Fluorescent nanoparticles

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

Technical Field

[0001] The present invention relates to fluorescent nanoparticles.

Background Art

[0002] In pathological diagnosis, staining targeting molecules for confirming the expression of molecular information of specimens, such as immunohistochemistry (IHC) and in situ hybridization (ISH), is performed to diagnose functional abnormalities such as abnormal expression of genes and proteins. For immunohistochemistry, for example, a dye staining method (such as DAB staining) using an enzyme is used.

[0003] However, staining by enzyme labeling such as DAB staining has a problem that it is difficult to estimate the actual amount of antigens and the like from the staining concentration because the staining concentration is greatly affected by environmental conditions such as temperature and time. Therefore, in immunological observation in pathological diagnosis, a fluorescence labeling method using a fluorescence label is also performed instead of staining by enzyme labeling.

[0004] This method is characterized by being superior in quantification compared to DAB staining. The fluorescence labeling method measures the amount of antigen by staining and observing the target antigen using an antibody modified with a fluorescent dye.

[0005] Conventionally, as a material used for fluorescence labeling, a staining solution containing fluorescent nanoparticles in which streptavidin as a substance that binds to a target substance is directly bound to resin particles containing a fluorescent dye is known. For example, Patent Document 1 discloses fluorescent nanoparticles to which streptavidin is bound.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] In order to perform highly accurate staining using fluorescent nanoparticles as described in Patent Document 1 above, it is preferable that the fluorescent nanoparticles do not aggregate and have good dispersibility. An object of the present invention is to provide fluorescent nanoparticles having good dispersibility.

Means for Solving the Problems

[0008] The 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 a binding substance that binds to a target substance and is bonded to the nanoparticles via the sugar.

Advantages of the Invention

[0009] According to the present invention, fluorescent nanoparticles having good dispersibility can be provided.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

Embodiments for Carrying Out the Invention

[0011] [Fluorescent Nanoparticles] FIG. 1A is a diagram schematically showing fluorescent nanoparticles 10 according to an embodiment of the present invention. The fluorescent nanoparticles 10 include nanoparticles 40 containing a resin 20 and a fluorescent dye 30 encapsulated in the resin 20, a sugar 50 having a carboxyl group or a salt thereof bonded to the nanoparticles 40, and a binding substance 60 bonded to the nanoparticles 40 via the sugar 50 and binding to a target substance 100 (see FIG. 1B). The fluorescent nanoparticles 10 are used for fluorescently staining (fluorescently labeling) an observation target.

[0012] FIG. 1B is a schematic diagram showing an example of labeling with the fluorescent nanoparticles 10. In FIG. 1B, a primary antibody 80 is bound to an antigen in a cell 70, and a secondary antibody 90 is bound to the primary antibody 80. The secondary antibody 90 is modified with a target substance 100.

[0013] The binding substance 60 of the fluorescent nanoparticles 10 binds to the above-mentioned target substance 100 to label an observation target (for example, an antigen). Since the labeling by the fluorescent nanoparticles 10 can be observed as bright spots, it is suitable for quantitatively evaluating the observation target. 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. Hereinafter, each component of the fluorescent nanoparticles will be described.

[0014] (Nanoparticles) The nanoparticles are the matrix of the fluorescent nanoparticles. The nanoparticles include a resin serving as a matrix and a fluorescent dye.

[0015] The type of the resin constituting the nanoparticles is not particularly limited. For example, as the resin constituting the nanoparticles, the following thermoplastic resins or thermosetting resins can be used. Examples of the thermoplastic resins include polystyrene, polyacrylonitrile, polyfuran, or resins similar thereto. Examples of the thermosetting resins include polyxylene, polylactic acid, glycidyl methacrylate, melamine resin, polyurethane, polybenzoguanamine, polyamide, phenol resin, polysaccharides, or resins similar thereto.

[0016] Among these resins, melamine resins and urea resins are particularly preferred. In particular, melamine resins are preferable in that they can suppress the elution of fluorescent dyes encapsulated in nanoparticles even by treatments such as dehydration, penetration, and encapsulation using organic solvents such as xylene.

[0017] The 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 when binding various substances in the technical field to which the present invention pertains can be used. For example, epoxy groups or amino groups are preferable.

[0018] The method for preparing nanoparticles having functional groups is not particularly limited. For example, as a monomer for synthesizing a thermoplastic resin or a thermosetting resin constituting the nanoparticles, a (co)monomer having a predetermined functional group in the side chain in advance is (co)polymerized, or after the synthesis of the thermoplastic resin or the thermosetting resin, a method of converting the functional group of the resin monomer unit constituting it into the predetermined functional group by reagent treatment can be used.

[0019] Examples of embodiments when producing nanoparticles using a thermoplastic resin include an embodiment of producing nanoparticles of a polystyrene-based resin having an epoxy group on the surface by copolymerizing glycidyl methacrylate as a monomer with styrene, or an embodiment of producing nanoparticles of a polystyrene-based resin having a carboxylic acid or a sulfonic acid on the surface by copolymerizing styrene carboxylic acid or styrene sulfonic acid with styrene, or an embodiment of producing nanoparticles of a polystyrene-based resin having an amino group on the surface by copolymerizing aminosulfonic acid with styrene. Note that the epoxy group of glycidyl methacrylate can also be converted into an amino group by a predetermined treatment.

[0020] On one hand, examples of embodiments for manufacturing nanoparticles using a thermosetting resin include embodiments of manufacturing melamine-based resin nanoparticles by copolymerizing a melamine resin raw material (e.g., MX-035 manufactured by Sanwa Chemical Co., Ltd.) as a monomer.

[0021] (Fluorescent dye) The fluorescent dye is encapsulated in the nanoparticles. The fluorescent dye is encapsulated in the nanoparticles by polymerizing the monomer of the above resin in a solvent containing the fluorescent dye.

[0022] The fluorescent dye encapsulated in the nanoparticles is considered to be bound to the nanoparticles by physical or chemical forces.

[0023] The amount of the fluorescent dye is preferably 1% to 10% based on the total weight of the fluorescent nanoparticles. If the amount of the fluorescent dye is small, sufficient fluorescence cannot be ensured against autofluorescence of tissues and cells, etc., and fading during imaging becomes a problem. Also, if the amount of the fluorescent dye is large, concentration quenching occurs due to intermolecular interaction between fluorescent molecules and the brightness decreases.

[0024] Examples of the fluorescent dye include rhodamine-based dye molecules, BODIPY-based dye molecules, squarylium-based dye molecules, aromatic hydrocarbon-based dye molecules, semiconductor nanoparticles, or combinations thereof.

[0025] Fluorescent dyes such as rhodamine-based dye molecules are preferable because they have relatively high light resistance. Among them, perylene, pyrene, and perylene diimide belonging to aromatic hydrocarbon-based dye molecules are preferable.

[0026] Examples of rhodamine-based 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 their derivatives.

[0027] Examples of BODIPY-based pigment 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 (manufactured by Invitrogen above), their derivatives, and the like.

[0028] Examples of squarylium-based pigment 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-(dibutyliminio)-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, their derivatives, and the like.

[0029] Examples of aromatic hydrocarbon-based pigment 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-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]perylen-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, 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, etc. are included.

[0030] The type of semiconductor constituting the semiconductor nanoparticles is not particularly limited as long as it can emit fluorescence. The semiconductor constituting the semiconductor nanoparticles is, for example, a Group II - VI compound semiconductor, a Group III - V compound semiconductor, or a Group IV semiconductor. Examples of the semiconductor constituting the semiconductor nanoparticles include CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, and Ge.

[0031] (Sugar) The sugar is bound to the surface of the nanoparticles. As shown in Fig. 1A, since the sugar 50 has a hydroxyl group (-OH), this is presumably one of the reasons for obtaining fluorescent nanoparticles with good dispersibility.

[0032] Also, 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. Thereby, a repulsive force can be generated between the fluorescent nanoparticles. This is also presumably one of the reasons for obtaining fluorescent nanoparticles with good dispersibility.

[0033] Also, the carboxyl group or a salt thereof is also useful for binding the sugar 50 to the nanoparticles 40 and for binding the binding substance 60 to the sugar 50 (see Fig. 1A). Specifically, a bond is formed between the carboxyl group or a salt thereof of the sugar and the functional group of the nanoparticles / binding substance. Examples of the bond include an amide bond.

[0034] 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.

[0035] (Binding substance) As shown in Fig. 1A, the fluorescent nanoparticle 10 according to the present embodiment has a binding substance 60 bound to the nanoparticle 40 via a sugar 50. The type of the binding substance 60 is not particularly limited as long as it can bind to a target substance 100 directly or indirectly bound to the observation target.

[0036] From the viewpoint of sufficiently binding to the target substance, the amount of the binding substance is preferably about 50 to 1000 per molecule of the fluorescent nanoparticle, although it also depends on the particle size and the size of the binding substance.

[0037] Examples of the binding substance include avidin, streptavidin, neutravidin, antibody, and nucleic acid.

[0038] Examples of the combination of the target substance and the binding substance binding 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, the specific binding can be interpreted as a general term in the technical field to which the present invention belongs. However, it can also be defined by the binding constant (K A ) or the dissociation constant (K D ), which is the reciprocal thereof.

[0039] That is, the binding substance used in the present invention has a binding constant (K A) is preferably in the range of 1×10 5 ~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.

[0040] (Average particle diameter) From the viewpoint that the average particle diameter of the fluorescent nanoparticles enables favorable observation of the bright spots even with a general-purpose fluorescence microscope, it is preferably 30 to 300 nm, more preferably 40 nm to 200 nm. When the average particle diameter exceeds 300 nm, the number of bright spots per cell decreases during the post-staining observation, making it difficult to observe the bright spots. Conversely, when the average particle diameter is less than 30 nm, the number of bright spots per cell increases, also making it difficult to observe the bright spots.

[0041] Also, from the viewpoint of improving the dispersibility (the viewpoint that the PdI, which is an index of dispersibility described later, becomes 0.1 or less), the average particle diameter of the fluorescent nanoparticles 10 is preferably 30 nm or more, and more preferably 40 nm or more.

[0042] The average particle diameter can be measured as the average value of the major axis lengths of each of the particles (100 or more) shown in the image taken by a scanning electron microscope (SEM image).

[0043] (Effect) The fluorescent nanoparticles according to the present embodiment have a high dispersibility because a sugar having a carboxyl group or its salt is bound to the surface of the nanoparticles. Therefore, by using the fluorescent nanoparticles according to the present embodiment, it becomes possible to perform highly accurate fluorescence staining (fluorescent labeling), and it also becomes possible to highly accurately observe or quantify the observation target. [Examples]

[0044] Hereinafter, the invention according to the present embodiment will be described in detail with reference to examples, but the invention according to the present embodiment is not limited by these examples.

[0045] [Production of Fluorescent Nanoparticles] Figure 2 schematically shows a method for manufacturing fluorescent nanoparticles according to an embodiment. The manufacturing of fluorescent nanoparticles will be described with reference to Figure 2.

[0046] The manufacturing of fluorescent nanoparticles was carried out as follows.

[0047] (Preparation of seed particles) To a solution obtained by dissolving 4.4 mg of perylene diimide, a fluorescent dye, in 20 mL of water, 2 mL of a 5 mass% aqueous solution of "Emulgen (registered trademark) 430" (polyoxyethylene oleyl ether, manufactured by Kao Corporation), an emulsifier for emulsion polymerization, was added. After heating this solution to 70°C while stirring on a hot stirrer, 0.14 g of "Niclac MX-035" (manufactured by Nippon Carbide Industries Co., Ltd.), a melamine resin raw material, was added to this solution as a solid content. Further, 0.70 mL of a 10 mass% aqueous solution of dodecylbenzenesulfonic acid (manufactured by Kanto Chemical Co., Inc.), an acid catalyst, was added to this solution, heated to 70°C, stirred for 50 minutes, and further heated to 90°C and stirred for 20 minutes to synthesize a melamine resin.

[0048] Separation of resin particles from the obtained dispersion of resin particles and further washing to remove impurities such as excess resin raw materials and fluorescent dyes adhering to the resin particles were carried out as follows. The above dispersion was centrifuged at 20000G for 90 minutes using a centrifuge (Micro Cooling Centrifuge 3740 manufactured by Kubota Corporation), and after removing the supernatant, ultrapure water was added to the separated particles, and ultrasonic irradiation was performed for redispersion. The processes of centrifugation, supernatant removal, and redispersion in ultrapure water were repeated 5 times. The obtained resin particles were used as seed particles 1. The average particle diameter of the seed particles was 20 nm, and the coefficient of variation of the particle diameter was 12%.

[0049] (Manufacturing of fluorescent nanoparticles targeting 50 nm) To a solution obtained by dissolving 1.9 mg of perylene diimide, a fluorescent dye, in 20 mL of water, 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. While stirring this solution on a hot stirrer, the temperature was raised to 70 °C. Then, 5.0×10 14 particles of seed particle 1 were added to this solution, and 0.06 g of "Niclac MX-035" (manufactured by Nippon Carbide Industries Co., Ltd.), which is a raw material for melamine resin, was added as a solid content to the resulting dispersion. To this 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., Inc.), which is an acid catalyst, and a 10% by mass aqueous solution of dodecylbenzenesulfonic acid, which is an acid catalyst, in a ratio of 1:3 was added, heated to 70 °C, stirred for 50 minutes, further heated to 90 °C, and stirred for 20 minutes to synthesize melamine resin.

[0050] Separation of resin particles from the resulting dispersion of resin particles and further washing to remove impurities such as excess resin raw materials and fluorescent dyes adhering to the resin particles were performed as follows. The above dispersion was centrifuged at 20000G for 90 minutes using a centrifuge (Micro Cooling Centrifuge 3740, manufactured by Kubota Corporation). After removing the supernatant, ultrapure water was added to the separated particles, and ultrasonic irradiation was performed for redispersion. The processes of centrifugation, supernatant removal, and redispersion in ultrapure water were repeated 5 times. In this way, as shown in the upper part of Figure 2, nanoparticles 40 encapsulating fluorescent dye 30 were obtained.

[0051] [Measurement of average particle diameter of fluorescent nanoparticles] The dispersion of fluorescent nanoparticles was left standing on a substrate at room temperature overnight for drying, and observation and imaging were performed using SEM (S-4800, manufactured by Hitachi High-Tech Corporation). The particle diameters of 1000 arbitrary particles were measured from the obtained images, and the average particle diameter and the coefficient of variation of the particle diameter were calculated. The particle diameter of the nanoparticles obtained as described above was 53.6 nm, and the coefficient of variation of the particle diameter was 12%.

[0052] (Production of fluorescent nanoparticles targeting 65 nm) In the above “(Production of Fluorescent Nanoparticles Aimed at 50 nm)”, fluorescent nanoparticles were prepared in the same manner except that the amount of the fluorescent dye perylene diimide was 2.5 mg, the amount of a 5% by mass aqueous solution of Emulgen 430 was 2.6 mL, and the amount of Niclac MX-035 was 0.08 g. The average particle diameter of the nanoparticles measured by the same method as above was 65.9 nm, and the coefficient of variation of the particle diameter was 10%.

[0053] (Production of Fluorescent Nanoparticles Aimed at 80 nm) In the above “(Production of Fluorescent Nanoparticles Aimed at 50 nm)”, fluorescent nanoparticles were prepared in the same manner except that the amount of the fluorescent dye perylene diimide was 3.0 mg, the amount of a 5% by mass aqueous solution of Emulgen 430 was 3.2 mL, and the amount of Niclac MX-035 was 0.1 g. The average particle diameter of the nanoparticles measured by the same method as above was 83 nm, and the coefficient of variation of the particle diameter was 9%.

[0054] (Production of Fluorescent Nanoparticles Aimed at 130 nm) In the above “(Production of Fluorescent Nanoparticles Aimed at 50 nm)”, fluorescent nanoparticles were prepared in the same manner except that the amount of the fluorescent dye perylene diimide was 4.9 mg, the amount of a 5% by mass aqueous solution of Emulgen 430 was 5.2 mL, and the amount of Niclac MX-035 was 0.16 g. The average particle diameter of the nanoparticles measured by the same method as above was 132.6 nm, and the coefficient of variation of the particle diameter was 9%.

[0055] Surface modification of the fluorescent nanoparticles was carried out as follows.

[0056] Example 1 Production of Dextran × Streptavidin-Modified Nanoparticles The middle part of Fig. 2 shows a state where the sugar 50 is bound to the surface of the nanoparticle 40, and further, the carboxyl group of the sugar 50 is activated by N-hydroxysuccinimide (NHS). Nanoparticles in such a state were obtained as follows.

[0057] 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. In this way, the carboxyl group of CMD was activated with NHS.

[0058] Next, 1 mg of the nanoparticles prepared above was added to the CMD having the activated carboxyl group and stirred at room temperature. By doing so, 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 nanoparticles, and dextran was bound to the surface of the nanoparticles. It is considered that among the carboxyl groups activated with NHS, those that did not bind to the nanoparticles exist on the surface of the nanoparticles as shown in the middle row of Fig. 2.

[0059] Binding of the conjugate The lower row of Fig. 2 shows the fluorescent nanoparticles 10 having the conjugate 60 bound to the nanoparticles via the sugar 50. Such fluorescent nanoparticles were obtained as follows.

[0060] 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 centrifugation was performed 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, 0.15 mg of streptavidin was added, and stirred at room temperature. By doing so, an amide bond was formed between the carboxyl group activated with NHS and the amino group of streptavidin, and streptavidin was bound to the nanoparticles. Then, 1 mL of Tris-HCl buffer was added, and centrifugation was performed 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.

[0061] Example 2 Production of Cellulose×Streptavidin-Modified Nanoparticles In the production of the dextran-modified nanoparticles of Example 1, they were prepared in the same manner except that CMD was changed to 1 mg of carboxymethyl cellulose (CMC, manufactured by Tokyo Chemical Industry Co., Ltd.).

[0062] Example 3 Production of Dextran×Nucleic Acid-Modified Nanoparticles In the production of the dextran-modified nanoparticles of Example 1, after the binding of CMD and after the washing operation with MES buffer, 30-mer Poly (dC) modified with an amino group at the 5'-end was added to a concentration of 2 μM and stirred at room temperature for 1 hour. Then, 1 mL of Tris-HCl buffer was added, and centrifugation was performed again at 15,000 rpm for 20 minutes to remove the supernatant and collect only the precipitate. After repeating this operation twice, the fluorescent nanoparticles of Example 3 were obtained by storing them in PBS containing 1% BSA.

[0063] (Fluorescent Nanoparticles of Comparative Example 1) Comparative Example 1 was fluorescent nanoparticles to which no sugar and binding substance were bound.

[0064] (Fluorescent Nanoparticles of Comparative Example 2) The fluorescent nanoparticles of Comparative Example 2 are different 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.

[0065] 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.

[0066] Next, the reaction solution was centrifuged at 15,000 rpm for 20 minutes to remove the supernatant and only the precipitate was collected. Then, 1 mL of water was added, and centrifugation was performed again at 15,000 rpm for 20 minutes to remove the supernatant and only the precipitate was collected. This operation was performed two more times, for a total of three water washings. Next, 1 mL of THF was added to the precipitate, and centrifugation was performed again at 15,000 rpm for 20 minutes to remove the supernatant and the precipitate was collected.

[0067] The obtained fluorescent nanoparticles were adjusted to 3 nmol / L using THF.

[0068] NHS-PEG12-maleimide was mixed into the nanoparticle solution to a final concentration of 10 mmol / L and reacted at room temperature for 1 hour.

[0069] This reaction solution was centrifuged at 15,000 rpm for 20 minutes to remove the supernatant and only the precipitate was collected. Then, PBS containing 2 mmol / L of EDTA was added to disperse the precipitate. And then, centrifugation was performed again at 15,000 rpm for 20 minutes to remove the supernatant and only the precipitate was collected. The washing of the nanoparticles by a series of operations from the dispersion treatment of the precipitate to centrifugation was performed three more times. And nanoparticles with maleimide or NHS groups 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 it was stirred at room temperature. By doing so, an amide bond was formed between the maleimide group or NHS group and the amino group possessed by streptavidin, and streptavidin was bound to the nanoparticles. Then, 1 mL of phosphate buffer was added, and centrifugation was performed again at 15,000 rpm for 20 minutes to remove the supernatant and only the precipitate was collected. After repeating this operation twice, the fluorescent nanoparticles of Comparative Example 2 were obtained by storing them in PBS containing 1% BSA.

[0070] [Evaluation of Fluorescent Nanoparticles] The dispersibility of the fluorescent nanoparticles of the examples and comparative examples obtained as described above was evaluated as follows.

[0071] (Evaluation of Dispersibility by PdI) For each of the fluorescent nanoparticles in the examples and comparative examples, PdI was measured by dynamic light scattering using a 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 for the evaluation of dispersibility.

[0072] PdI = (standard deviation (σ) / average particle size (μ)) 2

[0073] When the variation in the particle size 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. Generally, if PdI is 0.1 or less, it is considered that the dispersibility of the particles is good.

[0074] The measurement of PdI was specifically carried out as follows. The dispersion of the fluorescent nanoparticles was diluted with 10 mM phosphate buffer (pH 7.2), and 750 μL was added to a disposable cell (DTS1070) manufactured by Malvern Panalytical. The disposable cell was set on the Zetasizer Nano manufactured by the same company, and the measurement was carried out with the measurement angle set at 173°. The number of measurements was 3 times, and the average value and standard deviation of the 3 times were calculated.

[0075] Table 1 is a table showing the relationship between PdI measured as described above and the average particle size of the particles by SEM.

[0076]

Table 1

[0077] As can be seen from Table 1, for the fluorescent nanoparticles of Examples 1, 2, and 3, PdI was 0.1 or less for any average particle diameter, and the dispersibility was good. This represents the degree of dispersion in the neutral buffer used in actual immunostaining, suggesting that it is less likely to aggregate during immunostaining. Also, 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 Example 1 and Example 3, it was found that the binding substance 60 is not limited to proteins, and particles with good dispersibility can be obtained even when nucleic acids are modified.

[0078] On the other hand, in Comparative Example 1, PdI exceeded 0.1 for any particle size, suggesting poor dispersibility in the neutral buffer. Also, for the fluorescent nanoparticles of Comparative Example 2, PdI was 0.1 or less only when the average particle diameter was 132.6 nm, and the dispersibility tended to improve by surface-modifying with PEG, but the result was that the dispersibility was not sufficient.

[0079] Generally, the smaller the average particle diameter of particles, the larger the specific surface area, and thus the easier it is to aggregate. For the PEG surface-modified fluorescent nanoparticles of Comparative Example 2, PdI exceeded 0.1 when the average particle diameter was 90 nm or less, and the particles had aggregated, but for the fluorescent nanoparticles of the examples, even when the average particle diameter was 90 nm or less, PdI was 0.1 or less and the particles hardly aggregated.

[0080] (Evaluation of dispersibility by immunostaining) Fluorescent immunostaining was performed using the fluorescent nanoparticles of the examples and comparative examples, and the dispersibility of the fluorescent nanoparticles was evaluated.

[0081] Specifically, immunostaining was performed according to the following procedure.

[0082] ≪IHC staining using fluorescent nanoparticles≫ An antibody reagent (staining reagent for pathological diagnosis) having fluorescent nanoparticles and a biotin-labeled secondary antibody prepared as follows was prepared, and immunostaining was performed using this staining reagent.

[0083] <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 a 50 mmol / L Tris-HCl solution (pH 7.5). A DTT (dithiothretol) solution was mixed with this solution so that the final concentration became 3 mmol / L. Then, the solution was reacted at 37°C for 30 minutes. Thereafter, the secondary antibody reduced with DTT was purified using a desalting column. 200 μL of the total amount of the 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-PEG6-NH-Mal” (manufactured by 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.

[0084] 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 (“F-7000” manufactured by Hitachi) 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 solution of the biotin-labeled secondary antibody.

[0085] <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 (dithiothretol) solution was mixed with this solution to a final concentration of 3 mmol / L. Then, the solution was reacted at 37 °C for 30 minutes. Thereafter, the secondary antibody reduced with DTT was purified using a desalting column. 200 μL of the total amount of the purified antibody was dissolved in 50 mmol / L Tris-HCl solution (pH 7.5) to obtain an antibody solution. On the other hand, 30-mer Poly (dG) labeled at the 5'-end with maleimide 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.

[0086] This reaction solution was subjected to purification using a desalting column "Zeba Spin Desalting Columns". The absorbance at a wavelength of 300 nm of the desalted reaction solution was measured with a spectrophotometer ("F-7000" manufactured by Hitachi) to calculate the amount of protein contained in the reaction solution. The reaction solution was adjusted to 250 μg / mL with 50 mmol / L Tris solution, and this solution was used as a labeled secondary antibody solution.

[0087] <Fluorescent immunostaining method> (1) Deparaffinization treatment step Using the above biotin-labeled secondary antibody, etc., immunostaining and morphological observation staining of human breast cancer-derived cultured cells ZR-75-1 were performed as follows. As a slide for staining, a HER2 IHC positive control slide (manufactured by Pathology Institute, hereinafter referred to as a section slide) was used. This section slide was subjected to deparaffinization treatment.

[0088] (2) Activation treatment step After the section slide was subjected to deparaffinization treatment, washing was performed to replace it with water. The washed section slide was autoclaved at 121 °C for 15 minutes in 10 mmol / L citrate buffer (pH 6.0) to perform antigen activation treatment. The section slide after activation treatment was washed with PBS, and blocking treatment was performed on the washed section slide for 1 hour using PBS containing 1% BSA.

[0089] (3) Immunostaining process (3-1) Primary antibody reaction A solution of "Anti-HER2 rabbit monoclonal antibody (4B5)" manufactured by Roche was reacted with the above-mentioned blocked section slides at 4 °C overnight. The section slides after the reaction were washed with PBS. (3-2) Secondary antibody reaction After washing the section slides that had undergone the primary antibody reaction with PBS, they were reacted with the above-mentioned biotin-labeled secondary antibody or nucleic acid-labeled secondary antibody diluted to 2 μg / mL with PBS containing 1% BSA at room temperature for 30 minutes. The section slides after the reaction were washed with PBS. (3-3) Reaction with fluorescent nanoparticles To the section slides that had undergone the secondary antibody reaction, the above-mentioned fluorescent nanoparticles diluted to 0.12 nmol / L with PBS containing 1% BSA were reacted under neutral pH environment (pH 6.9 - 7.4) and room temperature conditions for 2 hours. The section slides after the reaction were washed with PBS.

[0090] (4) Morphological observation staining process 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.

[0091] (5) Mounting process For the sections that had completed the immunostaining process and the morphological observation staining process, the operation of immersing in pure ethanol for 5 minutes was performed 4 times for washing and dehydration. Subsequently, the operation of immersing in xylene for 5 minutes was performed 4 times for clearing. Finally, using a mounting agent ("Marinol" manufactured by Mutoh Chemical Co., Ltd.), the tissue sections were mounted to obtain slides for observation.

[0092] (6) Observation The slide that had completed the encapsulation process was irradiated with a predetermined excitation light to emit fluorescence. The slide in that state was observed and imaged using a fluorescence microscope (Olympus "BX53") and a digital camera for microscope (Olympus "DP80"). The wavelength of the above excitation light was set to 575 - 600 nm by passing it through an optical filter. Also, the wavelength of the fluorescence to be observed was set to 612 - 692 nm by passing it through an optical filter. The conditions of the excitation wavelength 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 when excited 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 did not saturate, and then imaging was performed.

[0093] The imaging results are shown in FIGS. 3A to 3E. FIGS. 3A, 3B, and 3C respectively show the imaging results when using the fluorescent nanoparticles of Examples 1, 2, and 3, and FIGS. 3D and 3E respectively show the imaging results when using the fluorescent nanoparticles of Comparative Examples 1 and 2.

[0094] In FIGS. 3A, 3B, and 3C, aggregation of bright spots due to aggregation of fluorescent nanoparticles was not observed. On the other hand, as indicated by the arrow in FIG. 3E, when using the fluorescent nanoparticles of the comparative example, aggregation of bright spots was observed. Since FIG. 3D shows the particles before surface modification, no fluorescence was detected at all.

Industrial Applicability

[0095] The fluorescent nanoparticles according to this embodiment have good dispersibility and are thus useful for fluorescence imaging and the like.

Explanation of Signs

[0096] 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

1. Nanoparticles comprising a resin and a fluorescent dye encapsulated in the resin, a sugar having a carboxyl group or a salt thereof, which is bound to the nanoparticles, a binding substance that binds to a target substance, which is bound to the nanoparticles via the sugar, and having, fluorescent nanoparticles.

2. The fluorescent nanoparticles according to Claim 1, wherein the resin is a melamine resin or a urea resin.

3. 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.

4. 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.

5. The fluorescent nanoparticles according to any one of Claims 1 to 4, wherein PdI is 0.1 or less.

6. The fluorescent nanoparticles according to any one of Claims 1 to 5, wherein the average particle diameter is 30 nm or more and 300 nm or less.

Images