WO2019172213A1 - Nanoparticle, nanoparticle production method, and anti-tumor agent - Google Patents

Nanoparticle, nanoparticle production method, and anti-tumor agent Download PDF

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WO2019172213A1
WO2019172213A1 PCT/JP2019/008494 JP2019008494W WO2019172213A1 WO 2019172213 A1 WO2019172213 A1 WO 2019172213A1 JP 2019008494 W JP2019008494 W JP 2019008494W WO 2019172213 A1 WO2019172213 A1 WO 2019172213A1
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core
shell
nanoparticle
glucose
mfe
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French (fr)
Japanese (ja)
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優子 一柳
達哉 橋本
孝明 千本松
秀吉 田中
阿部　真之
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国立大学法人横浜国立大学
学校法人埼玉医科大学
国立研究開発法人情報通信研究機構
国立大学法人大阪大学
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Priority to JP2020505032A priority Critical patent/JP7401864B2/en
Publication of WO2019172213A1 publication Critical patent/WO2019172213A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/26—Iron; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/52—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an inorganic compound, e.g. an inorganic ion that is complexed with the active ingredient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a nanoparticle, a method for producing the nanoparticle, an antitumor agent containing the nanoparticle as an active ingredient, and a method for producing a core-shell nanoparticle for producing the nanoparticle.
  • Nanoparticles which are ultrafine particles on the nanometer scale, bring about new and unique physical properties that have not been possible in the past, and high performance as functional materials can be expected, so various substances have been studied.
  • the magnetic material is made into fine particles, single domain particles having no domain wall are generated, and the coercive force is expected to increase.
  • the present inventors have found that the surface has been successful in the preparation of the magnetic nanoparticles of magnetite coated with SiO 2, and have reported a technique of introducing a functional group through the SiO 2 (Patent Document 1, 2). With this functional group, it is possible to easily modify the magnetic fine particles with a drug or the like and to easily take the magnetic fine particles into cells or tissues.
  • cancer cells are known to take up 3 to 8 times more glucose per unit time than normal cells. It is also known that the EPR effect, that is, the blood vessel wall around the cancer cells easily permeates the polymer drug, and the permeated polymer drug easily accumulates in or around the tumor tissue.
  • a structure similar to glucose can be introduced on the surface of the nanoparticle, it can be expected that a property that is easy to accumulate in or around the tumor tissue can be obtained.
  • the present invention relates to a nanoparticle chemically modified with glucose or a glucose derivative, a method for producing the same, an antitumor agent containing the nanoparticle as an active ingredient, and a method for producing a core-shell nanoparticle for producing the nanoparticle. It is an issue to provide.
  • M represents a transition metal
  • MFe 2 O 4 core consisting of the steps of forming a, a core-shell nanoparticles having a shell comprising a functional group that covers the core, The method for producing nanoparticles according to any one of [1] to [4], further comprising reacting glucose or a glucose derivative with the functional group.
  • M represents a transition metal
  • MFe 2 core consisting of O 4 forming the, core-shell nanoparticles having a shell of a polyalkylene glycol to cover the core , Oxidizing the terminal hydroxymethyl group of the polyalkylene glycol to a carboxyl group; And the step of reacting glucose or a glucose derivative with the carboxyl group.
  • An antitumor agent comprising the nanoparticle according to any one of [1] to [4] as an active ingredient.
  • the present invention provides a nanoparticle, a method for producing the nanoparticle, an antitumor agent containing the nanoparticle as an active ingredient, and a method for producing a core-shell nanoparticle for producing the nanoparticle.
  • the shell is chemically modified with glucose or a glucose derivative, it can be expected that the property of being easily accumulated in a tumor tissue can be obtained.
  • the nanoparticle of the present invention has a core composed of MFe 2 O 4 , it can be expected to be used for magnetic hyperthermia (cancer hyperthermia).
  • FIG. 1 shows measurement results of X-ray diffraction (XRD) of the cobalt ferrite (CoFe 2 O 4 ) magnetic nanoparticles obtained in Comparative Example 1.
  • FIG. 1 (bottom) is a measurement result of X-ray diffraction (XRD) of the core-shell nanoparticle obtained in Example 1.
  • FIG. 2 (upper) shows the FT-IR measurement results of the core-shell nanoparticle obtained in Example 1.
  • FIG. 2 (bottom) shows the FT-IR measurement results of a cobalt ferrite (CoFe 2 O 4 ) standard sample.
  • FIG. 3 shows the results of MS measurement of the core-shell nanoparticle obtained in Example 1.
  • FIG. 1 shows measurement results of X-ray diffraction (XRD) of the cobalt ferrite (CoFe 2 O 4 ) magnetic nanoparticles obtained in Comparative Example 1.
  • FIG. 1 (bottom) is a measurement result of X-ray diffraction (XRD) of the core-shell nano
  • FIG. 4 shows the FT-IR measurement results of the core-shell nanoparticle obtained in Example 2.
  • FIG. 4 (bottom) shows the FT-IR measurement results of the core-shell nanoparticle obtained in Example 1.
  • FIG. 5 shows the FT-IR measurement results of the nanoparticles obtained in Example 3.
  • FIG. 5 (bottom) shows the FT-IR measurement results of the core-shell nanoparticle obtained in Example 2. It is a TEM image which shows that the nanoparticle of Example 3 was taken in in the HeLa cell. It is a measurement result of FT-IR of core-shell type nano fine particles. It is a measurement result of the X-ray diffraction (XRD) of a core-shell type nanoparticle. It is a measurement result of FT-IR of core-shell type nano fine particles.
  • XRD X-ray diffraction
  • Nanoparticles of this embodiment (at the MFe 2 O 4, M represents a transition metal) core made of MFe 2 O 4 and has a shell that covers the core, the shell is glucose or glucose derivative It is chemically modified with
  • the nanoparticle 1 of the following formula (1) is an example of the structure of the nanoparticle of the present embodiment.
  • the nanoparticle 1 has a core 10 (M represents a transition metal) made of MFe 2 O 4 and a shell 20 that covers the core 10, and the shell 20 has a glucose derivative Glc via a divalent linking group X. It is chemically modified with '.
  • X is a divalent linking group.
  • the glucose derivative Glc ′ refers to a compound having a glucose skeleton.
  • the glucose derivative Glc ′ is preferably a glucose derivative having an amino group.
  • a specific example of the glucose derivative Glc ' is glucosamine.
  • nanoparticles 1 of the formula (1) those whose shell is chemically modified with glucosamine include nanoparticles represented by the following formula (1-1).
  • the nanoparticle 2 of the following formula (2) is an example of the structure of the nanoparticle of the present embodiment.
  • the nanoparticle 2 has a core 10 made of MFe 2 O 4 (M represents a transition metal) and a shell 20 that covers the core 10, and the shell 20 is chemically formed with glucose via a divalent linking group X. It is qualified.
  • X is a divalent linking group.
  • nanoparticle 2 of formula (2) include nanoparticles represented by the following formula (2-1), formula (2-2), and formula (2-3).
  • the shell covering the core is chemically modified with glucose or a glucose derivative.
  • the shell may cover the entire core or may partially cover the core.
  • An inclusion structure including two or more cores may be formed inside the shell.
  • the core made of MFe 2 O 4 is covered with the shell because the glucose or glucose derivative reacts after undergoing a reaction step of chemically modifying glucose or glucose derivative to the particle. It can be confirmed by chemical modification of the sex shell.
  • the material to be the shell may be any material that can be chemically modified with glucose or a glucose derivative, and may be composed of polyalkylene glycol. Nanoparticles having polyalkylene glycol as a shell can be produced, for example, by a “method for producing core-shell nanoparticle” described later.
  • the material for the shell may be made of amorphous SiO 2 , and the shell may form an amorphous SiO 2 network (network film) covering the core.
  • the core may be separated by an amorphous SiO 2 network.
  • the “network film” include those in which amorphous SiO 2 surrounds the periphery of each core and amorphous SiO 2 is continuous, but is not limited thereto.
  • the confirmation that the core of the nanoparticle is covered with the shell made of amorphous SiO 2 is that the diffraction lines of amorphous SiO 2 and magnetite are observed by X-ray diffraction, and the primary particle diameter of the nanoparticle is It can also be carried out because of the value expected from the half width of the X-ray diffraction.
  • the core according to the present embodiment is made of MFe 2 O 4 .
  • M represents a transition metal.
  • the transition metal is preferably one that becomes divalent when ionized, and examples thereof include Cr, Mn, Fe, Co, Ni, Cu, and Zn. Two or more of these transition metals may be used in combination.
  • M is Fe
  • MFe 2 O 4 is, for example, Fe 3 O 4 .
  • M is preferably Co.
  • MnZn is preferable.
  • the nano fine particles according to the present embodiment may include the core and a shell covering the core, and the shell may be made of amorphous SiO 2 chemically modified with glucose or a glucose derivative.
  • amorphous SiO 2 for example, using a coupling agent such as 3-isocyanatopropyltriethoxysilanesilane, 3-glycidoxypropyltriethoxysilane, etc., the formulas (2-1) and (2-2) Glucose or a glucose derivative can be chemically modified like the nanoparticle represented by these.
  • the shell may form an amorphous SiO 2 network (network film) covering the core.
  • the nano fine particles according to the present embodiment may form an inclusion structure including two or more cores inside the shell.
  • the MFe 2 O 4 molar ratio of the SiO 2 to (SiO 2 / MFe 2 O 4 ) is preferably 0.1 to 5, 0.3 or higher It is more preferably 4 or less, and further preferably 0.4 or more and 2 or less. It is preferable SiO 2 / MFe 2 O 4 is 0.1 to 5. Also, when the SiO 2 / MFe 2 O 4 falls within the above range is preferred since it is excellent in dispersibility of the nanoparticles.
  • Nanoparticles of the present embodiment for example, (in the MFe 2 O 4, M represents a transition metal) core made of MFe 2 O 4 core-shell nano with a, a, a shell comprising a functional group that covers the core It can be produced according to a method having a step of forming fine particles and a step of reacting glucose or a glucose derivative with the functional group.
  • a method in accordance with Patent Document 1 Japanese Patent Laid-Open No. 2001-261334 and Patent Document 2 (Japanese Patent Laid-Open No. 2007-269770) can be employed.
  • the method described in “Production method of mold nanoparticle” can be employed.
  • Nanoparticles whose surfaces are coated with SiO 2 formed by a method in accordance with Patent Document 1 (Japanese Patent Laid-Open No. 2001-261334) and Patent Document 2 (Japanese Patent Laid-Open No. 2007-269770) further include glucose or glucose
  • a functional group may be introduced with a silane coupling agent.
  • —CH 2 The core-shell nanoparticle having a —COOH group on the surface may be obtained by oxidizing the OH group.
  • M represents a transition metal
  • core made of MFe 2 O 4 and consists of a polyalkylene glycol to cover the core
  • the step of reacting glucose or a glucose derivative with the functional group can be performed by a known method depending on the combination of the functional group and glucose or the glucose derivative.
  • the step of causing the glucose derivative to react with the carboxyl group can be represented by the following formula (4).
  • the method of manufacturing the core-shell nanoparticle of the present embodiment includes a step of heating and mixing polyalkylene glycol, a transition metal chloride containing iron, and an alkali to obtain a hydroxide; Heating and baking the reaction product containing the hydroxide.
  • polyalkylene glycol examples include polyethylene glycol and polypropylene glycol. These polyalkylene glycols preferably have a mass average molecular weight of 200 to 8000, more preferably 300 to 2000.
  • a reagent of MCl 2 .6H 2 O (M represents a transition metal) or FeCl 2 .4H 2 O can be used as a transition metal chloride containing iron.
  • M represents a transition metal
  • FeCl 2 .4H 2 O FeCl 2 .4H 2 O
  • a neutralization reaction occurs to obtain a hydroxide.
  • the temperature for the neutralization reaction is preferably 40 to 100 ° C, more preferably 60 to 80 ° C.
  • the neutralization reaction time is preferably 30 minutes to 2 hours.
  • the core-shell nanoparticle can be precipitated in the liquid polyalkylene glycol by heating and baking the reaction product containing hydroxide.
  • the firing temperature is preferably 120 to 280 ° C, more preferably 160 to 220 ° C.
  • the firing time is preferably 3 hours or longer.
  • the volume average particle diameter of the magnetic part of the core-shell nanoparticle of the present embodiment is preferably 1 nm or more and 50 nm or less, and more preferably 2 nm or more and 20 nm or less.
  • the volume average particle diameter is a primary particle diameter obtained for the magnetic part of one core of the core-shell nanoparticle.
  • the volume average particle diameter is an arithmetic average diameter.
  • the volume average particle diameter of the magnetic part of the core of the core-shell nanoparticle can be determined by X-ray diffraction (XRD).
  • the core-shell nanoparticle is produced by mixing the aqueous solution and firing, the higher the firing temperature and the longer the firing time, the larger the core-shell nanoparticle grows and the particle size increases. By adjusting the firing time, the particle size of the core-shell nanoparticle can be controlled.
  • XRD analysis was performed using a powder X-ray diffractometer (Rigaku: MiniFlex II).
  • the secondary particle size (mode diameter and median diameter) is measured from the particle size distribution of the nanoparticles in an aqueous solution using a laser diffraction / scattering type particle size distribution analyzer (Horiba: LA-950V2). did. All samples were dispersed in an aqueous solution, subjected to dispersion treatment by ultrasonic treatment, and then measured in a circulation system. The outline of the device is shown below.
  • the precipitate dried for about 40 hours in a thermostat at about 50 ° C. was pulverized in a mortar. Then, it baked at about 800 degreeC for about 16 hours, and obtained the nanoparticle sample of the comparative example.
  • the composition of the obtained nanoparticle was CoFe 2 O 4 .3SiO 2 .
  • the average particle size (primary particle size) was 10 nm.
  • the mode diameter was 3.63 ⁇ m
  • the median diameter was 2.27 ⁇ m.
  • Example 1 ⁇ Preparation of CoFe 2 O 4 Nanoparticles Using PEG400>
  • 5 mmol of FeCl 2 .4H 2 O, 2.5 mmol of CoCl 2 .6H 2 O, and 15 mmol of sodium hydroxide were mixed at 70 ° C. for 1 hour for neutralization reaction.
  • the reaction solution is placed in a box oven as it is in a reaction vessel, heated at 200 ° C. for 16 hours, precipitated with a centrifuge (3500 rpm, 10 min) against the reaction solution after furnace cooling, and three times with ethanol. The washing process was repeated three times. Dried to obtain a powder of core-shell nanoparticles of the CoFe 2 O 4 and the core.
  • the average particle diameter (primary particle diameter) of the magnetic part of the obtained core-shell nanoparticle was 12 nm.
  • FIG. 2 shows the FT-IR measurement results of the core-shell nanoparticle of Example 1.
  • FIG. 2 (bottom) shows the FT-IR measurement results of the CoFe 2 O 4 standard sample.
  • the core-shell nanoparticle produced by this production method is washed with ethanol, and if there is a PEG400 alone, it is removed in the washing process.
  • the measurement result in FIG. 2 (upper) has a bond peak peculiar to saturated hydrocarbons such as COC, CO, and CC. Therefore, the core shell having CoFe 2 O 4 produced using PEG400 as the core.
  • the type nanoparticle is considered that the main chain of PEG derived from PEG400 is chemically bonded to the core of CoFe 2 O 4 .
  • the secondary particle diameter of the CoFe 2 O 4 nanoparticle produced using PEG400 was a mode diameter of 475 nm and a median diameter of 430 nm.
  • the colloid diameter in the liquid was reduced by an order of magnitude, confirming excellent dispersibility. .
  • Example 2 ⁇ Carboxylation of hydroxyl group of PEG400 terminal>
  • 0.2 g of the CoFe 2 O 4 nanoparticle powder obtained in Example 1 was placed, 4 ml of an aqueous hydrogen peroxide reagent (30 W / V%) was placed, and the mixture was stirred at room temperature for 10 hours. After washing with pure water three times, it was dried to obtain a powder.
  • FIG. 4 shows the FT-IR measurement results of the core-shell nanoparticle of Example 2.
  • FIG. 4 (lower) shows the FT-IR measurement results of the core-shell nanoparticle of Example 1. From the measurement result of FIG. 4 (upper), a peak derived from a ketone at 1660 nm and a peak derived from OH spreading in a short wavelength direction from 2500 to 3300 nm were observed. It was confirmed that carboxylation of the hydroxyl group was correctly performed according to the formula (5).
  • Example 3 ⁇ Chemical modification of glucosamine to PEG400>
  • 0.05 g of the core-shell nanoparticle powder obtained in Example 2 and 0.5 g of glucosamine hydrochloride were added, and the mixture was stirred and heated at 130 ° C. for 25.5 h while cooling with Liebig. Thereafter, it was washed with pure water and dried to obtain a powder.
  • FIG. 5 shows the FT-IR measurement results of the nanoparticle of Example 3.
  • FIG. 5 (bottom) shows the FT-IR measurement result of the nanoparticle of Example 2.
  • the peak near 1600 nm it can be seen that after the reaction, the peak is broad and the vertex is moved to the left. This is presumably because the COOH group on the side of the core-shell nanoparticle and the amino group of glucosamine were dehydrated and condensed to form an amide bond, so that peaks near 1550 nm were superimposed. Moreover, since the peak intensity of CH near 2880 nm is increased and the peak of OH is broadened by the influence of sugar having OH in various states, it can be considered that the sugar is modified. From the above measurement results of FIG. 5 (upper), it was confirmed that the chemical modification of glucosamine to PEG400 was correctly performed according to the equation (6).
  • nanoparticle of Example 3 is chemically modified with glucosamine having a glucose skeleton in the shell, it can be expected that the nanoparticle of Example 3 can easily accumulate in the tumor tissue. Also, nanoparticles of Example 3, because it has a core made of MFe 2 O 4, can be expected the use of the magnetic hyperthermia (cancer hyperthermia).
  • Example 4 (material) -Medium: DMEM containing 10% (vol / vol) calf serum was used. Hereinafter, it is referred to as a medium (DMEM / 10% CS).
  • DMEM / 10% CS a medium obtained from Riken Cell Bank were subcultured in a medium (DMEM / 10% CS), seeded in a 35 mm glass bottom dish, and then 24 hours later were used for experiments. The number of cells at the time of seeding was 2 ⁇ 10 5 cells / dish.
  • PBS (pH 7.4) was added to the nanoparticulate powder of Example 3 to form a uniform suspension by sonication, and finally diluted with PBS (pH 7.4) as appropriate.
  • a suspension with a concentration of 10 mg / ml was prepared.
  • a nanoparticle suspension diluted 100 times with a medium (DMEM / 10% CS) was prepared.
  • FIG. 6 is a TEM image showing that the nanoparticle of Example 3 was taken up into HeLa cells.
  • the aggregates of the nanoparticle of Example 3 can be confirmed in the endosome / lysosome, and it was confirmed that the nanoparticle of Example 3 was taken into HeLa cells 24 hours after the dropping. did it.
  • the antitumor agent containing the nanoparticle whose shell is chemically modified with glucose or a glucose derivative as an active ingredient can be used for magnetic hyperthermia. Be expected.
  • the average particle diameter (primary particle diameter) of the magnetic part of the obtained core-shell nanoparticle was 12 nm. Since it was confirmed from FT-IR, TG-DTA (differential thermal analysis) and the like that a sufficient amount of PEG was present after washing, it is considered that PEG coated the particle periphery.
  • glucose uptake also changes the uptake into cancer cells, it is considered that glucose is modified by PEG containing particles and that the amount of uptake of particles is different. It is considered that about 6000 PEGs are attached to one core particle, and it is considered that the core particle is covered with a thickness of about 2 nm.
  • Example 5-2 In 50 ml of PEG400, 5 mmol FeCl 2 .4H 2 O, 2.0 mmol MnCl 2 .4H 2 O, 0.5 mmol ZnCl 2 (anhydrous), and 15 mmol sodium hydroxide at 70 ° C. for 1 hour They were mixed and neutralized.
  • the reaction solution is placed in a box oven as it is in the reaction vessel, heated at 160 ° C. for 16 hours, precipitated with a centrifuge (3500 rpm, 10 min) against the reaction solution after furnace cooling, and twice with ethanol. And washed twice with pure water. Thereafter, drying was performed to obtain a powder of core-shell nano particles having Mn 0.8 Zn 0.2 Fe 2 O 4 as a core.
  • the average particle diameter (primary particle diameter) of the magnetic part of the obtained core-shell nanoparticle was 6 nm. There was a tendency that the smaller the amount of water added during the production, the narrower the particle size distribution, and it was possible to obtain almost monodisperse nanoparticles with an average particle size of 6 nm.
  • Example 5-3 In a mixture of 50 ml PEG 400 and 4 ml pure water, 5 mmol FeCl 2 .4H 2 O, 2.0 mmol MnCl 2 .4H 2 O, 0.5 mmol ZnCl 2 (anhydride), and 15 mmol water Sodium oxide was mixed at 70 ° C. for 1 hour to carry out a neutralization reaction. The reaction solution is placed in a box oven as it is in the reaction vessel, heated at 160 ° C. for 16 hours, precipitated with a centrifuge (3500 rpm, 10 min) against the reaction solution after furnace cooling, and twice with ethanol. And washed twice with pure water.
  • a centrifuge 3500 rpm, 10 min
  • the average particle diameter (primary particle diameter) of the magnetic part of the obtained core-shell nanoparticle was 10 nm.
  • the primary particle size of the oxide in the core portion is 6 to 30 nm and can be controlled by the production conditions (water amount, firing temperature).
  • Example 6 The core-shell nanoparticle obtained in Example 5 was further subjected to carboxylation of the hydroxyl group at the PEG 400 end as in Example 2 and chemical modification of glucosamine to PEG 400 in the same manner as in Example 3.
  • FIG. 7 shows the FT-IR measurement result of the MnFe 2 O 4 standard sample.
  • the FT-IR measurement result of Mn 0.8 Zn 0.2 Fe 2 O 4 nanoparticle using PEG400 of Example 5 is shown in FIG. 7 (middle).
  • 1105 C-O-C antisymmetric stretching vibration of ⁇ 55cm -1, CH 2 symmetric deformation vibration of 1485 ⁇ 15cm -1, 3400 O- H between stretching movement of ⁇ 200 cm -1 was observed.
  • FIG. 7 (bottom) shows the FT-IR measurement results of the nanoparticles after chemical modification with glucosamine of Example 6.
  • Example 7 ⁇ Preparation of CoFe 2 O 4 Nanoparticles Using PEG2000> 1/300 mol of FeCl 2 .4H 2 O and 1/600 mol of CoCl 2 .6H 2 O were dissolved in 1 ml of water and added to 30 g of PEG2000, which was liquefied at 55 ° C. After stirring for 10 hours, NaOH dissolved in 0.5 ml water was added. The mixture was stirred for 10 hours and then heated at 160 ° C. for 16 hours. Centrifugation, washing with ethanol and water, and drying at 50 ° C. gave core-shell nanoparticulate powders with CoFe 2 O 4 as the core. The average particle diameter (primary particle diameter) of the magnetic part of the obtained core-shell nanoparticle was 27 nm.
  • FT-IR measurement result (FT-IR measurement result) Subsequently, FT-IR measurement of the obtained core-shell nanoparticle was performed.
  • the FT-IR measurement result of the core-shell nanoparticle of Example 7 is shown in FIG.
  • the thick line “Std. (MnFe)” in FIG. 9 is the FT-IR measurement result of the MnFe 2 O 4 standard sample
  • the dotted line “400” in FIG. 9 is the CoFe 2 O 4 nanoparticle using the PEG 400 of Example 1. It is a FT-IR measurement result of fine particles.

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Abstract

This nanoparticle (1) has a core (10) of MFe2O4 (where M represents a transition metal) and a shell (20) covering the core (10). The shell (20) is chemically modified with glucose or a glucose derivative Glc'. X is a divalent linking group. The nanoparticle (1) can be produced by the steps of: forming a core-shell-type nanoparticle having a core (10) of MFe2O4 and a shell having a functional group covering the core (10); and reacting glucose or a glucose derivative with the functional group.

Description

ナノ微粒子、及びナノ微粒子の製造方法、並びに抗腫瘍剤Nanoparticle, method for producing nanoparticle, and antitumor agent
　本発明は、ナノ微粒子、及びナノ微粒子の製造方法、このナノ微粒子を有効成分として含有する抗腫瘍剤、並びにこのナノ微粒子を製造するためのコアシェル型ナノ微粒子の製造方法に関する。
　本願は、２０１８年３月５日に、日本に出願された特願２０１８－０３８９８９号に基づき優先権を主張し、その内容をここに援用する。 The present invention relates to a nanoparticle, a method for producing the nanoparticle, an antitumor agent containing the nanoparticle as an active ingredient, and a method for producing a core-shell nanoparticle for producing the nanoparticle.
This application claims priority based on Japanese Patent Application No. 2018-038989 filed in Japan on March 5, 2018, the contents of which are incorporated herein by reference.
　ナノメートルスケールの超微粒子であるナノ微粒子は、従来にない新たな特異な物性をもたらし、機能材料としての高性能化が期待できることから、種々の物質について検討がされている。特に、磁性材料を微粒子化すると、磁壁を持たない単磁区粒子が生じ、保磁力が大きくなることが期待されている。 Nanoparticles, which are ultrafine particles on the nanometer scale, bring about new and unique physical properties that have not been possible in the past, and high performance as functional materials can be expected, so various substances have been studied. In particular, when the magnetic material is made into fine particles, single domain particles having no domain wall are generated, and the coercive force is expected to increase.
　本発明者らは、表面をＳｉＯ２により被覆されたマグネタイト等の磁気ナノ微粒子の調製に成功しており、このＳｉＯ２を介して官能基を導入した技術を報告している（特許文献１、２参照）。この官能基により、磁気微粒子への薬剤等の修飾や、細胞、組織内へ磁気微粒子を容易に取り込むことが可能である。 The present inventors have found that the surface has been successful in the preparation of the magnetic nanoparticles of magnetite coated with SiO 2, and have reported a technique of introducing a functional group through the SiO 2 (Patent Document 1, 2). With this functional group, it is possible to easily modify the magnetic fine particles with a drug or the like and to easily take the magnetic fine particles into cells or tissues.
　一方、がん細胞は正常細胞に比べて単位時間当たり３～８倍のグルコースを取り込むことが知られている。また、ＥＰＲ効果、すなわち、がん細胞の周りの血管壁は高分子薬剤を透過しやすく、透過した高分子薬剤は腫瘍組織中あるいはその周辺に集積しやすいことも知られている。 On the other hand, cancer cells are known to take up 3 to 8 times more glucose per unit time than normal cells. It is also known that the EPR effect, that is, the blood vessel wall around the cancer cells easily permeates the polymer drug, and the permeated polymer drug easily accumulates in or around the tumor tissue.
特開２００１－２６１３３４号公報JP 2001-261334 A 特開２００７－２６９７７０号公報JP 2007-269770 A
　ナノ微粒子の表面に、グルコースに近似した構造を導入することができれば、腫瘍組織中あるいはその周辺に集積しやすい性質が得られると期待できる。 If a structure similar to glucose can be introduced on the surface of the nanoparticle, it can be expected that a property that is easy to accumulate in or around the tumor tissue can be obtained.
　本発明は、グルコース又はグルコース誘導体で化学修飾されたナノ微粒子、及びその製造方法、このナノ微粒子を有効成分として含有する抗腫瘍剤、並びにこのナノ微粒子を製造するためのコアシェル型ナノ微粒子の製造方法を提供することを課題とする。 The present invention relates to a nanoparticle chemically modified with glucose or a glucose derivative, a method for producing the same, an antitumor agent containing the nanoparticle as an active ingredient, and a method for producing a core-shell nanoparticle for producing the nanoparticle. It is an issue to provide.
　本発明は、以下の態様を有する。
［１］　ＭＦｅ２Ｏ４からなるコア（前記ＭＦｅ２Ｏ４において、Ｍは遷移金属を表す）と、
　前記コアを覆うシェルと、を有し、
　前記シェルがグルコース又はグルコース誘導体で化学修飾されているナノ微粒子。 The present invention has the following aspects.
[1] A core composed of MFe 2 O 4 (in the MFe 2 O 4 , M represents a transition metal);
A shell covering the core,
Nanoparticles in which the shell is chemically modified with glucose or a glucose derivative.
［２］　前記シェルがグルコース又はグルコース誘導体で化学修飾されたポリアルキレングリコールからなる、前記［１］に記載のナノ微粒子。 [2] The nanoparticle according to [1], wherein the shell is made of polyalkylene glycol chemically modified with glucose or a glucose derivative.
［３］　前記シェルがグルコース又はグルコース誘導体で化学修飾されたアモルファスＳｉＯ２からなる、前記［１］に記載のナノ微粒子。 [3] The nanoparticle according to [1], wherein the shell is made of amorphous SiO 2 chemically modified with glucose or a glucose derivative.
［４］　前記グルコース誘導体がアミノ基を有する、前記［１］～［３］のうちいずれか一項に記載のナノ微粒子。 [4] The nanoparticle according to any one of [1] to [3], wherein the glucose derivative has an amino group.
［５］　ＭＦｅ２Ｏ４からなるコア（前記ＭＦｅ２Ｏ４において、Ｍは遷移金属を表す）と、前記コアを覆う官能基を含むシェルと、を有するコアシェル型ナノ微粒子を形成する工程と、
　前記官能基にグルコース又はグルコース誘導体を反応させる工程と、を有する、前記［１］～［４］のうちいずれか一項に記載のナノ微粒子の製造方法。 [5] (in the MFe 2 O 4, M represents a transition metal) MFe 2 O 4 core consisting of the steps of forming a, a core-shell nanoparticles having a shell comprising a functional group that covers the core,
The method for producing nanoparticles according to any one of [1] to [4], further comprising reacting glucose or a glucose derivative with the functional group.
［６］　ＭＦｅ２Ｏ４からなるコア（前記ＭＦｅ２Ｏ４において、Ｍは遷移金属を表す）と、前記コアを覆うポリアルキレングリコールからなるシェルと、を有するコアシェル型ナノ微粒子を形成する工程と、
　前記ポリアルキレングリコールの末端のヒドロキシメチル基を酸化させて、カルボキシル基にする工程と、
　前記カルボキシル基にグルコース又はグルコース誘導体を反応させる工程と、を有する、前記［５］に記載のナノ微粒子の製造方法。 [6] (in the MFe 2 O 4, M represents a transition metal) MFe 2 core consisting of O 4 forming the, core-shell nanoparticles having a shell of a polyalkylene glycol to cover the core ,
Oxidizing the terminal hydroxymethyl group of the polyalkylene glycol to a carboxyl group;
And the step of reacting glucose or a glucose derivative with the carboxyl group. The method for producing nanoparticles according to [5] above.
［７］　前記［１］～［４］のうちいずれか一項に記載のナノ微粒子を有効成分として含有する抗腫瘍剤。
［８］　ポリアルキレングリコール、鉄を含む遷移金属の塩化物、及びアルカリを加熱し混合して、水酸化物を得る工程と、
　前記水酸化物を含む反応物を加熱し焼成する工程と、を有するコアシェル型ナノ微粒子の製造方法。 [7] An antitumor agent comprising the nanoparticle according to any one of [1] to [4] as an active ingredient.
[8] A step of heating and mixing polyalkylene glycol, chloride of transition metal containing iron, and alkali to obtain a hydroxide;
And a step of heating and baking the reaction product containing the hydroxide.
　本発明は、ナノ微粒子、及びナノ微粒子の製造方法、このナノ微粒子を有効成分として含有する抗腫瘍剤、並びにこのナノ微粒子を製造するためのコアシェル型ナノ微粒子の製造方法を提供する。本発明のナノ微粒子は、シェルがグルコース又はグルコース誘導体で化学修飾されているので、腫瘍組織中に集積しやすい性質が得られると期待できる。また、本発明のナノ微粒子は、ＭＦｅ２Ｏ４からなるコアを有しているので、磁気ハイパーサーミア（癌温熱療法）への利用も期待できる。 The present invention provides a nanoparticle, a method for producing the nanoparticle, an antitumor agent containing the nanoparticle as an active ingredient, and a method for producing a core-shell nanoparticle for producing the nanoparticle. In the nanoparticle of the present invention, since the shell is chemically modified with glucose or a glucose derivative, it can be expected that the property of being easily accumulated in a tumor tissue can be obtained. Moreover, since the nanoparticle of the present invention has a core composed of MFe 2 O 4 , it can be expected to be used for magnetic hyperthermia (cancer hyperthermia).
図１（上）は、比較例１で取得されたコバルトフェライト（ＣｏＦｅ２Ｏ４）磁気ナノ微粒子のＸ線回折（ＸＲＤ）の測定結果である。図１（下）は、実施例１で取得されたコアシェル型ナノ微粒子のＸ線回折（ＸＲＤ）の測定結果である。FIG. 1 (upper) shows measurement results of X-ray diffraction (XRD) of the cobalt ferrite (CoFe 2 O 4 ) magnetic nanoparticles obtained in Comparative Example 1. FIG. 1 (bottom) is a measurement result of X-ray diffraction (XRD) of the core-shell nanoparticle obtained in Example 1. 図２（上）は、実施例１で取得されたコアシェル型ナノ微粒子のＦＴ－ＩＲの測定結果である。図２（下）は、コバルトフェライト（ＣｏＦｅ２Ｏ４）標準試料のＦＴ－ＩＲの測定結果である。FIG. 2 (upper) shows the FT-IR measurement results of the core-shell nanoparticle obtained in Example 1. FIG. 2 (bottom) shows the FT-IR measurement results of a cobalt ferrite (CoFe 2 O 4 ) standard sample. 図３は、実施例１で取得されたコアシェル型ナノ微粒子のＭＳ測定の結果である。FIG. 3 shows the results of MS measurement of the core-shell nanoparticle obtained in Example 1. 図４（上）は、実施例２で取得されたコアシェル型ナノ微粒子のＦＴ－ＩＲの測定結果である。図４（下）は、実施例１で取得されたコアシェル型ナノ微粒子のＦＴ－ＩＲの測定結果である。FIG. 4 (upper) shows the FT-IR measurement results of the core-shell nanoparticle obtained in Example 2. FIG. 4 (bottom) shows the FT-IR measurement results of the core-shell nanoparticle obtained in Example 1. 図５（上）は、実施例３で取得されたナノ微粒子のＦＴ－ＩＲの測定結果である。図５（下）は、実施例２で取得されたコアシェル型ナノ微粒子のＦＴ－ＩＲの測定結果である。FIG. 5 (upper) shows the FT-IR measurement results of the nanoparticles obtained in Example 3. FIG. 5 (bottom) shows the FT-IR measurement results of the core-shell nanoparticle obtained in Example 2. 実施例３のナノ微粒子が、ＨｅＬａ細胞内に取り込まれたことを示すＴＥＭ像である。It is a TEM image which shows that the nanoparticle of Example 3 was taken in in the HeLa cell. コアシェル型ナノ微粒子のＦＴ－ＩＲの測定結果である。It is a measurement result of FT-IR of core-shell type nano fine particles. コアシェル型ナノ微粒子のＸ線回折（ＸＲＤ）の測定結果である。It is a measurement result of the X-ray diffraction (XRD) of a core-shell type nanoparticle. コアシェル型ナノ微粒子のＦＴ－ＩＲの測定結果である。It is a measurement result of FT-IR of core-shell type nano fine particles.
≪ナノ微粒子≫
　以下、本発明の実施形態について説明する。
　本実施形態のナノ微粒子は、ＭＦｅ２Ｏ４からなるコア（前記ＭＦｅ２Ｏ４において、Ｍは遷移金属を表す）と、前記コアを覆うシェルと、を有し、前記シェルがグルコース又はグルコース誘導体で化学修飾されている。 ≪Nanoparticles≫
Hereinafter, embodiments of the present invention will be described.
Nanoparticles of this embodiment, (at the MFe 2 O 4, M represents a transition metal) core made of MFe 2 O 4 and has a shell that covers the core, the shell is glucose or glucose derivative It is chemically modified with
　下記式（１）のナノ微粒子１は、本実施形態のナノ微粒子の構造の一例である。ナノ微粒子１は、ＭＦｅ２Ｏ４からなるコア１０（Ｍは遷移金属を表す）と、コア１０を覆うシェル２０と、を有し、シェル２０が２価の連結基Ｘを介してグルコース誘導体Ｇｌｃ’で化学修飾されている。Ｘは２価の連結基である。 The nanoparticle 1 of the following formula (1) is an example of the structure of the nanoparticle of the present embodiment. The nanoparticle 1 has a core 10 (M represents a transition metal) made of MFe 2 O 4 and a shell 20 that covers the core 10, and the shell 20 has a glucose derivative Glc via a divalent linking group X. It is chemically modified with '. X is a divalent linking group.
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
　ここで、グルコース誘導体Ｇｌｃ’とは、グルコース骨格を有する化合物をいう。グルコース誘導体Ｇｌｃ’としてアミノ基を有するグルコース誘導体が好ましい。グルコース誘導体Ｇｌｃ’の具体例として、グルコサミンが挙げられる。 Here, the glucose derivative Glc ′ refers to a compound having a glucose skeleton. The glucose derivative Glc ′ is preferably a glucose derivative having an amino group. A specific example of the glucose derivative Glc 'is glucosamine.
　式（１）のナノ微粒子１のうち、シェルがグルコサミンで化学修飾されたものとして、下記式（１－１）で表されるナノ微粒子が挙げられる。 Among the nanoparticles 1 of the formula (1), those whose shell is chemically modified with glucosamine include nanoparticles represented by the following formula (1-1).
Figure JPOXMLDOC01-appb-C000002
 （ｋは１以上の整数を表す。）
Figure JPOXMLDOC01-appb-C000002
 (K represents an integer of 1 or more.)
　下記式（２）のナノ微粒子２は、本実施形態のナノ微粒子の構造の一例である。ナノ微粒子２は、ＭＦｅ２Ｏ４からなるコア１０（Ｍは遷移金属を表す）と、コア１０を覆うシェル２０と、を有し、シェル２０が２価の連結基Ｘを介してグルコースで化学修飾されている。Ｘは２価の連結基である。 The nanoparticle 2 of the following formula (2) is an example of the structure of the nanoparticle of the present embodiment. The nanoparticle 2 has a core 10 made of MFe 2 O 4 (M represents a transition metal) and a shell 20 that covers the core 10, and the shell 20 is chemically formed with glucose via a divalent linking group X. It is qualified. X is a divalent linking group.
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000003
　式（２）のナノ微粒子２の具体例としては、下記式（２－１）、式（２－２）、式（２－３）で表されるナノ微粒子が挙げられる。 Specific examples of the nanoparticle 2 of formula (2) include nanoparticles represented by the following formula (2-1), formula (2-2), and formula (2-3).
Figure JPOXMLDOC01-appb-C000004
Figure JPOXMLDOC01-appb-C000004
Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000006
 （ｋは１以上の整数を表す。）
Figure JPOXMLDOC01-appb-C000006
 (K represents an integer of 1 or more.)
（シェル）
　コアを覆うシェルはグルコース又はグルコース誘導体で化学修飾されている。シェルはコアの全部を覆うものであってもよく、部分的に覆うものであっていてもよい。シェルの内部に２個以上のコアを包摂した包摂構造を形成していてもよい。 (shell)
The shell covering the core is chemically modified with glucose or a glucose derivative. The shell may cover the entire core or may partially cover the core. An inclusion structure including two or more cores may be formed inside the shell.
　本実施形態のナノ微粒子において、ＭＦｅ２Ｏ４からなるコアが、シェルで覆われていることは、微粒子に対してグルコース又はグルコース誘導体を化学修飾する反応工程を経た後、グルコース又はグルコース誘導体が反応性のシェルに化学修飾されたことで確認することができる。 In the nanoparticle of the present embodiment, the core made of MFe 2 O 4 is covered with the shell because the glucose or glucose derivative reacts after undergoing a reaction step of chemically modifying glucose or glucose derivative to the particle. It can be confirmed by chemical modification of the sex shell.
　シェルとなる材料は、グルコース又はグルコース誘導体で化学修飾され得るものであればよく、ポリアルキレングリコールからなるものであってもよい。ポリアルキレングリコールをシェルとするナノ微粒子は、例えば、後述する「コアシェル型ナノ微粒子の製造方法」により製造することができる。 The material to be the shell may be any material that can be chemically modified with glucose or a glucose derivative, and may be composed of polyalkylene glycol. Nanoparticles having polyalkylene glycol as a shell can be produced, for example, by a “method for producing core-shell nanoparticle” described later.
　シェルとなる材料は、アモルファスＳｉＯ２からなるものであってもよく、シェルは、コアを覆うアモルファスＳｉＯ２ネットワーク（網状膜）を形成していてもよい。例えば、アモルファスＳｉＯ２の網状膜によって、コアが分離された状態であってもよい。ここで「網状膜」とは、アモルファスＳｉＯ２が個々のコアの周囲を取り囲み、且つアモルファスＳｉＯ２が連なっているものが例示されるが、これに限定されない。 The material for the shell may be made of amorphous SiO 2 , and the shell may form an amorphous SiO 2 network (network film) covering the core. For example, the core may be separated by an amorphous SiO 2 network. Here, examples of the “network film” include those in which amorphous SiO 2 surrounds the periphery of each core and amorphous SiO 2 is continuous, but is not limited thereto.
　なお、ナノ微粒子のコアが、アモルファスＳｉＯ２からなるシェルで覆われていることの確認は、Ｘ線回折によりアモルファスＳｉＯ２とマグネタイトの回折線が観測されること、及びナノ微粒子の一次粒子径が上記Ｘ線回折の半値幅から予想される程度の値であること、からも行うことができる。 The confirmation that the core of the nanoparticle is covered with the shell made of amorphous SiO 2 is that the diffraction lines of amorphous SiO 2 and magnetite are observed by X-ray diffraction, and the primary particle diameter of the nanoparticle is It can also be carried out because of the value expected from the half width of the X-ray diffraction.
（コア）
　本実施形態に係るコアは、ＭＦｅ２Ｏ４からなる。本実施形態に係るナノ微粒子のコアとしてフェライトを採用することで、磁性材料としての利用が可能となる。
　前記ＭＦｅ２Ｏ４において、Ｍは遷移金属を表す。遷移金属としては、イオン化したときに２価になるものが好ましく、例えば、Ｃｒ、Ｍｎ、Ｆｅ、Ｃｏ、Ｎｉ、Ｃｕ、Ｚｎが挙げられる。また、これらの遷移金属を２種以上併せて用いても良い。前記ＭがＦｅである場合、ＭＦｅ２Ｏ４は、例えばＦｅ３Ｏ４である。これらのなかでも、ＭはＣｏであることが好ましい。また、２種以上併せて用いる場合には、ＭｎＺｎが好ましい。 (core)
The core according to the present embodiment is made of MFe 2 O 4 . By using ferrite as the core of the nanoparticle according to this embodiment, it can be used as a magnetic material.
In the MFe 2 O 4 , M represents a transition metal. The transition metal is preferably one that becomes divalent when ionized, and examples thereof include Cr, Mn, Fe, Co, Ni, Cu, and Zn. Two or more of these transition metals may be used in combination. When M is Fe, MFe 2 O 4 is, for example, Fe 3 O 4 . Among these, M is preferably Co. Moreover, when using together 2 or more types, MnZn is preferable.
（アモルファスＳｉＯ２をシェルとするナノ微粒子）
　本実施形態に係るナノ微粒子は、前記コアとそれを覆うシェルとを有し、前記シェルがグルコース又はグルコース誘導体で化学修飾されたアモルファスＳｉＯ２からなるからなるものであってもよい。アモルファスＳｉＯ２に対しては、例えば、３－イソシアネートプロピルトリエトキシシランシラン、３－グリシドキシプロピルトリエトキシシラン等のカップリング剤を用いて、式（２－１）、式（２－２）で表されるナノ微粒子のように、グルコース又はグルコース誘導体を化学修飾することができる。 (Nanofine particles with amorphous SiO 2 as shell)
The nano fine particles according to the present embodiment may include the core and a shell covering the core, and the shell may be made of amorphous SiO 2 chemically modified with glucose or a glucose derivative. For amorphous SiO 2 , for example, using a coupling agent such as 3-isocyanatopropyltriethoxysilanesilane, 3-glycidoxypropyltriethoxysilane, etc., the formulas (2-1) and (2-2) Glucose or a glucose derivative can be chemically modified like the nanoparticle represented by these.
　シェルは、コアを覆うアモルファスＳｉＯ２ネットワーク（網状膜）を形成していてもよい。本実施形態に係るナノ微粒子は、シェルの内部に２個以上のコアを包摂した包摂構造を形成していてもよい。 The shell may form an amorphous SiO 2 network (network film) covering the core. The nano fine particles according to the present embodiment may form an inclusion structure including two or more cores inside the shell.
　アモルファスＳｉＯ２をシェルとするナノ微粒子において、前記ＭＦｅ２Ｏ４に対する前記ＳｉＯ２のモル比（ＳｉＯ２／ＭＦｅ２Ｏ４）は、０．１以上５以下であることが好ましく、０．３以上４以下であることがより好ましく、０．４以上２以下であることがさらに好ましい。ＳｉＯ２／ＭＦｅ２Ｏ４が０．１以上５以下であることが好ましい。また、ＳｉＯ２／ＭＦｅ２Ｏ４が上記範囲内の値であると、ナノ微粒子の分散性に優れるため好ましい。 In nanoparticles of amorphous SiO 2 and the shell, the MFe 2 O 4 molar ratio of the SiO 2 to (SiO 2 / MFe 2 O 4 ) is preferably 0.1 to 5, 0.3 or higher It is more preferably 4 or less, and further preferably 0.4 or more and 2 or less. It is preferable SiO 2 / MFe 2 O 4 is 0.1 to 5. Also, when the SiO 2 / MFe 2 O 4 falls within the above range is preferred since it is excellent in dispersibility of the nanoparticles.
≪ナノ微粒子の製造方法の製造方法≫
　本実施形態のナノ微粒子は、例えば、ＭＦｅ２Ｏ４からなるコア（前記ＭＦｅ２Ｏ４において、Ｍは遷移金属を表す）と、前記コアを覆う官能基を含むシェルと、を有するコアシェル型ナノ微粒子を形成する工程と、前記官能基にグルコース又はグルコース誘導体を反応させる工程と、を有する方法に沿って製造できる。 ≪Production method of nanoparticle production process≫
Nanoparticles of the present embodiment, for example, (in the MFe 2 O 4, M represents a transition metal) core made of MFe 2 O 4 core-shell nano with a, a, a shell comprising a functional group that covers the core It can be produced according to a method having a step of forming fine particles and a step of reacting glucose or a glucose derivative with the functional group.
　コアシェル型ナノ微粒子を形成する工程は、特許文献１（特開２００１－２６１３３４号公報）や特許文献２（特開２００７－２６９７７０号公報）に沿った方法を採用することができ、後述する≪コアシェル型ナノ微粒子の製造方法≫において説明された方法を採用することができる。特許文献１（特開２００１－２６１３３４号公報）や特許文献２（特開２００７－２６９７７０号公報）に沿った方法で形成した、表面をＳｉＯ２により被覆されたナノ微粒子では、さらに、グルコース又はグルコース誘導体で化学修飾し易いように、シランカップリング剤で官能基を導入してもよい。 For the step of forming the core-shell nanoparticle, a method in accordance with Patent Document 1 (Japanese Patent Laid-Open No. 2001-261334) and Patent Document 2 (Japanese Patent Laid-Open No. 2007-269770) can be employed. The method described in “Production method of mold nanoparticle” can be employed. Nanoparticles whose surfaces are coated with SiO 2 formed by a method in accordance with Patent Document 1 (Japanese Patent Laid-Open No. 2001-261334) and Patent Document 2 (Japanese Patent Laid-Open No. 2007-269770) further include glucose or glucose In order to facilitate chemical modification with a derivative, a functional group may be introduced with a silane coupling agent.
　後述する≪コアシェル型ナノ微粒子の製造方法≫に沿った方法で形成した、表面に－ＣＨ２ＯＨ基を有するコアシェル型ナノ微粒子では、さらに、下記式（３）に示されるように、－ＣＨ２ＯＨ基を酸化して、表面に－ＣＯＯＨ基を有するコアシェル型ナノ微粒子としてもよい。 In the core-shell nanoparticle having a —CH 2 OH group on the surface formed by a method in accordance with “Method for producing core-shell nanoparticle” described later, as shown in the following formula (3), —CH 2 The core-shell nanoparticle having a —COOH group on the surface may be obtained by oxidizing the OH group.
Figure JPOXMLDOC01-appb-C000007
 （ｋは１以上の整数を表す。）
Figure JPOXMLDOC01-appb-C000007
 (K represents an integer of 1 or more.)
　このコアシェル型ナノ微粒子を用いるナノ微粒子の製造方法は、一例として、ＭＦｅ２Ｏ４からなるコア（前記ＭＦｅ２Ｏ４において、Ｍは遷移金属を表す）と、前記コアを覆うポリアルキレングリコールからなるシェルと、を有するコアシェル型ナノ微粒子を形成する工程と、前記ポリアルキレングリコールの末端のヒドロキシメチル基を酸化させて、カルボキシル基にする工程と、前記カルボキシル基にグルコース又はグルコース誘導体を反応させる工程と、を有する。 Method for producing nanoparticles using the core-shell type nanoparticles, as an example, (in the MFe 2 O 4, M represents a transition metal) core made of MFe 2 O 4 and consists of a polyalkylene glycol to cover the core Forming a core-shell nanoparticle having a shell, oxidizing a hydroxymethyl group at a terminal of the polyalkylene glycol to form a carboxyl group, and reacting glucose or a glucose derivative with the carboxyl group Have.
　前記官能基にグルコース又はグルコース誘導体を反応させる工程は、官能基とグルコース又はグルコース誘導体との組み合わせに応じて、公知の方法で行うことができる。例えば、前記官能基がカルボキシル基であって、前記グルコース誘導体がグルコサミンであるとき、前記カルボキシル基にグルコース誘導体を反応させる工程は、下記式（４）で示すことができる。 The step of reacting glucose or a glucose derivative with the functional group can be performed by a known method depending on the combination of the functional group and glucose or the glucose derivative. For example, when the functional group is a carboxyl group and the glucose derivative is glucosamine, the step of causing the glucose derivative to react with the carboxyl group can be represented by the following formula (4).
Figure JPOXMLDOC01-appb-C000008
 （ｋは１以上の整数を表す。）
Figure JPOXMLDOC01-appb-C000008
 (K represents an integer of 1 or more.)
≪コアシェル型ナノ微粒子の製造方法≫
　本実施形態のコアシェル型ナノ微粒子の製造方法は、ポリアルキレングリコール、鉄を含む遷移金属の塩化物、及びアルカリを加熱し混合して、水酸化物を得る工程と、
　前記水酸化物を含む反応物を加熱し焼成する工程と、を有する。 ≪Method for producing core-shell nanoparticle≫
The method of manufacturing the core-shell nanoparticle of the present embodiment includes a step of heating and mixing polyalkylene glycol, a transition metal chloride containing iron, and an alkali to obtain a hydroxide;
Heating and baking the reaction product containing the hydroxide.
　ポリアルキレングリコールとしては、ポリエチレングリコール、ポリプロピレングリコール等を挙げることができる。これらのポリアルキレングリコールの質量平均分子量は２００～８０００であるものが好ましく、３００～２０００であるものがより好ましい。例えば、質量平均分子量が約３８０～４２０の市販のＰＥＧ４００、質量平均分子量が約５６０～６４０の市販のＰＥＧ６００、質量平均分子量が約９００～１１００の市販のＰＥＧ１０００、質量平均分子量が１８５０～２１５０の市販のＰＥＧ２０００、質量平均分子量が約４００の市販のＰＰＧ４００、等を用いることができる。ＰＥＧ２０００を用いると立体障害が大きいため、コアシェル型ナノ微粒子の分散性が良くなると考えられる。 Examples of the polyalkylene glycol include polyethylene glycol and polypropylene glycol. These polyalkylene glycols preferably have a mass average molecular weight of 200 to 8000, more preferably 300 to 2000. For example, a commercially available PEG 400 having a mass average molecular weight of about 380 to 420, a commercially available PEG 600 having a mass average molecular weight of about 560 to 640, a commercially available PEG 1000 having a mass average molecular weight of about 900 to 1100, and a commercially available PEG 1000 having a mass average molecular weight of 1850 to 2150 PEG2000, commercially available PPG400 having a weight average molecular weight of about 400, and the like can be used. Since steric hindrance is large when PEG2000 is used, it is considered that the dispersibility of the core-shell nanoparticle is improved.
　鉄を含む遷移金属の塩化物としては、ＭＣｌ２・６Ｈ２Ｏ（Ｍは遷移金属を表す）、ＦｅＣｌ２・４Ｈ２Ｏの試薬を用いることができる。ＭＣｌ２・６Ｈ２Ｏ及びＦｅＣｌ２・４Ｈ２Ｏを、Ｍ：Ｆｅ＝１モル：２モルの比で混合して用いてもよく、ＦｅＣｌ２・４Ｈ２Ｏを単独で用いてもよい。 As a transition metal chloride containing iron, a reagent of MCl 2 .6H 2 O (M represents a transition metal) or FeCl 2 .4H 2 O can be used. MCl 2 · 6H 2 O and FeCl 2 · 4H 2 O may be mixed and used at a ratio of M: Fe = 1 mol: 2 mol, or FeCl 2 · 4H 2 O may be used alone.
　ポリアルキレングリコール、鉄を含む遷移金属の塩化物、及びアルカリを加熱し混合すると、中和反応し、水酸化物を得ることができる。中和反応の温度としては、４０～１００℃が好ましく、６０～８０℃が好ましい。中和反応の時間は、３０分～２時間が好ましい。さらに、水酸化物を含む反応物を加熱し焼成することで、コアシェル型ナノ微粒子を液体のポリアルキレングリコール中に沈殿させることができる。焼成温度は、１２０～２８０℃が好ましく、１６０～２２０℃がより好ましい。焼成時間は３時間以上とすることが好ましい。 When a polyalkylene glycol, a transition metal chloride containing iron, and an alkali are heated and mixed, a neutralization reaction occurs to obtain a hydroxide. The temperature for the neutralization reaction is preferably 40 to 100 ° C, more preferably 60 to 80 ° C. The neutralization reaction time is preferably 30 minutes to 2 hours. Furthermore, the core-shell nanoparticle can be precipitated in the liquid polyalkylene glycol by heating and baking the reaction product containing hydroxide. The firing temperature is preferably 120 to 280 ° C, more preferably 160 to 220 ° C. The firing time is preferably 3 hours or longer.
　本実施形態のコアシェル型ナノ微粒子の磁性体部分の体積平均粒子径は、１ｎｍ以上５０ｎｍ以下であることが好ましく、２ｎｍ以上２０ｎｍ以下であることがより好ましい。
　ここで、体積平均粒子径は、コアシェル型ナノ微粒子の１個のコアの磁性体部分に対して求められた一次粒子径である。体積平均粒子径は算術平均径である。
　コアシェル型ナノ微粒子のコアの磁性体部分の体積平均粒子径は、Ｘ線回折（ＸＲＤ）により求めることができる。 The volume average particle diameter of the magnetic part of the core-shell nanoparticle of the present embodiment is preferably 1 nm or more and 50 nm or less, and more preferably 2 nm or more and 20 nm or less.
Here, the volume average particle diameter is a primary particle diameter obtained for the magnetic part of one core of the core-shell nanoparticle. The volume average particle diameter is an arithmetic average diameter.
The volume average particle diameter of the magnetic part of the core of the core-shell nanoparticle can be determined by X-ray diffraction (XRD).
　上記コアシェル型ナノ微粒子を、上記水溶液を混合し焼成して製造する場合、焼成温度が高くなるほど、又焼成時間が長いほど、コアシェル型ナノ微粒子が成長して粒径が大きくなるので、焼成温度又は焼成時間を調整することで、コアシェル型ナノ微粒子の粒径を制御できる。 When the core-shell nanoparticle is produced by mixing the aqueous solution and firing, the higher the firing temperature and the longer the firing time, the larger the core-shell nanoparticle grows and the particle size increases. By adjusting the firing time, the particle size of the core-shell nanoparticle can be controlled.
　以下、具体的実施例により、本発明についてさらに詳しく説明する。ただし、本発明は、以下に示す実施例に何ら限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples.
＜ＸＲＤ分析＞
　粉末Ｘ線回折測定機器（リガク：ＭｉｎｉＦｌｅｘ ＩＩ）を用いてＸＲＤ分析を行った。 <XRD analysis>
XRD analysis was performed using a powder X-ray diffractometer (Rigaku: MiniFlex II).
＜平均粒径（一次粒子径）の測定＞
　粉末Ｘ線回折測定機器（リガク：ＭｉｎｉＦｌｅｘ ＩＩ）を用いて、コアシェル型ナノ微粒子の磁性体部分の平均粒径（一次粒子径）を測定した。数値は、Ｃｒｙｓｔａｌ　Ｓｉｚｅ　Ｄｉｓｔｒｉｂｕｔｉｏｎ　Ａｎａｌｙｓｉｓ（ＣＳＤＡ）を用いて体積平均粒子径として算出した。 <Measurement of average particle diameter (primary particle diameter)>
Using a powder X-ray diffraction measuring instrument (Rigaku: MiniFlex II), the average particle size (primary particle size) of the magnetic part of the core-shell nanoparticle was measured. The numerical value was computed as a volume average particle diameter using Crystal Size Distribution Analysis (CSDA).
＜ＭＳ測定＞
　ＤＨＢをマトリクスとして、Ａｕｔｏｆｌｅｘ　ｓｐｅｅｄ－ＴＯＦ－ＭＳ装置（Ｂｒｕｋｅｒ　Ｄａｌｔｏｎｉｃｓ）により、ナノ微粒子の質量分析測定を行った。装置の仕様を以下に示す。
レーザー周波数；　　　　　１ｋ　Ｈｚ
質量分解能（ＦＷＨＭ）；　Ｍａｘ：　２５００
質量精度　；　　　　　　　ｍｉｎ：　２　ｐｐｍ
レーザー焦点径；ｍａｘ：　１０　μｍ
　　レーザー；　　　　　　Ｎｄ：Ｙａｇ　ｌａｓｅｒ　（３５５　ｎｍ）
　最大パワー　　　　　　　＜１７０ｋＷ
　パルスエネルギー　　　　＜５００μＪ
　パルス幅　　　　　　　　＞３ｎｓ
　平均パワー　　　　　　　＜１．５Ｗ <MS measurement>
Using DHB as a matrix, mass spectrometric measurement of nanoparticles was performed using an Autoflex speed-TOF-MS apparatus (Bruker Daltonics). The specifications of the device are shown below.
Laser frequency: 1 kHz
Mass resolution (FWHM); Max: 2500
Mass accuracy; min: 2 ppm
Laser focal spot diameter; max: 10 μm
Laser; Nd: Yag laser (355 nm)
Maximum power <170kW
Pulse energy <500μJ
Pulse width> 3ns
Average power <1.5W
＜二次粒径（モード径及びメジアン径）の測定＞
　得られたナノ微粒子について、レーザー回折・散乱型粒度分布計（堀場製作所製：ＬＡ－９５０Ｖ２）を用いて水溶液中におけるナノ微粒子の粒度分布から、二次粒径（モード径及びメジアン径）を測定した。サンプルは全て水溶液中に分散させ、超音波処理にて分散処理を行った後、循環系にて測定を行った。装置の概要を以下に示す。 <Measurement of secondary particle size (mode diameter and median diameter)>
For the obtained nanoparticles, the secondary particle size (mode diameter and median diameter) is measured from the particle size distribution of the nanoparticles in an aqueous solution using a laser diffraction / scattering type particle size distribution analyzer (Horiba: LA-950V2). did. All samples were dispersed in an aqueous solution, subjected to dispersion treatment by ultrasonic treatment, and then measured in a circulation system. The outline of the device is shown below.
　堀場製作所製　ＬＡ－９５０Ｖ２
　・測定原理：　　　Ｍｉｅ散乱理論
　・測定レンジ：　 ０．０１～３０００ μｍ
　・レーザー光源： 半導体レーザー（６５０ ｎｍ）
　　　　　　　　　 発光ダイオード（４０５ ｎｍ）
　・検出器：　　　 リング上６４分割シリコンフォトダイオード×１個
　　　　　　　　　 ４ｃｈアレイデテクタ×５個
　　　　　　　　　 シリコンフォトデテクタ×３個 LA-950V2 manufactured by HORIBA, Ltd.
・ Measurement principle: Mie scattering theory ・ Measurement range: 0.01 to 3000 μm
・ Laser light source: Semiconductor laser (650 nm)
Light emitting diode (405 nm)
・ Detector: 64 split silicon photodiodes on the ring x 1 4ch array detector x 5 Silicon photo detector x 3
［比較例１］
＜ナノ微粒子の作製＞
　ＣｏＣｌ２・６Ｈ２Ｏ水溶液と、ＦｅＣｌ２・４Ｈ２Ｏ水溶液と、Ｎａ２ＳｉＯ３・９Ｈ２Ｏ水溶液をそれぞれモル比（ＣｏＣｌ２：ＦｅＣｌ２：Ｎａ２ＳｉＯ３＝１：２：３）で添加し、１５分間撹拌し、その後混合させ希釈したＮａＯＨを用いてｐＨを８．０に調整した。ｐＨ調整後の液をさらに１５分間撹拌し、遠心分離機により３５００ｒｐｍで１５分間遠心した後、上澄み液を除去し、沈殿物を純水にて洗浄する動作を３回繰り返した。約５０℃の恒温槽で約４０時間乾燥させた沈殿物を乳鉢にて粉砕した。その後、約８００℃で約１６時間焼成を行い、比較例のナノ微粒子試料を得た。ＸＲＤ分析の結果を図１（上）に示す。指数づけしたピークから、単相のスピネル構造を持ったＣｏＦｅ２Ｏ４ [No. 000-022-1086、Name: Cobalt Iron Oxide、Quality Mark: S、Cell (8.392, 8.392, 8.392)] が同定でき、２θ＝２０～３０°の位置にアモルファスＳｉＯ２のブロードなピークが観察された。得られたナノ微粒子の組成は、ＣｏＦｅ２Ｏ４・３ＳｉＯ２であった。平均粒径（一次粒子径）は１０ｎｍであった。二次粒径は、モード径が３．６３μｍ、メジアン径は２．２７μｍであった。 [Comparative Example 1]
<Preparation of nano particles>
CoCl 2 · 6H 2 O aqueous solution, FeCl 2 · 4H 2 O aqueous solution and Na 2 SiO 3 · 9H 2 O aqueous solution in molar ratio (CoCl 2 : FeCl 2 : Na 2 SiO 3 = 1: 2: 3), respectively. Added and stirred for 15 minutes, after which the pH was adjusted to 8.0 using mixed and diluted NaOH. The pH-adjusted solution was further stirred for 15 minutes and centrifuged at 3500 rpm for 15 minutes using a centrifuge, and then the supernatant was removed and the precipitate was washed with pure water three times. The precipitate dried for about 40 hours in a thermostat at about 50 ° C. was pulverized in a mortar. Then, it baked at about 800 degreeC for about 16 hours, and obtained the nanoparticle sample of the comparative example. The result of XRD analysis is shown in FIG. CoFe 2 O 4 with a single-phase spinel structure [No. 000-022-1086, Name: Cobalt Iron Oxide, Quality Mark: S, Cell (8.392, 8.392, 8.392)] can be identified from the indexed peaks. A broad peak of amorphous SiO 2 was observed at a position of 2θ = 20 to 30 °. The composition of the obtained nanoparticle was CoFe 2 O 4 .3SiO 2 . The average particle size (primary particle size) was 10 nm. Regarding the secondary particle size, the mode diameter was 3.63 μm, and the median diameter was 2.27 μm.
［実施例１］
＜ＰＥＧ４００を用いたＣｏＦｅ２Ｏ４ナノ微粒子の作製＞
　５０ｍｌのＰＥＧ４００の中で、５ｍｍｏｌのＦｅＣｌ２・４Ｈ２Ｏ、２．５ｍｍｏｌのＣｏＣｌ２・６Ｈ２Ｏ、及び１５ｍｍｏｌの水酸化ナトリウムを７０℃で１時間混合して、中和反応させた。反応液を、反応容器のまま箱型オーブンの中に入れ、２００℃で１６時間加熱し、炉冷後の反応液に対して、遠心分離機（３５００ｒｐｍ, １０ｍｉｎ）で沈殿させ、エタノールで三回洗浄する工程を３回繰り返した。乾燥して、ＣｏＦｅ２Ｏ４をコアとするコアシェル型ナノ微粒子の粉末を得た。得られたコアシェル型ナノ微粒子の磁性体部分の平均粒径（一次粒子径）は１２ｎｍであった。 [Example 1]
<Preparation of CoFe 2 O 4 Nanoparticles Using PEG400>
In 50 ml of PEG400, 5 mmol of FeCl 2 .4H 2 O, 2.5 mmol of CoCl 2 .6H 2 O, and 15 mmol of sodium hydroxide were mixed at 70 ° C. for 1 hour for neutralization reaction. The reaction solution is placed in a box oven as it is in a reaction vessel, heated at 200 ° C. for 16 hours, precipitated with a centrifuge (3500 rpm, 10 min) against the reaction solution after furnace cooling, and three times with ethanol. The washing process was repeated three times. Dried to obtain a powder of core-shell nanoparticles of the CoFe 2 O 4 and the core. The average particle diameter (primary particle diameter) of the magnetic part of the obtained core-shell nanoparticle was 12 nm.
（ＸＲＤ分析結果）
　ＸＲＤ分析の結果を図１（下）に示す。指数づけしたピークから、単相のスピネル構造を持ったＣｏＦｅ２Ｏ４ [No. 000-022-1086、Name: Cobalt Iron Oxide、Quality Mark: S、Cell (8.392, 8.392, 8.392)] が同定でき、不純物のピークを持たないことから、目的のＣｏＦｅ２Ｏ４が作成できた。湿式混合法で作ったＣｏＦｅ２Ｏ４をコアとするコアシェル型ナノ微粒子のＸＲＤ分析の結果の図１（上）に比べて、図１（下）のＰＥＧ４００を用いたコアシェル型ナノ微粒子のＸＲＤ分析の結果は、２０～３０°の位置にアモルファスのブロードなピークがないことが確認された。 (Results of XRD analysis)
The results of XRD analysis are shown in FIG. 1 (bottom). CoFe 2 O 4 [No. 000-022-1086, Name: Cobalt Iron Oxide, Quality Mark: S, Cell (8.392, 8.392, 8.392)] with a single-phase spinel structure can be identified from the indexed peak. The target CoFe 2 O 4 could be produced because it did not have an impurity peak. XRD analysis of core-shell nanoparticle using PEG400 of FIG. 1 (bottom) compared to FIG. 1 (top) of the result of XRD analysis of core-shell nanoparticle with CoFe 2 O 4 core made by wet mixing method As a result, it was confirmed that there was no amorphous broad peak at a position of 20 to 30 °.
（ＦＴ－ＩＲ測定結果）
　続いて、得られたコアシェル型ナノ微粒子のＦＴ－ＩＲ測定をした。実施例１のコアシェル型ナノ微粒子のＦＴ－ＩＲ測定結果を図２（上）に示す。ＣｏＦｅ２Ｏ４標準試料のＦＴ－ＩＲ測定結果を図２（下）に示す。本製法にて作製したコアシェル型ナノ微粒子は、エタノールにて洗浄したものであり、仮にＰＥＧ４００単体で存在しているものがあるとすれば洗浄過程で取り除かれる。図２（上）の測定結果は、ＣＯＣ、ＣＯや、ＣＣ等の飽和炭化水素に特有の結合のピークを有していることから、ＰＥＧ４００を用いて作製したＣｏＦｅ２Ｏ４をコアとするコアシェル型ナノ微粒子は、ＰＥＧ４００に由来するＰＥＧの主鎖がＣｏＦｅ２Ｏ４のコアと化学結合しているものと考えられる。 (FT-IR measurement result)
Subsequently, FT-IR measurement of the obtained core-shell nanoparticle was performed. FIG. 2 (upper) shows the FT-IR measurement results of the core-shell nanoparticle of Example 1. FIG. 2 (bottom) shows the FT-IR measurement results of the CoFe 2 O 4 standard sample. The core-shell nanoparticle produced by this production method is washed with ethanol, and if there is a PEG400 alone, it is removed in the washing process. The measurement result in FIG. 2 (upper) has a bond peak peculiar to saturated hydrocarbons such as COC, CO, and CC. Therefore, the core shell having CoFe 2 O 4 produced using PEG400 as the core. The type nanoparticle is considered that the main chain of PEG derived from PEG400 is chemically bonded to the core of CoFe 2 O 4 .
（ＭＳ測定結果）
　さらに、ＰＥＧ４００を用いて作製した実施例１のコアシェル型ナノ微粒子について、ＤＨＢをマトリクスとする質量分析測定を行った。測定結果を図３に示す。ピーク間隔が４４であるＰＥＧ４００由来のピークが観測されたことからも、本コアシェル型ナノ微粒子は、ＰＥＧ４００に由来するＰＥＧの主鎖が化学結合していることが確認された。ナノ微粒子とＰＥＧとの結合はレーザーによって切れたと考えられる。ピーク間隔が４４であるということは、ＰＥＧ４００の分子内の結合は反応によって不飽和結合などに形を変えていないことがわかる。 (MS measurement result)
Furthermore, the mass spectrometry measurement using DHB as a matrix was performed on the core-shell nanoparticle of Example 1 produced using PEG400. The measurement results are shown in FIG. From the fact that a peak derived from PEG400 with a peak interval of 44 was observed, it was confirmed that the main chain of the core-shell nanoparticle was chemically bonded to the main chain of PEG derived from PEG400. It is thought that the bond between the nanoparticle and PEG was broken by the laser. The fact that the peak interval is 44 indicates that the intramolecular bond of PEG400 has not changed into an unsaturated bond or the like due to the reaction.
（二次粒径測定結果）
　ＰＥＧ４００を用いて作製したＣｏＦｅ２Ｏ４ナノ微粒子の二次粒径はモード径が４７５ｎｍ、メジアン径は４３０ｎｍであった。比較例のナノ微粒子の二次粒径（モード径３．６３μｍ、メジアン径２．２７μｍ）に比べて液中でのコロイド径は１桁ほど小さくなっており、分散性に優れることを確認できた。 (Secondary particle size measurement result)
The secondary particle diameter of the CoFe 2 O 4 nanoparticle produced using PEG400 was a mode diameter of 475 nm and a median diameter of 430 nm. Compared to the secondary particle size (mode diameter 3.63 μm, median diameter 2.27 μm) of the nanoparticle of the comparative example, the colloid diameter in the liquid was reduced by an order of magnitude, confirming excellent dispersibility. .
［実施例２］
＜ＰＥＧ４００末端の水酸基のカルボキシル化＞
　純水１５０ｍｌに実施例１で得られたＣｏＦｅ２Ｏ４ナノ微粒子の粉末０．２ｇを入れ、４ｍｌの過酸化水素水溶液試薬（３０Ｗ／Ｖ％）を入れ、室温で１０ｈ撹拌した。純水で３回洗浄してから乾燥し、粉末とした。 [Example 2]
<Carboxylation of hydroxyl group of PEG400 terminal>
In 150 ml of pure water, 0.2 g of the CoFe 2 O 4 nanoparticle powder obtained in Example 1 was placed, 4 ml of an aqueous hydrogen peroxide reagent (30 W / V%) was placed, and the mixture was stirred at room temperature for 10 hours. After washing with pure water three times, it was dried to obtain a powder.
Figure JPOXMLDOC01-appb-C000009
 （ｋは１以上の整数を表す。）
Figure JPOXMLDOC01-appb-C000009
 (K represents an integer of 1 or more.)
（ＦＴ－ＩＲ測定結果）
　得られた実施例２のコアシェル型ナノ微粒子のＦＴ－ＩＲ測定をした。実施例２のコアシェル型ナノ微粒子のＦＴ－ＩＲ測定結果を図４（上）に示す。比較のため、実施例１のコアシェル型ナノ微粒子のＦＴ－ＩＲ測定結果を図４（下）に示す。図４（上）の測定結果から、１６６０ｎｍのケトン由来のピーク、２５００～３３００ｎｍの短波長方向に広がったＯＨ由来のピークが観測されたことによって、過酸化水素が酸化剤として働いて、ＰＥＧ４００末端の水酸基のカルボキシル化が、式（５）に沿って正しく行われたことが確認できた。 (FT-IR measurement result)
The obtained core-shell nanoparticle of Example 2 was subjected to FT-IR measurement. FIG. 4 (upper) shows the FT-IR measurement results of the core-shell nanoparticle of Example 2. For comparison, FIG. 4 (lower) shows the FT-IR measurement results of the core-shell nanoparticle of Example 1. From the measurement result of FIG. 4 (upper), a peak derived from a ketone at 1660 nm and a peak derived from OH spreading in a short wavelength direction from 2500 to 3300 nm were observed. It was confirmed that carboxylation of the hydroxyl group was correctly performed according to the formula (5).
［実施例３］
＜ＰＥＧ４００へのグルコサミンの化学修飾＞
　純水１５０ｍｌに実施例２で得られたコアシェル型ナノ微粒子の粉末０．０５ｇと、グルコサミン塩酸塩０．５ｇを入れ、リービッヒ冷却をしながら１３０℃で２５．５ｈ攪拌加熱した。その後、純水で洗浄してから乾燥し、粉末にした。 [Example 3]
<Chemical modification of glucosamine to PEG400>
In 150 ml of pure water, 0.05 g of the core-shell nanoparticle powder obtained in Example 2 and 0.5 g of glucosamine hydrochloride were added, and the mixture was stirred and heated at 130 ° C. for 25.5 h while cooling with Liebig. Thereafter, it was washed with pure water and dried to obtain a powder.
Figure JPOXMLDOC01-appb-C000010
 （ｋは１以上の整数を表す。）
Figure JPOXMLDOC01-appb-C000010
 (K represents an integer of 1 or more.)
（ＦＴ－ＩＲ測定結果）
　得られたナノ微粒子についてＦＴ－ＩＲ測定をした。実施例３のナノ微粒子のＦＴ－ＩＲ測定結果を図５（上）に示す。比較のため、実施例２のナノ微粒子のＦＴ－ＩＲ測定結果を図５（下）に示す。 (FT-IR measurement result)
The obtained nano fine particles were subjected to FT-IR measurement. FIG. 5 (upper) shows the FT-IR measurement results of the nanoparticle of Example 3. For comparison, the FT-IR measurement result of the nanoparticle of Example 2 is shown in FIG. 5 (bottom).
　１６００ｎｍ付近のピークに関して反応後にピークがブロードに、さらに頂点が左に移動していることがわかる。これは、コアシェル型ナノ微粒子の側のＣＯＯＨ基とグルコサミンのアミノ基が脱水縮合してアミド結合が形成されたことで、１５５０ｎｍ付近のピークが重ね合わせられたことによるものと考えられる。また、２８８０ｎｍ付近のＣＨのピーク強度が上がっていること、様々な状態でＯＨを持つ糖の影響によってＯＨのピークが広がっていることから、糖が修飾されたと考えることができる。以上の図５（上）の測定結果から、式（６）に沿って、ＰＥＧ４００へのグルコサミンの化学修飾が正しく行われたことを確認できた。 As for the peak near 1600 nm, it can be seen that after the reaction, the peak is broad and the vertex is moved to the left. This is presumably because the COOH group on the side of the core-shell nanoparticle and the amino group of glucosamine were dehydrated and condensed to form an amide bond, so that peaks near 1550 nm were superimposed. Moreover, since the peak intensity of CH near 2880 nm is increased and the peak of OH is broadened by the influence of sugar having OH in various states, it can be considered that the sugar is modified. From the above measurement results of FIG. 5 (upper), it was confirmed that the chemical modification of glucosamine to PEG400 was correctly performed according to the equation (6).
　実施例３のナノ微粒子は、シェルがグルコース骨格を有するグルコサミンで化学修飾されているので、腫瘍組織中に集積しやすい性質が得られると期待できる。また、実施例３のナノ微粒子は、ＭＦｅ２Ｏ４からなるコアを有しているので、磁気ハイパーサーミア（癌温熱療法）への利用も期待できる。 Since the nanoparticle of Example 3 is chemically modified with glucosamine having a glucose skeleton in the shell, it can be expected that the nanoparticle of Example 3 can easily accumulate in the tumor tissue. Also, nanoparticles of Example 3, because it has a core made of MFe 2 O 4, can be expected the use of the magnetic hyperthermia (cancer hyperthermia).
［実施例４］
（材料）
・培地：ＤＭＥＭ ｃｏｎｔａｉｎｉｎｇ １０％ （ｖｏｌ/ｖｏｌ） ｃａｌｆ ｓｅｒｕｍを用いた。以下、培地（ＤＭＥＭ／１０％ＣＳ）という。
・ＨｅＬａ細胞：理研セルバンクから入手したＨｅＬａ細胞を、培地（ＤＭＥＭ／１０％ＣＳ）で継代培養していたものを、３５ｍｍ ｇｌａｓｓ ｂｏｔｔｏｍ ｄｉｓｈに播種した後、２４ｈ後のものを実験に使用した。播種時の細胞数は、２×１０５ ｃｅｌｌｓ/ ｄｉｓｈであった。 [Example 4]
(material)
-Medium: DMEM containing 10% (vol / vol) calf serum was used. Hereinafter, it is referred to as a medium (DMEM / 10% CS).
-HeLa cells: HeLa cells obtained from Riken Cell Bank were subcultured in a medium (DMEM / 10% CS), seeded in a 35 mm glass bottom dish, and then 24 hours later were used for experiments. The number of cells at the time of seeding was 2 × 10 5 cells / dish.
（ナノ微粒子懸濁液の調製）
　実施例３のナノ微粒子の粉末にＰＢＳ（ｐＨ７．４）を添加し、超音波処理することによって均一な懸濁液とした後、適宜ＰＢＳ（ｐＨ７．４）で希釈することによって、最終的に１０ｍｇ/ｍｌの濃度の懸濁液を調製した。更に、培地（ＤＭＥＭ／１０％ＣＳ）で１００倍希釈したナノ微粒子懸濁液を調製した。 (Preparation of nanoparticle suspension)
PBS (pH 7.4) was added to the nanoparticulate powder of Example 3 to form a uniform suspension by sonication, and finally diluted with PBS (pH 7.4) as appropriate. A suspension with a concentration of 10 mg / ml was prepared. Further, a nanoparticle suspension diluted 100 times with a medium (DMEM / 10% CS) was prepared.
（実験）
　ｄｉｓｈ上のＨｅＬａ細胞に対して、培地（ＤＭＥＭ／１０％ＣＳ）で１００倍希釈したナノ微粒子懸濁液を添加し、３７℃のＣＯ２インキュベータで２４ｈ静置した。その後、２．５％（ｗ/ｖ）グルタルアルデヒド／ＰＢＳ（ｐＨ７．４）中で室温１ｈ固定した後、ＴＥＭ観察用のサンプル調製を行った。ＴＥＭ観察には、ＪＥＭ－１４００（日本電子株式会社）を使用した。 (Experiment)
To the HeLa cells on the dish, a nanoparticulate suspension diluted 100-fold with a medium (DMEM / 10% CS) was added and allowed to stand in a CO 2 incubator at 37 ° C. for 24 hours. Thereafter, the sample was fixed in 2.5% (w / v) glutaraldehyde / PBS (pH 7.4) for 1 h at room temperature, and then a sample for TEM observation was prepared. JEM-1400 (JEOL Ltd.) was used for TEM observation.
　図６は、実施例３のナノ微粒子が、ＨｅＬａ細胞内に取り込まれたことを示すＴＥＭ像である。図６に示すように、エンドソーム／リソソーム内に、実施例３のナノ微粒子の凝集体を確認でき、滴下から２４時間後には実施例３のナノ微粒子が、ＨｅＬａ細胞内に取り込まれたことが確認できた。
　実施例３のナノ微粒子ががん細胞内に取り込まれていることから、シェルがグルコース又はグルコース誘導体で化学修飾されているナノ微粒子を有効成分として含有する抗腫瘍剤は、磁気ハイパーサーミアへの利用が期待される。 FIG. 6 is a TEM image showing that the nanoparticle of Example 3 was taken up into HeLa cells. As shown in FIG. 6, the aggregates of the nanoparticle of Example 3 can be confirmed in the endosome / lysosome, and it was confirmed that the nanoparticle of Example 3 was taken into HeLa cells 24 hours after the dropping. did it.
Since the nanoparticle of Example 3 is incorporated into cancer cells, the antitumor agent containing the nanoparticle whose shell is chemically modified with glucose or a glucose derivative as an active ingredient can be used for magnetic hyperthermia. Be expected.
［実施例５］
＜ＰＥＧ４００を用いたＭｎ０．８Ｚｎ０．２Ｆｅ２Ｏ４ナノ微粒子の作製＞
　５０ｍｌのＰＥＧ４００の中で、５ｍｍｏｌのＦｅＣｌ２・４Ｈ２Ｏ、２．０ｍｍｏｌのＭｎＣｌ２・４Ｈ２Ｏ、０．５ｍｍｏｌのＺｎＣｌ２（無水物）、及び１５ｍｍｏｌの水酸化ナトリウムを７０℃で１時間混合して、中和反応させた。反応液を、反応容器のまま箱型オーブンの中に入れ、２００℃で１６時間加熱し、炉冷後の反応液に対して、遠心分離機（３５００ｒｐｍ, １０ｍｉｎ）で沈殿させ、エタノールで３回洗浄する工程を３回繰り返した。乾燥して、Ｍｎ０．８Ｚｎ０．２Ｆｅ２Ｏ４をコアとするコアシェル型ナノ微粒子の粉末を得た。得られたコアシェル型ナノ微粒子の磁性体部分の平均粒径（一次粒子径）は１２ｎｍであった。ＦＴ-ＩＲ, ＴＧ-ＤＴＡ（示差熱分析）などから洗浄後も十分な量のＰＥＧが存在することが確認できたことから、ＰＥＧが粒子周囲をコーティングしていると考えられる。 [Example 5]
<Preparation of Mn 0.8 Zn 0.2 Fe 2 O 4 Nanoparticles Using PEG400>
In 50 ml of PEG400, 5 mmol FeCl 2 .4H 2 O, 2.0 mmol MnCl 2 .4H 2 O, 0.5 mmol ZnCl 2 (anhydrous), and 15 mmol sodium hydroxide at 70 ° C. for 1 hour They were mixed and neutralized. The reaction solution is placed in a box oven as it is in a reaction vessel, heated at 200 ° C. for 16 hours, precipitated with a centrifuge (3500 rpm, 10 min) against the reaction solution after furnace cooling, and 3 times with ethanol. The washing process was repeated three times. Dried to obtain a powder of core-shell nanoparticles of core Mn 0.8 Zn 0.2 Fe 2 O 4 . The average particle diameter (primary particle diameter) of the magnetic part of the obtained core-shell nanoparticle was 12 nm. Since it was confirmed from FT-IR, TG-DTA (differential thermal analysis) and the like that a sufficient amount of PEG was present after washing, it is considered that PEG coated the particle periphery.
　さらにグルコース修飾によりがん細胞への取り込みも変化することからも、粒子を包含したＰＥＧにグルコースが修飾され、粒子の取り込み量に差が出ていると考えられる。１つのコア粒子に約６０００個のＰＥＧが付いていると考えられ、一周、２ｎｍ程度の薄さで覆っていると考えられる。 Furthermore, since glucose uptake also changes the uptake into cancer cells, it is considered that glucose is modified by PEG containing particles and that the amount of uptake of particles is different. It is considered that about 6000 PEGs are attached to one core particle, and it is considered that the core particle is covered with a thickness of about 2 nm.
［実施例５－２］
　５０ｍｌのＰＥＧ４００の中で、５ｍｍｏｌのＦｅＣｌ２・４Ｈ２Ｏ、２．０ｍｍｏｌのＭｎＣｌ２・４Ｈ２Ｏ、０．５ｍｍｏｌのＺｎＣｌ２（無水物）、及び１５ｍｍｏｌの水酸化ナトリウムを７０℃で１時間混合して、中和反応させた。反応液を、反応容器のまま箱型オーブンの中に入れ、１６０℃で１６時間加熱し、炉冷後の反応液に対して、遠心分離機（３５００ｒｐｍ, １０ｍｉｎ）で沈殿させ、エタノールで２回、純水で２回洗浄した。その後乾燥させ、Ｍｎ０．８Ｚｎ０．２Ｆｅ２Ｏ４をコアとするコアシェル型ナノ微粒子の粉末を得た。得られたコアシェル型ナノ微粒子の磁性体部分の平均粒径（一次粒子径）は６ｎｍであった。
　作製時に加える水量が少ないほど粒径分布が狭くなるという傾向があり、ほぼ単分散の平均粒径６ｎｍのナノ微粒子を得ることができた。 [Example 5-2]
In 50 ml of PEG400, 5 mmol FeCl 2 .4H 2 O, 2.0 mmol MnCl 2 .4H 2 O, 0.5 mmol ZnCl 2 (anhydrous), and 15 mmol sodium hydroxide at 70 ° C. for 1 hour They were mixed and neutralized. The reaction solution is placed in a box oven as it is in the reaction vessel, heated at 160 ° C. for 16 hours, precipitated with a centrifuge (3500 rpm, 10 min) against the reaction solution after furnace cooling, and twice with ethanol. And washed twice with pure water. Thereafter, drying was performed to obtain a powder of core-shell nano particles having Mn 0.8 Zn 0.2 Fe 2 O 4 as a core. The average particle diameter (primary particle diameter) of the magnetic part of the obtained core-shell nanoparticle was 6 nm.
There was a tendency that the smaller the amount of water added during the production, the narrower the particle size distribution, and it was possible to obtain almost monodisperse nanoparticles with an average particle size of 6 nm.
［実施例５－３］
　５０ｍｌのＰＥＧ４００及び４ｍｌの純水の混合液の中で、５ｍｍｏｌのＦｅＣｌ２・４Ｈ２Ｏ、２．０ｍｍｏｌのＭｎＣｌ２・４Ｈ２Ｏ、０．５ｍｍｏｌのＺｎＣｌ２（無水物）、及び１５ｍｍｏｌの水酸化ナトリウムを７０℃で１時間混合して、中和反応させた。反応液を、反応容器のまま箱型オーブンの中に入れ、１６０℃で１６時間加熱し、炉冷後の反応液に対して、遠心分離機（３５００ｒｐｍ, １０ｍｉｎ）で沈殿させ、エタノールで２回、純水で２回洗浄した。その後乾燥させ、Ｍｎ０．８Ｚｎ０．２Ｆｅ２Ｏ４をコアとするコアシェル型ナノ微粒子の粉末を得た。得られたコアシェル型ナノ微粒子の磁性体部分の平均粒径（一次粒子径）は１０ｎｍであった。コア部分の酸化物の１次粒径は、６～３０ｎｍで、作製条件（水の量、焼成温度）で制御することができる。 [Example 5-3]
In a mixture of 50 ml PEG 400 and 4 ml pure water, 5 mmol FeCl 2 .4H 2 O, 2.0 mmol MnCl 2 .4H 2 O, 0.5 mmol ZnCl 2 (anhydride), and 15 mmol water Sodium oxide was mixed at 70 ° C. for 1 hour to carry out a neutralization reaction. The reaction solution is placed in a box oven as it is in the reaction vessel, heated at 160 ° C. for 16 hours, precipitated with a centrifuge (3500 rpm, 10 min) against the reaction solution after furnace cooling, and twice with ethanol. And washed twice with pure water. Thereafter, drying was performed to obtain a powder of core-shell nano particles having Mn 0.8 Zn 0.2 Fe 2 O 4 as a core. The average particle diameter (primary particle diameter) of the magnetic part of the obtained core-shell nanoparticle was 10 nm. The primary particle size of the oxide in the core portion is 6 to 30 nm and can be controlled by the production conditions (water amount, firing temperature).
［実施例６］
　実施例５で得られたコアシェル型ナノ微粒子について、更に、実施例２と同様に、ＰＥＧ４００末端の水酸基のカルボキシル化と、実施例３と同様に、ＰＥＧ４００へのグルコサミンの化学修飾を行った。 [Example 6]
The core-shell nanoparticle obtained in Example 5 was further subjected to carboxylation of the hydroxyl group at the PEG 400 end as in Example 2 and chemical modification of glucosamine to PEG 400 in the same manner as in Example 3.
（ＦＴ－ＩＲ測定結果）
　グルコサミンで化学修飾された実施例６のナノ微粒子のＦＴ－ＩＲ測定をした。ＭｎＦｅ２Ｏ４標準試料のＦＴ－ＩＲ測定結果を図７（上）に示す。実施例５のＰＥＧ４００を用いたＭｎ０．８Ｚｎ０．２Ｆｅ２Ｏ４ナノ微粒子のＦＴ－ＩＲ測定結果を図７（中）に示す。１１０５ ± ５５ｃｍ－１のＣ－Ｏ－Ｃ逆対称伸縮運動、１４８５ ± １５ｃｍ－１のＣＨ２対称変角振動、３４００ ± ２００ｃｍ－１のＯ－Ｈ間伸縮運動が観測された。実施例６のグルコサミンで化学修飾後のナノ微粒子のＦＴ－ＩＲ測定結果を図７（下）に示す。１０９５ ± ２５ｃｍ－１のＣ－Ｏ－Ｃ伸縮運動、１１７５ ± ２５ｃｍ－１のＣ－Ｏ伸縮運動、１２６５ ± ５５ｃｍ－１のＣ－Ｏ伸縮、Ｏ－Ｈ変角振動、１６７０ ± ４０ｃｍ－１のアミド結合によるＣ＝Ｏ伸縮、３４００ ± ２００ｃｍ－１のＯ－Ｈ間伸縮運動が観測された。 (FT-IR measurement result)
FT-IR measurement of the nanoparticle of Example 6 chemically modified with glucosamine was performed. FIG. 7 (upper) shows the FT-IR measurement result of the MnFe 2 O 4 standard sample. The FT-IR measurement result of Mn 0.8 Zn 0.2 Fe 2 O 4 nanoparticle using PEG400 of Example 5 is shown in FIG. 7 (middle). 1105 C-O-C antisymmetric stretching vibration of ± 55cm -1, CH 2 symmetric deformation vibration of 1485 ± 15cm -1, 3400 O- H between stretching movement of ± 200 cm -1 was observed. FIG. 7 (bottom) shows the FT-IR measurement results of the nanoparticles after chemical modification with glucosamine of Example 6. C—O—C stretching motion of 1095 ± 25 cm −1 , C—O stretching motion of 1175 ± 25 cm −1 , C—O stretching motion of 1265 ± 55 cm −1 , O—H bending vibration, 1670 ± 40 cm −1 C = O stretching due to an amide bond, stretching movement between OH of 3400 ± 200 cm −1 was observed.
［実施例７］
＜ＰＥＧ２０００を用いたＣｏＦｅ２Ｏ４ナノ微粒子の作製＞
　１／３００ｍｏｌのＦｅＣｌ２・４Ｈ２Ｏ、及び１／６００ｍｏｌのＣｏＣｌ２・６Ｈ２Ｏを、１ｍｌの水に溶解させ、５５℃で液状にした３０ｇのＰＥＧ２０００に加えた。１０時間撹拌させた後、０．５ｍｌの水に溶解させたＮａＯＨを加えた。１０時間撹拌させた後、１６０℃で１６時間加熱した。遠心分離してエタノール、水で洗浄し、５０℃で乾燥させて、ＣｏＦｅ２Ｏ４をコアとするコアシェル型ナノ微粒子の粉末を得た。得られたコアシェル型ナノ微粒子の磁性体部分の平均粒径（一次粒子径）は２７ｎｍであった。 [Example 7]
<Preparation of CoFe 2 O 4 Nanoparticles Using PEG2000>
1/300 mol of FeCl 2 .4H 2 O and 1/600 mol of CoCl 2 .6H 2 O were dissolved in 1 ml of water and added to 30 g of PEG2000, which was liquefied at 55 ° C. After stirring for 10 hours, NaOH dissolved in 0.5 ml water was added. The mixture was stirred for 10 hours and then heated at 160 ° C. for 16 hours. Centrifugation, washing with ethanol and water, and drying at 50 ° C. gave core-shell nanoparticulate powders with CoFe 2 O 4 as the core. The average particle diameter (primary particle diameter) of the magnetic part of the obtained core-shell nanoparticle was 27 nm.
（ＸＲＤ分析結果）
　ＸＲＤ分析の結果を図８に示す。指数づけしたピークから、単相のスピネル構造を持ったＣｏＦｅ２Ｏ４ [No. 000-022-1086、Name: Cobalt Iron Oxide、Quality Mark: S、Cell (8.392, 8.392, 8.392)] が同定できたことから、目的のＣｏＦｅ２Ｏ４が作成できたことを確認した。湿式混合法で作ったＣｏＦｅ２Ｏ４をコアとするコアシェル型ナノ微粒子のＸＲＤ分析の結果の図１（上）に比べて、図８のＰＥＧ２０００を用いたコアシェル型ナノ微粒子のＸＲＤ分析の結果は、２０～３０°の位置にアモルファスのブロードなピークがないことが確認された。 (Results of XRD analysis)
The result of the XRD analysis is shown in FIG. CoFe 2 O 4 [No. 000-022-1086, Name: Cobalt Iron Oxide, Quality Mark: S, Cell (8.392, 8.392, 8.392)] with a single-phase spinel structure can be identified from the indexed peak. From this, it was confirmed that the target CoFe 2 O 4 could be prepared. Compared to FIG. 1 (top) of the result of XRD analysis of the core-shell nanoparticle having CoFe 2 O 4 as a core made by the wet mixing method, the result of XRD analysis of the core-shell nanoparticle using PEG2000 of FIG. , It was confirmed that there was no amorphous broad peak at a position of 20 to 30 °.
（ＦＴ－ＩＲ測定結果）
　続いて、得られたコアシェル型ナノ微粒子のＦＴ－ＩＲ測定をした。実施例７のコアシェル型ナノ微粒子のＦＴ－ＩＲ測定結果を図９に、実線「２０００」に示す。図９の太線「Ｓｔｄ．（ＭｎＦｅ）」は、ＭｎＦｅ２Ｏ４標準試料のＦＴ－ＩＲ測定結果であり、図９の点線「４００」は、実施例１のＰＥＧ４００を用いたＣｏＦｅ２Ｏ４ナノ微粒子のＦＴ－ＩＲ測定結果である。
　実施例７のコアシェル型ナノ微粒子からは、１１０５ ± ５５ｃｍ－１のＣ－Ｏ－Ｃ逆対称伸縮運動、１４８５ ± １５ｃｍ－１のＣＨ２対称変角振動、３４００ ± ２００ｃｍ－１のＯ－Ｈ間伸縮運動が観測された。
　実施例７のコアシェル型ナノ微粒子のＦＴ－ＩＲ測定結果は、ＣＯＣ、ＣＨ２や、Ｏ－Ｈ等の飽和炭化水素に特有の結合のピークを有していることから、ＰＥＧ２０００を用いて作製したＣｏＦｅ２Ｏ４をコアとするコアシェル型ナノ微粒子は、ＰＥＧ２０００に由来するＰＥＧの主鎖がＣｏＦｅ２Ｏ４のコアと化学結合しているものと考えられる。 (FT-IR measurement result)
Subsequently, FT-IR measurement of the obtained core-shell nanoparticle was performed. The FT-IR measurement result of the core-shell nanoparticle of Example 7 is shown in FIG. The thick line “Std. (MnFe)” in FIG. 9 is the FT-IR measurement result of the MnFe 2 O 4 standard sample, and the dotted line “400” in FIG. 9 is the CoFe 2 O 4 nanoparticle using the PEG 400 of Example 1. It is a FT-IR measurement result of fine particles.
From the core-shell nanoparticles of Example 7, C-O-C antisymmetric stretching vibration of 1105 ± 55cm -1, CH 2 symmetric deformation vibration of 1485 ± 15cm -1, 3400 between O-H of ± 200 cm -1 Stretching motion was observed.
Since the FT-IR measurement result of the core-shell nanoparticle of Example 7 has a bond peak peculiar to saturated hydrocarbons such as COC, CH 2 and OH, it was prepared using PEG2000. core-shell nanoparticles of the CoFe 2 O 4 and the core is believed to the main chain of the PEG derived from PEG2000 is bound core and chemistry of CoFe 2 O 4.
　１・・・ナノ微粒子、１０・・・コア、２０・・・シェル、Ｇｌｃ・・・グルコース、Ｇｌｃ’・・・グルコース誘導体 1 ... nano fine particle 10 ... core 20 ... shell Glc ... glucose Glc '... glucose derivative

Claims (8)

  1. 　ＭＦｅ２Ｏ４からなるコア（前記ＭＦｅ２Ｏ４において、Ｍは遷移金属を表す）と、
    　前記コアを覆うシェルと、を有し、
    　前記シェルがグルコース又はグルコース誘導体で化学修飾されているナノ微粒子。     A core composed of MFe 2 O 4 (in the MFe 2 O 4 , M represents a transition metal);
    A shell covering the core,
    Nanoparticles in which the shell is chemically modified with glucose or a glucose derivative.
  2. 　前記シェルがグルコース又はグルコース誘導体で化学修飾されたポリアルキレングリコールからなる、請求項１に記載のナノ微粒子。 The nanoparticle according to claim 1, wherein the shell is made of polyalkylene glycol chemically modified with glucose or a glucose derivative.
  3. 　前記シェルがグルコース又はグルコース誘導体で化学修飾されたアモルファスＳｉＯ２からなる、請求項１に記載のナノ微粒子。 The nanoparticle according to claim 1, wherein the shell is made of amorphous SiO 2 chemically modified with glucose or a glucose derivative.
  4. 　前記グルコース誘導体がアミノ基を有する、請求項１～３のうちいずれか一項に記載のナノ微粒子。 The nanoparticle according to any one of claims 1 to 3, wherein the glucose derivative has an amino group.
  5. 　ＭＦｅ２Ｏ４からなるコア（前記ＭＦｅ２Ｏ４において、Ｍは遷移金属を表す）と、前記コアを覆う官能基を含むシェルと、を有するコアシェル型ナノ微粒子を形成する工程と、
    　前記官能基にグルコース又はグルコース誘導体を反応させる工程と、を有する、請求項１～４のうちいずれか一項に記載のナノ微粒子の製造方法。     Forming a core-shell nanoparticle having a core composed of MFe 2 O 4 (in the MFe 2 O 4 , M represents a transition metal) and a shell including a functional group covering the core;
    The method for producing nanoparticles according to any one of claims 1 to 4, further comprising reacting glucose or a glucose derivative with the functional group.
  6. 　ＭＦｅ２Ｏ４からなるコア（前記ＭＦｅ２Ｏ４において、Ｍは遷移金属を表す）と、前記コアを覆うポリアルキレングリコールからなるシェルと、を有するコアシェル型ナノ微粒子を形成する工程と、
    　前記ポリアルキレングリコールの末端のヒドロキシメチル基を酸化させて、カルボキシル基にする工程と、
    　前記カルボキシル基にグルコース又はグルコース誘導体を反応させる工程と、を有する、請求項５に記載のナノ微粒子の製造方法。     (In the MFe 2 O 4, M represents a transition metal) MFe 2 core consisting of O 4 forming the, core-shell nanoparticles having a shell of a polyalkylene glycol to cover the core,
    Oxidizing the terminal hydroxymethyl group of the polyalkylene glycol to a carboxyl group;
    The method for producing nanoparticles according to claim 5, further comprising reacting glucose or a glucose derivative with the carboxyl group.
  7. 　請求項１～４のうちいずれか一項に記載のナノ微粒子を有効成分として含有する抗腫瘍剤。 An antitumor agent comprising the nanoparticle according to any one of claims 1 to 4 as an active ingredient.
  8. 　ポリアルキレングリコール、鉄を含む遷移金属の塩化物、及びアルカリを加熱し混合して、水酸化物を得る工程と、
    　前記水酸化物を含む反応物を加熱し焼成する工程と、を有するコアシェル型ナノ微粒子の製造方法。     Heating and mixing polyalkylene glycol, transition metal chloride including iron, and alkali to obtain a hydroxide;
    And a step of heating and baking the reaction product containing the hydroxide.
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