JP2009280630A - Aqueous near-infrared fluorescent material and multimodal aqueous near-infrared fluorescent material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an aqueous near-infrared fluorescent material with high quantum yield which emits near-infrared light and is used in an in vivo imaging technique, and to provide a multimodal aqueous near-infrared fluorescent material with high quantum yield which is used in a multimodal in vivo imaging technique using an optical (fluorescence) imaging, MRI, SPECT, etc. <P>SOLUTION: The aqueous near-infrared fluorescent material has a semiconductor quantum dot having an emission band in the near-infrared region and a coating layer coating the surface of the semiconductor quantum dot, wherein the coating layer is a layer composed of coated peptides selected from the group consisting of cysteine and consisting of peptides which are composed of 2-8 amino acid residues and contain one or more cysteine residues in amino acid residues. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a water-soluble near-infrared fluorescent material having a high quantum yield and a multimodal water-soluble near-infrared fluorescent material that emits near-infrared light and can be used in in vivo imaging methods.

  The field of immunology is one of the life sciences fields that has developed greatly over the last few years. Conventionally, an immune reaction has been studied using cells separately taken out from a living body. On the other hand, recently, a technique (in vivo imaging method) in which an immune reaction is visualized in a living body and an actual living body immune reaction is examined without separately removing cells has been studied. MRI (nuclear magnetic resonance imaging), X-ray CT, PET (positron tomography), SPECT (single-photon emission tomography), etc. are methods for cancer imaging in the medical field. Used for diagnosis or angiography. Recently, a light (fluorescence) imaging method using near-infrared light has attracted attention as a highly sensitive measurement method.

In these in vivo imaging methods, in order to visualize cancer tissues or organs by contrasting them with cancer tissues or organs existing in the body, and contrasting with other parts, it is often contrasted An agent is used. For example, fluorescent fine particles or compounds are used as contrast agents in optical imaging, and paramagnetic substances such as gadolinium or iron ions that affect the relaxation time of the nucleus are used as contrast agents in MRI. , Positron decaying compounds are used as contrast agents, and PET uses radioactive isotopes such as ⁹⁹ Tc, ⁶⁷ Ga, and ⁶⁸ Ga as contrast agents. As described above, in these imaging techniques, it is necessary to use contrast agents having different chemical structures and characteristics in accordance with the characteristics of each imaging technique depending on the imaging technique. Contrast agents for specifically detecting cancer tissue have already been put to practical use in MRI, PET, SPECT and the like. On the other hand, in optical imaging, there is a problem that the development of contrast agents is very late.

  On the other hand, the reliability of information obtained from images is greatly improved by multimodal imaging of tumor sites such as cancer using multiple imaging technologies such as optical (fluorescence) imaging, MRI, PET, and SPECT. (Nature Photonics, 487-489, 2007). In such multimodal imaging, it can be used in common in all imaging measurement methods such as optical (fluorescence) imaging, MRI, PET, SPECT, that is, the target object (such as cancer tissue) is specific to all imaging measurement methods. It is necessary to use a multimodal contrast agent that can be contrasted and visualized.

  By the way, optical imaging using a fluorescent probe has an excellent characteristic that, in principle, detection at a single molecule level is possible and the sensitivity is very high. Further, an optical imaging apparatus used in optical imaging has advantages that it is inexpensive and compact and can be moved relatively freely. Therefore, the potential versatility in clinical fields such as hospital use is very high. However, at present, optical (fluorescence) imaging devices have not been put into practical use at the clinical level. Technical conditions for practical application of optical (fluorescence) imaging devices at the clinical level include improved detection sensitivity and three-dimensional imaging, which requires a highly sensitive optical contrast agent. Yes. Further, in the use of optical imaging in a living body, a reduction in spatial resolution due to light scattering becomes a problem. Therefore, when optical imaging technology is used in vivo, it is considered desirable to use it together with MRI, PET, or SPECT. By using these techniques in combination, accurate three-dimensional position information in the living body can be obtained, and a target object such as a cancer tissue can be detected more accurately.

  Most of the multimodal contrast agents reported so far that can be used in optical imaging, MRI, PET, and SPECT use fluorescent materials that emit light in the visible region. However, light in the visible region is not suitable for use in in vivo optical imaging methods. This is because the living body contains a large number of endogenous dyes that absorb visible light, such as hemoglobin, and therefore hardly transmits visible light. The only light that can be transmitted through the living tissue is 700-900 nm near infrared light.

  As the near-infrared fluorescent material, cyanine-based organic dyes (particularly indocyanine) are generally used. However, cyanine-based organic dyes have an extremely low quantum yield of fluorescence in water of about 1%. Here, the quantum yield is a value indicating a rate at which one molecule absorbs one photon in a photochemical reaction, and thereby a molecule reacts (emits light). For this reason, in optical imaging, it is desired to develop a near-infrared fluorescent material with higher luminance that can be visualized with a small amount of use. Cyanine-based organic dyes also have the disadvantage of fading due to excitation light.

A semiconductor quantum dot is considered to be the most promising candidate for a fluorescent material having higher luminance and fading resistance. Here, the “semiconductor quantum dot” is a crystal having a size of several nanometers to several tens of nanometers, and has a property of confining electrons in the crystal. The semiconductor quantum dot is sometimes referred to as a semiconductor nanocrystal. In recent years, research on synthesizing contrast agents using semiconductor quantum dots has become active. For example, the following documents 1) to 9) are papers on contrast agents using semiconductor quantum dots.
1) WB Beng and Y. Zang, "Multifunctional quantum-dot-based magnetic chitosan particles", Adv. Materials, 17, 23756-2380 (2005);
2) H. Gu et al., "Direct synthesis of a bimodal nanosponge based on FePt and ZnS", Small, 4, 402-406 (2005);
3) S. Santra et al., "Synthesis of water-dispersible fluorescent, radio-opaque, and paramagnrtic CdS: Mn / ZnS quantum dots: a multifunctional probe for bioimaging", J. Am. Chem. Soc., 127, 1656 -1657 (2005);
4) WJM Mulder et al., "Quantum dots with a paramagnetic coating as a bimodal molecular imaging probe", Nano Lett., 6, 1-6 (2006);
5) H. Yang et al., "GdIII-functionalized fluorescent quantum dots as multimodal imaging probes", Adv. Materials, 18, 2890-2894 (2006);
6) GAF van Tilborg et al., "Annexin A5-conugated quantum dots with a paramagnetic lipidic coating for the multimodal detection of apoptic cells, Bioconjugate Chem., 17, 865-868 (2006);
7) L. Prinzen et al., "Optical and magnetic resonance imaging of cell deth and plateket activation using annexin A5-functionalized quantum dots", Nano Lett, 7, 93-100 (2007);
8) WB Tan and Y. Zhang, "Multi-functional chitosan nanoparticles encapsulating quantum dots and Gd-DTPA as imaging probes for bio-applications", J. Nanosci. Nonotechnol. 7, 2389-2393 (2007);
9) S. Wang et al., “Core / shell quantum dots with high relaxivity and photoluminescence for multimodality imaging, J. Am. Chem. Soc., 129, 3848-3856 (2007);
However, the quantum dots disclosed in the literatures 1) to 9) are quantum dots that emit light in the visible region. Such quantum dots are very difficult to use for in vivo optical imaging methods. This is because, as described above, since a large amount of endogenous dye such as hemoglobin or flavin that absorbs visible light exists in the living body, visible light (400 to 650 nm) hardly transmits. The documents 1) to 9) do not disclose a multimodal contrast agent using near-infrared light emitting quantum dots that have good biological permeability.

On the other hand, hydrophobic near-infrared semiconductor quantum dots that can be used as starting materials in the production of multimodal contrast agents are disclosed in the following documents 10) to 19).
10) MA Hines, GD Scholes, "Colloidal PbS Nanocrystals with size-tunable near-infrared emission: observation of post-synthesis self-narrowing of the particle size distribution", Adv. Materials, 21, 1844-1849 (2003);
11) RE Bailey, S. Nie, "Alloyed semiconductor quantum dots: tuning the optical properties without changing the particle size", J. Am. Chem. Soc., 125 7100-7106 (2003);
12) S. Kim et al., "Type II quantum dots: CdTe / CdSe (core / shell) and CdSe / ZnTe (core / shell) heterostructures", J. Am. Chem. Soc., 125 11466-11467 (2003 );
13) S. Kim et al., "Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping", Nat. Biotech., 22, 93-96 (2004);
14) RE Baily et al., "A new class of far-red and near-infrared biological labels based on alloyed semiconductor quantum dots", J. Nanosci. Nanotech., 6, 569-574 (2004);
15) S.-W. Kim, et al., "Engineering InAsxP1-x / InP / ZnS III-V alloyed core / shell quantum dots for the near-infrared", J. Am. Chem. Soc., 127 10526- 10523 (2005);
16) W. Jiang et al., "Optimizing the synthesis of red- to near-IR-emitting CdS-capped CdTexSe1-x alloyed quantum dots for biomedical imaging", Chem. Mater., 18, 4845-4854 (2006);
17) B.-R. Hyu, et al., "Near-infrared fluorescence imaging with water-soluble lead salt quantum dots", J. Phys. Chem. B, 111, 5726-5730 (2007);
18) H. Qian et al., "High-quality and water-soluble near-infrared photoluminescence CdHgTe / CdS quantum dots prepared by adjusting size and composition", J. Phys. Chem. C, 111, 7918-7923 (2007) ;
19) S. Hinds et al., "Nir-emitting colloidal quantum dots having 26% luminescence quantum yield in buffer solution", J. Am. Chem. Soc., 129, 7218-7219 (2007);

  Thus, the above-mentioned documents 10) to 19) disclose hydrophobic near-infrared semiconductor quantum dots. On the other hand, there are few examples in which hydrophobic near-infrared quantum dots are water-solubilized and applied as an optical contrast agent for in vivo imaging. As an example of water-solubilizing hydrophobic near-infrared quantum dots, the group of Bawendi et al. In 2004 developed a photocontrast agent for lymph node imaging by coating type II near-infrared quantum dots with phosphine oligomers. (Ref. 13)). In 2004, Nie et al. Obtained a water-soluble high-intensity quantum dot by coating the surface of an alloy-type near-infrared quantum dot with mercaptoacetic acid (Reference 14). There is no mention of imaging data). However, all of these methods use phosphine or mercaptoacetic acid, whose toxicity is a problem, as a coating agent. Therefore, in order to use the quantum dots obtained by these methods for in vivo imaging, biotoxicity becomes a problem.

  US Patent Application Publication No. 2005/0220714 (Patent Document 1) describes paramagnetic ion-doped semiconductor nanoparticles having optical and MRI contrast functions. This is a dual-modal quantum dot that emits visible light. On the other hand, this patent document 1 does not describe quantum dots that emit light in the near infrared region.

  US Patent Application Publication No. 2005/0265922 (Patent Document 2) describes the conceptual contents regarding the structure and manufacturing method of multimodal nanoparticles having an imaging function such as light, MRI, PET, and SPECT. . On the other hand, Patent Document 2 does not show an example in which such multimodal nanoparticles are specifically synthesized.

  US Patent Application Publication No. 2007/0269382 (Patent Document 3) describes a paramagnetic ion-doped multifunctional contrast agent. On the other hand, this patent document 3 does not describe quantum dots that emit light in the near infrared region.

US Patent Application Publication No. 2005/0220714 US Patent Application Publication No. 2005/0265922 US Patent Application Publication No. 2007/0269382

  The present invention solves the above-mentioned conventional problems, and an object of the present invention is to provide a water-soluble near-infrared fluorescent material with a high quantum yield that emits near-infrared light and can be used in in vivo imaging methods. Furthermore, another object of the present invention is to provide a multimodal water-soluble near-infrared fluorescent material having a high quantum yield that can be used in a multimodal in vivo imaging method using optical (fluorescence) imaging, MRI, SPECT and the like.

The present invention
A semiconductor quantum dot having an emission band in the near infrared region, and a coating layer covering the surface of the semiconductor quantum dot;
A water-soluble near-infrared fluorescent material having
This covering layer is composed of cysteine and covering peptides selected from the group consisting of 2 to 8 amino acid residues and a peptide containing one or more cysteine residues in the amino acid residues. The layer
A water-soluble near-infrared fluorescent material is provided, and the above object is achieved.

It is preferable that the semiconductor quantum dot is a semiconductor quantum dot having a core-shell structure including CdSe _1-x Te _x core / CdS shell (where x = 0.2 to 1).

  In addition, the coated peptides are cysteine, glutathione, γ-Glu-benzyl-Cys-Val, β-Asp-Cys-Gly, Glu-Cys-Gly, Asp-Cys-Gly, γ-Glu-Gly-Cys- Peptides selected from the group consisting of Gly, β-Asp-Gly-Cys-Gly, Glu-Gly-Cys-Gly, Asp-Gly-Cys-Gly are preferred.

  The water-soluble near-infrared fluorescent material preferably has an average particle diameter of 2 to 50 nm and is monodispersed.

The present invention also provides
A semiconductor quantum dot whose surface is coated with a coordinating organic compound and having an emission band in the near-infrared region; cysteine, and composed of 2 to 8 amino acid residues and one or more in this amino acid residue Coating peptides selected from the group consisting of peptides containing cysteine residues of
A method for producing a water-soluble near-infrared fluorescent material is also provided.

  The present invention also provides a water-soluble near-infrared fluorescent material produced by the method for producing a water-soluble near-infrared fluorescent material.

The present invention further includes
A semiconductor quantum dot having an emission band in the near infrared region,
A coating selected from the group consisting of cysteine and a peptide composed of 2 to 8 amino acid residues and containing one or more cysteine residues in the amino acid residues, covering the surface of the semiconductor quantum dot A coating layer, which is a layer composed of peptides,
Elemental inclusion groups chemically bonded to the coating layer, and Gd (III), Mn (II), Mn (III), Fe (II), Fe (III), which are included in the element inclusion group, Cu (II), Cr (III), Co (II), Ni (II), Dy (III), Tb (III), Nd (III), Fe ₃ O ₄ , ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ^{99 m} From Tc, ⁹⁰ Y, ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁶⁵ Dy, ¹¹¹ In, ²⁰¹ Tc, ¹⁸ F, ⁷⁷ Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹²⁶ I, ¹³¹ I, ²⁰¹ Tl An element selected from the group consisting of
A multimodal water-soluble near-infrared fluorescent material is also provided.

The present invention further includes
An element that chemically bonds the reactive functional group present in the coating layer and the element inclusion group-containing compound of the water-soluble near-infrared fluorescent material obtained by the method for producing the water-soluble near-infrared fluorescent material. Inclusion group introduction process,
Introduced element inclusion groups include Gd (III), Mn (II), Mn (III), Fe (II), Fe (III), Cu (II), Cr (III), Co (II), Ni (II), Dy (III), Tb (III), Nd (III), Fe ₃ O ₄ , ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ^99m Tc, ⁹⁰ Y, ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁸⁶ Re, ¹⁸⁸ Re ¹⁶⁵ Dy, ¹¹¹ In, ²⁰¹ Tc, ¹⁸ F, ⁷⁷ Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹²⁶ I, ¹³¹ I, ²⁰¹ Tl, introducing an element selected from the group consisting of:
And a method for producing a multimodal water-soluble near-infrared fluorescent material.

  The present invention further provides a multimodal water-soluble near-infrared fluorescent material produced by the method for producing a multimodal water-soluble near-infrared fluorescent material.

The water-soluble near-infrared fluorescent material of the present invention has semiconductor quantum dots that have higher brightness than organic dyes. And it has the advantage that the dispersibility in water is favorable and has high stability, and also has the advantage of high biocompatibility because it is coated with peptides. Therefore, the water-soluble near-infrared fluorescent material of the present invention is a high-intensity near-infrared contrast agent in in-vivo optical imaging that can be suitably used in in-vivo imaging. The water-soluble near-infrared fluorescent material of the present invention is further advantageous in that the production process is simple and mass synthesis is possible.
In the present invention, a multimodal water-soluble near-infrared fluorescent material can be prepared using this water-soluble near-infrared fluorescent material. This multimodal water-soluble near-infrared fluorescent material is a multimodal contrast agent that enables simultaneous measurements such as optical imaging, MRI, and SPECT. The multimodal water-soluble near-infrared fluorescent material of the present invention enables realization of multimodal imaging by light, MRI, and SPECT, and greatly contributes to the development of in vivo imaging technology.

Water-soluble near-infrared fluorescent material The water-soluble near-infrared fluorescent material of the present invention is
A semiconductor quantum dot having an emission band in the near infrared region, and a coating layer covering the surface of the semiconductor quantum dot;
Have Hereinafter, each configuration will be sequentially described.

Semiconductor quantum dot having a light emission band in the near infrared region As the semiconductor quantum dot contained in the water-soluble fluorescent material of the present invention, a semiconductor quantum dot having a light emission band in the near infrared region is used. The near-infrared semiconductor quantum dot in the present invention is composed of a metal compound having a band gap in the near-infrared region. In semiconductor quantum dots, the electronic state has a band structure composed of a valence band and a conduction band, and electrons occupy the valence band in the ground state. By absorbing light with energy larger than the band gap (energy difference between the valence band and the conduction band), electrons in the valence band are excited to the conduction band, and a pair of electrons and holes (exciton) in the particle Is formed. It is the fluorescence in the semiconductor quantum dot that is emitted as light when the electron-hole pair recombines. The fluorescence wavelength depends on the size of the band gap of the semiconductor quantum dot. As a semiconductor quantum dot having a light emission band in the near infrared region, a semiconductor quantum dot having a so-called core-shell structure having a semiconductor quantum dot core and a shell portion covering the semiconductor quantum dot core is preferably used.

  As the semiconductor quantum dot core, a II-VI group compound semiconductor, a III-V group compound semiconductor, or a group IV semiconductor is preferably used. Examples of the III-V group compound semiconductor include GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, AlAs, AlP, AlSb, and AlS. Examples of the II-VI group compound semiconductor include ZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe, CdTe, HgO, HgS, HgSe, HgTe, and the like. Examples of the group IV semiconductor include Ge, Si, Pb, PbS, PbSe, and the like. These semiconductors may be used alone or in combination of two or more. Further, these semiconductors may be alloys made of two or more kinds of semiconductors, or may be mixed crystals.

As the semiconductor quantum dot core, it is more preferable to use a II-VI group compound semiconductor, and it is more preferable to use CdSe, CdTe, or a mixture, alloy or mixed crystal thereof. This is because these semiconductors have excellent crystal grain size controllability and high light emission performance. In particular, it is preferable to use a mixture, alloy or mixed crystal of CdSe and CdTe. This is because these mixtures, alloys or mixed crystals can adjust the emission band to a higher wavelength region by adjusting the ratio of Se and Te. In particular, when the semiconductor quantum dot core is CdSe _1-x Te _x (where x = 0.2 to 1, more preferably 0.2 to 0.8), the range of the emission band Is more suitable for optical (fluorescence) imaging, and is more preferable.

Examples of the method for preparing the semiconductor quantum dot core include the following methods.
(1) A method in which a raw material aqueous solution is present as reverse micelles in a nonpolar organic solvent, and crystals are grown in a reverse micelle phase (reverse micelle method). For example, B. S. Zou et al .; Int. J. et al. Quant. Chem. 72, 439 (1999). Since it is possible to use a relatively inexpensive and chemically stable salt as a raw material, and at a relatively low temperature that does not exceed the boiling point of water, this method is suitable for industrial production. However, the current technology may be inferior in light emission characteristics as compared with the following hot soap method.
(2) A method of injecting a thermally decomposable raw material into a high-temperature liquid phase organic medium to grow crystals (hot soap method). For example, J. E. B. This is a known method described in the literature by Katari et al. Compared with the reverse micelle method, semiconductor crystal particles having excellent particle size distribution and purity can be obtained, and the resulting product is excellent in luminescent properties and usually soluble in an organic solvent. For the purpose of desirably controlling the reaction rate in the process of crystal growth in the liquid phase in the hot soap method, a coordinating organic compound having a coordinating power suitable for a semiconductor constituent element is used to form a liquid phase component (solvent and ligand). Is also selected). Examples of such coordinating organic compounds include trialkylphosphines, trialkylphosphine oxides, ω-aminoalkanes such as dodecylamine, tetradecylamine, hexadecylamine, and octadecylamine.
(3) A method (sol-gel) suitable for industrial production in which a semiconductor crystal and its precursor are produced at a relatively low temperature of about 100 ° C. or less in a protic solvent such as water or ethanol by using an acid-base reaction as a driving force. Law).

  Of the above preparation methods, (2) the hot soap method is preferably used. This is because it is possible to prepare a semiconductor quantum dot core having more excellent emission characteristics. L. Qum and X. Peng, J. Am. Chem. Soc., 124, 2049 (2002) also discloses a method for preparing semiconductor quantum dots coated with trialkylphosphine oxides.

  The semiconductor quantum dot core preferably has a particle size of about 1 nm to about 50 nm, more preferably has a particle size of about 1 nm to about 20 nm, and even more preferably has a particle size of about 1 nm to about 5 nm. By having a particle size in the above range, a water-soluble fluorescent material having good light emission characteristics can be obtained.

  The shell part covering the semiconductor quantum dot core includes a semiconductor having a band gap (forbidden band width) larger than the band gap of the semiconductor quantum dot core. By using such a semiconductor for the shell portion, an energy barrier is formed in the semiconductor quantum dots, and thereby good light emission performance can be obtained. Specifically, since the band gap of the semiconductor in the shell portion is larger than the band gap of the core portion, pairs of electrons and holes generated in the core are trapped in the core portion, and thus do not come out on the semiconductor surface. For this reason, electrons and holes generated by light absorption are recombined and emitted as fluorescence without being quenched on the semiconductor surface, thereby obtaining good light emission performance. For example, in the case of a quantum dot having only a core part without a shell part, it is considered that electrons and holes are quenched by oxygen or water molecules on the semiconductor surface, thereby reducing fluorescence intensity and luminance.

  The semiconductor preferably used for the shell portion depends on the band gap of the semiconductor quantum dot core used, but for example, ZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe, CdTe, MgS, MgSe, GaAs, GaN, GaP One or more semiconductors selected from the group consisting of GaAs, GaSb, HgO, HgS, HgSe, HgTe, InAs, InN, InP, InSb, AlAs, AlN, AlP, and AlSb, or alloys or mixed crystals thereof Is preferably used.

More preferably, the semiconductor quantum dots having an emission band in the near infrared region are CdSe _1-x Te _x core / CdS shell (where x = 0.2 to 1), CdTe core / CdSe shell, CdSe core. Examples thereof include semiconductor quantum dots having a core / shell structure including a / CdS shell, a CdSe core / ZnS shell, an InAs core / CdSa shell, a CdS core / CdTeSe shell, and a CdHgTe core / CdS shell. This is because these semiconductor quantum dots have a light emission band in a range more suitable for use in the in vivo imaging method.

  Semiconductor quantum dots having a core-shell structure are preferably prepared by a two-step method. This can form the shell part which coat | covers a semiconductor quantum dot core by adding the solution of the semiconductor contained in a shell part to the semiconductor quantum dot core obtained by the said method. This preparation method includes BO Dabbousi, J. Rodriguez-Viejo, FV Mikulec, JR Heine, H. Mattoussi, R. Ober, KF Jensen, MG Bawendi, J. Phys. Chem. B, 101, 9463 (1997), etc. And is publicly known.

  The semiconductor quantum dots having a core-shell structure preferably have a particle size of about 1 nm to about 100 nm, more preferably have a particle size of about 1 nm to about 40 nm, and have a particle size of about 1 nm to about 10 nm. Further preferred. By having a particle size in the above range, an emission band in the near infrared range can be obtained.

  The emission band of a semiconductor quantum dot having an emission band in the near infrared region used in the present invention is preferably in the wavelength range of 700 to 1400 nm, and more preferably in the wavelength range of 700 to 900 nm. This is because light having a wavelength in the above range, particularly light having a wavelength in the range of 700 to 900 nm, exhibits excellent permeability to a living body. When the wavelength is less than 700 nm, it is absorbed by a living endogenous dye that absorbs visible light such as hemoglobin and cannot pass through the living body. On the other hand, when the wavelength exceeds 900 nm, vibrational absorption of water molecules occurs, and the transmittance tends to decrease again. And if it exceeds 1400 nm, it will hardly permeate | transmit. The wavelength of the light emission band of the semiconductor quantum dot having a light emission band in the near infrared region used in the present invention is in the above range, so that it can be used favorably in in vivo imaging methods using light. A high-intensity photocontrast agent that emits fluorescence is obtained.

  In the present invention, a semiconductor quantum dot whose surface is coated with a coordinating organic compound is used. Examples of the coordinating organic compound include trialkylphosphine oxides such as trioctylphosphine oxide (TOPO), trialkylphosphines such as trioctylphosphine (TOP) and tributylphosphine (TBP), and ω such as hexadecylamine. -Aminoalkanes etc. are mentioned. Since the surface of semiconductor quantum dots has a very high reactivity, the surface is generally covered and stabilized by a coordinating organic compound such as trioctylphosphine oxide (TOPO) or trioctylphosphine (TOP). However, semiconductor quantum dots coated with a coordinating organic compound are very hydrophobic and can only be dispersed in organic solvents such as chloroform and hexane. And the semiconductor quantum dot coat | covered with the coordinating organic compound which cannot be disperse | distributed to water in this way can be disperse | distributed to water by being coat | covered with a coating layer in this invention.

  In the present invention, a core-shell type semiconductor quantum dot is preferably used because of its high light emission performance in the near infrared region, but it is limited to a core-shell type if it is a semiconductor quantum dot having high light emission performance in the near infrared region. Is not to be done. For example, a doped semiconductor quantum dot or the like may be used.

Coated peptides As described above, semiconductor quantum dots having an emission band in the near-infrared region have a high hydrophobicity because a coordinating organic compound such as trioctylphosphine oxide or a long-chain alkylamine is bonded to the surface. Insoluble in water. Therefore, in order to use this semiconductor quantum dot in a living body, it is necessary to make the semiconductor quantum dot water-soluble so as to make it water-soluble, thereby improving the dispersion stability in the living body. Furthermore, since it is a material used in a living body, the treatment agent used for the water-solubilization treatment needs to have a low toxicity.

  In the present invention, in order to solubilize a semiconductor quantum dot having an emission band in the near-infrared region, the coating is composed of cysteine, which is an amino acid, and 2 to 8 amino acid residues. Alternatively, cysteine or peptides (coated peptides) selected from the group consisting of peptides containing more cysteine residues were used. The “peptides” and “coating peptides” in this specification include the above peptides and cysteine. By using these coated peptides as a coating treatment for semiconductor quantum dots having a light emission band in the near infrared region, water-soluble near-infrared fluorescent materials with high dispersibility in water, high biocompatibility, and low biotoxicity Was obtained.

  The coated peptides used in the present invention have a cysteine residue, whereby a thiol group in the cysteine residue is coordinated to the surface of a semiconductor quantum dot having an emission band in the near infrared region, thereby Will cover the surface of the semiconductor quantum dots. Since the coated peptides have some hydrophilic groups in addition to the thiol group, the water dispersibility is improved by coating the surface of the semiconductor quantum dots with the coated peptides. Since the coated peptides are also peptides, they have high biocompatibility and high safety to living bodies. Furthermore, since the amino group and carboxyl group of the peptides are present on the surface of the semiconductor quantum dot coating layer, it has an excellent advantage that various biomolecules such as antibodies or proteins can be modified.

The peptide having a cysteine residue is more preferably a peptide composed of 1 to 4 amino acid residues and including one cysteine residue in the amino acid residue. Specific examples of peptides composed of 2 to 8 amino acid residues and containing one or more cysteine residues in the amino acid residues include, for example, the following peptides:
γ-Glu-Cys-Gly (glutathione (reduced form)),
γ-Glu-benzyl-Cys-Val,
β-Asp-Cys-Gly,
Glu-Cys-Gly,
Asp-Cys-Gly,
γ-Glu-Gly-Cys-Gly,
β-Asp-Gly-Cys-Gly,
Glu-Gly-Cys-Gly,
Asp-Gly-Cys-Gly,
Such. In the present invention, cysteine or glutathione (reduced form) is more preferably used as the coating peptides. This is because cysteine is one of the essential amino acids, is highly safe and inexpensive, and is particularly suitable for use in in vivo optical imaging. In addition, glutathione (reduced form) is a peptide derived from a living body, is highly safe, is inexpensive among peptides, and has protease resistance, and thus is particularly suitable for use in in vivo optical imaging.

Water-soluble near-infrared fluorescent material and preparation thereof The near-infrared fluorescent material of the present invention has a structure in which the surface of a semiconductor quantum dot having an emission band in the near-infrared region is covered with a coating layer. This near-infrared fluorescent material is composed of a semiconductor quantum dot whose surface is coated with a coordinating organic compound and having an emission band in the near-infrared region, cysteine, and 2 to 8 amino acid residues, and an amino acid residue. It can be prepared by mixing a coated peptide selected from the group consisting of peptides containing one or more cysteine residues in the group in a solvent (peptide coating step). The mixing method is not particularly limited, and any mixing method can be used.

  The temperature at which the semiconductor quantum dots whose surfaces are coated with a coordinating organic compound and have a light emission band in the near-infrared region and the coating peptides are not particularly limited. For example, it can be mixed well even under mild conditions such as 0 to 70 ° C., preferably 10 to 60 ° C., more preferably room temperature to 60 ° C. The mixing time can be appropriately selected from 2 seconds to 30 minutes, for example, when sonication is used. When sonication is not used, it can be appropriately selected, for example, between 10 minutes and 24 hours.

  As the solvent used for mixing, an arbitrary organic solvent, a mixed solvent of an organic solvent and an aqueous solvent, and the like can be used. Specific examples of organic solvents that can be used include, for example, aromatic solvents such as toluene and xylene; ketone solvents such as methyl ethyl ketone, acetone, methyl isobutyl ketone, and cyclohexanone; diethyl ether, isopropyl ether, tetrahydrofuran, dioxane, and ethylene. Ether solvents such as glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether, propylene glycol monomethyl ether, anisole and phenetol; ester solvents such as ethyl acetate, butyl acetate, isopropyl acetate and ethylene glycol diacetate; dimethylformamide Amide solvents such as diethylformamide, N-methylpyrrolidone; Ruserosorubu, ethyl cellosolve, cellosolve solvents such as butyl cellosolve; methanol, ethanol, alcohol solvents such as propanol; and the like; dichloromethane, halogenated solvents such as chloroform. These organic solvents may be used alone or in combination of two or more. Of these, ketone solvents, ether solvents, ester solvents and the like are particularly preferably used from the viewpoint of workability.

  The weight ratio between the semiconductor quantum dots used for mixing and the coated peptides depends on the particle size of the semiconductor nanocrystals used, but is generally about 1: 1 to 1: 100, and 1: 1 to 1:50. More preferably.

  The product obtained by mixing the semiconductor quantum dots and the coated peptides may be subjected to a purification operation (such as dialysis or filtration) as necessary. In addition, for the product obtained by mixing the semiconductor quantum dots and the coated peptides, potassium t-butoxide, lithium diisopropylamide, potassium hexamethyldisilazide, lithium 2,2,6,6-tetramethyl A strong base such as piperidide may be added. This is because by adding these strong bases, the C-terminal carboxylic acid (COOH) of the coated peptides can be deprotonated, whereby it can be easily dissolved and dispersed in neutral water. Even when these strong bases are not used, the obtained product can be dissolved and dispersed in an alkaline aqueous solution (for example, a 0.1 M NaOH aqueous solution or a KOH aqueous solution).

  The water-soluble near-infrared fluorescent material of the present invention preferably has a particle size of about 2 nm to about 100 nm, and more preferably has a particle size of about 2 nm to about 50 nm. When the average particle diameter of the water-soluble near-infrared fluorescent material is in the above range, good light emission characteristics can be obtained. The particle diameter of the water-soluble fluorescent material of the present invention is determined by using a dynamic light scattering method using a laser or fluorescence correlation spectroscopy (FCS). Here, the dynamic light scattering method is a method of detecting the Brownian motion of particles in a solution by the light scattering method and calculating the size of the particles. The fluorescence correlation spectroscopy is a method for evaluating the size of the molecule from the autocorrelation of the fluctuation of fluorescence intensity. By measuring the particle diameter of the water-soluble fluorescent material using fluorescence correlation spectroscopy (FCS), there is an advantage that the size of the water-soluble fluorescent material itself including the coating layer can be evaluated. As the dynamic light scattering device, a dynamic light scattering device manufactured by Malvern or Symex Corporation can be used. As the FCS measuring device, for example, a fluorescence correlation spectroscopic device manufactured by Hamamatsu Photonics Co., Ltd. can be used.

  The water-soluble near-infrared fluorescent material of the present invention is more preferably monodispersed. In the present specification, “the water-soluble near-infrared fluorescent material is monodispersed” means particles having an average particle diameter measured by the dynamic light scattering method and an average particle diameter measured by fluorescence correlation spectroscopy (FCS). The diameter peak shows one peak, and the average particle diameter measured by the dynamic light scattering method and the average particle diameter measured by fluorescence correlation spectroscopy (FCS) coincide with each other at a high rate. Means. In addition, when the water-soluble near-infrared fluorescent material is not monodispersed, that is, when the variation in particle diameter is large, there is a problem that the emission spectrum becomes broad and the luminance is inferior.

In the water-soluble near-infrared fluorescent material of the present invention, the semiconductor quantum dots contained in the water-soluble near-infrared fluorescent material are CdSe _1-x Te _x core / CdS shell (wherein x = 0.2-1). It is particularly preferable that the semiconductor quantum dot having a core-shell structure is a cysteine or glutathione (reduced type). Some semiconductor quantum dots have an emission band in the visible region. However, when a semiconductor quantum dot having a light emission band in the visible region is coated with cysteine or glutathione, there is a problem that the quantum yield of fluorescence is reduced to 1/10 to 1/20 compared to before coating. . In contrast, a semiconductor quantum dot having a core-shell structure composed of a CdSe _1-x Te _x core / CdS shell (where x = 0.2 to ₁₎ having an emission band in the near infrared region is used as cysteine or glutathione. In the case of coating with, the quantum yield of fluorescence is only reduced to about 6/10 compared to before coating, and it was revealed that the fluorescence quantum yield can be maintained at a high rate. When the semiconductor quantum dots having the above-mentioned core-shell structure having an emission band in the visible region are coated with cysteine or glutathione, the fluorescence quantum yield is compared with the case of coating the semiconductor quantum dots having an emission band in the visible region with cysteine or glutathione. The reason why does not decrease is not clear. However, the result of this fluorescence quantum yield is that the combination of a core-shell structure semiconductor quantum dot composed of CdSe _1-x Te _x core / CdS shell with cysteine or glutathione is useful for the preparation of water-soluble near-infrared fluorescent materials. The data confirms that it is very suitable.

  The water-soluble near-infrared fluorescent material of the present invention has a light-emitting band in the near-infrared region that can be used in vivo, and has high light-emitting performance, high brightness, and excellent fading resistance. It has the characteristics. The surface of the water-soluble near-infrared fluorescent material of the present invention is further coated with coating peptides. And since this covering peptide has some hydrophilic groups, it has the property of being highly water-dispersible. In addition, since the coated peptides are amino acids or peptides, they have high biocompatibility and high safety to living bodies. In particular, the water-soluble near-infrared fluorescent material of the present invention is more sensitive and excited because the luminance is about 10 to 100 times higher than that of conventionally used indocyanine-based near-infrared fluorescent reagents. Since it does not fade by light, it has an excellent advantage of enabling long-time measurement of optical (fluorescence) imaging. The reason why the brightness of the water-soluble near-infrared fluorescent material of the present invention is about 100 times higher than the brightness of the indocyanine-based near-infrared fluorescent reagent is as follows. (1) Indocyanine-based near-infrared While the quantum yield of the fluorescent reagent is about 1%, the quantum yield of the water-soluble near-infrared semiconductor quantum dots of the present invention is 10% or more, and (2) It is mentioned that the molar extinction coefficient of the infrared semiconductor quantum dot is about 10 times that of the indocyanine-based near infrared fluorescent reagent. Therefore, [(1) quantum yield is 10 times] × [(2) molar extinction coefficient is 10 times] = [luminance is 100 times], and the water-soluble near-infrared fluorescence of the present invention. The brightness of the material is about 100 times higher than that of the indocyanine-based near-infrared fluorescent reagent. By using the water-soluble near-infrared fluorescent material of the present invention having such excellent characteristics as a contrast agent for optical (fluorescence) imaging, there is an advantage that it is inexpensive and compact and can be moved relatively freely. The detection sensitivity of optical (fluorescence) imaging can be improved. Therefore, the water-soluble near-infrared fluorescent material of the present invention greatly contributes to practical application and spread of optical (fluorescence) imaging at the clinical level. The water-soluble near-infrared fluorescent material of the present invention can be used as a near-infrared contrast agent for in vivo imaging in small animals such as mice for which human pathological models are being actively created by genetic modification. In the future, the water-soluble near-infrared fluorescent material of the present invention is also considered to be used in in vivo imaging in the medical diagnostic field for humans such as detection of breast cancer or thyroid cancer and imaging of sentinel lymph nodes. .

The water-soluble near-infrared fluorescent material of the present invention further contains various components such as antibodies, ligands, and drugs in the water-soluble near red color because the amino groups and carboxyl groups of the coated peptides are present on the surface of the semiconductor quantum dot coating layer. It also has an excellent advantage that it can be modified to an outer fluorescent material. For example, there is an advantage that light (fluorescence) imaging of a tumor or a specific tissue can be performed in vivo by introducing a water-soluble near-infrared fluorescent material by binding a ligand for an antibody or a receptor. In addition, for example, there is an advantage that light (fluorescence) imaging of pharmacokinetics in a living body can be achieved by binding and introducing a drug into a water-soluble near-infrared fluorescent material. The introduction of these antibodies, ligands or drugs into water-soluble near-infrared fluorescent materials can be carried out simply by using a condensing agent that can be used in the following element inclusion group introduction procedures, similarly to the element inclusion group introduction procedure. It can be carried out. Furthermore, a multimodal water-soluble near-infrared fluorescent material can also be obtained by introducing an inclusion group-containing group into the water-soluble near-infrared fluorescent material and then introducing a specific element. This will be described in detail in “ Multimodal water-soluble near-infrared fluorescent material and preparation thereof ” below.

  In addition, as a prior art, since semiconductor quantum dots are used as a fluorescent probe, studies have been conducted to obtain water-soluble semiconductor quantum dots by chemically modifying the surface of the semiconductor quantum dots with a hydrophilic functional group. Examples of such chemical modification methods include the following two types. The first type is a method in which a hydrophobic coating agent on the surface of a semiconductor quantum dot is replaced with a thiol-based amphiphilic compound to make it water-soluble, and the second type is a semiconductor quantum dot using an amphiphilic polymer. In this method, the surface is coated and water-solubilized. In the first type of method, although water-soluble semiconductor quantum dots having a small particle size can be obtained, there is a drawback that the light emission efficiency is remarkably lowered. In addition, the water-soluble semiconductor quantum dots obtained by the second type method generally have higher luminous efficiency than the first type method, but have a problem of increasing the particle size. Furthermore, in the first type of method, it is necessary to use a thiol-based reagent having toxicity or corrosiveness in the production process, and therefore there is a problem that the cost of production equipment is increased. In the second type of method, there are also problems such that the polymer-based coating used is expensive and that it takes a long time to prepare the water-soluble semiconductor quantum dots.

  On the other hand, in the present invention, first, a semiconductor quantum dot having a light emission band in the near-infrared region and having high luminance was prepared in a state where it was covered with the coordination organic compound, and then obtained. A semiconductor quantum dot is prepared by coating with the above-described coating peptides. As a result, the obtained peptide-coated semiconductor quantum dots have a structure in which the surface is covered with a monomolecular layer of peptides, have a light emission band in the near infrared region, and have excellent luminous efficiency and high brightness. The performance is achieved.

  As described above, in the present invention, a semiconductor quantum dot having a light emission band in the near infrared region and high brightness is prepared in a state where it is coated with the coordination organic compound, and then the obtained semiconductor quantum Dots are prepared by coating with the above-described coating peptides. As another example of the method for preparing the peptide-coated semiconductor quantum dots, for example, a method of directly coating the peptides at the time of preparing the quantum dots without undergoing coating with a coordinating organic compound is also conceivable. However, at present, there has been no report that a peptide-coated semiconductor quantum dot having an emission band in the near infrared region has been prepared by such a method. Furthermore, such a method of directly coating peptides when preparing quantum dots has a problem that it is difficult unless the quantum dots are core-structured quantum dots. That is, it is difficult to directly coat the peptides in the core-shell structure quantum dots when preparing the quantum dots. Furthermore, peptides coated quantum dots (for example, peptides coated quantum dots having a light emission band in the visible region) obtained by a method of directly coating peptides during quantum dot preparation have a wide half-width of the fluorescence spectrum of quantum dots. And there is a performance disadvantage of low brightness.

On the other hand, in the present invention, as described above, a semiconductor quantum dot having a light emission band in the near infrared region and high brightness is prepared in a state of being coated with a coordinating organic compound, and then obtained. The semiconductor quantum dots are prepared by coating with coated peptides. As a result, the obtained peptide-coated semiconductor quantum dots have a structure in which the surface is covered with a monomolecular layer of peptides, have a light emission band in the near infrared region, and have excellent luminous efficiency and high brightness. The performance is achieved. The advantages of the method according to the present invention are as follows.
1) Synthesis of near-infrared semiconductor quantum dots coated with peptides is possible.
2) Bright water-soluble near-infrared semiconductor quantum dots (indocyanine green has a quantum yield of about 1%, whereas the water-soluble near-infrared semiconductor quantum dots of the present invention have a quantum yield of 10%. The above can be synthesized.
3) The size of the semiconductor crystal can be easily controlled by the reaction time and temperature, and a near-infrared semiconductor quantum dot having an emission wavelength peak at 700 to 850 nm can be synthesized.
4) The reaction precursor used for synthesis is easy to prepare and the synthesis procedure is simple.
5) Large-scale synthesis of water-soluble near-infrared semiconductor quantum dots is easy.

Multi-modal water-soluble near-infrared fluorescent material and preparation thereof The multi-modal water-soluble near-infrared fluorescent material in this specification refers to a multi-modal water-soluble near-infrared fluorescent material, which is based on a plurality of (two or more) imaging techniques such as optical (fluorescence) imaging, MRI, and SPECT. It means a water-soluble near-infrared fluorescent material that can be used for modal imaging. And the multimodal water-soluble near-infrared fluorescent material of the present invention is
A semiconductor quantum dot having an emission band in the near infrared region,
A coated peptide selected from the group consisting of cysteine and a peptide composed of 2 to 8 amino acid residues and containing one or more cysteine residues in the amino acid residues, covering the surface of the semiconductor quantum dot A coating layer, which is a layer composed of
An element inclusion group chemically bonded to the coating layer, and an element included in the element inclusion group,
Have That is, an element inclusion group is further introduced into the above water-soluble near-infrared fluorescent material in which a semiconductor quantum dot having an emission band in the near infrared region is coated with a coating layer, and this element inclusion group It has a structure in which elements are included. And this multi-modal water-soluble near-infrared fluorescent material has the following steps:
An element inclusion group introduction step of chemically bonding the reactive functional group present in the coating layer of the water-soluble near-infrared fluorescent material and the element inclusion group-containing compound;
An element introduction step for introducing an element into the introduced element inclusion group;
Can be prepared.

  The element inclusion group-containing compound used for introduction of the element inclusion group has an element inclusion group and reacts with a reactive functional group (such as an amino group or a carboxylic acid group) present in the coating layer. If it is a compound which has group which carries out, it can use without a restriction | limiting in particular. Specific examples of the element inclusion group-containing compound that can be used in the present invention include the following compounds. The element inclusion group-containing compound can be appropriately selected according to the type and size of the element as a contrast agent to be introduced later.

  All of the above element inclusion group-containing compounds have a carboxylic acid group. This carboxylic acid group undergoes a condensation reaction with a reactive functional group (amino group) present in the coating layer to form an amide bond. Thereby, the reactive functional group is chemically bonded to the coating layer. The “element inclusion group” in this specification refers to an element inclusion group-containing compound when the element inclusion group-containing compound and a reactive functional group (amino group) present in the coating layer are chemically bonded. A group having the property of confining an element in a three-dimensional space formed by the element inclusion group.

  A commonly known condensing agent can be used for the reaction in which the reactive functional group present in the coating layer is chemically bonded to the element inclusion group-containing compound to introduce the element inclusion group. As condensing agents that can be used, carbodiimide condensing agents such as 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 4- ( Triazine condensing agents such as 4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholine hydrochloride, aminium condensing agent, phosphonium condensing agent, dihydroquinone condensing agent Etc. can be used. In these reactions, dehydration condensation additives such as 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), N-hydroxysuccinimide (HOSu) may be used.

  The element inclusion group-containing compound may be a derivative such as an active ester. For example, when the element inclusion group-containing compound is DOTA, DOTA-NHS-ester (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester) ) And the like can be used. By using such an active ester, there is an advantage that an element inclusion group can be more easily introduced into the coating layer without using a condensing agent.

  Examples of the solvent used in the element inclusion group introduction step include water or a mixture of water and an organic solvent as necessary. Specific examples of the organic solvent include, for example, ketone solvents such as methyl ethyl ketone, acetone, methyl isobutyl ketone, and cyclohexanone; diethyl ether, isopropyl ether, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether, diethylene glycol diethyl ether Ether solvents such as ethyl acetate, butyl acetate, isopropyl acetate, and ethylene glycol diacetate; amides such as dimethylformamide, diethylformamide, dimethylsulfoxide, and N-methylpyrrolidone System solvents; and the like. These solvents may be used alone or in combination. Reaction conditions such as reaction temperature and reaction time can be appropriately selected depending on the type of the coated peptide. For example, it can select suitably in the range of reaction temperature 0-40 degreeC and reaction time 5 minutes-24 hours.

A multimodal water-soluble infrared fluorescent material can be obtained by including an element that can be used as a contrast agent in the element inclusion group thus introduced. The type of element to be introduced is not particularly limited as long as it is an element that can be used as a contrast agent in each imaging method such as MRI (nuclear magnetic resonance imaging), X-ray CT, and SPECT (single photon emission tomography). . For example, as elements that can be used as contrast agents in MRI, Gd (III), Mn (II), Mn (III), Fe (II), Fe (III), Cu (II), Cr (III), Co ( II), Ni (II), Dy (III), Tb (III), Nd (III), and paramagnetic elements such as Fe ₃ O ₄ or ferromagnetic elements.
For example, as elements that can be used as a contrast agent in SPECT, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ^{99 m} Tc, ⁹⁰ Y, ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁶⁵ Dy, ¹¹¹ In, ²⁰¹ Tc, ¹⁸ And radioisotopes such as F, ⁷⁷ Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹²⁶ I, ¹³¹ I, and ²⁰¹ Tl.
The introduction of these elements as a contrast agent can be easily carried out by adding an aqueous solution of the above element salt to a solution in which a water-soluble near-infrared fluorescent material into which element inclusion groups have been introduced is dispersed in water. Can be introduced.

  The conceptual structural formula of the multimodal water-soluble infrared fluorescent material of the present invention is shown below. Note that the following formulas are conceptual structural formulas only, and the number of element inclusion groups is not limited to the following conceptual structural formulas.

  The multimodal water-soluble near-infrared fluorescent material of the present invention has the advantages of the above-mentioned water-soluble near-infrared fluorescent material (having a light emission band in the near-infrared region, high luminous performance, high brightness, and fading resistance) In addition, the optical (fluorescence) imaging method, MRI (nuclear magnetic resonance imaging), X-ray CT or SPECT (single photon emission tomography), etc. And has an excellent advantage that it can be used as a contrast agent for multimodal imaging. By using the multimodal water-soluble near-infrared fluorescent material of the present invention as a contrast agent in multimodal imaging, accurate three-dimensional position information in a living body can be obtained, and target objects such as cancer tissues can be more targeted. There is an advantage that it can be detected accurately. As described above, the multimodal water-soluble near-infrared fluorescent material of the present invention enables multimodal imaging combining a light (fluorescence) imaging method with MRI, X-ray CT, SPECT, or the like. This greatly contributes to the development of imaging technology.

  The following examples further illustrate the present invention, but the present invention is not limited thereto. In the examples, “parts” and “%” are based on weight unless otherwise specified.

Example 1
Preparation of cysteine-coated water-soluble near-infrared semiconductor quantum dots (water-soluble near-infrared fluorescent material) Hydrophobic near-infrared semiconductor quantum dots CdSe _0.75 Te _0.25 / CdS (core / shell structure) were prepared by RE Bailey, The method of JB Strausburg, S. Nie, J. Nanosci. Nanotech. 4 569 (2004) was modified and synthesized in one step.
20 mg of cadmium oxide (CdO) and 250 mg of stearic acid were placed in a 25 ml three-necked flask and heated to 300 ° C. under an argon atmosphere to dissolve cadmium oxide. Once the temperature was lowered to 150 ° C., 2 g of trioctylphosphine oxide (TOPO) and 2 g of hexadecylamine (HAD) were added. The mixture was again heated to 300 ° C. and left for 5 minutes. Next, 0.5 ml of Se / Te precursor (tetrabutylphosphine solution in which 25 mg of Se and 13 mg of Te were dissolved) was quickly added with a syringe while the solution was stirred. The temperature of the solution was immediately lowered to 250 ° C., and the progress of crystal growth was monitored from the fluorescence spectrum. After about 5 minutes, a semiconductor crystal having a peak in the vicinity of a wavelength of 770 to 790 nm was formed. After confirming the formation of semiconductor crystals by the fluorescence spectrum, the temperature was lowered to 200 ° C. in order to stop the reaction.
Next, formation reaction of the CdS shell part was performed. While stirring the solution at 200 ° C., 0.25 ml of Cd / S precursor (10 ml of a trioctylphosphine solution containing 150 mg of CdO and 40 mg of sulfur) was slowly added dropwise. After completion of the dropwise addition, the temperature of the solution was lowered to 100 ° C. and stirred for 1 hour. Thereafter, the temperature was lowered to 60 ° C. and 20 ml of chloroform was added. Further, 20 ml of methanol was added to precipitate a CdSe _0.75 Te _0.25 / CdS semiconductor crystal. The obtained semiconductor crystal is highly hydrophobic and does not dissolve in water. The crystals were separated by a centrifuge and dried at room temperature.
5 mg of the obtained CdSe _0.75 Te _0.25 / CdS crystals were dissolved in 2 ml of tetrahydrofuran, and 1 ml of an aqueous cysteine solution (20 mg / ml) was added dropwise thereto at room temperature. After completion of dropping, the temperature of the solution was set to 60 ° C. and left for about 5 minutes. The resulting precipitate was separated by a centrifuge and the supernatant solution was removed. To the resulting precipitate, 10 mg of potassium t-butoxide and 2 ml of water were added, and sonicated for 30 seconds to dissolve. The solution became a black-brown aqueous solution. This solution was dialyzed against borate buffer (pH 8.2) to remove excess potassium t-butoxide. An aqueous solution of a water-soluble near-infrared fluorescent material that is a monodisperse cysteine-coated near-infrared semiconductor quantum dot (CdSe _0.75 Te _0.25 / CdS) by filtering the dialyzed solution through a 0.2-micron filter. Got.

Fluorescence spectrum change by cysteine coating When water-solubilizing hydrophobic quantum dots coated with a coordinating organic compound such as trioctylphosphine oxide (TOPO), a significant decrease in luminance is a problem. . For example, when CdSe / ZnS quantum dots are coated with a thiol-based compound such as mercaptopropionic acid, it has been reported that the fluorescence luminance decreases from about 1/10 to 1/20 (Chem. Commun. , 2829-2931 (2005)). In order to confirm the effectiveness of the coating method in the present invention, the fluorescence spectrum change of near-infrared semiconductor quantum dots (CdSe _0.75 Te _0.25 / CdS) when coated with cysteine was measured by JASCO FP-6200 ( FIG. 8). As a result, it became clear that the fluorescence brightness after coating was maintained at a high rate of about 65% compared to before coating. The fluorescence peak was only shifted to a short wavelength of 3 nm, and the spectrum width hardly changed. This indicates that the fluorescence spectrum characteristics of the near-infrared semiconductor quantum dots before coating were retained during water solubilization by cysteine coating.

Example 2
Preparation of glutathione-coated water-soluble near-infrared semiconductor quantum dots (water-soluble near-infrared fluorescent material) 5 mg of CdSe _0.75 Te _0.25 / CdS crystals prepared in the same manner as in Example 1 were dissolved in 2 ml of tetrahydrofuran, At room temperature, 1 ml of a reduced glutathione aqueous solution (20 mg / ml) was added dropwise thereto. After completion of dropping, the temperature of the solution was set to 60 ° C. and left for about 5 minutes. The resulting precipitate was separated by a centrifuge and the supernatant solution was removed. To the resulting precipitate, 10 mg of potassium t-butoxide and 2 ml of water were added, and sonicated for 30 seconds to dissolve. The solution became a black-brown aqueous solution. This solution was dialyzed against borate buffer (pH 8.2) to remove excess potassium t-butoxide. An aqueous solution of a water-soluble near-infrared fluorescent material that is a monodispersed glutathione-coated near-infrared semiconductor quantum dot (CdSe _0.75 Te _0.25 / CdS) by filtering the dialyzed solution through a 0.2-micron filter. Got.

Fluorescence spectrum of water-soluble near-infrared semiconductor quantum dots coated with glutathione The change in fluorescence spectrum of near-infrared semiconductor quantum dots (CdSe _0.75 Te _0.25 / CdS) when glutathione was coated was measured by JASCO FP-6200 ( FIG. 9). Also in this case, it was confirmed that the fluorescence luminance was maintained at a high rate of about 65% as in the case of coating with cysteine. Further, after the glutathione coating, the peak of the fluorescence wavelength shifts to a long wavelength of 5 nm, but the spectrum width hardly changes. From this, it can be concluded that in the case of glutathione coating, as in the case of cysteine coating, the fluorescence spectral characteristics of the near-infrared semiconductor quantum dots before coating are retained upon water solubilization.

Evaluation of particle size and dispersibility in water The size and dispersibility of glutathione-coated near-infrared semiconductor quantum dots were measured using a dynamic light scattering apparatus (Malvern, Nano-ZS) and a fluorescence correlation spectrometer (Hamamatsu Photonics, C9413-01MOD). Was used to evaluate. The hydrodynamic particle size of glutathione-coated near-infrared semiconductor quantum dots in water was measured by dynamic light scattering using a 633 nm He-Ne laser. As a result, the particle size was about 10 nm (FIG. 1). The dispersibility in water was evaluated by a fluorescence correlation spectroscopy apparatus. The fluorescence correlation curve of glutathione-coated near-infrared semiconductor quantum dots in water can be approximated by a correlation function based on almost single component translational diffusion, and the diffusion time was 0.4 ms (FIG. 2). It can be concluded that the hydrodynamic particle size of the glutathione-coated near-infrared semiconductor quantum dots estimated from this value almost coincides with the result obtained by the dynamic light scattering method and exists as monodisperse particles.

Example 3
Modification of Bovine Serum Albumin to Glutathione-Coated Near-Infrared Semiconductor Quantum Dots 4 ml of phosphate buffer (1 μM, pH = 7.4) of glutathione-coated near-infrared semiconductor quantum dots of Example 2 is a cross-coupling reagent 50 μl (10 mM) of EDC (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride) was added. Ten minutes later, 1 ml of bovine serum albumin (10 mg / ml) was added and incubated for 30 minutes. Thereafter, a glutathione-coated near-infrared semiconductor quantum dot in which bovine serum albumin was surface-modified was obtained by removing excess EDC and bovine serum albumin using a dialysis membrane (molecular weight: 100,000).

Example 4
Modification of antibody to glutathione-coated near-infrared semiconductor quantum dots 20 μl of phosphate buffer (0.1 μM, pH = 7.4) of glutathione-coated near-infrared semiconductor quantum dots of Example 2 is a cross-coupling reagent 2.5 μl (0.1 mM) of EDC (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride) was added. Ten minutes later, 20 μl of Anti-UCAM-1 antibody (70 μg / ml) was added and incubated for 30 minutes. Thereafter, using a dialysis membrane (molecular weight 200,000), excess EDC and antibodies were removed, and glutathione-coated near-infrared semiconductor quantum dots whose surfaces were modified with antibodies were obtained.

Example 5
Synthesis of dual-modal (light / MRI) contrast agent (multimodal water-soluble near-infrared fluorescent material) by modification of Gd-DOTA complex to glutathione-coated near-infrared semiconductor quantum dots Glutathione-coated near-infrared semiconductor quantum of Example 2 Dot CdSe _0.75 Te _0.25 / CdS 1 μM solution in 5 ml (25 mM borate buffer, pH = 8.2), DOTA-NHS-ester (1,4,7,10-tetraazacyclododecane-1 , 4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester; US, macrocyclics, USA) 1 ml of an aqueous solution (20 mg / ml) was added at room temperature and reacted for 30 minutes with stirring. Thereafter, the solution was dialyzed against 25 mM borate buffer (pH = 8.2) to remove unreacted DOTA-NHS-ester. To this solution, 1 mL of 0.1 mM GdCl ₂ aqueous solution was added dropwise. To remove excess Gd ²⁺ ions, dialyzed again with 25 mM borate buffer (pH = 8.2). In order to stabilize the Gd-DOTA complex-modified glutathione-coated near-infrared semiconductor quantum dots near neutrality, the solution was exchanged by dialysis against a phosphate buffer containing 1% bovine serum albumin. A glutathione-coated near-infrared semiconductor quantum dot (multimodal water-soluble near-infrared fluorescent material) modified with a Gd-DOTA complex was prepared by the above procedure.

Absorption and Fluorescence Characteristics FIG. 3 shows the results of measuring the absorption spectrum and fluorescence spectrum of glutathione-coated near-infrared semiconductor quantum dots and indocyanine green (ICG) modified with the Gd-DOTA complex obtained in Example 5. Since the absorption spectrum of the glutathione-coated near-infrared semiconductor quantum dot shows continuous absorption from the near-infrared region to the visible region, excitation at all wavelengths in this region is possible. On the other hand, indocyanine green shows an absorber having a peak at 780 nm, and the excitation wavelength is also limited to the region of 780 nm.
For example, when excited at 745 nm, the quantum efficiency of light emission of a glutathione-coated near-infrared semiconductor quantum dot is about 10 times higher than that of indocyanine green. The quantum yield of indocyanine green (Photochem. Photobio. 72, 392-398 (2000)) is about 1%, while that of glutathione-coated near-infrared semiconductor quantum dots is estimated to be about 12%. It is. As for the photobleaching property, as shown in FIG. 4, the glutathione-coated near-infrared semiconductor quantum dots are excited by a 785 nm semiconductor laser as compared with indocyanine green (ICG), which fades with time. It has excellent fluorescence characteristics that it hardly fades even when irradiated.

To confirm the optical imaging effect of Gd-DOTA complexes modified near infrared semiconductor quantum dots by light contrasting effect present invention in mice was subjected to fluorescence imaging in mice. A Clavivo OPT special model manufactured by Shimadzu Corporation was used for fluorescence imaging. An 845 nm interference filter was used to receive a 758 nm semiconductor laser as an excitation light source, and exposure was performed for 10 seconds. FIG. 5 shows the result of fluorescence imaging of mouse lymph nodes and femoral arteriovenous blood vessels when the glutathione-coated near-infrared semiconductor quantum dots modified with the Gd-DOTA complex obtained in Example 4 were used at a concentration of 10 μM. Is shown. FIG. 5 confirms the effectiveness of the multimodal water-soluble near-infrared fluorescent material of the present invention as an optical contrast agent in mice.

To confirm the MRI contrast effect of Gd-DOTA complexes modified near infrared semiconductor quantum dots by MRI contrast effect present invention, it was obtained _{T 1} -weighted images. The imaging device was AVANCE 500WB (11.7T) manufactured by Bruker, and the imaging was performed by the gradient echo method. FIG. 6 shows a manganese-doped quantum dot and a halotransferrin (iron ion-containing) modified quantum dot reported by a conventional method and a glutathione-coated near-infrared modified with the Gd-DOTA complex obtained in Example 4. It showed T ₁ -weighted images of the semiconductor quantum dots. The measurement conditions were 2.0 cm for the measurement area, 256 × 256 for the in-plane division, 0.5 mm for the slice thickness, 47 ms for the repetition time, 5.3 ms for the echo time, 32 times for the integration time, and the required time was 6 minutes. As is clear from FIG. 6, the glutathione-coated near-infrared semiconductor quantum dots modified with the Gd-DOTA complex in the present invention were found to have an extremely high MRI contrast effect as compared with quantum dots produced by the prior art.

Dual-modal imaging by near-infrared fluorescence and MRI in mice In order to confirm the dual-modal (light / MRI) contrast function of Gd-DOTA complex-modified near-infrared semiconductor quantum dots according to the present invention, a phantom containing this is placed in the abdominal cavity of mice. Dual modal imaging by embedding, near infrared light and MRI was performed. The obtained results are shown in FIG. As is clear from FIG. 7, the use of the glutathione-coated near-infrared semiconductor quantum dots obtained by modifying the Gd-DOTA complex obtained in Example 4 makes it possible to clearly display the mouse in both near-infrared light and MRI imaging methods. The presence of the phantom in the abdominal cavity was confirmed. In near-infrared light imaging, three-dimensional accurate position information cannot be obtained due to light scattering in a living body. However, the position of the phantom within the mouse abdominal cavity can be accurately determined by imaging with MRI. These results show that dual modal imaging of a living body is possible by using the multimodal near-infrared semiconductor quantum dots according to the present invention.

The water-soluble near-infrared fluorescent material of the present invention has a light-emitting band in the near-infrared region that can be used in vivo, and has high light-emitting performance, high luminance, and excellent fading resistance. Furthermore, it has the advantages of high water dispersibility, high biocompatibility, and high safety for the living body. Therefore, the water-soluble near-infrared fluorescent material of the present invention can be suitably used as a contrast agent for optical (fluorescence) imaging.
In addition, the multimodal water-soluble near-infrared fluorescent material of the present invention has the advantages of the above-described water-soluble near-infrared fluorescent material (having a light emission band in the near-infrared region, high luminous performance, high luminance, Excellent photobleaching, high biocompatibility and high biosafety), optical (fluorescence) imaging, MRI (nuclear magnetic resonance imaging), X-ray CT or SPECT (single photon emission tomography) ) And the like, and can be used as a contrast agent for multimodal imaging. By using the multimodal water-soluble near-infrared fluorescent material of the present invention as a contrast agent in multimodal imaging, accurate three-dimensional position information in a living body can be obtained, and target objects such as cancer tissues can be more targeted. There is an advantage that it can be detected accurately. The multimodal water-soluble near-infrared fluorescent material of the present invention enables multimodal imaging combining a light (fluorescence) imaging method with MRI, X-ray CT, SPECT, etc. It greatly contributes to development.

It is a graph which shows the measurement result of the hydrodynamic particle size in water of the water-soluble near-infrared fluorescent material of Example 2. It is a graph which shows the fluorescence correlation curve by the fluorescence correlation spectroscopy apparatus of the water-soluble near-infrared fluorescent material of Example 2. It is a graph which shows the absorption spectrum and fluorescence spectrum of the multimodal water-soluble near-infrared fluorescent material (Gd-DOTA complex modification near-infrared semiconductor quantum dot) of Example 5, and indocyanine green (ICG). It is a graph which shows the photobleaching property of the multimodal water-soluble near-infrared fluorescent material (Gd-DOTA complex modification near-infrared semiconductor quantum dot) obtained from Example 5, and indocyanine green (ICG). An image obtained by fluorescence imaging of mouse lymph nodes and femoral arteriovenous blood vessels when the multimodal water-soluble near-infrared fluorescent material of Example 5 (Gd-DOTA complex-modified near-infrared semiconductor quantum dots) is used at a concentration of 10 μM It is. And a reported manganese-doped quantum dots and halo transferrin (iron ion-containing) modified quantum dots in a conventional manner, a T ₁ -weighted images of the water-soluble near infrared fluorescent material of Example 2. It is the image which carried out dual modal imaging by near infrared light and MRI which embedded the multimodal water-soluble near-infrared fluorescent material of Example 5 in the mouse | mouth abdominal cavity. 6 is a graph showing changes in fluorescence spectrum by cysteine coating of near-infrared semiconductor quantum dots in Example 1. It is a graph which shows the change of the fluorescence spectrum by the glutathione coating | cover of the near-infrared semiconductor quantum dot in Example 2. FIG.

Claims (13)

A semiconductor quantum dot having an emission band in the near infrared region, and a coating layer covering the surface of the semiconductor quantum dot;
A water-soluble near-infrared fluorescent material having
The coating layer is composed of cysteine and coated peptides selected from the group consisting of 2 to 8 amino acid residues and a peptide including one or more cysteine residues in the amino acid residues. Is a layer,
Water-soluble near-infrared fluorescent material. The water-soluble near-red color according to claim 1, wherein the semiconductor quantum dot is a semiconductor quantum dot having a core-shell structure composed of CdSe _1-x Te _x core / CdS shell (where x = 0.2 to 1). Outside fluorescent material.   The coated peptides are cysteine, glutathione, γ-Glu-benzyl-Cys-Val, β-Asp-Cys-Gly, Glu-Cys-Gly, Asp-Cys-Gly, γ-Glu-Gly-Cys-Gly, The water-soluble near-infrared fluorescence according to claim 1 or 2, which is a peptide selected from the group consisting of β-Asp-Gly-Cys-Gly, Glu-Gly-Cys-Gly, Asp-Gly-Cys-Gly. material.   The water-soluble near-infrared fluorescent material according to any one of claims 1 to 3, wherein the water-soluble near-infrared fluorescent material has an average particle diameter of 2 to 50 nm and is monodispersed. A semiconductor quantum dot whose surface is coated with a coordinating organic compound and having an emission band in the near infrared region; cysteine, and composed of 2 to 8 amino acid residues, and one or more in the amino acid residues Coating peptides selected from the group consisting of peptides containing cysteine residues of
A method for producing a water-soluble near-infrared fluorescent material. The water-soluble near-red color according to claim 5, wherein the semiconductor quantum dot is a core-shell semiconductor quantum dot composed of CdSe _1-x Te _x core / CdS shell (where x = 0.2 to 1). A method for producing an outer fluorescent material.   The coated peptides are cysteine, glutathione, γ-Glu-benzyl-Cys-Val, β-Asp-Cys-Gly, Glu-Cys-Gly, Asp-Cys-Gly, γ-Glu-Gly-Cys-Gly, The water-soluble near-infrared fluorescence according to claim 5 or 6, which are peptides selected from the group consisting of β-Asp-Gly-Cys-Gly, Glu-Gly-Cys-Gly, Asp-Gly-Cys-Gly. Material manufacturing method.   The water-soluble near-infrared fluorescent material manufactured by the manufacturing method of the water-soluble near-infrared fluorescent material in any one of Claims 5-7. A semiconductor quantum dot having an emission band in the near infrared region,
A coating selected from the group consisting of cysteine and a peptide composed of 2 to 8 amino acid residues and containing one or more cysteine residues in the amino acid residues, covering the surface of the semiconductor quantum dots A coating layer, which is a layer composed of peptides,
Elemental inclusion groups chemically bonded to the coating layer, and Gd (III), Mn (II), Mn (III), Fe (II), Fe (III), which are included in the element inclusion group, Cu (II), Cr (III), Co (II), Ni (II), Dy (III), Tb (III), Nd (III), Fe ₃ O ₄ , ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ^{99 m} From Tc, ⁹⁰ Y, ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁶⁵ Dy, ¹¹¹ In, ²⁰¹ Tc, ¹⁸ F, ⁷⁷ Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹²⁶ I, ¹³¹ I, ²⁰¹ Tl An element selected from the group consisting of
A multimodal water-soluble near-infrared fluorescent material. The multimodal water-soluble compound according to claim 9, wherein the semiconductor quantum dot is a semiconductor quantum dot having a core-shell structure composed of CdSe _1-x Te _x core / CdS shell (where x = 0.2 to 1). Near-infrared fluorescent material.   The coated peptides are cysteine, glutathione, γ-Glu-benzyl-Cys-Val, β-Asp-Cys-Gly, Glu-Cys-Gly, Asp-Cys-Gly, γ-Glu-Gly-Cys-Gly, The multimodal water-soluble near-red color according to claim 9 or 10, which is a peptide selected from the group consisting of β-Asp-Gly-Cys-Gly, Glu-Gly-Cys-Gly, Asp-Gly-Cys-Gly. Outside fluorescent material. The element which chemically bonds the reactive functional group which exists in a coating layer, and the element inclusion group containing compound of the water-soluble near-infrared fluorescent material obtained by the method in any one of Claims 5-7. Inclusion group introduction process,
Introduced element inclusion groups include Gd (III), Mn (II), Mn (III), Fe (II), Fe (III), Cu (II), Cr (III), Co (II), Ni (II), Dy (III), Tb (III), Nd (III), Fe ₃ O ₄ , ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ^99m Tc, ⁹⁰ Y, ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁸⁶ Re, ¹⁸⁸ Re ¹⁶⁵ Dy, ¹¹¹ In, ²⁰¹ Tc, ¹⁸ F, ⁷⁷ Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹²⁶ I, ¹³¹ I, ²⁰¹ Tl, introducing an element selected from the group consisting of:
A method for producing a multimodal water-soluble near-infrared fluorescent material, comprising:   A multimodal water-soluble near-infrared fluorescent material produced by the method for producing a multimodal water-soluble near-infrared fluorescent material according to claim 12.

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