KR20100030264A - Fluorescent magnetic nanohybrids and method for preparing the same - Google Patents

Abstract

PURPOSE: Fluorescence magnetic nanocomposites and a method for manufacturing the same are provided to show excellent magnetic properties and high fluorescence by coating magnetic nanoparticles with fluorescent amphipathic compounds. CONSTITUTION: Magnetic nanocomposites include magnetic nanoparticles and amphiphilic compounds in which one or more hydrophobic part and one or more hydrophilic part and fluorescent material are included. The amphipathic compounds surround the magnetic nanoparticles. The magnetic nanoparticles are metals, magnetic materials, or magnetic alloys. The magnetic materials are selected from Co, Mn, Fe, Ni, Gd, Mo, MM'2O4, and MxOy(M and M' indicate Co, Fe, Ni, Mn, Zn, Gd, or Cr, respectively).

Description

Fluorescent magnetic nanocomposites and method for preparing the same {Fluorescent magnetic nanohybrids and method for preparing the same}

The present invention relates to a fluorescent magnetic nanocomposite and a method for manufacturing the same, and more particularly, to include a fluorescent amphiphilic compound layer surrounding magnetic nanoparticles, wherein the amphipathic compound layer is bound to a tissue specific binding component. As a nanocomposite, a fluorescent magnetic nanocomposite having a target orientation, which is stable in aqueous solution, has excellent magnetic properties and high fluorescence, and can be used as a multimodal contrast agent composition by enabling targeting according to the tissue-specific binding component. It relates to a manufacturing method.

Nanotechnology is a technology that controls and controls materials at the atomic and molecular level, and is suitable for the creation of new materials or new devices, and its applications are diverse in electronics, materials, communication, machinery, medicine, agriculture, energy, and environment.

Currently, nanotechnology is developing variously and can be divided into three fields. First, the synthesis of new materials and materials of extremely small size with nanomaterials. Second, a technology for manufacturing a device exhibiting a certain function by combining or arranging nano-sized materials and nano-sized materials. Third, a technology that applies nanotechnology, called nano-bio, to biotechnology.

In particular, in the field of nano-bio, magnetic nanoparticles have a wide range of applications such as separation of biomaterials, magnetic resonance imaging diagnostic probes, biosensors including giant magnetoresistance sensors, microfluidic sensors, drug / gene delivery, and magnetic pyrotherapy. It is used throughout.

Specifically, the magnetic nanoparticles may be used as diagnostic probes (contrast agents) of molecular magnetic resonance imaging. Magnetic nanoparticles have been widely used in resonance imaging until now, because they reduce the spin-spin relaxation time of hydrogen atoms in water molecules around the nanoparticles and amplify the magnetic resonance imaging signals.

In addition, the magnetic nanoparticles may act as a probe material of a giant magnetic resistance (GMR) sensor. When the magnetic nanoparticles detect and bind the biomolecules patterned on the surface of the giant magnetoresistive biosensor, the current signal of the giant magnetoresistive sensor is changed by the magnetic particles, and the biomolecules can be selectively detected by using the magnetic particles (US 6,452,763 B1; US 6,940,277 B2; US 6,944,939 B2; US 2003/0133232 A1).

Magnetic nanoparticles can also be applied to the separation of biomolecules. For example, when a cell expressing a specific biomarker and various other cells are mixed, the magnetic nanoparticles selectively bind to the specific biomarker, and when the external magnetic field is applied, only the desired cell is separated in the direction of the magnetic field. (Whitehead et al . US patent 4,554,088, US 5,665,582, US 5,508,164, US 2005/0215687 A1). In addition to the separation of cells, it can be applied to the separation of various biomolecules such as proteins, antigens, peptides, DNA, RNA and viruses. Magnetic nanoparticles can also be applied to magnetic microfluidic sensors to separate and detect biomolecules. By creating tiny channels on the chip and flowing magnetic nanoparticles into them, they can be detected and separated in microfluidic systems.

On the other hand, magnetic nanoparticles can also be used for biological treatment through drug or gene delivery. By chemically binding or adsorption to magnetic nanoparticles, drugs or genes are loaded and moved to a desired location using an external magnetic field, thereby releasing drugs and genes at specific sites, thereby bringing about selective therapeutic effects (US). 6,855,749).

Another example of the application of magnetic nanoparticles to biotherapy is high temperature treatment using magnetic spin energy (US 6,530,944 B2, US 5,411,730). Magnetic nanoparticles emit heat through spin flipping when the AC current flows from outside. At this time, if the temperature around the nanoparticles is more than 40 ℃ cells are killed by high heat can selectively kill the diseased cells.

Magnetic nanoparticles must have excellent magnetic properties, be stably transported and dispersed in vivo, that is, in an aqueous environment, and can be easily combined with bioactive materials. To this end, various technologies have been developed to date.

US Pat. No. 6,274,121 relates to paramagnetic nanoparticles comprising a metal, such as iron oxide, comprising a binding site capable of coupling with a tissue specific binding agent, a diagnostic or pharmaceutical active material on the surface of the nanoparticle. The present invention discloses nanoparticles having an inorganic substance attached thereto. US Pat. No. 6,638,494 relates to paramagnetic nanoparticles comprising a metal, such as iron oxide, and discloses a method of attaching specific carboxylic acids to the surface of the nanoparticles to prevent nanoparticles from agglomerating and sedimenting in gravity or magnetic fields. have. As the specific carboxylic acid, aliphatic dicarboxylic acids such as maleic acid, tartaric acid and glucaric acid or aliphatic polydicarboxylic acids such as citric acid, cyclohexane and tricarboxylic acid were used. US Patent Publication No. 2004/58457 relates to functional nanoparticles enclosed by a monolayer, wherein a bifunctional peptide is attached to the monolayer, and various biopolymers including DNA and RNA are bound to the peptide. Can be. GB 223,127 relates to a method for the preparation of a magnetic nanoparticle component comprising a magnetic nanoparticle formation step in a protein template and describes a method for encapsulating magnetic nanoparticles in apoferritin. US 2003 / 190,471 describes a method for forming nanoparticles with manganese zinc oxide in a bi-micellear vesicle, and describes nanoparticles exhibiting improved properties through heat treatment of the formed magnetic nanoparticles. . US 2005 / 130,167 discloses the synthesis of water-soluble magnetic nanoparticles surrounded by 16-mercaptohexadecanoic acid and intracellularly using TAT peptides as phase transfer agents to the synthesized magnetic nanoparticles. Intracellular magnetic labeling describes the detection of viruses and mRNA in experimental mice. Korean Patent Application No. 10-1998-0705262 discloses particles comprising superparamagnetic iron oxide core particles with a starch coating and any polyalkylene oxide coating and an MRI contrast agent comprising the same.

However, the water-soluble nanoparticles prepared by the above methods have the following disadvantages. That is, the nanoparticles disclosed in the above-mentioned documents are mainly synthesized in an aqueous solution. In this case, it is difficult to control the size of the nanoparticles, and the synthesized nanoparticles exhibit non-uniform size distribution. In addition, since they are synthesized at low temperatures, the crystallinity of the nanoparticles is low, and non-stoichiometric compounds tend to be formed. Therefore, the nanoparticles prepared by the above methods have a problem in that the colloidal stability is poor in aqueous solution, and they show agglomeration and large non-selective bonds in a biological application.

An object of the present invention is to solve the disadvantages of the prior art, to provide a magnetic nanocomposite and a method for producing a magnetic nanocomposite that is stable in aqueous solution and excellent magnetic properties.

Another object of the present invention to provide a contrast agent composition, or multiple diagnostic probes comprising the magnetic nanocomposite having the excellent physical properties.

In order to achieve the above object, the present invention

Magnetic nanoparticles; And

Amphiphilic compounds having at least one hydrophobic region and at least one hydrophilic region comprising a fluorescent material,

It provides a magnetic nanocomposite (I) characterized in that the amphiphilic compound surrounds the magnetic nanoparticles.

The invention also

Magnetic nanoparticles; And

Amphiphilic compounds having at least one hydrophobic region and at least one hydrophilic region comprising a fluorescent material,

The amphiphilic compound surrounds the magnetic nanoparticles,

The hydrophilic region provides a magnetic nanocomposite (II) characterized in that it is bound to a tissue specific binding component.

The invention also

Magnetic nanoparticles; And

Amphiphilic compounds having at least one hydrophobic region and at least one hydrophilic region comprising a fluorescent material,

The amphiphilic compound surrounds the magnetic nanoparticles,

The hydrophilic region is associated with a tissue specific binding component,

It provides a magnetic nanocomposite (III) characterized in that the pharmaceutically active ingredient is bound or enclosed in the hydrophobic region.

The invention also

Synthesizing magnetic nanoparticles in a solvent;

Hydrophilic compounds; And combining an hydrophobic compound or a hydrophobic fluorescent material to synthesize an amphiphilic compound; And

Adding an amphiphilic compound to the surface of the magnetic nanoparticles to prepare a magnetic nanocomposite

It provides a method for producing a magnetic nanocomposite comprising a.

The invention also

Magnetic nanocomposites (I) of the present invention; And

Provided is a contrast agent composition comprising a pharmaceutically acceptable carrier.

The invention also

Magnetic nanocomposites (II) of the present invention; And

It provides a target-oriented intelligent contrast agent composition comprising a pharmaceutically acceptable carrier.

The invention also

Magnetic nanocomposites (III) of the present invention; And

It provides a contrast diagnostic composition for the simultaneous diagnosis and treatment comprising a pharmaceutically acceptable carrier.

The invention also

Magnetic nanocomposites of the present invention; And

Provided are multiple diagnostic probes, including diagnostic probes.

The magnetic nanocomposite of the present invention is coated with a fluorescent amphiphilic compound, which is stable in aqueous solution, has excellent magnetic properties and high fluorescence, and can be targeted to enable multimodal contrast agent composition, drug carrier, or multimodal diagnosis. Probes and the like.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

The present invention

Magnetic nanoparticles; And

Amphiphilic compounds having at least one hydrophobic region and at least one hydrophilic region comprising a fluorescent material,

It relates to a magnetic nanocomposite, characterized in that the amphiphilic compound surrounds the magnetic nanoparticles.

In the magnetic nanocomposite of the present invention, firstly, a compound having both fluorescence and amphiphilicity can stably disperse water-insoluble nanoparticles in an aqueous medium to maximize bioavailability, and second, tissue-specific binding to a functional group of the amphiphilic compound. Since the components are combined, the nanocomposite may have target orientation, and third, the pharmaceutically active ingredient may be bound or encapsulated in the amphipathic compound.

In addition, the magnetic nanocomposite of the present invention is an emulsion type magnetic nanocomposite in which at least one magnetic nanoparticle is surrounded by a fluorescent amphipathic compound, or a suspension type in which one magnetic nanoparticle is combined with a fluorescent amphiphilic compound. Magnetic nanocomposites may be included.

The magnetic nanocomposite may be prepared through a substitution method described in Korean Patent Publication No. 10-0604976 or an addition method described in Korean Patent Publication No. 10-0819378, but preferably, an additional method. Fluorescent amphiphilic compounds can be prepared by binding to the magnetic nanoparticles.

That is, in the magnetic nanocomposite according to the present invention, an amphiphilic compound is added to the surface of the nanoparticle, and the hydrophobic region of the amphiphilic compound is bonded to the surface of the nanoparticle, and the hydrophilic region of the amphiphilic compound is distributed at the outermost portion of the nanocomposite. I'm doing it. Here, the hydrophobic region of the amphiphilic compound is bonded to the surface of the nanoparticles by physical bonding such as hydrogen bonds, van der Waals forces, and polar attraction forces. Thus, the hydrophobic region not only distributes the nanoparticles in the matrix of the hydrophobic region or binds to the surface of the nanoparticle, but also physically encapsulates the drug in the matrix of the hydrophobic region, or at one end of the hydrophobic region. The drug can be chemically bound to. On the other hand, the hydrophilic region of the amphiphilic compound is distributed in the outermost part of the nanocomposite to stabilize the water-insoluble nanoparticles in the aqueous medium to maximize the bioavailability.

The magnetic nanocomposite is preferably 5 to 200 nm in diameter. More preferably, the emulsion type nanocomposite is 30 to 200 nm, and the suspension type nanocomposite is 10 to 50 nm.

The magnetic nanoparticles are not particularly limited as long as they are magnetic and have a diameter of 1 to 1000 nm, more preferably 2 to 100 nm. Most preferably a metal, magnetic material, or magnetic alloy having the diameter is preferred.

The metal is not particularly limited, but Pt, Pd, Ag, Cu, or Au may be used alone or in combination of two or more.

The magnetic material is not particularly limited, but Co, Mn, Fe, Ni, Gd, Mo, MM ' ₂ O ₄ , Or M _x O _y (M and M 'each independently represent Co, Fe, Ni, Mn, Zn, Gd, or Cr, and x and y satisfy the expressions "0 <x? 3" and "0 <y? 5", respectively). .) May be used alone or in combination of two or more.

The magnetic alloy is not particularly limited, but CoCu, CoPt, FePt, CoSm, NiFe, or NiFeCo may be used alone or in combination of two or more.

In addition, the magnetic nanoparticles may be combined with an organic surface stabilizer to stabilize the binding with the amphiphilic compound. The combination of the magnetic nanoparticles and the organic surface stabilizer is achieved by forming a complex by coordinating the organic surface stabilizer to the precursor of the magnetic nanoparticles.

The organic surface stabilizer refers to an organic functional molecule capable of stabilizing the state and size of the nanoparticles, for example, a surfactant.

The surfactant may be a cationic surfactant including 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, but are not limited thereto. In particular, considering the stabilization and uniform size distribution of the nanoparticles, preference is given to using saturated or unsaturated fatty acids and / or alkylamines.

In addition, the amphiphilic compound may distribute the nanoparticles in the matrix, or may bind to the surface of the nanoparticles, and may physically enclose the pharmaceutically active ingredient or chemically bond to one end of the polymer as necessary.

The amphiphilic compound may be an amphiphilic compound having at least one hydrophobic region and at least one hydrophilic region including a fluorescent material.

The hydrophobic region includes a fluorescent material or a hydrophobic compound to which a fluorescent material is bound. The hydrophobic compound may be used alone or in combination of two or more saturated fatty acids, unsaturated fatty acids or hydrophobic polymers.

The saturated fatty acid is not particularly limited, but butyric acid, caproic acid, caprylic acid, capric acid, lauric acid (dodecyl acid), myristic acid, palmitic acid, stearic acid, eicosanoic acid, docosanoic acid, etc. May be used alone or in combination of two or more.

The unsaturated fatty acid is not particularly limited, but oleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosaptanoic acid, docosahexanoic acid, erric acid, or the like may be used alone or in combination.

The hydrophobic polymer is not particularly limited, but polyphosphazene, polylactide, polylactide-co-glycolide, polycaprolactone, polyanhydride, polymalic acid or derivatives thereof, polyalkylcyanoacrylate, poly Hydroxybutylate, polycarbonate, polyorthoester, hydrophobic polyamino acid, hydrophobic vinyl series polymer, etc. may be used alone or in combination of two or more.

The hydrophobic fluorescent material is not particularly limited, but may be used alone or two or more of pyrene, Texas red, Floresceamine, Neil Blue, Neil Red, or quantum dots.

The hydrophilic region may include polyalkylene glycol (PAG), polyetherimide (PEI), polyvinylpyrrolidone (PVP), hydrophilic polyamino acid (PAA), hydrophilic vinyl polymer, or hydrophilic acrylic polymer alone or two It can be used above.

The invention also

Magnetic nanoparticles; And

Amphiphilic compounds having at least one hydrophobic region and at least one hydrophilic region comprising a fluorescent material,

The amphiphilic compound surrounds the magnetic nanoparticles,

The hydrophilic region relates to a magnetic nanocomposite (II) characterized in that it is bound to a tissue specific binding component.

Magnetic nanocomposite according to the present invention can provide a target orientation to the magnetic nanocomposite by introducing a tissue-specific binding component in the hydrophilic region.

The tissue-specific binding component is not particularly limited, but antigen, antibody, RNA, DNA, hapten, avidin (avidin), streptavidin (neutravidin), protein A, protein G, lectin (lectin), selectin, radioisotope labeling components, substances capable of specifically binding tumor markers, and the like, may be used alone or in combination of two or more.

The nanocomposites of the present invention diagnose and / or treat various diseases associated with tumors, such as gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer and cervical cancer. Can be used.

Such tumor cells express and / or secrete certain substances which are produced little or no in normal cells and are generally termed “tumor markers”. Nanocomposites made by binding a substance capable of specifically binding such a tumor marker to the active ingredient binding region of the water-soluble nanoparticles can be usefully used for tumor diagnosis. Various tumor markers are known in the art, as well as materials capable of specifically binding to them.

In the present invention, tumor markers may be classified into ligands, antigens, receptors, and nucleic acids encoding them according to a mechanism of action.

When the tumor marker is a ligand, a substance capable of specifically binding to the ligand may be introduced as an active ingredient of the nanocomposite according to the present invention, but the receptor may specifically bind to the ligand. Or the antibody will be suitable. Examples of ligands and receptors that can specifically bind to the present invention include synaptotagmin C2 (synaptotagmin C2) and phosphatidylserine, annexin V and phosphatidylserine, integrin and its Receptors, Vascular Endothelial Growth Factor (VEGF) and its receptors, angiopoietin and Tie2 receptors, somatostatin and its receptors, vasointestinal peptides and their receptors. It is not.

When the tumor marker is an antigen, a substance capable of specifically binding to the antigen may be introduced as an active ingredient of the nanocomposite according to the present invention, and an antibody capable of specifically binding to the antigen may be suitable. Examples of antigens and antibodies that specifically bind to the present invention include carcinoembryonic antigens (colon cancer marker antigens), Herceptin (Genentech, USA), and HER2 / neu antigens (HER2 / neu antigens-breast cancer markers). Antigen) and Herceptin, prostate-specific membrane antigen (prostate cancer marker antigen) and rituxan (IDCE / Genentech, USA).

A representative example where the tumor marker is a "receptor" is a folic acid receptor expressed in ovarian cancer cells. A substance capable of specifically binding to the receptor (folic acid in the case of folic acid receptor) may be introduced as an active ingredient of the nanocomposite according to the present invention, and a ligand or an antibody capable of specifically binding to the receptor may be suitable. will be.

As mentioned above, antibodies are particularly preferred active ingredients in the present invention. Antibodies have properties that selectively and stably bind only to specific targets, and -NH _{2 of} lysine, -SH of cysteine, -COOH of aspartic acid and glutamic acid in the Fc region of the antibody are the active component binding region functional groups of the water-soluble nanocomposites. Because it can be useful to combine with.

Such antibodies can be obtained commercially or prepared according to methods known in the art. Generally mammals (eg, mice, rats, goats, rabbits, horses or sheep) are immunized one or more times with an appropriate amount of antigen. After a period of time when the titer reaches an appropriate level, it is recovered from the serum of the mammal. The recovered antibody can be purified using known processes if desired and stored in a frozen buffered solution until use. Details of this method are well known in the art.

On the other hand, "nucleic acid" includes RNA and DNA encoding the above-described ligand, antigen, receptor or a portion thereof. Nucleic acid having a specific base sequence can be detected using a nucleic acid having a base sequence complementary to the base sequence because the nucleic acid has a feature that forms a base pair between complementary sequences as known in the art . A nucleic acid having a nucleotide sequence complementary to the nucleic acid encoding the enzyme, ligand, antigen, receptor can be used as an active ingredient of the nanocomposite according to the present invention.

In addition, the nucleic acid has a functional group such as -NH ₂ , -SH, -COOH at the 5'- and 3'- terminal may be useful for binding to the functional group of the active ingredient binding region.

Such nucleic acids can be synthesized by standard methods known in the art, for example using automated DNA synthesizers (such as those available from BioSearch, Applied Biosystems, etc.). By way of example, phosphorothioate oligonucleotides are described in Stein et. al . Nucl . Acids Res . 1988, vol. 16, p. 3209). Methylphosphonate oligonucleotides can be prepared using controlled free polymer supports (Sarin et. al . Proc . Natl . Acad . Sci . USA 1988, vol. 85, p.7448).

The invention also

Magnetic nanoparticles; And

Amphiphilic compounds having at least one hydrophobic region and at least one hydrophilic region comprising a fluorescent material,

The amphiphilic compound surrounds the magnetic nanoparticles,

The hydrophilic region is associated with a tissue specific binding component,

It relates to a magnetic nanocomposite (III) characterized in that the pharmaceutically active ingredient is bound or enclosed in the hydrophobic region.

That is, the pharmaceutical active ingredient may be physically enclosed in the hydrophobic region of the amphiphilic compound of the magnetic nanocomposite (I) of the present invention or chemically bonded to one end thereof.

The pharmaceutically active ingredient is not particularly limited, but anticancer drugs, antibiotics, hormones, hormonal antagonists, interleukin, interferon, growth factor, tumor necrosis factor, endotoxin, lymphokoxy, urokinase, streptokinase, tissue plasminogen activator, protease inhibitor, Alkylphosphocholine, a component labeled with a radioisotope, a cardiovascular drug, a gastrointestinal drug, or a nervous system drug may be used alone or in combination of two or more thereof.

On the other hand, the pharmaceutically active ingredient present in the hydrophobic region may be physically encapsulated, chemically encapsulated, or a combination of both. During the preparation of the magnetic nanocomposites by the emulsion method and the suspension method, the drug is encapsulated through the physical combination of the hydrophobic active ingredient of the amphiphilic polymer and the anticancer agent. In addition, in the case of an anticancer agent capable of chemically bonding with the binding region of the hydrophobic active component of the amphiphilic polymer constituting the magnetic nanocomposite, an appropriate crosslinking agent may be used to bind the hydrophobic active component binding region of the amphiphilic polymer to the anticancer agent. Enclosure of the drug may be made.

Anticancer agents that can be used in the treatment method according to the present invention include, but are not limited to, epirubicin, docetaxel, gemcitabine, paclitaxel, cisplatin, carboplatin (carboplatin), taxol, procarbazine, cyclophosphamide, diactinomycin, daunorubicin, etoposide, tamoxifen Doxorubicin, mitomycin, bleomycin, plicomycin, transplatinum, vinblastin and methotrexate.

The invention also

Hydrophilic compounds; And combining an hydrophobic compound or a hydrophobic fluorescent material to synthesize an amphiphilic compound; And

Adding an amphiphilic compound to the surface of the magnetic nanoparticles to prepare a magnetic nanocomposite

It relates to a method for producing a magnetic nanocomposite comprising a.

The method for producing the magnetic nanocomposite of the present invention will be described in detail as follows.

Synthesizing the magnetic nanoparticles is a step of reacting the magnetic nanoparticle precursor and the organic surface stabilizer in a solvent.

a) mixing the magnetic nanoparticle precursor with the organic surface stabilizer in the presence of a solvent; And

b) pyrolyzing the magnetic nanoparticle precursor by heating the mixture.

Step a) is a step of mixing the nanoparticle precursor in a solvent containing a surface stabilizer.

Specific types of the magnetic nanoparticles and the organic surface stabilizer are as described above.

The magnetic nanoparticle precursor may use a metal compound in which a metal and -CO, -NO, -C ₅ H ₅ , alkoxide, or other known ligand are combined. For example, iron pentacarbonyl, Fe Metal carbonyl compounds such as (CO) ₅ ), ferrocene, or manganese carbonyl (Mn ₂ (CO) ₁₀ ); Or various organometallic compounds such as metal acetylacetonate-based compounds such as iron acetylacetonate (Fe (acac) ₃ ).

In addition, the magnetic nanoparticle precursor may use a metal salt including a metal ion bonded to a metal and a known anion such as Cl ⁻ , or NO ₃ ⁻ , and the like, for example, iron trichloro (FeCl ₃ ), iron dichlorochloride ( FeCl ₂ ), or iron nitrate (Fe (NO ₃ ) ₃ ) may be used.

In addition, in the synthesis of alloy nanoparticles and composite nanoparticles, it is possible to use a mixture of precursors of two or more metals described above.

In addition, the solvent usable in step a) preferably has a high breaking point close to the thermal decomposition temperature of the complex compound coordinated with the organic surface stabilizer on the nanoparticle surface, for example octyl ether, butyl ether (butyl ether compounds such as ether, hexyl ether, or decyl ether; Heterocyclic compounds such as pyridine or tetrahydrofuran (THF); Aromatic compounds such as toluene, xylene, mesitylene, or benzene; Sulfoxide compounds such as dimethyl sulfoxide (DMSO); Amide compounds such as dimethylformamide (DMF); Alcohols such as octyl alcohol or decanol; Hydrocarbons such as pentane, hexane, heptane, octane, decane, dodecane, tetradecane, or hexadecane; Or water etc. can be used individually or in 2 or more types.

The reaction conditions of step a) are not particularly limited and may be appropriately adjusted according to the kind of precursor and surface stabilizer of the magnetic nanoparticles. The reaction may be formed even at room temperature or below, but is usually preferred to be heated and maintained in the range of 30 to 200 ° C.

Step b) is a step of growing nanoparticles by pyrolyzing the magnetic nanoparticle precursor.

In this case, metal nanoparticles having a uniform size and shape may be formed according to the reaction conditions, and the thermal decomposition temperature may also be appropriately adjusted according to the type of the magnetic nanoparticle precursor and the surface stabilizer. Preferably it is reacted at 50-500 degreeC.

The nanoparticles prepared in step b) can be separated and purified through known means.

Synthesizing the amphiphilic compound

c) combining the hydrophilic compound and the hydrophobic compound to synthesize an amphiphilic compound;

d) providing a binding region of the fluorescent material to the hydrophobic compound portion of the amphiphilic compound using a crosslinking agent; And

e) it is preferable to include the step of binding the fluorescent material binding region and the fluorescent material of the amphiphilic compound.

Alternatively, the step of synthesizing the amphiphilic compound

f) providing a binding region of the hydrophobic phosphor to the hydrophilic compound moiety using a crosslinking agent; And

and g) combining the hydrophobic fluorescent material binding region with a hydrophobic fluorescent material.

Specific types of hydrophilic polymer, hydrophobic polymer, or hydrophobic fluorescent substance used in the above steps are as described above.

In the method of manufacturing the magnetic nanocomposite of the present invention, the manufacturing step of the magnetic nanocomposite is a step of bonding a fluorescent amphiphilic compound to the surface of the nanoparticles prepared in the above step, by an emulsion method and a suspension method Can be divided.

Preparation of the magnetic nanocomposite according to the method by the emulsion

h) dissolving the magnetic nanoparticles in an organic solvent to prepare an oil phase;

i) dissolving the amphiphilic compound to prepare an aqueous phase;

j) mixing the oil phase and the water phase to form an emulsion; And

k) preferably separating the oil phase from the emulsion to produce an emulsion type magnetic nanocomposite.

By going through this step, one or more magnetic nanocomposites can be prepared in which the magnetic nanocomposite is fluorescent by amphipathic compound coating.

The step of preparing a magnetic nanocomposite according to the method by the suspension

l) dispersing the magnetic nanoparticles and the amphiphilic compound in a solvent to prepare a suspension; And

m) preferably separating the solvent from the suspension to prepare a suspension-type magnetic nanocomposite.

By going through the above steps, one magnetic nanoparticle may be combined with an amphiphilic compound to produce a fluorescent magnetic nanocomposite.

In addition, the method may further include physically encapsulating the pharmaceutically active ingredient in the amphipathic compound or chemically binding to one end thereof.

Chemically binding the pharmaceutically active ingredient

n) providing a binding region of the pharmaceutically active ingredient to a portion of the amphiphilic compound using a crosslinking agent; And

o) preferably comprising the step of binding the pharmaceutically active ingredient with the binding region of the pharmaceutically active ingredient.

In addition, in the step of physically encapsulating the pharmaceutically active ingredient, it is preferable to dissolve the pharmaceutically active ingredient and the magnetic nanoparticles together.

In the method of manufacturing a magnetic nanocomposite of the present invention, the step of binding the tissue-specific binding component to the surface of the nanocomposite comprises chemically binding the tissue-specific binding component to the surface of the fluorescent magnetic nanocomposite using a crosslinking agent. Improving target efficiency.

The step of binding the tissue specific binding component to the surface of the nanocomposite

p) providing an active component binding region to the hydrophilic compound moiety using a crosslinker; And

q) it is preferable to include the step of binding a tissue specific binding component to the surface of the nanocomposite comprising the step of binding the active ingredient binding region and the tissue specific binding component.

Specific types of tissue specific binding components available in the above steps are as described above.

Further, in the above steps d), f), n) and p), the crosslinking agent available is not particularly limited, but N, N, N ', N', N ''-pentamethyldiethylenetriamine (N , N, N ', N', N ''-pentamethyldiethylenetriamine (PMDETA), 1,4-diisothiocyanatobenzene, 1,4-phenyline diisocyanate (1,4-Phenylene diisocyanate), 1,6-diisocyanatohexane, 4- (4-maleimidophenyl) butyric acid normal-hydroxysuccinimide ester (4- (4-Maleimidophenyl) butyric acid N -hydroxysuccinimide ester), phosgene solution, 4- (maleimido) phenyl isocyanate, 1,6-hexanediamine, p-nitrophenylchloroformate (p-Nitrophenyl chloroformate), N-Hydroxysuccinimide, 1,3-dicyclohexylcarbodiimide, 1,1′-carbonyldiimidazole (1 , 1′-Carbony ldiimidazole), 3-maleimidobenzoic acid N-hydroxysuccinimide ester, ethylenediamine, bis (4-nitrophenyl) carbonate (Bis (4-nitrophenyl) carbonate), succinyl chloride, N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride, N, N ′ -Disuccinimidyl carbonate (N, N′-Disuccinimidyl carbonate), N-succinimidyl 3- (2-pyridyldithio) propionate (N-Succinimidyl 3- (2-pyridyldithio) propionate) Or succinic anhydride may be used alone or in combination of two or more thereof.

The crosslinker reacts with a portion of the surface of the amphipathic compound and the fluorescent magnetic nanocomposite to react with -COOH, -CHO, -NH ₂ , -SH, -CONH ₂ , -PO ₃ H, -PO ₄ H, -SO ₃ H, _{-SO 4 H, -OH, -NR 4} + X -, - providing the group with the bond area of the same active ingredient-sulfonate, - nitrates,-phosphonate-succinimidyl group, - a maleimide group, or do.

The binding of the active ingredient binding region on the surface of the fluorescent magnetic nanocomposite of step q) and the active ingredient of the tissue-specific binding ingredient may be changed according to the type of each active ingredient and its chemical formula. Shown in

The method for producing the magnetic nanocomposite of the present invention may further comprise the step of separating the magnetic nanocomposite, which is generally produced as a precipitate, through a conventional method, for example, by centrifugation or filtration.

The manufacturing method according to the present invention as described above can be carried out according to the contents described in Korean Patent Publication No. 10-0819378, which is cited as part of the present specification.

The invention also relates to the magnetic nanocomposites (I), magnetic nanocomposites (II) or magnetic nanocomposites (III) of the present invention; And

It relates to a contrast agent composition comprising a pharmaceutically acceptable carrier.

The magnetic nanocomposite of the present invention includes magnetic nanoparticles and fluorescence at the same time by chemically binding or physically encapsulating a pharmaceutically active ingredient, and a tissue-specific binding component is bound to the surface of the nanocomposite so that targeting is possible. It can be used as a contrast agent capable of imaging the target site through a magnetic resonance and an optical imaging device.

Such pharmaceutically acceptable carriers include carriers and vehicles commonly used in the medical arts, and specifically include ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer materials (eg, Various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, hydrogen carbonate, sodium chloride and zinc salts) Silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substrates, polyethylene glycols, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene glycols or wool, and the like.

In addition, the contrast agent composition of the present invention may further include a lubricant, a humectant, an emulsifier, a suspending agent, or a preservative in addition to the above components.

In one embodiment, the contrast agent composition according to the present invention may be prepared as an aqueous solution for parenteral administration, preferably a buffered solution such as Hanks' solution, Ringer's solution or physically buffered saline. 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 contrast composition 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.

In addition, the sterile injectable preparation may be a sterile injectable solution or suspension (eg, a solution in 1,3-butanediol) in a nontoxic parenterally acceptable diluent or solvent. 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 contrast agent composition of the present invention can be used to obtain an image by detecting a signal emitted from the fluorescent magnetic nanocomposite by administering to tissues or cells isolated from a diagnosis target.

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

A magnetic resonance imaging apparatus places 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. Since magnetism or propagation is not obstructed by bone, clear three-dimensional tomograms can be obtained at longitudinal, transverse, and arbitrary angles around solid bones or tumors of the brain or bone marrow. In particular, the magnetic resonance imaging apparatus is preferably a T2 spin-spin relaxation magnetic resonance imaging apparatus.

 The fluorescent magnetic nanocomposites of the present invention can be used for nano probes, drugs, or delivery vehicles for separation, diagnosis, or treatment of biological molecules.

As a representative example of a biological diagnosis using a magnetic nanocomposite, molecular magnetic resonance imaging or a magnetic relaxation sensor may be mentioned. Magnetic nanocomposites show a larger T2 contrast effect as their size increases, which can be used as a sensor to detect biomolecules. That is, when a specific biomolecule induces agglomeration of the magnetic nanocomposite, the T2 magnetic resonance imaging effect is increased. This difference is used to detect biomolecules.

In addition, the fluorescent magnetic nanocomposite of the present invention can be used as a diagnostic material for a giant magnetic resistance (GMR) sensor. Magnetic nanocomposites have better magnetic properties, colloidal stability in aqueous solutions, lower than conventional micrometer ( ^10-6 m) size beads (US 6,452,763 B1; US 6,940,277 B2; US 6,944,939 B2; US 2003/0133232 A1) Since non-selective bonds can be represented, there is a possibility to greatly increase the detection limit of the existing large magnetoresistive biosensor.

In addition, the fluorescent magnetic nanocomposites of the present invention can be used for separation and detection using magnetic microfluidic sensors, delivery of drugs or genes, and magnetic high temperature therapy.

The present invention also provides a magnetic nanocomposite of the present invention; And

A multiple diagnostic probe comprising a diagnostic probe is provided.

The diagnostic probe may use a T1 magnetic resonance imaging diagnostic probe, an optical diagnostic probe, a CT diagnostic probe, or a radioisotope.

For example, when the T1 magnetic resonance imaging diagnostic probe is coupled to a water-soluble magnetic nanocomposite, the multiple diagnostic probe may simultaneously perform T2 magnetic resonance imaging and T1 magnetic resonance imaging, and when the optical diagnostic probe is combined, the magnetic resonance imaging and the optical Imaging can be performed simultaneously. Combined with CT diagnostic probes, MRI and CT diagnosis can be performed simultaneously. In addition, when combined with radioisotopes, magnetic resonance imaging, PET, and SPECT can be simultaneously diagnosed.

The T1 magnetic resonance imaging diagnostic probe may include a Gd compound or a Mn compound, and the optical diagnostic probe may be an organic fluorescent dye (dye), a quantum dot, or a dye labeled inorganic support (eg SiO ₂ , Al _2). O ₃ )), CT diagnostic probes include I (iodine) compounds, or gold nanoparticles, and radioisotopes include In, Tc, F, and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are only intended to illustrate the invention and do not limit the invention in any sense.

Preparation Example 1 Preparation of High Sensitivity Magnetic Nanoparticles Using Saturated Fatty Acids

7 nm of magnetite (MnFe ₂ O ₄ ) was heated with 0.6 moles of dodecyl acid and dodecyl amine in a benzyl ether solvent at 215 ° C. for heating iron triacetylacetonate and manganese triacetylacetonate (Aldrich) for 2 hours, and 315 Synthesis was carried out by thermal decomposition at 1 ° C. for thermal decomposition.

The 12 nm magnetite nanoparticles were composed of dodecyl acid (0.2 mole), dodecyl amine (0.1 mole), the 7 nm magnetite nanoparticles (10 mg / mL), and a benzyl ether solution containing iron triacetylacetonate and manganese triacetylacetonate. It prepared by heating at 30 degreeC, 2 hours at 215 degreeC, and 1 hour at 315 degreeC.

A transmission electron micrograph of the prepared magnetic nanoparticles is shown in FIG. 2A.

Preparation Example 2 Polymerization of Fluorescent Amphiphilic Compounds in which a Hydrophobic Fluorescent Compound was Bonded to the Hydrophobic Part of the Amphiphilic Compound

In the first step, 3.44 g (0.0175 mol) of 2-hydroxyethyl-2'-bromopropionate (2-hydroxyethyl 2 ') in 90 mL of distilled toluene to synthesize HO-PCL-Br. -bromopropionate) was azeotropically dried and 30 g of epsilon -caprolactone was added to the hydroxyethyl-2'-bromopropionate solution. 0.14 g (0.35 mmol) of stationary octoate (Sn (Oct) ₂ ) was added as an initiator at 120 ° C. to initiate the polymerization, and the reaction mixture was stirred for 24 hours in a nitrogen atmosphere. The reaction was precipitated from toluene with 1 L of hexane (n-hexane) to polymerize HO-PCL-Br in 93% yield.

Second, 3 g (11.8 mmol) of HO-PCL-Br and 0.49 g (7.1 mmol) of Cooper (I) bromide (Cu (I) Br) were added to the heat-dried round flask as a polymer initiator. 10 mL of toluene and tert-butyl methacrylate (tBMA) were degassed with nitrogen (N ₂ ) gas and added to the flask, followed by 1.48 g (7.1 mmol) of N, N, N ', N ', N''-pentamethyldiethylenetriamine (N, N, N', N ', N''-pentamethyldiethylenetriamine, PMDETA) was added as crosslinking agent. The polymerization was carried out for 15 hours while maintaining the reaction temperature at 85 ° C. The polymerized copolymer was passed through a silica column, and then precipitated from tetrahydrofuran (THF) into hexane (n-hexane). 1,3-dicyclohexylcarbodiimide (DCC) was used to bind pyrene (pyrene) to the terminal of the PCL block by binding pyrene-PCL-b-polytertbutyl meth Pyrene-labeled PCL-b-poly (tert-butyl methacrylate, Py-PCL-b-PtBMA) was synthesized.

As a final step, blocking of Py-PCL-b-PtBMA by adding 3 g of Py-PCL-b-PtBMA polymer and 10 mL of trifluoroacetic acid (TFA) to 10 mL of methylene chloride (MC) Activated carboxyl structure. Dialysis was performed on a water phase using a dialysis membrane (molecular weight cut off (MWCO): 1000) for 24 hours, followed by freeze drying under reduced pressure.

It was confirmed by using gel permeation chromatography (GPC) that the relative molecular weight of the prepared Py-PCL-b-PMAA was 4,200 Da. The Py-PCL-b-PMAA prepared by the nuclear magnetic resonance (NMR) showed a peak of PLC near 3.97 ppm and a pyrenyl peak near 7.9-8.3 ppm, and the peak of the tert butyl ester disappeared at 1.49 ppm. Confirmed.

Preparation Example 3 Polymerization of Fluorescent Amphiphilic Compounds with a Hydrophobic Fluorescent Compound Bonded to a Part of the Hydrophilic Polymer

5 g (0.001 mol) of hydrophilic monomethoxy polyethylene glycol (Mw: 5,000 Da) to synthesize Pyrene-PEG, 0.288 g (0.001 mol) of 1-pyrenicbutyric acid (Mw: 288.34 Da) as a hydrophobic fluorescent substance, 1.86 g (0.009 mol) of N, N'-dicyclohexylcarbodiimide and 1.01 g (0.009 mol) of 4-dimethylaminopyridine are dissolved in 100 mL of methylene chloride and 1.25 mL of triethylene 0.009 mol) was added and reacted in a nitrogen atmosphere at room temperature for 48 hours. Dicyclohexyl urea in the reaction was removed with a 200 nm pore size cellulose acetate filter. After removal of the organic solvent using a rotary evaporator, dichloromethane was added and precipitated in ether to purify the synthesized Py-PEG.

Ester bonds of Py-PEG prepared by FT-IR were found at 1,737 cm ⁻¹ , at 3,500 cm ⁻¹ and 1,695 cm ⁻¹ It was confirmed that the hydroxyl group of monomethoxy polyethylene glycol and the carboxy group of 1-pyrenic butyric acid disappeared (FIG. 3).

Nuclear magnetic resonance (NMR) confirmed the peak of monomethoxy polyethylene glycol near 3.65 ppm and the peak of 1-pyrenicbutyric acid near 7.82 and 8.12 ppm (FIG. 4).

It was confirmed that the molecular weight of Py-PEG was 5,288 Da using gel permeation chromatography (GPC) (FIG. 5).

In particular, the critical micelle concentration of Py-PEG, the solubility and the degree of fluorescence in the aqueous phase and the organic solvent were measured with a fluorescence spectrometer to confirm that it was a fluorescent amphiphilic compound (FIGS. 6 and 7).

Example 1 Preparation of Emulsion-Type Fluorescent Magnetic Nanocomposites

5 mg of the magnetic nanoparticles prepared in Preparation Example 1 and 50 mg of the fluorescent amphiphilic compound prepared in Preparation Example 2 were dissolved in 10 mL of dichloromethane in an oil phase. This oil phase was mixed with 20 mL of aqueous phase and then the mixture was saturated for 10 minutes by 450 W of ultrasound. The emulsion was stirred for 12 hours to evaporate the oil phase and prepare a fluorescent magnetic nanocomposite free of impurities through centrifugation (RPM: 20,000). The prepared particles were redispersed in 2 mL of PBS (pH 7.4) solution, and the size and zeta potential were confirmed using dynamic laser light scattering. In addition, it was confirmed using a transmission electron microscope, which is shown in Figure 2b respectively. In addition, the synthesis of the fluorescent amphiphilic compound through FT-IR is shown in FIG. 9A, and the crystallinity integrity of the magnetic nanoparticles in the fluorescent magnetic nanocomposite was confirmed by X-ray diffraction analysis (XRD). Shown. A vibration sample magnetometer (VSM) was used to confirm superparamagnetism and this is illustrated in FIG. 10A.

Example 2 Preparation of Emulsion-Type Fluorescent Magnetic Nanocomposites

50 mg of the magnetic nanoparticles prepared in Preparation Example 1 dissolved in 10 mL of hexane (n-hexane), which is an oil phase, and 200 mg of the fluorescent amphipathic compound prepared in Preparation Example 3 dissolved in a 20 mL aqueous phase were mixed. The mixture was saturated for 10 minutes by 450 W of ultrasound. The emulsion was stirred for 12 hours to evaporate the oil phase, and a fluorescent magnetic nanocomposite was prepared from which impurities were removed by centrifugation (RPM: 18,000) for 30 minutes. The prepared particles were redispersed in 10 mL of deionized water (DW) (pH 7.4) solution, and the size and zeta potential were confirmed using dynamic laser light scattering. This was confirmed using a transmission electron microscope, which is shown in FIG. 2C, respectively. In addition, the loading ratio and crystalline integrity of the magnetic nanoparticles in the fluorescent magnetic nanocomposite were confirmed by a thermogravimetric analyzer (TGA) and an X-ray diffraction (XRD). Shown. A vibration sample magnetometer (VSM) was used to confirm the superparamagnetism and this is illustrated in FIG. 10B. It was confirmed that the fluorescent magnetic nanoparticles maintain the fluorescence and ensure stability not only in the ultrapure solution, but also in aqueous solutions of various pH and sodium chloride concentration, and are shown in FIGS. 11 and 12.

Example 3 Preparation of Fluorescent Magnetic Nanocomposites Having Target Orientation

1 mg of fluorescent magnetic nanocomposites prepared in Example 1, 1 mg of Cetuximab, and 1 mg of human IgG were dispersed in 0.4 mL of PBS solution, respectively. N-hydroxysuccinimide (2.0 mM) and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (2.0 mM) were added to the solution, and after 4 hours, the mixture was combined using a Sephacryl S-300 column and an ultra centrifugal filter. Cetuximab was not isolated. The target orientation of the fluorescent magnetic nanocomposites was confirmed through cells overexpressing the Cetuximab receptor and control cells.

Experimental Example 1 Analysis of Magnetic Properties (Magnetic History Curve and Saturation Magnetization) of Fluorescent Magnetic Nanocomposites

In order to evaluate the magnetic sensitivity of the fluorescent magnetic nanocomposite under the magnetic field, the magnetic hysteresis loop and the saturation of magnetization of the fluorescent magnetic nanocomposites prepared in Examples 1 and 2 were subjected to the vibration sample magnetometer. (VSM, Model 7300, Lakershore) was used to evaluate. 10 is a diagram illustrating the measured hysteresis curve.

As shown in FIG. 10, the hysteresis loop of the magnetic nanoparticles observed at 300K was a shape showing superparamagnetic behavior in the absence of magnetic hysteresis. In addition, the fluorescent magnetic nanoparticles prepared in Example 1 had a saturation magnetization value of 52.5 emu / g at 1.5T (FIG. 10A), and the fluorescent magnetic nanoparticles prepared in Example 2 had 41.9 emu at 0.8T. It has a saturation magnetization of / g. Due to the presence of the fluorescent amphiphilic compound layer, the saturation magnetization value of the fluorescent magnetic nanoparticles was lower than that of the magnetic nanoparticles (FIG. 10B).

Experimental Example 2 MR Contrast Analysis of Fluorescent Magnetic Nanocomposites

In order to confirm the applicability of the fluorescent magnetic nanocomposites prepared in Examples 1 and 2 as MRI contrast agents, the MR contrast effect of the magnetic nanocomposites was examined by measuring r ₂ (T2 relaxivity coefficients).

Specifically, MR imaging tests were performed using a 1.5 T clinical MRI instrument (Intera, Philips Medical System) with a Micro-47 surface coil. The r ₂ (T2 relaxivity coefficients with unit of mM ⁻¹ s ⁻¹ ) values of the magnetic nanocomposites were measured using a Carr-Purcell-Meiboom-Gill (CPMG) sequence at room temperature (TR = 10s, 32 echoes). with 12 ms even echo space, number of acquisitions = 1, point resolution of 156 × 156 μm, section thickness of 0.6 mm).

As shown in FIG. 13, as the concentration of the fluorescent magnetic nanocomposites increased at 1.5T, r ₂ clearly increased. Although the saturation magnetization value is small, the high r ₂ is due to the clustering effect of the magnetic nanoparticles. The value was obtained. In addition, as the concentration was increased, a significantly darker MR contrast was provided. Accordingly, it was confirmed that the fluorescent magnetic particles as MR imaging probes had sufficient magnetic properties for molecular imaging.

Experimental Example 3 Simultaneous Confirmation of Fluorescence and Magnetic Properties of Fluorescent Magnetic Nanocomposites

The fluorescent magnetic nanocomposites prepared in Examples 1 and 2 are composed of magnetic nanoparticles at the center of the fluorescent amphiphilic compound layer to which fluorescent materials (pyrene, Pyrene) are bonded. Fluorescent magnetic nanocomposites were sensitively aligned by an external magnetic field (Nd-B-Fe magnet, 0.35T), and it was confirmed in FIGS. 14 and 15 that fluorescence of pyrene was shown. FIG. 14A shows the suspended particle state without the external magnetic field acting, and FIGS. 14B and 14C show the particle states aligned under the external magnetic field action. In addition, FIG. 15 shows the aligned particle state 8 seconds after the external magnetic field is applied in the state where the external magnetic field is not applied (t: 0 sec).

Experimental Example 4 Target Orientation of Fluorescent Magnetic Nanocomposites Having Target Orientation

The target directivity of the fluorescent magnetic nanocomposite conjugated with the tissue specific binding component prepared in Example 3 was tested.

The fluorescent magnetic nanocomposites were treated with 1 × 10 ⁶ cells (A431) and control cells (MCF-7) overexpressed with Cetuximab receptors, and then cultured for 30 minutes and washed three times with PBS. After washing with PBS, 400 μl of PBS was added, and after treatment with a target anti-human IgG (FATC) -bound fluorescent anti-human nanocomposite (FITC) -bound fluorescent magnetic nanocomposite to quantitatively analyze target orientation Incubate for 45 min at 4 ° C. in the dark. Flow cytometry was then performed with FACScan (Beckton-Dickinson, Sunnyvale, CA, USA) with CellQuest software.

As shown in FIG. 16, the fluorescent magnetic nanocomposite exhibited high FITC fluorescence intensity in the cells overexpressing the Cetuximab receptor, thereby confirming that it was targeted to specific cells.

This is a microscope for cells treated with fluorescent magnetic nanocomposites, MR. It was further confirmed through optical imaging.

As shown in FIG. 17, the fluorescence of the fluorescent magnetic nanocomposite pyrene fluorescence and the FITC of the secondary tissue specific binding component were confirmed through microscopic imaging.

In addition, as shown in FIG. 18, target-directed cells (A431) had dark MR imaging and high r ₂ compared to control cells (MCF-7). It was observed to indicate a value.

In addition, as shown in FIG. 19, the FITC of the secondary tissue specific binding component was higher in fluorescence than the MCF-7 under A431.

Through the above results, it was confirmed that the fluorescent magnetic nanocomposite is effectively targeted.

Test Example 5 Confirmation of Cell Affinity of the Fluorescent Magnetic Nanocomposites

The cell affinity test of the magnetic nanocomposites prepared in Example 2 was carried out. Six-well cells (NIH 3T6.7) were dispensed 1 × 10 ⁶ per well, incubated for 12 hours and washed three times with PBS. After washing with PBS, the fluorescent magnetic nanocomposite at a concentration of 2.7 µg / mL was treated and further incubated for 24 hours. After washing, PBS, trypsin-fluorescent magnetic nanoparticles with a complex treatment the cells with non-treated cells, each 1 × 10 ⁶ cells of the microscope, MR. It was further confirmed through UV imaging. Fluorescent magnetic nanocomposite pyrene fluorescence was confirmed by microscopic imaging and plotted in FIG. 20.

As shown in Figure 20, cells treated with the fluorescent magnetic nanocomposite were bright UV imaging and dark MR imaging, high r ₂ compared to the cells untreated The value was obtained. Through this, it was confirmed that the fluorescence and magnetism of the fluorescent magnetic nanocomposite were maintained in the cell affinity and intracellular.

1 is a schematic diagram showing a method for producing a fluorescent magnetic nanocomposite of the present invention.

2 is a transmission electron microscope (TEM) photograph of the magnetic nanoparticles or fluorescent magnetic nanocomposites of the present invention.

Figure 3 shows the FT-IR results of the fluorescent amphipathic compound of the present invention.

Figure 4 shows the nuclear magnetic resonance results of the fluorescent amphipathic compound of the present invention.

Figure 5 shows the gel permeation chromatography results of the fluorescent amphipathic compound of the present invention.

Figure 6 shows the critical micelle concentration of the fluorescent amphipathic compound of the present invention.

7 shows solubility and fluorescence intensity in the aqueous phase and the organic solvent phase of the fluorescent amphiphilic compound of the present invention.

8 is a photograph showing the stability of the water-soluble phase of the fluorescent magnetic nanocomposite of the present invention.

9 is a graph showing the FT-IR 'results (a), X-ray diffraction patterns (b, d) and thermal analysis results (c) of the fluorescent magnetic nanocomposite according to the embodiment of the present invention.

Figure 10 shows the magnetic properties of the fluorescent magnetic nanocomposite of the present invention.

Figure 11 shows the fluorescence intensity in the ultrapure water of the fluorescent magnetic nanocomposite of the present invention.

Figure 12 shows the fluorescence intensity in the water phase according to the pH and the concentration of sodium chloride of the fluorescent magnetic nanocomposite of the present invention.

Figure 13 shows the MR image test results of the fluorescent magnetic nanocomposite of the present invention.

14 and 15 are fluorescence micrographs showing the mobility in the magnetic field of the fluorescent magnetic nanocomposite of the present invention.

Figure 16 shows the FITC results in cells treated with the fluorescent magnetic nanocomposites of the present invention.

FIG. 17 is a fluorescence micrograph (a to d) of the fluorescent magnetic nanocomposite having the target orientation of the present invention, and e is a graph showing the fluorescence intensity according to the wavelength of the fluorescent magnetic nanocomposite and the FITC.

FIG. 18 is a graph showing the target orientation of the fluorescent magnetic nanocomposite having target orientation according to the present invention through magnetic resonance imaging and relaxation (R2) for each cell.

19 is a graph showing the target orientation of the fluorescent magnetic nanocomposite having the target orientation of the present invention through fluorescence image and fluorescence intensity for each cell.

FIG. 20 shows changes in optical microscope, magnetic resonance imaging, and relaxation (R ₂ ) in cells treated with the fluorescent magnetic nanocomposites of the present invention.

Claims (25)

Magnetic nanoparticles; And Amphiphilic compounds having at least one hydrophobic region and at least one hydrophilic region comprising a fluorescent material, Magnetic nanocomposite, characterized in that the amphiphilic compound surrounds the magnetic nanoparticles. The method of claim 1, Magnetic nanoparticles are magnetic nanocomposites, characterized in that the metal, magnetic material or magnetic alloy. The method of claim 2, Magnetic materials include Co, Mn, Fe, Ni, Gd, Mo, MM ' ₂ O ₄ , And M _x O _y (M and M 'each independently represent Co, Fe, Ni, Mn, Zn, Gd, or Cr, and x and y satisfy the expressions "0 <x? 3" and "0 <y? 5", respectively). Magnetic nanocomposite, characterized in that at least one selected from the group consisting of. The method of claim 1, Magnetic nanoparticles are magnetic nanocomposites, characterized in that combined with an organic surface stabilizer. The method of claim 1, The hydrophobic region is a fluorescent material; Or magnetic nanocomposite, characterized in that the hydrophobic compound is bonded to the fluorescent material. The method of claim 5, Hydrophobic compound is a magnetic nanocomposite, characterized in that the saturated fatty acid, unsaturated fatty acid or hydrophobic polymer. The method of claim 6, Hydrophobic polymers include polyphosphazenes, polylactides, polylactide-co-glycolides, polycaprolactones, polyanhydrides, polymalic acids or derivatives thereof, polyalkylcyanoacrylates, polyhydroxybutylates, poly Magnetic nanocomposite, characterized in that at least one member selected from the group consisting of carbonate, polyorthoester, hydrophobic polyamino acid and hydrophobic vinyl series polymer. The method of claim 5, The fluorescent substance is a magnetic nanocomposite, characterized in that at least one selected from the group consisting of pyrene, Texas red, Floresceinamine, Neil Blue, Neil Red and quantum dots. The method of claim 1, Hydrophilic region is one selected from the group consisting of polyalkylene glycol (PAG), polyetherimide (PEI), polyvinylpyrrolidone (PVP), hydrophilic poly amino acid (PAA), hydrophilic vinyl polymer and hydrophilic acrylic polymer The magnetic nanocomposite characterized by the above. Magnetic nanoparticles; And Amphiphilic compounds having at least one hydrophobic region and at least one hydrophilic region comprising a fluorescent material, The amphiphilic compound surrounds the magnetic nanoparticles, Magnetic nanocomposite, characterized in that the hydrophilic region is bound to a tissue specific binding component. The method of claim 10, Tissue specific binding components include antigens, antibodies, RNA, DNA, hapten, avidin, streptavidin, neutravidin, protein A, protein G, lectin, selectin ( selectin), a radioisotope labeling component, and a magnetic nanocomposite, characterized in that at least one selected from the group consisting of substances capable of specifically binding to tumor markers. Magnetic nanoparticles; And Amphiphilic compounds having at least one hydrophobic region and at least one hydrophilic region comprising a fluorescent material, The amphiphilic compound surrounds the magnetic nanoparticles, The hydrophilic region is associated with a tissue specific binding component, A magnetic nanocomposite characterized in that a pharmaceutically active ingredient is bound or enclosed in the hydrophobic region. The method of claim 12, Pharmaceutically active ingredients include anticancer agents, antibiotics, hormones, antagonists, interleukins, interferons, growth factors, tumor necrosis factors, endotoxins, lymphotoxins, urokinase, streptokinase, tissue plasminogen activators, protease inhibitors, alkylphosphocholines, radiation Magnetic nanocomposite, characterized in that at least one member selected from the group consisting of isotopically labeled components, cardiovascular drugs, gastrointestinal drugs and nervous system drugs. Synthesizing magnetic nanoparticles in a solvent; Hydrophilic compounds; And combining an hydrophobic compound or a hydrophobic fluorescent material to synthesize an amphiphilic compound; And Adding an amphiphilic compound to the surface of the magnetic nanoparticles to prepare a magnetic nanocomposite Method of producing a magnetic nanocomposite comprising a. The method of claim 14, wherein synthesizing the magnetic nanoparticles a) mixing the magnetic nanoparticle precursor with the organic surface stabilizer in the presence of a solvent; And b) pyrolyzing a magnetic nanoparticle precursor by heating the mixture. The method of claim 14, wherein synthesizing the amphiphilic compound c) combining the hydrophilic compound and the hydrophobic compound to synthesize an amphiphilic compound; d) providing a binding region of the fluorescent material to the hydrophobic compound portion of the amphiphilic compound using a crosslinking agent; And e) combining the fluorescent material binding region and the fluorescent material of the amphiphilic compound, or f) providing a binding region of the hydrophobic phosphor to the hydrophilic compound moiety using a crosslinking agent; And g) combining the hydrophobic fluorescent material binding region with a hydrophobic fluorescent material Method of producing a magnetic nanocomposite comprising a. The method of claim 14, wherein preparing the magnetic nanocomposite h) dissolving the magnetic nanoparticles in an organic solvent to prepare an oil phase; i) dissolving the amphiphilic compound to prepare an aqueous phase; j) mixing the oil phase and the water phase to form an emulsion; And k) separating the oil phase from the emulsion to prepare an emulsion type magnetic nanocomposite, l) dispersing the magnetic nanoparticles and the amphiphilic compound in a solvent to prepare a suspension; And m) A method of producing a magnetic nanocomposite, comprising the steps of preparing a suspension-type magnetic nanocomposite by separating a solvent from the suspension. The method of claim 14, The step of preparing the magnetic nanocomposite further comprises the step of binding or encapsulating the pharmaceutical active ingredient in the amphipathic compound. The method of claim 18, wherein the step of binding the pharmaceutically active ingredient n) providing a binding region of the pharmaceutically active ingredient to a portion of the amphiphilic compound using a crosslinking agent; And o) method of producing a magnetic nanocomposite comprising the step of combining the pharmaceutically active ingredient with the binding region of the pharmaceutically active ingredient. The method of claim 18, wherein the encapsulating the pharmaceutically active ingredient comprises dissolving the pharmaceutically active ingredient and the magnetic nanoparticle together. The method of claim 14, p) providing an active component binding region to the hydrophilic compound moiety using a crosslinker; And q) The method of manufacturing a magnetic nanocomposite further comprises the step of binding a tissue-specific binding component to the surface of the nanocomposite comprising the step of binding the active component binding region and a tissue-specific binding component. Magnetic nanocomposite according to claim 1; And A contrast agent composition comprising a pharmaceutically acceptable carrier. Magnetic nanocomposite according to claim 10; And Target-oriented intelligent contrast agent composition comprising a pharmaceutically acceptable carrier. Magnetic nanocomposite according to claim 12; And Concurrent diagnostic and therapeutic contrast composition comprising a pharmaceutically acceptable carrier. Magnetic nanocomposite according to claim 1; And Multiple diagnostic probes, including diagnostic probes.

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