JP2007269770A - Functional magnetic ultra-nanoparticles and their applications - Google Patents

Abstract

Magnetic ultra-nano-particles that have functional groups such as amino groups covalently modified on the surface of the magnetic ultra-nano-particles have been developed to modify nucleic acids, peptides (proteins), drugs, etc. And a functional material using the same.
[Solution]
Functional magnetic ultra-nanoparticles obtained by modifying the surface of fine particles coated with silica with functional groups such as amino groups by silanization, etc., and binding complexes of DNA, proteins (peptides), drugs, etc. using the same, and , Novel drug transport agents and transport systems thereof.
[Selection] Figure 2

Description

    The present invention relates to a functional magnetic ultra-nanoparticle having a functional group introduced on the surface of a shell, a method for producing the same, a composite containing the same, and a method for using the fine particles and the composite.

In recent years, with the improvement of the fine particle production technology, it has become possible to easily prepare fine particles with a very small monodisperse size (average particle diameter of about 1 nm to several hundreds nm) (Non-patent Document 1). On the other hand, magnetic fine particles in which fine particles are made magnetic have also been developed (Non-Patent Documents 2 to 5), which do not require work such as centrifugation when collecting the fine particles, and place them in a place where magnetism is applied. Separation is very easy because of the concentration of fine particles, and the possibility of crushing the fine particles due to excessive centrifugal speed, that is, gravitational acceleration (g) during centrifugation can be avoided. Has a problem that the particle size is as large as micrometer due to its preparation method, and nonspecifically magnetic substances such as iron oxide are adsorbed on the outside of the fine particles. Fine particles having a nanometer order are called “nano fine particles”, and nano particles having an average particle diameter of 3 nm or less are sometimes called “ultra-nano particles.” The fine particles may be referred to as “magnetic nanoparticle” or “magnetic ultra-nanoparticle”.
When trying to prepare nanometer-order magnetic fine particles, there are dry methods such as sputtering, vacuum deposition, and gas reaction, and wet methods such as coprecipitation and pyrolysis colloid. In general, the fine particles have a tendency to agglomerate and become bulky, and thus have problems in the separation method and the technique of uniform dispersion in an arbitrary medium. In the preparation of the fine particles, the above problem was solved by using a wet precipitation method. For example, Ichiyanagi et al. Succeeded in preparing magnetic ultra-nanoparticles coated with silica on the surface from a mixed solution of FeCl ₂ · 4H ₂ O and Na ₂ SiO ₃ · 9H ₂ O. Nanoparticles existed stably, and the average particle diameter was 10 nm or less. (Patent Documents 1 to 3 and Non-Patent Documents 3 and 5).

    As for the method of using nano-particles, a method of separating only a target substance from nucleic acids and proteins, which are biological substances, using the micro-particles has been developed (Patent Document 4 and Non-Patent Documents 6 and 7). For example, Patent Document 4 and Non-Patent Document 7 include a repeating unit obtained by reacting a monomer having two amino groups and one carboxyl group with diglycidyl ether by the W / O miniemulsion method. A system is described in which pH-responsive zwitterionic microparticles having two amino groups, one carboxyl group, and two hydroxyl groups are prepared and nucleic acid can be incorporated and released by only pH change. ing. Moreover, the composite_body | complex of nanoparticle and desired substances, such as a drug, can also be produced. However, in order to introduce nanoparticles or nanoparticle composites into cells, it is necessary to treat the nanoparticles with a cation to make them cationic, and in addition to having toxicity due to cations, There was a defect that the cells stayed in the cells without being metabolized and the cells were killed. Furthermore, introduction of a drug or the like into a cell via a membrane protein (folate receptor (Non-patent Document 8), amino acid transporter (Non-patent Documents 9 and 10), etc.) that is highly expressed on the surface of cancer cells It has already been reported (Patent Documents 6 and 7, Non-Patent Document 11). By applying this to folic acid modification on the surface of fine particles, a cancer cell-specific delivery method can be established and used in gene delivery methods (Non-Patent Document 12) and imaging methods (Non-Patent Document 13). However, in this case, the preparation of nano-particles is complicated because it is multi-staged, and the nano-particles are not efficient because of their large particle size. That is, it was difficult to accumulate nanoparticles or nanoparticle composites in a predetermined tissue or cell.

Regarding the method of using the nanoparticle composite, in addition to the above, a peptide on the surface of the microparticle,
A method of modifying a ligand such as a nucleic acid and identifying an intracellular protein that interacts with the ligand is also known, but this method involves adding a microparticle complex after cell disruption (in vitro) (non-in vitro). Patent Document 6).
In addition, J. Won et al. Modified a substance that interacts with intracellular proteins into cation-coated nanoparticles and incorporated them into living cells to identify target proteins (Non-patent Document 14). However, even in this case, the particle size of the nanoparticle is large, and in order to introduce the nanoparticle into the cell, a treatment to make the particle cationic is necessary (Non-Patent Documents 14 and 15), and is not completely nontoxic. . A solution to detoxification has not been established. In addition, it is difficult to supplement a target substance from cells that cannot be cultured in vitro with existing techniques, and it is necessary to locally introduce a nontoxic substance into a biological solid.
Furthermore, a substance and a method for recovering cells by magnetic force by modifying an antibody that interacts with a cell membrane protein on the surface of the magnetic nanoparticle have been reported, but in this case, the particle-antibody complex is not incorporated into the cell ( Patent Document 5).
As described above, the method for modifying the surface of a nanoparticle or magnetic nanoparticle and the nanoparticle or magnetic nanoparticle with a modified surface are known, but the magnetic ultrananoparticle with a modified surface is known. It was not done.
JP 2003-252618 A JP 2001-261334 A JP 2005-200437 A JP 2006-077037 A Special table 2003-533363 gazette Japanese translation of PCT publication No. 2004-529642 International Publication No. 2004/032966 Pamphlet Du YZ, Tomohiro T, Zhang G, Nakamura K, Kodaka M. Chem Commun 2004. 5, 616-617. Yamazaki N .; Du Y.-Z .; Nagai M .; Omi S. Colloids and Surfaces B: Biointerfaces, 2003. 29, 159-169 Y. Ichiyanagi, T. Uozumi, Y Kimisima. Ttansactions of the Materials Research Society of Japan. 2001, 279, 1097-1100 Nakayama H., Arakaki A., Maruyama K., Takeyama H., MatsunagaT. Biotech. And Bioeng., 2003, 84, 96-102 Y. Ichiyanagi, Y Kimisima. J. Thermal Analysis and Colorimetry. 2002, 69, 919-923 Tomohiro, T. et al., Bioconjugate Chem .; 2002; 13 163-166 S. Taira, YZ Dong, M. Kodaka, Biotechnology and Bioengineering, 2006 93, 396-400 SD Weitman, RH Lark, LR Coney, DW Fort, V. Frasca, VR Zurawski Jr and BA Kamen, Cancer Res. 1992, 52, 3396-3401 N. Gupta, PD. Prasad, S. Ghamande, P. Moore-Martin, AV. Herdman, RG. Martindale, R. Podolsky, S. Mager, ME. Ganapathy, V. Ganapathy, Gynecol Oncol. 2006, 100, 8 -13. N. Gupta, S. Miyauchi, RG. Martindale, AV. Herdman, R. Podolsky, K. Miyake, S. Mager, PD. Prasad, ME. Ganapathy, V. Ganapathy, Biochim. Biophys. Acta. 2005, 30, 215-23. T. Hatanaka, M. Haramura, YJ Fei, S. Miyauchi, CC Bridges, PS Ganapathy, SB Smith, V. Ganapathy, ME Ganapathy. J. Pharmacol. Exp. Ther. 2004, 308, 1138-1147 (2004). G. Zuber, L. Zammut-Italiano, E. Dauty and JP Behr, Angew. Chem. Int. Ed. 2006, 42, 2666-2669 C. Sun, R. Sze, M. Zhang, J. Biomed Mater. Res. A, 2006, 78, 550-557 J Won, M Kim, Yong-Weon Yi, YH Kim, N. Jung, TK Kim, Science 2005, 309, 121-125 Y. Jun, Y. Huh, J. Choi, J. Lee, H. Song, S. Kim, S. Yoon, K. Kim, J. Shin, J. Suh, J. Cheon JACS, 5732-5733

  An object of the present invention is to provide a functional magnetic ultra-nanoparticle having a functional group introduced on the surface of a shell, and a method for producing the same. It is another object of the present invention to provide a composite containing the functional magnetic ultra-nano fine particles and a method for using the fine particles and the composite.

As a result of intensive studies to solve the above-mentioned problems, the present inventor obtained functional groups such as amino groups by silanization into magnetic ultra-nanoparticles prepared monodisperse on the order of 1.3 to 3 nm using a wet precipitation method. Was found to be capable of preparing functional magnetic ultra-nanoparticles. And it discovered that a drug can be delivered to a predetermined tissue or cell in a subject and its delivery can be monitored by concentrating magnetism by using a complex of the functional magnetic ultra-nanoparticles and a drug. Further, it has been found that the distribution of a desired substance in a predetermined tissue or cell can be imaged using a complex of the functional magnetic ultra-nanoparticles and a ligand for the desired substance. Furthermore, by applying magnetism, the above complex can be directly introduced into living cells and tissues in the subject, and these cells and tissues can be allowed to survive, without applying magnetism to cultured cells, Moreover, it discovered that it could introduce | transduce into a cell, without using a cation. We also found cell-selective delivery by modifying the substrate that binds to membrane proteins on the surface to further functionalize the functional magnetic ultra-nanoparticles. Based on these findings, we established a method for selecting a drug target substance from living cells and tissues using the complex and a method for selecting a desired substance that specifically interacts with a ligand.
The present invention has been accomplished based on these findings.

That is, according to the present invention, the following inventions are provided.
(1) (a) a core made of a metal oxide,
(B) a shell made of an amorphous silica network covering the core;
(C) Functional magnetic ultra-nanoparticles having an average particle size of 1.3 to 3 nm having functional groups covalently introduced to the surface of the shell.
(2) The functional magnetic ultra-nanoparticle according to (1), wherein the functional group is introduced via a silane coupling agent.
(3) The functional group is at least one selected from the group consisting of an amino group, an isocyanate group, a mercapto group, a vinyl group, an epoxy group, a methacryloxy group, a ureido group, a chloropropyl group, and a sulfide group, (1) or (2) Functional magnetic ultra-nanoparticles.
(4) A silane coupling agent on the surface of fine particles having (a) a core made of a metal oxide, and (b) a shell made of an amorphous silica network covering the core, the shell having a hydroxyl group on the surface. A method for producing functional magnetic ultra-nanoparticles according to any one of (1) to (3), wherein a functional group is covalently introduced through the hydroxyl group.
(5) A drug comprising the functional magnetic ultra-nanoparticle according to any one of (1) to (3) and a drug, wherein the drug is bound via a functional group on the surface of the microparticle Binding complex.
(6) The drug-binding complex according to (5), wherein the functional magnetic ultra-nanoparticles or drug is labeled with a labeling substance.
(7) The functional magnetic ultra-nanoparticle according to any one of (1) to (3) and a ligand that specifically interacts with a desired substance, and the ligand is bonded via a functional group on the surface of the particle. A ligand-binding complex characterized by being bound to a functional magnetic ultra-nanoparticle.
(8) The ligand-binding complex according to (7), wherein the functional magnetic ultra-nanoparticle or ligand is labeled with a labeling substance.
(9) (a) administering the drug binding complex of (5) or (6) to a non-human subject,
(B) Concentrating magnetism from outside the non-human subject to the site of the non-human subject where a predetermined tissue or cell exists, and accumulating or retaining the drug-binding complex in the site to cause the tissue or cell to Delivering the drug-binding complex to a predetermined tissue or cell.
(10) (a) administering the drug binding complex of (6) to a non-human subject,
(B) Concentrating magnetism from outside the non-human subject to the site of the non-human subject where a predetermined tissue or cell exists, and accumulating or retaining the drug-binding complex in the site to cause the tissue or cell to Delivering a drug binding complex;
(C) detecting the presence of a labeling substance localized in the tissue or cell, and monitoring the delivery of the drug to a predetermined tissue or cell. (11) (a) administering the ligand-binding complex of (8) to a non-human subject,
(B) Concentrating magnetism from the outside of the non-human subject to the site of the non-human subject where a predetermined tissue or cell exists, and accumulating or retaining the ligand-binding complex in the site to Delivering a ligand binding complex;
(C) Presence of a labeling substance localized or distributed in the tissue or cell by dispersing or metabolizing the drug-binding complex that has accumulated or stayed at the site after being demagnetized or delivered to the tissue or cell Detecting a distribution of a desired substance in a predetermined tissue or cell.
(12) (a) administering the drug-binding complex of (5) or (6) to a non-human subject,
(B) Concentrating magnetism from outside the non-human subject to the site of the non-human subject where a predetermined tissue or cell exists, and accumulating or retaining the drug-binding complex in the site to cause the tissue or cell to Delivering a drug binding complex to bind the drug binding complex in vivo with a drug target substance present in the tissue or cell;
(C) A method for selecting a target substance of a drug, comprising a step of recovering a binding substance of the target substance and the drug-binding complex by magnetically adsorbing from the tissue or cells.
(13) (a) adding the drug-binding complex of (5) or (6) to cultured cells, and binding the drug-binding complex to a drug target substance present in the cultured cells;
(B) A method of selecting a target substance for a drug, which comprises a step of recovering the bound substance of the target substance and the drug-binding complex by magnetically adsorbing from the cultured cells.
(14) (a) administering the ligand-binding complex of (7) or (8) to a non-human subject,
(B) Concentrating magnetism from the outside of the non-human subject to the site of the non-human subject where a predetermined tissue or cell exists, and accumulating or retaining the ligand-binding complex in the site to Delivering a ligand binding complex to bind the ligand binding complex in vivo with a desired substance present in the tissue or cell;
(C) a step of recovering the binding substance of the substance and the ligand-binding complex from the tissue or cells by magnetically adsorbing the desired substance that specifically interacts with the ligand, How to choose.
(15) A step of adding the ligand-binding complex of (a) (7) or (8) to the cultured cell and binding the desired substance present in the cultured cell with the ligand-binding complex,
(B) recovering the bound substance of the substance and the ligand-binding complex from the cultured cells by magnetically adsorbing the desired substance that specifically interacts with the ligand, How to choose.
(16) A medicament comprising the drug complex of (5) or (6).

  The functional magnetic ultra-nanoparticles of the present invention exhibit a functional group such as an amino group, an isocyanate group, or a mercapto group on the surface, and are therefore very suitable for modifying other substances covalently. In addition, since the functional magnetic ultra-nanoparticles have magnetism, it is easy to concentrate on a single point by applying magnetism from a state where they are uniformly diffused in the solution, which is very suitable for recovery of the target substance. ing. In addition, since the magnetic ultra-nanoparticles can be taken into living cells, a substance such as a protein that interacts with these ligands can be identified in vivo by modifying the surface of the magnetic ultra-nanoparticles with a ligand. Moreover, since it can introduce | transduce into a cultured cell, without applying magnetism, without using a cation, substances, such as a protein which interacts with the ligand in a cultured cell, can also be identified easily. Furthermore, by modifying the drug on the surface of the magnetic ultra-nanoparticle, it becomes a drug-containing magnetic ultra-nanoparticle, and after oral administration, the drug can be concentrated intensively by applying magnetism locally to the affected area. Therefore, it is possible to develop a drug with enhanced immediate effect selectively.

  The present invention will be described in detail below, but these descriptions are examples (representative examples) of embodiments of the present invention, and do not limit the scope of the present invention.

(1) Magnetic ultra-nano fine particles First, the magnetic ultra-nano fine particles used as a material when producing the functional magnetic ultra-nano fine particles of the present invention will be described.
The magnetic ultra-nano fine particles have a form having (a) a core made of a metal oxide, and (b) a shell made of an amorphous silica network (a network film) covering the core. Although an average particle diameter should just be 3 nm or less, Preferably it is 1.3-3 nm. The average particle diameter can be determined by, for example, X-ray diffraction measurement or transmission electron microscope (TEM) measurement (Japanese Patent Laid-Open No. 2003-252618).

上記式中、Mは遷移金属または稀土類金属を示し、好ましくはNiまたはFeであり、より好ましくはFeである。XはF, Cl, Br, Iから選ばれるハロゲン元素を示す。pは２または３、nは０から９までの整数、mは９または０である。xおよびyはともに１未満の正数である。磁性を発揮するコアおよびその表面修飾層としてのシェルの役割分担の観点から、ｘ＞ｙ、かつ、ｘ／ｙとしては、通常１〜１００、好ましくは２〜２０程度の範囲から選定される。
より具体的には、磁気超ナノ微粒子 [xM(OH)₂・ySiO₂]の構造は、図１のように模式的に表すことができる。 The magnetic ultra-nanoparticles are preferably prepared using a wet precipitation method (Japanese Patent Laid-Open No. 2003-252618). For example, magnetic ultra-nanoparticles [xM (OH) ₂ · ySiO ₂ ] given by the following formula (I) can be mentioned. Here, xM (OH) ₂ represents a core and ySiO ₂ represents a shell.

In the above formula, M represents a transition metal or a rare earth metal, preferably Ni or Fe, and more preferably Fe. X represents a halogen element selected from F, Cl, Br, and I. p is 2 or 3, n is an integer from 0 to 9, and m is 9 or 0. x and y are both positive numbers less than 1. From the viewpoint of role sharing of the core exhibiting magnetism and the shell as the surface modification layer, x> y and x / y are usually selected from the range of about 1 to 100, preferably about 2 to 20.
More specifically, the structure of the magnetic ultrananoparticle [xM (OH) ₂ · ySiO ₂ ] can be schematically represented as shown in FIG.

(2) Method for preparing functional magnetic ultra-nanoparticles The functional magnetic ultra-nanoparticles of the present invention have functional groups covalently introduced onto the surface of the shell of the magnetic ultrananoparticles prepared in (1). If it is. That is, the magnetic ultra-nanoparticle of the present invention comprises (a) a core made of a metal oxide, (b) a shell made of an amorphous silica network covering the core, and (c) a functional group covalently introduced onto the surface of the shell. It has the form which has a group. Although an average particle diameter should just be 3 nm or less, Preferably it is 1.3-3 nm.
The functional group to be introduced may be any functional group as long as it has reactivity or affinity so that it can form a complex with a drug or a ligand after being introduced to the surface of the shell. An amino group, an isocyanate group, a mercapto group, etc. are mentioned.
These functional groups can be introduced covalently through, for example, a silane coupling agent. More specifically, for example, the structure of the shell surface of the functional magnetic ultra-nanoparticle into which an amino group is introduced can be expressed as shown in FIG.
As described above, the silane coupling agent preferably has a functional group having reactivity or affinity that can form a complex with a drug or a ligand. For example, as the silane coupling agent used for introducing an amino group, any compound having this functional group may be used. Specifically, 3-aminopropyltriethoxysilane (γ-APTES), N-2 (aminoethyl) 3-aminopropylmethyldimethoxysilane, N-2 (aminoethyl) 3-aminopropyltrimethoxysilane, N-2 (aminoethyl) 3-aminopropyltriethoxysilane, 3-aminopropyltri Mention may be made of methoxysilane, 3-triethoxysilyl-N- (1,3-dimethyl-butylidene) propylamine, N-phenyl-3-aminopropyltrimethoxysilane. In particular, γ-APTES is preferably used.
As the silane coupling agent used for introducing an isocyanate group, any compound having this functional group may be used, and specific examples include 3-isocyanatopropyltriethoxysilane.
As the silane coupling agent used for introducing a mercapto group, any compound having this functional group may be used, and specific examples include 3-mercaptopropylmethyldimethoxysilane and 3-mercaptopropyltrimethoxysilane. Can be mentioned.
The silane coupling agent used for introducing the vinyl group may be any compound having this functional group, and specifically, vinyltrichlorosilane, vinyltrimethoxysilane, vinyltriethoxysilane. Can be mentioned.
Furthermore, as the silane coupling agent used for introducing the epoxy group, any compound having this functional group may be used. Specifically, 2- (3,4-epoxycyclohexyl) ethyltrimethoxy Examples include silane, 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropyltriethoxysilane, and styryl p-styryltrimethoxysilane.
In addition, as the silane coupling agent used for introducing a methacryloxy group, any compound having this functional group may be used. Specifically, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxy Mention may be made of propyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, acryloxy-3-acryloxypropyltrimethoxysilane.
As the silane coupling agent used for introducing the ureido group, any compound having this functional group may be used, and specific examples include 3-ureidopropyltriethoxysilane.
As the silane coupling agent used for introducing the chloropropyl group, any compound having this functional group may be used, and specific examples include 3-chloropropyltrimethoxysilane.
As a silane coupling agent used for introducing a sulfide group, any compound having this functional group may be used, and specific examples thereof include bis (triethoxysilylpropyl) tetrasulfide.
Hereinafter, the introduction of the functional group through the silane coupling agent as described above may be referred to as “silanization” or “silanization treatment”.
Before performing the silanization treatment, it is preferable that the surface of the magnetic ultra-nano fine particles is washed to remove organic substances and expose many hydroxyl groups that can react with the silane coupling agent on the fine particle surfaces. Specifically, cleaning with an organic solvent such as ethanol, UV cleaning (JP-A-1-146330), plasma treatment, and the like can be given.

(3) Complex of functional magnetic ultra-nanoparticle and drug The functional magnetic ultra-nanoparticle obtained in (2) above has a functional group on its surface, so that a desired drug can be obtained via these functional groups. Can be combined to form a complex. Here, the bond between the functional magnetic ultra-nanoparticle and the desired drug is any bond such as a covalent bond, an ionic bond, or a hydrogen bond as long as it is via a functional group introduced on the surface of the functional magnetic ultra-nanoparticle. May be.
The desired drug is, for example, a pharmacologically active substance, physiologically active substance, and / or diagnostic substance that is pharmaceutically acceptable depending on the purpose of diagnosis and / or treatment.
The type of drug for treatment is not particularly limited as long as it does not impair the formation and stability of the complex, but includes nucleic acids, polynucleotides, genes and analogs thereof, glycosaminoglycans and derivatives thereof, oligosaccharides, polysaccharides and Their derivatives, vitamins and their derivatives, physiologically active substances such as proteins and peptides, and low molecular weight compounds with pharmacological activity. More specifically, anticancer agents, antibiotics, enzyme agents, antioxidants, lipid initiatives Inhibitor, Hormonal agent, Anti-inflammatory agent, Steroid agent, Vasodilator, Angiotensin receptor antagonist, Angiotensin converting enzyme inhibitor, Smooth muscle cell proliferation / migration inhibitor, Platelet aggregation inhibitor, Anticoagulant, Chemical mediator Release inhibitor, vascular endothelial cell proliferation or inhibitor, aldose reductase inhibitor, mesangi Cell growth inhibitors, lipoxygenase inhibitors, immunosuppressants, immunostimulants, antiviral agents, Maillard reaction inhibitors, amyloidosis inhibitors, NOS inhibitors, AGEs (Advanced glycation endproducts) inhibitors and radical scavengers Can be mentioned.
The diagnostic agent is not particularly limited as long as the formation of the complex is not impaired, and examples thereof include in-vivo diagnostic agents such as an X-ray contrast agent, a radioisotope-labeled nuclear medicine diagnostic agent, and a diagnostic agent for nuclear magnetic resonance diagnosis. Examples of X-ray contrast agents include meglumine amidotrizoate, sodium iotaramate, meglumine iotalamate, gastrografin, iodamid meglumine, lipiodol ultrafluid, adipiodon meglumine, oxaglic acid, meglumine iotroxate, iotrolan, iopanoic acid, iopamidol Iohexol, ioversol, iopodate sodium, iomeprol, isopaque, iodoxamic acid and the like.

(4) Complex of functional magnetic ultra-nanoparticle and ligand The functional magnetic ultra-nanoparticle obtained in (2) above has a functional group on its surface. It can bind to a ligand to form a complex. Here, the bond between the functional magnetic ultra-nanoparticle and the desired ligand may be any bond as long as it is via a functional group introduced on the surface of the functional magnetic ultra-nanoparticle, for example, a covalent bond, Examples include ionic bonds and hydrogen bonds.
The type of ligand is not particularly limited as long as it has a specific interaction with a desired substance. For example, proteins such as transferrin, CEA, EGF, and AFP; peptides such as insulin; monoclonal antibodies and the like Low molecular weight compounds such as antibodies such as tumor antigens, hormones, transmitters, carbohydrates such as Lewis X and ganglioside, amino acids and derivatives thereof, vitamins such as folic acid, and derivatives thereof. The ligand exemplified above may be used as a whole, or a fragment obtained by enzyme treatment or the like may be used. Further, it may be an artificially synthesized peptide or peptide derivative. The ligand is preferably an antibody, and when used for humans, a mouse-human chimeric antibody, a humanized antibody, or a human antibody is more preferable.

(5) Labeling of the complex The complex of (3) or (4) can be labeled with a labeling substance. Any of the functional magnetic ultra-nanoparticles, drugs, and ligands of the complex may be labeled. Examples of the labeling substance include a fluorescent substance, a radioactive substance, a non-radioactive labeling substance, and the like, and a fluorescent substance is preferable.
When labeling with a fluorescent substance, any fluorescent substance may be used. For example, FITC, BODIPI, Dansyl chloride (DNS), coumarin, Alexa series (Alexa-353, Alexa-488, etc .: Invitrogen), Rhodamine red, Tetramethyrhodamine, Cye dye series (Cye3, Cye 5, etc .: Amersham), Oregon green 514 (Invitrogen), Q-dot series (Quantam dot), DAPI, Dio and the like. Particularly preferred are those having a carboxyl group at the terminal and activated by N-hydroxysuccinimide or the like. In particular, 5 (6) -Carboxy-rhodamine N-succinimidylester is preferable.

(6) Drug delivery method, drug target substance selection method and drug delivery monitoring method using the drug binding complex of the present invention Using the functional magnetic ultra-nanoparticles and drug drug binding complex of the present invention The drug can be delivered to a predetermined tissue or cell of a non-human specimen. The drug binding complex may be labeled with a labeling substance. That is, after administration of the drug-binding complex to a non-human subject, the magnetism is concentrated from outside the non-human subject to the site of the non-human subject where a predetermined tissue or cell exists, and the drug-binding complex is applied to the site. Accumulate or retain the body. Since the drug-binding complex has an average particle size of 1.3 to 3 nm and concentrated magnetism, the drug-binding complex is specifically and efficiently incorporated into the tissue or cells at the accumulated or retained site. Thereafter, when the magnetism is released, the drug-binding complex accumulated at the site is dispersed, and the drug-binding complex taken up by the tissue or cells is metabolized to the outside of the tissue or outside the cell, that is, outside the subject. Can be discharged. Examples of means for concentrating magnetism include a method using a magnet and a method using a magnetic generator. Non-human subjects include, but are not limited to, laboratory animals such as mice, rats, rabbits, and dogs.
The above method can be used for evaluation of therapeutic effects and pharmacokinetics of drug candidate substances.
The drug-binding complex can also be used as a medicament for treating or diagnosing humans. That is, the drug-binding complex can be administered to humans alone or in combination with a pharmaceutically acceptable carrier, and the drug complex can be delivered specifically to the target tissue by concentrating the magnetism on the target tissue. Since the medicine using the drug conjugate of the present invention can be applied regardless of the kind of drug as an active ingredient, it can be applied to treatment and diagnosis of various diseases. Examples of diagnostic drugs include in-vivo diagnostic agents such as X-ray contrast agents, radioisotope-labeled nuclear medicine diagnostic agents, and diagnostic agents for nuclear magnetic resonance diagnosis.
The administration method and dose of the drug-binding complex of the present invention can be appropriately selected according to the type of drug bound, the nature of the functional magnetic ultra-nanoparticle, and the purpose of administration. Is preferably used for administration routes such as intravascular administration, intravesical administration, intraperitoneal administration, and local administration.

In addition, the drug-binding complex is specifically and efficiently taken into the tissue or cells at the site where the magnetism is concentrated or accumulated in the same manner as described above, and is further present in the drug-binding complex and the tissue or cells. After binding the target substance of the drug in vivo, damage the cell or tissue by magnetically adsorbing and recovering the drug binding complex and the target substance conjugate from the tissue or the cell. The target substance of the drug can be selected. Here, in vivo means that a cell or tissue is alive.
In addition, when selecting a drug target substance using cultured cells, the magnetic ultra-nanoparticles of the present invention can be transferred into cultured cells without applying magnetism or using an introduction agent such as a cation. Therefore, the drug target substance can be selected more easily.
The drug target substance is not particularly limited as long as it is present in the living body and specifically interacts with the drug bound to the magnetic ultra-nanoparticles, and examples thereof include proteins, peptides, and sugar chains.
When recovering the bound product, it is preferable to recover it by magnetically adsorbing it after performing treatment such as crushing the target tissue or target cells.
Examples of the method for analyzing the target substance after recovering the bound substance include electrophoresis, various types of chromatography, mass spectrometry, and amino acid sequence analysis when the target substance is a protein.

  Further, when the labeled drug-binding complex of the present invention is used, the labeled drug-binding complex can be specifically and efficiently incorporated into the tissue or cells at the site where the magnetism is concentrated or accumulated as described above. By later detecting the presence of the labeling substance, the delivery of the drug to a given tissue or cell can be monitored.

(7) Method for selecting a substance that interacts with a ligand using the ligand-binding complex of the present invention and imaging method for distribution of the substance Using the functional magnetic ultra-nanoparticle of the present invention and a ligand-binding complex of a ligand, a predetermined method is used. The desired substance that specifically interacts with the ligand can be selected from any tissue or cell. The ligand binding complex may be labeled with a labeling substance. That is, after administration of the ligand-binding complex to a non-human subject, the magnetism is concentrated from outside the non-human subject to the site of the non-human subject where a predetermined tissue or cell exists, and the ligand-binding complex is applied to the site. Accumulate or retain the body. Since the ligand-binding complex has an average particle size of 1.3 to 3 nm and concentrated magnetism, it is specifically and efficiently taken up into the tissue or cells at the site where it accumulates or stays.
Further, after binding the ligand-binding complex and a desired substance that specifically interacts with the ligand in vivo, magnetically adsorbing and recovering the ligand-binding complex from the tissue or the cell, A desired substance that specifically interacts with a ligand can be selected without damaging cells or tissues.
In addition, when selecting a substance that specifically interacts with a ligand using cultured cells, the magnetic ultra-nanoparticles of the present invention are not magnetized, and without using an introduction agent such as a cation. Since it can be transferred, it is possible to more easily select a substance that specifically interacts with the ligand.
The substance that specifically interacts with the ligand is not particularly limited as long as it exists in the living body and specifically interacts with the ligand. However, proteins such as receptors, antibodies, peptides, antigens, sugar chains, etc. And other physiologically active substances.
When recovering the bound product, it is preferable to recover it by magnetically adsorbing it after performing treatment such as crushing the target tissue or target cells.
Examples of the method for analyzing the target substance after recovering the bound substance include electrophoresis, amino acid sequence analysis, various chromatographies, and mass spectrometry.
The method for selecting a substance that specifically interacts with the ligand is preferably used when searching for an unknown substance that interacts with the ligand, or when purifying a substance that has an affinity for the ligand. be able to.
In addition, when the labeled ligand-binding complex of the present invention was used, the labeled ligand-binding complex was specifically and efficiently incorporated into the tissue or cells at the site where the magnetism was concentrated or accumulated as described above. Subsequently, the drug-binding complex that has accumulated or stayed at the site after being released from magnetism or has been delivered to the tissue or cell is dispersed or metabolized, and the presence of the labeling substance is detected, thereby detecting the predetermined tissue or cell. The distribution of the desired substance in can be imaged.

  Next, specific examples of the present invention will be shown, but the present invention is not limited to these examples.

Example 1: Preparation of functional magnetic ultra-nano fine particles (1) Preparation of magnetic ultra-nano fine particles The fine particles used a wet precipitation method in which fine particles are obtained in a monodisperse form. At the time of preparation, FeCl ₂ .4H ₂ O and Na ₂ SiO ₃ .9H ₂ O were employed as materials to prepare magnetic ultra-nano particles.
19.9 g of FeCl ₂ .4H ₂ O (0.1 mol) and 28.4 g of Na ₂ SiO ₃ .9H ₂ O (0.1 mol) are weighed and dissolved in 500 mL of distilled water. An aqueous solution was obtained. After mixing the two with sufficient agitation at room temperature for about 5 hours to complete the reaction, throw the supernatant into a centrifuge for 20 minutes, discard the supernatant, re-disperse it with distilled water, and then apply to the centrifuge to remove the supernatant. The operation of discarding was repeated several times to wash the precipitate. Next, after drying in a thermostatic bath maintained at about 350K, pulverizing in a mortar and placing on an alumina boat, firing in an electric furnace in an air atmosphere at a temperature range of 373-873K for 4-10 hours Magnetic ultra-nanoparticles were obtained. The average particle diameter could be determined to be 1.3 to 3 nm in both X-ray diffraction measurement and transmission electron microscope (TEM) measurement.
FIG. 1 shows a chemical prediction diagram of the magnetic ultra-nanoparticles obtained.

(2) Introduction of hydroxyl group on the surface of magnetic ultra-nano fine particle As the fine particle surface cleaning method, since the fine particle surface is silica-coated, either ethanol cleaning or plasma treatment may be used. It is preferable because washing is simple.
Specifically, 500 μL of ethanol was used, gently stirred at room temperature for 1 minute, centrifuged at 5,000 rpm for 3 minutes, the supernatant was removed, and washed with 500 μL of ultrapure water in the same manner three times. Then, it was made to dry for 30 minutes at 100 degreeC, and the water | moisture content was removed completely.

(3) Introduction of amino group on the surface of magnetic ultra-nano fine particle γ-APTES (γ-aminopropyltriethoxysilane) was introduced into the hydroxyl-introduced magnetic ultra-nano fine particle obtained in (2) above by silanization. That is, a large excess (4M) γ-APTES was added to 20 mg of the washed magnetic ultra-nanoparticles and reacted at 130 ° C. for 20 hours. After the reaction, unreacted γ-APTES was washed 3 times with 3 mL of ethanol and 3 times with 3 mL of ultrapure water.
The obtained fine particles were observed using an IR measuring device (HORIBA model) manufactured by HORIBA. The measurement method used is the Attenuated Total Refraction method (ATR method). IR results are shown in FIG. 3A. Si-O (800~1100cm ^-1), a peak of OH (3500~3900cm ^-1) were observed.
FIG. 3B shows the IR of the magnetic ultrananoparticle after aminosilanization. Since the peak of CH (2982 to 2822 cm ⁻¹ ) derived from γ-APTES was observed, the amino group could be modified by silanization on the surface of the magnetic ultra-nanoparticle.
The same characteristic stretching peak was obtained for other functional groups.
The average particle size of the magnetic ultra-nanoparticles modified by functional groups and functionalized can be determined to be 3 nm in both X-ray diffraction measurement and transmission electron microscope (TEM) measurement. It was suggested that there was no influence.
An electron micrograph of the resulting aminated magnetic ultra-nanoparticle is shown in FIG.
The diameter was 3 nm, and it was confirmed that fine particles were prepared in monodisperse.

(4) Modification of fluorescent substance The aminated magnetic ultra-nanoparticle was modified with 5 (6) -Carboxy-rhodamine N-succinimidylester, which is a fluorescent substance. Using 5 mM of a fluorescent substance, 5 mg of aminated magnetic ultra-nanoparticles were mixed and reacted at room temperature for 10 minutes. In order to eliminate the influence of the unreacted amino group, 0.1M paraformaldehyde is added after rhodamine modification, and the remaining amino group is crushed by reacting for 60 minutes, and the Schiff base formed by the reaction between the aldehyde compound and the amino group is a reducing agent ( Reduction with 0.1M NaBH ₄ ).
The resulting fluorescent modified magnetic ultra-nanoparticles were confirmed to be modified with a fluorescent scanner. The scanner used is Typhoon 9400 manufactured by Biorad. From the aminated magnetic ultra-nanoparticles, rhodamine fluorescence was observed even after washing. From the non-aminated magnetic ultra-nanoparticles, no rhodamine fluorescence was observed after washing. From these results, it was confirmed that rhodamine was covalently modified via an amino group (FIG. 5).

Example 2: Introduction of functional magnetic nano-particles into cells Functional magnetic ultra-nano particles were introduced into cells. The cells used are rat kangaroo kidney-derived PtK2 cells.
After culturing PtK2 cells on a 10 cm petri dish for 24 hours, functional magnetic ultra-nanoparticles (fluorescent substance-modified magnetic ultra-nanoparticles) or fluorescent beads (average particle size 200 nm; Invitrogen) as a control from the above solution Each diluted 50,000-fold was introduced into the culture medium. After 24 hours, the inside of the petri dish was washed three times with the culture solution, and non-specifically adsorbed cells or fine particles (fluorescent beads) adsorbed on the petri dish were removed. As a result, fluorescence was observed from inside the cells into which the functional magnetic ultra-nanoparticles were introduced (FIG. 6A). In contrast, no fluorescence was observed from the cells into which the fluorescent beads were introduced (the arrow in FIG. 6B confirming the presence of the fluorescent beads in a place where no cells exist due to nonspecific adsorption to the substrate). In addition, as a result of breeding the cells into which the functional magnetic ultra-nanoparticles were introduced as they were and observing them after 5 days, the cells proliferated without disappearing, and the fluorescence of the functional magnetic ultra-nanoparticles was also observed (FIG. 6A). When the fluorescence intensity was measured with respect to the samples FIG. 6A and FIG. 6B after 24 hours, the ratio of the intensity per cell area was 8: 1, which was stronger in the cells into which the functional magnetic ultra-nanoparticles were introduced. From these facts, it was confirmed that the functional magnetic ultra-nanoparticles easily enter cells. As an example correlating with this, an electron microscopic image of cells after introduction of functional magnetic ultra-nanoparticles (FIG. 6C) or control particles (fluorescent beads: average particle size 200 nm) (FIG. 6D) is shown. From FIG. 6C, aggregates of functional magnetic ultra-nanoparticles (FIG. 6C: wavy circles) are observed, whereas control fine particles (average particle size 200 nm) are not observed from FIG. 6D. In addition, ribosomes (arrows in FIG. 6C) and microtubules (FIG. 6C: wavy squares) are observed. This indicates that normal cell activity is still performed after the functional magnetic ultra-nanoparticle is introduced into the cell. Both examples show that the functional magnetic ultra-nanoparticles are not cytotoxic.

Example 3: Effect of increasing the concentration of local functional magnetic ultra-nanoparticles by magnetism Introduction of functional magnetic ultra-nanoparticles into a living body and concentration of the fine particles at one point of the tissue by magnetism. A mixed solution of functional magnetic ultra-nanoparticles (rhodamine modifying substance) and petroleum jelly at a ratio of 1: 1 was applied to the outer ear portion of the mouse. The coated side was the surface. A circular magnet having a diameter of 7 mm was fixed to the center of the back surface of the outer ear with tape. Functional magnetic ultra-nanoparticles (rhodamine modifier) and petrolatum were applied to the outer ear surface in the above ratio, and aluminum foil was fixed to the back surface with tape, and fluorescent fine particles (Invitrogen) with an average particle size of 200 nm were applied. Two types of things were used as controls. After 24 hours, mouse ear shell tissue sections were prepared, and the fluorescence of rhodamine possessed by the functional magnetic ultra-nanoparticles was observed. From the results, the fluorescence of rhodamine was observed from the ear shell section of the object to which the functional magnetic ultra-nanoparticles were applied and the magnet was fixed (FIG. 7A). In the ear shell section to which the magnet was fixed, fine particles were concentrated on the part to which the magnet was fixed (FIG. 7A). However, almost no fluorescence was observed from the sections where no magnet was used (FIG. 7B). The fluorescence of the fine particles was not observed even from the section coated with the 200 nm fluorescent fine particles (FIG. 7C). The results of quantifying these results in fluorescence intensity are shown in FIG. As a result of testing each of the magnets fixed (FIG. 7A) and those not using magnets (FIG. 7B) or those coated with 200 nm fluorescent particles (FIG. 7C), it was shown that there was a significant difference. . From these facts, the concentration could be locally improved by using magnetic force.

Example 4: Modification of crosslinking agent on aminated magnetic ultra-nanoparticle surface In order to modify amino acid (L-cysteine) via a crosslinking agent on the aminated magnetic ultra-nanoparticle obtained in Example 1 (3), First, a crosslinking agent was modified on aminated magnetic ultra-nanoparticles. That is, 5 mg of the washed aminated magnetic ultra-nanoparticles dispersed in 100 μL of DMF (dimethylformamide) and 7.2 mg of the crosslinking agent Sulfo-EMCS (N- [ε-maleimidocaproyloxy] sulfosuccinimide ester) A solution dissolved in 100 μL of DMF was reacted at 4 ° C. for 30 minutes and at room temperature for 1 hour. After the reaction, unreacted Sulfo-EMCS was washed three times with 3 mL of DMF, and then the obtained fine particles were observed using an IR measuring device. The measurement method used is the ATR method. The result of IR is shown in FIG. FIG. 9-A shows the state before modification with the cross-linking agent, and FIG. 9-B shows the state after the modification. Since the peak of C = O (1604 to 1688 cm ^-1 ) was observed only from the graph after the modification of the crosslinking reagent, the crosslinking reagent could be modified to the amino group of the aminated magnetic ultra-nanoparticle.

Example 5: Modification of L-cysteine on the surface of the crosslinking reagent-modified magnetic ultra-nanoparticles Next, modification of L-cysteine was attempted on the magnetic ultrananoparticles modified with the crosslinking reagent obtained in Example 4. Cross-linking reagent-modified magnetic ultra-nanoparticles 5 mg dispersed in 100 μL buffer (0.1 M K-PIPES; pH 6.8) and 2.2 mg L-cysteine in 100 μL buffer (0.1 M K-PIPES) The dissolved product was reacted at 4 ° C. for 30 minutes and at room temperature for 1 hour. After the reaction, unreacted L-cysteine was washed 3 times with 3 mL of the same buffer as above. A predicted chemical structure of the resulting amino acid-modified magnetic ultra-nanoparticle is shown in FIG. 10, and a TEM photograph is shown in FIG. The diameter was about 3 nm, and the amino acid modification did not affect the morphology of the magnetic ultra-nanoparticles, and it was confirmed that the amino acid-modified magnetic ultrananoparticles were present in monodisperse.

Fluorescent substance modification In order to confirm the introduction of amino acid (L-cysteine) into the crosslinking agent-modified magnetic ultra-nanoparticle, the amino acid-modified magnetic ultra-nanoparticle was modified with Dansyl chloride (DNS), which is a fluorescent substance.
First, before reacting with DNS, 0.175M paraformaldehyde is added to the cross-linking reagent-modified magnetic ultra-nanoparticles dispersed in 1 mL of DMF in order to eliminate the influence of residual amino groups on the surface of the cross-linking reagent-modified magnetic ultra-nanoparticles. The remaining amino group was methylated by reacting at room temperature for 15 minutes. As the Schiff base formed by the reaction between the aldehyde compound and the amino group, a crosslinking agent-modified magnetic ultra-nanoparticle reduced by a reducing agent (0.175M NaBH ₄ ) was used. Then, it was washed 3 times with DMF (3 mL).
Next, amino acid-modified magnetic ultra-nanoparticles were prepared in the same procedure as in the above-mentioned “Modification of L-cysteine on the surface of the crosslinking reagent-modified magnetic ultra-nanoparticle”. 5 mg of amino acid-modified magnetic ultra-nanoparticles were dispersed in a solvent in which 100 μL of 20 mM citrate buffer (pH 8) and 80 μL of acetone were mixed. 20 μL of 0.175M DNS dissolved in acetone was added thereto and reacted at 50 ° C. for 15 minutes. Unreacted DNS was washed three times with 3 mL of the same solvent as described above to obtain fluorescent substance-labeled amino acid-modified magnetic ultra-nanoparticles.
A suspension of two types of fluorescent substance-labeled amino acid-modified magnetic ultra-nanoparticles and aminated magnetic ultra-nanoparticles in DMF (1 mL) was observed with a confocal microscope (Carl Zeiss: LSM-5). . As a result, the fluorescence of DNS was confirmed only from amino acid-modified magnetic ultra-nanoparticles (FIG. 12A). Note that the observed microparticles are aggregated, and are not a single microparticle observation. On the other hand, no fluorescence is obtained from the aminated magnetic ultra-nanoparticles (FIG. 12B). This indicates that the fine particles themselves do not have fluorescence. That is, it shows that DNS has been covalently modified through the amino group of the amino acid present in the amino acid-modified magnetic ultra-nanoparticle, and that amino acid has been introduced into the cross-linking agent-modified magnetic ultra-nanoparticle.

Example 6: Preparation of folic acid-modified magnetic ultra-nanoparticles The aminated magnetic ultra-nanoparticles obtained in Example 1 (3) were modified with folic acid and a fluorescent substance (coumarin). Specifically, 25 mg of aminated magnetic ultra-nanoparticles were weighed and suspended in 10 mL of DMF using ice with a stirrer, and 12.7 mg of folic acid (2.3 mM), 0.57 mg of coumarin (0.23 mM). 149.5 mg of PyBop (Nacalai Tesque: 23 mM), 38.3 mg of HoBT (1-Hydroxy-1H-benzotriazole: 23 mM), and 1.48 μL of DIEA (N, N-diisopropylethylamine: 2.3 mM) Added to the microparticle suspension. The reaction was completed while slowly stirring at room temperature for about 21 hours while shielding from light, then centrifuged at 9000 rpm, 4 ° C. for 20 minutes, the supernatant was discarded, and DMF was poured to redispersed, followed by 9000 rpm, 4 ° C. The operation of centrifuging for 15 minutes and discarding the supernatant was repeated 3 times to wash the precipitate. Next, in order to methylate unreacted amino groups on the fine particles, formaldehyde (0.1 M) is added to a solution in which the washed precipitate is suspended in 2 mL of methanol and stirred well. Further, the Schiff base is added to NaBH. ₄ (0.1M) was reacted and reduced at 4 ° C. Finally, the operation of centrifuging with methanol at 15000 rpm for 5 minutes and discarding the supernatant was washed three times to obtain magnetic ultra-nanofine particles modified with folic acid. A TEM photograph of the obtained folic acid-modified magnetic ultra-nanoparticle is shown in FIG. The diameter was about 3 nm, and folic acid modification did not affect the morphology of the fine particles, and it was confirmed that the fine particles were present in a monodisperse form.
In the same manner, fine particles modified only with fluorescence (coumarin) without mixing folic acid were also obtained.

Confirmation of folic acid modification By IR, aminated magnetic ultra-nanoparticles before folic acid modification (FIG. 14A) and aminated magnetic ultra-nanoparticles after folic acid modification (FIG. 14B) were measured. Folic acid modification was confirmed since a peak specific to folic acid (p-aminobenzoic acid) 1419 cm ⁻¹ was obtained only from the aminated magnetic ultra-nanoparticles after folic acid modification.

Example 7: Introduction of functional (aminated) magnetic ultra-nanoparticles modified with folic acid into cells The folic acid-modified magnetic ultra-nanoparticles were introduced into cells. The cells used were KB cells derived from human pharyngeal cancer.
After culturing KB cells on a petri dish for 48 hours, the above-mentioned folic acid-modified magnetic ultra-nanoparticles or a microparticle modified only with a fluorescent substance (coumarin) as a control suspended in 2 mL of DMF is centrifuged at 6000 rpm for 20 seconds. Then, 10 μL of the supernatant was introduced into each culture solution (2 mL). Two hours later, the medium was replaced with a solution (0.2 μg / L) obtained by diluting a reagent for cell staining carbocyanine dye (DiI) with medium, and staining was performed for about 1 hour. Then, it was confirmed whether the microparticles | fine-particles were taken in into the cell using the confocal microscope. For folic acid-modified magnetic ultra-nanoparticles, the same operation was performed using PtK2 cells (Potorous tridactylis-derived epithelial cells). In addition, for the folic acid-modified magnetic ultra-nanoparticles, a large excess amount of folic acid (1.1 mM) was simultaneously added when introduced into KB cells.

The results are shown in FIG.
From the cells into which the folic acid-modified magnetic ultra-nanoparticles were introduced, the fluorescence of coumarin similarly modified from the inside of the cells to the magnetic ultra-nanoparticles was observed (FIG. 15A). On the other hand, no fluorescence was observed from the inside of the cells into which coumarin-modified magnetic ultra-nanoparticles not modified with folic acid were introduced. Slight fluorescence was observed from the cell surface (FIG. 15B). Note that FIG. 15C shows an unmanipulated cell, and no coumarin fluorescence is observed. This indicates that there is no autofluorescence of the cells. In addition, when folic acid-modified magnetic ultra-nanoparticles were introduced into cells (PtK2 cells) in which no folate receptor was expressed on the cell surface, no fluorescence was observed from the cell surface layer or inside. The presence of PtK2 on the cell surface was observed in the folic acid-unmodified magnetic ultra-nanoparticles. The observation of coumarin-modified magnetic ultrananoparticles without folic acid from the cell surface is consistent with KB cells. In other words, the surface of the folic acid-modified magnetic ultra-nanoparticle is anionic due to the influence of α-carboxyl group of folic acid (confirmed by electrophoresis). In general, since the cell surface is anionic, it is supported by the electrostatic repulsion that the ultrafine nanoparticles modified with folic acid cannot be brought close to the cell surface. Furthermore, no fluorescence was observed from the cell surface layer and from the inside when a large excess of folic acid and folic acid-modified magnetic ultra-nanoparticles were simultaneously introduced into KB cells. From these results, it was confirmed that folic acid-modified magnetic ultra-nanoparticles were selectively introduced into the cell via folate receptors on the cell membrane.

  In the functional magnetic ultra-nanoparticle of the present invention, a functional group such as an amino group, an isocyanate group, or a mercapto group is covalently modified on the surface of the fine particle. The functional magnetic ultra-nanoparticles of the present invention can be modified not only with peptides but also with general drugs. Since it can be easily taken into cells and tissues and the local concentration can be controlled by using magnetic force, it can be used in a novel drug delivery system. Furthermore, the functional magnetic ultra-nanoparticles of the present invention can also construct a novel protein purification system because the target intracellular substance can be captured in a sample or cell modified with a drug or a specific protein interacting substance.

The functional magnetic ultra-nanoparticle of this invention is shown typically. ● represents Si, ○ represents oxygen, and the center represents a metal oxide. Functional conceptual diagram of magnetic ultra-nanoparticles. A: IR spectrum of magnetic ultra-nanoparticles. B: IR spectrum of an aminated magnetic ultra-nanoparticle. Electron micrograph of aminated magnetic ultra-nanoparticle (scale bar is 5 nm). Fluorescent photograph of fluorescent material modified or unmodified magnetic ultra-nanoparticles. A: Intracellular introduction of functional magnetic ultra-nanoparticles (photo). B: Intracellular introduction of control fine particles (photo). C: Intracellular electron micrograph of functional magnetic ultra-nanoparticles. D: Intracellular electron micrograph of control fine particles. Wavy circles indicate the corresponding fine particles, arrows indicate ribosomes, and wavy squares indicate microtubules. A: Introduction drawing (photograph) of functional magnetic ultra-nanoparticles using magnetism to mouse outer ear. B: Introduction diagram (photograph) of functional magnetic ultra-nanoparticles not using magnetism in mouse outer ear. C: Introduction (control) (photograph) of a fluorescent fine particle having an average particle diameter of 200 nm on a mouse outer ear slice without using magnetism. The figure which shows the fluorescence intensity of rhodamine in an outer ear slice. Tested using t-test. The vertical axis represents the relative fluorescence intensity of the fluorescence intensity. A: IR spectrum of aminated magnetic ultra-nanoparticles. B: IR spectrum of the cross-linking agent-modified magnetic ultra-nanoparticle. Functional diagram of amino acid-modified magnetic ultra-nanoparticles. An electron micrograph of an amino acid-modified magnetic ultra-nanoparticle (scale bar is 5 nm). A: Fluorescent image (photograph) of fluorescent substance-labeled amino acid-modified magnetic ultra-nanoparticles. B: Fluorescent image (photograph) of aminated magnetic ultra-nanoparticles. Electron micrograph of folic acid-modified magnetic ultra-nanoparticle (scale bar is 5 nm). A: IR spectrum of aminated magnetic ultra-nanoparticles. B: IR spectrum of folic acid-modified magnetic ultra-nanoparticles. A: Photograph of KB cell with folic acid-modified magnetic ultra-nanoparticles introduced. B: Coumarin-modified magnetic ultra-nanoparticle-introduced KB cell photograph. C: KB cell picture. Images taken from the top (1) KB cells and (2) fluorescence images, respectively. A (3) KB cell and (4) a fluorescence image are seen from the side.

Claims (16)

(A) a core made of a metal oxide,
(B) a shell made of an amorphous silica network covering the core;
(C) Functional magnetic ultra-nanoparticles having an average particle size of 1.3 to 3 nm having functional groups covalently introduced to the surface of the shell.   The functional magnetic ultra-nanoparticle according to claim 1, wherein the functional group is introduced via a silane coupling agent.   The functional group is at least one selected from the group consisting of an amino group, an isocyanate group, a mercapto group, a vinyl group, an epoxy group, a methacryloxy group, a ureido group, a chloropropyl group, and a sulfide group. Functional magnetic ultra-nano particles.   A silane coupling agent is used on the surface of fine particles having (a) a core made of a metal oxide, and (b) a shell made of an amorphous silica network covering the core, the shell having a hydroxyl group on the surface thereof. The method for producing functional ultrafine magnetic particles according to any one of claims 1 to 3, wherein a functional group is covalently introduced through the hydroxyl group.   A drug binding comprising the functional magnetic ultra-nanoparticle according to any one of claims 1 to 3 and a drug, wherein the drug is bound via a functional group on a surface of the microparticle. Complex.   The drug-binding complex according to claim 5, wherein the functional magnetic ultra-nanoparticle or drug is labeled with a labeling substance.   The functional magnetic ultra-nanoparticle according to any one of claims 1 to 3 and a ligand that specifically interacts with a desired substance, wherein the ligand functions through a functional group on the surface of the particle. Ligand-binding complex, characterized in that it is bound to a magnetic ultrafine nanoparticle.   The ligand-binding complex according to claim 7, wherein the functional magnetic ultra-nanoparticle or ligand is labeled with a labeling substance. (A) administering the drug-binding complex according to claim 5 or 6 to a non-human subject;
(B) Concentrating magnetism from outside the non-human subject to the site of the non-human subject where a predetermined tissue or cell exists, and accumulating or retaining the drug-binding complex in the site to cause the tissue or cell to Delivering the drug-binding complex to a predetermined tissue or cell. (A) administering the drug-binding complex according to claim 6 to a non-human subject;
(B) Concentrating magnetism from outside the non-human subject to the site of the non-human subject where a predetermined tissue or cell exists, and accumulating or retaining the drug-binding complex in the site to cause the tissue or cell to Delivering a drug binding complex;
(C) detecting the presence of a labeling substance localized in the tissue or cell, and monitoring the delivery of the drug to a predetermined tissue or cell. (A) administering the ligand-binding complex according to claim 8 to a non-human subject;
(B) Concentrating magnetism from the outside of the non-human subject to the site of the non-human subject where a predetermined tissue or cell exists, and accumulating or retaining the ligand-binding complex in the site to Delivering a ligand-binding complex, (c) dispersing or metabolizing the drug-binding complex that has accumulated or stayed at the site after being released from magnetism, or has been delivered to the tissue or cell; Detecting the presence of a labeling substance localized in the method, and imaging a distribution of the desired substance in a predetermined tissue or cell. (A) administering the drug-binding complex according to claim 5 or 6 to a non-human subject;
(B) Concentrating magnetism from outside the non-human subject to the site of the non-human subject where a predetermined tissue or cell exists, and accumulating or retaining the drug-binding complex in the site to cause the tissue or cell to Delivering a drug binding complex to bind the drug binding complex in vivo with a drug target substance present in the tissue or cell;
(C) A method for selecting a target substance of a drug, comprising a step of recovering a binding substance of the target substance and the drug-binding complex by magnetically adsorbing from the tissue or cells. (A) adding the drug-binding complex according to claim 5 or 6 to cultured cells, and binding the drug-binding complex to a target substance of a drug present in the cultured cells;
(B) A method of selecting a target substance for a drug, which comprises a step of recovering the bound substance of the target substance and the drug-binding complex by magnetically adsorbing from the cultured cells. (A) administering the ligand-binding complex according to claim 7 or 8 to a non-human subject,
(B) Concentrating magnetism from the outside of the non-human subject to the site of the non-human subject where a predetermined tissue or cell exists, and accumulating or retaining the ligand-binding complex in the site to Delivering a ligand binding complex to bind the ligand binding complex in vivo with a desired substance present in the tissue or cell;
(C) a step of recovering the binding substance of the substance and the ligand-binding complex from the tissue or cells by magnetically adsorbing the desired substance that specifically interacts with the ligand, How to choose. (A) adding the ligand-binding complex according to claim 7 or 8 to cultured cells to bind the desired substance present in the cultured cells to the ligand-binding complex;
(B) recovering the bound substance of the substance and the ligand-binding complex from the cultured cells by magnetically adsorbing the desired substance that specifically interacts with the ligand, How to choose.   A medicament comprising the drug complex according to claim 5 or 6.

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