JP7704040B2 - Fluorescent Nanoparticles - Google Patents

Description

The present invention relates to fluorescent nanoparticles.

In pathological diagnosis, a technique called immunohistochemistry (IHC) or in situ hybridization (ISH) is used to stain molecules to confirm the expression of molecular information in the specimen, and observations are made to diagnose functional abnormalities such as abnormal expression of genes or proteins. For example, an enzyme-based dye staining method (DAB staining, etc.) is used for immunohistochemistry.

However, staining using enzyme labels such as DAB staining has the problem that the staining concentration is greatly affected by environmental conditions such as temperature and time, making it difficult to estimate the actual amount of antigens, etc. from the staining concentration. For this reason, in immunological observation in pathological diagnosis, a fluorescent labeling method using fluorescent labels is also used instead of staining using enzyme labels.

This method has the advantage of being more quantitative than DAB staining. The fluorescent labeling method measures the amount of antigen by staining the target antigen using an antibody modified with a fluorescent dye and observing it.

Conventionally, a staining solution containing fluorescent nanoparticles in which streptavidin, a substance that binds to a target substance, is directly bound to resin particles containing a fluorescent dye has been known for use in fluorescent labeling. For example, Patent Document 1 discloses fluorescent nanoparticles bound to streptavidin.

Special Publication No. 2008-543982

In order to perform highly accurate dyeing using fluorescent nanoparticles such as those described in Patent Document 1, it is preferable that the fluorescent nanoparticles do not aggregate and have good dispersibility. The object of the present invention is to provide fluorescent nanoparticles that have good dispersibility.

The fluorescent nanoparticles according to the 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 bound to the nanoparticles, and a binding substance that binds to a target substance and is bound to the nanoparticles via the sugar.

The present invention can provide fluorescent nanoparticles with good dispersibility.

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 is a diagram showing 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 an embodiment of the present invention, and FIGS. 3D and 3E are diagrams showing the results of staining using fluorescent nanoparticles according to a comparative example.

[Fluorescent nanoparticles]
1A is a schematic diagram of a fluorescent nanoparticle 10 according to an embodiment of the present invention. The fluorescent nanoparticle 10 has a nanoparticle 40 including a resin 20 and a fluorescent dye 30 encapsulated in the resin 20, a sugar 50 having a carboxyl group or a salt thereof bound to the nanoparticle 40, and a binding substance 60 that binds to a target substance 100 (see FIG. 1B) bound to the nanoparticle 40 via the sugar 50. The fluorescent nanoparticle 10 is used to fluorescently stain (fluorescently label) an object to be observed.

Figure 1B is a schematic diagram showing an example of labeling with fluorescent nanoparticles 10. In Figure 1B, a primary antibody 80 binds to an antigen in a cell 70, and a secondary antibody 90 binds to the primary antibody 80. The 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 the object of observation (e.g., an antigen). The labeling by the fluorescent nanoparticles 10 can be observed as bright spots, making it suitable for quantitative evaluation of the object of observation. If such fluorescent nanoparticles 10 have poor dispersibility and tend to aggregate, the bright spots will be observed in an aggregated state, which will hinder quantitative evaluation. Each component of the fluorescent nanoparticles will be described below.

(Nanoparticles)
The nanoparticles are the base material for the fluorescent nanoparticles. The nanoparticles contain a resin that serves as a matrix and a fluorescent dye.

The type of resin constituting the nanoparticles is not particularly limited. For example, the following thermoplastic resins or thermosetting resins can be used as the resin 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 resin, polyurea, polybenzoguanamine, polyamide, phenolic resin, polysaccharide, or similar resins.

Among these resins, melamine resin and urea resin are particularly preferred. Melamine resin is particularly preferred because it can suppress the elution of fluorescent dyes encapsulated in nanoparticles even when subjected to processes such as dehydration, clearing, and encapsulation using an organic solvent such as xylene.

Nanoparticles have sugars and binding substances directly or indirectly on their surfaces. Therefore, it is preferable that the nanoparticles have functional groups for binding these. As such functional groups, the same functional groups as those used to bind various substances in the technical field to which the present invention pertains can be used, but for example, epoxy groups or amino groups are preferred.

The method for preparing nanoparticles having functional groups is not particularly limited, but for example, a method can be used in which a (co)monomer having a specific functional group on its side chain is (co)polymerized as a monomer for synthesizing the thermoplastic resin or thermosetting resin that constitutes the nanoparticles, or a method can be used in which, after the thermoplastic resin or thermosetting resin is synthesized, the functional group of the resin monomer unit that constitutes it is treated with a reagent to convert it to the specific functional group.

Examples of embodiments in which nanoparticles are produced using a thermoplastic resin include an embodiment in which polystyrene-based resin nanoparticles having epoxy groups on the surface are produced by copolymerizing glycidyl methacrylate as a monomer with styrene, an embodiment in which styrene carboxylic acid or styrene sulfonic acid is copolymerized with styrene to produce polystyrene-based resin nanoparticles having carboxylic acid or sulfonic acid on the surface, and an embodiment in which aminosulfonic acid is copolymerized with styrene to produce polystyrene-based resin nanoparticles having amino groups on the surface. The epoxy groups of the glycidyl methacrylate can also be converted to amino groups by a specified treatment.

On the other hand, an example embodiment for producing nanoparticles using a thermosetting resin includes an embodiment in which nanoparticles of a melamine-based resin are produced by copolymerizing a melamine resin raw material (e.g., MX-035 manufactured by Sanwa Chemical Co., Ltd.) as a monomer.

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

The fluorescent dyes encapsulated in the nanoparticles are thought to be bound to the nanoparticles by physical or chemical forces.

The amount of fluorescent dye is preferably 1% to 10% of the total weight of the fluorescent nanoparticles. If the amount of fluorescent dye is small, sufficient fluorescence cannot be obtained against the autofluorescence of tissues and cells, and fading during imaging becomes an issue. Furthermore, if the amount of fluorescent dye is large, concentration quenching occurs due to intermolecular interactions between fluorescent molecules, resulting in reduced brightness.

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 dye molecules are preferred because they have relatively high light resistance, and among them, perylene, pyrene, and perylene diimide, which belong to aromatic hydrocarbon dye molecules, are preferred.

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, and derivatives thereof.

Examples of BODIPY 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 630/650, BODIPY 650/665 (all manufactured by Invitrogen), and derivatives thereof.

Examples of squarylium 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-e nolate, 2-(4-(Dibutylamino)-2-hydroxyphenyl)-4-(4-(dibutyl minio)-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, and their derivatives.

Examples of aromatic hydrocarbon dye molecules include N,N-Bis-(2,6-diisopropylphenyl)-1,6,7,12-(4-tert-butylphenoxy)-perylene-3,4,9,10-tetracarbonacid diimide, N,N'-Bis(2,6-diisopropylphenyl)-1,6,7,12-tetraphenoxyperylene-3,4:9,10-tetracarboxdiimide, N,N'-Bis(2,6-dii sopropylphenyl) 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]perylen-2,10-diyl)dioxy]dipropylamine, 1 7-BIS (Octyloxy) Anthra[9,1,2-cde-]Benzo[RST]Pentaphene-5-10-Dione, Octadecanoid, 5,10-dihydro-5,10-dioxoanthra[9,1,2-cde]benzo[rst]pentaphe ne-16,17-diylester, Dihydroxydibenzanthrone, 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-] and their derivatives.

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

(sugar)
The sugar is bound to the surface of the nanoparticle. As shown in FIG. 1A, the sugar 50 has a hydroxyl group (—OH), which is presumably one of the reasons why fluorescent nanoparticles with good dispersibility are obtained.

In addition, in this embodiment, the sugar has a carboxyl group or a salt thereof. The carboxyl group or a salt thereof can be negatively charged depending on the conditions. This can generate a repulsive force between the fluorescent nanoparticles. This is presumably one of the reasons why fluorescent nanoparticles with good dispersibility can be obtained.

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

The type of sugar is not particularly limited as long as it is a sugar having a carboxyl group or a salt thereof. Examples of sugars having a carboxyl group or a salt thereof include carboxyalkyl dextran, carboxymethyl dextran, carboxyethyl dextran, carboxymethyl cellulose, carboxymethyl starch, carboxymethyl chitin, carboxymethyl chitosan, succinyl chitosan, succinyl carboxymethyl chitosan, or salts thereof.

(binding substance)
1A, a fluorescent nanoparticle 10 according to this embodiment has a binding substance 60 bound to a nanoparticle 40 via a sugar 50. The type of binding substance 60 is not particularly limited as long as it can bind to a target substance 100 that is directly or indirectly bound to an object of observation.

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

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

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

That is, the binding substance used in the present invention preferably has a binding constant (K _A ) for the target substance possessed by the subject to be stained in the range of 1×10 ⁵ to 1×10 ^15. When 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 size 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 fluorescent microscope. If the average particle size exceeds 300 nm, the number of bright spots per cell decreases during observation after staining, making it difficult to observe the bright spots, whereas if the average particle size is less than 30 nm, the number of bright spots per cell increases, making it difficult to observe the bright spots.

In addition, from the viewpoint of improving dispersibility (PdI, an index of dispersibility described below, being 0.1 or less), the average particle size of the fluorescent nanoparticles 10 is preferably 30 nm or more, and more preferably 40 nm or more.

The average particle size can be calculated by measuring the long diameter of each particle (100 or more particles) shown in an image taken with a scanning electron microscope (SEM image) and averaging the results.

(effect)
The fluorescent nanoparticles according to the present embodiment have high dispersibility because sugars having carboxyl groups or salts thereof are bound to the surface of the nanoparticles. Therefore, by using the fluorescent nanoparticles according to the present embodiment, it is possible to perform highly accurate fluorescent staining (fluorescent labeling), and it is also possible to observe or quantify the observation target with high accuracy.

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

[Production of fluorescent nanoparticles]
2 is a schematic diagram showing a method for producing fluorescent nanoparticles according to an embodiment of the present invention. The production of fluorescent nanoparticles will be described with reference to FIG.

Fluorescent nanoparticles were produced as follows:

(Preparation of seed particles)
4.4 mg of perylene diimide, a fluorescent dye, was dissolved in 20 mL of water to obtain a solution, to which 2 mL of a 5% by mass aqueous solution of "EMULGEN (registered trademark) 430" (polyoxyethylene oleyl ether, manufactured by Kao Corporation), an emulsifier for emulsion polymerization, was added. After the solution was heated to 70°C while stirring on a hot stirrer, 0.14 g of "NIKALAC MX-035" (manufactured by Nippon Carbide Industries Co., Ltd.), a raw material for melamine resin, was added to the solution as a solid content. Furthermore, 0.70 mL of a 10% by mass aqueous solution of dodecylbenzenesulfonic acid (manufactured by Kanto Chemical Co., Ltd.), an acid catalyst, was added to the solution, 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 obtained dispersion of resin particles, and further washed to remove impurities such as excess resin raw material and fluorescent dye adhering to the resin particles, as follows. The above dispersion was centrifuged at 20,000 G for 90 minutes in a centrifuge (Kubota Corporation, Micro Refrigerated Centrifuge 3740) and the supernatant was removed. Ultrapure water was added to the separated particles, and they were redispersed by ultrasonic irradiation. The process of centrifugation, removal of the supernatant, and redispersion in ultrapure water was repeated five times. The obtained resin particles were designated as seed particles 1. The average particle size of the seed particles was 20 nm, and the coefficient of variation of the particle size was 12%.

(Production of fluorescent nanoparticles aiming at 50 nm)
1.9 mg of perylene diimide, a fluorescent dye, was added to 20 mL of water and dissolved to obtain a solution, to which 2 mL of a 5% by mass aqueous solution of "EMULGEN (registered trademark) 430" (polyoxyethylene oleyl ether, manufactured by Kao Corporation), an emulsifier for emulsion polymerization, was added. After this solution was heated to 70°C while stirring on a hot stirrer, 5.0 x 10 ¹⁴ seed particles 1 were added to this solution, and 0.06 g of "NIKALAC MX-035" (manufactured by Nippon Carbide Industries Co., Ltd.), a raw material for melamine resin, was added as a solid content to the obtained dispersion. 0.70 mL of an acid solution obtained by mixing a 2.47% by mass aqueous solution of sulfamic acid (manufactured by Kanto Chemical Co., Ltd.), an acid catalyst, and a 10% by mass aqueous solution of dodecylbenzenesulfonic acid, an acid catalyst, in a ratio of 1:3 was added to this dispersion, and the mixture was 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 obtained resin particle dispersion, and further washed to remove impurities such as excess resin raw material and fluorescent dye adhering to the resin particles, as follows. The above dispersion was centrifuged at 20,000 G for 90 minutes in a centrifuge (Kubota Corp. Micro Refrigerated Centrifuge 3740) and the supernatant was removed. Ultrapure water was added to the separated particles, and they were redispersed by ultrasonic irradiation. The process of centrifugation, removal of the supernatant, and redispersion in ultrapure water was repeated five times. In this way, nanoparticles 40 encapsulating fluorescent dye 30 were obtained, as shown in the upper part of Figure 2.

[Measuring the average particle size of fluorescent nanoparticles]
The dispersion of fluorescent nanoparticles was left to dry overnight at room temperature on the substrate, and was observed and photographed using a SEM (S-4800, Hitachi High-Technologies Corporation). The particle sizes of 1,000 random particles were measured from the obtained image, and the average particle size and the coefficient of variation of the particle size were calculated. The particle size of the nanoparticles obtained as described above was 53.6 nm, and the coefficient of variation of the particle size was 12%.

(Production of fluorescent nanoparticles targeting 65 nm)
Fluorescent nanoparticles were produced in the same manner as in the above "(Production of fluorescent nanoparticles aiming at 50 nm)" except that the amount of fluorescent dye perylene diimide was 2.5 mg, the amount of 5 mass% aqueous solution of Emulgen 430 was 2.6 mL, and the amount of Nikalac MX-035 was 0.08 g. The average particle size of the nanoparticles measured by the same method as above was 65.9 nm, and the coefficient of variation of the particle size was 10%.

(Production of fluorescent nanoparticles aiming at 80 nm)
Fluorescent nanoparticles were produced in the same manner as in the above "(Production of fluorescent nanoparticles aiming at 50 nm)" except that the amount of fluorescent dye perylene diimide was 3.0 mg, the amount of 5 mass% aqueous solution of Emulgen 430 was 3.2 mL, and the amount of Nikalac MX-035 was 0.1 g. The average particle size of the nanoparticles measured by the same method as above was 83 nm, and the coefficient of variation of the particle size was 9%.

(Production of fluorescent nanoparticles targeting 130 nm)
Fluorescent nanoparticles were produced in the same manner as in the above "(Production of fluorescent nanoparticles aiming at 50 nm)" except that the amount of fluorescent dye perylene diimide was 4.9 mg, the amount of 5% by mass aqueous solution of Emulgen 430 was 5.2 mL, and the amount of Nikalac MX-035 was 0.16 g. The average particle size of the nanoparticles measured by the same method as above was 132.6 nm, and the coefficient of variation of the particle size was 9%.

Surface modification of fluorescent nanoparticles was carried out as follows.

Example 1: Preparation of dextran x streptavidin modified nanoparticles The middle part of Fig. 2 shows a state in which sugar 50 is bound to the surface of nanoparticle 40 and the carboxyl group of sugar 50 is activated by N-hydroxysuccinimide (NHS). Nanoparticles in this state were obtained as follows.

First, 1 mg of carboxymethyldextran (CMD, 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. In this way, the carboxyl group of CMD was activated with NHS.

Next, 1 mg of the nanoparticles produced above was added to the CMD having an activated carboxyl group and stirred at room temperature. In this way, an amide bond was formed between the carboxyl group activated with NHS and the amino group derived from the melamine resin on the surface of the nanoparticle, and dextran was bound to the surface of the nanoparticle. It is considered that the carboxyl groups activated with NHS that were not bound to the nanoparticle are present on the surface of the nanoparticle, as shown in the middle of Figure 2.

2 shows fluorescent nanoparticles 10 having a binding substance 60 bound to the nanoparticle via a sugar 50. Such fluorescent nanoparticles were obtained as follows.

The reaction solution of the nanoparticles obtained above was centrifuged at 15,000 rpm for 20 minutes, the supernatant was removed, and only the precipitate was collected. Then, 1 mL of MES buffer was added, and centrifuged again at 15,000 rpm for 20 minutes, the supernatant was removed, and only the precipitate was collected. After repeating this operation twice, 1 mL of acetate buffer was added to the precipitate, and 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 carboxyl group activated by NHS and the amino group of streptavidin, and streptavidin was bound to the nanoparticles. Then, 1 mL of Tris-HCl buffer was added, and centrifuged again at 15,000 rpm for 20 minutes, the supernatant was removed, and only the precipitate was collected. After repeating this operation twice, the fluorescent nanoparticles of Example 1 were obtained by storing them in PBS containing 1% BSA.

Example 2 Production of Cellulose×Streptavidin-Modified Nanoparticles Dextran-modified nanoparticles were produced in the same manner as in Example 1, except that CMD was changed to 1 mg of carboxymethylcellulose (CMC, Tokyo Chemical Industry Co., Ltd.).

Example 3: Preparation of dextran x nucleic acid modified nanoparticles In the preparation of dextran modified nanoparticles in Example 1, after CMD binding, washing with MES buffer, 30mer Poly (dC) modified with an amino group at the 5' end was added to 2 uM and stirred at room temperature for 1 hour. Then, 1 mL of Tris-HCl buffer was added, and centrifuged again at 15,000 rpm for 20 minutes, the supernatant was removed, and only the precipitate was collected. This operation was repeated twice, and then 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 a fluorescent nanoparticle to which neither sugar nor a binding substance was 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.

Conjugation 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 precipitate was collected. After that, 1 mL of water was added, and centrifuged again at 15,000 rpm for 20 minutes, the supernatant was removed, and only the precipitate was collected. This operation was repeated two more times, for a total of three water washes. Next, 1 mL of THF was added to the precipitate, and centrifuged again at 15,000 rpm for 20 minutes, the supernatant was removed, and the precipitate was collected.

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

NHS-PEG12-maleimide was mixed into the nanoparticle solution to a final concentration of 10 mmol/L, and the mixture was allowed to react 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 precipitate was collected. Then, PBS containing 2 mmol/L of EDTA was added to disperse the precipitate. Then, centrifugation was again performed at 15,000 rpm for 20 minutes to remove the supernatant and collect only the precipitate. The nanoparticles were washed three times by a series of operations from dispersion treatment of the precipitate to centrifugation. Then, nanoparticles on which the maleimide or NHS group was present on the particle surface were obtained. 1 mL of phosphate buffer was added to this precipitate, and 0.15 mg of streptavidin was added and stirred at room temperature. In this way, 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, and centrifugation was again performed at 15,000 rpm for 20 minutes, the supernatant was removed, and only the precipitate was collected. This procedure was repeated twice, and then the fluorescent nanoparticles of Comparative Example 2 were obtained by storing them in PBS containing 1% BSA.

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

(Evaluation of Dispersibility by PdI)
For each of the fluorescent nanoparticles of the examples and comparative examples, PdI was measured by dynamic light scattering using a Zetasizer Nano manufactured by Malvern Panalytical Corp. PdI is a dimensionless index that indicates the spread of the particle size distribution expressed by the following formula, and can be used to evaluate dispersibility.

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

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

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

Table 1 shows the relationship between PdI measured as above and the average particle size 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 for all average particle sizes, and had good dispersibility. This represents the degree of dispersion in the neutral buffer used in actual immunostaining, and suggests the possibility that aggregation may be difficult during immunostaining. In addition, the results of Examples 1 and 2 show that the type of sugar is not limited to a specific type, and that particles with good dispersibility can be obtained if the sugar is carboxylated. Furthermore, the results of Examples 1 and 3 show that the binding substance 60 is not limited to proteins, and particles with good dispersibility can be obtained even if the nucleic acid is modified.

On the other hand, in Comparative Example 1, the PdI was significantly greater than 0.1 for all particle sizes, suggesting poor dispersibility in neutral buffer solutions. In addition, in the fluorescent nanoparticles of Comparative Example 2, the PdI was 0.1 or less only when the average particle size was 132.6 nm, and although the dispersibility tended to improve by surface modification with PEG, the results did not indicate that the dispersibility was sufficient.

In general, the smaller the average particle size of particles, the larger the specific surface area, and therefore the more likely they are to aggregate. In the fluorescent nanoparticles surface-modified with PEG in Comparative Example 2, when the average particle size was 90 nm or less, the PdI exceeded 0.1, causing the particles to aggregate. However, in the fluorescent nanoparticles of the Example, even when the average particle size was 90 nm or less, the PdI was 0.1 or less, and the particles hardly aggregated at all.

(Evaluation of dispersibility by immunostaining)
Fluorescent immunostaining was carried out using the fluorescent nanoparticles of the Examples and Comparative Examples, and the dispersibility of the fluorescent nanoparticles was evaluated.

Specifically, immunostaining was performed using 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 described below was prepared, and immunostaining was performed using this staining reagent.

<Preparation of biotin-modified secondary antibody>
The 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 50 mmol/L Tris-HCl solution (pH 7.5). DTT (dithiothreitol) 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 secondary antibody reduced with DTT was then purified using a desalting column. 200 μL of the total amount of purified antibody was dissolved in 50 mmol/L Tris-HCl solution (pH 7.5) to obtain an antibody solution. Meanwhile, a linker reagent "(+)-Biotin-PEG ₆ -NH-Mal" (PurePEG, product number 2461006-250) with a spacer length of 30 angstroms was adjusted to 0.4 mmol/L using DMSO. 8.5 μL of this solution was added to the antibody solution, mixed, and reacted at 37° C. for 30 minutes.

This reaction solution was purified by passing it through a desalting column "Zeba Spin Desalting Columns". The absorbance of the desalted reaction solution at a wavelength of 300 nm was measured using 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 using 50 mmol/L Tris solution, and this solution was used as a solution of biotin-labeled secondary antibody.

<Preparation of nucleic acid-modified secondary antibody>
The secondary antibody used in Example 3 was prepared as follows.
First, 50 μg of anti-rabbit IgG antibody was dissolved in 50 mmol/L Tris-HCl solution (pH 7.5). A DTT (dithiothreitol) 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 secondary antibody reduced with DTT was then purified using a desalting column. 200 μL of the total amount of purified antibody was dissolved in 50 mmol/L Tris-HCl solution (pH 7.5) to obtain an antibody solution. Meanwhile, 30mer 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 reacted at 37°C for 30 minutes.

This reaction solution was purified by passing it through a desalting column "Zeba Spin Desalting Columns". The absorbance of the desalted reaction solution at a wavelength of 300 nm was measured using 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 using 50 mmol/L Tris solution, and this solution was used as the labeled secondary antibody solution.

<Fluorescent immunostaining method>
(1) Deparaffinization process Using the biotin-labeled secondary antibody and the like, human breast cancer-derived cultured cell ZR-75-1 was subjected to immunostaining and morphological observation staining as follows. HER2 IHC Posicon Slide (manufactured by Pathology Research Institute, hereafter referred to as section slide) was used as the slide for staining. This section slide was deparaffinized.

(2) Activation Treatment Step After the section slide was deparaffinized, it was washed by replacing with water. The washed section slide was autoclaved in 10 mmol/L citrate buffer (pH 6.0) at 121° C. for 15 minutes to perform antigen activation treatment. The section slide after activation treatment was washed with PBS, and the washed section slide was subjected to blocking treatment for 1 hour using PBS containing 1% BSA.

(3) Immunostaining step (3-1) Primary antibody reaction The section slide that had been subjected to the blocking treatment described above was reacted with a solution of "anti-HER2 rabbit monoclonal antibody (4B5)" manufactured by Roche overnight at 4° C. After the reaction, the section slide was washed with PBS.
(3-2) Secondary Antibody Reaction After the section slides subjected to the primary antibody reaction were washed with PBS, they were reacted with the biotin-labeled secondary antibody or nucleic acid-labeled secondary antibody diluted to 2 μg/mL with PBS containing 1% BSA for 30 minutes at room temperature. After the reaction, the section slides were washed with PBS.
(3-3) Reaction with fluorescent nanoparticles The section slides that had been subjected to the secondary antibody reaction were reacted with the aforementioned fluorescent nanoparticles diluted to 0.12 nmol/L with PBS containing 1% BSA for 2 hours at room temperature in a neutral pH environment (pH 6.9 to 7.4). After the reaction, the section slides were washed with PBS.

(4) Morphological Observation Staining Step After immunostaining, hematoxylin staining was performed. The immunostained sections were stained with Mayer's hematoxylin solution for 5 minutes to perform hematoxylin staining. Then, the section slides were washed with running water for 3 minutes.

(5) Mounting process The sections that had been subjected to the immunostaining process and the morphological observation staining process were immersed in pure ethanol for 5 minutes four times to wash and dehydrate them. Then, they were immersed in xylene for 5 minutes four times to clear them. Finally, the tissue sections were mounted using a mounting medium ("Marinol" manufactured by Muto Chemical Industries, Ltd.) to prepare slides for observation.

(6) Observation The slide after the mounting process was irradiated with a predetermined excitation light to emit fluorescence. The slide in this state was observed and imaged using a fluorescence microscope (Olympus "BX53") and a digital camera for a microscope (Olympus "DP80"). The wavelength of the excitation light was set to 575-600 nm by passing it through an optical filter. The wavelength of the fluorescence to be observed was also set to 612-692 nm by passing it through an optical filter. The excitation wavelength conditions during microscope observation and image acquisition were such that the irradiation energy near the center of the field of view was 900 W/cm ² for excitation at 580 nm. The exposure time during image acquisition was arbitrarily set (for example, set to 4000 μs) so that the brightness of the image would not be saturated.

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

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

The fluorescent nanoparticles according to this embodiment have good dispersibility and are therefore useful for fluorescent imaging, etc.

10 Fluorescent nanoparticle 20 Resin 30 Fluorescent dye 40 Nanoparticle 50 Sugar 60 Binding substance 70 Cell 80 Primary antibody 90 Secondary antibody 100 Target substance

Claims (6)

Nanoparticles including 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 nanoparticle via the sugar;
having
Fluorescent nanoparticles. The fluorescent nanoparticles according to claim 1, wherein the resin is a melamine resin or a urea resin. The fluorescent nanoparticles according to claim 1 or 2, wherein the sugar is carboxymethyl dextran, carboxymethyl cellulose, carboxymethyl starch, carboxymethyl chitin, carboxymethyl chitosan, succinyl chitosan, succinyl carboxymethyl chitosan, or a salt thereof. The 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. The 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.