JP2023109473A - fluorescent nanoparticles - Google Patents

Abstract

To provide a fluorescent nanoparticles with good dispersibility.SOLUTION: A fluorescent nanoparticle is provided, comprising a nanoparticle containing a resin and fluorescent dye encapsulated in the resin, a sugar with a carboxyl group or salt thereof bound to the nanoparticle, a binding substance bound to the nanoparticle via the sugar and destined to bind to a target substance.SELECTED DRAWING: Figure 1

Description

The present invention relates to fluorescent nanoparticles.

In pathological diagnosis, immunohistochemistry (IHC) and in situ hybridization (ISH), which are called immunohistochemistry (IHC) and in situ hybridization (ISH), are performed to target molecules for confirming the expression of molecular information in specimens, and functional abnormalities such as gene and protein expression abnormalities are detected. Observations are being made to diagnose For immunostaining, for example, a dye staining method using an enzyme (DAB staining, etc.) is used.

However, staining with an enzyme label such as DAB staining has the problem that the staining concentration is greatly affected by environmental conditions such as temperature and time, so it is difficult to estimate the actual amount of antigen etc. from the staining concentration. Therefore, in immunological observation in pathological diagnosis, a fluorescent labeling method using fluorescent labeling is also performed instead of staining with enzyme labeling.

This method is characterized by superior quantification compared to DAB staining. The fluorescence labeling method measures the amount of the antigen by staining the target antigen with an antibody modified with a fluorescent dye and observing the stain.

Conventionally, staining solutions containing fluorescent nanoparticles, which are obtained by directly binding streptavidin as a substance that binds to a target substance to resin particles containing a fluorescent dye, have been known for use in fluorescent labeling. For example, US Pat. No. 6,200,001 discloses streptavidin-conjugated fluorescent nanoparticles.

Japanese Patent Publication No. 2008-543982

In order to perform high-precision staining using the fluorescent nanoparticles described in Patent Document 1, it is preferable that the fluorescent nanoparticles do not aggregate and have good dispersibility. It is an object of the present invention to provide fluorescent nanoparticles with good dispersibility.

Fluorescent nanoparticles according to an embodiment of the present invention include nanoparticles containing a resin and a fluorescent dye encapsulated in the resin, a sugar having a carboxyl group or a salt thereof bonded to the nanoparticles, and and a binding substance that binds to a target substance and is bound to the nanoparticles by means of a binding substance.

According to the present invention, fluorescent nanoparticles having good dispersibility can be provided.

FIG. 1A is a schematic diagram showing fluorescent nanoparticles, and FIG. 1B is a schematic diagram showing an example of labeling with fluorescent nanoparticles. FIG. 2 shows a method for producing fluorescent nanoparticles according to an embodiment of the present invention. 3A to 3C are diagrams showing the results of staining using fluorescent nanoparticles according to Examples, and FIGS. 3D and 3E are diagrams showing the results of staining using fluorescent nanoparticles according to Comparative Examples.

[Fluorescent nanoparticles]
FIG. 1A is a diagram schematically showing fluorescent nanoparticles 10 according to an embodiment of the present invention. Fluorescent nanoparticles 10 are composed of nanoparticles 40 containing resin 20 and fluorescent dye 30 encapsulated in resin 20, sugar 50 having a carboxyl group or a salt thereof bound to nanoparticles 40, and nanoparticles via sugar 50. 40 and a binding substance 60 that binds to the target substance 100 (see FIG. 1B). The fluorescent nanoparticles 10 are used for fluorescently staining (fluorescently labeling) an observation target.

FIG. 1B is a schematic diagram showing an example of labeling with fluorescent nanoparticles 10. FIG. In FIG. 1B, antigen in cell 70 is bound by primary antibody 80 and primary antibody 80 is bound by secondary antibody 90 . A secondary antibody 90 is modified with a target substance 100 .

The binding substance 60 of the fluorescent nanoparticles 10 binds to the target substance 100 described above, thereby labeling an observation target (for example, an antigen). Since the label by the fluorescent nanoparticles 10 can be observed as a bright spot, it is suitable for quantitative evaluation of the observed object. If such fluorescent nanoparticles 10 have poor dispersibility and tend to aggregate, the bright spots are observed in an aggregated state, which hinders quantitative evaluation. Each component of the fluorescent nanoparticles will be described below.

(Nanoparticles)
Nanoparticles are the matrix of fluorescent nanoparticles. The nanoparticles contain a matrix resin and a fluorescent dye.

The type of resin that constitutes the nanoparticles is not particularly limited. For example, the following thermoplastic resins or thermosetting resins can be used as resins constituting the nanoparticles. Examples of thermoplastic resins include polystyrene, polyacrylonitrile, polyfuran, or similar resins. Examples of thermosetting resins include polyxylene, polylactic acid, glycidyl methacrylate, melamine resins, polyureas, polybenzoguanamines, polyamides, phenolic resins, polysaccharides or the like.

Among these resins, melamine resins and urea resins are particularly preferred. In particular, the melamine resin is preferable because it can suppress the elution of the fluorescent dye encapsulated in the nanoparticles even by treatments such as dehydration, penetration, and encapsulation using an organic solvent such as xylene.

Nanoparticles have sugars and binding substances directly or indirectly on their surface. The nanoparticles are therefore preferably provided with functional groups for binding them. As such a functional group, the same functional group as in the technical field to which the present invention belongs can be used for binding various substances, but for example, an epoxy group or an amino group is preferable.

The method for preparing nanoparticles having functional groups is not particularly limited. (co)polymerizing the (co)monomers in the above, or after synthesizing the thermoplastic resin or thermosetting resin, treating the functional groups of the resin monomer units constituting it with reagents to form the predetermined functional groups A method of conversion can be used.

An example of an embodiment in the case of producing nanoparticles using a thermoplastic resin is to produce nanoparticles of a polystyrene-based resin having epoxy groups on the surface by copolymerizing styrene and glycidyl methacrylate as monomers. An embodiment, or an embodiment in which styrene carboxylic acid or styrene sulfonic acid is copolymerized with styrene to produce polystyrene resin nanoparticles having carboxylic acid or sulfonic acid on the surface, or an amino sulfonic acid is copolymerized with styrene. An embodiment in which polystyrene-based resin nanoparticles having amino groups on the surface are produced by using a method is included. In addition, the epoxy group of the glycidyl methacrylate can be converted to an amino group by a predetermined treatment.

On the other hand, in an embodiment of producing nanoparticles using a thermosetting resin, a melamine resin raw material (for example, MX-035 manufactured by Sanwa Chemical Co., Ltd.) is used as a monomer and copolymerized to obtain a melamine-based Included are embodiments that produce nanoparticles of resin.

(Fluorescent dye)
A fluorescent dye is encapsulated in the nanoparticles. The fluorescent dye is encapsulated in the nanoparticles by polymerizing the above resin monomer in a solvent containing the fluorescent dye.

It is believed that the fluorescent dyes encapsulated in the nanoparticles are bound to the nanoparticles by physical or chemical forces.

The amount of fluorescent dye is preferably 1% to 10% with respect to the total weight of the fluorescent nanoparticles. If the amount of fluorescent dye is too small, sufficient fluorescence cannot be ensured against the autofluorescence of tissues and cells, and color fading during imaging becomes a problem. Further, when the amount of the fluorescent dye is large, concentration quenching occurs due to intermolecular interaction between fluorescent molecules, resulting in a decrease in luminance.

Examples of fluorescent dyes include rhodamine-based dye molecules, BODIPY-based dye molecules, squarylium-based dye molecules, aromatic hydrocarbon-based dye molecules, semiconductor nanoparticles, or combinations thereof.

Fluorescent dyes such as rhodamine-based dye molecules are preferable because they have relatively high light resistance, and among them, perylene, pyrene, and perylene diimide belonging to aromatic hydrocarbon-based dye molecules are preferable.

Examples of rhodamine dye molecules include 5-carboxy-rhodamine, 6-carboxy-rhodamine, 5,6-dicarboxy-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine, Texas Red, Spectrum Red, LD700 PERCHLORATE, Derivatives thereof and the like are included.

Examples of BODIPY-based dye molecules include BODIPY FL, BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 6 30/650, BODIPY 650/665 (manufactured by Invitrogen), derivatives thereof and the like are included.

Examples of squarylium-based dye molecules include SRfluor 680-Carboxylate, 1,3-Bis[4-(dimethylamino)-2-hydroxyphenyl]-2,4-dihydroxycyclobutenediylium dihydroxide, bis, 1,3-Bis[4-( dimethylamino ) phenyl]-2,4-dihydroxycyclobutenediylium dihydroxide, bis, 2-(4-(Diethylamino)-2-hydroxyphenyl)-4-(4-(diethyliminio)-2-hydroxycyclohexa-2,5 -dienylidene) -3-oxocyclobut -1-enolate, 2-(4-(Dibutylamino)-2-hydroxyphenyl)-4-(4-(dibutylimino)-2-hydroxycyclohexa-2,5-dienylidene)-3-oxocyclobut-1-enolate, 2-( 8-Hydroxy-1,1,7,7-tetramethyl-1,2,3,5,6,7-hexahydropyrido[3,2,1-ij]quinolin-9-yl)-4-(8-hydroxy- 1,1,7,7-tetramethyl-2,3,6,7-tetrahydro-1H-pyrido[3,2,1-ij]quinolinium-9(5H)-ylidene)-3-oxocyclobut-1-enolate, Derivatives thereof and the like are included.

Examples of aromatic hydrocarbon dye molecules include N,N-Bis-(2,6-diisopropylphenyl)-1,6,7,12-(4-tert-butylphenyl)-perylene-3,4,9, 10-tetracarbonacid diimide, N,N'-Bis (2,6-diisopropylphenyl)-1,6,7,12-tetraphenylene-3,4: 9,10-tetracarboxdiimide, N,N'-Bis(2,6- diisopropylphenyl)perylene-3,4,9,10-bis(dicarbimide), 16,N,N'-Bis(2,6-dimethylphenyl)perylene-3,4,9,10-tetracarboxylic diimide, 4,4'- [(8,16-Dihydro-8,16-dioxodibenzo[a,j]perylene-2,10-diyl)dioxy]dibutyric acid, 2,10-Dihydroxy-dibenzo[a,j]perylene-8,16-dione , 2,10-Bis(3-aminopropoxy)dibenzo[a,j]perylene-8,16-dione, 3,3′-[(8,16-Dihydro-8,16-dioxodibenzo[a,j]perylene- 2,10-diyl)dioxy]dipropylamine, 17-BIS(Octyloxy)Anthra[9,1,2-cde-]Benzo[RST]Pentaphene-5-10-Dione, Octadecanoicacid, 5,10-dihydro-5,10 -dioxoanthra[9,1,2-cde]benzo[rst]pentaphene-16,17-diylester, Dihydroxydibenzothrone, Benzenesulfonic acid, 4,4',4'',4'''-[[2,9-bis[ 2,6-bis(1-methylethyl)phenyl]-1,2,3,8,9,10-hexahydro-1,3,8,10-tetraoxoanthra[2,1,9-def: 6,5,10 -d'e'f']diisoquinoline-5,6,12,13-tetrayl]tetrakis(oxy)]tetrakis-, Benzeneethanaminium, 4,4',4'',4'''-[[2,9- bis[2,6-bis(1-methylethyl)phenyl]-1,2,3,8,9,10-hexahydro-1,3,8,10-tetraoxoanthra[2,1,9-def:6,5 , 10-d'e'f']diisoquinoline-5,6,12,13-tetrayl]tetrakis(oxy)]tetrakis[N,N,N-trimethyl-], derivatives thereof and the like.

The type of semiconductor that constitutes the semiconductor nanoparticles is not particularly limited as long as it can emit fluorescence. Semiconductors constituting semiconductor nanoparticles are, for example, II-VI group compound semiconductors, III-V group compound semiconductors, or IV group semiconductors. Examples of semiconductors that make up the semiconductor nanoparticles include CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si and Ge.

(sugar)
Sugars are attached to the surface of the nanoparticles. As shown in FIG. 1A, the sugar 50 has a hydroxyl group (—OH), which is presumed to be a factor in obtaining fluorescent nanoparticles with good dispersibility.

Also, in this embodiment, the sugar has a carboxyl group or a salt thereof. A carboxyl group or a salt thereof can be negatively charged depending on the conditions. Thereby, a repulsive force can be generated between the fluorescent nanoparticles. This is also presumed to be a factor in obtaining fluorescent nanoparticles with good dispersibility.

The carboxyl group or salt thereof also serves to bind sugar 50 to nanoparticle 40 and bind substance 60 to sugar 50 (see FIG. 1A). Specifically, a bond is formed between the carboxyl group of the sugar or its salt and the functional group of the nanoparticle/binding substance. Examples of bonds include amide bonds.

The type of sugar is not particularly limited as long as it has a carboxyl group or a salt thereof. Examples of sugars having a carboxyl group or a salt thereof include carboxyalkyldextran, carboxymethyldextran, carboxyethyldextran, carboxymethylcellulose, carboxymethylstarch, carboxymethylchitin, carboxymethylchitosan, succinylchitosan, succinylcarboxymethylchitosan, or these. contains salt of

(binding substance)
As shown in FIG. 1A, fluorescent nanoparticles 10 according to this embodiment have binding substances 60 bound to nanoparticles 40 via sugars 50 . The type of binding substance 60 is not particularly limited as long as it can bind to the target substance 100 directly or indirectly bound to the observation target.

From the viewpoint of sufficient binding to the target substance, the amount of the binding substance is preferably about 50 to 1000 per fluorescent nanoparticle molecule, depending on the particle size and the size of the binding substance.

Examples of binding substances include avidin, streptavidin, neutravidin, antibodies, nucleic acids.

Examples of combinations of target substances and binding substances that bind thereto include biotin-avidin, biotin-streptavidin, biotin-neutravidin, antigen-antibody, nucleic acid-nucleic acid, antibody-nucleic acid combinations. The binding between the target substance and the binding substance is preferably specific binding. Here, the specific binding, which can be interpreted as a general term in the technical field to which the present invention belongs, is defined by the association constant (K _A ) or its reciprocal dissociation constant (K _D ). is also possible.

That is, the binding substance used in the present invention preferably has a binding constant (K _A ) for the target substance of the object to be stained in the range of 1×10 ⁵ to 1×10 ¹⁵ . If the binding constant (K _A ) is within this range, the target substance can be treated as a substance that specifically binds to the binding substance.

(Average particle size)
The average particle diameter of the fluorescent nanoparticles is preferably 30 to 300 nm, more preferably 40 to 200 nm, from the viewpoint of enabling suitable observation of bright spots even with a general-purpose fluorescence microscope. If the average particle diameter exceeds 300 nm, the number of bright spots per cell decreases during observation after staining, making it difficult to observe the bright spots. This is because the number of bright spots increases, making it difficult to observe the bright spots.

In addition, the average particle diameter of the fluorescent nanoparticles 10 is preferably 30 nm or more from the viewpoint of improving the dispersibility (the viewpoint that PdI, which is an index of dispersibility described later, is 0.1 or less), It is more preferably 40 nm or more.

The average particle size can be obtained by measuring the long axis of each particle (100 or more particles) shown in an image (SEM image) taken with a scanning electron microscope and taking the average value.

(effect)
The fluorescent nanoparticles according to the present embodiment have high dispersibility because sugars having a carboxyl group or a salt thereof are bound to the surfaces of the nanoparticles. Therefore, by using the fluorescent nanoparticles according to the present embodiment, it becomes possible to perform fluorescent staining (fluorescent labeling) with high precision, and it is possible to observe or quantify the observation target with high precision.

Hereinafter, the invention according to this embodiment will be described in detail with reference to examples, but the invention according to this embodiment is not limited to these examples.

[Production of fluorescent nanoparticles]
FIG. 2 schematically shows a method for producing fluorescent nanoparticles according to an example. The production of fluorescent nanoparticles will be described with reference to FIG.

The production of fluorescent nanoparticles was carried out as follows.

(Preparation of seed particles)
To a solution obtained by dissolving 4.4 mg of perylene diimide, which is a fluorescent dye, in 20 mL of water, 5 of "Emulgen (registered trademark) 430" (polyoxyethylene oleyl ether, manufactured by Kao Corporation), which is an emulsifier for emulsion polymerization, was added. 2 mL of a mass % aqueous solution was added. After heating this solution to 70° C. while stirring it on a hot stirrer, 0.14 g of solid content of “Nikalac MX-035” (manufactured by Nippon Carbide Industry Co., Ltd.), which is a raw material for melamine resin, is added to this solution. rice field. Furthermore, 0.70 mL of a 10% by mass aqueous solution of dodecylbenzenesulfonic acid (manufactured by Kanto Kagaku Co., Ltd.), which is an acid catalyst, was added to this solution, heated to 70°C, stirred for 50 minutes, and further heated to 90°C. The mixture was stirred for 20 minutes to synthesize a melamine resin.

The resin particles were separated from the resulting resin particle dispersion, and the resin particles were washed to remove excess resin raw materials and impurities such as fluorescent dyes adhering to the resin particles as follows. The above dispersion is centrifuged at 20000 G for 90 minutes in a centrifuge (Micro refrigerated centrifuge 3740 manufactured by Kubota Corporation), and after removing the supernatant, ultrapure water is added to the separated particles, and ultrasonic irradiation is performed. Went and re-dispersed. The treatment by centrifugation, supernatant removal and redispersion in ultrapure water was repeated five times. The obtained resin particles were referred to as seed particles 1. The seed particles had an average particle size of 20 nm and a coefficient of variation of the particle size of 12%.

(Production of fluorescent nanoparticles aiming at 50 nm)
Emulgen (registered trademark) 430 (polyoxyethylene oleyl ether, manufactured by Kao Corporation), an emulsifier for emulsion polymerization, was added to a solution obtained by adding 1.9 mg of perylene diimide, a fluorescent dye, to 20 mL of water and dissolving it. 2 mL of a 5% by mass aqueous solution of was added. After raising the temperature of this solution to 70° C. while stirring it on a hot stirrer, 5.0×10 ¹⁴ seed particles 1 are added to this solution, and the resulting dispersion liquid is a raw material for melamine resin, “Nicalac MX- 035" (manufactured by Nippon Carbide Industry Co., Ltd.) was added in an amount of 0.06 g as a solid content. This dispersion was mixed with a 2.47% by mass aqueous solution of sulfamic acid (manufactured by Kanto Kagaku Co., Ltd.) as an acid catalyst and a 10% by mass aqueous solution of dodecylbenzenesulfonic acid as an acid catalyst at a ratio of 1:3. 0.70 mL of the acid solution obtained above was added, heated to 70° C. and stirred for 50 minutes, and further heated to 90° C. and stirred for 20 minutes to synthesize a melamine resin.

The resin particles were separated from the resulting resin particle dispersion, and the resin particles were washed to remove excess resin raw materials and impurities such as fluorescent dyes adhering to the resin particles as follows. The above dispersion is centrifuged at 20000 G for 90 minutes in a centrifuge (Micro refrigerated centrifuge 3740 manufactured by Kubota Corporation), and after removing the supernatant, ultrapure water is added to the separated particles, and ultrasonic irradiation is performed. Went and re-dispersed. The treatment by centrifugation, supernatant removal and redispersion in ultrapure water was repeated five times. In this way, nanoparticles 40 encapsulating the fluorescent dye 30 were obtained as shown in the upper part of FIG.

[Measurement of average particle size of fluorescent nanoparticles]
The dispersion of fluorescent nanoparticles was allowed to stand overnight on the substrate at room temperature to dry, and then observed and imaged using an SEM (S-4800, manufactured by Hitachi High-Tech Co., Ltd.). The particle diameter of 1000 arbitrary particles was measured from the obtained image, and the average particle diameter and the coefficient of variation of the particle diameter were calculated. The nanoparticles obtained as described above had a particle diameter of 53.6 nm and a coefficient of variation of the particle diameter of 12%.

(Production of fluorescent nanoparticles aimed at 65 nm)
In the above "(Production of fluorescent nanoparticles aimed at 50 nm)", the amount of fluorescent dye perylene diimide was 2.5 mg, the amount of 5% by mass Emulgen 430 aqueous solution was 2.6 mL, and the amount of Nicalac MX-035 was 0.5 mg. Fluorescent nanoparticles were made in the same manner, except that they were used in 08g. The nanoparticles had an average particle size of 65.9 nm and a variation coefficient of 10%, as measured by the same method as above.

(Production of fluorescent nanoparticles aimed at 80 nm)
In the above "(Production of Fluorescent Nanoparticles Aimed at 50 nm)", the amount of the fluorescent dye perylene diimide was 3.0 mg, the amount of the 5% by mass Emulgen 430 aqueous solution was 3.2 mL, and the amount of Nicalac MX-035 was 0.2 mL. Fluorescent nanoparticles were produced in the same manner except that 1 g was used. The nanoparticles had an average particle size of 83 nm and a variation coefficient of 9%, which were measured in the same manner as above.

(Production of fluorescent nanoparticles aimed at 130 nm)
In the above "(Production of fluorescent nanoparticles aimed at 50 nm)", the amount of the fluorescent dye perylene diimide was 4.9 mg, the amount of the 5 mass% aqueous solution of Emulgen 430 was 5.2 mL, and the amount of Nicalac MX-035 was 0.9 mg. Fluorescent nanoparticles were made in the same way, except that 16 g was used. The nanoparticles had an average particle size of 132.6 nm and a variation coefficient of 9%, which were measured by the same method as above.

Surface modification of the fluorescent nanoparticles was performed as follows.

Example 1 Production of Dextran×Streptavidin-Modified Nanoparticles The middle part of FIG. It shows how it is converted. Nanoparticles in such a state were obtained as follows.

First, 1 mg of carboxymethyl dextran (CMD, manufactured by Meito Sangyo Co., Ltd.) was dissolved in 1 ml of MES buffer. Next, 11.5 mg of N-hydroxysuccinimide and 19.2 mg of 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride were added and stirred at room temperature. Thus, the carboxyl groups of CMD were activated with NHS.

Next, 1 mg of the nanoparticles produced above was added to CMD having an activated carboxyl group and stirred at room temperature. By doing so, an amide bond is formed between the NHS-activated carboxyl group and the amino group derived from the melamine resin on the surface of the nanoparticles, and the dextran is bound to the surface of the nanoparticles. rice field. It is considered that those of the NHS-activated carboxyl groups that did not bind to the nanoparticles are present on the surfaces of the nanoparticles as shown in the middle of FIG.

Binding Substance Binding The bottom row of FIG. 2 shows a fluorescent nanoparticle 10 having a binding substance 60 bound to the nanoparticle via a sugar 50 . Such fluorescent nanoparticles were obtained as follows.

The nanoparticle reaction solution obtained above was centrifuged at 15000 rpm for 20 minutes, the supernatant was removed, and only the sediment was recovered. Then, 1 mL of MES buffer was added, centrifugation was performed again at 15000 rpm for 20 minutes, and the supernatant was removed to collect only the sediment. After repeating this operation twice, 1 mL of acetate buffer was added to the precipitate, 0.15 mg of streptavidin was added, and the mixture was stirred at room temperature. In this way, an amide bond was formed between the NHS-activated carboxyl group and the amino group of streptavidin, and streptavidin was bound to the nanoparticles. After that, 1 mL of Tris-HCl buffer was added, centrifugation was performed again at 15000 rpm for 20 minutes, and the supernatant was removed to collect only the sediment. After repeating this operation twice, the fluorescent nanoparticles of Example 1 were obtained by storing in PBS containing 1% BSA.

Example 2 Production of Cellulose x Streptavidin-Modified Nanoparticles Production of dextran-modified nanoparticles in Example 1 was carried out in the same manner, except that CMD was changed to 1 mg of carboxymethyl cellulose (CMC, manufactured by Tokyo Chemical Industry Co., Ltd.). .

Example 3 Production of Dextran x Nucleic Acid-Modified Nanoparticles In the production of dextran-modified nanoparticles in Example 1, after CMD binding, after washing with MES buffer, 30-mer Poly (dC ) was added to 2 uM and stirred at room temperature for 1 hour. After that, 1 mL of Tris-HCl buffer was added, centrifugation was performed again at 15000 rpm for 20 minutes, and the supernatant was removed to collect only the sediment. After repeating this operation twice, the fluorescent nanoparticles of Example 3 were obtained by storing in PBS containing 1% BSA.

(Fluorescent nanoparticles of Comparative Example 1)
Comparative Example 1 was fluorescent nanoparticles to which sugars and binding substances were not bound.

(Fluorescent nanoparticles of Comparative Example 2)
The fluorescent nanoparticles of Comparative Example 2 differ from the fluorescent nanoparticles of Example 1 in that they have polyethylene glycol instead of sugar 50 . Such fluorescent nanoparticles of Comparative Example 2 were obtained as follows.

Binding of Polyethylene Glycol 1 mg of nanoparticles was suspended in 1 mL of pure water, mixed with 20 μL of 1,2-Bis(2-aminoethoxy)ethane (BAEE), and reacted at 70° C. for 1 hour.

Next, this reaction solution was centrifuged at 15,000 rpm for 20 minutes, the supernatant was removed, and only the sediment was recovered. After that, 1 mL of water was added, centrifugation was performed again at 15000 rpm for 20 minutes, and the supernatant was removed to collect only the sediment. This operation was carried out two more times for a total of three water washings. Next, 1 mL of THF was added to the sediment, centrifugation was performed again at 15000 rpm for 20 minutes, and the supernatant was removed to collect the sediment.

The resulting fluorescent nanoparticles were adjusted to 3 nmol/L using THF.

NHS-PEG12-maleimide was mixed with the nanoparticle solution to a final concentration of 10 mmol/L, and reacted at room temperature for 1 hour.

This reaction solution was centrifuged at 15,000 rpm for 20 minutes, the supernatant was removed, and only the sediment was recovered. After that, PBS containing 2 mmol/L of EDTA was added to disperse the sediment. Then, centrifugation was performed again at 15000 rpm for 20 minutes to remove the supernatant and collect only the sediment. The nanoparticles were further washed three times by a series of operations from dispersing the sediment to centrifugation. Then, nanoparticles were obtained in which the maleimide or NHS groups were present on the particle surface. 1 mL of phosphate buffer was added to the precipitate, 0.15 mg of streptavidin was added, and the mixture was stirred at room temperature. By doing so, an amide bond was formed between the maleimide group or NHS group and the amino group of streptavidin, and streptavidin was bound to the nanoparticles. Then, 1 mL of phosphate buffer was added, centrifugation was performed again at 15000 rpm for 20 minutes, the supernatant was removed, and only the sediment was recovered. After repeating this operation twice, the fluorescent nanoparticles of Comparative Example 2 were obtained by storing in PBS containing 1% BSA.

[Evaluation of fluorescent nanoparticles]
The dispersibility of the fluorescent nanoparticles of Examples and Comparative Examples obtained as described above was evaluated as follows.

(Evaluation of dispersibility by PdI)
For each of the fluorescent nanoparticles of Examples and Comparative Examples, PdI was measured by a dynamic light scattering method using Zetasizer Nano manufactured by Malvern Panalytical. PdI is a dimensionless index indicating the spread of the particle size distribution represented by the following formula, and can be used to evaluate dispersibility.

PdI = (standard deviation (σ) / average particle size (μ)) ²

When the particle size variation of the fluorescent nanoparticles is small, PdI indicates the degree of dispersibility of the particles, and the smaller the value, the more the particles are dispersed without aggregation. In general, when PdI is 0.1 or less, the dispersibility of particles is considered to be good.

PdI was specifically measured as follows.
The dispersion of fluorescent nanoparticles was diluted with 10 mM phosphate buffer (pH 7.2), and 750 uL of the diluted solution was added to a disposable cell (DTS1070) manufactured by Malvern Panalytical. The disposable cell was set in Zetasizer Nano manufactured by the same company, and the measurement was performed at a measurement angle of 173°. The number of measurements was 3, and the average value and standard deviation of the 3 measurements were calculated.

Table 1 is a table showing the relationship between PdI measured as described above and the average particle size of particles measured by SEM.

As can be seen from Table 1, the fluorescent nanoparticles of Examples 1, 2, and 3 had a PdI of 0.1 or less at any average particle size, and had good dispersibility. This indicates the degree of dispersion in a neutral buffer solution used in actual immunostaining, and suggests the possibility of less aggregation during immunostaining. Moreover, from the results of Examples 1 and 2, it was found that the type of sugar is not limited to a specific type, and particles with good dispersibility can be obtained as long as the sugar is carboxylated. Furthermore, from the results of Examples 1 and 3, it was found that even if the binding substance 60 is not limited to proteins, and nucleic acids are modified, particles with good dispersibility can be obtained.

On the other hand, in Comparative Example 1, the PdI was much higher than 0.1 at any particle size, suggesting poor dispersibility in a neutral buffer solution. In addition, in the fluorescent nanoparticles of Comparative Example 2, the PdI was 0.1 or less only when the average particle diameter was 132.6 nm, and the dispersibility tended to be improved by surface modification with PEG. However, the results were not sufficient.

In general, the smaller the average particle diameter, the larger the specific surface area of the particles, which makes them more likely to agglomerate. In the fluorescent nanoparticles surface-modified with PEG of Comparative Example 2, when the average particle diameter was 90 nm or less, PdI exceeded 0.1 and the particles aggregated. Even if the diameter was 90 nm or less, the PdI was 0.1 or less, and the particles hardly aggregated.

(Evaluation of dispersibility by immunostaining)
Fluorescent immunostaining was performed using the fluorescent nanoparticles of Examples and Comparative Examples to evaluate the dispersibility of the fluorescent nanoparticles.

Specifically, immunostaining was performed by the following procedure.

<<IHC staining using fluorescent nanoparticles>>
An antibody reagent (staining reagent for pathological diagnosis) containing fluorescent nanoparticles and a biotin-labeled secondary antibody prepared as follows was prepared, and immunostaining was performed using this staining reagent.

<Preparation of biotin-modified secondary antibody>
Secondary antibodies used in Comparative Examples 1 and 2 and Examples 1 and 2 were prepared as follows.
First, 50 μg of anti-rabbit IgG antibody was dissolved in a 50 mmol/L Tris-HCl solution (pH 7.5). A DTT (dithiothretol) solution was mixed with the solution to a final concentration of 3 mmol/L. The solution was then reacted at 37°C for 30 minutes. The DTT-reduced secondary antibody was then purified using a desalting column. 200 μL of the total purified antibody was dissolved in a 50 mmol/L Tris-HCl solution (pH 7.5) to obtain an antibody solution. On the other hand, a linker reagent "(+)-Biotin-PEG ₆ -NH-Mal" (manufactured by PurePEG, product number 2461006-250) having a spacer length of 30 angstroms was added to 0.4 mmol/ml using DMSO. Adjusted to be L. 8.5 μL of this solution was added to the antibody solution, mixed and allowed to react at 37° C. for 30 minutes.

This reaction solution was subjected to a desalting column "Zeba Spin Desalting Columns" for purification. The absorption of the desalted reaction solution at a wavelength of 300 nm was measured with a spectrophotometer (Hitachi "F-7000") to calculate the amount of protein contained in the reaction solution. The reaction solution was adjusted to 250 μg/mL with a 50 mmol/L Tris solution, and this solution was used as a biotin-labeled secondary antibody solution.

<Preparation of nucleic acid-modified secondary antibody>
The secondary antibody used in Example 3 was produced as follows.
First, 50 μg of anti-rabbit IgG antibody was dissolved in a 50 mmol/L Tris-HCl solution (pH 7.5). A DTT (dithiothretol) solution was mixed with the solution to a final concentration of 3 mmol/L. The solution was then reacted at 37°C for 30 minutes. The DTT-reduced secondary antibody was then purified using a desalting column. 200 μL of the total purified antibody was dissolved in a 50 mmol/L Tris-HCl solution (pH 7.5) to obtain an antibody solution. On the other hand, 30-mer Poly (dG) labeled with maleimide at the 5' end was adjusted to 0.4 mmol/L using DMSO. 8.5 μL of this solution was added to the antibody solution, mixed and allowed to react at 37° C. for 30 minutes.

This reaction solution was subjected to a desalting column "Zeba Spin Desalting Columns" for purification. The absorption of the desalted reaction solution at a wavelength of 300 nm was measured with a spectrophotometer (Hitachi "F-7000") to calculate the amount of protein contained in the reaction solution. The reaction solution was adjusted to 250 μg/mL with a 50 mmol/L Tris solution, and this solution was used as a labeled secondary antibody solution.

<Fluorescent immunostaining method>
(1) Deparaffinization Step Immunostaining and morphological observation staining of human breast cancer-derived cultured cells ZR-75-1 were performed using the above biotin-labeled secondary antibody and the like as follows. As a slide for staining, HER2 IHC positive control slide (manufactured by Pathology Institute, hereinafter referred to as section slide) was used. The sectioned slides were deparaffinized.

(2) Activation Treatment Step After deparaffinization of the section slide, washing was performed by substituting with water. Antigen retrieval was performed by autoclaving the washed section slides in a 10 mmol/L citrate buffer (pH 6.0) at 121° C. for 15 minutes. After the activation treatment, the section slides were washed with PBS, and the washed section slides were blocked with PBS containing 1% BSA for 1 hour.

(3) Immunostaining process (3-1) Primary antibody reaction A solution of Roche's "anti-HER2 rabbit monoclonal antibody (4B5)" was allowed to react with the above-described blocked section slide at 4°C overnight. rice field. Section slides after the reaction were washed with PBS.
(3-2) Secondary antibody reaction After washing the section slide subjected to the primary antibody reaction with PBS, the biotin-labeled secondary antibody or nucleic acid-labeled secondary antibody diluted to 2 μg/mL with PBS containing 1% BSA and reacted at room temperature for 30 minutes. Section slides after the reaction were washed with PBS.
(3-3) Reaction with Fluorescent Nanoparticles The above-described fluorescent nanoparticles diluted to 0.12 nmol/L with PBS containing 1% BSA were added to the section slides subjected to the secondary antibody reaction at a neutral pH. The reaction was carried out for 2 hours under ambient conditions (pH 6.9 to 7.4) and room temperature. Section slides after the reaction were washed with PBS.

(4) Morphological Observation Staining Step After immunostaining, hematoxylin staining was performed. Hematoxylin staining was performed by staining the immunostained sections with Mayer's hematoxylin solution for 5 minutes. Section slides were then washed under running water for 3 minutes.

(5) Encapsulation Treatment Step The sections that had undergone the immunostaining step and the morphological observation staining step were washed and dehydrated by immersing them in pure ethanol for 5 minutes four times. Subsequently, an operation of immersion in xylene for 5 minutes was performed four times to carry out penetration. Finally, a mounting medium (“Marinol” manufactured by Muto Kagaku Co., Ltd.) was used to mount the tissue section to obtain a slide for observation.

(6) Observation A predetermined excitation light was applied to the slide that had undergone the encapsulation process to cause fluorescence to be emitted. The slide in that state was observed and photographed with a fluorescence microscope ("BX53" manufactured by Olympus) and a digital camera for microscope ("DP80" manufactured by Olympus). The wavelength of the excitation light was set to 575-600 nm by passing it through an optical filter. The wavelength of fluorescence to be observed was also set to 612 to 692 nm by passing through an optical filter. The conditions for the excitation wavelength during microscopic observation and image acquisition were such that the irradiation energy near the center of the field of view was 900 W/cm ² at excitation of 580 nm. The exposure time during image acquisition was arbitrarily set (for example, set to 4000 μsec) so as not to saturate the brightness of the image.

The imaging results are shown in FIGS. 3A to 3E. 3A, 3B, and 3C show the imaging results when using the fluorescent nanoparticles of Examples 1, 2, and 3, respectively, and FIGS. 3D and 3E show the imaging results when using the fluorescent nanoparticles of Comparative Examples 1 and 2, respectively. shows the results.

In Figures 3A, 3B, and 3C, no aggregation of bright spots due to aggregation of fluorescent nanoparticles was observed. On the other hand, as indicated by the arrow in FIG. 3E, aggregation of bright spots was observed when the fluorescent nanoparticles of the comparative example were used. Since FIG. 3D shows the particles before surface modification, no fluorescence was detected.

The fluorescent nanoparticles according to the present embodiment are useful for fluorescence imaging and the like because they have good dispersibility.

10 fluorescent nanoparticles 20 resin 30 fluorescent dye 40 nanoparticles 50 sugar 60 binding substance 70 cells 80 primary antibody 90 secondary antibody 100 target substance

Claims (6)

Nanoparticles containing a resin and a fluorescent dye encapsulated in the resin;
a sugar having a carboxyl group or a salt thereof bound to the nanoparticles;
a binding substance that binds to a target substance and is bound to the nanoparticles via the sugar;
having
fluorescent nanoparticles. Fluorescent nanoparticles according to claim 1, wherein the resin is melamine resin or urea resin. Fluorescent nanoparticles according to claim 1 or 2, wherein the sugar is carboxymethyldextran, carboxymethylcellulose, carboxymethylstarch, carboxymethylchitin, carboxymethylchitosan, succinylchitosan, succinylcarboxymethylchitosan or salts thereof. Fluorescent nanoparticles according to any one of claims 1 to 3, wherein the binding substance is avidin, streptavidin, neutravidin, an antibody or a nucleic acid. Fluorescent nanoparticles according to any one of claims 1 to 4, wherein PdI is 0.1 or less. The fluorescent nanoparticles according to any one of claims 1 to 5, having an average particle size of 30 nm or more and 300 nm or less.