WO2019088266A1 - Aggregated nanoparticles and fluorescent labeling material - Google Patents

Abstract

The purpose of the present invention is to provide aggregated nanoparticles and a fluorescent labeling material that are of high brightness and exhibit superior light resistance. The aggregated nanoparticles according to the present invention are formed through aggregation of aggregation-induced luminescent molecules, and have a hydrophilic group disposed on the surface thereof, and exhibit a particle-size variation coefficient of at most 30%.

Description

Aggregated nanoparticles and fluorescent labeling material

The present invention relates to aggregated nanoparticles and fluorescent labels.

In recent years, molecular imaging technology has attracted high attention in the clinical field and basic research. Molecular imaging is a technology that visualizes the movement of molecules in the living body that could not be visualized so far, and for example, analysis of biomolecules at the molecular level, research on the dynamics of viruses and bacteria that cause disease, drugs It is widely used for various purposes, such as the evaluation of the action of the compound on the living body. In particular, due to its excellent detection sensitivity and operability, fluorescent imaging performed using a fluorescent substance is widely used to detect a trace substance in a living body.

In diagnosis and research using fluorescence imaging, a method has been proposed in which a fluorescent substance is bound to a biological substance to be detected as a labeling reagent and the fluorescence of the labeling reagent is detected with high sensitivity by irradiating a predetermined excitation light. ing. With the fluorescence signal obtained by such fluorescence imaging, quantification of biomolecular interactions, long-term dynamic observation of biomolecules, ultra-high sensitivity observation, etc. There is a need for a fluorescent labeling material that combines two properties.

The fluorescent labeling materials conventionally used include, for example, commercially available organic fluorescent dyes, etc., but even if the quantum yield is high, the brightness per molecule bound to the target molecule is low, and the use of At the time, due to the aggregation of the dye molecules, the functions such as the light emission efficiency, the color forming property, the photosensitivity and the photosensitivity are remarkably reduced, and there is a disadvantage that the inherent properties of the fluorescent dye are limited.

Still another fluorescent labeling material is a quantum dot which is a nanoparticle having a high quantum yield and a high light resistance (Patent Document 1). However, the composition of a quantum dot having a relatively high quantum yield has a problem that it can not be used for living cells or living organisms because it is a composition containing Cd having high biotoxicity. In addition, since the quantum dots are unstable in fluorescence, such as causing an unpredictable flicker phenomenon, and are particles with relatively large specific gravities, they interfere with the dynamics of labeled biological substances and interactions with other substances. For example, there is a drawback that accurate observation and quantification of biomolecules are difficult.

In order to solve these problems, recently, aggregated nanoparticles in which aggregation-induced luminescent molecules are aggregated have been developed (Patent Document 2). The aggregated nanoparticles have the advantages of higher brightness than conventional fluorescent labeling materials and lower cytotoxicity. Generation of high brightness of aggregation-induced luminescent molecules has been described in various ways, but the fine particles of aggregation-induced luminescent molecules are packed at a high density to suppress rotation of the partial structure of dye molecules, conversion to vibration, thermal energy, etc. The mechanism by which excitation light energy is effectively used for the light emission path and the quantum yield is improved, and the quantum yield is improved because packing is performed in such a configuration that regular layer stacks of molecules do not emit light. Mechanism etc. are considered.

JP 2011-530187 gazette US Patent Application Publication No. 2013/089889

However, when the inventors conducted additional experiments on the aggregated nanoparticles described in Patent Document 2 mentioned above, unevenness in brightness occurs at the time of dyeing, and the brightness and light resistance after dyeing when used after storage for a certain period of time The problem was low. In order to clarify the cause of this, when the present inventors further tried, the particle size distribution due to particle aggregation starts to spread with the lapse of time after producing the above-mentioned aggregated nanoparticles, and the particle diameter variation coefficient is It was found that it exceeded 60%, and it was also found that the increase in particle size distribution due to the aggregation was correlated with the decrease in light resistance.

As a result of intensive investigations by the inventors of the present invention, it was found that the increase in particle size distribution with time was remarkable as the particle size variation coefficient at the time of production was larger. Furthermore, the aggregation nanoparticles are likely to cause secondary aggregation due to interaction because the surface is hydrophobic, and as a result, excitation light absorption of aggregation-induced luminescent molecules located near the aggregation centers of the nanoparticles is insufficient. It was considered that sufficient brightness could not be obtained due to the fact that the fluorescence caused by the aggregation-induced luminescent molecule was not detected due to multiple scattering of light emission inside the nanoparticle aggregate. In addition, the secondary aggregation of the nanoparticles causes the absorption of excitation light to exhibit anisotropy, so that the fluorescence intensity is not uniform (brightness unevenness occurs), and the aggregation-induced luminescence of the particle surface adjacent to each other due to aggregation. Interaction between molecules causes repositioning to generate voids and the like on the surface of the nanoparticles, and oxygen molecules, which become a deterioration factor of aggregation-induced luminescent molecules, are more likely to act, which results in deterioration of light resistance. I thought it was the cause.

The present invention relates to aggregated nanoparticles that are brighter and have higher light resistance than fluorescent labeling materials conventionally used.

The inventors of the present invention conducted intensive studies to solve the above problems, and as a result, made the surface of the aggregation nanoparticles, which are nanoparticles of aggregation-induced light emitting molecules, hydrophilic, and the particle size variation coefficient of the aggregation nanoparticles. By making it 30% or less, the above problems are solved and the present invention is completed.

That is, the present invention provides, for example, the following aggregated nanoparticles and fluorescent labeling material.
[Item 1]
Aggregated nanoparticles formed by the aggregation of aggregation-induced luminescent molecules,
The aggregated nanoparticles have a hydrophilic group on the surface of the particles,
Aggregated nanoparticles, wherein the particle size variation coefficient of the aggregated nanoparticles is 30% or less.
[Section 2]
The aggregated nanoparticles, wherein the hydrophilic group has one / nm ² or more on the particle surface, agglomeration nanoparticles according to claim 1.
[Section 3]
The aggregated nanoparticle according to item 1 or 2, wherein an average particle diameter of the aggregated nanoparticle is 1 nm or more and 100 nm or less.
[Section 4]
The aggregated nanoparticle according to any one of Items 1 to 3, wherein the aggregation-induced luminescent molecule does not have a carbon / carbon double bond that does not contribute to the formation of a ring structure.
[Section 5]
The aggregated nanoparticle according to any one of Items 1 to 4, which has a central nucleus.
[Section 6]
A fluorescent labeling material comprising the aggregated nanoparticle according to any one of Items 1 to 5, wherein the fluorescent labeling material has a targeting ligand on the surface of the aggregated nanoparticle.
[Section 7]
7. The fluorescence according to item 6, wherein the targeting ligand is one or more molecules selected from the group consisting of an antibody, a protein having a binding property with a sugar chain, an organelle affinity substance, and a peptide. Signs.
[Section 8]
Item 8. A fluorescent label dispersion comprising the fluorescent label according to item 6 or 7 and a buffer solution.
[Section 9]
5. The method for producing aggregated nanoparticles according to any one of Items 1 to 4, comprising the step (A) of bringing a solution of aggregation-induced luminescent molecules into contact with a poor solvent to aggregate the aggregation-induced luminescent molecules.
[Section 10]
10. The aggregated nanoparticle according to item 9, wherein the step (A) is a step of bringing a solution of the aggregation-induced luminescent molecule into contact with a poor solvent in the presence of a central nucleus to aggregate the aggregation-induced luminescent molecule. Manufacturing method.
[Item 11]
A method for producing a fluorescent labeling material, comprising the step of binding a targeting ligand to the aggregated nanoparticle according to any one of Items 1 to 5.

The “aggregated nanoparticles” of the present invention have a higher quantum yield and higher luminance than conventional nanoparticles containing aggregation-induced luminescent molecules, and the above-described decrease in luminance due to secondary aggregation and aggregation-induced luminescent molecules By suppressing the deterioration due to the repositioning, the light resistance over time is dramatically improved.

In addition, since a strong and uniform fluorescence signal can be stably obtained by using the fluorescent labeling material containing the aggregated nanoparticles of the present invention for labeling of biomolecules, quantification of biological substances, dynamic observation over a long period, And, it becomes possible to perform ultra-sensitive observation. Furthermore, since the aggregated nanoparticles and the fluorescent labeling material of the present invention have relatively small particle sizes and specific gravities, they are less likely to interfere with the interaction between the labeled biological material and other materials, so the interaction between biological materials can be quantified. It will also be possible to evaluate.

The “aggregated nanoparticles” of the present invention are particles formed by the aggregation of aggregation-induced light emitting molecules, and the surface of the particles has a hydrophilic group and has a particle size variation coefficient of 30% or less. It is characterized by
<Aggregation-induced luminescent molecule>
The aggregation-induced luminescent molecules used in the present invention do not emit fluorescence because the quantum yield is low when each molecule is dissolved or dispersed without aggregation in a dilute solution, or the fluorescence emission intensity is weak. Although it is a substance, it is a fluorescent substance having the property of increasing the quantum yield by aggregating to form an aggregate, emitting strong fluorescence, or increasing the fluorescence intensity. As aggregation-induced light-emitting molecules, known aggregation-induced light-emitting molecules and the like can be used without particular limitations, but usually, molecules having at least one of a hydrocarbon-based aromatic ring and a heteroaromatic ring are used. Specifically, for example, maleimide-based aggregation-induced luminescent molecules, aminobenzopyranoxanthene (ABPX) -based aggregation-induced luminescent molecules, benzofuroxazolo-carbazole-based aggregation-induced luminescent molecules, carborane-based aggregation-induced luminescent molecules, Rhodamine-based aggregation-induced luminescent molecules, tetraphenylethylene-based aggregation-induced luminescent molecules, silole-based aggregation-induced luminescent molecules, aromatic ring-containing metal complex-based compounds, BODIPY-based boronimine complex aggregation-induced luminescent molecules, other hetero compounds, etc. However, the present invention is not limited thereto.
<1. Benzofuro-oxazolo-carbazole aggregation-induced luminescent molecule>
The benzofuro oxazolo carbazole-based aggregation inducing light emitting molecule refers to an aggregation inducing light emitting molecule having a benzofuro oxazolo carbazole skeleton, and the benzofuro oxazolo carbazole skeleton is represented by the following formula (1) Ru. That is, the benzofuro-oxazolo-carbazole-based aggregation inducing light emitting molecule is an aggregation inducing light emitting molecule having a skeleton represented by the following formula (1) in the molecule. Examples of the benzofuro-oxazolo-carbazole aggregation-induced luminescent molecule include a compound represented by the following formula (2).

Each of R ₁ to R ₃ independently represents a hydrogen atom, an organic group or an organic metal group.

R ₁ and R ₂ may be the same or different and are preferably an alkyl group or a substituted alkyl group, and as a substituent of the substituted alkyl group, a sulfonic acid group, a carboxyl group, a phosphoric acid group, It is preferable that it is at least one kind of substituent selected from a phosphorous acid group, a hydroxyl group, an amino group, an isocyanate group, a silyl group and a halogen atom.

The substituent represented by R ₃ is preferably an aromatic group, an aliphatic group or a hydrogen atom, and the aromatic group may be an aromatic hydrocarbon group or an aromatic heterocyclic group, and preferred examples thereof And phenyl group, naphthyl group, anthranyl group, phenanthryl group, pyrenyl group, pyridyl group, pyrimidyl group, triazinyl group and the like. Examples of the aliphatic group include an alkyl group, a cycloalkyl group, and an unsaturated aliphatic group. In the aromatic group and the aliphatic group, a hydrogen atom may be substituted with a substituent, and as the substituent, a sulfone may be mentioned. At least one substituent selected from an acid group, a carboxyl group, a phosphate group, a phosphorous group, a hydroxyl group, an amino group, an isocyanate group, a silyl group and a halogen atom is preferable.

Y represents an electron withdrawing group, and specifically, cyano, nitro, methoxy, tosyl, mesyl, halogen, phenyl, acyl, keto, carboxyl, aldehyde, ethoxycarbonyl Groups, methoxycarbonyl group, pyridyl group, pyrimidyl group, triazinyl group, triazolyl group, tetrazolyl group, dicyanomethyl group, cyanamide group and the like.

The benzofuro-oxazolo-carbazole-based aggregation-inducing light emitting molecule is preferably a compound represented by the following formulas (3) -1 to (3) -3.

<2. Carborane-based aggregation-induced luminescent molecule>
The carborane-based aggregation-induced light emitting molecule is a compound having a carborane skeleton composed of C ₂ B ₁₀ , preferably a compound having an ortho-carborane skeleton. Examples of the carborane-based aggregation-induced light emitting molecules include compounds represented by the following formula (4).

In the formula (4), open circles represent carbon atoms and black dots represent BH. BH which can not be illustrated from the viewpoint of three-dimensional structure is omitted.

In Formula (4), R ₁ and R ₂ can each be independently selected from a hydrogen atom, an organic group or an organometallic group. The substituents represented by R ₁ and R ₂ may be the same or different, and may be condensed with each other to form a cyclic structure. Preferably, one of R ₁ and R ₂ is an aromatic hydrocarbon group or an aromatic heterocyclic group, in which case the other is an aliphatic hydrocarbon group even if it is an aromatic hydrocarbon group or an aromatic heterocyclic group. It may be a hydrogen group or a hydrogen atom.

Examples of the aromatic hydrocarbon group include phenyl group, naphthyl group, anthranyl group, phenanthryl group, pyrenyl group and the like, and the aromatic heterocyclic group includes pyridyl group, pyrimidyl group, triazinyl group, pyrrole group, imidazole group And pyrazole, triazole, oxadiazole, oxazole, thiadiazole, thiazole and the like. Among the aliphatic hydrocarbon groups, the saturated aliphatic hydrocarbon group includes an alkyl group and a cycloalkyl group, and the unsaturated aliphatic group includes an alkenyl group, an alkynyl group, a cycloalkenyl group and a cycloalkynyl group. Be Further, the aliphatic hydrocarbon group includes an aralkyl group in which one of the hydrogen atoms of the aralkyl group is substituted with an aryl group.

The aromatic hydrocarbon group, the aromatic heterocyclic group, and the aliphatic hydrocarbon group may be substituted with any alkali metal group, and at least one selected from a hydrophilic group and a reactive functional group It may be substituted by a group. As at least one group selected from the hydrophilic group and the reactive functional group, for example, a sulfonic acid group, a carboxyl group, a phosphoric acid group, a phosphorous acid group, a hydroxyl group, an amino group, an isocyanate group, a silyl group and A halogen atom etc. are mentioned.

When R ₁ and R ₂ are the same, it is preferable that R ₁ and R ₂ be selected from the following formula (4) -1.

Moreover, it is also preferable that said R ₁ and R ₂ are a combination selected from the following formula (4) -2.

<3. Maleimide-based aggregation-induced luminescent molecule>
The maleimide aggregation-induced light emitting molecule is a compound having a maleimide skeleton, and examples thereof include a compound represented by the following formula (5) -1 or (5) -2.

In the above formula (5) -1 or (5) -2, R, R ′ and R ′ ′ can each independently be selected from a hydrogen atom, an organic group or an organometallic group and is an aromatic group Is preferable, and a phenyl group, a naphthyl group, an anthranyl group, a pyridyl group, a pyrimidyl group and a triazinyl group are more preferable.

The substituents R ′ and R ′ ′ may be the same or different, and may be fused to each other to form a saturated ring, an unsaturated ring or an aromatic ring.

In addition, as the maleimide-based aggregation-induced light emitting molecule, one or more of hydrogen atoms at any position of the compound represented by the formula (5) -1 or (5) -2 may be a sulfonic acid group or a carboxyl group It is preferably substituted by at least one selected from phosphoric acid group, phosphorous acid group, hydroxyl group, amino group, isocyanate group, silyl group and halogen atom.

Specific examples of the compound suitably used as the maleimide aggregation-induced light emitting molecule include the following compounds.

<4. Aminobenzopyranoxanthene aggregation-induced luminescent molecule>
The aminobenzopyranoxanthene-based aggregation inducing luminescent molecule means an aggregation inducing luminescent molecule having an aminobenzopyranoxanthene skeleton, and examples thereof include a compound represented by the following formula (6).

In the formula (6), R ₁ to R ₄ can be each independently selected from a hydrogen atom, an organic group or an organic metal group. It is preferable that it is an alkyl group and a substituted alkyl group, and as a substituent of the substituted alkyl group, a sulfonic acid group, a carboxyl group, a phosphoric acid group, a phosphorous acid group, a hydroxyl group, an amino group, an isocyanate group, a silyl group and a halogen atom It is more preferable that it is at least one selected from

Furthermore, R ₁ and R ₂ , and R ₃ and R ₄ may be combined to form a ring. In addition, a salt may be formed by coordination of the anion to the cation of the amino group. Further, R ₁ and R ₂ may be each independently bonded to another carbon atom of a ring containing a carbon atom to which a nitrogen atom to which R ₁ and R ₂ are bonded is bonded to form a ring, R ₃ And R ₄ may be each independently bonded to another carbon atom of a ring containing a carbon atom to which the nitrogen atom to which R ₃ and R ₄ are bonded is bonded to form a ring.

Specifically, the aminobenzopyranoxanthene-based aggregation inducing light emitting molecule is preferably the following compound.

<5. Rhodamine-based aggregation-induced luminescent molecule>
The rhodamine-based aggregation-induced light emitting molecule refers to an aggregation-induced light emitting molecule having a rhodamine skeleton, and the rhodamine skeleton is represented by the following formula (7). The rhodamine aggregation-induced light emitting molecule is preferably an aminobenzopyroxanthene dye, and more preferably a compound represented by the following formula (7) -1. In the compound represented by the formula (7) -1, two R ^a to R ^g and two m exist, respectively, and those represented by the same symbol may be the same or different. And are preferably identical.

In the formula (7) -1, R ^a and R ^b are each independently a hydrogen atom or a hydrocarbon group having 1 to 20 carbon atoms, preferably a hydrocarbon group having 1 to 12 carbon atoms.

The R ^c and R ^d each independently represent a hydrogen atom, a halogen atom or a hydrocarbon group having 1 to 20 carbon atoms, preferably a hydrogen atom.

The R ^e is independently an amino group or —COOR ^h , and the R ^h is a hydrogen atom or a hydrocarbon group having 1 to 20 carbon atoms, preferably a hydrogen atom.

R ^f independently represents a hydrogen atom, a halogen atom, an amino group, an amide bond-containing group which may have a protecting group, or a hydrocarbon group having 1 to 20 carbon atoms, preferably a hydrogen atom.

^R.sup.g is independently a hydrogen atom, a halogen atom, a nitro group, a carboxyl group, an amino group or a hydrocarbon group having 1 to 20 carbon atoms, preferably a hydrogen atom.

M is an integer of 1 to 4, preferably 1.

The hydrocarbon group in each of R ^a , R ^b , R ^c , R ^d , R ^f , R ^g and R ^h may be partially substituted by a nitrogen atom, an oxygen atom or a sulfur atom.

The R ^a and R ^c and / or R ^b and R ^d may be bonded to each other to form a heterocyclic group having 5 or 6 atoms including at least one nitrogen atom. The heterocyclic group includes, for example, pyrrolidine, pyrrole, imidazole, pyrazole, piperidine, pyridine, piperazine, pyridazine, pyrimidine and pyrazine. Of these, piperidine is preferred.

The heterocyclic group may have a substituent R ^j . The R ^j is independently a hydrocarbon group having 1 to 12 carbon atoms or a group in which part of the hydrocarbon group is substituted with a nitrogen atom, an oxygen atom or a sulfur atom. Examples of such a compound include compounds represented by the following formulas (7) -2 and (7) -3. At this time, n is independently an integer of 0 to 3, preferably an integer of 0 to 2.

[In the formulas (7) -2 and (7) -3, R ^a , R ^c , R ^e , R ^f , R ^g and m are each independently R ^a , R ^c in the formula (7) -1, It is synonymous with R ^e , R ^f , R ^g and m. ]
The R ^e and R ^f may be bonded to each other to form a heterocyclic group having 5 or 6 atoms including at least one nitrogen atom. Examples of the heterocyclic ring include γ-butyrolactone, β-lactam and γ-lactam, and in particular γ-butyrolactone is preferable. The heterocyclic group may have a substituent, and as the substituent, for example, a hydrocarbon group having 1 to 12 carbon atoms which may be substituted with a nitrogen atom, an oxygen atom or a sulfur atom, hydroxyl Groups, amino groups, pyridinyl groups, furyl groups, or thienyl groups. As such a compound, for example, a compound represented by the following formula (7) -4 can be mentioned.

[In the formula ^{(7) -4, R a ~} R d, R g and m are independently the same as R ^a ~ R ^d, R ^g and m of formula (7) in -1. ]
The compounds represented by the above formula (7) -4 also include those having a resonance structure as in the following formula (3A) and those ionized as in the following formula (3B).

[Repletion based on rule 26 03.12.2018]

<6. Tetraphenylethylene-based aggregation-induced luminescent molecule>
The tetraphenylethylene-based aggregation-induced light emitting molecule means an aggregation-induced light emitting molecule having a tetraphenylethylene skeleton, and examples thereof include a compound represented by the following formula (8).

In the formula (8), a to d are each independently an integer of 0 to 5. It is also preferable that a to d = 0, that is, the compound represented by the above formula (8) is tetraphenylethylene. When a to d are 2 or more, a plurality of R ¹ to R ⁴ may be the same or different, and a plurality of R ^{1 's} , R ^{2' s} , R ^{3 's} or R ^{4' s} It may combine to form a ring. R ¹ to R ⁴ may be the same or different, and R ¹ and R ² , R ² and R ⁴ , R ³ and R ⁴ , and R ³ and R ¹ may be condensed to form a saturated ring, It may form a saturated ring or an aromatic ring.

In the above formula (8), R ¹ to R ⁴ are each independently an organic group or an organic metal group, preferably an aromatic ring-containing organic group, more preferably an aromatic group, a phenyl group or a naphthyl group. An anthranyl group, a pyridyl group, a pyrimidyl group and a triazinyl group are particularly preferred.

The following compounds may be mentioned as an example of the compound represented by the formula (8).

In addition, as a tetraphenylethylene aggregation-induced light emitting molecule, in the compound represented by the formula (8), a sulfonic acid group, a carboxyl group, a phosphoric acid group, a phosphorous acid group, a hydroxyl group may be provided at any position in the molecule. It is also preferable to use a compound having one or more of an amino group, an isocyanate group, a silyl group and a halogen atom.
<7. Silole-based aggregation-induced luminescent molecule>
The silole-based aggregation-induced light emitting molecule means an aggregation-induced light emitting molecule having a silole ring, and is not particularly limited, but is preferably a compound represented by the following formula (2). In addition, although the group represented with the same code | symbol in following formula (9) may be same or different, respectively, it is preferable that it is the same.

The R ^A is independently a hydrogen atom or a hydrocarbon group having 1 to 12 carbon atoms, preferably a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, and more preferably a hydrogen atom or 1 to 4 carbon atoms Is a hydrocarbon group, more preferably a hydrogen atom.

The a is independently an integer of 1 to 5, preferably 1 or 2.

The R ^B is independently an organic group such as an aromatic ring-containing organic group, and as the aromatic ring, a benzene ring, a naphthalene ring, a pyrrole ring, an imidazole ring, an imidazoline ring, a pyrazole ring, a pyridine ring, a pyrazine ring, a furan ring And a thiophene ring, an oxazole ring, a thiazole ring and the like.

Wherein R ^B preferably contains at least one benzene ring, more preferably a phenyl group.

The R ^C is independently an organic group, preferably an aromatic ring-containing organic group or a hydrocarbon group having 1 to 20 carbon atoms.

The hydrocarbon group having 1 to 20 carbon atoms is preferably a hydrocarbon group having 1 to 12 carbon atoms.

The R ^C is preferably a hydrocarbon group having 1 to 12 carbon atoms, and more preferably a phenyl group or an alkyl group having 1 to 12 carbon atoms.

The organic group in R ^B and R ^C may be a group consisting only of carbon atoms and hydrogen atoms, or may be a group containing a hetero atom such as a nitrogen atom, an oxygen atom, a sulfur atom or a silicon atom. Specifically, it may have at least one selected from a sulfonic acid group, a carboxyl group, a phosphoric acid group, a phosphorous acid group, a hydroxyl group, an amino group, an isocyanate group, a silyl group and a halogen atom.

Also, the R ^B and R ^C may be combined to form a ring. Examples of compounds in which R ^B and R ^C form a ring include the following compounds.

Moreover, the following are mentioned as a preferable example as a combination of said R ^<A> , R <^B> and R < ^C >.

Specific examples of the compound suitably used as the silole aggregation-induced light emitting molecule include the following compounds.

The compound represented by the formula (9) has a structure in which two benzene rings are bonded to a silole ring and further has at least two aromatic ring-containing organic groups, etc. It is believed that the light emission is suppressed. That is, the compound represented by the formula (9) is not particularly limited as long as two benzene rings are bonded to the silole ring, and more preferably, it has at least two aromatic ring-containing organic groups, Various groups can be introduced depending on the application. For example, one or at least one selected from a sulfonic acid group, a carboxyl group, a phosphoric acid group, a phosphorous acid group, a hydroxyl group, an amino group, an isocyanate group, a silyl group and a halogen atom at any position in the molecule It is preferable to have two or more.

The compounds represented by the above formula (9) also include those having an ionized structure. For example, when the R ^C is a group represented by —C ₆ H ₄ —CH ₂ —N (C ₂ H ₅ ) ₂ , in the compound represented by the formula (9), the moiety is Also included are those that are C ₆ H ₄ -CH ₂ -N ⁺ (C ₂ H ₅ ) ₂ .

<8. Aromatic ring-containing metal complex molecule>
The aromatic ring-containing metal complex molecule is an aggregation-induced light emitting molecule having an aromatic ring and a metal complex part. The metal atom constituting the metal complex part is not particularly limited, and examples thereof include Pt, Pd, Co, Cu, Ni, Fe, Ru, Mo, Zr, Cr, Re, Mn, Rh, V, W, Ti, and Ta. , Nb, Ir and the like.

The aromatic ring-containing metal complex molecule is not particularly limited, and examples thereof include the following.

<9. BODIPY-based boronimine complex aggregation-induced light emitting molecule>
The BODIPY-based boronimine complex aggregation-induced light emitting molecule refers to an aggregation-induced light emitting molecule having a boron atom and an imine structure, and is not particularly limited, but it is a compound represented by the following formula (10) Are more preferable, and compounds represented by the following formulas (10) -1 and (10) -2 are more preferable.

In the above formulas (10), (10) -1 and (10) -2, Y represents an electron withdrawing group or an electron donating group, and the emission wavelength or intensity of the aggregation-induced light emitting molecule is represented by this substituent. The fluorescence characteristics such as In the formula (10), X is S, O or N. Note that R ₄ does not exist when XXO or S in the formula.

Examples of the electron donating group include methoxy group, alkoxy group, amino group alkylamino group, dialkylamino group, trialkylamino group, alkyl group and aromatic group having a methoxy group moiety.

R ₁ , R ₂ , R ₃ and R ₄ each independently represent a hydrogen atom, an organic group or an organic metal, which may be the same or different, and may be mutually condensed to form a ring structure.

Specific examples of the compound suitably used as the BODIPY-based boronimine complex aggregation-inducing light emitting molecule include the following compounds.

The aggregation induced light emitting molecule preferably does not have a carbon / carbon double bond which does not contribute to the formation of a ring structure from the viewpoint of light resistance. In other words, it is preferable that all of the carbon-carbon double bonds form a part of a ring structure from the viewpoint of light resistance in the aggregation-induced light-emitting molecule.

The aggregation induced light emitting molecule may be a commercially available product, and a conventionally known method such as US Patent Application Publication No. 2012/299474, US Patent Application Publication No. 2013/177991 and US Patent Application A compound synthesized by the method described in Publication No. 2013/89889, S. Kamino, et. Al., Chem. Commun., 2010, 46, 9013-9015 may be used.

The aggregation-induced light emitting molecule may further have a mechanochromic property capable of adjusting the fluorescence property such as the light emission wavelength or the luminance.

The aggregation inducing luminescent molecule used for preparation of aggregation nanoparticles can be selected to emit fluorescence of a desired wavelength (color) in a captured fluorescence image. When two or more types of target substances to be targeted for fluorescent labeling are used, a combination of aggregation-induced luminescent molecules emitting fluorescence of different wavelengths corresponding to each may be selected to produce aggregated nanoparticles. When using such two or more types of aggregation-induced light-emitting molecules, it is preferable to select one having the emission wavelength peaks separated by 100 nm or more.

<Aggregated nanoparticles>
The aggregated nanoparticle of the present invention is a particle formed by the aggregation of the aggregation-induced light emitting molecule, has a hydrophilic group on the particle surface, and has a particle size variation coefficient of 30% or less.

The type of hydrophilic groups aggregated nanoparticles have on its surface of the present invention is not particularly limited, for example -OH, -SH, -COOH, -S ( = O) 2 OH, -S (= O) NH 2, - _{S (= O) 2 NH 2} , -P (= O) (OH) 3, -P (= O) R (OH) 2, -P (= O) R 2 (OH), - P (OH) 3 , -P (= O) (NH 2) 3, -P (= O) R (NH 2) 2, -P (= O) R 2 (NH 2), - P (NH 2) 3, -O ( _{C = O) OH, -NH 2} , -NHR, -NHCONH 2, -NHCONHR, -NHCOOH, -Si (OH) 3, -Si (R) (OH) 2, -Si (R) 2 OH, -Ge _{(OH) 3, -Ge (R} ) (OH) 2, -Ge (R) 2 OH, -Ti (OH) 3, -Ti (R) (OH) 2, -Ti (R) 2 OH, -Si (NH _{2) 3,} -Si ( _{) (NH 2) 2, -B} (OH) 2, -O-B (OH) 2, -B (NH 2) 2, -NHB (OH) 2 and the like. Each of R's independently represents hydrogen or an alkyl group having 1 to 12 carbon atoms.

The method for producing the aggregated nanoparticles of the present invention is not particularly limited, but, for example, by aggregating aggregation-induced light emitting molecules in which hydrophilic groups have been previously introduced at arbitrary positions by aggregation by any method, Aggregated nanoparticles having hydrophilic groups on the surface can be prepared. Also, aggregation-induced light emitting molecules having no hydrophilic group are aggregated by hydrophobic interaction to form nanoparticles, and then a hydrophilic group is introduced to the particle surface by chemical synthesis, intermolecular interaction or complex formation. May be used. The method for introducing the hydrophilic group onto the surface of the aggregated nanoparticle is not particularly limited, but specific examples include a method for treating an aggregation-induced luminescent molecule with an acid such as sulfuric acid or nitric acid.

Aggregated nanoparticles of the invention preferably has one / nm ² or more hydrophilic groups on the particle surface, and more preferably has 1.5 / nm ² or more. Further, preferably it has a hydrophilic group 6.0 pieces / nm ² or less to the particle surface, and more preferably has 4.5 or / nm ² or less. The number of hydrophilic groups is the number of hydrophilic groups per 1 nm ² of the surface area of the aggregated nanoparticles.

The introduced amount of the hydrophilic group is determined by measuring each electron energy loss value by STEM-EELSE (electron energy loss spectroscopy using a scanning transmission electron microscope) according to the reference sample and the aggregation-induced light emitting molecule to be evaluated. It can be calculated based on Lambert-Veil's law.

<Average particle size and particle size variation coefficient>
As the particle diameter of the aggregated nanoparticles becomes smaller as the particle diameter decreases, the binding strength with the target substance is increased, and it becomes possible to excite all aggregation-induced luminescent molecules contained in the particles. The average particle diameter is preferably 1 nm or more and 100 nm or less, and particularly preferably 10 nm or more and 70 nm or less from the viewpoint of being correlated with the particle diameter and the brightness. The average particle size of the aggregated nanoparticles can be made to fall within a desired range by adjusting the conditions in the production method.

The particle size of the aggregated nanoparticles can be measured by photographing an image of the particles using a scanning electron microscope (SEM). The particle size of each of the aggregated nanoparticles contained in a sufficient number (for example, 100) of the population is measured, and the average particle size is calculated as its arithmetic mean.

The particle size variation coefficient of the aggregated nanoparticles is calculated by the formula: 100 × standard deviation of particle size / average particle size. The smaller the particle size variation coefficient, the smaller the luminance unevenness when used for dyeing, and the quantitativeness of the dyeing result is improved. The particle size variation coefficient of the agglomerated nanoparticles according to the present invention is preferably 30% or less, and more preferably 15% or less. The lower limit of the particle size variation coefficient is not particularly limited, but is usually 4% or more. The average particle diameter and particle diameter variation coefficient of the aggregated nanoparticles can be kept within a predetermined range, for example, by appropriately adjusting the production conditions according to the production method. For example, a method of dropping a dispersion of aggregation-induced luminescent molecules into a high dilution, high rotation number of poor solvent, or a method of bringing a poor solvent into contact with a dispersion of aggregation-induced luminescent molecules at high speed using a micromixer An aggregation having a small coefficient of variation of particle diameter by a method (eg, JP-A-2005-238342) of performing a laser ablation on a dispersion in which crystals of relatively large aggregation-induced luminescent molecules are dispersed in a poor solvent. Nanoparticles can be produced.

When the laser ablation is performed, various known lasers can be used as the laser, and a YAG laser, an excimer laser, a titanium-sapphire laser or the like is preferably used. As an irradiation laser, it is preferable to apply a pulse wave. Further, in order to prepare aggregated nanoparticles having a more uniform particle size distribution, it is preferable to adjust the concentration of the dispersion before performing laser application to 0.1 mg / L to 500 mg / L. The irradiation power, pulse width, wavelength and irradiation time can be appropriately adjusted according to the type and size of the aggregation-induced luminescent molecule of interest and the mixing ratio with the poor solvent, and the aggregation nano size is more uniform. For preparing the particles, for example, the power is 0.5 to 500 mJ / cm ² , the pulse width is 1 to 100 femtoseconds, the pulse width is 0.01 to 500 Hz, the irradiation time is 0.5 minutes to 5 hours, It is preferable to select in the range of and irradiate a laser.

As the poor solvent, use may be made of water, alcohol solvents such as methanol and ethanol, aliphatic solvents such as pentane, hexane and heptane, aromatic solvents such as benzene and toluene, or a mixed solvent of two or more of them. But not limited thereto. The laser ablation method can be performed, for example, with an apparatus set up by the method described in The Review of Laser Engineering, 33, 41-46.

The aggregated nanoparticles may have a central nucleus in addition to the aggregation inducing luminescent molecule as a component thereof. The central nucleus is a substance serving as a nucleus for aggregating and causing the aggregation-induced luminescent molecules to grow when the aggregation nanoparticles are produced from the aggregation-induced luminescent molecules as described later.

The substance used as the central nucleus is not particularly limited, and for example, fine particles of organic molecules such as polystyrene and latex, and inorganic molecules such as silica are suitably used. The nature and size of the central core can be selected according to the desired particle size of the aggregated nanoparticles and the nature of the aggregation-induced luminescent molecule used to make it. As the central nucleus, one having an average particle diameter of 1 nm or more and 20 nm or less and a particle diameter variation coefficient of 5% or less is preferable. It is preferable for the average particle size and the particle size variation coefficient of the central core to be in the above-mentioned range, since it is easy to make the average particle size and the particle size variation coefficient of the obtained agglomerated nanoparticles into the above-mentioned range.

The aggregated nanoparticles may have nonspecific adsorption preventing molecules on the surface thereof. Although the binding mode of the aggregation nanoparticles and the nonspecific adsorption preventing molecule is not particularly limited, for example, they can be coupled by covalent bonding or electrostatic interaction via hydrophilic groups on the surface of the aggregation nanoparticles. Although the nonspecific adsorption preventing molecule is not particularly limited, for example, a polymer such as polyethylene glycol is preferable.

The form of the aggregated nanoparticles of the present invention, for example, the form at the time of production, storage, and distribution is not particularly limited, but for example, a form of dispersion using a known buffer such as PBS as a dispersion medium preferable.

<Fluorescent labeling material>
The fluorescent labeling material according to one embodiment of the present invention contains aggregated nanoparticles and has a targeting ligand on the particle surface. The aggregated nanoparticle and the targeting ligand may be directly linked or may be linked via a linker or the like. Alternatively, the targeting ligand can be bound by introducing a binding group to the surface of the aggregated nanoparticle by a known method. As the bonding group, for example, a hydrophilic group contained in the above-mentioned aggregated nanoparticle may be used.

The form of the fluorescent labeling material, for example, the form at the time of production, storage, and distribution is not particularly limited, but is preferably in the form of a dispersion using a known buffer such as PBS as a dispersion medium. One embodiment of the present invention is in the form of a dispersion, that is, a fluorescent label dispersion, and the fluorescent label dispersion contains a fluorescent label and a buffer.

<Targeting ligand>
The targeting ligand used in the present invention is a substance that specifically recognizes and binds to the target substance, for example, a target biological substance that is a biological substance contained in tissues and cells collected from an animal etc. It is preferably a substance that specifically recognizes and binds. The target biological substance is not particularly limited, and examples include proteins, nucleic acids, sugar chains, lipids and the like. The target biological material is preferably a biological material associated with any disease. Specifically, for example, marker proteins (for example, cancer-specific proteins, vascular endothelial cell-specific proteins, phosphorylated proteins, etc.) specifically expressed in cancer cells, inflammation-related proteins, etc., and immune-related proteins can be mentioned. .

For example, when the target biological substance is a protein specifically expressed in a tumor tissue or a cancer cell, antibodies against these are preferably selected as a targeting ligand. When the target biological substance is a glycoprotein, a protein (for example, lectin) having a binding property with a sugar chain is preferably selected as the target-directed molecule.

Other target-directed molecules include, for example, organelle compatible substances, peptides and the like.

When an antibody is selected as the targeting ligand, it is usually IgG or IgM, and IgG is preferably used. The antibody may be a natural antibody such as full-length IgG, as long as it has the ability to specifically recognize and bind a target protein or a lower antibody, Fab, Fab ', F (ab' ₂ ) It may be a non-naturally occurring antibody such as an artificial antibody which has been multifunctionalized (multivalented or multispecificized) using antibody fragments such as ₂ , Fv and scFv, or antibody fragments thereof . A primary antibody that recognizes and binds to a unique epitope on an antigen is preferably used. In the case of using a secondary antibody which is an antibody which recognizes and binds a unique epitope to a primary antibody as a targeting ligand, a target biological substance to which a primary antibody is bound in advance is used as a target substance.

The manner of binding to the targeting ligand and the surface of the aggregated nanoparticle is not particularly limited, and for example, it can be bound via covalent bond, ionic bond, hydrogen bond, coordinate bond, physical adsorption, chemical adsorption and the like. In particular, it is preferable that the hydrophilic group present on the surface of the aggregated nanoparticle and the functional group possessed by the targeting ligand be bound by covalent bonding. In this case, the hydrophilic group on the surface of the aggregated nanoparticle is lost by the binding with the targeting ligand, but the surface of the fluorescent labeling material of the present invention is hydrophilic because it is complemented by the hydrophilic group possessed by the targeting ligand. Keep in mind that

In addition, when the aggregated nanoparticles have nonspecific adsorption molecules on their surface, the nonspecific adsorption preventing molecule may be used as a linker for binding to a target-directed ligand. Examples of nonspecific adsorption preventing molecules used for such purpose include MPC polymers and polyethylene glycol derivatives having various terminal groups such as NHS or maleimide.

<Method for producing aggregated nanoparticles>
The aggregated nanoparticles of the present invention preferably include a step (A) of bringing a poor solvent into contact with a solution of aggregation-induced luminescent molecules and aggregating the aggregation-induced luminescent molecules, wherein the step (A) comprises a core In the presence of the aggregation-induced luminescent molecule, the solution of the aggregation-induced luminescent molecule may be brought into contact with the poor solvent to aggregate the aggregation-induced luminescent molecule. The step other than the step (A) is not particularly limited, and, for example, a step of introducing a hydrophilic group into the aggregation-induced light emitting molecule, a step of introducing a hydrophilic group into the surface of the aggregated nanoparticle, and the like are appropriately performed. By performing the step (A) in the presence of the central nucleus, it is preferable to control the particle size variation coefficient and the average particle size of the aggregated nanoparticles, which is preferable. The central core may be premixed in the solution of aggregation-induced luminescent molecules, or may be premixed in the poor solvent.

The aggregation nanoparticles in the present invention use a solvent (good solvent) capable of dissolving aggregation-induced luminescent molecules, prepare a solution of aggregation-induced luminescent molecules, and then induce aggregation in the solution of aggregation-induced luminescent molecules. It can be prepared by the reprecipitation method of precipitating aggregated nanoparticles by mixing the luminescent molecule with the poor solvent. By using such a reprecipitation method, it is possible to produce particles densely packed with aggregation-induced luminescent molecules. Specifically, for example, a reprecipitation method using a mixer with a small inner diameter called a micromixer, and pumping a good solvent and a poor solvent of aggregation-induced luminescent molecules into the micromixer, both rapidly and uniformly The method (flow-through method) to which microparticles | fine-particles are deposited is mentioned by mixing to (1).

As a micromixer that can be suitably used, the inner diameter of the flow path of the mixing section for mixing the minute solution of aggregation-induced luminescent molecules and the poor solvent (when the cross section of the flow path is not circular, the cross-sectional area of the flow path The diameter of the circle having the same area as that of the above is preferably 2 mm or less, and in order to mix the solution and the poor solvent more rapidly, the inner diameter of the flow path is preferably 1 mm or less. Further, in order to prevent the clogging of the flow path by the fine particles and to reduce the pressure loss inside the flow path, the inner diameter of the flow path is preferably 0.05 mm or more.

The good solvent of the present invention is not particularly limited as long as it exhibits good solubility in aggregation-induced light emitting molecules, and it is preferable to select one having good compatibility with the poor solvent described later. Specifically, for example, ether solvents such as tetrahydrofuran and dioxane, ketone solvents such as acetone and methyl ethyl ketone, and amides such as 1-methyl-2-pyrrolidinone, 1,3-dimethylimidazolinone and N, N-dimethylformamide A system solvent, a sulfur-containing solvent such as dimethyl sulfoxide, or a mixed solvent of two or more of these can be suitably used. Moreover, it is preferable to use a good solvent having a boiling point lower than the boiling point of the poor solvent from the viewpoint of preventing redispersion of aggregation-induced light emitting molecules. Moreover, you may dissolve an inorganic compound, a dispersing agent, etc. as needed.

The poor solvent of the present invention is not particularly limited as long as it has relatively low solubility in the aggregation-induced light emitting molecule, and it is preferable to select one having good compatibility with the above-mentioned good solvent. For example, water or an aqueous solution is preferable, and alcohol solvents such as methanol and ethanol, aliphatic solvents such as pentane, hexane and heptane, aromatic solvents such as benzene and toluene, or mixed solvents of two or more of them are used. Although it can be, it is not limited to these. From the viewpoint of facilitating removal, those having a relatively low boiling point (eg, 40 ° C. to 120 ° C.) are preferable. Moreover, you may dissolve an inorganic compound etc. as needed.

The reaction conditions for the reaction time and reaction temperature are not particularly limited as long as the aggregated nanoparticles satisfying the above-mentioned conditions are produced, but in order to efficiently form the aggregation-induced light-emitting property into nanoparticles, Preferably, the turbulent mixing conditions are such that, for example, the Reynolds number is 4,000 or more.

<Method for producing fluorescent labeling material>
One embodiment of the present invention includes a method for producing a fluorescent labeling material, which comprises the step of binding a targeting ligand to aggregated nanoparticles. There is no particular limitation on the method of binding the targeting ligand to the aggregated nanoparticle, and therefore, in the above-mentioned production method, the aggregated nanoparticle and the targeting ligand may be directly reacted, or the aggregated nanoparticle and the targeting ligand may be used. And the step of introducing an optional binding group to the aggregated nanoparticles before reacting them. The method may further include the step of performing non-specific adsorption preventing treatment to bind the non-specific adsorption preventing molecule to the surface of the aggregated particle before attaching the targeting nanoparticle to the aggregated nanoparticle.

Hereinafter, preferred embodiments of the present invention will be more specifically described based on examples, but the present invention is not limited to these examples.
[Preparation Example 1]

<Preparation of aggregation-induced luminescent molecules>
Add 5 mL of concentrated nitric acid and 5 mL of concentrated sulfuric acid to 0.1 mol of 1,1,2,3,4,5-hexaphenyl-1H-silole (hereinafter referred to as AIE 0) having no hydrophilic group and stir for 1 hour The nitro group was introduced into the aromatic ring possessed by AIE0 by Subsequently, after liquid-phase purification with toluene / water, AIE0 into which one nitro group was introduced and AIE0 into which two nitro groups were introduced were separated and separated using column chromatography.

0.1 g of tin powder and 10 mL of concentrated hydrochloric acid were added to AIE0 introduced with one 10.1 g of nitro group, and stirred for 1 hour. Subsequently, 50 mL of 1 N aqueous sodium hydroxide solution was added, and the mixture was stirred for 10 minutes. Subsequently, liquid separation purification was performed with water / ethyl acetate, and drying under reduced pressure was performed to obtain a silole aggregation-induced light emitting molecule (hereinafter referred to as AIE1) having one amino group in one molecule.

Similarly, a silole-based aggregation-induced light emitting molecule (hereinafter referred to as AIE2) having two amino groups in one molecule was synthesized from AIE0 introduced with two nitro groups.

In addition, tetraphenylethene (hereinafter referred to as AIE3) into which one amino group was introduced was obtained by the same production method as AIE1 using tetraphenylethene instead of 1,2,3,4,5-hexaphenyl-1H-silole . AIE 3 has an olefin skeleton in which an aromatic ring is bonded to a carbon-carbon double bond.

A silole aggregation-induced luminescent molecule was produced by the synthesis method described in Dalton Trans, 2013, 42, 3646-3652 (hereinafter referred to as AIE 4).

A carborane-based aggregation-induced luminescent molecule was produced by the synthesis method described in Macromolecules 2009, 42, 1418-1420 (hereinafter referred to as AIE 5).

A benzofuro-oxazolo-carbazole aggregation-induced luminescent molecule was prepared by the synthetic method described in New J. Chem., 2007, 31, 2076-2082 (hereinafter referred to as AIE 6).

A maleimide aggregation-induced luminescent molecule was produced by the synthesis method described in Chem. Lett. 2012, 41, 1445-1447 (hereinafter referred to as AIE 7).

A silole aggregation-induced light emitting molecule was produced by the synthesis method described in Organometallics, 2016, 35 (14), pp 2327-2332. (Hereinafter, referred to as AIE8).

The BODIPY-based boronimine complex aggregation-induced light emitting molecule was prepared by the synthesis method described in Chem. Eur. J, 2013, 19, 4506-4512 (hereinafter referred to as AIE 9).

Each of the AIE 1 to AIE 9 has the following structure.

[Production Example 2]
<Biotin modified secondary antibody>
100 μg of anti-rabbit IgG antibody was dissolved in 50 mM Tris-HCl solution (pH 7.5). The solution was mixed with DTT (Dithiothretol) solution to a final concentration of 3 mM. Thereafter, the solution was allowed to react at 37 ° C. for 30 minutes, and the reduced secondary antibody was purified using a desalting column. 200 μL of the total amount of the purified solution was dissolved in 50 mM Tris-HCl solution (pH 7.5) to obtain an antibody solution. On the other hand, a linker reagent "(+)-Biotin-PEG6-NH-Mal" (PurePEG, product number 2461006-250) having a spacer length of 30 Å is prepared to be 0.4 mM using DMSO. did. 8.5 μL of this solution was added to the above antibody solution, mixed and allowed to react at 37 ° C. for 30 minutes.

This reaction solution was purified using a desalting column (Zaba Spin Desalt Spin Columuns) (Thermo Scientific Co., Ltd. 89882). The absorption at a wavelength of 300 nm of the desalted reaction solution was measured with a spectrophotometer (F-7000 manufactured by Hitachi, Ltd.). The amount of protein contained in the reaction solution was calculated by measuring “.” The reaction solution was adjusted to 250 μg / mL with a 50 mM Tris solution to obtain a solution of a biotin-modified secondary antibody.
Example 1
<Flocculated nanoparticles 1>
What was melt | dissolved in the tetrahydrofuran so that it might become 1 mM of AIE1 produced by the preparation example 1 was adjusted. The above solution is sent at a flow rate of 1.0 mL / min using a pump (PU-1580, JASCO Corporation) in a stainless steel T-shaped micro mixer (MT1XCS6, manufactured by Valco) equipped with a flow path with an inner diameter of 0.15 mm. The solution is mixed, and the two solutions are mixed in the micromixer by feeding ultrapure water at a flow rate of 74.0 mL / min using another pump (NS-KX-500, Japan Precision Science Co., Ltd.). The particles of AIE1 were precipitated. The pressure at the time of mixing was 4 to 5 MPa, and clogging of the flow path by AIE1 particles did not occur. The Reynolds number at mixing was calculated to be about 12,000.

0.1 g of the obtained particles and 10 mL of nitric acid are mixed and stirred at 50 ° C. for 1 hour to hydrophilize the particle surface, and then treated with a centrifugal separator at 10000 rpm for 30 minutes to remove the supernatant. The aggregate nanoparticles 1 were obtained by washing.

With respect to the obtained aggregated nanoparticles 1, arbitrary 100 particles are observed by TEM to obtain an average particle diameter, and the particle diameter variation coefficient (CV) is calculated based on the formula; 100 × (standard deviation of particles / average particle diameter) ) Was calculated.

With respect to the obtained aggregated nanoparticles 1, the amount of introduced hydrophilic groups [number / nm ² ] to the particle surface was calculated by the following method.

A reference sample solution (100 μM, 100 μL) having a hydrophilic group of 3 / nm ^{2 on} the surface was dropped into a φ3 mm grid for TEM, dried uniformly, and the loss value was measured by STEM-EELSE measurement. The loss value of the STEM-EELSE measurement at the wavelength derived from the absorption to the hydrophilic group or the ligand at this time was used as a reference value.

The area of 1 nm ^{2 on} the particle surface was specified by STEM-EELSE measurement for the aggregated nanoparticles 1, and the loss value was measured. From the Lambert-Beer law, the amount of introduced hydrophilic groups (number / nm ² ) was calculated based on the loss value of aggregation-induced light emitting molecule 1 nm ^{2 and} the loss value of the reference.

The particle size variation coefficient and the amount of introduced hydrophilic group were calculated in the same manner for the aggregated nanoparticles 1 to 21 obtained in each of the examples and comparative examples.
<Fluorescent labeling material 1>
SM (PEG) 12 (succinimidyl-[(N-maleimidopropionamido) -dodecaneethyleneglycol] ester; in a dispersion prepared by adjusting aggregated nanoparticles 1 to 3 nM with PBS containing 2 mM EDTA to a final concentration of 10 mM; thermo Scientific) were mixed and allowed to react at 5 ° C. for 1 hour.

The dispersion was centrifuged at 10,000 rpm for 20 minutes, and after removing the supernatant, it was added to a phosphate buffer containing 2 mM of EDTA to disperse the precipitate, and centrifugation was performed again. The same operation was performed three times to obtain aggregated nanoparticles 1 modified with a maleimide group.

After adding 40 μL of streptavidin adjusted to 1 mg / mL to 210 μL of borate buffer, add 70 μL of 2-iminothiolane hydrochloride (manufactured by Sigma Aldrich) adjusted to 64 mg / mL, and react at room temperature for 1 hour. A thiol group was introduced to the amino group of avidin. The streptavidin solution was desalted with a gel filtration column (Zaba Spin Desalting Columuns, Funakoshi Co., Ltd.) to prepare streptavidin capable of binding to the aggregated nanoparticles 1 having amination on the surface.

Using PBS containing 0.04 mg of this streptavidin and 2 mM of EDTA, the mixture was mixed with 740 μL of aggregated nanoparticles 1 having amination on the surface adjusted to 0.67 nM above, and reacted at room temperature for 1 hour. The reaction was stopped by adding 10 mM mercaptoethanol. The resulting solution was concentrated using a centrifugal filter, and unreacted streptavidin and the like were removed with a gel filtration column for purification, to obtain a fluorescent labeling material 1 which is a streptavidin-conjugated aggregated nanoparticle.

Example 2
<Aggregated nanoparticles 2>
Aggregated nanoparticles 2 were obtained in the same manner as aggregated nanoparticles 1 except that AIE1 was changed to AIE2.
<Fluorescent labeling material 2>
A fluorescent labeling material 2 was obtained in the same manner as the fluorescent labeling material 1 except that the aggregation nanoparticles 1 were changed to aggregation nanoparticles 2.

[Example 3]
<Flocculated nanoparticles 3>
1 mL of an aqueous dispersion of aggregated nanoparticles 2 adjusted to 10 w% and 10 mL of sulfuric acid are mixed and stirred at 50 ° C. for 1 hour to hydrophilize the particle surface, followed by a centrifuge for 30 minutes at 10000 rpm Aggregated nanoparticles 3 were obtained by processing, removing the supernatant and washing.
<Fluorescent labeling material 3>
A fluorescent labeling material 3 was obtained in the same manner as the fluorescent labeling material 1 except that the aggregation nanoparticles 1 were changed to aggregation nanoparticles 3.

Example 4
<Flocculated nanoparticles 4>
The aggregated nanoparticles 4 were obtained in the same manner as the aggregated nanoparticles 3 except that the treatment time with sulfuric acid was 3 hours.
<Fluorescent labeling material 4>
A fluorescent labeling material 4 was obtained in the same manner as the fluorescent labeling material 1 except that the aggregation nanoparticles 1 were changed to aggregation nanoparticles 4.

[Example 5]
<Flocculated nanoparticles 5>
Aggregated nanoparticles 5 were obtained in the same manner as the aggregated nanoparticles 3 except that the flow rate of ultrapure water was fed into the flow path of the micromixer to 40.0 mL / min.
<Fluorescent labeling material 5>
A fluorescent labeling material 5 was obtained in the same manner as the fluorescent labeling material 1 except that the aggregation nanoparticles 1 were changed to aggregation nanoparticles 5.

[Example 6]
<Flocculated nanoparticles 6>
Aggregated nanoparticles 6 were obtained in the same manner as the aggregated nanoparticles 3 except that the tetrahydrofuran solution concentration of AIE 2 fed into the flow path of the micromixer was changed to 0.1 mM.
<Fluorescent labeling material 6>
A fluorescent labeling material 6 was obtained in the same manner as the fluorescent labeling material 1 except that the aggregation nanoparticles 1 were changed to aggregation nanoparticles 6.

[Example 7]
<Aggregated nanoparticles 7>
Aggregated nanoparticles 7 were obtained in the same manner as the aggregated nanoparticles 3 except that the tetrahydrofuran solution concentration of AIE 2 fed into the flow path of the micromixer was changed to 3 mM.
<Fluorescent labeling material 7>
A fluorescent labeling material 7 was obtained in the same manner as the fluorescent labeling material 1 except that the aggregation nanoparticles 1 were changed to aggregation nanoparticles 7.

[Example 8]
<Flocculated nanoparticles 8>
Aggregated nanoparticles 8 were obtained in the same manner as the aggregated nanoparticles 3 except that the tetrahydrofuran solution concentration of AIE 2 fed into the flow path of the micromixer was changed to 0.07 mM.
<Fluorescent labeling material 8>
A fluorescent labeling material 8 was obtained in the same manner as the fluorescent labeling material 1 except that the aggregation nanoparticles 1 were changed to aggregation nanoparticles 8.

[Example 9]
<Flocculated nanoparticles 9>
Aggregated nanoparticles 9 were obtained in the same manner as the aggregated nanoparticles 3 except that the concentration of the tetrahydrofuran solution of AIE 2 fed into the flow path of the micromixer was changed to 6 mM.
<Fluorescent labeling material 9>
A fluorescent labeling material 9 was obtained in the same manner as the fluorescent labeling material 1 except that the aggregation nanoparticles 1 were changed to aggregation nanoparticles 9.

[Example 10]
<Aggregated nanoparticles 10>
What dissolved AIE2 in tetrahydrofuran so that it might be 0.1 mM was adjusted. Subsequently, 50 mL of the prepared AIE 2 solution is dropped at a pace of 1 mL per minute in 1000 mL of poor solvent water stirred at 1000 rpm using a high-speed stirring granulator (SPG-25TG, manufactured by DALTON). Agglutinated nanoparticles 10 were obtained by continuing the stirring at 1000 rpm for 30 minutes.
<Fluorescent labeling material 10>
A fluorescent labeling material 10 was obtained in the same manner as the fluorescent labeling material 1 except that the aggregation nanoparticles 1 were changed to aggregation nanoparticles 10.

[Example 11]
<Flocculated nanoparticles 11>
When mixing a tetrahydrofuran solution of AIE 2 adjusted to 1 mM with a micromixer with water, add 2 mL of zirconia particle dispersion liquid (SZR-M, Sakai Chemical Industry Co., Ltd.) having an average particle diameter of 3 nm as a central nucleus Were obtained in the same manner as for the aggregated nanoparticles 3 to obtain aggregated nanoparticles 11.
<Fluorescent labeling material 11>
A fluorescent labeling material 11 was obtained in the same manner as the fluorescent labeling material 1 except that the aggregation nanoparticles 1 were changed to aggregation nanoparticles 11.

[Example 12]
<Flocculated nanoparticles 12>
Aggregated nanoparticles 12 were obtained in the same manner as aggregated nanoparticles 3 except that AIE1 was changed to AIE3.
<Fluorescent labeling material 12>
A fluorescent labeling material 12 was obtained in the same manner as the fluorescent labeling material 1 except that the aggregation nanoparticles 1 were changed to aggregation nanoparticles 12.

Comparative Example 1
<Aggregated nanoparticles 13>
As described in US Patent Application Publication No. 2013/089889, 100 mg of AIE 2 is completely dissolved in a solvent of methanol: THF = 1: 1, and then aggregated nanoparticles 13 by recrystallization in a cool dark place for 3 days. I got
<Fluorescent labeling material 13>
A fluorescent labeling material 13 was obtained in the same manner as the fluorescent labeling material 1 except that the aggregation nanoparticles 1 were changed to aggregation nanoparticles 13.

Comparative Example 2
<Flocculated nanoparticles 14>
Aggregated nanoparticles 14 were obtained in the same manner as the aggregated nanoparticles 3 except that AIE1 was changed to AIE0 and the surface of the particles was not subjected to a hydrophilization treatment.
<Fluorescent labeling material 14>
A fluorescent labeling material 14 was obtained in the same manner as the fluorescent labeling material 1 except that the aggregation nanoparticles 1 were changed to aggregation nanoparticles 14.

[Example 13]
<Aggregated nanoparticles 15>
Aggregated nanoparticles 15 were obtained in the same manner as aggregated nanoparticles 3 except that AIE1 was changed to AIE4.
<Fluorescent labeling material 15>
100 mg of aggregated nanoparticles 15, 30 mg of sodium amide, 28 w% ammonia water, referring to the amination reaction of an aromatic sulfo-acid salt with sodium amide (Japan Chemical Society Journal 1974, (8), P. 1522, Nara et al.) The sulfonic acid groups on the particle surface were substituted with amino groups by adding 0.5 mL of H 2 O, 5 mL of water, and reacting at 60 ° C. for 4 hours. Subsequently, purification was performed by ultrafiltration with YM-100 (manufactured by Millipore) using pure water. Streptavidin was introduced by the same method as the fluorescent labeling material 1 using the aggregated nanoparticles 15 after purification, and the fluorescent labeling material 15 was obtained.

Example 14
<Aggregated nanoparticles 16>
Aggregated nanoparticles 16 were obtained in the same manner as aggregated nanoparticles 3 except that AIE1 was changed to AIE5.
<Fluorescent labeling material 16>
A fluorescent labeling material 16 was obtained in the same manner as the fluorescent labeling material 1 except that the aggregation nanoparticles 1 were changed to aggregation nanoparticles 16.

[Example 15]
<Aggregated nanoparticles 17>
Aggregated nanoparticles 17 were obtained in the same manner as aggregated nanoparticles 3 except that AIE1 was changed to AIE6.
<Fluorescent labeling material 17>
A fluorescent labeling material 17 was obtained in the same manner as the fluorescent labeling material 15 except that the aggregation nanoparticles 15 were changed to aggregation nanoparticles 17.

[Example 16]
<Flocculated nanoparticles 18>
Aggregated nanoparticles 18 were obtained in the same manner as aggregated nanoparticles 3 except that AIE1 was changed to AIE7.
<Fluorescent labeling material 18>
A fluorescent labeling material 18 was obtained in the same manner as the fluorescent labeling material 15 except that the aggregation nanoparticles 15 were changed to aggregation nanoparticles 18.

[Example 17]
<Flocculated nanoparticles 19>
Aggregated nanoparticles 19 were obtained in the same manner as aggregated nanoparticles 3 except that AIE1 was changed to AIE8.
<Fluorescent labeling material 19>
A fluorescent labeling material 19 was obtained in the same manner as the fluorescent labeling material 15 except that the aggregation nanoparticles 15 were changed to aggregation nanoparticles 19.

[Example 18]
<Aggregated nanoparticles 20>
Aggregated nanoparticles 20 were obtained in the same manner as aggregated nanoparticles 3 except that AIE1 was changed to AIE9.
<Fluorescent labeling material 20>
A fluorescent labeling material 20 was obtained in the same manner as the fluorescent labeling material 15 except that the aggregation nanoparticles 15 were changed to aggregation nanoparticles 20.

[Example 19]
<Flocculated nanoparticles 21>
The aggregated nanoparticles 3 were referred to as aggregated nanoparticles 21 in Example 19.
<Fluorescent labeling material 21>
One hundred μg of anti-PD-L1 rabbit monoclonal antibody (Cell signaling Technology, Inc .; No. E1L3N) was dissolved in 100 μL of PBS. To this antibody solution, 0.002 mL (0.2 × 10 ^-5 mol) of 1 M 2-mercaptoethanol was added, and the reaction solution reacted at pH 8.5 and room temperature for 30 minutes was passed through a gel filtration column and excess Removal of 2-mercaptoethanol gave a solution of thiolated anti-PD-L1 rabbit monoclonal antibody.

In the same manner as in Example 1 except that the aggregation nanoparticles 1 were replaced with aggregation nanoparticles 21, aggregation nanoparticles 21 modified with a maleimide group were obtained. Example 1 and Example 1 except that the aggregated nanoparticle 1 modified with maleimide group is changed to the aggregated nanoparticle 21 modified with maleimide group and further changed to streptavidin, and a thiolated anti-PD-L1 rabbit monoclonal antibody is used In the same manner, a fluorescent labeling material 21 which is an anti-PD-L1 rabbit monoclonal antibody-bound aggregated nanoparticle was obtained.

[Example 20]
<Aggregated nanoparticles 22>
AIE2 was prepared with methanol so as to be 100 mg / L, and ultrasonication was performed for 3 minutes to obtain a dispersion of AIE2. Subsequently, the dispersion is placed in a transparent quartz container, and dispersed using a titanium sapphire laser (TiF-100, manufactured by Tokyo Instruments) under conditions of 100 mJ / cm ² , pulse width 100 femtosecond, and irradiation time 20 minutes. The solution was irradiated with a laser to obtain aggregated nanoparticles 22.
<Fluorescent labeling material 21>
A fluorescent labeling material 21 was obtained in the same manner as the fluorescent labeling material 1 except that the aggregation nanoparticles 1 were changed to aggregation nanoparticles 22.

[Evaluation 1]
(Change in luminance over time)
Dispersions in which aggregated nanoparticles 1 to 20 were dispersed in PBS such that the particle molar concentration was 0.01 mmol / L were prepared. The fluorescence intensities at an excitation wavelength of 450 nm and a fluorescence maximum wavelength of 630 nm were measured for the dispersion immediately after adjustment and the dispersion after storage for 7 days at room temperature using a fluorometer (Hitachi High Technologies, Inc .; F-7000). The change in luminance over time was evaluated based on the following criteria.
AA: “brightness after storage for 7 days at room temperature” / “brightness immediately after dispersion preparation” is 95% or more; BB: “brightness after storage for 7 days at room temperature” / “brightness immediately after dispersion preparation” is 80 % Or more and less than 95% CC: “brightness after storage for 7 days at room temperature” / “brightness immediately after dispersion preparation” is 70% or more and less than 80% DD: “brightness after storage for 7 days at room temperature” / “ "Brightness immediately after dispersion preparation" is less than 70%

[Example of experiment]
(Preprocessing)
In order to evaluate the fluorescent labeling materials 1 to 21 prepared in the above Examples and Comparative Examples, fluorescent staining was performed using a tissue array slide (Cosmo Bio Inc .; CB-A 712). The deparaffinized tissue array slide was washed with water, and was subjected to activation treatment by autoclaving at 121 ° C. in 10 mM citric acid buffer solution (pH 6.0) for 15 minutes. The activated tissue array slide was washed with phosphate buffer, and blocking treatment was performed using phosphate buffer containing 1% of BSA for 1 hour.

(Staining: fluorescent labeling materials 1 to 20)
The anti-HER2 rabbit monoclonal antibody (Ventana Co., Ltd .; 4B5 was adjusted to 0.05 nM using PBS containing 1% of BSA) and reacted overnight at 4 ° C. against the blocking tissue array slide mentioned above. The tissue array slide was washed with PBS and then reacted with the biotin-modified secondary antibody prepared in Preparation Example 2 diluted with PBS containing 1% of BSA to 6 μg / mL for 30 minutes at room temperature. After washing the tissue array slide after reaction with antibody with PBS, fluorescent labeling materials 1 to 20 diluted to 0.02 nM with PBS containing 1% of BSA and neutral environment (pH 6.9 to 7.4, respectively) Reaction was performed for 3 hours under room temperature conditions Each tissue array slide after reaction was washed with PBS.

(Staining: fluorescent labeling material 21)
The fluorescent labeling material 15 diluted to 0.02 nM with PBS containing 1% of BSA and the above-mentioned blocking-treated tissue array slide are reacted for 3 hours under neutral conditions (pH 6.9 to 7.4) at room temperature conditions I did. Each tissue array slide after reaction was washed with PBS.

(Post-processing, brightness observation)
For each tissue array slide after the staining, the operation of immersing in pure ethanol for 5 minutes was repeated 4 times to carry out washing and dehydration. Subsequently, an operation of immersing in xylene for 5 minutes was performed four times to perform clearing. Finally, a sealing agent (manufactured by Merck & Co., Inc., Entelanu) was used for sealing treatment to obtain a tissue array slide for observation.

With respect to each tissue array slide which has been subjected to the encapsulation process, irradiation of excitation light and observation and imaging of fluorescence emission were performed using a fluorescence microscope (manufactured by Olympus, DP 73). At this time, the wavelength of excitation light was set to 575 to 600 nm using the optical filter for excitation light provided in a fluorescence microscope, and the wavelength of fluorescence to be observed was set to 612 to 692 nm using the optical filter for fluorescence.

The intensity of excitation light at the time of observation with a fluorescence microscope and image photography was such that the irradiation energy in the vicinity of the center of the field of view was 900 W / cm ² . The exposure time at the time of photographing the image was adjusted in a range where the luminance of the image was not saturated, and was set to, for example, 4000 μsec.

Photographing was performed at 400 ×, and the obtained image was subjected to image processing using image processing software “Image J” (open source). The brightness score of 100 cells was measured by Find Maxima (detection of maximum point) which is the processing menu of Image J, and the average value was calculated.

The luminance was calculated by designating one particle region in the acquired image. Mean values and standard deviations were calculated for the bright spots of 100 particles.

[Evaluation 2]
(Light resistance)
The observation tissue array slide prepared in the experimental example is irradiated with a UV lamp of 365 nm and 8 W for 1 hour, and the average luminance value per one bright spot before and after irradiation is determined, and the average luminance after irradiation relative to the average luminance value before irradiation The rate of decrease of the value was calculated, and the light resistance was evaluated based on the following criteria.
AA: Average luminance decrease after UV lamp irradiation is less than 5% BB: Average luminance decrease of particles after UV lamp irradiation is 5% or more but less than 10% CC: Average luminance value of particles after UV lamp irradiation Decrease: 10% or more and less than 15% DD: The decrease in average brightness of particles after UV lamp irradiation is 15% or more

[Evaluation 3]
(Uneven brightness)
In the image acquired about the tissue array slide for observation produced by the experiment example, the brightness nonuniformity was evaluated by calculating | requiring the standard deviation about the luminescent point of 100 particle | grains, and evaluating by the following references | standards.
AA: standard deviation of 100 bright spot luminances is less than 10% BB: standard deviation of 100 bright spot luminances is 10% or more, less than 20% CC: standard deviation of 100 bright spot luminances is 20 % Or more and less than 30% DD: The standard deviation of the brightness of 100 bright spots is 30% or more For each aggregated nanoparticle and fluorescent label, it is shown in Table 2 below.

Claims (11)

1. Aggregated nanoparticles formed by the aggregation of aggregation-induced luminescent molecules,
   The aggregated nanoparticles have a hydrophilic group on the surface of the particles,
   Aggregated nanoparticles, wherein the particle size variation coefficient of the aggregated nanoparticles is 30% or less.
2. The aggregated nanoparticles, wherein the hydrophilic group has one / nm ² or more on the particle surface, agglomeration nanoparticles according to claim 1.
3. The aggregated nanoparticle according to claim 1 or 2, wherein an average particle diameter of the aggregated nanoparticle is 1 nm or more and 100 nm or less.
4. The aggregated nanoparticle according to any one of claims 1 to 3, wherein the aggregation induced light emitting molecule does not have a carbon / carbon double bond which does not contribute to the formation of a ring structure.
5. Aggregated nanoparticles according to any of the preceding claims, having a central core.
6. A fluorescent labeling material comprising the aggregated nanoparticle according to any one of claims 1 to 5, wherein the fluorescent labeling material has a targeting ligand on the surface of the aggregated nanoparticle.
7. The target targeting ligand according to claim 6, wherein the targeting ligand is one or more molecules selected from the group consisting of an antibody, a protein having a binding property with a sugar chain, an organelle affinity substance, and a peptide. Fluorescent labeling material.
8. A fluorescent labeling material dispersion comprising the fluorescent labeling material according to claim 6 and a buffer solution.
9. The method for producing aggregated nanoparticles according to any one of claims 1 to 4, comprising a step (A) of bringing a solution of aggregation-induced luminescent molecules into contact with a poor solvent to aggregate the aggregation-induced luminescent molecules. .
10. The aggregation nano according to claim 9, wherein the step (A) is a step of bringing a solution of the aggregation inducing luminescent molecule into contact with a poor solvent in the presence of a central nucleus to aggregate the aggregation inducing luminescent molecule. Method of producing particles.
11. A method for producing a fluorescent labeling material, comprising the step of binding a targeting ligand to the aggregated nanoparticle according to any one of claims 1 to 5.