KR100848643B1 - Nanohybrid Particles for Diagnosis and Treatment of Tumor - Google Patents

Abstract

In the present invention, the nanoparticles are surrounded by a multifunctional ligand including an attachment region, a cross-linking region and an active ingredient binding region, wherein the cross-linking region of the multi-functional ligand is cross-linked with the cross-linking region of another multi-functional ligand. Tumor diagnostic or therapeutic nanohybrid particles, tumor diagnostics comprising the nanohybrid particles and a pharmaceutically acceptable carrier are characterized in that the active ingredient binding region is bound to a substance that can specifically bind to a tumor marker. Or injecting the therapeutic composition and the composition into a living body or a sample and detecting a signal emitted by the nanohybrid particles from the living body or the sample to diagnose tumor presence and selectively killing tumor cells in some cases. About diagnosis or treatment of tumor It is.

Multifunctional Ligands, Nanohybrid Particles for Diagnosing or Treating Tumors

Description

Nanohybrid Particles for Diagnosis and Treatment of Tumor

1 is a schematic diagram of a method for producing and diagnosing nanohybrid particles for diagnosing or treating tumors according to the present invention.

Figure 2 shows various embodiments of the nanohybrid particles for diagnostic or therapeutic tumors according to the present invention.

Figure 3 shows the results confirmed by the dynamic laser light scattering method (DLS) whether the water-soluble iron oxide nanoparticles and Herceptin is combined to form nanohybrid particles.

Figure 4 shows the results confirmed by the flow cytometry that the nanohybrid particles according to the invention selectively binds to cells expressing breast cancer marker antigen in vitro (expression of breast cancer marker antigen is control <1.Bx-PC3 < 2.MDA-MB-231 ≤ 3.BT-474 <4.NIH3T6.7 in order).

Figure 5 shows the results confirmed by magnetic resonance imaging that the nanohybrid particles according to the invention selectively binds to cells expressing breast cancer marker antigen in vitro (expression of breast cancer marker antigen is control <1.Bx-PC3 < 2.MDA-MB-231 ≤ 3.BT-474 <4.NIH3T6.7 in order).

Figure 6 shows the results confirmed by magnetic resonance imaging that the nanohybrid particles according to the present invention selectively binds to cells expressing a breast cancer marker antigen in vivo.

Figure 7 shows the results confirmed by the tissue immunostaining method that the nanohybrid particles according to the invention selectively binds to cells expressing breast cancer marker antigen in vivo.

Figure 8 shows the results confirmed by magnetic resonance imaging that the nanohybrid particles according to the invention selectively binds to cells expressing the ovarian cancer marker receptor in vitro.

9 is a graph showing that the nanohybrid particles according to the present invention can selectively kill cells expressing breast cancer marker antigens.

The present invention relates to nanohybrid particles, and more particularly, wherein the nanoparticles are surrounded by a multifunctional ligand including an attachment region, a cross-linking region, and an active ingredient-binding region, wherein the cross-linking region of the multi-functional ligand is another multi-functional ligand. It is cross-linked with the cross-linking region of the active ingredient binding region for the diagnostic or therapeutic nano-hybrid particles, characterized in that the material that can specifically bind to the tumor marker, the diagnostic or therapeutic comprising the same The present invention relates to a composition and a method for diagnosis and treatment using the same.

In the field of biotechnology, nanoparticles are used for cancer cell specific killing, boosting immune response, cell fusion, gene or drug delivery, and diagnosis. In order to be used for these applications, the nanoparticles must not only have a portion capable of attaching the active ingredient, but also must be smoothly transported and dispersed in vivo, that is, in an aqueous environment. Various techniques have been developed in the art to satisfy these conditions.

US Pat. No. 6,274,121 discloses paramagnetic particles made of inorganic or organic materials comprising paramagnetic particles and binding sites capable of coupling with tissue specific binding substances, diagnostic or pharmaceutically active substances on the surface of the particles. It is starting.

US Pat. No. 6,649,138 discloses a method of improving the water solubility of nanoparticles by forming multiple amphiphilic dispersant layers on the surface of hydrophobic nanoparticles comprising semiconductor or metal materials. As the amphiphilic dispersant, (1) a hydrophobic backbone having a hydrophilic side chain, (2) a hydrophilic backbone having a hydrophobic side chain, or (3) a hydrophobic or hydrophilic backbone having both hydrophilic and hydrophobic side chains was used.

US Patent Publication US 2003/0092029 relates to nanoparticles comprising a binding moiety, wherein the binding moiety is an oligonucleotide, polypeptide or polysaccharide. The nanoparticles can be used to detect target molecules such as nucleic acids and proteins in vitro or as contrast agents for diagnosing diseases in vivo.

However, U.S. Patent No. 6,274,121 and U.S. Patent Publication No. 2003/0092029 synthesize nanoparticles 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 a low temperature, nanoparticles tend to have low crystallinity and non-stoichiometric compounds are formed. In addition, the surface of the nanoparticles is coated with a single molecule interfacial stabilizer has a disadvantage in that the stability of the aqueous solution is poor because the bonding strength with the nanoparticles is not large. The water-soluble nanoparticles mentioned in US Pat. No. 6,649,138 are surrounded by amphiphilic polymers and have the problem of greatly increasing diameters compared to inorganic nanoparticles.

On the other hand, as a specific conventional technology in which nanoparticles are used as a contrast agent, Korean Patent Application No. 10-1998-0705262 relates to composite nanoparticles used to extend blood stay, and more specifically, oxidation-starved starch coating and arbitrary functionalization. Disclosed is a particle comprising a superparamagnetic iron oxide core particle with a polyalkylene oxide coating and an MRI contrast agent comprising the same.

However, the nanoparticles not only have no tumor specificity, but because starch coating greatly increases the size of the nanoparticles, the half-life in the blood is reduced, and they are fed by Cooper's cells. In addition, as described above, nanoparticles that are unstable in an aqueous phase may not be diffused and dispersed smoothly in vivo, and thus, may not only take a long time to diagnose, but also may not be able to penetrate deep into tissues when they are aggregated. There is a concern.

In addition, WO 01/56546 discloses a composition containing a magnetoliposome for treating a target site of a biological tissue and a treatment method using the same. The treatment method is determined after determining a target site through a separate diagnosis process. By administering the magnetoposome-containing composition and applying a magnetic field to the target site, the magnetoposome may be concentrated on the target site, thereby selectively treating only the target site. However, in the prior art, the diagnosis and the treatment are separated, which is a cumbersome disadvantage.

Therefore, the inventors of the present invention not only accurately and efficiently diagnose tumors with tumor diagnostic nanohybrid particles made by binding a substance capable of binding tumor markers to the active ingredient binding region of water-soluble nanoparticles that are stable in the water phase and are easily dispersed and diffused. At the same time, it was confirmed that only tumor sites can be treated selectively, and thus the present invention has been completed.

In one aspect, the nanoparticles are surrounded by a multifunctional ligand comprising an attachment region, a crosslinking region and an active ingredient binding region, wherein the crosslinking region of the multifunctional ligand is crosslinked with the crosslinking region of another multifunctional ligand. The active ingredient binding region provides a nanohybrid particle for tumor diagnosis or treatment, characterized in that the material that can specifically bind to the tumor marker is bound.

In another aspect, the present invention provides a tumor diagnostic or therapeutic composition comprising the nanohybrid particles and a pharmaceutically acceptable carrier.

In another aspect (1) injecting the diagnostic or therapeutic composition into a living body or a sample; And (2) diagnosing the presence of a tumor by sensing a signal emitted by the nanohybrid particles from the living body or sample; Or (3) selectively killing the identified tumor cells.

In the present invention, the "water-soluble nanoparticle" is surrounded by a multifunctional ligand, wherein the nanoparticle includes an attachment region, a cross-linking region and a binding region with an active ingredient, wherein the cross-linking region of the multi-functional ligand is a polyfunctional ligand. Particles characterized by being cross-linked with the cross-linked region, which is particularly stable in water-soluble environments.

In the present invention, "nanoparticles" are particles having a diameter of 1 nm to 1000 nm, preferably 2 nm to 100 nm, including a magnetic material, a magnetic alloy material or a multicomponent hybrid structure containing them. Means.

Examples of magnetic materials include Co, Mn, Fe, Ni, Gd, MM ' ₂ O ₄ , M _x O _y (M or M '= Co, Fe, Ni, Mn, Zn, Gd, Cr, 0 <x≤3, 0 <y≤5) and the like, and examples of magnetic alloys include CoCu, CoPt, FePt, CoSm, NiFe, NiFeCo and the like. Meanwhile, the multicomponent hybrid structure refers to a particle including two or more components selected from the magnetic material and the magnetic alloy.

In the present invention, "multifunctional ligand" means a material including (a) an attachment region, (b) a cross-linking region, and (c) an active ingredient binding region. Hereinafter, the multifunctional ligand will be described in more detail.

The "attachment region" means a portion of a multifunctional ligand including a functional group capable of attaching nanoparticles, preferably a terminal. Therefore, the attachment region preferably includes a functional group having high affinity with the material forming the nanoparticles, and may be variously selected according to the material forming the nanoparticles. The attachment region may include, for example, -COOH, -NH ₂ , -SH, -CONH ₂ , -PO ₃ H, -PO ₄ H, -SO ₃ H, -SO ₄ H or -OH.

By "crosslinking region" is meant a portion, preferably central, of a multifunctional ligand comprising a functional group capable of crosslinking with an adjacent multifunctional ligand. By "crosslinking" is meant that one polyfunctional ligand is coupled in an intermolecular interaction with another polyfunctional ligand in close proximity. The kind of intermolecular attraction (e.g., hydrophobic attraction, hydrogen bond, covalent bond (e.g. disulfide bond), van der Waals bond, ionic bond, etc.) is not particularly limited. It can be variously selected according to. The cross linking region may include, for example, -SH, -NH ₂ , -COOH, -OH, -epoxy, -ethylene or -acetylene as a functional group.

The "active component binding region" means a portion of a multifunctional ligand including a functional group attachable to the active component, preferably a terminal located opposite to the attachment region. The functional group of the active ingredient binding region may vary depending on the type of active ingredient and its chemical formula (see Table 1). Active ingredient-binding region in this invention _{-SH, -COOH, -NH 2, -OH} , -NR 4 + X - include nitrates (nitrate) or phosphonate (phosphonate) -, - sulfonate (sulfonate), It may, but is not limited to.

Such water-soluble nanoparticles can be prepared according to the method disclosed in Korean Patent Application No. 10-2004-0070303. The preparation method is included in the present invention as a reference.

Examples of functional groups of active ingredient binding regions that can be included in multifunctional ligands 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 the active ingredient binding region of the multifunctional ligand, II: active ingredient,

III: binding example according to reaction of I and II)

In one aspect, the present invention provides a nanohybrid particle having a substance capable of diagnosing and / or treating a tumor in an active ingredient binding region of the water-soluble nanoparticle. That is, in the present invention, "nanohybrid particles" are nanoparticles are surrounded by a multifunctional ligand comprising an attachment region, a cross-linking region and an active ingredient binding region, the cross-linking region of the multi-functional ligand of the other multi-functional ligand It refers to particles that are cross-linked with the cross-linked region and the active component binding region is bound to a substance capable of specifically binding to the tumor marker.

The nanohybrid particles of the present invention are used to 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". Nanohybrid particles 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 substances that can specifically bind to them. Meanwhile, in the specification of the present invention, a substance capable of specifically binding to a tumor marker has the same meaning as "active ingredient", and they may be used interchangeably.

Tumor markers in the present invention can be classified into ligands, antigens, receptors, and nucleic acids encoding them according to the mechanism of action (see Table 2).

Examples of Tumor Markers and Active Ingredients Kinds Examples of Tumor Markers Examples of active ingredients 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 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 as an active ingredient of the nanohybrid particles 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 nanohybrid particle 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 nanohybrid particles according to the present invention, and a ligand or an antibody capable of specifically binding to the receptor is suitable. something to do.

As mentioned above, antibodies are particularly preferred active ingredients 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 are active ingredient binding region functional groups of water-soluble nanoparticles. Because it can be useful to combine with.

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, 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 aforementioned ligand, antigen, receptor or part 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 nanohybrid particles 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.). 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).

In another aspect, the present invention provides a composition for diagnosing or treating tumors comprising the nanohybrid particles according to the present invention and a pharmaceutically acceptable carrier.

Tumor diagnostic or therapeutic compositions according to the invention may be administered via routes commonly used in the medical arts, and parenteral administration is preferred, for example via intravenous, intraperitoneal, intramuscular, subcutaneous or topical routes. May be administered.

Carriers used in the composition for diagnosing or treating tumors according to the present invention include carriers and vehicles commonly used in the medical field. Pharmaceutically acceptable carriers that may be used in the diagnostic or therapeutic compositions of the invention include, but are not limited to, ion exchange, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer materials (E.g. various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts or electrolytes (e.g. , Colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose based substrates, polyethylene glycol, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene glycols and wool, and the like. The diagnostic or therapeutic composition of the present invention may further include lubricants, wetting agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components.

In one embodiment, the tumor diagnostic or therapeutic composition according to the invention may be prepared in an aqueous solution for parenteral administration. Preferably, buffer solutions 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.

Preferred tumor diagnostic or therapeutic compositions of the invention may be in the form of sterile injectable preparations as sterile injectable aqueous or oily suspensions. Such suspensions can 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.

In another aspect, the present invention comprises the steps of (1) injecting a tumor diagnostic or therapeutic composition according to the invention into a living body or a sample; And (2) diagnosing the presence of a tumor by sensing a signal emitted by the nanohybrid particles from the living body or sample; Or (3) selectively killing the identified tumor cells.

The "sample" refers to tissue or cells isolated from a subject to be diagnosed. Through such a tumor diagnosis method, it is possible to diagnose tumors in vivo and in vitro.

In the diagnostic method, the signal emitted by the nanohybrid particles may be detected by various devices using a magnetic field. Such a device may include a magnetic resonance imaging diagnostic apparatus (MRI).

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 and make 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.

When it is confirmed that the tumor is present in the living body through the above-described diagnostic method, a treatment method may be performed including selectively killing only tumor cells.

One aspect of the treatment method according to the present invention is based on tumor specific hyperthermia, which is called "thermotherapy" from an external magnetic field applied to the magnetic nanoparticles in an alternating magnetic field at radio frequency, It releases heat through the relaxation (Neel relaxation). Heat generated at this time affects the ambient temperature of the magnetic nanoparticles, and it means a treatment that kills cells by raising the biological temperature of 37 ℃ to 40-45 ℃. do. After injecting a diagnostic and therapeutic composition according to the present invention into a living body and confirming a portion (tumor) that emits a signal, a magnetic field is applied to the portion to generate heat, thereby selectively killing only tumor cells.

Another aspect is a treatment method that can be selected when the substance itself that can specifically bind a tumor marker has a cytotoxic effect. Specifically, the antibodies described above, such as Herceptin and Rituxan, can not only bind tumor cells specifically but can also kill tumor cells by themselves. Therefore, if the substance that can specifically bind the tumor marker is an antibody such as Herceptin or Rituxan can not only diagnose the tumor, but also kill the tumor cells to treat the tumor.

Another embodiment is a case where the nanohybrid particles according to the present invention are used as carriers of an anticancer agent, and the nanohybrid particles are preferably combined with an anticancer agent to a substance capable of specifically binding to a tumor marker. Particles can concentrate on tumor cells to diagnose tumors, as well as to selectively kill only tumor cells without affecting normal cells by focusing anticancer agents on the tumor site.

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

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

<Example 1>

Preparation of Iron Oxide-HERCEPTIN NanoHybrid Particles for Breast Cancer Diagnosis

100 μl of Herceptin [(10 mg / ml, in 10 mM phosphate buffer, pH7.2) Genentech, Inc., South San Francisco, CA, USA] was taken in an Eppendorf tube and 0.2 mg of sulfo-SMCC [40 ( N-Maleimidomethyl) cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxy-succimide ester] was added and reacted for 30 minutes at room temperature to replace the lysine residue of Herceptin with a maleimide group. Excess sulfo-SMCC molecules were removed through the Sephadex G-25 column, and then Herceptin substituted with Malade group was dissolved in a solution containing 200 μl of water-soluble iron oxide nanoparticles (10 mM PB, pH 7.2, 2 mg / ml) for 24 hours. The reaction was carried out at room temperature. After the reaction, the unreacted Herceptin and iron oxide water-soluble nanoparticles were removed through the Sephacryl S-300 column, and then concentrated to a concentration of about 2 mg / ml using a centricon filtration kit. Nanohybrid particles were prepared.

The binding of the water-soluble iron oxide nanoparticles and Herceptin was confirmed using a dynamic laser light scattering method (Brookheaven, BI-200SM). Specifically, each of the water-soluble iron oxide nanoparticles and the water-soluble iron oxide nanoparticles bound to Herceptin were diluted in 10 ml of water, passed through a 500 nm PTFE syringe filter to remove impurities, and then loaded into a DLS device and scanned 60 times. This scanning result is shown in FIG. 3. As shown in FIG. 3, as the water-soluble iron oxide nanoparticles bind with Herceptin, its size increased from 9 nm to 28 nm.

<Example 2>

Confirmation of Tumor Cell Selectivity of Iron Oxide-Herceptin Nanohybrid Particles in Vitro

In vitro magnetic resonance imaging and flow cytometry were performed simultaneously to analyze the binding specificity and efficiency of the HER2 / neu antigen, the breast cancer marker antigen, of the iron oxide-Herceptin nanohybrid particles prepared in Example 1 above.

The process of reacting the iron oxide-Herceptin nanohybrid particles to HER2 / neu antigen non-expressing, expression and overexpressing cell lines was as follows. The cell lines were first isolated in culture dishes using 0.25% trypsin / EDTA at room temperature. Iron oxide-Herceptin nanohybrid particles were added to 50 μl PBS buffer containing 10 ⁷ cells at a concentration of 20 μg / ml on an antibody basis. It was left on ice for about 30 minutes and washed three times. In order to find the nanohybrid particles in which the primary antibody (Herceptin) and the iron oxide water-soluble nanoparticles were combined, the reaction was performed with the FITC polymerized secondary antibody for about 20 minutes. On the other hand, as a control, nanohybrid particles containing an anti-HMEN-B3 antibody (hereinafter referred to as "control nanohybrid particles") were used.

A. Flow Cytometry

Antigen specificity and efficiency of iron oxide-Herceptin nanohybrid particles were measured using a FACScan flow cytometer (Becton Dickinson, San Diego, Calif.). Ten thousand events were measured for each cell line. The fluorescent index was used as the average value and the median range of the fluorescence intensity distribution. Iron oxide-Herceptin nanohybrid particles and control nanohybrid particles were treated with cell lines expressing HER2 / neu receptors, respectively, and reacted with FITC polymerized secondary antibodies as described above to determine whether fluorescence was detected using fluorescence microscopy. Confirmed and the results are shown in FIG.

As shown in Figure 4, as the expression level of the HER2 / neu receptor was increased it was confirmed that the fluorescence expression intensity also increased. In the BX-PC-3 cell line with low HER2 / neu receptor expression level (FIG. 4), the fluorescence expression intensity was slightly increased compared with the control nanohybrid particles (control of FIG. 4). As the expression level increased (BX-PC-3 cell line <MDA-MB-231 cell line ≤ BT-474 cell line <NIH3T6.7 cell line) it was confirmed that the fluorescence expression intensity gradually increased.

B. Analysis by Magnetic Resonance Imaging

In order to analyze the antigen specificity of the iron oxide-Herceptin nanohybrid particles using magnetic resonance imaging, each cell was transferred to a PCR tube and centrifuged to sink the cells. 1.5 T (Intera; Philips Medical Systems, Best, The Netherlands) system was used to see the contrast effect of magnetic resonance imaging according to antigen specificity of each cell line, and micro-47 coil was used. Coronal images were obtained with Fast Field Echo (FFE) pulse trains (FIG. 5). Specific parameters were as follows: resolution 156 x 156 mu m, section thickness 0.6 mm, TE = 20 ms, TR = 400 ms, image excitation count 1, image acquisition time 6 minutes. T2 map was performed to quantitatively evaluate the magnetic resonance imaging contrast effect on antigen specificity. Specific parameters were as follows: resolution 156 x 156 μm, section thickness 0.6 mm, TR = 4000 ms, TE = 20, 40, 60, 80, 100, 120, 140, 160 ms, image excitation count 2, image acquisition time 4 minutes .

The results of FIG. 5 were consistent with the fluorescence expression results shown in FIG. 4, and as the degree of HER2 / neu receptor expression was gradually increased, the magnetic resonance imaging signal gradually became gray and black. In the case of BX-PC-3 cell line with low expression level (1 in FIG. 4), it was found to turn slightly darker than when using the control nanohybrid particles (control in FIG. 4), and as the expression level of the receptor was increased (BX -PC-3 cell line <MDA-MB-231 cell line <BT-474 cell line <NIH3T6.7 cell line) It was confirmed that the black color gradually. The iron oxide-herceptin nanohybrid particles selectively show magnetic resonance imaging signals by selectively binding to cell lines expressing the HER2 / neu receptor. The iron oxide-herceptin nanohybrid particles of the present invention can be used for diagnosing in vitro breast cancer. It could be confirmed.

<Example 3>

Confirmation of Tumor Cell Selectivity in Vivo of Iron Oxide-Herceptin Nanohybrid Particles

Iron oxide-Herceptin nanohybrid (400 μg Fe, n = 5) particles were administered through the tail vein to perform target imaging of a mass formed by a cell line overexpressed with HER2 / neu antigens formed in the proximal thigh of the nude mouse. Immediately, images were taken at 1, 2 and 4 hours. At this time, the control nanohybrid particles were imaged according to the same method was used as a control.

The specific imaging method was as follows. A 1.5 T (Intera; Philips Medical Systems, Best, The Netherlands) system was used and a micro-47 coil was used. Coronal images were obtained with Fast Field Echo (FFE) pulse trains (FIG. 6). Specific parameters were as follows: resolution 234 x 234 μm, section thickness 0.6 mm, TE = 20 ms, TR = 400 ms, image excitation count 4, image acquisition time 6 minutes. T2 map was performed to quantitatively evaluate the magnetic resonance imaging contrast effect on the antigen specificity. Specific parameters were as follows: resolution 234 x 234 μm, section thickness 3 mm, TR = 4000 ms, TE = 20, 40, 60, 80, 100, 120, 140, 160 ms, image excitation count 2, image acquisition time 4 minutes.

As shown in FIG. 6, it can be observed that magnetic control signals of breast cancer marker overexpressing tumors hardly change before and after nanohybrid particle administration when the control nanohybrid particles are used (FIG. 6A), whereas iron oxide-Herceptin nanohybrid It can be seen that the magnetic image signal is gradually reduced when the particles are used (B of FIG. 6). Nuclear spin-spin relaxation (T2) values were compared to quantify these results (FIG. 6C). The control nanohybrid particles showed no change in T2 values before and after administration, whereas T2 after administration of iron oxide-Herceptin nanohybrid particles. It was confirmed that the value continued to decrease. This indicates that the iron oxide-herceptin nanohybrid particles selectively bind to tumors expressing breast cancer markers, and it was confirmed that the iron oxide-herceptin nanohybrid particles according to the present invention can be used for diagnosing breast cancer.

<Example 4>

Tissue Immunostaining Verification of Tumor Cell Selectivity in Vivo of Iron Oxide-Herceptin Nanohybrid Particles

Masses in nude mice (NIH3T6.7) were isolated and fixed with 4% paraformaldehyde. After fixation, tissues were sealed using O.C.T compound (Sakura, Tokyo, Japan), sections of about 10 ㎛ were prepared, and immunostaining was performed. The primary antibody for immunostaining was Herceptin, and the secondary antibody was a fluorescein-conjugated goat IgG fraction (ICN Biomedicals, Inc. Aurora, OH 44202) for the Fc portion of human immunoglobulins (IgG, IgA, IgM). . For immunostaining of vascular endothelial cells, purified anti-mouse CD31 (PECAM-1) (A Becton Dickinson Co.) was used as the primary antibody, and the secondary antibody was Cy5 goat anti-rat IgG + IgM (Biomeda corp). .) Was used. Immunofluorescence was analyzed using confocal microscopy (Olympus Optical, Tokyo, Japan) and the results are shown in the figure.

As shown in FIG. 7, when the cancer cells express green fluorescence, it was confirmed that the iron oxide-Herceptin nanohybrid particles according to the present invention can be selectively bound to breast cancer antigen overexpressing tumor cells and used for diagnosing breast cancer.

<Example 5>

Preparation of Iron Oxide-PEG-Folic Acid Nanohybrid Particles for Ovarian Cancer Diagnosis

Take 100 μl of folic acid [(10mg / ml, in 10mM phosphate buffer, pH7.2) Genentech, Inc., South San Francisco, CA, USA], put into eppendorf tube, add 0.2mg PEG at room temperature The reaction was carried out for 30 minutes to replace one amine group of PEG having an amine group at both ends with folic acid. Excess folic acid molecules were removed through a Sephadex G-25 column, and then the terminal amine group was substituted with a maleimide group by addition of sulfo-SMCC to the resulting PEG-folic acid. The excess sulfo-SMCC was removed through a Sephadex G-25 column and reacted with a solution containing 200 μl of water-soluble iron oxide nanoparticles (10 mM PB, pH 7.2, 2 mg / ml) at room temperature for 24 hours. After the reaction, the unreacted PEG-folic acid and water-soluble iron oxide nanoparticles were removed through a Sephacryl S-300 column, and then concentrated to a concentration of about 2 mg / ml using a centricon filtration kit. -PEG-folic acid nanohybrid particles were prepared.

<Example 6>

In Vitro Ovarian Cancer Diagnosis Using Iron Oxide-PEG-Folic Acid Nanohybrid Particles

In vitro magnetic resonance imaging was performed to analyze the binding specificity and efficiency of the iron oxide-PEG-folic acid nanohybrid particles prepared in Example 5 to the folate receptor.

The reaction of the iron oxide-PEG-folic acid nanohybrid particles to the folate receptor unexpressed, expressed and overexpressed cell lines was as follows. The cell lines were first isolated in culture dishes using 0.25% trypsin / EDTA at room temperature. Iron oxide-PEG-folic acid nanohybrid particles were added to 50 μl PBS buffer containing 10 ⁷ cells at a concentration of 20 μg / ml based on iron. It was left on ice for about 30 minutes and washed three times.

In order to analyze the antigen specificity of the nanohybrid particles using the magnetic resonance imaging, each cell was transferred to a PCR tube and centrifuged to sink the cells. 1.5 T (Intera; Philips Medical Systems, Best, The Netherlands) system was used to see the contrast effect of magnetic resonance imaging according to antigen specificity of each cell line, and micro-47 coil was used. Coronal images were obtained with Fast Field Echo (FFE) pulse trains (FIG. 8). Specific parameters were as follows: resolution 156 x 156 mu m, section thickness 0.6 mm, TE = 20 ms, TR = 400 ms, image excitation count 1, image acquisition time 6 minutes. T2 map was performed to quantitatively evaluate the magnetic resonance imaging contrast effect on antigen specificity. Specific parameters were as follows: resolution 156 x 156 μm, section thickness 0.6 mm, TR = 4000 ms, TE = 20, 40, 60, 80, 100, 120, 140, 160 ms, image excitation count 2, image acquisition time 4 minutes .

As shown in FIG. 8, the magnetic resonance imaging signal of the folate receptor expression (A431 cell line <SKOV cell line <OVCAR cell line) gradually turned black. This means that the iron oxide-PEG-folic acid nanohybrid particles selectively show magnetic resonance imaging signals in black by selectively binding to cell lines expressing the folic acid receptor. The iron oxide-PEG-folic acid nanohybrid particles according to the present invention are ex vivo. It can be seen that it can be used to diagnose ovarian cancer.

<Example 7>

Confirmation of Breast Cancer Cell Killing Effects of Iron Oxide-Herceptin Nanohybrid Particles

Trypan Blue Exclusion Assay was performed to determine whether the iron oxide-Herceptin nanohybrid particles prepared in Example 1 can selectively kill cells in breast cancer.

Trypan blue staining was performed at 12.5, 25, 50, 100, 200 μg / ml concentrations in culture containing ^{7 7} HER2 / neu antigen-expressing cells (NIH3T6.7) or unexpressed cells (HeLa). Is shown in FIG. 9.

As shown in FIG. 9, the iron oxide-Herceptin nanohybrid particles did not have any effect on the HER2 / neu antigen-expressing cells, whereas the HER2 / neu antigen-expressing cells were killed at a concentration of 25 µg / ml or higher. .

The nanohybrid particles according to the present invention can be stably dispersed and diffused in an aqueous solution, thereby improving the efficiency and accuracy of tumor diagnosis and treatment. In addition, the nanohybrid particles according to the present invention can be used to diagnose and treat tumors at once.

Claims (14)

Co, Mn, Fe, Ni, Gd, MM ' ₂ O ₄ , M _x O _y , magnetic material selected from the group consisting of (M or M '= Co, Fe, Ni, Mn, Zn, Gd, Cr 0 <x≤3, 0 <y≤5), CoCu, CoPt, FePt , Nanoparticles having a diameter of 1 to 1000 nm comprising a magnetic alloy material selected from the group consisting of CoSm, NiFe and NiFeCo or a multicomponent hybrid structure comprising two or more components selected from the magnetic material and the magnetic alloy. An attachment region for binding to a nanoparticle comprising a functional group selected from the group consisting of NH ₂ , —SH, —CONH _2, —PO ₃ H, —PO ₄ H, —SO ₃ H, —SO ₄ H, and —OH, And -SH, -COOH, -NH ₂ , -OH, -NR ₄ ⁺ X and a crosslinking region comprising a functional group selected from the group consisting of SH, -NH ₂ , -COOH, -OH, epoxy, -ethylene and -acetylene ^-, - sulfonate, - nitrate and phosphonate groups can be specific binding to the tumor marker containing functional groups selected from the consisting of It is surrounded by a multifunctional ligand comprising an active ingredient binding region including an active ingredient, wherein the cross linking region is cross-linked with the cross linking region of another multifunctional ligand, and the active ingredient binding region specifically includes a tumor marker. A substance capable of binding is bound, and a substance capable of specifically binding to the tumor marker is C2, annexin V, integrin, VEGF, angiopoietin 1, angiopoietin 2, synaptotagmine I, For diagnosing or treating tumors, characterized in that it is a substance capable of specifically binding to a tumor marker selected from the group consisting of somatostatin, vasointestinal peptide, cancerous fetal antigen, HER2 / neu antigen, prostate specific antigen and folic acid receptor Nanohybrid Particles. delete delete delete delete delete delete delete delete A tumor diagnostic or therapeutic composition comprising the nanohybrid particles for diagnostic or therapeutic use according to claim 1 and a pharmaceutically acceptable carrier. 11. The tumor diagnostic or therapeutic composition according to claim 10, formulated for intravenous, intraperitoneal, intramuscular, subcutaneous or topical administration. (1) injecting the tumor diagnostic or therapeutic composition according to claim 10 into a living body or a sample; And (2) diagnosing the presence of a tumor by sensing a signal emitted by the nanohybrid particles from the living body or sample; Or (3) a method for diagnosing or treating tumors in mammals other than humans, including selectively killing identified tumor cells. The method for diagnosing or treating tumors in mammals other than humans according to claim 12, wherein a magnetic resonance imaging apparatus is used to detect the signals. The method for diagnosing or treating tumors in mammals other than humans according to claim 12, characterized in that only a tumor cell is selectively killed by applying a magnetic field to a portion where a signal is emitted to generate heat.

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