WO2019111952A1 - Hydrophilized dye-containing particles, dye-containing particles, method for producing hydrophilized dye-containing particles, and method for in vivo observation - Google Patents

Abstract

An embodiment of the present invention relates to hydrophilized dye-containing particles, dye-containing particles, a method for producing hydrophilized dye-containing particles, and a method for in vivo observation. The hydrophilized dye-containing particles are for imaging, and contain a hydrophilic polymer and a fluorescent dye that emits fluorescent light having a wavelength within a range of 900 to 1700 nm.

Description

Hydrophilized dye-containing particles, dye-containing particles, method of producing hydrophilicized dye-containing particles and method of observing in vivo

One embodiment of the present invention relates to a hydrophilized dye-containing particle, a dye-containing particle, a method of producing the hydrophilized dye-containing particle, and a method of observing in vivo.

Fluorescence imaging is frequently used for microscopic observation of cells, tissue sections, etc., in vivo imaging, etc., and there are no restrictions on its simplicity, handling of radioactive materials, etc. Because it can be used, its clinical use is also expanding.

In fluorescence imaging, in order to clearly observe blood vessels, tissues, organs and the like in the deep part of the body, use a near-infrared region (a first biological window) having a wavelength of 700 to 899 nm, which is higher in biological permeability than visible light. In particular, clinical applications using indocyanine green, which emits fluorescence at a wavelength of 800 to 899 nm, are in progress. However, in this wavelength region, light scattering is still large, resulting in haze in the observed image. Therefore, in consideration of the light loss of the living tissue, it is desirable to use the near-infrared region (the window of the second living body) having a wavelength of 900 to 1700 nm in order to observe the deep part more clearly. A probe that emits fluorescence is desired.

Metal / inorganic nanomaterials such as semiconductor nanoparticles and carbon nanotubes are known as probes that emit fluorescence at the window of the second living body (Patent Document 1), but when these materials are used in the living body, Biotoxicity is a concern. Therefore, development of a probe using a fluorescent dye (organic molecule) that emits fluorescence at the window of the second living body has been performed. However, since these fluorescent dyes are generally insoluble in water and can not emit fluorescence in water, an approach of incorporating them in polymer micelles and the like has been attempted (Patent Document 2).

JP, 2014-178155, A US Patent Application Publication No. 2015/0056142

However, polymer micelles are self-assemblies utilizing hydrophobic interactions and ionic interactions, and are not rigid, so their ionic strength is high and various contaminants are present in vivo and in vitro, especially in body fluids. In living tissues, it has been difficult to maintain fluorescence intensity for a long time due to water intrusion into micelles, dye outflow, and micelle collapse.

One embodiment of the present invention provides a hydrophilized dye-containing particle for imaging capable of maintaining fluorescence intensity for a long time in body fluid, in living tissue, and the like.

As a result of intensive studies to solve the above problems, the present inventor has found that the problems can be solved according to the following configuration example, and the present invention has been completed.
The structural example of this invention is as follows.

<1> A hydrophilized dye-containing particle for imaging, comprising a hydrophilic polymer and a fluorescent dye that emits fluorescence having a wavelength in the range of 900 to 1700 nm.

<2> The hydrophilized dye-containing particle according to <1>, which comprises the base particle and the fluorescent dye.

The hydrophilized pigment | dye containing particle | grain as described in <1> or <2> which has <3> said hydrophilic polymer in at least one part of the surface.

<4> The hydrophilized dye-containing particle according to any one of <1> to <3>, wherein the fluorescent dye is a cationic compound.
<5> The hydrophilized dye-containing particle according to any one of <1> to <4>, wherein the fluorescent dye has a polymethine skeleton.

<6> a base particle and the fluorescent dye,
The hydrophilized dye-containing particle according to any one of <1> to <5>, wherein the base particle is an organic polymer particle.

<7> Base particles and the fluorescent dye,
The hydrophilized dye-containing particle according to any one of <1> to <6>, wherein the base particle is an organic polymer particle and contains a structural unit having an acidic group.
<8> The hydrophilized dye-containing particle according to <7>, wherein the content of the acidic group is 0.40 mmol or more per 1 g of the organic polymer particle.
The hydrophilized pigment | dye containing particle | grains as described in <7> or <8> whose structural unit which has <9> above-mentioned acidic group is 3 mass% or more with respect to 100 mass% of total structural units which comprise organic polymer particle.

<10> a base particle and the fluorescent dye,
The hydrophilized dye-containing particle according to any one of <1> to <9>, wherein a surface charge amount of the base particle is 0.05 to 6.0 mmol / g.

<11> a base particle and the fluorescent dye,
The hydrophilized dye-containing particle according to any one of <1> to <10>, wherein the base particle is an organic polymer particle and contains a hydrophobic structural unit.

<12> The hydrophilized dye-containing particle according to any one of <1> to <11>, which has a volume average particle size of 1 to 300 nm.

<13> The hydrophilized dye-containing particle according to any one of <1> to <12>, wherein the hydrophilic polymer is a polymer containing a polyalkylene glycol structure at least in part.

<14> The hydrophilized dye-containing particle according to any one of <1> to <13>, wherein the hydrophilic polymer is bound to at least a part of the particle via a covalent bond.

<15> The hydrophilized dye-containing particle according to any one of <1> to <14>, wherein the hydrophilic polymer has a polyalkylene glycol structure and at least one amine structure.

<16> The hydrophilized dye-containing particle according to any one of <1> to <15>, wherein the hydrophilic polymer has a structure derived from a polymer represented by Formula (8).

In the formula (8), n is an integer of 5 to 300. ]

<17> A pigment-containing particle comprising a base particle and the fluorescent dye, and a hydrophilic polymer on the surface of at least a part of the pigment-containing particle,
The hydrophilized dye-containing particle according to any one of <1> to <16>, wherein the density at which the hydrophilic polymer occupies the surface of the dye-containing particle is 0.01 string / nm ² or more.

<18> a base particle and the fluorescent dye,
The base particle is an organic polymer particle, and the amount of the fluorescent dye contained in the organic polymer particle is 0.01 to 30 mmol per 1 g of the organic polymer particle according to any one of <1> to <17> Hydrophilized dye-containing particles.

<19> an organic polymer particle and the fluorescent dye,
The hydrophilized dye-containing particle according to any one of <1> to <18>, wherein the amount of the fluorescent dye is 0.001 to 3.0 parts by mass with respect to 100 parts by mass of the organic polymer particles.

<20> Base particles and a fluorescent dye that emits fluorescence having a wavelength in the range of 900 to 1700 nm,
The base particle comprises a structural unit having an acidic group,
Dye-containing particles for imaging.
<21> A method of producing a hydrophilized dye-containing particle for imaging, which is the following production method I or production method II.
Production method I: Step 1 of obtaining a dye-containing particle comprising a base particle and a fluorescent dye that emits fluorescence having a wavelength in the range of 900 to 1700 nm, and
Contacting the dye-containing particles obtained in step 1 with a hydrophilic polymer to obtain hydrophilized dye-containing particles having the dye-containing particles and the hydrophilic polymer;
Production method II: contacting the base particle and the hydrophilic polymer, and
Contacting the particles obtained in step 3 with a fluorescent dye that emits fluorescence having a wavelength in the range of 900 to 1700 nm to obtain hydrophilized dye-containing particles 4;
<22> The manufacturing method according to <21>, which is the manufacturing method I.

<23> Process A of irradiating excitation light of the said fluorescent dye to the test substance which contains in a body the hydrophilized pigment containing particle | grains in any one of <1>-<19>, or the pigment | dye containing particle | grains as described in <20> ,and,
Detecting fluorescence in a wavelength range of 900 to 1700 nm emitted from the subject,
A method of observing in vivo, including

According to one embodiment of the present invention, it is possible to obtain a fluorescent polymer particle for imaging capable of maintaining the fluorescence intensity for a long time in a body fluid, a living tissue or the like.

FIG. 1 shows the ratio of the amount of fluorescent dye to the amount of organic polymer particles used in the production of dye-containing particles (the amount of fluorescent dye × 100 / the amount of organic polymer particles) (% by mass) and the fluorescence intensity of the obtained dye-containing particles FIG. FIG. 2 is a view showing the relationship between the content (mmol / g) of acidic groups per 1 g of base particles (organic polymer particles) and the fluorescence intensity of the obtained dye-containing particles. FIG. 3 shows the absorption spectrum of the dye-containing particle obtained in the synthesis example. FIG. 4 shows fluorescence pictures of mice observed in the in vivo imaging of the example.

Hereinafter, preferred embodiments according to the present invention will be described in detail. It is to be understood that the present invention is not limited to only the embodiments described below, but also includes various modifications implemented within the scope of the present invention. In the present specification, the descriptions of “A to B” and the like representing numerical ranges are synonymous with “A or more and B or less”, and include A and B within the numerical ranges.
Furthermore, in the present specification, “-(meth) acrylate” is a concept encompassing both “-acrylate” and “-methacrylate”. Similar descriptions have similar meanings.

«Hydrophilized dye-containing particles for imaging»
The hydrophilized dye-containing particles for imaging according to one embodiment of the present invention (hereinafter also referred to as "the hydrophilized particles") are a hydrophilic polymer and a fluorescent dye which emits fluorescence having a wavelength in the range of 900 to 1700 nm (hereinafter This fluorescent dye is also referred to as "fluorescent dye A".
In the present specification, a particle which is a particle and which is a part of the particle other than the fluorescent dye A and the hydrophilic polymer is referred to as a "base particle" and a particle containing the fluorescent dye A and the base particle is a "dye-containing particle". The particles having the fluorescent dye A and the hydrophilic polymer are referred to as "hydrophilized dye-containing particles". For example, in the following (Method 1-1) and (Method 1-3), the particles before contacting with the fluorescent dye A are base particles, and as in the following (Method 1-2), they are granulated. The particle formed using the fluorescent dye A is a dye-containing particle (a particle composed of a component other than the fluorescent dye A of the dye-containing particle is a base particle) containing the fluorescent dye A and the base particle.
Also, "particles" in the present specification do not include micelles and liposomes.

Such a hydrophilized particle can maintain fluorescence intensity for a long time in body fluid, in living tissue, etc. including in vitro and in vivo, has low biotoxicity, and is stable and dispersed in body fluid, living tissue, etc. It is excellent in sex, can emit fluorescence even in body fluids and living tissues, and has biocompatibility. In addition, according to one embodiment of the present invention, particles with high fluorescence intensity can be produced. The hydrophilized particles are preferably used in vivo.
Further, by using the present hydrophilized particles, noise due to auto-fluorescence of the living tissue can be suppressed, and blood vessels, tissues, organs and the like in the deep region can be observed more clearly.

It is more preferable that the present hydrophilized particles contain the fluorescent dye A and the base particles from the viewpoint of being able to easily obtain particles capable of emitting fluorescence even in body fluids or biological tissues, etc., with low biotoxicity. .
Further, the hydrophilic polymer is preferably present on at least a part of the surface of the present hydrophilized particle, from the viewpoint that the effect of the present hydrophilized particle is more exhibited, etc., and the fluorescent dye A and the base are preferably used. More preferably, they are present on the surface of at least a part of the dye-containing particles containing particles. When the dye-containing particles are brought into contact with a hydrophilic polymer to obtain the present hydrophilized particles having them, it can usually be said that the hydrophilic polymer is present on at least a part of the surface of the dye-containing particles.

«Dye-containing particles for imaging»
Dye-containing particles for imaging according to an embodiment of the present invention (hereinafter also referred to as "the present dye-containing particles") include the fluorescent dye A and base particles, and the base particles preferably have a structure having an acidic group. The dye-containing particles, which contain units, are preferably fluorescent particles with a wavelength in the range of 900-1700 nm.
Such pigment-containing particles are low in biotoxicity and can emit fluorescence even in body fluids or living tissues. The present dye-containing particles can also be particles with high fluorescence intensity. Also, by having a negative charge, the dispersibility of particles due to charge repulsion is improved. Furthermore, electrostatic repulsion with cells that are generally negatively charged can suppress nonspecific adsorption to cells present in the body and entry into cells.
Hereinafter, the present hydrophilized particles and the present dye-containing particles are collectively referred to as present particles.

It is preferable that a part of the wavelength of emitted light of the present particles is in the range of 900 to 1700 nm, and the permeability of the particles is high, so that deep blood vessels, tissues, organs, etc. can be observed more clearly. From the point of point of view, the range of 1000 to 1700 nm is preferable, the range of 1000 to 1500 nm is more preferable, and the range of 1000 to 1200 nm is particularly preferable.
In addition, in view of high biopermeability and clear observation of deep blood vessels, tissues, organs and the like, the present particle has a maximum of fluorescence emitted in the above wavelength range (peak peak of fluorescence) Is preferred.
The fluorescence can be measured by the method described in the examples.

When this particle is used for in vivo imaging, it needs to be excited within the range that penetrates the body, and is excited within a range shorter than the wavelength at which the fluorescence peak is relatively obtained. Specifically, the excitation wavelength is preferably 700 to 1700 nm, more preferably 750 to 1700 nm, and still more preferably 800 to 1200 nm.

The present particles are preferably particles for in vivo imaging, and are used for imaging lesions such as blood vessels, living tissues, organs, tumors and the like. As a result, it can be utilized for non-invasive imaging which monitors in vivo processes such as disease discovery and progression processes, effects of cell activities and drugs, disease progression, and healing status from outside the body. It can also be used for disease research, drug discovery, clinical trial development and the like.
The imaging may be multiple imaging.

<Fluorescent dye A>
The fluorescent dye A is not particularly limited as long as it is a dye that emits fluorescence having a wavelength in the range of 900 to 1700 nm. The fluorescent dye A is more preferably a fluorescent dye that can emit fluorescence such that the emission wavelength of the present particle is in the same wavelength range as the emission wavelength of the fluorescent dye A itself.
The fluorescent dye A used in the present particles may be two or more types, but is usually one type.

The fluorescent dye A may have a part of the wavelength of emitted fluorescence in the range of 900 to 1700 nm, but has high bio-permeability and is easy to observe blood vessels, tissues, organs, etc. in a deep region more clearly. It is preferable to be in the range of 1000 to 1700 nm, more preferable to be in the range of 1000 to 1500 nm, and particularly preferable to be in the range of 1000 to 1200 nm, from the viewpoint that it can be used.
In addition, in view of high biopermeability and the ability to clearly observe deep blood vessels, tissues, organs, etc., the fluorescent dye A is a maximum of fluorescence that emits in the wavelength range (peak peak of fluorescence) Is preferred.
The wavelength of the fluorescence emitted by the fluorescent dye A can be measured with a near infrared fluorescence spectrophotometer such as Fluorolog-NIR manufactured by Horiba, Ltd., or the like.

The fluorescent dye A is preferably a water-insoluble fluorescent dye.
Here, water-insoluble means that the solubility of the fluorescent dye in 100 g of water at 25 ° C. is less than 1 g.
When the solubility is within the above range, the present particles having a high content of the fluorescent dye A can be easily obtained, and even when the dye-containing particles and the hydrophilized dye-containing particles are dispersed in water, the fluorescent dye A is leached The hydrophilized dye-containing particles (dispersion liquid) and the dye-containing particles (dispersion liquid) can be easily obtained which are stable and have high fluorescence intensity.

The fluorescent dye A is cationic in that it can enhance the bonding with organic polymer particles, particularly organic polymer particles containing a structural unit having an acidic group, and can further suppress the leaching (dropping) of the fluorescent dye A into water. It is preferably a compound.
Examples of such cationic compounds include compounds containing thiopyrylium ion, benzo [cd] indolinium cation, and the like.

The fluorescent dye A is preferably a compound having a polymethine skeleton, more preferably a compound having a heterocyclic ring at both ends of the polymethine skeleton, as represented by the following formula (A), from the viewpoint of exhibiting high fluorescence intensity etc. Compounds are particularly preferred.

The R ¹ and R ² are each independently an organic group having 1 to 30 carbon atoms including a heterocyclic ring. The organic group is preferably a group in which a heterocycle is bonded to a carbon atom to which R ¹ and R ² are bonded, and a hetero atom constituting one heterocycle of R ¹ or R ² is positively charged. preferable. Examples of the heterocyclic ring include a thiopyran ring, a benzo [cd] indole ring and the like.
R ³ independently represents a halogen atom or a hydrocarbon group having 1 to 12 carbon atoms, preferably a halogen atom or a hydrocarbon group having 1 to 6 carbon atoms.
The n is an integer of 1 to 4, preferably 1 or 2.

As the fluorescent dye A, 4- [2- [2-chloro-3-[(2,6-diphenyl-4H-thiopyran-4-ylidene) ethylidene] -1-cyclohexen-1-yl] ethenyl] -2, 6-diphenylthiopyrylium tetrafluoroborate (IR-1061, manufactured by Sigma Aldrich), 1-butyl-2- [2- [3-[(1-butyl-6-chlorobenzo [cd] indole-2 (1H) -Ylidene) ethylidene] -2-chloro-1-cyclohexen-1-yl] ethenyl] -6-chlorobenzo [cd] indolium tetrafluoroborate (IR-1048, manufactured by Sigma Aldrich), 4- [2- [3 -[(2,6-Diphenyl-4H-thiopyran-4-ylidene) ethylidene] -2-phenyl-1-cyclohexen-1-yl] ethene [L] -2,6-diphenylthiopyrylium tetrafluoroborate (IR-1040, manufactured by Sigma Aldrich), 1-butyl-2- [2- [3-[(1-butyl-6-chlorobenzo [cd] indole -2 (1H) -ylidene) ethylidene] -2-chloro-5-methyl-1-cyclohexen-1-yl] ethenyl] -6-chlorobenzo [cd] indolium tetrafluoroborate (IR-1050, manufactured by Sigma-Aldrich Co.) Etc.).

The content of the fluorescent dye A contained in the present dye-containing particles is preferably 0.001 to 3.0 parts by mass, more preferably 0.01 to 1.0 parts by mass, further preferably 100 parts by mass of the base particles. Preferably, it is 0.05 to 0.5 parts by mass.
Further, the content of the fluorescent dye A contained in the present hydrophilized particles is preferably 0.0009 to 2.7% by mass, more preferably 0.009 to 0.9 based on 100% by mass of the present hydrophilized particles. % By mass, more preferably 0.049 to 0.49 mass%.
When the content of the fluorescent dye A increases, the fluorescence intensity of the present particles tends to increase. However, when the content of the fluorescent dye A becomes too large, the fluorescence intensity of the obtained particles saturates and decreases due to the quenching phenomenon. May. For this reason, it is preferable that the content of the fluorescent dye A is in the above-described range in consideration of the point of the fluorescence intensity, the point of the cost, and the like.

<Base particle>
The base particle is not particularly limited as long as it has components other than the fluorescent dye A and the following hydrophilic polymer, but is relatively hydrophobic, is swellable with an organic solvent, and facilitates the dye-containing particle containing the fluorescent dye A It is preferable that it is organic polymer particle from the point of being able to prepare to, and being able to suppress the leaching (dropping out) of fluorescent dye A easily. The base particles are preferably particles made of a polymer other than the following hydrophilic polymer, particularly particles made of a polymer which dissolves less than 1 g per 100 g of pure water at normal temperature (25 ° C.) and 1 atm.
The number of base particles used in the present particles may be two or more, but usually one.

As the organic polymer particles, known particles can be used and are not particularly limited. However, the polymerizable unsaturated aromatic compound, the polymerizable unsaturated carboxylic acid compound, the polymerizable unsaturated sulfonic acid compound or a salt thereof, the polymerizable carboxylic acid Obtained by (co) polymerization using at least one monomer selected from the group consisting of ester compounds, polymerizable unsaturated carboxylic acid amide compounds, polymerizable unsaturated nitrile compounds, halogenated vinyl compounds, and conjugated diene compounds Particles which are preferably selected from the group consisting of polymerizable unsaturated aromatic compounds, polymerizable unsaturated carboxylic acid compounds, polymerizable unsaturated sulfonic acid compounds or salts thereof, and polymerizable carboxylic acid ester compounds. More preferably, they are particles obtained by (co) polymerizing one kind of monomer.

Specific examples of the monomer include styrene, chlorostyrene, α-methylstyrene, divinylbenzene, vinyltoluene, vinylnaphthalene, divinylnaphthalene, α-naphthyl (meth) acrylate, β-naphthyl (meth) acrylate and the like. Unsaturated aromatic compounds; polymerizable unsaturated carboxylic acid compounds such as (meth) acrylic acid, itaconic acid, maleic acid and fumaric acid; polymerizable unsaturated sulfonic acid compounds such as sodium styrene sulfonate or salts thereof; ) Methyl acrylate, ethyl (meth) acrylate, n-butyl (meth) acrylate, 2-hydroxyethyl (meth) acrylate, glycidyl (meth) acrylate, ethylene glycol di (meth) acrylate, (meth) ) Acrylic acid tribromophenyl, vinyl acetate, etc. Phosphate ester compounds; and the like.

Also, examples of the polymerizable unsaturated carboxylic acid amide compound, polymerizable unsaturated nitrile compound, halogenated vinyl compound, conjugated diene compound, etc. include acrylonitrile, methacrylonitrile, acrolein, methacrolein, (meth) acrylamide, N-methylol ( Meta) acrylamide, methylene bis (meth) acrylamide, butadiene, isoprene, vinyl pyridine, N-vinyl pyrrolidone, vinyl chloride, vinylidene chloride, vinyl bromide and the like.

The organic polymer particles may be crosslinked. Such crosslinked particles can be made, for example, using crosslinking monomers. Examples of the crosslinking monomer include compounds having two or more polymerizable groups among the above-mentioned monomers, and specifically, polyfunctional polymerizable unsaturated aromatic compounds such as divinylbenzene and ethylene glycol di (meth) acrylate And polyfunctional polymerizable carboxylic acid ester compounds and the like.

The organic polymer particles are preferably particles containing a structural unit having an acidic group. By using a particle containing a structural unit having an acidic group, in particular, by using it with a fluorescent dye A which is a cationic compound, and further by using it together with a fluorescent dye A which is hydrophobic and which is a cationic compound, The present particles having high fluorescence intensity can be easily obtained.

As an acidic group, a carboxy group, a sulfo group, a phosphoric acid group, or these salts etc. are mentioned. Examples of the salt include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; ammonium salt; organic ammonium salt and the like. Among these, a carboxy group is preferable in that a compound containing an amino group can be easily covalently bonded, and in particular, a particle surface-modified with a compound containing an amino group can be easily obtained.
The organic polymer particle may have one acidic group in one molecule, and may have two or more.

The content of the acidic group is preferably 0.40 mmol or more, more preferably 0.70 mmol or more, and still more preferably 1.40 mmol or more per 1 g of the organic polymer particles.
When the content of the acidic group is in the above range, the present particles having higher fluorescence intensity can be easily obtained. The reason why the fluorescence intensity can be increased is not necessarily clear, but is considered to be because the fluorescent dye A can be dispersed uniformly in the present particles due to the presence of the acidic group, and thus the quenching of the fluorescent dye A can be further suppressed.

The content of the acidic group is preferably 7.0 mmol or less, more preferably 5.6 mmol or less, and still more preferably 4.0 mmol or less, per 1 g of the organic polymer particles.
When the content of the acidic group in the organic polymer particle is excessive, the hydrophilicity is increased and the particle shape tends to be unable to be maintained, so the above range is preferable.

The content of the acidic group can be calculated, for example, when using a monomer having an acidic group, from the amount of the monomer having an acidic group to the total monomers used in the synthesis of the organic polymer.

The content of the structural unit having an acidic group is preferably 3% by mass or more, more preferably 5% by mass or more, and still more preferably 10% by mass or more based on 100% by mass of all structural units constituting the organic polymer particles. .
When the content of the structural unit having an acidic group is in the above range, the present particles having higher fluorescence intensity can be easily obtained. The reason why the fluorescence intensity can be increased is not necessarily clear, but is considered to be because quenching of the fluorescent dye A can be suppressed because the fluorescent dye A can be uniformly dispersed in the dye-containing particle due to the presence of the acidic group.

Further, the content of the structural unit having an acidic group is preferably 50% by mass or less, more preferably 40% by mass or less, still more preferably 30% by mass or less, based on 100% by mass of all structural units constituting the organic polymer particles. It is.
When the content of the acidic group in the organic polymer particle is excessive, the hydrophilicity is increased and the particle shape tends to be unable to be maintained, so the above range is preferable.

The content of the structural unit having an acidic group can be calculated, for example, when using a monomer having an acidic group, from the amount of the monomer having an acidic group to the total monomers used in the synthesis of the organic polymer.

Organic polymer particles containing a structural unit having an acidic group may be produced using a monomer having an acidic group, and produced by modifying the obtained particles so as to have an acidic group by a conventionally known method. It is also good.

Examples of the monomer having a carboxy group include polymerizable unsaturated carboxylic acid compounds such as (meth) acrylic acid, fumaric acid, maleic acid and itaconic acid. As a monomer having a sulfo group, sulfo group-containing polymerizable unsaturated monomers such as ethylene sulfonic acid, allyl sulfonic acid, methallyl sulfonic acid, 2-sulfoethyl (meth) acrylate, 2-acrylamido-2-methylpropane sulfonic acid, etc. Can be mentioned. Examples of the monomer having a phosphoric acid group include phosphoric acid group-containing polymerizable unsaturated monomers such as 2- (meth) acryloyloxyethyl acid phosphate and 2- (meth) acryloyloxypropyl acid phosphate. These monomers may be used alone or in combination of two or more.
Among these, acrylic acid and itaconic acid are preferable in terms of ease of introduction of the acid group, reactivity, and the like.

The surface charge amount of the base particles is preferably 0.05 to 6.0 mmol / g, more preferably 0.20 to 6.0 mmol / g, from the viewpoint that the present particles having higher fluorescence intensity can be obtained. More preferably, it is 0.28 to 3.8 mmol / g, particularly preferably 0.60 to 2.4 mmol / g.
The surface charge amount in the present specification is a value calculated by determining the amount of sulfuric acid titrated using the tangent of the obtained conductivity curve when measured by the potentiometric titrator, and the surface charge per 1 g of particles is calculated. Represents the quantity. It can be measured using a Titrino series (manufactured by Metrome) potentiometric titrator.

The organic polymer particles can be easily incorporated with the fluorescent dye A, particularly the fluorescent dye A which is hydrophobic, and can easily suppress the leaching (dropping out) of the fluorescent dye A, and so on. It is preferable that it is a particle containing a hydrophobic structural unit.
Particles containing a hydrophobic structural unit are produced, for example, by using one or more of the above-mentioned polymerizable unsaturated aromatic compounds and polymerizable carboxylic acid ester compounds as monomers used for the synthesis of organic polymer particles. Can.

The content of the hydrophobic structural unit is an organic compound from the viewpoint that the fluorescent dye A, particularly the fluorescent dye A which is hydrophobic, can be incorporated more easily and the leaching (dropping out) of the fluorescent dye A can be more easily suppressed. The amount is preferably 50 to 97% by mass, more preferably 60 to 95% by mass, and still more preferably 70 to 90% by mass with respect to 100% by mass of all structural units constituting the polymer particles.
The content of the hydrophobic structural unit can be calculated, for example, from the amount of the hydrophobic monomer used relative to all the monomers used in the synthesis of the organic polymer.

A commercial item may be used for organic polymer particle, and what was synthesize | combined by the conventionally well-known method may be used for it.
Examples of methods for producing organic polymer particles include emulsion polymerization, seed polymerization, soap-free polymerization, suspension polymerization, and precipitation polymerization. Among these, the emulsion polymerization method is preferable from the viewpoint that nano-sized particles can be easily produced.

The volume average particle size of the base particles is not particularly limited, but is preferably 1 to 300 nm, more preferably 5 to 200 nm, still more preferably 10 to 100 nm, from the viewpoint of being suitably used by in vivo and in vitro imaging. It is.
If the volume average particle size is 100 nm or less, the accumulation of the present particles in the spleen, the liver and the like can be reduced, and imaging of a tumor by the EPR (Enhanced Permeability and Retention effect) can be easily performed. In addition, when the volume average particle size is 10 nm or more, the clearance by the kidney can be reduced, and the residence time in the body can be extended, so that long-term in-vivo imaging can be easily performed.
The volume average particle size can be measured by the method described in the examples.
In addition, even if the base particles contain a fluorescent dye, and the surface of the base particles has a hydrophilic polymer, the volume average particle size does not largely change from the volume average particle size of the base particles. Accordingly, the volume average particle size of the present particles is also preferably in the above range.

<Hydrophilic polymer>
The present hydrophilized particles are not particularly limited as long as they have a hydrophilic polymer, but it is preferable to have a hydrophilic polymer on the surface of the dye-containing particle.
Thus, by having a hydrophilic polymer, it has biocompatibility and is excellent in dispersibility in a body fluid or a living tissue, in vivo, in vitro, and further, fluorescence in vivo, in vitro for a long period of time Hydrophilized dye-containing particles capable of maintaining strength can be obtained.
Although the hydrophilic polymer as a raw material of the hydrophilized dye-containing particle, specifically, the hydrophilic polymer before being physically adsorbed or chemically bonded to the surface of the pigment-containing particle is described below, it is present on the surface of the pigment-containing particle The hydrophilic polymer is substantially the same as the polymer described below except that the terminal physically adsorbed or chemically bonded to the surface of the dye-containing particle may be different from the polymer described below.

In the present specification, a hydrophilic polymer means that it has a strong affinity to water. Specifically, a polymer which dissolves by 1 g or more in 100 g of pure water at normal temperature (25 ° C.) and 1 atmospheric pressure is referred to as a hydrophilic polymer.

The hydrophilic polymer generally has a hydrophilic repeating unit, may have a hydrophilic repeating unit in the main chain, and may have a hydrophilic repeating unit in a side chain. In the present specification, when the hydrophilic repeating unit is a homopolymer consisting of only one kind of repeating unit (having a number average molecular weight of about 1,000 to 100,000), pure water at normal temperature (25.degree. C.) and 1 atm. When 1 g or more is dissolved in 100 g, the repeating unit is said to be a hydrophilic repeating unit. The hydrophilic polymer is preferably physically adsorbed to the pigment-containing particles or chemically bonded, but it is chemically preferable in that it can further suppress leaching (dropping) from the hydrophilized pigment-containing particles. Preferably it is attached to Examples of chemical bonds include covalent bonds, but there is less risk of detachment from dye-containing particles, adhesion of biorelated substances such as proteins can be further suppressed, and dispersibility of hydrophilized dye-containing particles is further enhanced. Covalent bonding is preferred in that it can be used.

In order to obtain a hydrophilized dye-containing particle in which a hydrophilic polymer is chemically bonded to the dye-containing particle, a polymer having a functional group capable of covalently bonding with (the functional group of) the dye-containing particle surface is used as the hydrophilic polymer. Is preferred. As such a functional group, an amino group etc. are mentioned when the pigment | dye containing particle | grain surface has an acidic group, A carboxy group etc. are mentioned when particle surface has an amino group. As described above, since organic polymer particles having an acidic group are preferable from the viewpoint that hydrophilicized dye-containing particles having high fluorescence intensity can be easily obtained, etc., amino acids can be effectively used, etc. Hydrophilic polymers having a group are preferred.

Examples of the polymer having a hydrophilic repeating unit in the main chain include polymers having a polyalkylene glycol structure or a polyvinyl alcohol structure in the main chain. A polymer having a polyalkylene glycol structure is preferable, and a polymer having a polyethylene glycol structure is more preferable, from the viewpoint of further suppressing the adhesion of a biological substance such as a protein and further enhancing the dispersibility of the hydrophilized dye-containing particles.
In addition, one end of the polyalkylene glycol is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and the other end is a group capable of physically adsorbing on the surface of the dye-containing particle, or a functional group capable of chemically bonding Polymers which are a group having a are preferred.

The hydrophilic polymer is preferably a polymer having a polyalkylene glycol structure and at least one amine structure. The amine structure is not particularly limited, but an oligoamine structure is preferable, and a polyethyleneimine structure is more preferable.
As such a hydrophilic polymer, a polymer represented by the following formula (8) may be mentioned, and specifically, Blockmaster CE510 or CE210 manufactured by JSR Life Science Co., Ltd. can be suitably used.

In the formula (8), n is an integer of 5 to 300. ]

As a polymer having a hydrophilic repeating unit having a hydrophilic group in the side chain, it has a polyalkylene glycol structure in the side chain, and the end of the side chain is composed of a hydrogen atom or an alkyl group having 1 to 4 carbon atoms A polymer having a repeating unit (A-1), a polymer having a repeating unit (A-2) having an ammonio alkyl phosphate group at a side chain end, and a repeating unit (A-3) having an amide group at a side chain end Polymer, polymer having a repeating unit (A-4) having a hetero ring containing nitrogen and oxygen at the side chain end, polymer having a repeating unit (A-5) having a lactam at the side chain end, and It is preferable that it is one or more selected from polymers having a repeating unit (A-6) having a betaine group.

The polymer having any one or more of the repeating units (A-1) to (A-6) is further reacted with a functional group on the surface of the particle to immobilize the hydrophilic polymer on the surface of the dye-containing particle by covalent bonding. It is preferable to have a terminal structure that can be used and a repeating unit (B). As a repeating unit (B), the repeating unit which has a functional group which reacts with the carboxy group of pigment | dye containing particle | grain surface, for example, forms a covalent bond in a side chain is preferable. As a functional group which reacts with a carboxy group and forms a covalent bond, an amino group, a mercapto group, etc. are mentioned.

The hydrophilic polymer may be a naturally occurring hydrophilic polymer, or may be BSA used as a blocking agent, a protein such as casein, a saccharide such as chitosan, or the like, and a carboxy group on the surface of the base particle or the dye-containing particle The compound which can form a covalent bond by a condensing agent etc. is preferable.

As a monomer for deriving the repeating unit (A-1), polyethylene glycol (meth) acrylate, polypropylene glycol (meth) acrylate, polyethylene glycol polypropylene glycol (meth) acrylate, polyethylene glycol polytetramethylene glycol (meth) acrylate, polypropylene glycol Polytetramethylene glycol (meth) acrylate, methoxy polyethylene glycol (meth) acrylate, ethoxy polyethylene glycol (meth) acrylate and the like can be mentioned. Among these, polyethylene glycol (meth) acrylate and methoxy polyethylene glycol (meth) acrylate are preferable.
Examples of the repeating unit (A-1) include repeating units obtained by using these singly or in combination of two or more.

As a monomer to derive the repeating unit (A-2), 2- (meth) acryloyloxyethyl-2 '-(trimethylammonio) ethyl phosphate (2- (meth) acryloyloxyethyl phosphorylcholine), 3- (meth) Acryloyloxypropyl-2 '-(trimethylammonio) ethyl phosphate, 4- (meth) acryloyloxybutyl-2'-(trimethylammonio) ethyl phosphate, 2- (meth) acryloyloxyethoxyethyl-2 '-(trimethyl) Ammonio) ethyl phosphate, 2- (meth) acryloyloxydiethoxyethyl-2 '-(trimethylammonio) ethyl phosphate, 2- (meth) acryloyloxyethyl-2'-(triethylammonio) ethyl phosphate, 2- (Meth) acryl Ruokishiechiru -2 '- (tributyl ammonio) ethyl phosphate.
Examples of the repeating unit (A-2) include repeating units obtained by using these singly or in combination of two or more.

As a monomer which derives a repeating unit (A-3), dimethyl (meth) acrylamide, diethyl (meth) acrylamide, N- isopropyl (meth) acrylamide, N- (hydroxymethyl) (meth) acrylamide, N- (2-) And hydroxyethyl) (meth) acrylamide and the like.
Examples of the repeating unit (A-3) include repeating units obtained by using these singly or in combination of two or more.

As a monomer for deriving the repeating unit (A-4), 4- (meth) acryloyl morpholine and the like can be mentioned.
Examples of the repeating unit (A-4) include repeating units obtained by using the above-mentioned monomers alone or two or more kinds.

Examples of the monomer for deriving the repeating unit (A-5) include 1-vinyl-2-pyrrolidone, N-vinyl-ε-caprolactam and the like.
Examples of the repeating unit (A-5) include repeating units obtained by using these singly or in combination of two or more.

As a monomer to derive the repeating unit (A-6), N- (meth) acryloyloxyethyl-N, N-dimethylammonium-α-N-methylcarboxybetaine, N- (meth) acryloyloxyethyl-N, N And (meth) acrylate monomers such as dimethylammonium-α-N-propylsulfobetaine.
Examples of the repeating unit (A-6) include repeating units obtained by using these singly or in combination of two or more.

The number average molecular weight (Mn) of the hydrophilic polymer is preferably 500 to 100,000, more preferably 1,000 to 10,000.
When the number average molecular weight is in the above range, hydrophilized dye-containing particles having a large content of hydrophilic polymer can be easily obtained, and in particular, the modification amount of the hydrophilic polymer on the surface of the dye-containing particle can be further increased. As a result, it is possible to further suppress the adhesion of a biorelated substance such as a protein, and to easily obtain a hydrophilized dye-containing particle having higher dispersibility.
The number average molecular weight can be measured by gel permeation chromatography.

The density at which the hydrophilic polymer occupies the surface of the dye-containing particle can suppress the adhesion of biorelated substances such as proteins, and can easily obtain hydrophilic pigment-containing particles having higher dispersibility. Is preferably 0.01 line / nm ² or more, more preferably 0.05 line / nm ² or more, and still more preferably 0.1 line / nm ² or more, and the upper limit is preferably 2.0 lines / nm ² is there.
The density is calculated from the ratio of the hydrophilic polymer to the dye-containing particles, the particle size and the specific gravity of the dye-containing particles, and can be specifically measured by the method described in the examples.

«Method for producing hydrophilized dye-containing particles»
Examples of the method for producing the hydrophilized dye-containing particles (hereinafter also referred to as “the present production method”) include the following production method I or production method II.
Production method I: Step 1 of obtaining dye-containing particles containing base particles and the fluorescent dye A, and
Contacting the dye-containing particles obtained in step 1 with a hydrophilic polymer to obtain hydrophilized dye-containing particles having the dye-containing particles and the hydrophilic polymer;
A method of manufacturing comprising a method of manufacturing II: a step 3 of bringing a base particle into contact with a hydrophilic polymer, and
Contacting the particles obtained in step 3 with the fluorescent dye A to obtain hydrophilized dye-containing particles;
Among these methods, among these methods, the method I is more preferable because the fluorescent dye A can be efficiently introduced, and in particular, the content of the fluorescent dye A can be dramatically increased.

<Step 1>
The step 1 is not particularly limited as long as dye-containing particles can be obtained, (Method 1-1) A method of immobilizing the fluorescent dye A on the surface of the base particle by covalent bonding, (Method 1-2) in the presence of the fluorescent dye A A method of incorporating fluorescent dye A into the particles in the process of granulating and forming into particles, (Method 1-3) base particles, preferably using a liquid containing organic polymer particles and fluorescent dye A, fluorescent dye A May be incorporated into base particles, preferably organic polymer particles. That is, the dye-containing particle may be a particle having the fluorescent dye A on the surface of the base particle, or may be a particle having the fluorescent dye A inside the base particle, and the surface of the fluorescent dye A on the base particle And particles inside it.
Among these, the production method of (Method 1-3) is preferable in that the present particles of high fluorescence intensity can be easily produced.

Hereinafter, a preferable example of the process 1 is given.
First, organic polymer particles are prepared. Preferred compositions, particle sizes and the like are as described above. As the organic polymer particles, commercially available products may be used, or those synthesized by a conventionally known method such as the method described above may be used.

Next, the organic polymer particles are dispersed in an aqueous medium to prepare an aqueous dispersion. The aqueous dispersion may contain an additive such as a surfactant. Examples of the surfactant include nonionic surfactants. Among them, polymeric surfactants are preferable in that a dispersion having excellent particle dispersion stability can be easily obtained, and the like, and Pluronic F-127 and Kolliphor are preferable. P407 (manufactured by BASF) is more preferable.

A dye solution in which the fluorescent dye A is dissolved in an organic solvent is added to the obtained aqueous dispersion of organic polymer particles, and the dye is dyed while being stirred (staining step). When the dye solution is added to the aqueous dispersion of organic polymer particles, the organic polymer particles swell, and the dye present in the solvent is gradually absorbed into the organic polymer particles.

The organic solvent for dissolving the fluorescent dye A is not particularly limited, and may be either a nonpolar or polar organic solvent, but a polar solvent miscible with water in an arbitrary ratio is preferable. For example, alcohols such as methyl alcohol and ethyl alcohol; ethers such as tetrahydrofuran, 1,3-dioxolane and dioxane, ketones such as acetone, dimethyl sulfoxide, dimethylformamide, N-methylpyrrolidone and the like, and fluorescent dyes used Depending on the type of A etc., one type or two or more types are used. Among these, dimethyl sulfoxide is preferable because the solubility of the fluorescent dye A is high.

The concentration of the fluorescent dye A in the dye solution is preferably 0.001 to 5 g / L, more preferably 0.01 to 3 g / L, and still more preferably 0.01 to 1 g / L.

The concentration of the organic polymer particles (the concentration of the organic polymer particles in the total of the aqueous dispersion and the dye solution) in the dyeing step is preferably 0.01 to 10% by mass, more preferably 0.05 to 5% by mass. More preferably, it is 0.1 to 3% by mass.
By using organic polymer particles at the above concentration, dye-containing particles in which aggregation is suppressed can be easily obtained with high productivity.

The concentration of the surfactant (the concentration of the surfactant in the total of the aqueous dispersion and the dye solution) in the dyeing step is preferably 0.005 to 10% by mass, more preferably 0.01 to 5% by mass. More preferably, it is 0.05 to 1% by mass.
By using a surfactant at the above concentration, dye-containing particles in which aggregation is further suppressed can be easily obtained.

The concentration of the organic solvent (the concentration of the organic solvent in the total of the aqueous dispersion and the dye solution) in the dyeing step is preferably 0.1 to 50% by mass, more preferably 1 to 25% by mass, and still more preferably 2 to It is 10% by mass.
By using the organic solvent at the above concentration, the organic polymer particles can be sufficiently swollen, and the fluorescent dye A is more easily incorporated into the base particles, which is preferable, and leaching of the fluorescent dye A from the obtained particles is preferable. (Dropping off) is reduced, and aggregation and shape change due to state change of the present particles can be suppressed.

The amount of the fluorescent dye A added to the organic polymer particles in the dyeing step is preferably such that the content of the fluorescent dye A per 1 g of the organic polymer particles in the obtained present particles is 0.01 to 30 mmol, more preferably 0.1 to The amount is preferably 15 mmol, more preferably 0.5 to 7 mmol.
When the amount of the fluorescent dye A used is large, the fluorescence intensity of the obtained particles tends to increase, but when the amount of the fluorescent dye A used is too large, the fluorescence intensity of the obtained particles is saturated due to the quenching phenomenon.・ It tends to decrease. Therefore, in consideration of the point of the fluorescence intensity and the point of cost, it is preferable that the amount of the fluorescent dye A used is in the above range.

The temperature of the dyeing step is not particularly limited, but is preferably 0 to 50 ° C., more preferably 10 to 40 ° C., and still more preferably 20 to 30 ° C.
By setting the temperature in the above range, the fluorescent dye A can be efficiently contained in the organic polymer particles, and dye-containing particles in which aggregation is further suppressed can be easily obtained.

The time for which the fluorescent dye A is absorbed into the organic polymer particles (the time for stirring the aqueous dispersion of the organic polymer particles and the dye solution) is appropriately determined depending on the type of the fluorescent dye A used, the composition of the organic polymer particles, the particle size, etc. But preferably 1 to 60 minutes, more preferably 2 to 30 minutes, and still more preferably 3 to 10 minutes.

Step 1 may include the step of replacing the dispersion after the dyeing step with an aqueous dispersion. Methods for such replacement include centrifugation, dialysis, ultrafiltration and the like. Among these, ultrafiltration is preferable in that it can be easily replaced in a short time.

In Step 1, basically, a dye-containing particle containing almost all of the used fluorescent dye A can be obtained, and the amount of fluorescent dye used may be adjusted, but this production method can be obtained in the staining step. And the step of removing the free fluorescent dye A in the dispersion using a filter or the like.

<Step 2>
The step 2 is not particularly limited, and (Method 2-1) a method of causing a hydrophilic polymer to be physically adsorbed on a pigment-containing particle, particularly the particle surface by hydrophobic interaction or ionic interaction, (Method 2-2) hydrophilicity And the like. The functional group contained in the polymer and the functional group possessed by the surface of the dye-containing particle are covalently bonded to each other, and the hydrophilic polymer is covalently bonded to the dye-containing particle, particularly to the particle surface. Among these, hydrophilic polymer is difficult to be detached in vitro or in vivo, and it is possible to easily obtain a hydrophilized dye-containing particle which is excellent in dispersibility and can maintain high fluorescence intensity. The method of 2) is preferred.

Hereinafter, a preferable example of the process 2 is given.
The pigment-containing particles obtained in Step 1 are dispersed in an aqueous medium to prepare an aqueous dispersion. Alternatively, the aqueous dispersion of the dye-containing particles obtained in Step 1 may be used as it is. These aqueous dispersions may contain additives such as surfactants.

Next, a hydrophilic polymer aqueous solution and, if necessary, a condensing agent are added to the aqueous dispersion to modify the dye-containing particles, particularly the particle surfaces, with the hydrophilic polymer (modification step).

The condensing agent may be selected according to the functional group of the hydrophilic polymer and the dye-containing particle. For example, when the hydrophilic polymer contains an amino group and the dye-containing particle contains a carboxy group, a carbodiimide compound such as dicyclohexyl carbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, etc .; N, N ′ Imidazole compounds such as -carbonyldiimidazole; triazine compounds such as 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride n-hydrate; 1H Phosphonium compounds such as -benzotriazol-1-yloxy tris (dimethylamino) phosphonium hexafluorophosphate; O- (benzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium Uronium compounds such as hexafluorophosphoric acid can be used. Among these, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is preferable in view of easy handling and the like.

The concentration of the dye-containing particles in the modification step is preferably 0.05 to 10% by mass, more preferably 0.1 to 5% by mass, and still more preferably 0.3 to 3% by mass.
By using the pigment-containing particles at the above concentration, hydrophilized pigment-containing particles in which aggregation is suppressed can be easily obtained with high productivity.

The amount of the hydrophilic polymer added to 1 g of the pigment-containing particles in the modification step is preferably 0.001 to 10 g / g, more preferably 0.01 to 5.0 g / g, still more preferably 0.1 to 1.0 g It is / g.
When the addition amount of the hydrophilic polymer is in the above-mentioned range, it is possible to easily obtain a sufficiently surface-modified hydrophilized dye-containing particle at low cost.

The amount of the condensing agent added to 1 g of the pigment-containing particles in the surface modification step is preferably 0.0001 to 1 g / g, more preferably 0.001 to 0.5 g / g, still more preferably 0.01 to 0.3 g It is / g.
When the amount of the hydrophilic polymer added is in the above range, it is possible to easily obtain a hydrophilized dye-containing particle in which aggregation is suppressed and the surface is sufficiently modified with the hydrophilic polymer.

The modification step may be performed in a buffer solution. The buffer is not particularly limited as long as it does not inhibit the modification reaction. For example, when the hydrophilic polymer contains an amino group and the dye-containing particles contain a carboxy group, MES buffer of pH 4 to 7 is preferable .

<Step 3>
The step 3 is not particularly limited, and the base particle and the hydrophilic polymer may be brought into contact with each other. (Method 3-1) Hydrophilic polymer or hydrophilic polymer based particle by hydrophobic interaction or ionic interaction, particularly the particle Method of physically adsorbing to the surface, (Method 3-2) The functional group contained in the hydrophilic polymer is covalently bonded to the functional group possessed by the base particle surface, and the hydrophilic polymer is attached to the base particle, particularly to the particle surface. Methods of covalent bonding and the like can be mentioned. Among these, the production method of (Method 3-2) is preferable in that the hydrophilic polymer is difficult to be detached in vitro or in vivo, and particles having excellent dispersibility can be easily obtained.
As these (Method 3-1) and (Method 3-2), the same methods as those described in the above (Method 2-1) and (Method 2-2) but using a base particle instead of the dye-containing particle are listed. Be

<Step 4>
Step 4 is a step of bringing the particles obtained in step 3 into contact with the fluorescent dye A to obtain hydrophilized dye-containing particles.
The step 4 is not particularly limited as long as it is possible to obtain the hydrophilized dye-containing particles, but (Method 4-1) a method of immobilizing the fluorescent dye A on the particle surface obtained in the step 3 by covalent bonding, -3) Using a liquid containing the particles obtained in step 3 and fluorescent dye A, a method of incorporating fluorescent dye A into the particles obtained in step 3 and the like can be mentioned.
Among these, the production method of (Method 4-3) is preferable in that the present particles of high fluorescence intensity can be easily produced.
As these (Method 4-1) and (Method 4-3), the same method as in (Method 1-1) and (Method 1-3) above is used except that the particles obtained in Step 3 are used instead of the base particles. Method is mentioned.

«Method to observe in vivo»
The method of observing the inside of a living body (hereinafter also referred to as “the present observation method”) is
A step A of irradiating excitation light of the fluorescent dye A to an analyte containing the present hydrophilized particle or the present dye-containing particle in the body, and
Detecting fluorescence in a wavelength range of 900 to 1700 nm emitted from the subject,
including.

<Step A>
Specifically, the step A includes a step A1 of administering the present hydrophilized particle or the present dye-containing particle, preferably the present hydrophilized particle, and a step A2 of irradiating the analyte with the excitation light of the fluorescent dye A. And the steps involved.

<Step A1>
The administration method in step A1 may adopt any suitable means depending on the subject. For example, (a) oral administration, (b) parenteral administration can be mentioned.
These administrations may be local or topical administration, administration using a syringe, an infusion pump, a tube or the like, administration on the skin, transmucosal administration, inhalation administration, or buccal administration.

(A) For oral administration, the dispersion of the present particles may be directly orally administered, or a mixture containing the present particles and a pharmaceutically acceptable carrier and the like well known in the art may be administered. Good. The dosage form of the mixture is not particularly limited, and examples thereof include pills, dragees, capsules, solutions, gels, syrups, slurries and suspensions.

An excipient may be used when orally administering the particles. The excipient used preferably is not particularly limited, but, for example, sugars such as lactose, sucrose, mannitol and sorbitol, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, carboxy Cellulose preparations such as methylcellulose sodium, polyvinyl pyrrolidone (PVP).

In addition, a disintegrant may be used when orally administering the particles. The disintegrant is not particularly limited, and examples thereof include cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof (eg, sodium alginate).

The capsule may be a push-fit capsule made of gelatin, or a sealed soft capsule made of gelatin and a plasticizer (eg, glycerol or sorbitol).
The push-fit capsule may contain a filler (eg, lactose), a binder (eg, starch), a lubricant (eg, talc, magnesium stearate), a stabilizer and the like.
In the soft capsule, the particles may be dissolved or suspended in a suitable liquid such as fatty oil, liquid paraffin, liquid polyethylene glycol and the like.

The amount of the particles used for oral administration is preferably a dosage suitable for such administration.

(B) Parenteral administration includes administration other than oral, for example, rectal administration, vaginal administration, intraurethral administration, intraocular administration, intranasal administration or ear drops.

For parenteral administration, the particles may be in the form of a rectal composition, such as a suppository or retention enema. In this case, for example, conventional suppository bases (eg, cocoa butter, other glycerides) may be used.

Local and local administration includes, for example, direct injection in the kidney or heart region and depot transplantation.

Examples of administration using a syringe, infusion pump, etc. include subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, intradermal injection, intraorbital injection, intrathecal injection, intraspinal injection, intrasternal injection, etc. (Including infusion pump delivery). At the time of such administration, the present particles may be used as a suspension, if necessary, using an excipient and the like. Examples of suitable excipients include, but are not limited to, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, and cysteine hydrochloride. In addition, if desired, small amounts of nontoxic auxiliary substances, such as wetting agents, buffers, absorption enhancers and the like may be used.

For transmucosal administration, penetrants appropriate to the barrier to be permeated may be used.
For buccal administration, it may take the form of a tablet or lozenge containing the particles, formulated in a conventional manner.

For administration by inhalation, the particles can be loaded from a pressurized pack or a nebulizer with a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. May be coadministered with
When administered as a pressurized aerosol, the dosage may be controlled using a valve to release a metered amount.
In the case of administration using an inhaler, an inhaler or the like, a powder base (eg, powder (mixture) such as lactose and starch) may be used.

<Step A2>
The method for irradiating the subject with excitation light is not particularly limited, and irradiation may be performed from the outside of the subject using a light source, and the present particles can be made to emit light by emitting a luminescent substance administered to the subject separately from the present particles. Although irradiation may be performed, it is preferable to irradiate from the outside of the subject in terms of being able to adjust the intensity of excitation light.

The light source for irradiating excitation light from the outside of the subject is not particularly limited, and common excitation light sources such as various lasers (eg, ion laser, dye laser: semiconductor laser), halogen light source, xenon light source, etc. You may use.
The excitation light may, if desired, be illuminated only at the optimum excitation wavelength using various optical filters.

The wavelength of the excitation light is preferably 700 to 1700 nm, more preferably 750 to 1700 nm, and still more preferably 800 to 1200 nm, from the viewpoint of high biopermeability and clear observation of the deep part.

<Step B>
In the step B, there is no particular limitation as long as the fluorescence emitted from the subject, specifically, the fluorescence in the wavelength range of 900 to 1700 nm emitted from the present particles is detected. During this detection, various optical filters may be used to detect only light of the desired wavelength.

Although a detector for detecting fluorescence is not particularly limited, for example, a CCD camera can be used. More specifically, an InGaAs-CCD camera can be used. In addition, an optical CT apparatus, an endoscope, a fundus camera or the like may be used.

In the present observation method, after the fluorescence is detected, the detected fluorescence may be subjected to data processing as fluorescence information, and a recordable fluorescence image may be created based on this data.
Specifically, the fluorescence image may be created by irradiating excitation light to a wide region including a target tissue, detecting fluorescence with a CCD camera, and image-processing the obtained fluorescence information.

Step A2 and step B may be performed continuously in a dedicated device. Such an apparatus is not particularly limited, but SAI-1000 manufactured by Shimadzu Corporation can be suitably used.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited to these examples.

[Measurement of particle size]
The particle size (volume average particle size) of each particle is determined by using a dynamic light scattering nanotrack particle size analyzer (manufactured by Nikkiso Co., Ltd.), particle permeability: transmission, particle shape: spherical shape, particle refractive index: 1 It measured on condition of .59, particle density: 1.00, solvent: water, solvent refractive index: 1.333.

[Fluorescent measurement]
A quartz cell containing 2 mL of a 0.3% by mass particle dispersion was placed in a fiber-connected cuvette holder (Quantum North West, qpod 2e) managed at 25 ° C. Next, a 980 nm excitation light controlled by a precision LD driver with a temperature controller (manufactured by YAMAKI, KLD-6.5 ALT) is a laser diode (manufactured by LASER COMPONENTS.LTd., A high power fiber laser diode, SP-976-5-). The light was irradiated at 1015-7), and the fluorescence spectrum of each particle at a wavelength of 1000 to 1700 nm was measured using an optical fiber spectrometer (AVANTES, AvaSpec-NIR 256-1.7).

[Measurement of absorbance]
A quartz cell containing 2 mL of 0.3% by mass particle dispersion is placed in an ultraviolet-visible near-infrared spectrophotometer (V-770, manufactured by JASCO Corporation), and the absorption spectrum of each particle at a wavelength of 700 to 1300 nm is obtained. It was measured.

[Calculation of density at which the hydrophilic polymer occupies the surface of the dye-containing particle]
The hydrophilized dye-containing particles are dissolved in DMSO-d6, and NMR (manufactured by Bruker, AVANCEIIIHD, 700 MHz-cryo-DCH-NMR, nuclide: 13C (NS = 512, D1 = 15), temperature: 120 ° C.), organic The mass (α) of the hydrophilic polymer modified per 1 g of the hydrophilized dye-containing particle was determined from the integral ratio of the carbon peak derived from styrene of the polymer particle and the carbon peak derived from polyethylene glycol of the hydrophilic polymer.
Next, the number (β) of the hydrophilic polymer modified per 1 g of the hydrophilized dye-containing particles was determined from the number average molecular weight of the hydrophilic polymer. Next, the surface area (γ) per 1 g of the hydrophilized dye-containing particles is determined from the particle diameter and specific gravity of the hydrophilized dye-containing particles, and the hydrophilic polymer occupies the surface of the hydrophilized dye-containing particles by (β) / (γ). Density was calculated.

Synthesis Example 1-1
In a polymerization vessel, 330 parts by mass of water and 0.7 parts by mass of sodium dodecylbenzene sulfonate were charged, and the temperature was raised to 80 ° C. under a nitrogen atmosphere. After adding 0.3 parts by mass of potassium persulfate thereto, 80 parts by mass of water, 1.1 parts by mass of sodium dodecyl sulfate, 96 parts by mass of styrene, 3.8 parts by mass of acrylic acid and 0.2 parts by mass of itaconic acid Is added dropwise at 80.degree. C. over 2 hours, and then 0.1 parts by mass of potassium persulfate is added, followed by polymerization at 80.degree. C. for 3 hours to obtain organic polymer particles (A-1). Obtained. The surface charge of the resulting particles (A-1) was 0.24 mmol / g.

Synthesis Examples 1-2 to 1-6
The same procedure as in Synthesis Example 1-1 was repeated, except that the amounts of styrene, acrylic acid and itaconic acid were as listed in Table 1, to obtain organic polymer particles (A-2) to (A-6). .
In addition, "part" in Table 1 shows a "mass part."

Synthesis Example 2-1
In a 100 mL beaker, 50 mg of the organic polymer particles (A-1) are dispersed in 28.5 g of an aqueous solution in which 20 mg of Kolliphor P407 (manufactured by BASF Corporation) is dissolved, and 0.001 mass% of IR-1061 (4- [2 -[2-Chloro-3-[(2,6-diphenyl-4H-thiopyran-4-ylidene) ethylidene] -1-cyclohexen-1-yl] ethenyl] -2,6-diphenylthiopyrylium tetrafluoroborate Then, 1.5 mL of a dimethylsulfoxide solution of Sigma Aldrich) was added, and the mixture was stirred at room temperature for 5 minutes with a magnetic stirrer (manufactured by As One Corp., HS-30D, 100 rpm). After stirring, the dispersion medium was replaced with water by ultrafiltration (Amicon Ultra, 100,000 NMWL) to obtain dye-containing particles (B-1).

Synthesis Examples 2-2 to 2-6
The same procedure as in Synthesis Example 2-1 was repeated, except that the concentration of IR-1061 was changed as described in Table 2, to obtain Dye-containing particles (B-2) to (B-6).
The value of “IR-1061 / organic polymer particles [mass%]” in Table 2 is a value calculated from the preparation amount.

The fluorescence of the dye-containing particles (B-1) to (B-6) was measured, and the results of the fluorescence intensity at a wavelength of 1096 nm are shown in Table 2.
In addition, the fluorescence measurement of the dye-containing particles (B-1) to (B-6) is performed, and the mass ratio of the fluorescent dye A contained in the organic polymer particles (IR-1061 / organic polymer particles [mass%]) A semi-logarithmic graph was created with the axis (log scale) and the fluorescence intensity at a wavelength of 1096 nm as the vertical axis. The results are shown in FIG.

Synthesis Example 2-8 to 2-12
The same procedure as in Synthesis Example 2-4 was followed, except that particles (A-2) to (A-6) were used, to obtain dye-containing particles (B-8) to (B-12).

The fluorescence of the dye-containing particles (B-4) and (B-8) to (B-12) was measured, and the results of the fluorescence intensity at a wavelength of 1096 nm are shown in Table 3.
The fluorescence measurement of the pigment-containing particles (B-4) and (B-8) to (B-12) was carried out, and the content (mmol / g) of the acidic group per 1 g of the organic polymer particles (base particles) The fluorescence intensity at a wavelength of 1096 nm was plotted as the vertical axis. The results are shown in FIG.
In addition, content of the acidic group per 1 g of organic polymer particle (base particle) was computed from the usage-amount of the monomer which has an acidic group with respect to all the monomers used for the synthesis | combination of organic polymer particle. Specifically, the content (mmol / g) of the acidic group per 1 g of the organic polymer particles (A-1) can be determined by (3.8 / 72 + 2 × 0.2 / 130) / 100 × 1000. . Here, 3.8 / 72 represents the mass of the acidic group derived from acrylic acid when the total monomer is 100 g, and 2 × 0.2 / 130 represents the mass of the acidic group derived from itaconic acid.

The absorbances of the dye-containing particles (B-4) and (B-8) to (B-12) were measured. The results are shown in FIG.

It can be seen from FIGS. 2 and 3 that the fluorescence intensity and absorption characteristics change according to the proportion of the acidic groups of the organic polymer particles. From this, it is inferred that the acidic group of the organic polymer particle changes the state of the fluorescent dye A contained in the particle. The inventor infers that the reason for these changes is that the fluorescent dye A, which is cationic, can be uniformly distributed in the particles due to the presence of the acidic group.
It is believed that by changing the type of organic polymer particles used, the type of pigment and the amount of pigment, it is possible to obtain pigment-containing particles that emit stronger fluorescence.

Example 1
3 mL of an aqueous dispersion containing 150 mg of dye-containing particles (B-4) in a reaction vessel, and an aqueous solution of polyethylene glycol having an amino group at one end of 2.0 mass% (BSRMaster CE510, manufactured by JSR Life Science Co., Ltd.) MES buffer solution (0.1 M, pH 5.0) of 3.75 mL and 1.0% by mass of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Dojin Chemical Laboratory Co., Ltd.) ) 1.5 mL was added and stirred at room temperature for 2 hours.
Thereafter, the excess raw material was removed by dialysis, and the solvent was replaced with water to obtain hydrophilized dye-containing particles (C-1) having polyethylene glycol chains on the surface. The particle size was 70 nm, and the density at which the hydrophilic polymer occupied the surface of the dye-containing particles was 0.20 / nm ² .

Example 2
The same procedure as in Example 1 was carried out except using the dye-containing particles (B-9) to obtain hydrophilized dye-containing particles (C-2). The particle size was 82 nm, and the density at which the hydrophilic polymer occupied the surface of the dye-containing particle was 0.20 / nm ² .

[Example 3]
A hydrophilic dye-containing particle (C-3) was obtained by the same operation as in Example 1 except that polyethylene glycol (Blockmaster CE210, manufactured by JSR Life Sciences Ltd.) was used instead of Blockmaster CE510. . The particle diameter was 67 nm, and the density at which the hydrophilic polymer occupied the surface of the dye-containing particles was 0.29 / nm ² .

Example 4
The same operation as in Example 2 was carried out except using block master CE210 instead of block master CE510, to obtain hydrophilized dye-containing particles (C-4). The particle size was 79 nm, and the density at which the hydrophilic polymer occupied the surface of the dye-containing particles was 0.29 / nm ² .

Comparative Example 1
While stirring a mixed solution of PEG-b-PCL (manufactured by Montreal) in acetonitrile (0.4 mM, 1 mL) and IR-1061 in acetonitrile (130 mM, 1100 μL), 1 mL of water was added. Evaporation of acetonitrile gave a polymer micelle with near infrared fluorescence.

[Test Example 1]
10 μL of the aqueous dispersion containing 1 mg of the particles obtained in the example is placed in a 2 mL eppendorf tube, and 200 μL of human plasma (Cordin Bio Co., Ltd., normal human plasma pool, EDTA) is placed in each tube, 10 After vortexing for a second, centrifugation (2000 g, 10 seconds) was performed. The case where precipitation of particles was not confirmed visually was evaluated as ○, and the case where precipitation of particles was confirmed as x. The results are shown in Table 4.

[Test Example 2]
Dispersion immediately after dispersing 6 mg of particles or polymer micelles obtained in Examples or Comparative Examples in 2 mL of human plasma (Korjin Bio Co., Ltd., normal human plasma, pool, EDTA) and dispersion after standing for 3 days The fluorescence at a wavelength of 1096 nm, which was emitted by irradiating the liquid with excitation light, was measured, and the maintenance rate of the fluorescence intensity (fluorescence intensity after 3 days / fluorescence intensity immediately after dispersion [%]) was calculated. The results are shown in Table 4.
In addition, it can be said that the hydrophilized dye-containing particle having a high maintenance rate of the fluorescence intensity in this Test Example 2 can maintain the fluorescence intensity for a long time similarly in in vivo.

[Test Example 3]
200 μL of an aqueous dispersion containing the particles obtained in the example of 6.0% by mass was injected by tail vein injection into a mouse, and SAI-1000 (Filter: 1050 nm long pass filter) manufactured by Shimadzu Corporation. 5 minutes after administration, in vivo imaging was performed. The case where blood vessels and organs were clearly observed was evaluated as ○, the case where very clearly observed was ◎, and the case where particle aggregates were not observed but not clearly observed was evaluated as x. The results are shown in Table 4 and FIG.

Claims (23)

1. Hydrophilized dye-containing particles for imaging, comprising a hydrophilic polymer and a fluorescent dye that emits fluorescence having a wavelength in the range of 900 to 1700 nm.
2. The hydrophilized dye-containing particle according to claim 1, comprising a base particle and the fluorescent dye.
3. The hydrophilized dye-containing particle according to claim 1, wherein the hydrophilic polymer is on at least a part of the surface.
4. The hydrophilized dye-containing particle according to any one of claims 1 to 3, wherein the fluorescent dye is a cationic compound.
5. The hydrophilized dye-containing particle according to any one of claims 1 to 4, wherein the fluorescent dye has a polymethine skeleton.
6. A base particle and the fluorescent dye,
   The hydrophilized dye-containing particle according to any one of claims 1 to 5, wherein the base particle is an organic polymer particle.
7. A base particle and the fluorescent dye,
   The hydrophilized dye-containing particle according to any one of claims 1 to 6, wherein the base particle is an organic polymer particle and contains a structural unit having an acidic group.
8. The hydrophilized dye-containing particle according to claim 7, wherein the content of the acidic group is 0.40 mmol or more per 1 g of the organic polymer particle.
9. The hydrophilized dye-containing particle according to claim 7 or 8, wherein the structural unit having an acidic group is 3% by mass or more based on 100% by mass of all structural units constituting the organic polymer particle.
10. A base particle and the fluorescent dye,
    The hydrophilized dye-containing particle according to any one of claims 1 to 9, wherein a surface charge amount of the base particle is 0.05 to 6.0 mmol / g.
11. A base particle and the fluorescent dye,
    The hydrophilized dye-containing particle according to any one of claims 1 to 10, wherein the base particle is an organic polymer particle and contains a hydrophobic structural unit.
12. The hydrophilized dye-containing particle according to any one of claims 1 to 11, which has a volume average particle size of 1 to 300 nm.
13. The hydrophilized dye-containing particle according to any one of claims 1 to 12, wherein the hydrophilic polymer is a polymer containing a polyalkylene glycol structure at least in part.
14. The hydrophilized dye-containing particle according to any one of claims 1 to 13, wherein the hydrophilic polymer is bound to at least a part of the particle via a covalent bond.
15. The hydrophilized dye-containing particle according to any one of claims 1 to 14, wherein the hydrophilic polymer has a polyalkylene glycol structure and at least one amine structure.
16. The hydrophilized dye-containing particle according to any one of claims 1 to 15, wherein the hydrophilic polymer has a structure derived from a polymer represented by formula (8).
    

    

    In the formula (8), n is an integer of 5 to 300. ]
17. A base particle and a dye-containing particle comprising the fluorescent dye, and a hydrophilic polymer on the surface of at least a part of the dye-containing particle,
    The hydrophilized dye-containing particle according to any one of claims 1 to 16, wherein the density at which the hydrophilic polymer occupies the surface of the dye-containing particle is 0.01 string / nm ² or more.
18. A base particle and the fluorescent dye,
    The base particle is an organic polymer particle, The amount of the fluorescent dye contained in the organic polymer particle is 0.01 to 30 mmol per 1 g of the organic polymer particle according to any one of claims 1 to 17. Hydrophilized dye-containing particles.
19. An organic polymer particle and the fluorescent dye,
    The hydrophilized dye-containing particle according to any one of claims 1 to 18, wherein the amount of the fluorescent dye relative to 100 parts by weight of the organic polymer particles is 0.001 to 3.0 parts by weight.
20. A base particle and a fluorescent dye that emits fluorescence with a wavelength in the range of 900-1700 nm;
    The base particle comprises a structural unit having an acidic group,
    Dye-containing particles for imaging.
21. Method of producing hydrophilized dye-containing particles for imaging which is the following production method I or production method II:
    Obtaining a dye-containing particle comprising a base particle and a fluorescent dye that emits fluorescence having a wavelength in the range of 900 to 1700 nm;
    Contacting the dye-containing particles obtained in step 1 with a hydrophilic polymer to obtain hydrophilized dye-containing particles having the dye-containing particles and the hydrophilic polymer;
    Manufacturing method I,
    Contacting the base particle with the hydrophilic polymer;
    Contacting the particles obtained in step 3 with a fluorescent dye that emits fluorescence having a wavelength in the range of 900 to 1700 nm to obtain hydrophilized dye-containing particles 4;
    Manufacturing method II.
22. The production method according to claim 21, which is the production method I.
23. 20. A step A of irradiating the fluorescent dye with excitation light to an analyte containing the hydrophilized dye-containing particle according to any one of claims 1 to 19 or the dye-containing particle according to claim 20 in the body,
    Detecting fluorescence in a wavelength range of 900 to 1700 nm emitted from the subject,
    A method of observing in vivo, including

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