KR100924786B1 - A magnetic metal nano composite for the diagnosis and treatment - Google Patents

Abstract

The present invention relates to a multifunctional magnetic metal nanocomposite, specifically, dielectric particles containing a magnetic body; And a metal-containing coating layer formed on the surface of the dielectric particles, wherein the metal relates to a magnetic metal nanocomposite that generates heat when irradiated with electromagnetic waves. The nanocomposites are useful for the diagnosis and treatment of cellular diseases.

Nanoparticles, Magnetic Materials, Dielectrics, Silica, Gold

Description

Magnetic metal nanocomposite for diagnosis and treatment {A MAGNETIC METAL NANO COMPOSITE FOR THE DIAGNOSIS AND TREATMENT}

1 is a conceptual diagram of a magnetic metal nanocomposite according to an embodiment of the present invention.

2 is a graph showing transmission electron micrographs and magnetic properties of magnetic nanoparticles using saturated fatty acids according to an embodiment of the present invention.

3 is a graph illustrating transmission electron micrographs and magnetic properties of magnetic nanoparticles using unsaturated fatty acids according to another embodiment of the present invention.

4A and 4E are transmission electron micrographs showing the manufacturing process of the magnetic metal nanocomposite according to Example 1 from the magnetic nanoparticles of Preparation Example 1, and 4f is a scanning transcription micrograph of the magnetic metal nanocomposite according to Example 1; .

FIG. 5 is a graph showing UV-Vis absorbances of gold nanoparticles of Preparation Example 5, magnetic material-containing silica particles of Preparation Example 4, and magnetic metal nanocomposites of Example 1;

6 is a magnetic metal nanocomposite (a) of Example 1, magnetic material-containing silica particles (b) of Preparation Example 4, silica particles (c) prepared without a magnetic body in Preparation Example 4 and the magnetic nanoparticles of Preparation Example 1 ( It is a graph which shows the X-ray diffraction pattern for confirming the crystallinity of d).

7A and 7B are graphs showing changes in magnetic resonance images and relaxation degrees according to concentrations of the magnetic metal nanocomposites according to one embodiment of the present invention, and FIG. 7C is a graph showing a magnetic history curve of the magnetic metal nanocomposites. to be.

8a and 8b show the flow cytometry graph for confirming the target orientation of the magnetic metal nanocomposite, 8c is a bar graph compared with the relative value.

Figure 9a is a fluorescence microscope and magnetic resonance imaging showing the binding degree of the antibody-bound magnetic metal nanocomposite and target cancer cells according to an embodiment of the present invention, Figure 9b is the relaxation of the cells obtained through the magnetic resonance image It is a graph showing the change.

10 is a temperature increase graph (a) obtained by irradiating a laser to an aqueous solution in which the magnetic metal nanocomposite is dispersed according to an embodiment of the present invention, and a temperature increase graph (b) obtained by irradiating laser to pure water.

11 is a fluorescence micrograph and apoptosis graph showing the cancer cell treatment effect of the magnetic metal nanocomposite according to the present invention, respectively.

The present invention relates to a multifunctional magnetic metal nanocomposite, and more particularly, to a multifunctional magnetic metal nanocomposite which can be used for diagnosis and treatment of various cellular diseases, and preferably can provide a synergistic therapeutic effect by an antibody. It is about.

Nanoparticles, commonly referred to as metal nanocomposites, include spherical dielectric core nanoparticles; And a thin metal film surrounding the core. The metal nanocomposite can absorb electromagnetic waves by surface plasmon resonance, and the absorbed electromagnetic waves generate heat by collectively vibrating conductive electrons in the metal film. The wavelength range of the electromagnetic wave absorbed by the metal nanocomposite and the resulting heat generation can be easily controlled by controlling the type of metal used, the thickness of the dielectric core nanoparticles and the metal film. In addition, the electromagnetic wave has a characteristic that can penetrate the living tissue to some extent, although the degree varies depending on the wavelength, and this characteristic of the electromagnetic wave has been applied as a tool for diagnosing a disease in a clinic. For example, the pulse oximeter, optical coherence tomography (OCT), diffuse tomography, or laser Doppler imaging uses the penetration characteristics of electromagnetic waves. Based on the properties of such metal nanocomposites and electromagnetic waves, researches for applying metal nanocomposites to drug delivery, disease treatment, and the like are continuously performed in the art.

Japanese Patent Application No. 2001-510655 discloses a method for delivering a drug to a desired place using a metal nanocomposite. Specifically, the Japanese application provides a composition comprising a therapeutic drug, a thermosensitive material (eg, hydrogel), and a metal nanocomposite, wherein the electromagnetic wave is generated from the metal nanocomposite when irradiated with electromagnetic waves at a desired place after administering the composition. It is described that heat disrupts the thermosensitive material and releases the therapeutic drug. However, since the metal nanocomposite used in the Japanese application is used as one component of the composition, it is small in size and generates little heat, and heat treatment caused by electromagnetic wave radiation is used to disintegrate the thermosensitive material. Hard to expect.

L. R. Hirsch et al. Disclose a method for treating cancer cells with heat generated by surface plasmon resonance (PNAS 2003; 100; 13549-13554). Specifically, L. R. Hirsch et al. Investigated near-infrared radiation on gold-coated silica nanomaterials and suggested that the generated heat is useful for cancer cell death through in vivo and ex vivo experiments. However, the present invention only discloses that the gold-silica nanocomposite may be useful for the treatment of cancer, and suggests or suggests nothing about the simultaneous diagnosis and treatment using the nanocomposite as in the present invention. I'm not doing it. Moreover, since the method relies only on heat generated by surface plasmon resonance phenomena, it is difficult to expect an effective therapeutic effect for cancer cells located deeper in consideration of the fact that the penetration of near infrared rays decreases with depth.

Therefore, there is a continuous demand in the art for the development of a multifunctional metal nanocomposite capable of providing various functions while overcoming the above problems.

An object of the present invention is a multifunctional magnetic metal nanocomposite capable of diagnosing and treating cellular diseases; And a method for producing the same.

Another object of the present invention is a diagnostic and therapeutic pharmaceutical composition comprising the multifunctional magnetic metal nanocomposite and a pharmaceutically acceptable carrier; And it provides a method for diagnosing and / or treating a disease using the same.

The inventors have found that magnetic metal nanocomposites coated with metals containing dielectric particles containing magnetic bodies are useful for the diagnosis and treatment of cellular diseases. In addition, by attaching the antibody to the surface of the metal, the present inventors have found that they exhibit excellent target directivity and therapeutic effect on specific cells, thereby completing the present invention.

Accordingly, the present invention provides a dielectric particle containing a magnetic material; And

It includes a metal-containing coating layer formed on the surface of the dielectric particles, the metal provides a magnetic metal nanocomposite that generates heat upon electromagnetic wave irradiation.

Preferably, the magnetic metal nanocomposite further comprises a water-soluble polymer having an antibody attached thereto on the surface of the metal-containing coating layer.

The magnetic metal nanocomposite according to the present invention can be used as a contrast agent for diagnostic methods such as magnetic resonance imaging (MRI), and provides a useful therapeutic effect on abnormal cells, preferably cancer cells, in a patient. In the present invention, the part performing the diagnostic role and the therapeutic role is divided into magnetic material-containing dielectric particles and dielectric-metal layers of the magnetic metal nanocomposites, respectively. The magnetic nanoclusters of the magnetic body-containing dielectric particles serve as a contrast medium of the magnetic resonance image. The monodisperse magnetic material was prepared in a cluster form using an emulsion-suspension method to maximize the contrast agent effect. In addition, the dielectric-metal layer generates heat due to surface plasmon resonance during 800 nm laser irradiation, thereby exhibiting a therapeutic effect. Preferably, the magnetic metal nanocomposite according to the present invention is capable of further providing a therapeutic effect and a transfer to a desired place by using an antigen-antibody reaction. Hereinafter, the configuration will be described in detail.

Magnetic body-containing dielectric particles used as diagnostic tools

Since the magnetic metal nanocomposite according to the present invention contains a magnetic material in the dielectric particles, it may be usefully used in a diagnostic method using magnetic properties. Specifically, the diagnostic method using the magnetic properties include magnetic resonance imaging (MRI), the magnetic metal nanocomposite of the present invention can be used without limitation in such a diagnostic method. In the present invention, when the magnetic material is present in the form of a cluster, the magnetic material may exhibit a sensitivity when diagnosing the magnetic resonance imaging device.

Preferably, the magnetic material used in the present invention is magnetic nanoparticles (magnetic nanoparticles) or a collection of magnetic nanoclusters thereof, and may be present in a dispersed form or core form in dielectric particles. The magnetic nanoparticles may be used without limitation as long as the magnetic nanoparticles are magnetic and have a diameter of 1 nm to 1,000 nm, preferably 2 nm to 100 nm, but may be a metal material, a magnetic material, or a magnetic alloy. alloy).

The metal is not particularly limited, but is preferably 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 _x O _y (M and M 'each independently represent Co, Fe, Ni, Mn, Zn, Gd, or Cr, and are preferably selected from the group consisting of 0 <x ≦ 3, 0 <y ≦ 5).

In addition, the magnetic alloy is not particularly limited, but is preferably selected from the group consisting of CoCu, CoPt, FePt, CoSm, NiFe, and NiFeCo.

In addition, the metal, magnetic material, or magnetic alloy is preferably combined with an organic surface stabilizer. An organic surface stabilizer means an organic functional molecule capable of stabilizing the state and size of the nanoparticles of the present invention, and representative examples thereof include surfactants.

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 an alkylamine such as trialkylphosphine or trialkylphosphine oxide such as tributylphosphine, dodecylamine, oleic amine, trioctylamine, or octylamine neutral surfactants including alkyl amines, or 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 the present invention, the dielectric serves to stabilize the magnetic nanoclusters in a hydrophilic medium. Also, metals generally absorb light by absorbing light in the wavelength range of about 520 nm. When the metal is coated on the dielectric, the metal absorbs in the wavelength range of about 800 nm, and electrons are absorbed by surface plasmon resonance rather than excitation. Occurs and has a therapeutic effect.

The dielectric refers to a material having an induction electric dipole moment and generating induced polarization when an external electric field is applied to the material. Such materials include most materials other than metals and conductive polymers, and are well known in the art, and those skilled in the art can arbitrarily select a known dielectric in consideration of the purpose of use. Although not limited thereto, the dielectric may be preferably selected from the group consisting of silica, titania, polymer particles, alumina and mixtures thereof. The dielectric particles may be modified with functional groups such as carboxyl groups, thiol groups or amine groups to improve the adhesion of the dielectric particles and the metal-containing coating layer. Modification to the functional group can be carried out in various ways using a known method, but is not limited thereto, and has a functional group such as (3-aminopropyl) trimethoxysilane, (3-aminopropyl) triethoxysilane and the like. It can be easily carried out by reacting with an alkoxysilane.

The particle diameter of the dielectric particles is not particularly limited, but considering the therapeutic effect, it is preferably 80 nm to 120 nm, more preferably 95 nm to 105 nm.

Preferably, the magnetic metal nanocomposite according to the present invention can be used as a dual or multiple diagnostic probe by additionally containing other diagnostic probes, together with a magnetic body generating a magnetic field in the dielectric particles. For example, combining a T1 magnetic resonance imaging probe with a water-soluble magnetic metal nanocomposite enables simultaneous diagnosis of T2 magnetic resonance imaging and T1 magnetic resonance imaging, and combining the optical diagnostic probe allows simultaneous magnetic resonance imaging and optical imaging. By combining CT diagnostic probes, MRI and CT diagnosis can be performed simultaneously. Combined with radioisotopes, magnetic resonance imaging and PET or SPECT can be simultaneously diagnosed.

The T1 magnetic resonance imaging probe includes a Gd compound, a Mn compound, and the like, and the optical diagnostic probe includes a fluorescent 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.

Metal-containing coating layer to provide heat treatment

The thermal treatment effect according to the invention is provided by a metal-containing coating layer formed on the surface of the (dielectric-metal layer) dielectric particles. In general, the metal absorbs light to excite the electron, but in the present invention, the metal is composed of a dielectric and a layer to absorb light by surface plasmon resonance. Therefore, rather than absorbing light in the wavelength range of about 520nm, it absorbs about 800nm wavelength band. The metal contained in the coating layer generates heat due to surface plasmon resonance when radiated from electromagnetic waves, preferably laser, and emits heat to the surroundings. According to the experiments of the present inventors, when the water containing the magnetic metal nanocomposite according to the present invention was irradiated with a laser having a wavelength of about 800 nm for 5 minutes at an intensity of 30 w / cm ² , the temperature of the water was increased by about 15 ° C. ( Reference: Experimental Example 3). Therefore, if the magnetic metal nanocomposite is distributed throughout the patient's whole body, the electromagnetic wave is irradiated to a specific site, or if the magnetic metal nanocomposite is locally distributed, the electromagnetic wave is irradiated to a specific site or the whole body, thereby locally raising the temperature of the cell. It is possible. This temperature rise can raise the intracellular temperature locally to above 40 ° C. and is sufficient to kill cells by irreversibly denaturing the tertiary or quaternary structure of the protein. Thus, the magnetic metal nanocomposite according to the present invention can provide heat treatment.

The metal included in the metal-containing coating layer may be arbitrarily selected from known metals that may generate heat by surface plasmon resonance upon electromagnetic wave irradiation. The wavelength range of the electromagnetic wave absorbed by the metal and the heat generated by the surface plasmon resonance are based on the type of metal used; And the thickness of the dielectric particles and the metal-containing coating layer. Therefore, those skilled in the art will be able to easily select specific combinations of these by repeating experiments according to the purpose of use. Although not limited thereto, the metal may preferably be a noble metal, and more preferably may be selected from the group consisting of Au, Ag, and Pt. Although not limited to this, the thickness of the metal-containing coating layer is preferably 10 nm to 30 nm, more preferably 15 nm to 25 nm in consideration of the therapeutic effect. The range of wavelengths that can be absorbed by the metals varies and they can be used in the present invention without limitation. Although not limited thereto, preferably, the wavelength absorbed by the metal is a wavelength in the near infrared to mid-area infrared region showing a high penetration rate with little absorption by biological tissues, and more preferably by water and hemoglobin when applied to a living body. It is 700 nm to 1000 nm, which is a wavelength band that is not absorbed.

Hydrophilic Polymer with Tissue-Specific Binding Components

Preferably, the magnetic metal nanocomposite according to the present invention may further comprise a hydrophilic polymer on the surface of the metal-containing coating layer in order to improve stability in the hydrophilic medium. The hydrophilic polymer may be distributed outside the magnetic metal nanocomposite to stabilize the water-insoluble nanocomposite in a hydrophilic medium to maximize bioavailability. In other words, the magnetic metal nanocomposite is prevented from being degraded by the immune system or to the target site in vivo. In addition, the hydrophilic polymer mediates the connection between the metal-containing coating layer and the tissue-specific binding component described below through chemical bonding, and helps to avoid the immune system in vivo and reach the target point.

The hydrophilic polymer used in the present invention is not particularly limited as long as it does not adversely affect the physical properties of the formed magnetic metal nanocomposite, but is preferably polyethylene glycol, polypropylene glycol, or fluoronic series (polyethylene glycol-polypropylene glycol-polyethylene). Glycol copolymers), polyetherimide (PEI), polyvinylpyrrolidone (PVP), N-isopropylacrylamide (NIPAM) and mixtures thereof. The hydrophilic polymer includes one or more, preferably one to three, functional groups each capable of binding to a tissue-specific binding component and a metal-containing coating layer, and in the case where such functional groups are not present in the original structure of the hydrophilic polymer The necessary functional group can be added and used as desired. The hydrophilic polymer preferably has a weight average molecular weight of 100 to 100,000. If the weight average molecular weight is less than 100 shows biotoxicity, if it exceeds 100,000 it is difficult to apply.

The type of functional group that reacts with the tissue specific binding component in the hydrophilic polymer may be arbitrarily changed according to the type of tissue specific binding component used. Representative functional groups are -COOH, -CHO, -NH ₂ , -SH, _{-CONH 2, -PO 3 H, -PO} 4 H, -SO 3 H, -SO 4 H, -OH, -NR 4 + X -, - sulfonate, - nitrates,-phosphonate-succinimidyl It is preferable to include at least one functional group selected from the group consisting of a dill group, a -maleimide group, and a -alkyl group.

In addition, the type of functional group reacting with the metal-containing coating layer in the hydrophilic polymer is not limited thereto, but preferably includes at least one functional group selected from the group consisting of -SH and -NH ₂ . Although not limited thereto, the bond between the hydrophilic polymer and the metal-containing coating layer is an ester bond, a thioester bond, an amide bond and / or a thioamide bond, and an amide bond is more preferable in view of reaction stability and ease.

Since the metal-containing coating layer does not have a functional group for chemical bonding as described above, the metal-containing coating layer may react with the metal-containing layer after adding the functional group to the hydrophilic polymer. Methods of adding functional groups are well known in the art, but thiol groups can be added using reagents such as, but not limited to, 4-aminothiophenol, cystiamine.

More preferably, the magnetic metal nanocomposite according to the present invention may further include a tissue specific binding component on the surface of the hydrophilic polymer to improve target directivity and therapeutic effect. The tissue specific binding component contributes to the target directivity and biological therapeutic effect of the nanocomposite by interacting with specific tissues.

The tissue-specific binding component is antigen, antibody, RNA, DNA, hapten, avidin, streptavidin, neutravidin, protein A in consideration of target directivity and biological therapeutic effect Protein G, lectin, selectin, radioisotope labeled components, including but not limited to substances capable of specifically binding to tumor markers.

The magnetic metal 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. It can be used to Such tumor cells express and / or secrete certain substances which are produced little or no in normal cells and are generally termed “tumor markers”. Polymers or metal nanocomposites containing materials capable of specifically binding such tumor markers may be usefully used for the treatment of tumors. 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.

Kinds Examples of Tumor Markers Examples of Tissue Specific Binding Components Ligand C2 of Synaptotamine I Phosphatidylserine Annexin V Integreen Integrin receptor VEGF VEGFR Angiopoietins 1 and 2 Tie2 receptor Somatostatin Somatostatin Receptor Basointestinal Peptide Basointestinal Peptide Receptor antigen Cancerous Fetal Antigens Herceptin (Genentech, USA) HER2 / neu antigen EGFR 1 antigen Erbitux (MERCK, USA) Prostate-specific antigen Rituxan (Genentech, USA) Receptor Folic acid receptor Folic acid

When the tumor marker is a ligand, a substance capable of specifically binding to the ligand may be introduced into the magnetic metal nanocomposite according to the present invention, and 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 that can specifically bind to the antigen can be introduced as a tissue-specific binding component of the magnetic metal nanocomposite according to the present invention. An antibody that can specifically bind to the antigen is suitable. something to do. 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). Antigens), Herceptin, epidermal growth factor receptor 1 (EGFR1) antigens, Erbitux, (prostate-specific membrane antigens) and rituxan (IDCE / Genentech, USA).

Representative examples in which the tumor marker is a "receptor" include folic acid receptors 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 a tissue specific binding component of the magnetic metal nanocomposite according to the present invention, and a ligand capable of specifically binding to the receptor Or the antibody will be suitable.

As mentioned above, antibodies are particularly preferred tissue specific binding components in the present invention. The antibody has the property of selectively and stably binding only to a specific target, and -NH _{2 of} lysine, -SH of cysteine, -COOH of aspartic acid and glutamic acid in the Fc region of the antibody bind to the hydrophilic polymer of the magnetic metal nanocomposite. This is because it can be usefully used. Such antibodies can be obtained commercially or prepared according to methods known in the art. In general, a mammal (eg, mouse, rat, goat, rabbit, horse or sheep) is immunized one or more times with an appropriate amount of antigen. After a period of time, when the titer reaches an appropriate level, the antibody 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. It is also possible to produce a monoclonal antibody by extracting the spleen from the animal. Specific experimental conditions used in this method are well known in the art (see, eg, Nam, KS, et al. (1990) Biochim. Biophys. Acta 1046, 89-96.).

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 . Nucleic acid having a base sequence complementary to the nucleic acid encoding the enzyme, ligand, antigen, receptor can be used as a tissue specific binding component of the magnetic metal 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 can be usefully used to bind to the functional group of the hydrophilic polymer.

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.). As an example, phosphorothioate oligonucleotides can be synthesized by the methods described in Stein et al. Nucl. Acids Res. 1988, vol. 16, p. 3209. Methylphosphonate oligonucleotides can be prepared using a controlled free polymer support (Sarin et al. Proc. Natl. Acad. Sci. U.S.A. 1988, vol. 85, p.7448). The amount of tissue specific binding component is not particularly limited and may be appropriately selected by those skilled in the art.

Manufacturing method of magnetic metal nanocomposite

The invention also comprises the steps of A) adding a dielectric to the surface of the magnetic material to produce magnetic-containing dielectric particles; And

B) a method of manufacturing a magnetic metal nanocomposite comprising the step of adding a metal to the surface of the dielectric particles to form a metal-containing coating layer.

The method of manufacturing the magnetic metal nanocomposite according to the present invention optionally includes the steps of: C) adding a hydrophilic polymer to the surface of the metal-containing coating layer, or D) attaching a tissue specific binding component to the hydrophilic polymer. It can be included as.

Hereinafter, each step of the manufacturing method of the magnetic metal nanocomposite according to the present invention will be described in more detail.

In step A) of preparing the magnetic body-containing dielectric particles, the magnetic body is

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

b) magnetic nanoparticles prepared by the method comprising the step of pyrolyzing the reactants.

Step a) is a step of coordinating the organic surface stabilizer on the surface of the magnetic nanoparticles by introducing a magnetic nanoparticle precursor in a solvent containing the organic surface stabilizer.

The magnetic nanoparticles of step a) are preferably metals, magnetic materials, or magnetic alloys, and organic surface stabilizers include alkyl trimethylammonium halides, saturated or unsaturated fatty acids, and trialkylphosphine oxides. oxide, alkyl amine, alkyl thiol, sodium alkyl sulfate, sodium alkyl phosphate, and sodium alkyl phosphate. Specific types of the metal, the magnetic material, the magnetic alloy, and the organic surface stabilizer are as described above.

The magnetic nanoparticle precursor of step a) may use a metal compound in which a metal and -CO, -NO, -C ₅ H ₅ , an alkoxide or other known ligand are bound, specifically, iron pentacarbonyl ( metal carbonyl compounds such as iron pentacarbonyl, Fe (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 specifically, iron trichloride (FeCl ₃ ) and iron dichlorochloride (FeCl _2). ), Or iron nitrate (Fe (NO ₃ ) ₃ ) can be used. In addition, in the synthesis of alloy nanoparticles and composite nanoparticles, a mixture of two or more metal precursors mentioned above may be used.

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 surface of the magnetic nanoparticles, for example, an ether compound, a heterocyclic compound, and an aromatic compound. , Sulfoxide compounds, amide compounds, alcohols, hydrocarbons and water may be selected from the group consisting of water.

Specifically, the solvent may be an ether compound such as octyl ether, butyl 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 can be used.

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

Step b) is a step of growing magnetic nanoparticles by pyrolyzing a complex compound in which an organic surface stabilizer is coordinated on the magnetic nanoparticle surface. At this time, the magnetic nanoparticles having a uniform size and shape can be formed according to the reaction conditions, and the thermal decomposition temperature can also be appropriately adjusted according to the type of metal precursor and the surface stabilizer. Preferably, the reaction is performed at about 50 to 500 ° C. Magnetic nanoparticles prepared in step b) can be separated and purified through known means.

Preferably, the magnetic nanoparticles obtained in step b) may be used to prepare a magnetic nanocluster, which is a collection of the nanoparticles, using an oil phase / water phase (O / W) emulsion method. Since specific methods and conditions relating to the method are well known in the art, those skilled in the art will be able to easily select them by repeating the known methods. Specifically, the magnetic material used in step A),

c) dissolving the magnetic nanoparticles obtained in step b) in an organic solvent to prepare an oil phase;

d) dissolving the hydrophilic compound in an aqueous solvent to prepare an aqueous phase;

e) mixing the oil phase and the aqueous phase to form an emulsion; And

f) magnetic nanoclusters prepared by a process comprising the step of separating the oil phase from the emulsion.

The organic solvent in step c) is not particularly limited as long as it does not adversely affect the physical properties of the formed dielectric particles, preferably chloroform, hexane, heptane, methylene chloride, benzene, toluene, tetrahydrofuran, acetone and mixtures thereof It is selected from the group consisting of.

The hydrophilic compound in step d) is selected from the group consisting of, but not limited to, polyvinyl alcohol, fluoronic series, sodium dodecyl sulfite (SDS), hexadecyltrimethylammonium bromide (CTAB) and mixtures thereof .

Separating the oil phase in step e) may be carried out by a conventional method, preferably by stirring.

Preferably, after step f), the obtained magnetic nanocluster may further include a step g) of applying impurities to the centrifugation and gel filtration columns to remove impurities.

The addition of the dielectric to the surface of the magnetic body in step A) may be carried out using conventional methods known in the art depending on the specific type of dielectric used. For example, the magnetic material is C _{1 -} can be coated in the presence of a _6-alkoxy silane, preferably tetrakis (C _1-4 alkoxy) silane to the silica.

Forming the metal-containing coating layer on the surface of the dielectric particles obtained in step A) is well known in the art (see LR Hirsch, et al., PNAS 2003; 100; 13549-13554; SJ Oldenberg , et al., (1998) Chem . Phys . Lett . 28, 243-247; W. Stober, et al., (1968) J. Colloid Interface Sci . 26, 62-69). Although not limited thereto, the metal may be made of nanoparticles and attached to the surface of the dielectric particles. Since the nanoparticles attached to the surface of the dielectric particles act as nuclei for metal growth, it is possible to form a metal-containing coating layer having a desired thickness by growing the metal around the metal nanoparticles under reducing conditions in an aqueous metal solution. Do. If necessary, the growth process of the metal may be repeated several times.

More specifically, step B)

h) preparing metal nanoparticles by reducing metal ions;

i) modifying the surface by adding a functional group for bonding with the metal nanoparticles to the surface of the magnetic body-containing dielectric particles obtained in step A);

j) reacting the metal nanoparticles of step h) with the dielectric particles having modified surface of step i) to bond them; And

k) preferably using the metal nanoparticle of step j) as a nucleus to grow a metal. Preferably, step k) can be repeated several times depending on the thickness of the desired metal layer.

The method of reducing the metal ion in step h) is well known in the art, and preferably can be easily carried out using a conventional reducing agent. Gold nanoparticles can be prepared by reducing hydrogen tetrachloroaurate (III) (HAuCl ₄ ) to tetrakis (hydroxymethyl) phosphonium chloride, although not limited thereto.

For example, 3 nm gold spherical particles are added with NaOH solution in ultrapure water followed by THPC (tetrakis hydroxymethyl phosphonium chloride). Thereafter it may be prepared by the addition of hydrogen tetra chloroaurate (HAuCl ₄ ). In addition, the method for producing gold particles of 20nm may be prepared by adding hydrogen tetra chloroaurate (HAuCl ₄ ) in ultrapure water and raising the temperature and then trisodium citrate (trisodium citrate). In addition, gold nanoparticles of various sizes and shapes can be produced according to various other conditions.

In step i) the surface of the magnetic body-containing dielectric particles are modified for bonding with the metal nanoparticles, specifically, may be modified with a carboxyl group, thiol group or amine group. Modification to a thiol group or an amine group can be carried out in various ways using a known method, but is not limited thereto, succinimidyl group and thioamine group; It can be easily carried out by reacting with conventional reagents such as (3-aminopropyl) trimethoxysilane.

The method of bonding the surface-modified dielectric and the metal nanoparticles in step j) may be performed in various ways using a known method. This method is not an an a gold-coated silica is limited to a method to put the silica particles with formaldehyde and then into the hydrogen tetrachloroaurate (HAuCl ₄₎ in a state dissolved in a K ₂ CO ₃ in pure water.

The metal-containing coating layer in step k) can be easily carried out by reducing the metal ions in reducing conditions, preferably in the presence of a reducing agent. Reducing conditions of metal ions, reducing agents and the like are well known in the art.

In addition, the step C) of adding a hydrophilic polymer to the surface of the metal-containing coating layer or the step D) of binding a tissue-specific binding component to the hydrophilic polymer is preferably performed in the presence of a crosslinking agent.

The crosslinking agent may provide a hydrophilic polymer with a functional group capable of binding to the metal-containing coating layer or a tissue specific binding component.

Although not limited thereto, amine groups may be added using reagents such as 4-aminothiophenol and cystiamine.

The crosslinking agent is not particularly limited, but may be 1,4-diisothiocyanatobenzene, 1,4-phenylene diisocyanate, or 1,6-diisocyanato. Hexane (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, 1,6-Hexanediamine, p-Nitrophenyl chloroformate, normal-hydride N-Hydroxysuccinimide, 1,3-Dicyclohexylcarbodiimide, 1,1'-carbonyldiimidazole, 3-male Midobenzoic Acid Normal-Hydroxysuccinimide ester, Ethylenediamine, Non (4-nitrophenyl) carbonate (Bis (4-nitrophenyl) carbonate), Succinyl chloride, N- (3-dimethylaminopropyl) -N'-ethylcarbonide hydrochloride (N- ( 3-Dimethylaminopropyl) -N'-ethylcarbodiimide Hydrochloride), N, N'-disuccinimidyl carbonate, N-succinimidyl 3- (2-pyridyldithio) It is preferable to include at least one selected from the group consisting of N-Succinimidyl 3- (2-pyridyldithio) propionate, and succinic anhydride.

 The binding of the hydrophilic polymer and the tissue specific binding component may be changed according to the type of each compound, and specific examples thereof are shown in Table 2 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: Functional group of hydrophilic polymer II: Functional group of tissue specific binding component III: Example of binding according to reaction of I and II

Pharmaceutical Compositions and Diagnosis and Treatment Using the Same

The magnetic metal nanocomposite according to the present invention simultaneously provides a diagnostic effect using a magnetic field and a therapeutic effect using heat, and preferably additionally exhibits a biological therapeutic effect by a tissue-specific binding component, whereby cell death is required. Useful for diagnosing and / or treating a disease. Such cellular diseases include, but are not limited to, cancer, obesity, or inflammatory diseases. More preferably gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, skin cancer and cervical cancer. Therefore, the present invention is a magnetic metal nanocomposite defined above; And a pharmaceutically acceptable carrier, the composition for diagnosing or treating a cell disease requiring cell death. Preferably, the magnetic metal nanocomposite includes a tissue specific binding component.

Carriers used in the composition according to the present invention include carriers and vehicles commonly used in the medical field, and specifically, ion exchange, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer materials ( E.g. several 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) Colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose based substrates, polyethylene glycol, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene glycols 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 according to the invention can be prepared in an aqueous solution for parenteral administration. Preferably, a buffer solution such as Hanks' solution, Ringer's solution, or physically buffered saline may 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 compositions of the present invention may be in the form of sterile injectable preparations of 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 invention also comprises administering a pharmaceutical composition according to the invention to a living body or a sample; And

The present invention relates to a method for diagnosing a disease comprising detecting an image emitted by a magnetic metal nanocomposite from a living body or a sample and obtaining an image.

Preferably, the diagnostic method may further comprise detecting a signal emitted from a T1 or T2 magnetic resonance imaging diagnostic probe, an optical diagnostic probe, a CT diagnostic probe, and a radioisotope.

The invention also comprises administering a pharmaceutical composition according to the invention to a living body; And

The present invention relates to a method for diagnosing and treating cell diseases requiring cell death, including irradiating electromagnetic waves to the living body. Electromagnetic waves can be irradiated by dividing the whole body region or a local region determined as a lesion of the living body.

Preferably, the treatment method may further comprise the step of obtaining an image by sensing a signal emitted by the magnetic metal nanocomposite from the living body, before the step of irradiating the electromagnetic wave.

As used herein, the term "sample" refers to tissue or cells isolated from a subject to be diagnosed. In addition, the step of injecting the pharmaceutical composition into the living body may be administered via a route commonly used in the medical field, parenteral administration is preferred, for example, intravenous, intraperitoneal, intramuscular, subcutaneous or topical routes. It can be administered through.

In the diagnostic method, a signal emitted by the magnetic metal nanocomposite may be detected by various equipments using a magnetic field, and in particular, a magnetic resonance imaging apparatus (MRI) may be preferably used.

The magnetic resonance imaging apparatus puts a living body in a strong magnetic field and irradiates radio waves of a specific frequency so that atomic nuclei such as hydrogen in biological tissues absorb energy to make energy high, and then stops propagating the nuclear energy such as hydrogen. The energy is converted into a signal, processed by a computer, and imaged. 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 present invention also relates to a method of binding a magnetic metal nanocomposite coupled to a tissue-specific binding component with a target material; And

The present invention relates to a method for separating a target material comprising applying a magnetic field to a combination of the magnetic metal nanocomposite and the target material.

In the separation method according to the present invention, a preferred example of a target substance means a biomolecule, and more specifically, a cell, protein, antigen, peptide, DNA, RNA, or virus having affinity with a tissue-specific binding component. And the like, and the like. Representative examples of tissue specific binding components include antigen, antibody, RNA, DNA, hapten, avidin, streptavidin, neutravidin, protein A, protein G, lectin , Selectin, radioisotope labeled components, and substances capable of specifically binding to tumor markers.

Hereinafter, the present invention will be described in more detail according to the following Preparation Examples and Examples. However, these preparation examples and examples are merely to aid the understanding of the present invention, and are not intended to limit the scope of the present invention to them in any sense.

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

6 nm of magnetite (Fe ₃ O ₄ ) was thermally decomposed (0.6 minutes) by thermal decomposition of 0.6 moles of dodecyl acid, 0.6 moles of dodecyl amine and iron triacetylacetonate (Aldrich) at 290 ° C. in a benzyl ether solvent. Synthesized. The 12 nm iron oxide nanoparticles were heated at 290 ° C. for 30 minutes by heating a benzyl ether solution containing 0.2 mol of dodecyl acid, 0.1 mol of dodecyl 6amine, 10 mg / ml of the 6 nm iron oxide nanoparticles, and iron triacetylacetonate. Prepared. Manganese ferrite (MnFe ₂ O ₄ ) was prepared by the addition of manganese tuacetylacetonate to the above reaction. Transmission electron micrographs of the prepared magnetite and manganese ferrite are shown in FIGS. 2A and 2B, respectively. Magnetic properties of the magnetite and manganese ferrite were measured using a Vibrating Sample Magnetometer (VSM), which is shown in FIG. 2C with dotted and solid lines, respectively.

Production Example  2: Preparation of High Sensitivity Magnetic Nanoparticles Using Unsaturated Fatty Acids

6 nm of magnetite (Fe ₃ O ₄ ) was added with 0.6 moles of oleic acid and 0.6 moles of oleyl amine in a benzyl ether solvent. And Iron triacetylacetonate (Aldrich) was synthesized by thermal decomposition (30 minutes) at 290 ° C. 12 nm iron oxide nanoparticles were prepared by heating a benzyl ether solution containing 0.2 mol of oleic acid, 0.1 mol of oleyl amine, 10 mg / ml of the 6 nm iron oxide nanoparticles, and iron triacetylacetonate at 290 ° C. for 30 minutes. . Manganese ferrite (MnFe ₂ O ₄ ) was prepared by the addition of manganese tuacetylacetonate to the above reaction. Transmission electron micrographs of the prepared magnetite and manganese ferrite are shown in FIGS. 3A and 3B, respectively. Magnetic properties of the magnetite and manganese ferrite were measured using VSM, and these are shown in FIG. 3C with dotted and solid lines, respectively.

Production Example  3: Preparation of Magnetic Nanoclusters

Magnetic nanoclusters were prepared from the magnetic nanoparticles obtained in Preparation Examples 1 or 2 using an oil phase / water phase emulsion (O / W emulsion) method. Specifically, 600 mg of polyvinyl alcohol was dissolved in 20 ml of ultrapure water as an aqueous phase, and 100 mg of magnetic nanoparticles obtained in Preparation Example 1 was dissolved in chloroform as an oil phase. The emulsion was stirred for 6 hours to evaporate the oil phase, and centrifugation was repeated several times to remove impurities and to prepare a highly sensitive magnetic nanocluster clustered in a constant size. Transmission electron micrographs of the prepared magnetic nanoclusters are shown in FIG. 4b and used in the next step to coat with silica.

Production Example  4: Preparation of Magnetic Body-Containing Dielectric Particles (Coating of Magnetic Nanoclusters with Silica)

The magnetic nanoclusters obtained in Preparation Example 3 were redispersed in a mixture of 1 ml of ultrapure water, 4 ml of ethanol and 0.1 ml of ammonia water. Magnetic nano-clusters were coated with silica to slowly inject 0.1 ml of tetrakis (ethoxy) silane into the mixture to prepare magnetic-containing dielectric particles. A transmission electron micrograph of the dielectric particles is shown in FIG. 4C. In addition, the crystallinity of the dielectric particles was confirmed by X-ray diffraction, and the results are shown in FIG. 6.

Production Example  5: Preparation of Gold Nanoparticles

0.5 ml of 1 M NaOH solution was added to 45 ml of ultrapure water, and STIRRING was strongly performed for 1 minute. Then, THPC (1 ml (0.28 mmol) of tetrakis (hydroxymethyl) phosphonium chloride solution) was added thereto and stirred for 5 minutes. Thereafter, 2 ml of a 1% HAuCl4 solution was added thereto, followed by stirring for 2 minutes to prepare 3 nm gold nanoparticles.

Production Example  6: Preparation of Dielectric Particles Bonded with Gold Nanoparticles

The dielectric particles obtained in Preparation Example 4 were reacted with 2 µl of (3-aminopropyl) trimethoxysilane (APTMS) to modify the silica surface of the dielectric with an amine group (NH ₂ ). The surface-modified dielectric particles were mixed with 7 × 10 ¹⁵ gold nanoparticles having a particle size of 3 nm obtained in Preparation Example 5 to bind the amine group of the silica and the gold nanoparticles. Several centrifugations removed unreacted APTMS and gold nanoparticles. Gold nanoparticles on the silica surface acted as nuclei during gold growth. Using a transmission electron microscope, it was confirmed that the silica surface and the gold nanoparticles are bonded, the results are shown in Figure 4d.

Example  1: Preparation of Magnetic Metal Nanocomposites

Gold salt solution was prepared using 1.5 ml of 1% HAuCl ₄ solution, 25 mg of K ₂ CO ₃ and 100 ml of ultrapure water. The magnetic metal nanocomposite according to the present invention was prepared by reacting 4 ml of the gold salt solution, 80 μl of formaldehyde and 1 × 10 ¹² dielectric particles combined with gold nanoparticles obtained in Preparation Example 6 for a short time. A transmission electron micrograph of the prepared magnetic metal nanocomposite is shown in FIG. 4E. UV-Vis absorbance was used to confirm that the magnetic metal nanocomposite absorbed light of 800 nm wavelength by surface plasmon resonance. Infrared absorbance of the magnetic gold nanocomposites was compared with magnetic body-containing silica particles and gold nanoparticles, and the results are shown in FIG. 5.

Example  2: Polyethylene glycol  And binding of antibodies

Example 1 Sensitivity magnetic metal nanocomposite 1 × 10 ¹⁰ and one 1 × 10 obtained in ⁶ moles of di-COOH PEG and Yen samdayi dimethylaminopropyl-N-ethylcarbodiimide hydrochloride / N - hydroxy-succinimide MIDI (EDC / NHS) Was added in a 6-fold excess of the number of moles of PEG added and reacted. 1.5 hours after the reaction to remove the unreacted PEG and EDC / NHS through several times centrifugation in the following, the antibody as Erbitux (Erbitux) 3 × 10 ^- 6-fold amount of antibody mol is added with ¹⁰ moles EDC / NHS was added and reacted at 4 ° C. for about 10 hours. By removing the unreacted antibody and excess EDC / NHS through several centrifugation, a magnetic metal nanocomposite to which the antibody is bound was prepared.

Experimental Example  1: Target Directivity Verification by Antibody

In order to verify the target directivity by the antibody, each of the magnetic metal nanocomposites to which Erbitux and Illeibant antibody (IRR) attached according to the method of Example 2 were attached, respectively, EGFR 1 (epidermal growth factor receptor 1) The well-expressed A431 cells and MCF7 cells without EGFR 1 expression were treated. A431 cells are cells that express a large number of antigens capable of antigen-antibody binding with Erbitux. Flow cytometry (FACS) and fluorescence microscopy confirmed that Erbitux-bound magnetic metal nanocomposites specifically bind to A431 cells overexpressed EGFR 1. The results are shown in FIG. IRR-MGNCs represent magnetic metal nanocomposites bound to elelibant antibodies, and ERB-MGNCs represent magnetic metal nanocomposites bound to Erbitux.

Experimental Example  2: of magnetic metal nanocomposites MRI  Check function as contrast agent

In order to confirm that the magnetic metal nanocomposites prepared in Examples show magnetic resonance imaging effects, the magnetic metal nanocomposites were titrated at concentrations of 600, 800, 1000, 1200, 1400, 1600, 1800, and 2000 µg / ml. It was injected into the tube. Cortical plane images were obtained using Fast Field Echo (FFE) pulse trains, which are shown in FIGS. 9A and 9B. In order to confirm the cancer diagnostic effect of the magnetic metal nanocomposite using magnetic resonance imaging, the EGFR 1 overexpressed cells of the magnetic metal nanocomposite to which the Erbitux and erylicant antibodies prepared according to Example 2 were attached (A431 ) And non-expressed cells (MCF7) and magnetic resonance images were obtained. When comparing the magnetic resonance images, the image change between the normal and cancer cells was obvious (Fig. 9a).

Specific conditions used to obtain magnetic resonance imaging are as follows:

Instrument: 1.5T (Intera; Philips Medical Systems, Best, The Netherlands) system and micro-47 coil; Resolution 156 × 156 μm; Section thickness 0.6 mm; TE = 20 ms; TR = 400 ms; Image excitation count 1; Image acquisition time 6 minutes.

T2 maps were performed to quantitatively evaluate the magnetic resonance imaging contrast effect on antigen specificity. Specific parameters were as follows:

Resolution 156 × 156 μm; Section thickness 0.6 mm; TR = 4000 ms; TE = 20, 40, 60, 80, 100, 120, 140, 160 ms; Video here2; Video acquisition time 4 minutes.

Experimental Example  3: fever and treatment effect by laser irradiation

A laser having an 800 nm wavelength was irradiated with water having a wavelength of 30 w / cm ² for 5 minutes in the water and the magnetic water in which the magnetic metal nanocomposite obtained in Example 2 was dispersed. The results are shown in FIG. After the laser irradiation, the water in which the magnetic metal nanocomposite was dispersed had a temperature of 15 ° C. or more, but pure water had a temperature of about 2 ° C.

Erbitux-coupled magnetic metal nanocomposites were incubated with cells overexpressing EGFR 1 (A431) and cells without expression (MCF7), and then irradiated with a laser having an 800 nm wavelength at an intensity of 30 w / cm ² . The magnetic metal nanocomposites were uptake into cancer cells by antigen / antibody binding, and only cancer cells were clearly killed after laser irradiation. Uptake of the magnetic metal nanocomposite was confirmed by magnetic resonance imaging, FACS (flow cytometry experiment) and fluorescence microscope (Fig. 8, 9), cancer cell death was confirmed by fluorescence microscope and MTT (toxicity test) experiment (Fig. 11). ).

The present invention is a magnetic metal nanocomposite capable of diagnosing and treating cellular diseases; And the production method thereof.

The present invention also provides a diagnostic and / or therapeutic pharmaceutical composition comprising the magnetic metal nanocomposite and a pharmaceutically acceptable carrier; And providing a method for diagnosing and / or treating a disease using the same.

Claims (35)

Dielectric particles containing magnetic material; And Including a metal-containing coating layer formed on the surface of the dielectric particles, The magnetic material is a) reacting the magnetic nanoparticle precursor with the surfactant in the presence of a solvent selected from the group consisting of ether compounds, heterocyclic compounds, aromatic compounds, sulfoxide compounds, amide compounds, alcohols, hydrocarbons and water; And b) a magnetic nanocluster, which is a collection of magnetic nanoparticles prepared by a method comprising thermally decomposing a reactant of the magnetic nanoparticle precursor with a surfactant, present in the form of a core dispersed in a dielectric particle, The solvent is a solvent having a boiling point higher than the thermal decomposition temperature of the complex compound coordinated with the surfactant on the surface of the magnetic nanoparticles, The magnetic nanoparticle precursor is selected from the group consisting of metal compounds including metals and ligands, metal salts including metal ions with metals and anions, and mixtures thereof, The magnetic nanoparticle is a metal selected from the group consisting of Pt, Pd, Ag, Cu and Au; 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, the magnetic being selected from the group consisting of 0 <x ≦ 3, 0 <y ≦ 5) matter; Or a magnetic alloy selected from the group consisting of CoCu, CoPt, FePt, CoSm, NiFe and NiFeCo, The metal of the metal-containing coating layer is a magnetic metal nanocomposite is selected from the group consisting of Au, Ag and Pt. delete The method of claim 1, Magnetic nanoparticles are magnetic metal nanocomposites, characterized in that the diameter of 1nm to 1,000nm. delete delete delete delete The method of claim 1, Magnetic metal nanocomposite, characterized in that the magnetic nanoparticles are combined with a surfactant. The method of claim 8, The surfactant may be alkyl trimethylammonium halide, saturated or unsaturated fatty acid, trialkylphosphine, trialkylphosphine oxide, alkyl amine, alkyl thiol, Magnetic metal nanocomposite, characterized in that at least one selected from the group consisting of sodium alkyl sulfate, and sodium alkyl phosphate. The method of claim 1, Magnetic metal nanocomposite, characterized in that the dielectric is selected from the group consisting of silica, titania, polymer particles other than the conductive polymer, alumina and mixtures thereof. The method of claim 1, Magnetic metal nanocomposite, characterized in that the particle size of the dielectric is 80nm to 120nm. The method of claim 1, Magnetic metal nanocomposite, wherein the dielectric particles further comprise a T1 magnetic resonance imaging diagnostic probe, an optical diagnostic probe, a CT diagnostic probe or a radioisotope. delete The method of claim 1, Magnetic metal nanocomposite, characterized in that the thickness of the metal-containing coating layer is 10nm to 30nm. The method of claim 1, Magnetic metal nanocomposite, characterized in that it further comprises a hydrophilic polymer on the surface of the metal-containing coating layer. The method of claim 15, Hydrophilic polymers include polyethylene glycol, polypropylene glycol, fluoronic series (polyethylene glycol-polypropylene glycol-polyethylene glycol copolymer), polyetherimide (PEI), polyvinylpyrrolidone (PVP), N-isopropyl acrylamide (NIPAM) and mixtures thereof. Magnetic metal nanocomposite. The method of claim 16, The hydrophilic polymer is _{-COOH, -CHO, -NH 2, -SH} , -CONH 2, -PO 3 H, -PO 4 H, -SO 3 H, -SO 4 H, -OH, -NR 4 + X -, A magnetic metal nanocomposite comprising at least one functional group selected from the group consisting of -sulfonate, -nitrate, -phosphonate, -succinimidyl group, -maleimide group, and -alkyl group. The method of claim 15, Magnetic metal nanocomposite, characterized in that it further comprises a tissue specific binding component on the surface of the hydrophilic polymer. The method of claim 18, Tissue-specific binding components include antigen, antibody, RNA, DNA, hapten, avidin, streptavidin, neutravidin, protein A, protein G, lectin, selectin ( selectin), a radioisotope labeled component, and a magnetic metal nanocomposite, wherein the magnetic metal nanocomposite is selected from the group consisting of a substance capable of specifically binding to a tumor marker. The method of claim 19, A magnetic metal nanocomposite, wherein the tumor marker is selected from the group consisting of ligands, antigens, receptors, and nucleic acids encoding them. The method of claim 20, Tumor markers include C2, annexin V, integrin, VEGF, angiopoietin 1, angiopoietin 2, somatostatin, vasointestinal peptide, cancerous fetal antigen, HER2 / neu antigen, EGFR1, of synaptotamine I Magnetic metal nanocomposite, characterized in that selected from the group consisting of prostate specific antigen and folic acid receptor. The method of claim 19, A substance capable of specifically binding to a tumor marker is at least one selected from the group consisting of phosphatidylserine, VEGFR, integrin receptor, Tie2 receptor, somatostatin receptor, vasointestinal peptide receptor, Herceptin, Erbitux, Rituxan and Folic acid. Magnetic metal nanocomposite. A) adding a dielectric to the surface of the magnetic material to produce magnetic-containing dielectric particles; And B) adding a metal to the surface of the dielectric particles to form a metal-containing coating layer, The magnetic material is a) reacting the magnetic nanoparticle precursor with the surfactant in the presence of a solvent selected from the group consisting of ether compounds, heterocyclic compounds, aromatic compounds, sulfoxide compounds, amide compounds, alcohols, hydrocarbons and water; And b) a magnetic nanocluster, which is a collection of magnetic nanoparticles prepared by a method comprising thermally decomposing a reactant of the magnetic nanoparticle precursor with a surfactant, present in the form of a core dispersed in a dielectric particle, The solvent is a solvent having a boiling point higher than the thermal decomposition temperature of the complex compound coordinated with the surfactant on the surface of the magnetic nanoparticles, The magnetic nanoparticle precursor is selected from the group consisting of metal compounds including metals and ligands, metal salts including metal ions with metals and anions, and mixtures thereof, The magnetic nanoparticle is a metal selected from the group consisting of Pt, Pd, Ag, Cu and Au; 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, the magnetic being selected from the group consisting of 0 <x ≦ 3, 0 <y ≦ 5) matter; Or a magnetic alloy selected from the group consisting of CoCu, CoPt, FePt, CoSm, NiFe and NiFeCo, The metal of the metal-containing coating layer is selected from the group consisting of Au, Ag and Pt Method for producing a magnetic metal nanocomposite. delete The method of claim 23, Wherein the magnetic nanoparticle precursor of step a) is an organometallic compound in which a metal and -CO, -NO, -C ₅ H ₅ or an alkoxide are bonded. delete The method of claim 23 wherein the magnetic body of step A) c) dissolving the dielectric and magnetic nanoparticles obtained in step b) in an organic solvent to prepare an oil phase; d) dissolving the hydrophilic compound in an aqueous solvent to prepare an aqueous phase; e) mixing the oil phase and the aqueous phase to form an emulsion; And f) a magnetic nanocluster prepared by the method comprising the step of separating the oil phase from the emulsion. The method of claim 27, Wherein the hydrophilic compound of step d) is selected from the group consisting of polyvinyl alcohol, fluoronic series, sodium dodecylsulfite (SDS), hexadecyltrimethylammonium bromide (CTAB) and mixtures thereof. The method of claim 23, wherein step B) h) preparing metal nanoparticles by reducing metal ions; i) modifying the surface by adding a functional group for bonding with the metal nanoparticles to the surface of the magnetic body-containing dielectric particles obtained in step A); j) reacting the metal nanoparticles of step h) with the dielectric particles having modified surface of step i) to bond them; And k) growing the metal using the metal nanoparticle of step j) as a nucleus. The method of claim 23, Adding C to the hydrophilic polymer on the surface of the metal-containing coating layer. The method of claim 30, The method further comprises the step D) of binding the tissue specific binding component to the hydrophilic polymer. 32. The method of claim 30 or 31 wherein Step C) or D) is carried out in the presence of a crosslinking agent. Magnetic metal nanocomposite according to any one of claims 1, 3, 8 to 12, 14 to 22; And a pharmaceutically acceptable carrier, the pharmaceutical composition for diagnosing or treating cancer, obesity, or inflammatory disease. The method of claim 33, wherein A pharmaceutical composition, characterized in that the cell disease is cancer, obesity, or inflammatory disease. The method of claim 34, wherein A pharmaceutical composition, wherein the cellular disease is selected from the group consisting of 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.

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