WO2007097473A1 - Organic magnetic nanocomplex having functional molecule introduced therein - Google Patents

Abstract

Disclosed is an organic magnetic nanocomplex comprising: a magnetic metal oxide nanoparticle which is coated with a water-soluble carboxyalkyl-etherified polysaccharide and has an entire particle diameter ranging from 10 to 100 nm; and a site-specific functional molecule which is covalently bound to the polysaccharide through a linker. When administered to a living body, the organic magnetic nanocomplex can be accumulated in a target site specifically and efficiently. Therefore, the organic magnetic nanocomplex is useful as an MRI contrast agent for detecting or diagnosing a site having any one of various types of cancer and various diseases in a specific and safe manner or is also useful in magnetic field therapy.

Description

 Description Organic magnetic nanocomposite into which functional molecule is introduced Technical field 'The present invention is a nuclear magnetic resonance imaging (hereinafter abbreviated as MR I)', which specifically focuses on various lesions such as various diseases and cancers. Useful function as a contrast agent that can be detected, or in a treatment that raises the temperature of the lesion site by irradiation with a high-frequency magnetic field and selectively or specifically kills only the lesion site by Z or cell disruption The present invention relates to an organic magnetic nanocomposite in which a functional molecule is introduced. Background art

 Magnetic particles formulated in an appropriate form, such as dextran magnetite, are administered to a living body, and diagnostic imaging is performed to detect in vivo tissues and fluids at a specific site where they are concentrated by MR I. It is known that it can be used in hyperthermia that locally raises the temperature of dextran magnetite that is concentrated at a specific site by irradiating electromagnetic waves from a magnetic field, and some magnetic particles have already been commercialized, for example MR I It is used for clinical diagnosis as a contrast agent. However, since dextran magnetite is administered in vivo, most of it goes through vascular circulation and is taken into the reticuloendothelial system (RES) such as the liver. Therefore, it is used for screening for liver cancer. Recently, attempts have been made to use it as an angiographic or lymph node contrast agent, but there is still a problem that the target diseases are still limited to a very narrow range.

On the other hand, by introducing site-specific functional molecules, which are substances that specifically recognize biological tissue and lesions, onto the surface of magnetic nanoparticles typified by dextran magnetite, diagnostic imaging using MR I and others Several attempts have been made to make heat treatment more applicable to a wider range of diseases. For example, LG Remsenet. Al., American J. Neuroradiology, 17, 411 (1996), made sodium periodate on dextran-coated superparamagnetic monocrystalline iron oxide nanoparticles (MION). Then, the possibility of applying the complex bound to a brain tumor-specific monoclonal antibody to brain tumor-specific diagnosis by MR I is disclosed. Moreover, P. Reimeret. Al., Radiology , 193, 527 (19 ⁹ 4) is obtained by ultrasonic dextran coated monocrystalline iron oxide nanoparticles (MION) with Koreshisutoki nin (CCK) Application of MION labeled with CCK as a spleen-specific MR I contrast agent is disclosed. In addition, L. Josephson 'et. Al., Bioconjugate Chera., 10, 186 (1999) described dextran-coated superparamagnetic iron oxide nanoparticles (MION) by reacting them with epichlorohydrin and ammonia. After the introduction of the group, the complex formed by reacting with 3- (2-pyridyldithio) propionate N-succimidyl and then binding the peptide derived from HIV-tat protein to the intracellular magnetic label of the target cell Applications are disclosed.

 U.S. Patent Application Publication No. 2 0 0 3/0 0 9 2 0 2 9 states that when a complex of an oligonucleotide, a polypeptide or a polysaccharide and a magnetic nanoparticle binds to a target molecule, it aggregates and NMR relaxation occurs. It has been disclosed to detect a target molecule using the change in ability.

 On the other hand, Japanese Patent Application Laid-Open No. 11-115703 discloses a living body internal heating apparatus using a heat-sensitive heating element mainly composed of magnetic iron-based oxide fine particles surrounded by phospholipids in some cases in a ribosome form. It is disclosed.

 However, the above complex has problems in in vivo stability, safety to the living body, and efficiency to reach the target site, and it has not reached the level where it can be clinically applied as well as commercialized. Disclosure of the invention

 The main object of the present invention is to enable site-specific MR I diagnosis and / or magnetic field treatment by high-frequency magnetic field irradiation that can be administered to a living body and can reach a target site such as a lesion accurately and efficiently. It is to provide an organic magnetic nanocomposite in which a functional molecule is introduced.

As a result of intensive studies to achieve the above object, the present inventors have used magnetic metal oxide nanoparticles coated with a water-soluble carboxyalkyl etherified polysaccharide as magnetic nanoparticles. And by introducing a functional molecule to the polysaccharide via a linker, an organic magnetic nanocomposite with not only stability and safety, but also significantly improved specific reach efficiency to the target site is obtained. As a result, the present invention has been completed. In summary, according to the present invention, magnetic metal oxide nanoparticles having an overall diameter in the range of 10 to 100 nm coated with a water-soluble carboxyalkyl etherified polysaccharide, and the polysaccharide via a linker. Thus, an organic magnetic nanocomposite composed of a site-specific functional molecule covalently bonded to a force is provided. ''

 The organic magnetic nanocomposite of the present invention is useful as a contrast agent for MR I diagnosis, a local magnetic field therapeutic agent, etc. for various disease or cancer lesion sites. Brief Description of Drawings

FIGS. 1A to F show the KDR—BP—CMDM (complex number 1) obtained in Example 1 and the CMDM of Reference Example 1 added to KDR positive cells and negative cells, and MR after 12 hours. I (T ₂ weighted image) Α: Water only, B: CMDM only, C: Negative cells + CMDM, D: Positive cells + CMDM, E: Negative cells + KDR—BP—CMDM, F: Positive cells + KD R—BP—CMDM.

Figures 2A-F show the RB P-1 obtained in Example 2 (1 CMDM (complex number 2)) and the CMDM of Reference Example 1 added to RET positive cells and negative cells, and 12 hours later. I (T ₂ weighted image) Α: water only, B: CMDM only, C: negative cells + CMDM, D: positive cells + CM DM, E: negative cells + RBP—1 CMDM, F: positive cells + RBP— 1— CMDM. Figure 3 shows that KDR-BP-CMDM (complex number 1) obtained in Example 1 was injected into the mouse transplanted with KDR positive tumor (right) and negative tumor (left) via tail vein, 4 hours 20 minutes. It is a later MR I image photograph.

4A to F show C 45 D 18 -CMDM (complex number 6) obtained in Example 6 and Reference Example 1 This is a MR I image photograph 12 hours after adding CMDM to cultured cells. A: Water only, B: CMDM only, C: Cell + CMDM, D: Cell + C45D18 (2) —CMDM, E: Cell + C45D18 (6) 1 CMDM,: Cell + 145018 (10) 1 CMDM.

 FIG. 5 is a MR I image photograph 1 hour and 40 minutes after C45D 18-CMDM (complex number 6) obtained in Example 6 was injected into the tail vein of a mouse transplanted with a KDR-expressing tumor. The left mouse is before administration, and the right mouse is after administration. A child is recognized as the signal in the region indicated by the arrow in the right figure changes to black. '

 FIG. 6 shows C 45 D 18—CMDM (complex number 6) obtained in Example 6, the raw material C MDM of Reference Example 1 and the C 45D 18 peptide alone in He La cells and as Fe. FIG. 6 is a graph showing the degree of cell proliferation after irradiating a high-frequency magnetic field for 1 hour to cells after adding 800 μg / m 1 and incubating for 12 hours. Detailed description of the invention

 Hereinafter, the organic magnetic nanoparticle composite into which the site-specific functional molecule of the present invention has been introduced (hereinafter sometimes abbreviated as “introduced magnetic nanocomposite”) will be described in more detail.

 The introduced magnetic nanocomposite of the present invention comprises a magnetic nanoparticle coated with a water-soluble carboxyalkyl etherified polysaccharide as a raw material (hereinafter sometimes abbreviated as raw magnetic nanoparticle) via a linker. It can be produced by covalently binding a specific functional molecule. Raw magnetic nanoparticles

In addition to being stable, biosafety, and difficult to be incorporated into the reticuloendothelial system, raw magnetic nanoparticles bind site-specific functional molecules to the particle surface via a linker. In the present invention, as the raw magnetic nanoparticles satisfying the powerful requirements, magnetic metal oxide ultrafine particles coated with a water-soluble carboxyl alkyl etherated polysaccharide, for example, A composite of a carboxyalkyl ether of a polysaccharide and a magnetic metal oxide described in Japanese Patent No. 2726520 (European Patent No. 450092, US Pat. No. 5,204,457) is used.

 The raw material magnetic nanoparticles used in the present invention will be described in brief here instead of the detailed description with reference to Japanese Patent No. 272652◦. '' Magnetic metal oxide core particles, which are one component of the raw magnetic nanoparticles,

(1):

, (Μ ^π Ο), · Μ ₂ ^{! ΙΙ} 〇 ₃ ^··· (1)

Where Μ "represents a divalent metal atom, Μ" ¹ represents a trivalent metal atom, and 1 is a number in the range of 0 to 1,

Can be mentioned. In the above formula (1), the divalent metal atom Micromax ^11, for example, magnesium, calcium, manganese, iron, nickel, Kono Noreto, 'copper, zinc, strike opening Nchiumu, Roh helium, and the like. These Each can be used alone or in combination of two or more. Examples of the trivalent metal atom ¹¹¹ include, for example, aluminum, iron, yttrium, neodymium, samarium, europium, gadmium, etc., and these can be used alone or in combination of two or more thereof. A combination of the above can also be used.

Among the magnetic gold oxide core particles, the magnetic metal oxide in which M ¹¹¹ is trivalent iron in the above formula (1), that is, the following formula (2):

(M ⁿ O) _m · F e ₂ 0 ₃ · · · (2)

Wherein M ¹¹ is as defined above, m is a number in the range of 0 to 1,

Is preferable. Here, as M ¹¹ , the same metal atoms as exemplified in the above formula (1) can be mentioned. In particular, when M ¹¹ is divalent iron, the magnetic metal oxide of the above formula (2), that is, the following formula (3):

(F e〇) _n · F e ₂ 0 ₃ · · · (3) ' Where n is a number in the range 0-1;

The magnetic iron oxide represented by can be mentioned as a more preferable magnetic metal oxide in the present invention. In the above formula (3), in the case of n = 0 is a y- iron oxide (.gamma. F e ₂ 〇 ₃₎ In the case of n = l is Magunetai bets (F e ₃ 0 ₄₎ . In the present invention, the magnetic metal oxide includes a magnetic metal oxide having crystal water. Examples of other components constituting the raw magnetic nanoparticles, that is, water-soluble carboxyalkyl etherified polysaccharides (including reduced polysaccharides, the same shall apply hereinafter) for coating the magnetic metal oxide core particles include, for example, glucose Polymers such as dextran, starch, glycogen, cellulose, pullulan, curdlan, schizophyllan, lentinan, pestarotian Xylan, which is a xylose polymer; carboxyalkyl etherified products of polysaccharides such as arabinan, which is an L-arabinose polymer, and among others, carboxyalkyl etherified products of glucose polymers, A carboxyalkyl etherified product of stran, starch or pullulan is preferred, and a carboxyalkyl etherified dextran is particularly preferred. .

 The alkyl portion of the carboxyalkyl etherified polysaccharide can be lower alkyl, for example, methyl, ethyl, propyl, butyl, etc., preferably methyl. Further, the carboxyl group can take the form of a salt, and examples of the salt include alkali metal salts such as sodium salt and potassium salt, ammonium salt and amine salt, preferably sodium salt. It is. Furthermore, the degree of substitution of the carboxyalkyl etherified polysaccharide is generally in the range of 0.05 to 0.5, preferably 0.:! To 0.3, per monosaccharide unit.

Carboxyalkyl etherified polysaccharides have a low molecular weight, which reduces the stability of the complex, or increases the foreign body reaction in vivo. On the other hand, if the molecular weight is too high, the polysaccharide content of the conjugate is too high. Thus, in general, those having a number average molecular weight in the range of 1,000 to 100,000, preferably 3,000 to 50,000, more preferably 5,000 to 20,000 are suitable. The raw magnetic nanoparticles are prepared according to the method described in Japanese Patent No. 2 7 2 6 5 20, for example, a magnetic metal oxide core particle is prepared in advance, and then reacted with a carboxyalkyl etherified polysaccharide. Alternatively, it can be produced by a one-step method of synthesizing magnetic nanoparticles in the presence of a carboxyalkyl etherified polysaccharide, but in the present invention, a one-step method that can produce raw magnetic nanoparticles having a wide range of properties is provided. More preferred. Specifically, first, a mixed solution of a divalent metal mineral salt and a trivalent metal mineral salt is added in advance to an aqueous solution of a carboxyalkyl etherified polysaccharide, and then N is added at room temperature or under heating. a OH, KOH, to no neutral base such as NH ₄ 〇_H, added until weakly alkaline, and carboxyalkyl ether polysaccharide covering it with a metal oxide core by heating to reflux for about 1 hour A complex can be formed. The free polymer that is not bound to the metal oxide core may reduce the efficiency of introducing site-specific functional molecules, so after cooling, for example, fractional precipitation with organic solvents, ultrafiltration, gel filtration It is preferable to remove as much as possible together with by-product salts by a method such as '

 In the raw magnetic nanoparticles obtained by force, the magnetic metal oxide core particles usually have an average diameter in the range of 1 to 20 nm, preferably 2 to 10 nm, more preferably 3 to 8 nm. be able to. In the present specification, the particle diameter of the core of the raw magnetic nanoparticles is measured with a transmission electron microscope, and the average diameter is an average value of 200 particle diameters.

 The ratio of the carboxyalkyl etherified polysaccharide and magnetic metal oxide core constituting the raw magnetic nanoparticles is the weight ratio of the metal in the carboxyalkyl etherated polysaccharide Z magnetic metal oxide core, usually 1/10 0-4 Z l, preferably 2/10 to 2 Zl, more preferably 3 Z10 to 1: 1.

From the viewpoint of avoiding uptake by the liver, which is an organ with a developed reticuloendothelial system, the total particle size of the raw magnetic nanoparticles is preferably a small particle size. A range of 100 nm, particularly 15 to 50 nm, more particularly 15 to 40 nm is preferred. In the present specification, the total particle diameter of the raw magnetic particles “raw” is a value when measured by a laser light scattering measurement device according to a dynamic light scattering method. In addition, the T and T ₂ relaxation ability of the raw magnetic nanoparticles is preferably as high as possible from the viewpoint of improving detection sensitivity by MR I, and the T \ relaxation ability is generally 5 to 100 (mM · sec ) —Especially 10 to 100 (mM .sec) more particularly 10 to 50 (mM .sec) —within the range of ¹ , T ₂ relaxation capacity is generally 10 to 40 ° (mM · Sec) —especially within the range of 5 0 to 4 0 0 (raM · sec) ⁻¹ , more particularly 5 0 to 2 0 0 (raM · sec) ^—1 . In the present specification, the 1 and T ₂ relaxation abilities are measured by pulse NMR of 20 MH ζ (0.47 Tesla) Τ [or Τ ₂ relaxation times and the reciprocal of the obtained relaxation times, ie, 1 / T This is a value calculated from the slope of a straight line obtained by plotting the relationship between ₂ (sec " ¹ ) and the metal concentration (mM) in the measurement sample on the graph, and using the least squares method. The physical property values for which no measurement method is described were measured according to the method described in Japanese Patent No. 2 7 2 6 5 2 0. 'Site-specific functional molecule'

 The site-specific functional molecule (hereinafter sometimes abbreviated as functional molecule) that can be introduced into the raw magnetic nanoparticles according to the present invention is arbitrarily selected according to the type of target site in the living body. As long as the molecule has a function or property that specifically recognizes and binds to or accumulates on the target site, it can be used as a polymer or a low molecule.

 Specific examples of such functional molecules include, for example, antibodies, peptides, hormones, saccharides, lesion-specific antibodies that can specifically recognize and bind or accumulate target sites such as various cancers or disease-causing lesions. And other substances that can be metabolized and decomposed. Among these, from the viewpoints of stability and safety of the introduced magnetic nanocomposite, ease of production, etc., relatively low molecular weight functional molecules, especially peptides, hormones, saccharides, etc. having a molecular weight of 10 or less. Organic molecules having a site-specific functionality are preferred. Chemical synthesis is easy, its composition can be freely changed, the possibility of endotoxin is low, and the amount of binding with raw magnetic nanoparticles can be adjusted accurately and easily. Peptides are particularly suitable because of their many advantages. ·

Examples of the peptide that can be used in the present invention include, for example, A peptide that specifically binds to RET-expressing cells (hereinafter referred to as RBP-1) disclosed in Japanese Patent No. 94, and binds to the KDR receptor of VEGF disclosed in US Patent Application Publication No. 2005/154187 Peptide (hereinafter referred to as KDR-BP), NATURE MEDICINE, 9 (9), 1173-1179 (2003), a peptide that binds to a receptor expressed on 3 cells (hereinafter referred to as GLP) —1 and le), Biochem. Biophys. Res. Co thigh., 320, 18-26 (2004), a peptide having a nuclear translocation function (hereinafter referred to as C45D18), Nat. Biotechnol. 17, 768-774 (1999), a peptide that specifically binds to _ _ 2 (hereinafter referred to as MM, Ρ-2-BP), protein A disclosed in US Pat. No. 619.7927 Examples include, but are not limited to, partial peptides (hereinafter referred to as Z33).

 The above peptide can be synthesized by a peptide synthesis method known per se, such as the well-known solid phase synthesis method (Marrifield, J. Am. Chem. Soc., 85, 2149-2154, 1963), and using a peptide synthesizer that is automated and widely used on this principle, the desired peptide can be synthesized in a short time and easily. It can also be produced using a genetic engineering technique known per se. Manufacture of introduced magnetic nanocomposites

 The introduced magnetic nanocomposite of the present invention can be produced by covalently bonding the above-described raw magnetic nanoparticles and site-specific functional molecules via a linker.

As the linker, for example, one end has a functional group (a) that can be covalently bonded to a functional group in the carboxyalkyl ether polysaccharide on the surface of the raw magnetic nanoparticles, particularly a carboxyl group or a hydroxyl group, and the other end. In addition, a linear or branched organic compound having a functional group (b) that can be covalently bonded to a functional group in the functional molecule, such as an amino group, an SH group, a carboxyl group, or a hydroxyl group. Examples include aliphatic hydrocarbons, avidin, streptavidin and the like. Examples of the functional group (a) include a nucleophilic group that can be covalently bonded to a carboxyl group, particularly an amino group that can form a strong amide bond, an active ester group that can be covalently bonded to a hydroxyl group, and the like. Also, Examples of the functional group (b) include a pyridinyl disulfide group, a maleimide group, an olefin group, an active ester group, and an amino group. Among these functional groups (b), a pyridyl disulfide group, a maleimide group, Olefin group can be bonded to SH group, active ester group can be bonded to nucleophilic group such as amino group and hydroxy group, and amino group can be bonded to electrophilic group such as active ester group. . ■ As the functional group (b), since the majority of functional molecules are substances composed of amino acid units such as antibodies, hormones, peptides, etc., they do not substantially affect the functions of these substances. In view of this, it is particularly preferable to select a functional group having a small abundance in the amino acid, for example, a group that binds to the SH group. Maleimide groups, which are easily formed, are preferred [see Walker, Tetrahedron Letters, 35 (5), 665-668 (1994)]. Specific examples of linkers that can be used when SH groups can be used for the introduction of site-specific functional molecules into raw magnetic nanoparticles include the following formula (4):

H ₂ N— (CH ₂ ) n— X (4)

 In the formula, X is a group selected from a maleimide group, a pyridyldisulfide group, an olefin group and an active ester group, and n is :! ~ 15, preferably 2: 1: 10, especially 2.

Are preferred.

 Among the above formulas (4), in the linker represented by the following formula (5) where X is a maleimide group, the amino group forms an amide bond with the carboxyl group of the carboxyalkyldextran and binds firmly. Since the maleimide group forms a specific and strong C-S bond with the SH group in the functional molecule, it is particularly suitable from the viewpoint of the chemical stability of the introduced magnetic nanocomposite and the functional expression of the functional molecule. Can be used for

Therefore, the functional molecule and the raw magnetic nanoparticles are bonded using the linker of the above formula (5). In some cases, when no SH group is present in the functional molecule of the raw material, for example, peptides such as KD R—BP, RBP —1, and GLP—1, An SH group can be introduced in advance by attaching glycyl-glycyl-cysteine to the C-terminal side of the peptide. As a result, functional molecules can be easily introduced into the raw magnetic nanoparticles using the maleimide linker. ′ The functional molecule and the raw magnetic nanoparticles can be bonded through such a linker according to a method known per se. For example, first, a linker is bonded to the raw magnetic nanoparticles, and then the linker is bonded. A functional molecule may be bound to one, or conversely, a linker may be bound to the functional molecule, and then the raw magnetic nanoparticles may be bound to the linker.

 In addition, avidin and streptavidin bind specifically and firmly to the 4 molecules of piotin. Therefore, when SH groups cannot be used for introduction of site-specific functional molecules, avidin or streptavidin is used as a linker, and it is used as a force loxyl group on the raw magnetic nanoparticles. For example, the functional molecule can be introduced into the raw magnetic nanoparticles by bonding with an amide bond, while introducing a peatin at the end of the functional molecule and firmly bonding with an avidin-piotine bond. .

 The functional molecules that can be introduced into the raw magnetic nanoparticles are not limited to one type, and two or more functional molecules can be introduced depending on the application of the introduced magnetic nanoparticles. Thus, the range of diseases that can be detected and diagnosed can be widened.

 The binding amount of the linker to the raw magnetic nanoparticles can be in the range of 1 to 30, preferably 5 to 25, more preferably 5 to 20 per particle. The method for measuring the binding amount of the introduced linker varies depending on the structure and the introduction method, but the binding amount of the maleimide linker of the above formula (5) can be measured, for example, as follows. Method for measuring the amount of maleimide linker binding:

First, the buffer solution of the raw magnetic nanoparticles to which the linker is bound is diluted appropriately and the sample solution And Add excess known amount of dartathion to the sample solution and bind to the linker. The solution is then ultrafiltered to recover unbound dartathione. The amount of this unbound dartathione was quantified using an SH group detection reagent known as El 1 man reagent (see Ellman, GL, Arch. Biochem. Biophysic. 74, 443 (1958)). That is, the amount of linker is calculated. In addition, the amount of functional molecules introduced into the raw magnetic nanoparticles can be varied depending on the application of the introduced magnetic nanoparticles, but generally 1 to 30, preferably 5 to 1 per particle. It can be in the range of 25, more preferably 5-20. The method for measuring the introduction amount of a functional molecule differs depending on the structure and introduction method. In the case of a peptide having an SH group that is most preferably used in the present invention, for example, the introduction amount is as follows. Can be measured.

Method for measuring the amount of peptide having an S H group:

 The amount of peptide bound to the raw magnetic nanoparticles through a linker can be measured by applying a general method for determining SH groups. That is, after reacting a known amount of peptide to the raw magnetic nanoparticles to which the linker is bound, the unreacted peptide recovered and purified by ultrafiltration is quantified using the aforementioned El 1 man reagent and bound. The amount of peptide introduced can be determined by calculating back the amount of peptide.

 The introduction of functional molecules into the raw magnetic nanocomposite is further described, for example, in U.S. Pat. No. 4 4 5 2 7 7 3, EP 0 4 5 2 3 4 2, U.S. Pat. 4 9 2 8 14, Weissleder et. Al., Bioconjugate Chem,, 10, 186-191 (1999). Introduced magnetic nanocomposite;

The introduced magnetic nanocomposite obtained by force substantially maintains the physical and Z or chemical properties of the raw magnetic nanoparticles. In a preferred embodiment, the particle size of the magnetic metal oxide core, the carbo The weight ratio of the metal in the xyalkyl etherified polysaccharide / magnetic metal oxide core, the overall particle size, and the T ₂ relaxation capacity hardly fluctuate, and each should be maintained within a fluctuation range of 30% or less. Good. Here, the total particle size does not change greatly due to the wrinkle change of the solution of the original magnetic nanoparticles such as CMDM, but for the introduced magnetic nanocomposite, it does not change due to the wrinkle change of the solution. The numerical value may change greatly. This is a phenomenon that is commonly seen in colloidal particles such as proteins. However, by selecting the optimal ΡΗ, the value is almost the same as the value for the raw magnetic particles alone as described above, and the fluctuation is within 30%. Can be stopped. In the specification, the whole particle diameter of the introduced magnetic nanocomposite of the present invention means this. Usefulness

 The introduced magnetic nanocomposite provided by the present invention does not substantially aggregate when administered intravenously in the state of a physiologically acceptable aqueous magnetic sol, and is specific to the target site. It is extremely useful as a contrast agent for MR I diagnosis of various lesions or lesions such as cancer and a local magnetic field therapy agent by high frequency magnetic field irradiation.

 When the introduced magnetic nanocomposite of the present invention is used as an MR I contrast agent or magnetic field therapeutic agent, the introduced magnetic nanocomplex is preferably used in the form of an aqueous sol. The concentration of the introduced magnetic nanocomposite in the aqueous sol can be varied over a wide range depending on its application, etc., but is usually about 0.1 to about 2mo 1ZL, especially ◦ 3 to 2mo 1L in terms of metal. Within range is suitable. In addition, the aqueous sol contains, for example, inorganic salts such as sodium chloride; monosaccharides such as glucose; sugar alcohols such as mannitol and sorbitol; organics such as lactic acid, citrate and tartrate. Acid salts; Various physiologically acceptable auxiliaries such as phosphate buffer and tris buffer can also be added.

When the introduced magnetic nanocomposite of the present invention is used as an MR I contrast agent, the dosage varies depending on the diagnostic site and the like, but is usually about Ι μπιο 1 / kg (body weight) to about 10 mm o 1 / in metal. kg (body weight), preferably about 2 μmo 1 / kg (body weight) to about 1 mm o 1 / kg (body weight), more preferably about 5 μπιο 1 / kg (body weight) to about l OO / zmo l Within the range of / kg (weight). Throw The administration can be performed by, for example, intravenous injection, intraarterial injection, infusion, etc., but in some cases, oral administration, direct intestinal administration, intravesical administration, and the like are also possible.

 The introduced magnetic nanocomposite of the preferred form of the present invention is excellent in storage stability. For example, when intravenously administered, it is specifically and efficiently observed after several minutes to 24 hours depending on the nature of the introduced functional molecule. Accumulated in the target site, and the diagnosis of the lesion site is preferably performed by MR I imaging. On the other hand, when the introduced magnetic nanocomposite of the present invention is used as a magnetic field therapeutic agent, the dose varies depending on the severity of the symptoms of the patient to be treated, age, treatment site, etc., but usually about 10 in terms of metal μmo 1 / kg (body) to about 10 mm o 1 / kg (body weight), preferably about 20 μπιο 1 / kg (body weight) to about lmmo 1 / kg (body weight). Administration can be performed, for example, by intravenous or intraarterial injection, infusion, etc., as in the case of MR I contrast media described above, but in some cases, direct administration to the treatment site is also possible .

 The introduced magnetic nanocomposite of a preferred form of the present invention is excellent in storage stability. For example, when intravenously administered, the target magnetic nanocomposite is specifically and efficiently after several minutes to 24 hours depending on the nature of the introduced functional molecule. Treatment of a lesioned part is suitably performed by accumulating in the part and performing high-frequency magnetic field irradiation. The frequency of the high-frequency magnetic field to be irradiated is generally 20 to ζ to 10 MHz, preferably 50 KHz to 1 ΜΗζ, and more preferably 100 to 50 OKHz. The magnetic field strength is generally lmT or more, preferably 5 mT or more, more preferably 10 mT or more. Clinical significance of introduced magnetic nanocomposite of the present invention

 In recent years, it has become possible to perform high-frequency focal irradiation under the diagnosis of MR I, and pinpoint hyperthermia for lesions has been performed. Imaging of microscopic lesions by MR I using the introduced magnetic nanocomposite of the present invention is expected to become a navigator when performing this pin-boyt therapy, and to realize a more accurate treatment method. '

Several molecular species have been tried to bind target functional molecules to raw magnetic nanoparticles and try to image them with MRI. For example, specificity by using monoclonal antibodies Highly targeted. However, when clinically applying antibodies, it is essential to convert them into human-type antibodies, the number of monoclonal antibodies that can be used is limited, and the molecular weight per molecule is large. The system for reproducible binding to the raw magnetic nanoparticles must be complicated, and it is difficult to completely prevent endotoxin contamination when preparing antibodies. Many problems remain.

The present invention makes it possible to use peptides as target functional molecules, thereby solving the above problems all at once and succeeding in generalizing target imaging by MRI. Peptide by chemical synthesis ^Endo; can toxin acquires a large amount in a state that does not mix, or it is also possible to search for a different peptide having binding properties and characteristics of by changing freely Amino acid sequence The introduced magnetic nanocomposite of the present invention has a potentially great potential. In the following, the four peptides introduced into the raw magnetic nanoparticles according to the present invention, namely KDR—BP RBP—1 GLP—1 MMP—2—BP Z 33 and C45D 18, are outlined and expected for each peptide. The clinical applicability will be explained. KDR-B P

 It is known that the growth of cancer cells requires vegetative blood vessels. For example, metastatic foci that grow beyond a diameter of l 2 mm are known to involve the growth of new blood vessels (Folkman Nat. Med., 1, 27 -31, 1995) New blood vessels are formed by the proliferation of vascular endothelial cells. Vascular endothelial cells are proliferated by vascular endothelial growth factor (VEGF-2) and its receptor, VEGF-2 receptor ( KDR) is involved as an essential factor. Therefore, it is conceivable to target cancer metastasis by using KDR as a target functional molecule. In fact, the effectiveness of DNA vaccines targeting KDR was also reported (Nietha er et al., At. Med. 12, 1369-1375, 2002)

In the present invention, a peptide (KDR—BP) ′ (Hetian et al., J. Biol. Chera. 277, 43137-43142, 2002) that binds to KDR is bound to a raw magnetic nanoparticle, One of the objectives of imaging with I is clinical use of magnetic nanocomposites with KDR-BP. Therefore, it is possible to detect metastatic lesions, and to treat cancer patients with cancer metastasis while preserving the maximum QOL.

RB P— 1

 Among childhood cancers, neuroblastoma is raised as the worst prognosis cancer. In neuroblastoma, the receptor type synthin kinase RET is frequently expressed. RET gene expression is observed in all 29 tumor tissues (Nagao et at., Jpn. J. Cancer Res. 81, 309-312, 1990), whereas 11 out of 10 cell lines (Ikeda I, Oncogene, 9, 1291-1296, 1990). In addition, by using a monoclonal antibody that binds to RET, neuroblastoma cell-selective gene transfer has become possible (Yano et al., Human Gene Therapy 11, 995-1004, 2000). Thus, a peptide (RB P-1) that binds to the RET extracellular domain has been identified to enable a highly specific molecular target (Japanese Patent Laid-Open No. 2003-018994). RBP-1 is a peptide consisting of 8 amino acids (KAGRGRDR, amino acids are expressed in one letter), and a peptide in which the central arginine is substituted with alanine (KAGAGADR) does not show binding to RET. That is, it is known that a positively charged amino acid located in the center is essential for binding to RET.

 Thus, using a magnetic nanocomposite into which RB P — 1 has been introduced according to the present invention, neuroblastoma that is localized in the small pelvic cavity and cannot be treated surgically by MRI imaging, for example. It is expected that treatment will be possible after accurate diagnosis.

GL P- 1

Sputum cancer has already infiltrated the posterior abdominal wall at the time of symptom, and it is difficult to perform surgery, and it is positioned as a malignant tumor with a very poor prognosis. 0LP— 1 (Glucagon like peptide- 1) is a gastrointestinal peptide hormone whose receptor is expressed in spleen cells (Thorens, Proc. Natl. Acad. Sci. USA 89, 8641-8645, 1992 ) , If MR I diagnosis becomes possible by using a magnetic nanocomposite into which GLP-1 is introduced according to the present invention, it is expected that early diagnosis of knee cancer will be possible. It is also expected that changes in B spleen tissue can be detected in diabetic cases. MM P-2-BP, tumor cells generally express matrix metaprotease (MM P) on the cell surface. MM P works to degrade the basement membrane and infiltrate normal tissues. Therefore, the higher the expression, the more malignant the tumor is. Of these, MMP-2 is known to degrade type IV collagen, and Dariooma is known as a tumor that specifically expresses MMP-2. Thus, by using the magnetic nanocomposite into which MMP-2_BP is introduced according to the present invention, it is possible to early detect glioma, which is a cancer type with high malignancy and high mortality, by MR I.

Z 3 3

The molecule selected as a site-specific functional molecule is generally superior to an antibody when compared to the binding ability. In order to produce a complex of various antibodies and magnetic nanoparticles, a peptide having a function of binding to various antibodies is required, and Z 3 3 is known as such a peptide (AC Braisted et al., Proc. Natl. Acad. Sci., 93, 5688-5692, 1996). Z 33 is a partial peptide of protein A (amino acid 33 residues) and binds to the 0 fragment of 1 _§ 0. Can be used and can be advantageously used for the purposes of the present invention.

Therefore, if a magnetic nanocomposite into which Z 33 is introduced according to the present invention, it is possible to bind any antibody, and bind magnetic nanoparticles to the target antigen with a very high binding ability. It is possible to improve the detection sensitivity by MR I. C 4 5 D 1 8 This peptide consists of 27 amino acids derived from V pr (Viral protein R), one of the HIV-1 gene products. Vpr consists of 96 amino acids, and when added to cell culture medium, it is known that Vpr is taken up into cells within a few hours and transported into the nucleus. The region responsible for this phenomenon has been identified (C 4 5 D 1 8) in which the C-terminal 18 amino acids are deleted from the C-terminal 45 amino acids (Taguchi et al. , Biochem. Biophys. Res. Commun. 320, 18-26, 2004). Furthermore, it has been reported that gene transfer to resting macrophages has become possible using this function (Mizoguchi et al., Biochem. Biophys. Res. Commun. 338, 1499-1506, 2005). By using the property of being easily taken up by cells in this way, it is expected that imaging of the part where blood is stored in the body will be possible. A specific application example is imaging of cancer lesions. It is known that blood vessels in cancerous areas have increased permeability, and low molecular compounds and nanoparticles leak from the blood vessels.

Such a phenomenon is called EPR (Enhanced permeation and retension), and it is suggested that the blood vessels of the cancerous part have different properties from those of normal tissues. In recent years, DDS using nanoparticles has been attempted using this EPR effect, and effective therapeutic results have been reported.

 Therefore, if a magnetic nanocomposite into which C 4 5 D 18 is introduced according to the present invention is used, it leaks out of the blood vessel and is then quickly taken up into the cell, and the tumor lesion can be visualized by MR I. Expected to be possible. In addition, by using the magnetic nanocomposite into which the C 4 5 D 1 8 is introduced, not only the cancerous part, but also blood vessel lesions such as dissecting aneurysms, and the blood-retained lesions are imaged by MR I. Therefore, early diagnosis is possible and progress of symptoms can be prevented in advance, and the prognosis of patients is expected to be improved. Example

Hereinafter, the present invention will be described more specifically with reference examples, examples and test examples. Reference Example 1: Synthesis of raw magnetic nanoparticles Carboxymethyl dextran (hereinafter referred to as CMD) 5 1 7 g having a degree of carboxymethyl substitution of 0.20 and an average molecular weight of about 10 and 0 0 0 was dissolved in 1400 ml of water, and 1 M Add ferrous chloride tetrahydrate to an aqueous solution of ferrous chloride 1 0 10 0 m 1 in an atmosphere of nitrogen under a nitrogen stream and stir while stirring at about 80 ° C. Add 3N aqueous sodium hydroxide solution 1 9 4 4m I. Next, 6N hydrochloric acid is added to adjust the pH to 7.0, and the mixture is refluxed for 1 hour and 30 minutes. After cooling, centrifuge for 30 minutes at 1880 G, ultrafiltration of the supernatant (fractionated molecular weight 50 0, 0 00 Dalton), the desired force loxymethyldextran magnetite CMDM) Aqueous sol 1800 m 1 was obtained. .

Iron concentration: 4 4.2 mg / m 1 (iron yield 83%), magnetic iron oxide particle size: 5.1 nm, total particle size: 40 nm, CMDZ iron weight ratio: 0.7, T \ Relaxation capacity: 3 2 (rnM · sec) — T ₂ relaxation capacity: 1 2 1 (mM. Sec) — Reference example 2: Synthesis of dextran-coated magnetic nanoparticles

Carboxydextran (CD x) 5 1 7 g with intrinsic viscosity of 0.0 5 3 d 1 / g and number average molecular weight of about 2,700 is dissolved in water 1 4 0 Om l. Add aqueous solution of ferric chloride 'tetrahydrate 55.7 g dissolved in nitrogen stream to 70 ml of ferric aqueous solution, and further warm to about 80 ° C', while stirring. Add 9 85 ml of 3N aqueous sodium hydroxide solution. Next, 6N hydrochloric acid is added to adjust the pH to 7.1, and the mixture is heated to reflux for 1 hour and 30 minutes. After cooling, centrifuge for 30 minutes at 1880 G, and ultrafiltrate the supernatant (fractionated molecular weight: 50,00 0 Dalton) to obtain the target carboxydextran magnetite (ATDM) aqueous sol 7 3 6 ml were obtained. Iron concentration: 5 9 mg / ml (iron yield 90%), magnetic iron oxide particle size: 5.0 nm, total particle size: 68 nm, CD xZ iron weight ratio: 0.27, T _L relaxation capacity: 2 7 (mM · sec) — T ₂ relaxation capacity: 2 0 3 (mM · sec) ^ 'Example 1 KDR— Introduction of BP (1) To 8 ml of CMDM obtained in Reference Example 1 (iron concentration 20 mg 1), 1-ethyl 3- (3-dimethylaminopropyl) carpositimide hydrochloride (hereinafter referred to as E DC) 623 mg, N-hydroxysuccinimide ( Add NHS (187 mg) and N-ethylaminomaleimide 'trifluoroacetate (22 mg) in 0.2M sodium borate buffer solution (8 ml) in order, and react at room temperature. After 20 hours, ultrafiltration (fractionated molecular weight 50,000 danoleton) was performed to remove unnecessary reagents, and linker-binding CMDM was obtained.

 (2) Add 9.1 mg of KDR-BP to the aqueous linker-bonded CMDM solution obtained in (1) and react at room temperature. After 20 hours, P-extrafiltration (fractionated molecular weight 50,000 daltons) is performed, and unbound peptides are collected and quantified. Dilute the supernatant with water, add 1. Omg of cysteine, and react at room temperature. After 20 hours, ultrafiltration (fractionated molecular weight: 50,000 daltons) was performed, and the supernatant was diluted to 8 ml with 0.1 M sodium phosphate buffer to obtain an aqueous KDR-BP-CM DM sol ( Complex number 1).

Iron concentration: 7.3 mg / m 1, CMDZ iron weight ratio: 0.59, Total particle size: 36 nm (pH 4), 97 nm (pH 7), 73 nm (pH 9), T _t relaxation capacity: 28 ( mM · sec 广¹ , T ₂ relaxation Ability: 111 (raM · sec) ' ¹ , Number of peptides introduced per magnetic nanoparticle: 11 Example 2: Introduction of RB P-1

 In Example 1, except that the peptide to be added was R BP-1 9. lmg and the linker-binding CMDM was 0.1M sodium phosphate buffer solution, the same treatment as in Example 1 was carried out. A sol was obtained (complex number 2).

Iron concentration: 6 · 5 mg / m 1, CMDZ iron weight ratio: 0.54, total particle size: 45 nm (pH 7), T \ relaxation capacity: 27 (ηιΜ · sec) " ¹ , T ₂ relaxation capacity: 104 (mM · sec) — ¹ , Number of peptides introduced per magnetic nanoparticle: 13. Example 3: Introduction of GLP— 1 In Example 1, except that the peptide to be added was changed to GLP-1 5.8 mg, the same treatment as in Example 1 was carried out to obtain GLP-1 single CMDM aqueous sol (complex No. 3).

Iron concentration: 7.7 mg / ml, CMD / iron weight ratio: 0.53, total particle size: 39 nm (pH 4), 43 nm (pH 7), 43 nm (pH 9), T relaxation capacity: 3 ¾ (mM · Sec) ^-1 T ₂ relaxation capacity: 110 (mM · sec) " ¹ , number of peptides introduced per magnetic nanoparticle: 5.2 2 Example 4: MMP— 2— Introduction of BP

 In Example 1, except that the peptide to be added was MMP-2-BP 6.6 mg, the same treatment as in Example 1 was carried out to obtain an MMP-2-BP-CMDM aqueous sol (complex number 4).

Iron concentration: 7.3 mg / m K CMDZ Iron weight ratio: 0.53, Total particle size: 52 nm (pH 9), 1 \ Relaxation ability: 29 (mM 'sec 广¹ , T ₂ Relaxation ability: 105 ( raM · sec) " ¹ , Number of peptides introduced per magnetic particle: 11.6. 'Example 5: Introduction of Z 33

 In Example 1, except that the added peptide was Z 33 1 Omg, it was processed in the same manner as in Example 1 to obtain a Z 33-CMDM aqueous sol (Complex No. 5A).

Iron concentration: 7. '5mg / ml, CMDZ iron weight ratio: 0.57, total particle size: 51 nm (pH 9), T relaxation capacity: 30 (mM · sec) " ¹ , T ₂ relaxation capacity: 109 ( mM · sec) " ¹ , Number of peptides introduced per magnetic particle: 5.8.

 Further, Z 33 to be added was synthesized in the same manner as 5 mg to obtain the above aqueous solution in which the amount of Z 33 bound was reduced by half (complex number 5 B).

 Iron concentration: 7.5 mg / m 1, CMD / iron weight ratio: ◦ .56, Total particle size: 52 nm (pH

9) T \ relaxation capacity: 31 (mM-sec) " ¹ , T ₂ relaxation capacity: 113 '(mM · _sec , number of peptides introduced per magnetic particle: 3.2.

' Example 6: Introduction of C 45 D 18

 A C45D 18-CMDM aqueous sol was obtained in the same manner as in Example 1 except that the added peptide was C 45D 18 7.6 mg (complex number 6).

Iron concentration: 7.8 mg m 1 CMD / Iron weight ratio: 0.55, Total particle size: 109 nm (pH 4), 100 nm (pH 7), 46 nm (pH 9), T \ Relaxation ability: 30 (raM · sec) — ¹ , Ding ₂ relaxation capacity: 1 17 (mM · sec) ” ¹ , Number of peptides introduced per magnetic nanoparticle: 5.9 Example 7: Pretreatment with mouse administration ·

0.55 ml of KDR-BP-CMDM (complex number 1) obtained in Example 1 is diluted with phosphate buffered saline to 10 ml. This membrane filter (pore one Size: 0 · 2 _M m) was filled in a vial with sterile filtered at (iron concentration: 0. 4mg / mL). Example 8: Formulation example

KDR—BP—CMDM (complex number 1) obtained in Example 1 6. Oml was concentrated to 1.5 ml by centrifugal ultrafiltration (fractional molecular weight 50,000 daltons), and D-mannito 64 mg and 1 M_L monolactic acid 16 1 were added, and the pH was adjusted to 6.5 with 1 M hydrochloric acid. This main 'emissions membrane filter: with filter-sterilized at (pore one size 0. 2 / m) was filled into vial bottle was further filled with nitrogen. (Iron concentration: 28. 7mg / mL) ₀ Comparative Example 1 : KDR—Introduction of BP

(1) To 8 ml of ATDM (iron concentration 20 mg Zm 1) obtained in Reference Example 2, 91 mg of EDC, 27 mg of NHS and N-ethylaminomaleimide 'trifluoroacetate 6.3 mg of 0.2 M— Add 8 ml of sodium borate buffer solution sequentially and react at room temperature. After 20 hours, ultrafiltration (fractionated molecular weight: 50,000 daltons) was performed to remove unnecessary reagents, and linker group-bound ATDM was obtained. , (2) Add 4.2 mg of KDR-BP to the linker-bonded ATDM aqueous solution obtained in (1) and react at room temperature. After 20 hours, ultrafiltration (fractionated molecular weight 50,000 danoleton) is performed, and unbound peptide is recovered and quantified. Dilute the supernatant with water, add 1. Omg of cysteine, and react at room temperature. After 20 hours, ultrafiltration (fractionated molecular weight: 50,000 daltons) was performed, and the supernatant was diluted to 8 ml with 0.1 M sodium phosphate buffer to obtain an aqueous KDR-BP-AT DM sol (complex Body number 7).

Iron concentration: 7.3 mg / 1, CD x / iron weight ratio: 0.25, Total particle size: 6 7 nm (H 7), T relaxation capacity: 27 (mM-sec) " ¹ , T ₂ relaxation capacity : 1 8 9 (mM · sec) " ¹ , Number of peptides introduced per magnetic nanoparticle: 5.1. Comparative Example 2: Introduction of RBP-1

 In Comparative Example 1, treatment was performed in the same manner as in Comparative Example 1 except that RBP-1 5.2 mg and linker-bound ATDM was 0.1 M sodium phosphate buffer solution. An aqueous sol was obtained (complex number 8).

Iron concentration: 7.7mgZm 1, CDxZ iron weight ratio: 0.22, total particle size: 6 7 nm (pH 7), 1 \ relaxation ability: 25 (mM · sec) " ¹ , Τ relaxation ability: 205 ( raM · sec ", number of peptides introduced per magnetic nanoparticle: 8.5. The properties of composite numbers 1 to 8 obtained in Examples 1 to 6 and Comparative Examples 1 and 2 are summarized. If shown, it is as shown in Table 1 below. table 1

試験例 1 ： 安定性試験

Test example 1: Stability test

 In Example 1 and Comparative Example 1, a dartathione monomagnetic nanocomposite was obtained in the same manner as in Example 1 and Comparative Example 1 except that the peptide to be added was glutathione (complex number 9 , Ten) . After 5 ml of a 5 OmM calcium chloride solution was added to 5 ml of these composites (iron concentration 0.5 mgZml), the mixture was heat-treated with an autoclave, and the degree of aggregation of the solution was visually determined. In addition, the case where the solution was transparent was marked as ◯, the case where the solution was agglomerated, and the case where it was precipitated were marked as X. The results are shown in Table 2. Table 2

試験例 2 : KDR—BP i n v i t r o

Test example 2: KDR—BP invitro

KDR-BP-CMDM (complex number 1) obtained in Example 1 and the raw material CMDM of Reference Example 1 were added to KDR positive cultured cells and negative cultured cells, respectively, as 800 / igZml as Fe. . After 12 hours, rinsed with culture medium and then peeled off the cells from the plate, and embedded in acrylamide gel, 1 at 5 Tesla MR I system (SI EMENS Co.) were taken T ₂ -weighted images. The results are shown in Fig. 1. In the figure, Α: Water only, B: CMDM only, C: Negative cell + CMD M, D: Positive cell + CMDM, E: Negative cell + KDR—BP—CMDM, F: Positive cell + KD R—BP—CMDM It is.

 As a result, accumulation of magnetic substances was confirmed in the specimen (F in Fig. 1) in which KDR-BP-CMDM was allowed to act on KDR-positive cells. Test example 3: RBP—1 i n v i t r o

RBP-l-CMDM (complex No. 2) obtained in Example 2 and the raw material CMDM of Reference Example 1 were added as Fe to 800 ig / ml to RET positive cultured cells and negative cultured cells. After 12 hours, rinsed with culture medium and then peeled off the cells from the plate, and embedded in acrylamide gel, 1 at 5 Tesla MR I system (SI EMENS Co.) were taken T ₂ -weighted images. Figure 2 shows the results. In the figure, Α: Water only, B: CMDM only, C: Negative cell + CMDM, D: Positive cell + CMDM, E: Negative cell + KDR—BP—CMDM, F: Positive cell + KD R—BP—CM DM.

 As a result, accumulation of the magnetic substance was confirmed in the specimen (F in Fig. 2) in which RB P-1 CMDM was allowed to act on RET positive cells. Test Example 4: KDR-B PinVivo

KDR—BP—CMDM (complex number 1) obtained in Example 1 was intravenously injected into mice transplanted with a KDR positive tumor and a negative tumor at 5 g / g body weight as Fe, for 4 hours and 20 minutes. After 1- MR I was photographed with a 5 Tesla MR I device (manufactured by SI EMENS). The results are shown in Fig. 3. Figure 3 is a T ₂ -weighted images of the mouse whole body.

 As a result, it was confirmed that KDR-positive tumors (arrows) appear black compared to negative tumors, and magnetic substances are accumulated.

 Test Example 5: C45D18 i n v i t r o

 C 45 D 18—CMDM (complex number 6) obtained in Example 6 and the raw material CMDM of Reference Example 1 were added to He La cells at 800 μg / m 1 as Fe, and 12 hours later. MR I was photographed using a 1.5 Tesla MR I device (manufactured by SI EMENS). The results are shown in FIGS. In the figure, A: Water only, B: CMDM only, C: He La cell + CMDM, D: He B cell 3 + 45D 18 (2) — CMDM, E: He La cell + C45 D 18 ( 6) — CMDM, F: He La cells + C45D18 (10) One CMDM.

 As a result, it was shown that C45D 18-CMDM was taken up into cells as compared with CMDM alone, and that the amount taken up into cells was increased depending on the number of binding. Test example 6: C 45 D 18 i n v i v o

C45D 18-CMDM (complex number 6) obtained in Example 6 was intravenously injected at 15 μg / g body weight as Fe into a mouse transplanted with a KDR-expressing tumor, and 1 hour and 40 minutes later 1. MR I was photographed with a 5 Tesla MR I device (manufactured by SI EMENS). The results are shown in Fig. 5. Figure 5 is a T ₂ -weighted images of the mouse whole body, mouse before administration left, right is a mouse after administration. As a result, it was confirmed that the mouse tumor after administration (two points on the right arrow) was black in comparison with the mouse tumor before administration (left arrow), and the magnetic substance was accumulated.

C 45 D 18—CMDM (complex number 6) obtained in Example 6 and the raw material CMDM of Reference Example 1, And C 45 D 18 peptide alone is added to He La cells as 800 / ig / m 1 as Fe, and the cells after incubation for 12 hours are used to specify the frequency and magnetic field strength. For 1 hour. When the cell proliferation degree was examined, it was as shown in FIG. This indicates that Complex 6 has the effect of damaging cells by magnetic field irradiation. Test Example 8

 Z33-CMDM (complex number 5 A, B) obtained in Example 5 and M2 antibody (Sigma) were mixed and incubated at 37 ° C for 1 hour. Next, this liquid mixture is used as a surface plasmon resonance apparatus.

(BI ACORE ™, GST-FLAG protein, which is the target antigen, was bound on the sensor chip) and the ability to bind to the antigen was evaluated. Table 3 shows the results of the mixing ratio of the complex and antibody and the binding ability thereof.

Table 3

表 3の結果より、 コントロール I g Gを作用させた複合体群においては、 抗原との結合能を 全く示さないのに対し、 M 2抗体を作用させた複合体群においては、 目的抗原との結合を示し た。 また、 その結合量は、 磁性ナノ粒子に結合している Z 3 3の量および作用させる M 2抗体 の量に応じて向上した。

From the results in Table 3, the complex group treated with control IgG showed no binding ability to the antigen, whereas the complex group treated with M2 antibody showed no binding with the target antigen. Showed binding. In addition, the amount of binding increased with the amount of Z 3 3 bound to the magnetic nanoparticles and the amount of M 2 antibody to act.

Claims

The scope of the claims 1. Magnetic metal oxide nanoparticles coated with a water-soluble carboxyalkyl etherified polysaccharide and having an overall diameter in the range of 10 to 100 nm, covalently bonded to the polysaccharide via a linker. Organic magnetic nanocomposite consisting of site-specific functional molecules. 2. The complex according to claim 1, wherein the carboxyalkyl etherified polysaccharide is a carboxyalkyl ester of a glucose polymer and a teration product. 3. The complex according to claim 2, wherein the glucose polymer is dextran. 4. The composite according to claim 2, wherein the alkyl moiety of the carboxyalkyl ether compound is methyl. 5. The composite according to claim 1, wherein the core of the magnetic metal oxide nanoparticles is magnetic iron oxide and the diameter thereof is in the range of 1 to 20 nm. 6. The ratio of the carboxyalkyl etherified polysaccharide to the magnetic metal oxide core in the weight ratio of the metal in the carboxyalkyl etherified polysaccharide magnetic metal oxide core is within the range of 1/10 to 41. 5. The complex according to 5. . 7 magnetic metal oxide nanoparticles 5~ 1 0 0 (mM · sec ) - 1 in the ¹ range \ relaxation capability and 1 0~4 0 0 (raM · sec ) - T 2 relaxation in ^one of the ranges The complex according to claim 1. 8. The composite of claim 1, wherein the magnetic metal oxide nanoparticles coated with a carboxyalkyl etherified polysaccharide have an overall diameter in the range of 15-40 nm. 9. The complex according to claim 1, wherein the site-specific functional molecule is a peptide. 10. The peptide is KDR—BP, RBP—1, C45D18, GLP—  10. A complex according to claim 9, selected from BP and Z33. The linker is of formula (5):

 The complex according to claim 1, which is a compound represented by the formula: 12. An MR I contrast agent comprising the complex according to any one of claims 1 to 11 as an active ingredient. - 13. A magnetic field therapeutic agent comprising the complex according to any one of claims 1 to 11 as an active ingredient. 14. A method for diagnosing a lesion site, comprising administering the complex according to any one of claims 1 to 11 to a patient and then performing MR I imaging. 15. A method for treating a lesion site comprising administering the complex according to any one of claims 1 to 11 to a patient, and then irradiating a radio frequency magnetic field. 1 6. Use of the complex according to any one of claims 1-11 in an MR I contrast agent. 1 7. Use of the complex according to any one of claims 1 to 11 in a magnetic field therapeutic agent.