LIGHT-EMITTING NANOPARTICLES AND LIGHT-EMITTING LABELING MATERIAL FOR PATHOLOGICAL DIAGNOSIS

Abstract

The present invention addresses the problem of providing a luminescent labeling material for pathological diagnosis and luminescent nanoparticles for enabling high-sensitivity imaging that can avoid the negative impact of auto-fluorescence of cells on bioimaging. Provided are luminescent nanoparticles containing a luminescent dye, wherein the luminescent dye is a compound having a specific structure represented by Formula (1), and the luminescent nanoparticles have at least one of delayed luminesence or long Stokes shift luminescence.

Claims

1. Luminescent nanoparticles containing a luminescent dye, wherein the luminescent dye is a compound having a structure represented by the following Formula (1), and the luminescent nanoparticles have at least one of delayed luminescence or long Stokes shift luminescence. ##STR00079## in Formula (1), A represents an electron-withdrawing group; D represents an electron-donating group; L represents a linking group having a backbone containing 2 to 4 atoms; the 2 to 4 atoms are a carbon atom, an oxygen atom, a nitrogen atom, a silicon atom, a sulfur atom, a boron atom, or a phosphorus atom; and the linking group may have a hydrogen atom or a substituent.

2. The luminescent nanoparticle according to claim 1, wherein Formula (1) is represented by at least one of the following Formulas (2) to (6), ##STR00080## in Formulas (1) to (6), A represents an electron-withdrawing group; D represents an electron-donating group; X represents a cyclic saturated hydrocarbon group, a cyclic unsaturated hydrocarbon group, an aryl group or a heteroaryl group; Y and Z represent a carbon atom, an oxygen atom, a nitrogen atom, a silicon atom, a sulfur atom, a boron atom, or a phosphorus atom and may have a hydrogen atom or a substituent; and Y in Formulas (4) and (5) may be a linking group having a backbone containing two atoms.

3. The luminescent nanoparticles according to claim 1, having delayed luminescence.

4. The luminescent nanoparticles according to claim 1, having intramolecular exciplex luminescence.

5. The luminescent nanoparticles according to claim 1, having long Stokes shift luminescence.

6. The luminescent nanoparticles according to claim 2, wherein in Formulas (2) to (6), A represents “a carbonyl group which may be substituted”, “a sulfonyl group which may be substituted”, “a boryl group which may be substituted”, “a phosphine oxide group which may be substituted”, “an aryl group which may be substituted with an electron-withdrawing group”, “an electron-donating heterocyclic group substituted with a cyano group, a fluorine atom, an alkyl group substituted with a fluorine atom, or a nitrogen-containing aromatic six-membered ring-containing heterocyclic group”, or “an electron-withdrawing heterocyclic group which may be substituted”; D represents “an aryl group substituted with an electron-donating group”, “an electron-donating heterocyclic group which may be substituted with a substituent other than a cyano group, a fluorine atom, an alkyl group substituted with a fluorine atom, or a nitrogen-containing aromatic six-membered ring-containing heterocyclic group”, or “an amino group which may be substituted”; and X represents an aryl group or a heteroaryl group.

7. The luminescent nanoparticles according to claim 2, wherein in Formulas (4) to (6), A represents “a carbonyl group which may be substituted”, “a sulfonyl group which may be substituted”, “a boryl group which may be substituted”, “a phosphine oxide group which may be substituted”, “an aryl group which may be substituted with an electron-withdrawing group”, “an electron-donating heterocyclic group substituted with a cyano group, a fluorine atom, an alkyl group substituted with a fluorine atom, or a nitrogen-containing aromatic six-membered ring-containing heterocyclic group”, or “an electron-withdrawing heterocyclic group which may be substituted”; D represents “an aryl group substituted with an electron-donating group”, “an electron-donating heterocyclic group which may be substituted with a substituent other than a cyano group, a fluorine atom, an alkyl group substituted with a fluorine atom, or a nitrogen-containing aromatic six-membered ring-containing heterocyclic group”, or “an amino group which may be substituted”; X represents an aryl group or a heteroaryl group; and Y represents a carbon atom, an oxygen atom, or a silicon atom.

8. The luminescent nanoparticles according to claim 1, wherein the luminescent dye has a hydrophilic group.

9. The luminescent nanoparticles according to claim 1, containing a binder.

10. The luminescent nanoparticles according to claim 9, wherein the binder present on a surface of the luminescent nanoparticle among the binder has a hydrophilic group.

11. The luminescent nanoparticles according to claim 9, wherein the binder and the luminescent dye form a covalent bond.

12. A luminescent labeling material for pathological diagnosis, wherein a target-directed ligand is covalently bonded to a surface of the luminescent nanoparticle according to claim 1.

Description

EXAMPLES

[0134] Hereinafter, the present invention will be specifically described with examples, but the present invention is not limited thereto. In the examples, “part” or “%” is used. It indicates “part by mass” or “mass %” unless otherwise specified.

<<Luminescent Dye>>

<Luminescent Dyes Used in Comparative Example>

[0135] ##STR00071##

<Luminescent Dyes Used in Example 1>

[0136] ##STR00072## ##STR00073## ##STR00074##

<Luminescent Dyes Used in Example 2>

[0137] ##STR00075## ##STR00076##

<Luminescent Dyes Used in Example 3>

[0138] ##STR00077##

<Luminescent Dye Used in Example 4>

[0139] ##STR00078##

<<Preparation of Luminescent Nanoparticles>>

[Example 1 and Comparative Example] Preparation of Luminescent Nanoparticles Using Oil-Soluble Dyes

<Preparation of Luminescent Nanoparticles Nos. (1-1) to (1-22)>

[0140] To a solution of a luminescent dye (types and amounts added as listed in Table I) dissolved in 0.2 mL of dichloromethane, a surfactant (EMULGEN 430) was added to become 0.5 vol % by adding 20 mL of water. The solution was stirred on a hot stirrer and the temperature was raised to 40° C., after which 1.2 g of melamine resin was added.

[0141] The luminescent nanoparticles were prepared by adding 0.02 mmol of dodecylbenzenesulfonic acid to the solution. The resulting dispersion was centrifuged (20000 G for 90 minutes) to collect the particles and purified by washing with ultrapure water. The process was repeated five times by removing the supernatant after centrifugation and redispersing in ultrapure water to obtain luminescent nanoparticles Nos. (1-1) to (1-22) with an average particle size of 130 nm.

[Example 2] Preparation of Luminescent Nanoparticles Using Water-Soluble Dyes

<Preparation of Luminescent Nanoparticles Nos. (2-1) to (2-12)>

[0142] To a solution of a luminescent dye (types and amounts added as listed in Table I) dissolved in 0.2 mL of water, a surfactant (EMULGEN 409PV) was added to become 0.5 vol % by adding 20 mL of water. The solution was stirred on a hot stirrer and the temperature was raised to 80° C., and then 2.0 g of melamine resin was added. Except the change of the temperature and the amount of melamine resin, the same operation as in Example 1 was carried out to produce luminescent nanoparticles Nos. (2-1) to (2-12) with an average particle size of 130 nm.

[Example 3] Preparation of Luminescent Nanoparticles Having a Covalent Bond to a Binder

<Preparation of Luminescent Nanoparticles Nos. (3-1) to (3-4)>

[0143] The luminescent dye (types and amounts added as listed in Table I) was mixed with 48 mL of 99% ethanol, 0.6 mL of tetraethoxysilane (TEOS), 2 mL of ultrapure water, and 2.0 mL of 28 mass % ammonia water at 5° C. for 3 hours.

[0144] The mixture prepared in the above process was centrifuged at 10000 G for 20 minutes and the supernatant was removed. Ethanol was added to the precipitate to disperse the precipitate, and a rinse operation was performed to centrifuge the precipitate again. The same rinsing was repeated twice, and luminescent nanoparticles Nos. (3-1) to (3-4) (silica particles containing a luminescent dye) with an average particle size of 130 nm were prepared.

[Example 4] Preparation of Luminescent Nanoparticles Having a Covalent Bond to a Binder

<Preparation of Luminescent Nanoparticles Nos. (4-1) to (4-2)>

[0145] The luminescent nanoparticles No. (4-1) to (4-2) to (4-3) with an average particle size of 130 nm were prepared under the same conditions as in Example 1, except that the luminescent dye (types and amounts added shown in Table I) was changed.

<<Evaluation of Luminescent Nanoparticles>>

[0146] The luminescent nanoparticles with oil-soluble dyes prepared in Example 1, the luminescent nanoparticles with water-soluble dyes prepared in Example 2, and the luminescent nanoparticles a covalent bond with a binder prepared in Examples 3 and 4 were evaluated. In the evaluation of the luminescent nanoparticles, the quantum yield and luminance were measured, and their relative values were summarized in Table I. The relative values for each particle are values by setting the measured value of the luminescent nanoparticle No. 1 (1-1) to 100.

[0147] The quantum yields were measured as follows. Luminescent nanoparticles were dispersed in PBS so that the particle molar concentration was 0.01 mmol/L. The quantum yield of the dispersion was measured by excitation at the maximum absorption wavelength of each nanoparticle using an absolute PL quantum yield measurement device C9920-02 (Hamamatsu Photonics, Inc.).

[0148] The luminance was measured as follows. Luminescent nanoparticles were dispersed in PBS so that the particle molar concentration was 0.01 mmol/L. The luminance of the dispersion was measured with a fluorescence spectrophotometer (F-7000; Hitachi High-Technologies Corporation) at room temperature by excitation at the maximum absorption wavelength of each nanoparticle.

[0149] Since the luminescent nanoparticles of the present invention have solid-state luminescence, the quantum yield increased or was maintained when the amount of dye added was increased. Furthermore, since the absorbance also increased as the amount of dye added increased, the luminance increased in both cases. As a result, the luminescent nanoparticles of the present invention were superior to the luminescent nanoparticles of the comparative examples, and the effectiveness of the present invention was confirmed.

[0150] In the comparative example of luminescent nanoparticle No. (1-2), since the luminescent dye (C-1) has an aggregation-induced luminescence property, the quantum yield and luminance of the luminescent nanoparticles were increased with increasing the amount of dye added compared to No. (1-1), but the increase was limited in its range.

[0151] In the comparative example of luminescent nanoparticle No. (1-4), both quantum yield and luminance were decreased with increasing the amount of dye added compared to No. (1-3) due to concentration quenching when the amount of dye added was increased.

TABLE-US-00001 TABLE I Luminescent Added amount nanoparticles of dye Relative Relative No. Dye (μmol) quantum yield luminance Remarks 1-1 C-1 50 100 100 Comparative Example 1-2 C-1 200 155 210 Comparative Example 1-3 C-2 50 195 261 Comparative Example 1-4 C-2 200 71 141 Comparative Example 1-5 a103 50 223 301 Present Invention 1-6 a103 200 251 461 Present Invention 1-7 a106 50 270 360 Present Invention 1-8 a106 200 281 642 Present Invention 1-9 a310 50 265 377 Present Invention  1-10 a310 200 291 599 Present Invention  1-11 a89 50 309 409 Present Invention  1-12 a89 200 314 608 Present Invention  1-13 a143 50 299 400 Present Invention  1-14 a143 200 305 578 Present Invention  1-15 a153 50 345 421 Present Invention  1-16 a153 200 368 601 Present Invention  1-17 a223 50 327 406 Present Invention  1-18 a223 200 340 587 Present Invention  1-19 a254 50 401 467 Present Invention  1-20 a254 200 420 621 Present Invention  1-21 a318 50 290 381 Present Invention  1-22 a318 200 412 523 Present Invention 2-1 a116 50 301 410 Present Invention 2-2 a116 200 301 608 Present Invention 2-3 a130 50 251 374 Present Invention 2-4 a130 200 260 624 Present Invention 2-5 a128 50 245 437 Present Invention 2-6 a128 200 295 641 Present Invention 2-7 a126 50 223 360 Present Invention 2-8 a126 200 270 634 Present Invention 2-9 a131 50 260 403 Present Invention  2-10 a131 200 265 678 Present Invention  2-11 a241 50 241 398 Present Invention  2-12 a241 200 241 630 Present Invention 3-1 a320 50 294 347 Present Inventior 3-2 a320 200 315 460 Present Invention 3-3 a321 50 308 388 Present Invention 3-4 a321 200 343 500 Present Invention 4-1 a325 50 320 364 Present Invention 4-2 a325 200 350 603 Present Invention

[0152] The luminescent nanoparticles of the examples were dispersed in water, and absorption and emission spectra measurements and delayed luminescence measurements were carried out. The results are shown in Table II. In Table II, for long Stokes shift luminescence, the Stokes shift of 100 to 200 nm is indicated as “Circle”, and the Stokes shift of 200 nm or more is indicated as “Double circle”. The delayed luminescence properties were observed for all the luminescent nanoparticles in Table II. When the ratio of delayed to immediate luminescence intensity after 100 ns is 1/10 or more, it is indicated as “Double circle”, and when it is less than 1/10, it is indicated as “Circle”.

[0153] The delayed luminescence measurements were performed using a streak camera C4334 (Hamamatsu Photonics, Inc.) while the dispersion was excited by laser light.

TABLE-US-00002 TABLE II Luminescent Addedamount Long Stokes Luminance intensity nanoparticles of dye shift ratio of No. Dye (μmol) luminescence (Delayed/Immediate) Remarks 1-5 a103 50 ⊚ ⊚ Present Invention 1-7 a106 50 ⊚ ⊚ Present Invention 1-9 a310 50 ⊚ ⊚ Present Invention  1-12 a89 200 ⊚ ⊚ Present Invention  1-14 a143 200 ⊚ ⊚ Present Invention  1-16 a153 200 ⊚ ⊚ Present Invention  1-17 a223 50 ◯ ◯ Present Invention  1-19 a254 50 ◯ ◯ Present Invention  1-21 a318 50 ⊚ ⊚ Present Invention 2-1 a116 50 ⊚ ⊚ Present Invention 2-3 a130 50 ⊚ ⊚ Present Invention 2-5 a128 50 ⊚ ⊚ Present Invention 2-7 a126 50 ⊚ ⊚ Present Invention  2-10 a131 200 ⊚ ⊚ Present Invention  2-12 a241 200 ◯ ◯ Present Invention 3-1 a320 50 ◯ ◯ Present Invention 3-4 a321 200 ⊚ ⊚ Present Invention 4-1 a325 50 ⊚ ⊚ Present Invention

[0154] As shown in Table II, the long Stokes shift luminescence and delayed luminescence properties were confirmed in all the example particles. Among them, the long Stokes shift was more than 200 nm in the intramolecular exciplex dye-containing particles. Furthermore, the ratio of delayed to immediate luminescence intensity was higher for the intramolecular exciplex dye-containing particles than for the heat-active delayed fluorescent dye-containing particles.

<<Preparation of Luminescent Labeling Materials for Pathological Diagnosis>>

[Example 5] Luminescent Labeling Materials for Pathological Diagnosis Composed of Luminescent Nanoparticles Nos. (1-1) to (1-22), (2-1) to (2-12), and (4-1) to (4-2)

[0155] <Preparation of Luminescent Nanoparticles Surface-Modified with a PEG Chain Having a Maleimide Group at the End>

[0156] The above luminescent nanoparticles Nos. (1-1) to (1-22), (2-1) to (2-12), and (4-1) to (4-2), which are melamine particles containing luminescent dyes, each were taken in an amount of 0.1 mg, and dispersed in 1.5 mL of ethanol. Then, they were mixed with 2 μL of aminopropyltrimethoxysilane “LS-3150” (Shin-Etsu Chemical Co., Ltd.) and the reaction was carried out at room temperature with stirring for 8 hours to perform surface amination treatment. The concentration of luminescent nanoparticles with aminated surface was adjusted to 3 nM using PBS (phosphate buffered physiological saline) containing 2 mM of EDTA (ethylenediaminetetraacetic acid). To this solution was added the linker reagent “SM(PEG)12” (Thermo Scientific, Inc. Cat. No. 22112) and mixed to a final concentration of 10 mM, and the reaction was carried out at room temperature for 1 hour with stirring.

[0157] The reaction solution was centrifuged at 10,000 G for 20 minutes to remove the supernatant, and then PBS containing 2 mM of EDTA was added to disperse the sediment and centrifuged again under the same conditions. The luminescent nanoparticles surface-modified with a PEG chain having a maleimide group at the end were obtained by washing three times using the same procedure.

<Preparation of Streptavidin Introduced with a Thiol Group>

[0158] First, to 40 μL of an aqueous solution of streptavidin (Wako Pure Chemical Industries, Ltd.) adjusted to 1 mg/mL was added 70 μL of an aqueous solution of N-succinimidyl-S-acetyl thioacetate (SATA, Pirce Corporation) adjusted to 64 mg/mL. The reaction was allowed to proceed at room temperature for 1 hour to convert the amino group of the streptavidin to the protected thiol group (—NH—CO—CH.sub.2—S—CO—CH.sub.3).

[0159] Subsequently, hydroxylamine treatment was used to generate a free thiol group (—SH) from the protected thiol group to complete the process of introducing a thiol group (—SH) to streptavidin. The solution was then passed through a gel filtration column (Zaba Spin Desalting Columns: Funakoshi) to desalinate the solution to obtain a thiol group-introduced streptavidin.

<Preparation of Luminescent Nanoparticles Modified with Streptavidin>

[0160] The prepared luminescent nanoparticles surface-modified with a PEG chain having a maleimide groups at the end and the prepared streptavidin introduced with a thiol group were mixed in PBS containing 2 mM of EDTA and allowing them to react for 1 hour. Thus, streptavidin was bonded via a PEG chain to the luminescent nanoparticles. The reaction was stopped by adding 10 mM of mercaptoethanol to the reaction solution. The resulting solution was concentrated by centrifugal filtering, and unreacted material was removed using a purifying gel filtration column to obtain a luminescent labeling material (streptavidin-modified luminescent nanoparticles) for pathological diagnosis.

[Example 6] Luminescent Labeling Materials for Pathological Diagnosis Composed of Luminescent Nanoparticles Nos. (3-1) to (3-4)

[0161] <Preparation of Luminescent Nanoparticles with a Maleimide Group at the End>

[0162] For each of the above luminescent nanoparticles Nos. (3-1) to (3-4), which are silica particles containing luminescent dyes, the solution was adjusted to 3 nM using PBS containing 2 mM of EDTA (ethylenediaminetetraacetic acid). SM(PEG) 12 (manufactured by Thermo Scientific, succinimidyl-[(N-maleimidopropionamide)-dodecane ethylene glycol] ester) was mixed with this solution to make a final concentration of 10 mM and reacted at 5° C. for 1 hour.

[0163] This mixed solution was centrifuged at 10,000 G for 20 minutes, and after removing the supernatant, PBS containing 2 mM of EDTA was added to disperse the sediment, followed by centrifugation again. By performing washing by the same procedure three times, luminescent nanoparticles with a maleimide group at the end were obtained.

<Preparation of Streptavidin Introduced with a Thiol Group>

[0164] Streptavidin capable of binding to luminescent nanoparticles was prepared as follows.

[0165] First, 40 μL of streptavidin (Wako Pure Chemical Industries, Ltd.) adjusted to 1 mg/mL was L was added to 210 μL of borate buffer. Then, 70 μL of 2-iminothiolane hydrochloride (Sigma-Aldrich Co., Ltd.), adjusted to 64 mg/mL, was added. The mixture was allowed to react at room temperature for 1 hour. By this, the thiol group (—NH—C(═NH.sub.2.sup.+Cl.sup.−)—CH.sub.2—CH.sub.2—CH.sub.2—SH) was introduced to the amino group of streptavidin.

[0166] This streptavidin solution was then transferred to a gel filtration column (Zaba Spin Desalting Columns: Funakoshi) to obtain streptavidin that is capable of binding to the above silica particles.

<Preparation of Luminescent Nanoparticles Modified with Streptavidin>

[0167] The total amount of streptavidin (containing 0.04 mg) and 740 μL of the luminescent nanoparticles (silica particles) adjusted to 0.67 nM using PBS containing 2 mM of EDTA were mixed and reacted at room temperature for 1 hour. Further, 10 mM of mercaptoethanol was added to stop the reaction. The resulting solution was concentrated by centrifugal filtering, and unreacted streptavidin and other substances were removed using a purifying gel filtration column to obtain a luminescent labeling material for pathological diagnosis (streptavidin-modified luminescent nanoparticles (silica particles containing luminescent dye)).

Example 7: Evaluation of Luminescent Labeling Materials for Pathological Diagnosis

<Tissue Staining Process>

[Immunohistochemical Staining]

[0168] Immunostaining of human breast tissue was performed using a staining agent for tissue staining containing the luminescent labeling material for pathological diagnosis composed of the luminescent nanoparticles produced in Examples 5 and 6. Here, the staining agent for tissue staining was prepared using a PBS buffer containing 1% BSA. A tissue array slide (manufactured by Cosmo Bio, product number CB-A712) was used for the stained section. For the stained sections, the FISH score for each spot was calculated in advance using Pathvysion HER2 DNA probe kit (manufactured by Abbott). This FISH score was calculated according to the procedure described in the document attached to HER2 gene kit, Pathvysion™ HER2 DNA probe kit (Abbot Japan).

[0169] After the tissue array slide was deparaffinized, it was washed with water and autoclaved in a 10 mM citrate buffer (pH 6.0) for 15 minutes to activate the antigen. The tissue array slide after antigen retrieval treatment was washed with a PBS buffer, and anti-HER2 rabbit monoclonal antibody (4B5) diluted to 0.05 nM with a 1% BSA-containing PBS buffer was reacted with the tissue section for 2 hours. After washing with PBS, it was reacted with a biotin-labeled anti-rabbit antibody diluted with a 1% BSA-containing PBS buffer for 30 minutes. Furthermore, using the staining agent for tissue staining, that is, reacting with the luminescent labeling material for pathological diagnosis (luminescent nanoparticles having streptavidin) produced above for 2 hours, followed by washing, an immunohistochemical stained section was obtained. The obtained immunohistochemically stained section was fixed by immersing that in a 4% neutral paraformaldehyde aqueous buffer solution for 10 minutes.

[Morphological Staining]

[0170] HE staining was performed on the immunohistochemically stained section fixed as described above, and the stained section was dehydrated by immersion in ethanol, and the dehydrated section was further cleared by immersing in xylene and air-drying, resulting in a double-stained section.

[Sealing]

[0171] Entellan™ new (manufactured by Merck & Co., Ltd.), which is a xylene-based mounting medium, was added dropwise to the morphologically stained specimen, and the specimen was covered with a cover glass and sealed.

<Evaluation of Tissue Samples>

[0172] A luminescent labeling material for pathological diagnosis in which the HER2 protein expressed on the cell membrane is labeled by specifying the shape of the cell (the position of the cell membrane) by image processing using the stained image for morphological observation and superimposing it on the immunostained image. A bright spot representing the material (streptavidin-modified luminescent nanoparticles composed of luminescent nanoparticles) could be confirmed by microscopic observation by irradiation with excitation light. This result showed that the luminescent nanoparticles of the present invention can be used as a luminescent labeling material for pathological diagnosis.

INDUSTRIAL APPLICABILITY

[0173] The present invention may be used for luminescent nanoparticles and luminescent labeling materials for pathological diagnosis for enabling high-sensitivity imaging that can avoid adverse effects of autofluorescence of cells on bioimaging.