WO2019172213A1 - Nanoparticule, procédé de production de nanoparticule, et agent antitumoral - Google Patents

Nanoparticule, procédé de production de nanoparticule, et agent antitumoral 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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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/ja
Publication of WO2019172213A1 publication Critical patent/WO2019172213A1/fr

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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

La présente invention concerne une nanoparticule (1) possédant un cœur (10) constitué de MFe2O4 (M représentant un métal de transition) et une écorce (20) recouvrant le cœur (10). L'écorce (20) est chimiquement modifiée par du glucose ou un dérivé du glucose Glc'. X représente un groupe de liaison divalent. La nanoparticule (1) peut être produite au moyen des étapes consistant : à former une nanoparticule de type cœur-écorce possédant un cœur (10) constitué de MFe2O4 et une écorce possédant un groupe fonctionnel recouvrant le cœur (10) ; et à faire réagir du glucose ou un dérivé de glucose avec le groupe fonctionnel.
PCT/JP2019/008494 2018-03-05 2019-03-05 Nanoparticule, procédé de production de nanoparticule, et agent antitumoral WO2019172213A1 (fr)

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