JP7116563B2 - Composite particles - Google Patents

Description

The present invention relates to composite particles. More particularly, the present invention relates to composite particles, methods for producing the same, and optical imaging agents.

In the optical imaging method, the subject is irradiated with light, and the internal structure of the subject is imaged based on the intensity of the signal generated from the subject's endogenous light absorbers or the optical imaging agent introduced into the subject. It is a method to As optical imaging methods, for example, a photoultrasonic imaging method, a fluorescence imaging method, and the like have been proposed.

As an optical imaging agent used in the optical imaging method, the present inventors have proposed composite particles in which a hydrophilic coating is formed on the surface of manganese (II) oxide-containing particles (see, for example, Patent Document 1). .

Since the optical imaging method can image the internal structure of the subject without destroying it, it is used for purposes such as image diagnosis in vivo, for example. Therefore, even when the optical imaging method is applied to a living body, the development of an optical imaging agent that is excellent in dispersibility and stability in an aqueous solvent is desired from the viewpoint of accurately imaging the internal structure.

JP 2012-52271 A

The present invention has been made in view of the above prior art, and provides composite particles with excellent dispersibility and stability in aqueous solvents, a method for producing the same, and an optical imaging agent with excellent dispersibility and stability in aqueous solvents. The task is to

The present invention
(1) Composite particles containing Group 4 metal oxide fine particles, a luminescent material and a surfactant, wherein the Group 4 metal oxide fine particles aggregate primary particles made of the Group 4 metal oxide Composite particles, which are secondary particles and have a hydrodynamic average particle diameter of 30 to 300 nm,
(2) A method for producing composite particles containing Group 4 metal oxide fine particles, a luminescent material and a surfactant, wherein an alkali-soluble surface treatment layer is formed on the surface of the Group 4 metal oxide fine particles. After contacting the formed coated fine particles with an alkali metal hydroxide or alkaline earth metal hydroxide, the coated fine particles are brought into contact with a luminescent material, and the coated fine particles and a surfactant are further brought into contact. and (3) an optical imaging agent characterized by containing the composite particles described in (1) above.

INDUSTRIAL APPLICABILITY According to the present invention, composite particles excellent in dispersibility and stability in an aqueous solvent, a method for producing the same, and an optical imaging agent excellent in dispersibility and stability in an aqueous solvent are provided.

1 shows absorption spectra (visible-near infrared light absorption spectra) of the composite particles obtained in Example 1, the composite particles obtained in Comparative Example 1, and indocyanine green at a wavelength of 600 to 900 nm. (A) is the absorption spectrum (visible-near infrared light absorption spectrum) of the composite particles obtained in Example 1 contained in the sample immediately after preparation, and (B) is the example contained in the sample after standing for 24 hours. 1 is an absorption spectrum (visible-near infrared light absorption spectrum) of the composite particles obtained in 1. (A) is the particle size distribution of the composite particles obtained in Example 1 contained in the sample immediately after preparation, and (B) is the particles of the composite particles obtained in Example 1 contained in the sample after standing for 24 hours. diameter distribution. (A) is the particle size distribution of the composite particles obtained in Comparative Example 1 contained in the sample immediately after preparation, and (B) is the particles of the composite particles obtained in Comparative Example 1 contained in the sample after standing for 24 hours. diameter distribution. 4 is a graph showing the relationship between the type of composite particles and the change ΔF ₂ in oscillation frequency. (A) is a drawing-substituting photograph showing the results of observing the tumor-bearing part of a cancer-bearing mouse when the optical imaging agent obtained in Example 5 was used, and (B) is the optical imaging agent obtained in Comparative Example 2. (C) is a drawing substitute photograph showing the results of observing the tumor-bearing part of the tumor-bearing mouse when using the optical imaging agent obtained in Comparative Example 3. It is a drawing substitute photograph which shows the result which carried out. 4 is a graph showing temporal changes in fluorescence intensity (differential fluorescence intensity) in tumor-bearing regions of cancer-bearing mice. 2 is a graph showing the results of examining the relationship between the type of optical imaging agent and the fluorescence intensity in an organ containing a tumor.

As described above, the composite particles of the present invention are composite particles containing Group 4 metal oxide fine particles, a luminescent material and a surfactant, and the Group 4 metal oxide fine particles The secondary particles are aggregated primary particles, and the secondary particles have a hydrodynamic average particle size of 30 to 300 nm.

The composite particles of the present invention are excellent in dispersibility and stability in an aqueous solvent because the group 4 metal oxide fine particles, the luminescent material and the surfactant are used in combination.

Group 4 metal oxide fine particles are secondary particles in which primary particles made of a Group 4 metal oxide aggregate. Examples of Group 4 metal oxide fine particles include titanium dioxide fine particles, zirconium dioxide fine particles, hafnium dioxide fine particles, etc., but the present invention is not limited to these examples. When the composite particles of the present invention are applied to a living body, among these Group 4 metal oxide fine particles, titanium dioxide fine particles and zirconium dioxide fine particles are preferable from the viewpoint of improving stability in vivo, and zirconium dioxide fine particles are preferable. more preferred.

The hydrodynamic average particle size of the secondary particles is 30 nm or more, preferably 40 nm or more, more preferably 50 nm or more, from the viewpoint of reducing the accumulation of the composite particles of the present invention in living cells, tissues, etc. From the viewpoint of reducing the accumulation of the composite particles of the present invention in living cells, tissues, etc., and suppressing blockage of capillaries, the particle size is 300 nm or less, preferably 250 nm or less, more preferably 200 nm or less. Since the secondary particles are aggregated primary particles, the particle size of the secondary particles is usually larger than the particle size of the primary particles. The number average particle size of the primary particles is not particularly limited, but from the viewpoint of improving the industrial productivity of the composite particles of the present invention, it is preferably 5 nm or more, more preferably 10 nm or more. From the viewpoint of improving practical productivity, it is preferably 100 nm or less, more preferably 80 nm or less, and even more preferably 50 nm or less.

In the present specification, the "hydrodynamic average particle size" is obtained by suspending the particles to be measured in ultrapure water as a measurement sample, and using a zeta potential/particle size/molecular weight measuring device [Malvern product, trade name: Zetasizer nano ZS], the particle size distribution of the particles contained in the measurement sample is measured at 25 ° C. based on the dynamic light scattering method, and the harmonization by the scattered light intensity standard calculated using the cumulant method Average particle size.

Examples of the shape of the fourth metal oxide fine particles include spherical, ellipsoidal, cubic, rectangular parallelepiped, pyramidal, needle-like, rod-like, cylindrical, scale-like, and plate-like shapes. It is not limited only to such examples. Among the shapes of the fourth metal oxide fine particles, a spherical shape and a rod shape are preferable from the viewpoint of improving dispersibility in an aqueous solvent.

The composite particles of the present invention are obtained after contacting the coated fine particles in which an alkali-soluble surface treatment layer is formed on the surface of the group 4 metal oxide fine particles with an alkali metal hydroxide or an alkaline earth metal hydroxide. , can be produced by contacting the coated fine particles with a luminescent material, and further contacting the coated fine particles with a surfactant.

The group 4 metal oxide fine particles can be produced, for example, by heating a raw material compound of the group 4 metal oxide in the presence of water. The pressure when heating the raw material compound of the fourth metal oxide in the presence of water may be normal pressure or increased pressure. When the pressure is pressurization, for example, a reaction vessel for hydrothermal synthesis that can withstand pressure can be used.

The starting compound for the Group 4 metal oxide may be any compound that forms the Group 4 metal oxide by being heated in the presence of water. Examples of Group 4 metal oxides include titanium dioxide, zirconium dioxide, and hafnium dioxide, but the present invention is not limited to these examples. The Group 4 metal oxide may be an oxygen-deficient metal oxide in which some oxygen atoms in the crystal structure are missing. Examples of starting compounds for Group 4 metal oxides include hydroxides of Group 4 metals, chlorides of Group 4 metals, oxychlorides of Group 4 metals, nitrates of Group 4 metals, and Group 4 metals. metal oxynitrates, fourth metal sulfates, group 4 metal oxysulfates, group 4 metal organic acid salts, group 4 metal oxyorganic acid salts, group 4 metal carbonates, group 4 Alkoxides of group metals, complexes of group 4 metals and the like are included, but the present invention is not limited to such examples. Examples of Group 4 metals include titanium, zirconium, and hafnium, but the present invention is not limited to these examples.

When the composite particles of the present invention are applied to a living body, among the Group 4 metal oxides, from the viewpoint of being easily available and improving the stability in vivo, the raw material compound of titanium dioxide and the raw material compound of zirconium dioxide are used. is preferred.

Titanium dioxide raw material compounds include, for example, titanium chloride; titanium oxychloride; titanium oxynitrate; titanium sulfate; titanium organic acid salts such as titanium acetate, titanium octanoate and titanium 2-ethylhexanoate; Titanium oxyorganic acid salts such as titanium oxide, titanium stearate and titanium laurate; titanium alkoxides such as tetrabutoxytitanium and tetraisopropoxytitanium; titanium complexes such as acetylacetonatotitanium; , is not limited to such examples. Among these titanium dioxide raw material compounds, titanium chloride, titanium oxychloride, titanium oxysulfate, and acetic acid are easily available and easy to control the reaction rate in the hydrolysis and polycondensation reaction of the raw material compounds. Titanium, titanium 2-ethylhexanoate, tetrabutoxytitanium, tetraisopropoxytitanium, acetylacetonatotitanium are preferred, and titanium 2-ethylhexanoate, tetrabutoxytitanium, tetraisopropoxytitanium and acetylacetonatotitanium are more preferred.

Zirconium dioxide starting compounds include, for example, zirconium chloride; zirconium oxychloride; zirconium oxynitrate; zirconium sulfate; zirconium organic acid salts such as zirconium acetate, zirconium octanoate, and zirconium 2-ethylhexanoate; Zirconium oxyorganic acid salts such as zirconium oxide, zirconium stearate oxide, and zirconium laurate oxide; zirconium carbonate; zirconium alkoxides such as tetrabutoxy zirconium; . Among these zirconium dioxide starting compounds, zirconium 2-ethylhexanoate, zirconium oxychloride, oxy Zirconium sulfate and zirconium carbonate are preferred, and zirconium 2-ethylhexanoate is more preferred.

The water used for heating the starting compound of the Group 4 metal oxide may be mixed with an organic solvent. The organic solvent is preferably an organic solvent having a boiling point of 120° C. or higher under normal pressure from the viewpoint of sufficiently advancing the hydrothermal reaction during heating of the starting compound of the Group 4 metal oxide. The boiling point of the organic solvent under normal pressure is preferably 120° C. or higher, more preferably 180° C. or higher, and still more preferably 210° C., from the viewpoint of sufficiently advancing the hydrothermal reaction during heating of the raw material compound of the fourth metal oxide. °C or higher. Examples of organic solvents include hydrocarbon organic solvents such as decane, dodecane, tetradecane, xylene, trimethylbenzene, and toluene; octanol, decanol, cyclohexanol, terpineol, ethylene glycol, diethylene glycol, 1,2-propanediol, 1, alcohol-based organic solvents such as 3-propanediol, 1,4-butanediol, 2,3-butanediol, hexanediol, glycerin, and methanetrimethylol; ether-based organic solvents such as diethyl ether and tetrahydrofuran; ketone-based organic solvents; amide-based organic solvents such as dimethylformamide; sulfoxide-based organic solvents such as dimethylsulfoxide; In addition, when the mixture of water and the organic solvent is separated into two layers, the mixture may be used in a state of being separated into two layers. It may be used in a phase state or a suspended emulsified state.

The molar ratio between the starting compound of the Group 4 metal oxide and water used when heating the starting compound of the Group 4 metal oxide (water/starting compound of the Group 4 metal oxide) is From the viewpoint of shortening the time required for producing fine particles and suppressing the formation of coarse particles, the ratio is preferably 4/1 or more, more preferably 8/1 or more, and improves the industrial productivity of the composite particles of the present invention. From the viewpoint of improvement, it is preferably 100/1 or less, more preferably 50/1 or less.

The pressure at the time of heating the raw material compound of the group 4 metal oxide maintains the water used in the production of the group 4 metal oxide fine particles in a liquid state, thereby improving the industrial productivity of the composite particles of the present invention. It is preferably 0.5 MPa or more, more preferably 0.6 MPa or more, from the viewpoint of reducing the production cost of the composite particles of the present invention. It is more preferably 2 MPa or less, still more preferably 1.5 MPa or less, and even more preferably 1 MPa or less.

The heating temperature of the raw material compound of the Group 4 metal oxide is preferably 100° C. or higher from the viewpoint of shortening the time required for producing the composite particles of the present invention and improving the crystallinity of the Group 4 metal oxide fine particles. , more preferably 140 ° C. or higher, more preferably 160 ° C. or higher, and from the viewpoint of improving safety during production and reducing the production cost of the composite particles of the present invention, it is preferably 300 ° C. or lower, more preferably 250° C. or less, more preferably 220° C. or less.

The heating time of the starting compound of the Group 4 metal oxide varies depending on the type of the starting compound of the Group 4 metal oxide, etc., and cannot be determined indiscriminately. It is preferable to set appropriately according to such as. The heating time is usually 5 to 50 hours, preferably 10 to 40 hours. In this specification, the term "heating time of the source compound of the Group 4 metal oxide" means the time during which the source compound of the Group 4 metal oxide is maintained at the heating temperature.

From the viewpoint of improving safety during production, the atmosphere used for heating the source compound of the Group 4 metal oxide preferably contains an inert gas such as nitrogen gas or argon gas.

Next, an alkali-soluble surface treatment layer is formed on the surface of the group 4 metal oxide fine particles to obtain coated fine particles. Alkali-soluble surface treatment layers include, for example, silane coupling agent layers, carboxylic acid layers, phosphoric acid ester layers, and sulfuric acid ester layers, but the present invention is not limited to these examples.

Since the method for forming the alkali-soluble surface treatment layer differs depending on the type of the alkali-soluble surface treatment layer, etc., it cannot be determined unconditionally. preferably. The types and amounts of the compounds used for forming the alkali-soluble surface treatment layer, the conditions for forming the alkali-soluble surface treatment layer, etc. differ depending on the type of the alkali-soluble surface treatment layer, and so on. Therefore, it is preferable to determine it appropriately according to the type of the alkali-soluble surface treatment layer.

Among these alkali-soluble surface treatment layers, the silane coupling agent layer is preferable because it can be easily formed. The silane coupling agent layer can be formed, for example, by mixing a dispersion of group 4 metal oxide fine particles in a dispersion medium with a silane coupling agent and heating the resulting mixture. . A silane coupling agent layer is a layer containing a silane coupling agent. The silane coupling agent layer may contain a by-product or the like that is generated when the source compound of the group 4 metal oxide is heated.

Examples of the dispersion medium include hydrocarbon-based organic solvents such as benzene, toluene, xylene, and cyclohexane, but the present invention is not limited to these examples.

The content of the group 4 metal oxide fine particles in the dispersion liquid varies depending on the type of the fourth metal oxide fine particles, the type of the silane coupling agent, the application of the composite particles, etc., and cannot be determined indiscriminately. It is preferable to appropriately set according to the type of group 4 metal oxide fine particles, the type of silane coupling agent, the type of application of the composite particles, and the like. The content of the fourth metal oxide fine particles in the dispersion liquid is usually preferably 0.1% by mass or more from the viewpoint of reducing the production cost of the composite particles of the present invention, and is suitable for industrial production of the composite particles of the present invention. From the viewpoint of improving the properties, the content is preferably 70% by mass or less.

Silane coupling agents include, for example, formula (I):
-Si-OR ¹ (I)
(Wherein, R ¹ represents an alkyl group having 1 to 4 carbon atoms)
Examples include silane coupling agents having a hydrolyzable group represented by and the like, but the present invention is not limited to such examples.

In formula (I), R ¹ is an alkyl group having 1 to 4 carbon atoms. Examples of alkyl groups having 1 to 4 carbon atoms include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group and the like. The examples are not intended to be limiting.

Silane coupling agents having a hydrolyzable group represented by formula (I) include, for example, formula (II):
[ _R2- ( ^CH2 ) _m ] _{4-n- Si-(OR1)n} ⁽ II)
(In the formula, R ¹ is the same as above, R ² is a vinyl group, amino group, (meth)acryloyloxy group, mercapto group or glycidoxy group, m is an integer of 0 to 4, and n is an integer of 1 to 3. )
and alkoxysilanes, etc., but the present invention is not limited to such examples.

In formula (II), R ¹ is the same as R ¹ in formula (I). R ² is a vinyl group, amino group, (meth)acryloyloxy group, mercapto group or glycidoxy group. Examples of compounds represented by formula (II) include compounds in which R ² is a vinyl group, such as vinyltrimethoxysilane and vinyltriethoxysilane; 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, N -2-(aminoethyl)-3-aminopropylmethyldimethoxysilane, N-2-(aminoethyl)-3-aminopropylmethyltrimethoxysilane, etc., in which R ² is an amino group; 3-acryloyloxypropyltri methoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, etc. where R ² is a (meth)acryloyloxy group Compounds in which R ² is a mercapto group, such as 3-mercaptopropyltrimethoxysilane and 3-mercaptopropyltriethoxysilane; 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, 3-glycidoxy Examples include compounds in which R ² is a glycidoxy group, such as propyltrimethoxysilane, 3-glycidoxypropyltriethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, and 3-glycidoxypropylmethyldiethoxysilane. , the invention is not limited to such examples only.

Examples of alkoxysilanes include methyltrimethoxysilane, dimethyldimethoxysilane, trimethylmethoxysilane, propyltrimethoxysilane, butyltriethoxysilane, hexyltrimethoxysilane, hexyltriethoxysilane, octyltriethoxysilane, and decyltrimethoxysilane. and aryl group-containing alkoxysilanes such as phenyltrimethoxysilane, diphenyldimethoxysilane and p-styryltrimethoxysilane, but the present invention is not limited to these examples.

Silane coupling agents may be used alone or in combination of two or more. Among these silane coupling agents, from the viewpoint of reducing the manufacturing cost of the composite particles of the present invention and improving the dispersibility of the composite particles of the present invention in an ^aqueous solvent, R Certain compounds and alkyl group-containing alkoxysilanes are preferred, with 3-acryloyloxypropyltrimethoxysilane, 3-methacryloxypropyltrimethoxysilane, hexyltrimethoxysilane, octyltriethoxysilane and decyltrimethoxysilane being more preferred.

The amount of the silane coupling agent per 100 parts by mass of the group 4 metal oxide fine particles in the mixture varies depending on the type of the group 4 metal oxide fine particles, the type of the silane coupling agent, the application of the composite particles, etc., and is generally determined. Therefore, it is preferable to appropriately set according to the type of the Group 4 metal oxide fine particles, the type of the silane coupling agent, the type of application of the composite particles, and the like. The amount of the silane coupling agent per 100 parts by mass of the group 4 metal oxide fine particles in the mixture is usually preferably 0.1 parts by mass or more, more preferably 0.5 parts by mass, from the viewpoint of efficiently forming the surface treatment layer. It is at least 2 parts by mass, more preferably at least 2 parts by mass, and even more preferably at least 4 parts by mass. From the viewpoint of reducing the production cost of the composite particles of the present invention, it is preferably 50 parts by mass or less, more preferably 40 parts by mass. Below, more preferably 30 mass parts or less. Further, it is preferable to use an appropriate solvent when mixing the dispersion liquid in which the group 4 metal oxide fine particles are dispersed in the dispersion medium and the silane coupling agent. Since the solvent varies depending on the hydrolysis rate of the hydrolyzable functional group contained in the silane coupling agent, it cannot be determined unconditionally, so the hydrolysis rate of the hydrolyzable functional group contained in the silane coupling agent It is preferable to set appropriately according to. Examples of the solvent include hydrocarbon organic solvents such as toluene, xylene, and hexane; alcohols such as methanol, ethanol, propanol, isopropanol, and butanol; however, the present invention is limited only to such examples. is not. These solvents may be used alone or in combination of two or more. By using two or more solvents in combination, the hydrolysis rate can be easily controlled.

The heating temperature of the mixture varies depending on the type of group 4 metal oxide fine particles, the type of silane coupling agent, the application of the composite particles, the boiling point of the solvent, etc., and cannot be determined indiscriminately. It is preferable to appropriately set according to the type of the fine particles, the type of the silane coupling agent, the application of the composite particles, the boiling point of the solvent, and the like. The temperature for heating the mixture is usually preferably 30 to 180°C, more preferably 40 to 150°C, still more preferably 50 to 130°C.

The heating time of the mixture varies depending on the type of the Group 4 metal oxide fine particles, the type of the silane coupling agent, the application of the composite particles, etc., and cannot be determined unconditionally. , the type of silane coupling agent, the type of application of the composite particles, and the like. The heating time of the mixture is generally preferably 0.1 to 10 hours, more preferably 0.3 to 3 hours.

Next, the coated fine particles are brought into contact with an alkali metal hydroxide or an alkaline earth metal hydroxide.

Alkali metal hydroxides include, for example, lithium hydroxide, sodium hydroxide, potassium hydroxide and the like, but the present invention is not limited to these examples. Alkaline earth metal hydroxides include, for example, calcium hydroxide and magnesium hydroxide, but the present invention is not limited to these examples.

The contact of the coated fine particles with an alkali metal hydroxide or an alkaline earth metal hydroxide is carried out by, for example, adding a coated fine particle dispersion liquid in which the coated fine particles are dispersed in a dispersion medium to an alkali metal hydroxide solution or an alkaline earth metal hydroxide. It can be carried out, for example, by adding dropwise to the substance solution and stirring the resulting mixture. Examples of the alkali metal hydroxide solution include an aqueous alkali metal hydroxide solution and an alcoholic alkali metal hydroxide solution, but the present invention is not limited to these examples. The alkali metal hydroxide may be dissociated into ions in the alkali metal hydroxide solution upon contact between the coated fine particles and the alkali metal hydroxide. Alkaline earth metal hydroxide solutions include, for example, alkaline earth metal hydroxide aqueous solutions and alkaline earth metal hydroxide alcohol solutions, but the present invention is not limited to these examples. do not have. The alkaline earth metal hydroxide may be dissociated into ions in the alkali metal hydroxide solution upon contact between the coated fine particles and the alkaline earth metal hydroxide.

Examples of the dispersion medium used for the coated fine particle dispersion include water; alcohol-based organic solvents such as methanol, ethanol, n-propanol, isopropanol and ethylene glycol; ketone-based organic solvents such as methyl ethyl ketone, methyl isobutyl ketone and cyclohexanone; Ester-based organic solvents such as ethyl, propyl acetate, and propylene glycol monomethyl ether acetate; Ethers such as ethylene glycol monomethyl ether and diethylene glycol monobutyl ether; Modified ether-based organic solvents such as propylene glycol monomethyl ether acetate; Benzene, toluene, xylene, and ethylbenzene , trimethylbenzene, hexane, cyclohexane, methylcyclohexane, ethylcyclohexane, mineral spirits and other hydrocarbon organic solvents; dichloromethane, halogenated hydrocarbons such as chloroform; dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, etc. and oils such as mineral oil, vegetable oil, wax oil and silicone oil, but the present invention is not limited to these examples. Among these solvents, alcohol-based organic solvents, ketone-based organic solvents, and hydrocarbon-based organic solvents are preferable, and ketone-based organic solvents and hydrocarbon-based organic solvents are more preferable, because they are easy to handle. A solvent may be used independently and may use 2 or more types together.

The content of the coated fine particles in the coated fine particle dispersion liquid varies depending on the type of the coated fine particles, the type of the alkali metal hydroxide or alkaline earth metal hydroxide, the application of the composite particles, etc., and cannot be determined unconditionally. Therefore, it is preferable to appropriately set according to the type of the coated fine particles, the type of alkali metal hydroxide or alkaline earth metal hydroxide, the use of the composite particles, and the like. The content of the coated fine particles in the coated fine particle dispersion is usually preferably 0.1% by mass or more from the viewpoint of reducing the production cost of the composite particles of the present invention, and the industrial productivity of the composite particles of the present invention is improved. From the viewpoint of improvement, it is preferably 70% by mass or less.

The concentration of the alkali metal hydroxide in the alkali metal hydroxide solution varies depending on the type of the coated fine particles, the type of the alkali metal hydroxide, the application of the composite particles, etc., and cannot be determined unconditionally. It is preferable to appropriately set according to the type, the type of alkali metal hydroxide, the application of the composite particles, and the like. The alkali metal hydroxide concentration in the alkali metal hydroxide solution is generally preferably 1 to 50% by mass, more preferably 5 to 30% by mass. The concentration of the alkaline earth metal hydroxide in the alkaline earth metal hydroxide solution varies depending on the type of the coated fine particles, the type of the alkaline earth metal hydroxide, the application of the composite particles, etc., and cannot be determined unconditionally. Therefore, it is preferable to appropriately set according to the type of the coated fine particles, the type of the alkaline earth metal hydroxide, the application of the composite particles, and the like. The concentration of the alkaline earth metal hydroxide in the alkaline earth metal hydroxide solution is usually preferably 1 from the viewpoint of sufficiently reacting the coated fine particles with the alkali metal hydroxide or the alkaline earth metal hydroxide. to 50% by mass, more preferably 5 to 30% by mass.

Since the dropping rate of the coated fine particle dispersion solution varies depending on the type of coated fine particles, the type of alkaline earth metal hydroxide, the application of the composite particles, etc., it cannot be determined unconditionally. It is preferable to appropriately set according to the type of metal hydroxide, the application of the composite particles, and the like.

The stirring time and stirring speed of the resulting mixture vary depending on the type of coated fine particles, the type of alkaline earth metal hydroxide, the application of the composite particles, etc., and cannot be determined indiscriminately. It is preferable to appropriately set according to the type of alkaline earth metal hydroxide, the application of the composite particles, and the like.

Next, the coated fine particles after contact with the alkali metal hydroxide or alkaline earth metal hydroxide are brought into contact with a luminescent material. As a method of bringing the coated fine particles into contact with the luminescent material, for example, the coated fine particles after contact with the alkali metal hydroxide or alkaline earth metal hydroxide are dispersed in a dispersion medium, and the luminescent material is added as a solvent. and the like, but the present invention is not limited only to such examples.

Examples of the dispersion medium used in the dispersion liquid with which the luminescent material is brought into contact include water, methanol, ethanol, acetone, dimethylsulfoxide, and acetonitrile, but the present invention is not limited to these examples. .

The content of the coated fine particles in the dispersion liquid with which the luminescent substance is brought into contact varies depending on the type of the coated fine particles, the type of the luminescent substance, the type of surfactant, the application of the composite particles, etc., and cannot be determined indiscriminately. Therefore, it is preferable to appropriately set according to the type of the coated fine particles, the type of the luminescent material, the type of the surfactant, the application of the composite particles, and the like.

Luminescent substances include, for example, fluorescent substances and phosphorescent substances, but the present invention is not limited to such examples.

Examples of fluorescent substances include near-infrared fluorescent dyes such as cyanine dyes, but the present invention is not limited to such examples. Examples of cyanine dyes include indocyanine green, monomethine cyanine, trimethine cyanine, pentamethine cyanine, heptamethine cyanine, phthalocyanine, and naphthalocyanine, but the present invention is limited only to these examples. is not. Cyanine dyes are readily commercially available, examples of which include, for example, the trade names of IRDye (registered trademark) 800RS and IRDye (registered trademark) manufactured by LI-COR Biosciences. ) 800CW, product names of Lumiprobe: Cy3, Cy5 and Cy7, etc., but the present invention is not limited only to such examples.

Phosphorescent substances include, for example, zinc sulfide, iridium complexes, ruthenium complexes, osmium complexes, platinum complexes, rhenium complexes, molybdenum complexes, strontium aluminate, lanthanide ions (complex salts), etc., but the present invention provides only such examples. is not limited to

When the composite particles of the present invention are applied to a living body, among these luminescent substances, near-infrared light, which has excellent permeability to living bodies, can be used as excitation light. Cyanine dyes are preferred, and cyanine dyes are more preferred.

Examples of the solvent used for the luminescent substance solution include water and ethanol, but the present invention is not limited to these examples. Since the concentration of the luminescent substance in the solution of the luminescent substance varies depending on the use of the composite particles, it cannot be determined unconditionally.

The amount of the luminescent substance per 100 parts by mass of the coated fine particles when brought into contact with the luminescent substance varies depending on the type of the coated fine particles, the type of the luminescent substance, the application of the composite particles, etc., and cannot be determined unconditionally. Therefore, it is preferable to appropriately set according to the type of the coated fine particles, the type of the luminescent material, the application of the composite particles, and the like. The amount of the luminescent material per 100 parts by mass of the coated fine particles when brought into contact with the luminescent material is preferably 1 part by mass or more, more preferably 5 parts by mass, from the viewpoint of effectively contacting the coated fine particles of the luminescent material. From the viewpoint of improving industrial productivity, the amount is preferably 100 parts by mass or less, more preferably 50 parts by mass or less.

Next, composite particles can be obtained by bringing a surfactant into contact with the coated fine particles that have come into contact with the luminescent material. Examples of the method of bringing the surfactant into contact with the coated fine particles include a method of bringing the surfactant into contact with a mixture of the dispersion and the luminescent material, but the present invention is limited only to such examples. is not.

Surfactants include anionic surfactants, cationic surfactants, nonionic surfactants and amphoteric surfactants. These surfactants may be used alone or in combination of two or more.

The pH when the surfactant is dispersed in water adjusts the zeta potential when the composite particles of the present invention are dispersed in an aqueous solvent to a negative value, and when incorporated into cells, the load on the cells is reduced. From the viewpoint of reduction, it is preferably 2 to 7, more preferably 3 to 7. More specifically, the pH when the surfactant is dispersed in water is the pH when the surfactant is dispersed in ion-exchanged water so that the concentration is 10 wt %. A commercially available surfactant aqueous solvent solution can be used as a surfactant when contacting the coated fine particles. The pH of the surfactant aqueous solvent solution is preferably 2-7, more preferably 3-7. The surfactant concentration in the surfactant aqueous solvent solution is preferably 10% by mass or more from the viewpoint of improving industrial productivity, and preferably 80% by mass or less from the viewpoint of improving reactivity with coated microparticles. is.

Examples of anionic surfactants include polyoxyethylene chain-containing phospholipids such as distearoyl-N-monomethoxy-polyethyleneglycol-succinyl-phosphatidylethanolamine; polyoxyethylene chain-containing (meth)acrylic acid-based polymers alkylsulfonate salts such as ammonium dodecylsulfonate, sodium dodecylsulfate; alkylsulfonate salts such as ammonium dodecylsulfonate, sodium dodecylsulfonate, sodium alkyldiphenylether disulfonate; alkylarylsulfonate salts such as ammonium dodecylbenzenesulfonate, sodium dodecylnaphthalenesulfonate; Oxyethylene alkyl sulfonate salts; polyoxyethylene alkyl aryl ether sulfate salts such as polyoxyethylene alkyl sulfate salts; polyoxyethylene alkyl phenyl ether sulfate ammonium salts; dialkyl sulfosuccinates; aryl sulfonic acid-formalin condensates; fatty acid salts such as sodium stearate; bis(polyoxyethylene polycyclic phenyl ether) methacrylate sulfonate salts; propenyl-alkylsulfosuccinic acid ester salts; salts thereof; sulfuric acid ester salts of allyloxymethylalkoxyethylpolyoxyethylene; and polyoxyalkylenealkenyl ether ammonium sulfates, etc., but the present invention is not limited to these examples. Among these anionic surfactants, polyoxyethylene chain-containing phospholipid and Polyoxyethylene chain-containing (meth)acrylic acid-based polymers are preferred.

The weight-average molecular weight of the polyoxyethylene chain-containing phospholipid is preferably 2000 or more, more preferably 5000 or more, from the viewpoint of improving the dispersibility of the composite particles of the present invention in an aqueous solvent, and improves the stability of the composite particles. It is preferably 10,000 or less, more preferably 8,000 or less, from the viewpoint of The weight-average molecular weight of the polyoxyethylene chain-containing (meth)acrylic acid-based polymer is preferably 5000 or more, more preferably 10000 or more, from the viewpoint of improving the dispersibility of the composite particles of the present invention in an aqueous solvent. It is preferably 200,000 or less, more preferably 100,000 or less, from the viewpoint of improving the stability of and controlling the hydrodynamic average particle size.

In addition, in this specification, a weight average molecular weight means the polystyrene conversion weight average molecular weight calculated|required by the gel permeation chromatography (GPC).

Examples of nonionic surfactants include polyoxyethylene alkyl ethers, polyoxyethylene alkylaryl ethers, condensates of polyethylene glycol and polypropylene glycol, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, fatty acid monoglycerides, ethylene oxide and Condensates with aliphatic amines, allyloxymethylalkoxyethylhydroxypolyoxyethylenes, polyoxyalkylenealkenyl ethers, etc. may be mentioned, but the present invention is not limited to these examples.

Examples of cationic surfactants include alkylammonium salts such as dodecyl ammonium chloride, but the present invention is not limited to these examples.

Examples of amphoteric surfactants include alkylbetaine amphoteric surfactants, sulfobetaine amphoteric surfactants, alkylamidobetaine amphoteric surfactants, imidazoline amphoteric surfactants, and the like. It is not limited only to such examples.

Among these surfactants, from the viewpoint of improving the dispersibility and stability of composite particles in an aqueous solvent, anionic surfactants are preferred, and polyoxyethylene chain-containing phospholipids and polyoxyethylene chain-containing (meth) surfactants are preferred. Acrylic acid-based polymers are more preferred.

The amount of the surfactant per 100 parts by mass of the coated fine particles when brought into contact with the surfactant varies depending on the type of the coated fine particles, the type of the surfactant, the application of the composite particles, etc., and cannot be determined unconditionally. Therefore, it is preferable to appropriately set according to the type of the coated fine particles, the type of surfactant, the application of the composite particles, and the like. The amount of the surfactant per 100 parts by mass of the coated microparticles when brought into contact with the surfactant is preferably 0.2 parts by mass or more, more preferably 0.5 parts by mass or more, from the viewpoint of obtaining a stable dispersion. From the viewpoint of improving industrial productivity, it is preferably 50 parts by mass or less, more preferably 30 parts by mass or less.

When the luminescent material is brought into contact with the coated fine particles, a dispersion liquid in which the coated fine particles after contact with the alkali metal hydroxide or alkaline earth metal hydroxide are dispersed in a dispersion medium, and a luminescent material dissolved in the solvent are used. The composite particles can be separated from the product obtained by contacting the coated microparticles with the luminescent material and the surfactant.

The average hydrodynamic particle size of the composite particles of the present invention may be larger than the average hydrodynamic particle size of the secondary particles. The average hydrodynamic particle size of the composite particles of the present invention is preferably 40 nm or more, more preferably 50 nm, from the viewpoint of obtaining a clear emission signal when the composite particles of the present invention are used for fluorescence imaging, phosphorescence imaging, or the like. From the viewpoint of suppressing aggregation of the composite particles, it is preferably 400 nm or less, more preferably 200 nm or less. When the composite particles of the present invention are applied to a living body, the hydrodynamic average particle diameter of the composite particles of the present invention is also determined by the retention of the composite particles in blood when the composite particles of the present invention are used to detect tumors. is preferably 40 nm or more, more preferably 50 nm or more, from the viewpoint of improving the From the viewpoint of improvement, it is preferably 200 nm or less, more preferably 150 nm or less.

As described above, the composite particles of the present invention are excellent in dispersibility and stability in aqueous solvents, and therefore can be used, for example, as optical imaging agents applied to living organisms.

The optical imaging agent of the present invention is characterized by containing the composite particles. Since the optical imaging agent of the present invention contains composite particles, it is excellent in dispersibility and stability in aqueous solvents.

The optical imaging agent of the present invention may be composed only of the composite particles, or may contain additives. Examples of the additive include a dispersion medium for dispersing the composite particles, a stabilizer, and the like, but the present invention is not limited to such examples. When the optical imaging agent of the present invention contains an additive or the like, the content of the composite particles in the optical imaging agent of the present invention varies depending on the application of the composite particles and cannot be determined unconditionally. It is preferable to set appropriately according to the use of.

As described above, the optical imaging agent of the present invention is excellent in dispersibility and stability in aqueous solvents, and therefore can be used as an optical imaging agent for photoacoustic imaging, fluorescence imaging, and the like in living organisms. can. Therefore, the optical imaging agent of the present invention is expected to be used for, for example, bioimaging, diagnostic imaging, preferably diagnostic imaging of tumors.

EXAMPLES The present invention will be described in more detail below based on examples, but the present invention is not limited only to these examples. The meaning of each abbreviation is as follows.
<Explanation of abbreviations>
BSA: bovine serum albumin DSPE-PEG: distearoyl-N-monomethoxy-polyethyleneglycol-succinyl-phosphatidylethanolamine (polyethylene glycol chain-containing phospholipid)
FBS: fetal bovine serum ICG: indocyanine green PBS: phosphate buffered saline PEG: polyethylene glycol

Production example 1
(1) Production of coated zirconium dioxide particles (coated fine particles (1-1)) Zirconium 2-ethylhexanoate mineral spirit solution (manufactured by Daiichi Kigenso Kagaku Kogyo Co., Ltd., zirconium 2-ethylhexanoate content: 44 %) and 268 g of pure water were mixed to obtain a mixture. The resulting mixture was charged into an autoclave equipped with a stirrer, and the atmosphere inside the autoclave was replaced with nitrogen gas. The mixture in the autoclave was heated to 180° C. and maintained at 180° C. for 16 hours under a pressure of 0.94 MPa to produce a precipitate of zirconium dioxide particles. The precipitate was filtered off, washed with acetone, and dried. 100 g of the dried precipitate was dispersed in 800 mL of toluene to obtain a dispersion. Quantitative filter paper [manufactured by Advantech Toyo Co., Ltd., model: No. 5C] to remove coarse particles and the like. The resulting filtrate was concentrated under reduced pressure to remove toluene, thereby obtaining white coated zirconium dioxide particles (coated microparticles (1-1)).

X-ray diffraction of the coated zirconium dioxide particles was measured. An X-ray diffractometer (manufactured by Rigaku Co., Ltd., product number: RINT-TTRIII) was used for X-ray diffraction measurement. Measurement conditions are as follows.
(X-ray diffraction measurement conditions)
X-ray source: CuKα (wavelength: 0.154 nm)
X-ray output setting: 50kV, 300mA
Sampling width: 0.0200°
Scan speed: 10.0000°/min
Measurement range: 10° to 75°
Measurement temperature: 25°C

In addition, using integrated powder X-ray analysis software [manufactured by Rigaku Co., Ltd., product number: PDXL], by analyzing the intensity value of the diffraction peak according to the reference intensity ratio method, the ratio of tetragonal crystals to monoclinic crystals [tetragonal crystals/monoclinic crystals (mass ratio)] were quantified.

As a result, a diffraction peak attributed to the tetragonal crystal and a diffraction peak attributed to the monoclinic crystal were detected. Therefore, it can be seen that the zirconium dioxide particles have a crystal structure composed of a tetragonal system and a monoclinic system. It was also confirmed that the ratio of tetragonal crystals to monoclinic crystals [tetragonal crystals/monoclinic crystals (mass ratio)] was 54/46.

Under an ultra-high resolution field emission scanning electron microscope [manufactured by Hitachi High-Technologies Co., Ltd., product number: S-4800], the longitudinal length (major diameter) of any 100 coated zirconium dioxide particles is measured, The number average particle diameter of the coated zirconium dioxide particles (primary particles) was calculated by calculating the average value of the length in the major axis direction.

As a result, it was confirmed that the coated zirconium dioxide particles (primary particles) had a number average particle size of 12 nm.

In addition, an infrared absorption spectrum of the coated zirconium dioxide particles was measured using an infrared absorption spectrometer (manufactured by PerkinElmer, trade name: Spectrum One).

Absorption derived from CH and absorption derived from a carboxyl group were confirmed from the measurement results of the infrared absorption spectrum. Thus, it can be seen that the coated zirconium dioxide particles have a coating layer of 2-ethylhexanoic acid and/or a carboxylate derived from 2-ethylhexanoic acid.

Furthermore, using a thermogravimetric-indicative thermal analysis device [manufactured by Rigaku Co., Ltd., trade name: Thermo plus EVO II / TG-DTA], coating was performed from room temperature to 800 ° C. at a heating rate of 10 ° C./min under an air atmosphere. The temperature of the zirconium dioxide particles was raised, and the mass reduction rate of the coated zirconium dioxide particles was measured.

As a result, it was confirmed that the mass reduction rate of the coated zirconium dioxide particles was 12%. Therefore, it can be seen that the content of 2-ethylhexanoic acid and/or 2-ethylhexanoic acid-derived carboxylate in the coated zirconium dioxide particles is 12% by mass.

(2) Production of coated fine particles (1-2) by surface treatment of coated fine particles (1-1) 10 g of the coated zirconium dioxide particles obtained in Production Example 1 (1) were dispersed in 90 g of toluene to prepare a transparent dispersion. Obtained. 1.5 g of 3-acryloyloxypropyltrimethoxysilane was added to the resulting dispersion, and the mixture was heated to reflux at 90° C. for 1 hour. After heating under reflux, 300 g of n-hexane was added to the dispersion to aggregate the coated zirconium dioxide particles in the dispersion to obtain a cloudy liquid. Aggregated particles (secondary particles obtained by aggregating primary particles) were separated by filtration from the obtained cloudy liquid, and then dried at room temperature.

The crystal structure of the aggregated particles and the ratio of tetragonal crystals to monoclinic crystals [tetragonal crystals/monoclinic crystals (mass ratio)] were examined in the same manner as in Production Example (1). As a result, a diffraction peak attributed to the tetragonal crystal and a diffraction peak attributed to the monoclinic crystal were detected. Therefore, it can be seen that the aggregated particles have a crystal structure composed of a tetragonal crystal and a monoclinic crystal. It was also confirmed that the ratio of tetragonal crystals to monoclinic crystals [tetragonal crystals/monoclinic crystals (mass ratio)] was 54/46.

Furthermore, the infrared absorption spectrum of the aggregated particles was measured in the same manner as in Production Example 1(1). Absorption derived from C—H, absorption derived from a carboxyl group, and absorption derived from Si—O—C were confirmed from the measurement results of the infrared absorption spectrum. From this, it can be seen that the aggregated particles have a layer containing 2-ethylhexanoic acid and/or a carboxylate derived from 2-ethylhexanoic acid and a layer containing 3-acryloyloxypropyltrimethoxysilane. Recognize.

Furthermore, the mass reduction rate of the aggregated particles was measured in the same manner as in Production Example 1(1). As a result, it was confirmed that the mass reduction rate of the aggregated particles was 17%. Therefore, it can be seen that the total content of 2-ethylhexanoic acid and/or carboxylate derived from 2-ethylhexanoic acid and 3-acryloyloxypropyltrimethoxysilane in the aggregated particles is 17% by mass.

In addition, the content of 3-acryloyloxypropyltrimethoxysilane in the aggregated particles was determined by measuring the silicon content in the aggregated particles using a fluorescent X-ray spectrometer [manufactured by Rigaku Corporation, product number: ZSX Primus II]. asked for quantity. Furthermore, the total carbon content in the agglomerated particles was measured using an elemental analyzer [manufactured by J Science Lab Co., Ltd., product number: JM10]. Next, by subtracting the value of the amount of carbon derived from 3-acryloyloxypropyltrimethoxysilane from the value of the total carbon content in the aggregated particles, 2-ethylhexanoic acid and/or 2-ethylhexanoic acid derived The amount of carbon derived from carboxylate was calculated. Based on the calculated amount of carbon derived from 2-ethylhexanoic acid and/or 2-ethylhexanoic acid-derived carboxylates, 2-ethylhexanoic acid and/or 2-ethylhexanoic acid-derived carboxylates in the aggregate particles was determined. Next, based on the content of 3-acryloyloxypropyltrimethoxysilane in the aggregated particles and the content of 2-ethylhexanoic acid and/or carboxylate derived from 2-ethylhexanoic acid in the aggregated particles, 2- The abundance ratio (molar ratio) of 3-acryloyloxypropyltrimethoxysilane to carboxylate derived from ethylhexanoic acid and/or 2-ethylhexanoic acid was determined. As a result, it was confirmed that the abundance ratio (molar ratio) of 3-acryloyloxypropyltrimethoxysilane to 2-ethylhexanoic acid and/or carboxylate derived from 2-ethylhexanoic acid was 1.5.

The obtained aggregated particles [coated fine particles (1-2)] were dispersed in methyl ethyl ketone so that the concentration was 70% by mass to obtain a zirconium dioxide/methyl ethyl ketone dispersion.

Example 1
10 μL of the zirconium dioxide/methyl ethyl ketone dispersion obtained in Production Example 1 was added dropwise to 1 mL of a 5 M sodium hydroxide aqueous solution while stirring. After the dropwise addition was completed, the resulting mixture was stirred at room temperature for 30 minutes to obtain a dispersion liquid A. Next, using a centrifugal filter unit [manufactured by Merck Millipore, trade name: Amicon Ultra-4, molecular weight cut off: 3000] and ultrapure water, the solid content contained in the dispersion liquid A was centrifuged at 5000 x g. and for 30 minutes to obtain particles. The obtained particles were dispersed in 10 mL of ultrapure water to obtain dispersion B.

Using a zeta potential/particle size/molecular weight measuring device [manufactured by Malvern, trade name: Zetasizer nano ZS], the particle size distribution of the particles contained in the dispersion B is measured at 25°C based on the dynamic light scattering method, and a cumulant is obtained. The hydrodynamic average particle size of the aggregated particles (secondary particles) was determined based on the scattered light intensity using the method. As a result, the hydrodynamic average particle size (secondary particles) of the aggregated particles was 190 nm.

While the dispersion B was stirred, 1 mL of an ICG aqueous solution [ICG concentration: 2 mg/mL] was added dropwise to the dispersion B. After the dropwise addition was completed, the resulting mixture was stirred at room temperature for 12 hours to obtain a suspension. Next, using a centrifugal filter unit [manufactured by Merck Millipore, trade name: Amicon Ultra-4, molecular weight cut off: 10000] and ultrapure water, the particles contained in the suspension are centrifuged at 5000 x g. It was carried out under the conditions of 30 minutes. After confirming that the wash liquid did not contain ICG, the particles were recovered. The obtained particles were dispersed in 10 mL of ultrapure water to obtain dispersion C.

DSPE-PEG [manufactured by NOF Corporation, trade name: SUNBRIGHT DSPE-050CN] was added to give a DSPE-PEG aqueous solution at a concentration of 20 mg/mL. 2 mL of the resulting DSPE-PEG aqueous solution was added to Dispersion C to obtain a mixture. The resulting mixture was stirred at room temperature for 2 hours to obtain Dispersion D. Next, using a centrifugal filter unit [manufactured by Merck Millipore, trade name: Amicon Ultra-4, molecular weight cut off: 30000] and ultrapure water, the particles contained in the dispersion D are centrifuged and washed at 5000×g. A dispersion of composite particles containing zirconium dioxide fine particles, ICG and DSPE-PEG was obtained by carrying out for 20 minutes.

Comparative example 1
10 μL of the zirconium dioxide/methyl ethyl ketone dispersion obtained in Production Example 1 was added dropwise to 1 mL of a 5 M sodium hydroxide aqueous solution while stirring. After the dropwise addition was completed, the resulting mixture was stirred at room temperature for 30 minutes to obtain a dispersion liquid A. Next, using a centrifugal filter unit [manufactured by Merck Millipore, trade name: Amicon Ultra-4, molecular weight cut off: 3000] and ultrapure water, the solid content contained in the dispersion liquid A was centrifuged at 5000 x g. and for 30 minutes to obtain particles. The obtained particles were dispersed in 10 mL of ultrapure water to obtain dispersion B.

While the dispersion B was stirred, 1 mL of an ICG aqueous solution [ICG concentration: 2 mg/mL] was added dropwise to the dispersion B. After the dropwise addition was completed, the resulting mixture was stirred at room temperature for 12 hours to obtain a suspension. Next, using a centrifugal filter unit [manufactured by Merck Millipore, trade name: Amicon Ultra-4, molecular weight cut off: 10000] and ultrapure water, the particles contained in the suspension are centrifuged at 5000 x g. It was carried out under the conditions of 30 minutes. After confirming that the wash liquid did not contain ICG, the particles were recovered. The obtained particles were dispersed in 10 mL of ultrapure water to obtain dispersion C.

Next, using a centrifugal filter unit [manufactured by Merck Millipore, trade name: Amicon Ultra-4, molecular weight cut off: 30000] and ultrapure water, the particles contained in the dispersion liquid C are centrifugally washed at 5000 x g. A dispersion liquid of composite particles containing zirconium dioxide fine particles and ICG was obtained by carrying out for 20 minutes.

Test example 1
A sample for dynamic light scattering (DLS) measurement was obtained by diluting the dispersion liquid of the composite particles obtained in Example 1 by 10 times with ultrapure water and stirring. A DLS measurement sample was obtained in the same manner as described above, except that the composite particles obtained in Comparative Example 1 were used in place of the composite particles obtained in Example 1.

The composite particle dispersion obtained in Example 1 was diluted 20-fold with ultrapure water and stirred to obtain an electrophoretic light scattering (ELS) measurement sample. A sample for ELS measurement was obtained in the same manner as described above, except that the composite particles obtained in Comparative Example 1 were used in place of the composite particles obtained in Example 1.

Using a zeta potential/particle size/molecular weight measuring device [manufactured by Malvern, trade name: Zetasizer nano ZS], the particle size distribution of the particles contained in the DLS measurement sample is measured at 25 ° C. based on the dynamic light scattering method, and the cumulant is obtained. method was used to determine the hydrodynamic mean particle size of aggregated particles based on scattered light intensity. Furthermore, the zeta potential of the composite particles contained in the ELS measurement sample was measured at 25 ° C. based on the electrophoretic light scattering method using a zeta potential/particle size/molecular weight measuring device [manufactured by Malvern, trade name: Zetasizer nano ZS]. did.

As a result, the composite particles obtained in Example 1 had a hydrodynamic average particle diameter of 210 nm and a zeta potential of -24 mV. The composite particles obtained in Comparative Example 1 had a hydrodynamic average particle diameter of 200 nm and a zeta potential of -20 mV. Therefore, it can be seen that the absolute value of the zeta potential of the composite particles obtained in Example 1 is higher than the absolute value of the zeta potential of the composite particles obtained in Comparative Example 1. In Example 1, zirconium dioxide fine particles, ICG, and DSPE-PEG are used in combination, whereas in Comparative Example 1, DSPE-PEG is not used. Also, the absolute value of the zeta potential can be used as an index of dispersibility. These results show that the composite particles obtained in Example 1 have improved dispersibility compared to the composite particles obtained in Comparative Example 1 because the zirconium dioxide fine particles, ICG, and DSPE-PEG are used in combination. It can be seen that

Test example 2
The composite particles obtained in Example 1 were mixed with PBS at a concentration of 20 mM in terms of ICG, and the mixture was stirred at 37° C. for 30 minutes to obtain a sample. A sample was obtained in the same manner as described above, except that the composite particles obtained in Comparative Example 1 were used in place of the composite particles obtained in Example 1. Note that the composite particles in the sample are maintained in the same coexistence of ions as in the living body at the in vivo temperature (37° C.).

Samples were obtained by mixing ICG with PBS to a concentration of 20 mM and stirring at 37° C. for 30 minutes. The ICG in the sample is maintained in the same coexistence of ions as in the living body at the temperature of the living body (37° C.).

Using the sample and a spectrophotometer [manufactured by Hitachi High-Tech Co., Ltd., product number: UV-3010], the composite particles obtained in Example 1 under the same ion coexistence as in vivo at in vivo temperature (37 ° C.) , the composite particles and ICG obtained in Comparative Example 1 were measured for absorption spectra at a wavelength of 600 to 900 nm (also referred to as “optical absorption spectrum” or “visible-near infrared absorption spectrum”). These results are shown in FIG. In the figure, 1 is the absorption spectrum of the composite particles obtained in Example 1, 2 is the absorption spectrum of the composite particles obtained in Comparative Example 2, and 3 is the absorption spectrum of ICG. In the figure, λ1 indicates a wavelength of 779 nm and λ2 indicates a wavelength of 792 nm.

From the results shown in FIG. 1, the absorption peak wavelength of the absorption spectrum of the composite particles obtained in Example 1 is longer than the absorption peak wavelength of each absorption spectrum of the composite particles and ICG obtained in Comparative Example 1. It can be seen that there is a shift to the wavelength side. On the other hand, it can be seen that the absorption peak wavelength of the absorption spectrum of the composite particles obtained in Comparative Example 1 is similar to the absorption peak wavelength of the absorption spectrum of ICG. These results show that when ICG is liberated from composite particles containing zirconium dioxide fine particles, ICG, and DSPE-PEG, the absorption peak wavelength of the absorption spectrum shifts. Therefore, it can be seen that the presence or absence of liberation of ICG from composite particles containing zirconium dioxide fine particles, ICG and DSPE-PEG can be examined by using the absorption spectrum.

Test example 3
The composite particles obtained in Example 1 were mixed with PBS at a concentration of 20 mM in terms of ICG, and the mixture was stirred at 37° C. for 30 minutes to obtain a sample. The resulting sample was allowed to stand at 37°C for 24 hours. Note that the composite particles in the sample are maintained at the in vivo temperature (37° C.) in the same coexistence of ions and blood proteins as in the in vivo.

Using the sample and a spectrophotometer [manufactured by Hitachi High-Tech Co., Ltd., product number: UV-3010], the composite obtained in Example 1 contained in the sample immediately after preparation and the sample after standing at 37 ° C. for 24 hours By measuring the absorption spectrum of particles at a wavelength of 500 to 900 nm (also referred to as "optical absorption spectrum" and "visible-near infrared absorption spectrum"), the same ions as in the living body at the temperature in the living body (37 ° C.) The stability of the composite particles obtained in Example 1 under coexistence was investigated.

FIG. 2A shows the absorption spectrum of the composite particles obtained in Example 1 contained in the sample immediately after preparation, and the absorption spectrum of the composite particles obtained in Example 1 contained in the sample after standing for 24 hours is shown in FIG. 2(B). In the figure, the arrow indicates the absorption peak at a wavelength of 798 nm.

From the results shown in FIG. 2, the absorption spectrum of the composite particles obtained in Example 1 contained in the sample immediately after preparation shown in FIG. It can be seen that there is no clear difference between the absorption spectrum of the composite particles obtained in Example 1 contained in the sample after standing. From these results, the composite particles obtained in Example 1 stably retain ICG even when maintained in the same coexistence of ions as in vivo at an in vivo temperature (37°C). I understand. Therefore, it can be seen that the composite particles obtained in Example 1 exhibit excellent stability at in vivo temperature (37° C.) and in the same coexistence of ions as in vivo.

Test example 4
Sample A was obtained by mixing the composite particles obtained in Example 1 with a PBS solution containing 50% by mass of FBS so that the concentration in terms of ICG was 20 mM, and stirring the mixture at 37° C. for 30 minutes. The resulting sample was allowed to stand at 37°C for 24 hours. The composite particles in the sample A are maintained at the in vivo temperature (37° C.) in the same coexistence of ions and blood proteins as in the in vivo.

Further, sample B was obtained by mixing the composite particles obtained in Comparative Example 1 with a PBS solution containing 50% by mass of FBS so that the concentration in terms of ICG was 20 mM, and stirring the mixture at 37° C. for 30 minutes. The resulting sample was allowed to stand at 37°C for 24 hours. The composite particles in the sample B are maintained at the in vivo temperature (37° C.) in the same coexistence of ions and blood proteins as in the in vivo.

Using each obtained sample and a zeta potential/particle size/molecular weight measuring device [manufactured by Malvern, trade name: Zetasizer nano ZS], the composite particles obtained in Example 1 and Comparative Example 1 included in the sample immediately after preparation and the composite particles obtained in Example 1 and the composite particles obtained in Comparative Example 1 contained in the sample after standing for 24 hours, were measured for particle size distribution. Using the obtained particle size distribution, by comparing the particle size distribution of the composite particles contained in the sample immediately after preparation and the particle size distribution of the composite particles contained in the sample after standing for 24 hours, the composite particles and blood The presence or absence of interactions with medium proteins was investigated.

FIG. 3A shows the particle size distribution of the composite particles obtained in Example 1 contained in the sample immediately after preparation, and the particle size distribution of the composite particles obtained in Example 1 contained in the sample after standing for 24 hours. is shown in FIG. 3(B). Note that FIG. 3 shows the results of measuring the particle size distribution three times using the same sample.

4(A) shows the particle size distribution of the composite particles obtained in Comparative Example 1 contained in the sample immediately after preparation, and the particles of the composite particles obtained in Comparative Example 1 contained in the sample after standing for 24 hours. The diameter distribution is shown in FIG. 4(B). Note that FIG. 4 shows the results of measuring the particle size distribution three times using the same sample.

From the results shown in FIG. 3, the particle size distribution of the composite particles obtained in Example 1 contained in each sample immediately after preparation shown in FIG. Since no significant shift in the absorption peak was observed between the particle size distribution of the composite particles obtained in Example 1 and contained in each sample after standing for 24 hours, it was confirmed that there was almost no change. Recognize. From these results, the composite particles obtained in Example 1 interacted with blood proteins even when maintained in the same coexistence of ions and blood proteins as in vivo at in vivo temperature (37°C). Turns out it doesn't work. In the composite particles obtained in Example 1, zirconium dioxide fine particles, ICG and DSPE-PEG are used in combination. Therefore, it can be seen that the combined use of zirconium dioxide fine particles, ICG, and DSPE-PEG in the composite particles effectively suppresses the interaction with blood proteins in vivo.

On the other hand, from the results shown in FIG. 4, the particle size distribution of the composite particles obtained in Comparative Example 1 contained in each sample immediately after preparation shown in FIG. ) shows a large shift in the absorption peak between the particle size distribution of the composite particles obtained in Comparative Example 1 and contained in each sample after standing for 24 hours. It is understood that From these results, the composite particles obtained in Comparative Example 1 interacted with blood proteins when maintained in the same coexistence of ions and blood proteins as in vivo at in vivo temperature (37°C). I know you do.

Test example 5
The composite particles (ICG equivalent: 0.9 pmol) obtained in Example 1 were added to 5 μL of PBS. A sample was obtained by stirring the resulting mixture at room temperature for 30 minutes. In addition, the composite particles (ICG equivalent: 0.9 pmol) obtained in Comparative Example 1 were added to 5 μL of PBS. A sample was obtained by stirring the resulting mixture at room temperature for 30 minutes.

In PBS, 0.18 pmol of BSA was attached to the surface of a cell composed of a crystal oscillator of a physical property change/intermolecular interaction quantitative QCM device [manufactured by Initium Co., Ltd., trade name: Affinix QN Pro]. Next, by measuring the change in the oscillation frequency of the crystal oscillator before and after contact between the sample and BSA adhering to the surface of the cell at 37° C., the type of composite particles contained in the sample and the composite The relationship between the type of particles and the change in oscillation frequency due to interaction with BSA was investigated.

FIG. 5 shows the relationship between the type of composite particles and the change ΔF ₂ in oscillation frequency. In the figure, lane 1 shows the change in oscillation frequency when the composite particles obtained in Example 1 are used, and lane 2 shows the change in oscillation frequency when the composite particles obtained in Comparative Example 1 are used.

From the results shown in FIG. 5, the change in the oscillation frequency based on the interaction between the composite particles obtained in Example 1 and BSA is the same as the oscillation frequency based on the interaction between the composite particles obtained in Comparative Example 1 and BSA. It can be seen that the change in frequency is 1/2 or less. This result shows that the interaction between the composite particles obtained in Example 1 and BSA is weaker than the interaction between the composite particles obtained in Comparative Example 1 and BSA. It is known that a substance that interacts weakly with BSA is difficult to be recognized as a foreign substance in vivo and is difficult to excrete from the liver. Therefore, compared with the composite particles obtained in Comparative Example 1, the composite particles obtained in Example 1 are less likely to be recognized as foreign substances in vivo and are less likely to be excreted from the liver.

Examples 2-4
In Example 1, instead of using DSPE-PEG, PEG chain-containing methacrylic acid-based polymer [weight average molecular weight: 10000 (Example 2)], PEG chain-containing acrylic acid-based polymer [weight average molecular weight: 35000 (Example 3) ] and a PEG-acrylic acid graft polymer [weight average molecular weight: 10000 (Example 4)] were used, and composite particles were obtained in the same manner as in Example 1. The zirconium dioxide fine particles used in the composite particles obtained in Examples 2 to 4 were secondary particles in which primary particles made of zirconium dioxide aggregated.

The hydrodynamic mean particle size and zeta potential of each of the composite particles obtained in Examples 2-4 were determined in the same manner as in Example 1. Table 1 shows the results.

From the results shown in Table 1, composite particles using PEG chain-containing methacrylic acid-based polymer, PEG chain-containing acrylic acid-based polymer, or PEG-acrylic acid graft polymer had a hydrodynamic average particle size of 200 nm or less. and has a zeta potential of -40 to -20 mV. Therefore, it can be seen that each composite particle obtained in Examples 2 to 4 has excellent dispersibility in an aqueous solvent.

In addition, the stability of each of the composite particles obtained in Examples 2 to 4 in the same coexistence of ions as in vivo at an in vivo temperature (37° C.) is examined in the same manner as in Test Example 2. The results show that the composite particles obtained in Examples 2 to 4 have the same stability as the composite particles obtained in Example 1. Furthermore, the presence or absence of interaction between each of the composite particles obtained in Examples 2 to 4 and blood proteins is examined in the same manner as in Test Example 3. From the results, each composite particle obtained in Examples 2 to 4 was maintained in the same coexistence of ions and blood proteins as in vivo at in vivo temperature (37 ° C.), even when it was maintained in the same way as in in vivo. It can be seen that, like the composite particles obtained in 1., they do not interact with blood proteins.

In the above, even when other group 4 metal oxide fine particles are used in place of the zirconium dioxide fine particles, the same tendency as the composite particles obtained in Examples 1 to 4 is observed. In the above, even when composite particles using other luminescent substances instead of ICG were used, the same tendency as the composite particles obtained in Examples 1 to 4 was observed.

These results show that the composite particles obtained in each example are excellent in dispersibility and stability in an aqueous solvent, and also excellent in stability under conditions similar to those in living bodies. In addition, the composite particles obtained in each example have a hydrodynamic average particle size of 30 to 300 nm, and thus it is understood that the EPR effect is easily exhibited. From this, it can be seen that the composite particles obtained in each example are superior to the composite particles obtained in Comparative Example 1 in the ability to accumulate in tumors in vivo. Therefore, the composite particles of the present invention have excellent dispersibility and stability in aqueous solvents, excellent stability under conditions similar to those in living bodies, and excellent accumulation in tumors in living bodies. I understand.

Example 5
The composite particles (ICG equivalent: 40 nmol) obtained in Example 1 were added to 100 μL of purified water. The resulting mixture was stirred at room temperature for 30 minutes to obtain the optical imaging agent.

Comparative example 2
The composite particles (ICG equivalent: 40 nmol) obtained in Comparative Example 1 were added to 100 μL of purified water. The resulting mixture was stirred at room temperature for 30 minutes to obtain the optical imaging agent.

Comparative example 3
40 nmol of ICG was dissolved in 100 μL of purified water to obtain an optical imaging agent.

Preparation example 1
Mouse colon cancer cell line colon-26 (5×10 ⁶ cells) was inoculated into the abdomen near the lower leg of Balb-c nu/nu nude mice. By breeding the Balb-c nu/nu nude mice after inoculation for 6 days, the mouse colon cancer cell line colon-26 was engrafted in the Balb-c nu/nu nude mice to obtain tumor-bearing mice. .

Test example 6
100 μL of the optical imaging agent obtained in Example 5 was administered to the tail vein of the tumor-bearing mouse obtained in Preparation Example 1. Using the tumor-bearing mouse and a fluorescence imaging device (manufactured by PerkinElmer, trade name: IVIS lumina), the tumor-bearing part of the tumor-bearing mouse was observed according to the in vivo fluorescence imaging method. In addition, the fluorescence intensity in the tumor-bearing portion (boxed portion in FIG. 6) of the tumor-bearing mouse and the fluorescence intensity in the normal tissue portion on the opposite side of the tumor-bearing portion were measured over time. Using the fluorescence intensity in the tumor-bearing part of the tumor-bearing mouse and the fluorescence intensity in the normal tissue part opposite to the tumor-bearing part, formula (III):
[Differential fluorescence intensity]
= [fluorescence intensity in tumor-bearing area] - [fluorescence intensity in normal tissue area] (III)
The differential fluorescence intensity was determined accordingly. The same procedure as described above, except that the optical imaging agent obtained in Comparative Example 2 or the optical imaging agent obtained in Comparative Example 3 was used instead of the optical imaging agent obtained in Example 5. was performed to determine the differential fluorescence intensity.

FIG. 6A shows the result of observing the tumor-bearing part of the tumor-bearing mouse when the optical imaging agent obtained in Example 5 was used. FIG. 6(B) shows the result of observing the tumor-bearing part of the mouse, and FIG. 6(C) shows the result of observing the tumor-bearing part of the tumor-bearing mouse when the optical imaging agent obtained in Comparative Example 3 was used. . In the figure, the encircled part indicates the observation target part in the tumor-bearing part of the tumor-bearing mouse.

From the results shown in FIG. 6, the epifluorescence of the observation target area when using the optical imaging agent obtained in Example 5 is different from that when using each optical imaging agent obtained in Comparative Examples 2 and 3. It can be seen that it is stronger than the epi-fluorescence of the observed area.

In addition, FIG. 7 shows the results of examining changes over time in fluorescence intensity (differential fluorescence intensity) in tumor-bearing regions of tumor-bearing mice. In the figure, black circles indicate changes over time in fluorescence intensity (differential fluorescence intensity) in tumor-bearing regions of tumor-bearing mice when the optical imaging agent obtained in Example 5 was used, and black triangles indicate changes over time obtained in Comparative Example 2. Changes over time in fluorescence intensity (differential fluorescence intensity) in the tumor-bearing part of the tumor-bearing mouse when the optical imaging agent was used. FIG. 10 shows temporal changes in fluorescence intensity (differential fluorescence intensity) in tumor-bearing regions.

From the results shown in FIG. 7, 48 hours after the end of administration of the optical imaging agent, fluorescence intensity (difference Fluorescence intensity) is higher than the fluorescence intensity (differential fluorescence intensity) in the tumor-bearing portion of the tumor-bearing mouse when each optical imaging agent obtained in Comparative Examples 2 and 3 is used. These results show that the optical imaging agent obtained in Example 5 is superior to the optical imaging agents obtained in Comparative Examples 2 and 3 in tumor accumulation.

Comparative example 4
(1) Production of Composite Particles While stirring 1 mL of a 5 M aqueous sodium hydroxide solution, 10 μL of the zirconium dioxide/methyl ethyl ketone dispersion obtained in Production Example 1 was added dropwise to the aqueous sodium hydroxide solution. After the dropwise addition was completed, the resulting mixture was stirred at room temperature for 30 minutes to obtain a dispersion liquid A. Next, using a centrifugal filter unit [manufactured by Merck Millipore, trade name: Amicon Ultra-4, molecular weight cut off: 3000] and ultrapure water, the solid content contained in the dispersion liquid A was centrifuged at 5000 x g. and for 30 minutes to obtain particles. The obtained particles were dispersed in 10 mL of ultrapure water to obtain dispersion B.

DSPE-PEG [manufactured by NOF Corporation, trade name: SUNBRIGHT DSPE-050CN] was added to give a DSPE-PEG aqueous solution at a concentration of 20 mg/mL. While stirring 10 mL of the dispersion B, 2 mL of the DSPE-PEG aqueous solution was added to the dispersion B to obtain a mixture. The resulting mixture was stirred at room temperature for 2 hours to obtain Dispersion E. Next, using a centrifugal filter unit [manufactured by Merck Millipore, trade name: Amicon Ultra-4, molecular weight cut off: 30000] and ultrapure water, the particles contained in the dispersion D are centrifuged and washed at 5000×g. Composite particles containing zirconium dioxide fine particles and DSPE-PEG were obtained by carrying out for 20 minutes.

(2) Production of Optical Imaging Agent The composite particles (ICG equivalent: 40 nmol) obtained in Comparative Example 4(1) were added to 100 μL of purified water. The resulting mixture was stirred at room temperature for 30 minutes to obtain the optical imaging agent.

Test example 7
100 μL of the optical imaging agent obtained in Example 5, 100 μL of the optical imaging agent obtained in Comparative Example 2, and the light obtained in Comparative Example 3 were added to the tail vein of each tumor-bearing mouse obtained in the same manner as in Preparation Example 1. 100 μL of the imaging agent and 100 μL of the optical imaging agent obtained in Comparative Example 4 were administered, respectively. Forty-eight hours after the end of administration of the optical imaging agent, the tumor-bearing mice were euthanized, and the tumor-containing organs were excised. Using the excised organs and a fluorescence imaging device (manufactured by PerkinElmer, trade name: IVIS lumina), the fluorescence intensity in the organs was measured according to the in vivo fluorescence imaging method.

FIG. 8 shows the results of examining the relationship between the type of optical imaging agent and the fluorescence intensity in an organ containing a tumor. In the figure, lane 1 is the fluorescence intensity in the organ containing the tumor when the optical imaging agent obtained in Example 5 is used, and lane 2 is the tumor when the optical imaging agent obtained in Comparative Example 2 is used. Fluorescence intensity in organs, Lane 3 is fluorescence intensity in organs containing tumors when the optical imaging agent obtained in Comparative Example 3 is used, Lane 4 is the tumor when the optical imaging agent obtained in Comparative Example 4 is used. Fluorescence intensity in organs containing

From the results shown in FIG. 8, the fluorescence intensity in the tumor-containing organ when the optical imaging agent obtained in Example 5 was used was the same as that when each optical imaging agent obtained in Comparative Examples 2 to 4 was used. It can be seen that the fluorescence intensity is larger than that in the tumor-containing organ. From these results, it can be seen that the optical imaging agent obtained in Example 5 is superior to the optical imaging agents obtained in Comparative Examples 2 to 4 in the accumulation in organs including tumors.

Note that even when the optical imaging agent containing the fine particles obtained in Examples 2 to 4 is used instead of the composite particles obtained in Example 1, the composite particles obtained in Example 1 are also contained. A similar trend is seen with the optical imaging agent.

In addition, even when an optical imaging agent containing composite particles in which other Group 4 metal oxide fine particles were used instead of the zirconium dioxide fine particles used in Example 1, the A similar trend is seen when using an optical imaging agent containing composite particles.

Furthermore, even when an optical imaging agent containing composite particles in which another luminescent substance is used instead of ICG used in Example 1 is used, the light containing the composite particles obtained in Example 1 A similar tendency is observed as in the case of using an imaging agent.

As described above, since the optical imaging agent of the present invention contains the composite particles of the present invention, it is excellent in dispersibility and stability in an aqueous solvent, and is stable under conditions similar to those in a living body. It can be seen that it is excellent in the cytotoxicity and the accumulation in the tumor in vivo. Therefore, the optical imaging agent of the present invention can be suitably used for, for example, bioimaging, diagnostic imaging, preferably diagnostic imaging of tumors.

Claims (1)

A method for producing composite particles for use in in vivo fluorescence imaging, wherein the composite particles containing Group 4 metal oxide fine particles, a luminescent material and a surfactant are produced by Forming an alkali-soluble surface treatment layer on the surface of the metal oxide fine particles, bringing the coated fine particles on which the surface treatment layer is formed into contact with an alkali metal hydroxide or an alkaline earth metal hydroxide , The method is characterized in that the coated fine particles brought into contact with an alkali metal hydroxide or an alkaline earth metal hydroxide are brought into contact with a luminescent material, and the coated fine particles brought into contact with the luminescent material are brought into contact with a surfactant . A method for producing composite particles .

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