plates were treated with DMEM / F12 (Gibco, Grand Island) containing 1% insulin, transferrin, selenium (ITS; Gibco), penicillin (10 UmL ⁻¹ ), and streptomycin (10 mgmL ⁻¹ ). , NY, USA) media and treated with 5 × 10 ⁻⁷ M all-trans-RA (Sigma, St. Louis, MO, USA). P19 differentiation medium was replaced every 2 days with fresh RA-containing medium. Six days after inducing neural differentiation using the tomographic differentiation method, the pattern of neurites was observed.

Neuronal differentiation of P19 cells was verified using specific neuronal markers such as MAP2 (microtubule-associated protein-2) identified by immunofluorescence staining. Undifferentiated P19 cells were also identified using the oct4 antibody, a stem cell marker. For immunofluorescence staining, P19 cells were first fixed for 4 minutes with 4% formaldehyde or overnight. They were then washed three times for 10 minutes each with PBS and gently shaken during incubation. The blocking and permeation process was carried out simultaneously by adding 20% normal goat serum reaction mixture and 0.2% Triton X-100 to the cells for 60 minutes. Anti-Oct-4 antibody (Chemicon, Millipore, Watford, UK) or anti-MAP2 antibody (Santa Cruz Biotechnology, Heidelberg, Germany) with Oct-4 or MAP2 protein diluted 1: 1000 and detected at 4 ° C. Incubate overnight. After washing three times for each 10 minutes, Alexa Fluor secondary antibody conjugate was added and the mixture was incubated for 90 minutes. P19 cells were transferred to coverslips and mounted with an aqueous mounting solution containing DAPI (Vector Laboratories, Inc., CA, USA). As a result, as shown in Figure 5, the stem cell biomaker significantly reduced the expression of Oct4 gene on the 6th day after retinoic acid (RA) treatment of P19 cells, whereas the expression of MAP2 gene, a neuronal differentiation marker, was increased. It was confirmed that P19 cells were induced to differentiate into neurons by RA.

(3) maturity miR124a To detect qRT - PCR  analysis

To assess mature miR124a expression patterns during neuronal differentiation of P19 cells, quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed using small RNA extracts before and after differentiation of RA-treated P19 cells into neurons. Was performed.

First, whole small RNA extracted with nuclease-free water was isolated from P19 cells at every differentiation time point using mirVana miRNA separation kit (Ambion, Austin, TX, USA). To investigate the copy number of mature miR124a during neuronal differentiation, qRT-PCR was performed using mirVana qRT-PCR primer set (Ambion) and mirVana qRT-PCR miRNA detection kit (Ambion). Mature miR124a levels were verified by PCR amplification using iCycer (Bio-Rad) with SYBR Premix Ex Taq (2 ×) (Takara, Japan) (3 min at 95 ° C., 15 sec at 95 ° C., 30 at 60 ° C.). 40 amplification cycles per second). U6 snRNA primer set (Ambion, Austin, TX, USA) was used as an intrinsic control.

As a result, as can be seen in Figure 6a, the endogenous mature miR124a expression was confirmed to increase gradually after the induction of neuronal differentiation of P19 cells.

(4) MF - miR124a Beacon P19  Endogenous following introduction into cells miR124a  Expression change investigation

MF-miR124a beacons were introduced into undifferentiated and differentiated P19 cells to investigate for endogenous miR124a expression during differentiation of neurons.

P19 cells were dispensed into 6-well plates pre-coated with gelatin and covered with sterile cover slips. Cells were washed twice with phosphate-buffered saline (PBS). MF-miR124a beacons were transfected into undifferentiated and differentiated P19 cells and incubated at 37.8 ° C. for 4 hours. The size of the MF particles has been found to be suitable for passive cellular introduction by endocytosis [JS KIM, et al., J. vet . Sci . 2006, 7, 321. For comparison, MF particles without the introduction of miR124a beacons were also introduced into each cell group. For in vitro fluorescence analysis, cells transfected with MF-miR124a beacons were rinsed with PBS and harvested by trypsin treatment. The collected cells were transferred to dark 96-well microplates and their fluorescence intensities were measured with a Varioskan Flash microplate reader (Thermo Fisher Scientific, Vantaa, Finland).

As a result, as can be seen in Figure 6b, mature miR124a with progressively increased expression during neuronal differentiation attaches to the miR124a binding region of the MF-miR124a beacon, detaching the quencher oligo from the backbone of the beacon, and simultaneously fluorescence It was confirmed that the increase gradually. After 3 and 6 days of neuronal differentiation, the fluorescence activity was increased by about 42.3% and 84.1%, respectively, compared to undifferentiated P19 cells. In addition, pretreatment of anti-miR124a (miR124a inhibitor, 100 pmol) to P19 cells 6 days after neuronal differentiation showed that the quenching state of the MF-miR124a beacon was maintained.

(5) internalized MF  Particle Confocal  Microscopic observation

Confocal microscopy of MF-miR124a beacons was performed during induction of P19 cells into neurons to obtain in-vitro fluorescence images of intracellular expression of miR124a during neurogenesis. First, P19 cells were fixed using 3.7% formaldehyde solution (Sigma, St. Louis, MO, USA) and then rinsed three times with PBS for 10 minutes. After adding the mounting medium (Vector Laboratories, Inc., CA, USA) containing 4 ', 6-diamidino-2-phenylindoledihydrochloride (DAPI) solution, cover slips from the 6-well plate onto the glass slides. Transferred and observed under confocal microscope. As a result, similar to the quantitative fluorescence results shown in FIG. 6B, the confocal laser scanning microscopy results shown in FIG. 6C showed surprising fluorescence changes of the MF-miR124a beacon during neuronal differentiation of P19 cells. In undifferentiated P19 cells, fluorescence signal of MF-miR124a was hardly detected in the cell because little mature miR124a was expressed. In contrast, three and six days after induction of differentiation into neurons, significantly strong fluorescence brightness was clearly observed in P19 cells due to the high expression of mature miR124a. In addition, the fluorescence signal of MF-miR124a observed 6 days after neuronal differentiation returned to its original quenched state after treatment with anti-miR124a. These results demonstrate that miR124a beacon conjugated to fluorescent nanoparticles can successfully provide intracellular miRNA images with very high specificity.

Example  3: MF - miR124a Beacons  In vivo of Utilized Neural Differentiation monitoring

(One) MF -124a Beacon Intracellular  Magnetic property investigation

To monitor in vivo tracking of cells containing MF-miR124a beacons, the magnetic properties of MF particles conjugated with intracellular miR124a beacons were evaluated by moving beacons to P19 cells. It was observed that the magnetic signal intensity gradually decreased with increasing concentration of MF-miR124a. Figure 7 shows the result of reducing the T2 Weighted MR signal in proportion to the concentration after the P19 cells into the MF-miR124a beacon of various concentrations. As can be seen in Figure 7, the specific relaxivity was 53.39mM- ¹ s ^{- 1} . In addition to in vivo cell tracing of P19 cells by magnetic resonance imaging (MRI), plasmid constructs comprising the luciferase gene induced by the cytomegalovirus (CMV) promoter can be used for in vivo normalization of fluorescence intensity. Transfected with miR124a beacon into P19 cells as internal control. As a result, the presence of P19 cells introduced from the living body was confirmed by MRI, and the same number of cells were transferred to the left and right thighs by the luciferase reporter gene and confirmed that they survived.

(2) P19  Cells loaded PLLA Scaffold  Living transplant

On the other hand, poly-L-lactic acid (PLLA) scaffolds were used to help stably settle P19 cells in vivo. The PLLA scaffold was provided by S. J. Lee (Ewha Womans University College of Pharmacy). Fibrous PLLA scaffolds were prepared using wet-spin from spun PLLA microfibers. Prior to dispensing P19 cells, PLLA scaffolds were sterilized overnight with 70% isopropyl alcohol and rinsed three times with PBS. PLLA scaffolds were incubated with P19 culture medium for 12 hours prior to cell division. The use of poly-L-lactic acid (PLLA) scaffolds gained better fluorescence activity in cells into which MF-miR124a beacons were introduced and gastric-positive background magnetic resonance inside the scaffolds observed with white areas in T2 MR scanning (MR) signal was reduced [D. W. Hwang, et al., J. Nucl. Med. Mol. Imaging 2008, 35, 1887.].

For in vivo neuronal induction, 5 × 10 ⁶ P19 cells containing miR124a beacons were harvested from PBS, resuspended with 10 × 10 ⁻⁷ M RA and then introduced into the PLLA scaffold. The P19-scaffold complex was implanted into the subcutaneous tissue of both thighs and right shoulders of nude mice (male BALB / c, 7 weeks old).

(3) acquisition of in vivo fluorescent images

Three days after implantation of the P19-scaffold complex, for fluorescence imaging, the animals were placed in ketamine (Ketalar, Panpharma, Fougeres, France) / xylazine (Rompun, Bayer Pharma, Puteaux, France) solution (2: 1, 50 mL). Anesthetized with. Mice were scanned using a Maestro in vivo imaging system (CRi Inc., Woburn, MA, USA). Optical imaging was performed using a green filter set (503-555 nm, band-pass filter; 580 nm, long-pass filter). Images captured at constant exposure time (1 second) were acquired using a camera. For autofluorescence correction using Maestro software 2.6, pure autofluorescence signal spectra were obtained from fluorescence signals obtained from MF-miR124a beacon-introduced nude mice and pseudo-color fluorescence images of autofluorescence signals from normal mice. Created by not mixing the spectra. Rhodamine fluorescence spectra in mice with MF-miR124a were obtained by subtracting the autofluorescence spectra from the mixed spectra for mice introduced with MF-miR124a beacons. FIG. 8A shows that the CMV-Firefly luciferase optical image vector transfected into the transplanted P19 cells was transferred to the same cell through the same optical signal in the right (neural differentiation) thigh and the left thigh (pre-neural differentiation). Shows. In addition, FIG. 8B shows a result of confirming that the bio-optical image signal of the MF-miR124a beacon is increased from the left as miR-124a is increased by neuronal differentiation in the right thigh 2 days after P19 cell transplantation. As shown, after 3 days of implantation, the fluorescence image in the right thigh of the mouse treated with RA for neuronal differentiation resulted in high expression of intracellular miR124a in P19 cells as compared to the fluorescence image of the left thigh untreated with RA. Increased fluorescence images could be detected. Quantitative region of interest (ROI) analysis revealed approximately 82.8% fluorescence recovery in the RA-treated group compared to the fluorescence activity of the untreated group.

(4) acquisition of in vivo bioluminescence images

On the other hand, for bioluminescence images, mice were seded with O ₂ gas containing 2% isoflurane at a flow rate of 1 L min ⁻¹ through a nose cone and 3 mg / 0.1 mL / mouse luciferase Substrate luciferin was injected intraperitoneally to obtain bioluminescent images. Animals were transferred to an IVIS-200 imaging system equipped with a CCD camera (Xenogen, CA, USA). Bioluminescence images were acquired by collecting and integrating light for 15 minutes.

As a result, bioluminescent activity similar to fluorescence activity was found at both thigh sites, indicating that the improvement in fluorescence signal shown in FIGS. 8A and 8B was not due to the difference in cell number.

(5) Invivo T2 -emphasis MR  Image Acquisition

T2-weighted MR images were obtained from both thighs and right shoulders of mice to confirm whether the fluorescence activity was from cells containing MFmiR124a beacons. T2-weighted MR images at various concentrations of MFmiR124a beacons were obtained in an animal coil box using a 1.5-T MR imager (GE Medical Systems, Milwaukee, WI, USA). During the MRI experiments, animals were anesthetized with a ketamine / xylazine (2: 1, 50 mL) solution, and their breathing and body temperature were monitored using a rectal thermistor. In vivo MRI of nude mice was also performed with a 1.5-T whole-body MR scanner (GE Medical Systems, Milwaukee, WI, USA) to obtain T2 axial images. At this time, the sequence parameters for repetitive time (TR) and echo time (TE) were 1400 and 55.8 ms, respectively. As can be seen in FIG. 8C, dark signals were clearly visualized in both thighs implanted with PLLA scaffolds containing P19 cells into which MF-miR124a beacons were introduced as compared to the pre-transplantation region. In the right shoulder where only the empty scaffold was implanted, no magnetic signal was observed before and after transplantation. The implanted PLLA scaffold was carefully removed to confirm that the in vivo fluorescent image was obtained by the MF-miR124a beacon. As can be seen in FIG. 8D, the fluorescence activity detected in the PLLA scaffold into which P19 cells were differentiated was brighter compared to the fluorescence activity in the scaffold with only P19 cells prior to differentiation. Quantitative ROI analysis of scaffolds separated from the shoulder and both thighs showed approximately 91.2% fluorescence recovery in the RA treated group compared to the fluorescence activity of the untreated group of the left thigh. In addition, a similar fluorescence pattern from the isolated scaffold group was also found in the frozen sections of the P19-scaffold complex, showing a stronger fluorescence signal in the RA-treated complex compared to the RA-untreated complex.

As discussed above, molecular beacons that can tune fluorescent signals may be used as highly specific molecular imaging biosensors for identification of specific target biomarkers or for evaluation of intracellular genes. Nanoparticle-based molecular beacons can also be used to observe neuronal differentiation patterns by detection of endogenous target molecules as well as neuronal cell-related target molecules in living subjects for intracellular molecular targeting. The technique provides noninvasive information in living subjects and allows for repeated analysis. In addition, miRNA specific molecular beacon imaging systems for the detection of neuronal differentiation patterns have the potential to be an effective method for the diagnosis of neurological disorders at the genetic level.

Claims (22)

Magnetic nanoparticles having a coating layer comprising a fluorophore and a biocompatible material; And
Molecular beacons are coupled to the coating layer and comprises a nucleic acid probe comprising a region capable of hybridizing with the target molecule and a molecular beacon comprising a region capable of hybridizing with the nucleic acid probe and an oligomer to which a fluorescent control material is bound. Containing
Magnetic fluorescent nanoparticle imaging probe for detection of target molecules.
The method of claim 1,
The magnetic nanoparticles are magnetic fluorescent nanoparticle imaging probes of a metal material, magnetic material, or magnetic alloy.
The method of claim 2,
The metal material is at least one magnetic fluorescent nanoparticle imaging probe selected from the group consisting of Pt, Pd, Ag, Cu and Au.
The method of claim 2,
The magnetic material is Co, Mn, Fe, Ni, Gd, Mo, MM ' ₂ O ₄ , And M _p O _q (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 = 5", respectively). One or more magnetic fluorescent nanoparticle imaging probes selected from the group consisting of:.
The method of claim 2,
The magnetic alloy is at least one magnetic fluorescent nanoparticle imaging probe selected from the group consisting of CoCu, CoPt, FePt, CoSm, NiFe and NiFeCo.
The method of claim 1,
The fluorescent substance is rhodamine and its derivatives, fluorescein and its derivatives, coumarin and its derivatives, acridine and its derivatives, pyrene and its derivatives, erythrocin and its derivatives, eosin and its derivatives, and 4-acetamido- Magnetic fluorescent nanoparticle imaging probe selected from the group consisting of 4'-isothiocyanatostilbene-2,2'disulfonic acid.
The method of claim 1,
The biocompatible material is a magnetic fluorescent nanoparticle imaging probe comprising a material selected from the group consisting of silica, polymers, lipids and dextran.
The method of claim 1,
A magnetic fluorescent nanoparticle imaging probe having a larger region where a target molecule may hybridize with the nucleic acid probe than a region where the target molecule may hybridize with an oligomer to which a fluorescent control material is bound.
The method of claim 1,
Magnetic fluorescent nanoparticle imaging probe further comprises a linker at one end of the nucleic acid probe.
The method of claim 1,
The fluorescence 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. Magnetic fluorescent nanoparticle imaging probe that is selected.
The method of claim 1,
Magnetic fluorescent nanoparticle imaging probe wherein the target molecule is a microRNA, mRNA or protein.
The method of claim 1,
And one or more probes selected from the group consisting of a T1 magnetic resonance imaging diagnostic probe, an optical diagnostic probe, a CT diagnostic probe, and a radioisotope.
The method of claim 1,
The magnetic fluorescent nanoparticle imaging probe is further coupled to the intracellular delivery ligand to the magnetic fluorescent nanoparticle imaging probe.
The method of claim 11,
The intracellular delivery ligand is a protein transduction domain, a transmembrane domain, or a cell penetrating peptide.
The method of claim 1,
Cell or tissue targeting ligand is further coupled to the fluorescent nanoparticle imaging probe.
Forming a coating layer including a fluorophore and a biocompatible material on the magnetic nanoparticles;
On the coating layer, a molecular beacon comprising a nucleic acid probe including a region capable of hybridizing with a target molecule and an oligomer including a region capable of hybridizing with the nucleic acid probe and having a fluorescence control agent bound thereto is hybridized. Involving
A method of making a magnetic fluorescent nanoparticle imaging probe for the detection of a target molecule.
The method of claim 16,
The binding of the molecular beacon and the coating layer is a method of manufacturing a magnetic fluorescent nanoparticle imaging probe is made through a covalent bond between a functional group present on the coating layer and a functional group introduced on the nucleic acid probe of the molecular beacon.
The method of claim 17,
The functional groups present on the coating layer are -COOH, -CHO, -NH ₂ , -SH, -CONH ₂ , -PO ₃ H, -PO ₄ H, -SO ₃ H, -SO ₄ H, -OH, -sulfo Nate, -nitrate, -phosphonate, -succinimidyl group, -maleimide group, and -alkylli manufacturing method of the magnetic fluorescent nanoparticle imaging probe.
The method of claim 17,
The functional group introduced on the nucleic acid probe consists of -NH ₂ , -COOH, -COH, -NCO, -NCS, -COCH ₂ , -SH, -CON ₃ and -CH ₂ C (NH ^{2 +} ) OCH ₃ A method of making a magnetic fluorescent nanoparticle imaging probe.
The method of claim 17,
The functional group present on the coating layer is -COOH, and the functional group introduced on the nucleic acid probe is -NH _{2 The} method of manufacturing a magnetic fluorescent nanoparticle imaging probe.
The composition for detecting a target molecule comprising the magnetic fluorescent nanoparticle imaging probe of any one of claims 1 to 15.
Administering the composition of claim 21 to a living body or a sample;
Method of obtaining an image of a living body or a sample comprising the step of sensing the signal emitted by the fluorescent nanoparticle imaging probe from the living body or sample.

Images