Luminescent particles, labeling luminescent particles, method of manufacturing luminescent particles, evaluation method, oxygen concentration evaluation method, and activity evaluation method

Abstract

To provide luminescent particles which can be introduced into cells without using an organic solvent and without impairing inherent functionality of a luminescent dye contained therein, and to provide labeling luminescent particles, method of manufacturing the luminescent particles, evaluation method, oxygen concentration evaluation method, and activity evaluation method.SOLUTION: Luminescent particles of the present invention contain a luminescent dye and a polymer. The polymer is biomembrane-compatible.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to luminescent particles, luminescent particles for labeling, a method for producing luminescent particles, an evaluation method, an oxygen concentration evaluation method, and an activity evaluation method, and more particularly to luminescent particles and the like that can be introduced into cells without using an organic solvent and that do not impair the original function of the luminescent dye contained therein. [Background technology]

[0002] Luminescent dyes are excellent tools in various analytical methods. For example, they can be used to detect target substances with high sensitivity. Furthermore, differences in the luminescence intensity and spectral shape can provide information about the environment surrounding the luminescent dye. For this reason, luminescent dyes with various functions have been developed according to the purpose.

[0003] One promising application of luminescent dyes is their use in obtaining information about the intracellular environment. However, many luminescent dyes are hydrophilic and have low permeability through biological membranes, making them difficult to introduce into cells on their own.

[0004] Patent Documents 1 and 2 each disclose a technology for visualizing the oxygen concentration within a cell, and a technology for a luminescent dye whose luminescence intensity changes in response to the oxygen concentration.

[0005] In the technology disclosed in Patent Document 1, a luminescent dye that is poorly soluble in water is introduced into cells as an organic solvent solution, and the oxygen concentration within the cells is measured. The luminescent dye can be introduced into cells by forming an organic solvent solution. However, the organic solvent has a significant effect on the cells, and this effect cannot be ignored, so this technology cannot be said to be capable of accurately measuring the oxygen concentration within the cells.

[0006] In the technology disclosed in Patent Document 2, a luminescent dye that is poorly soluble in water is converted into a water-soluble luminescent dye by introducing a highly hydrophilic substituent. The luminescent dye is then introduced into cells to measure the oxygen concentration within the cells. However, this technology requires the introduction of a substituent and the synthesis of a new luminescent dye. Furthermore, the introduction of a substituent significantly changes the properties and functions of the luminescent dye, and therefore this technology cannot be said to be capable of accurately measuring the oxygen concentration within cells. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2008-281467 [Patent Document 2] Japanese Patent Application Laid-Open No. 2010-70494 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention has been made in consideration of the above problems and circumstances, and the problem to be solved is to provide luminescent particles, luminescent particles for labeling, methods for producing luminescent particles, evaluation methods, oxygen concentration evaluation methods, and activity evaluation methods that can be introduced into cells without using organic solvents and that do not impair the original functions of the luminescent dyes contained therein. [Means for solving the problem]

[0009] In order to solve the above-mentioned problems, the inventor investigated the causes of the above-mentioned problems and discovered that by containing a luminescent dye and a biomembrane-compatible polymer in luminescent particles, they can be introduced into cells without using organic solvents and without impairing the original function of the luminescent dye contained therein, thereby arriving at the present invention. That is, the above-mentioned problems of the present invention are solved by the following means.

[0010] 1. Luminescent particles containing a luminescent dye and a polymer, The polymer is a biomembrane-compatible polymer. A luminescent particle characterized by:

[0011] 2. The biomembrane-compatible polymer is water-soluble. 2. The luminescent particle according to claim 1,

[0012] 3. The luminescent dye is poorly soluble in water. 2. The luminescent particle according to claim 1,

[0013] 4. The luminescent particles are cell membrane permeable. 2. The luminescent particle according to claim 1,

[0014] 5. The luminescent dye is a phosphorescent dye. 2. The luminescent particle according to claim 1,

[0015] 6. The phosphorescent dye is an iridium complex 6. The luminescent particle according to claim 5,

[0016] 7. In the emission spectrum measured when irradiated with excitation light having the maximum absorption wavelength of the luminescent dye, the maximum luminescence intensity when the mass ratio of the luminescent dye to the biomembrane-compatible polymer is C is defined as I C , the I when the content mass ratio C of the luminescent dye is changed C The maximum value of I max , I C The value of I max When the content mass ratio of the luminescent dye is 80% of the value of (a), the smaller value is C1 and the larger value is C2. In the luminescent particle, the content mass ratio C of the luminescent dye to the biomembrane-compatible polymer is within the range of C1 to C2. 2. The luminescent particle according to claim 1,

[0017] 8. The biomembrane-compatible polymer has a structure derived from a phospholipid. 2. The luminescent particle according to claim 1,

[0018] 9. The biomembrane-compatible polymer is a polymer of 2-methacryloyloxyethyl phosphorylcholine. 2. The luminescent particle according to claim 1,

[0019] 10. The biomembrane-compatible polymer has a site capable of forming at least one of a dipole-dipole interaction, a CH-π interaction, and a π-π interaction with a molecule contained in the luminescent particle. 2. The luminescent particle according to claim 1,

[0020] 11. The average primary particle diameter of the luminescent particles measured by dynamic light scattering is within the range of 10 to 1000 nm. 2. The luminescent particle according to claim 1,

[0021] 12. Contains the luminescent particles described in item 1 A luminescent particle for labeling characterized in that:

[0022] 13. A method for producing luminescent particles, comprising: The luminescent particles are the luminescent particles according to any one of items 1 to 11, preparing a solution of the luminescent dye in an organic solvent; preparing an aqueous solution of the biomembrane-compatible polymer; a step of mixing and homogenizing the organic solvent solution of the luminescent dye and the aqueous solution of the biomembrane-compatible polymer; removing the organic solvent to form particles containing the luminescent dye and the biomembrane-compatible polymer. A method for producing luminescent particles, comprising:

[0023] 14. In the emission spectrum measured when the luminescent particle is irradiated with excitation light having the maximum absorption wavelength of the luminescent dye, the maximum luminescence intensity when the mass ratio of the luminescent dye to the biomembrane-compatible polymer is C is defined as I C , the I when the content mass ratio C of the luminescent dye is changed C The maximum value of I max When the above I C The value of I max The concentration of the luminescent dye in the organic solvent solution is adjusted so that the value of 14. The method for producing luminescent particles according to claim 13,

[0024] 15. An evaluation method for evaluating a cell or a microorganism, comprising: The luminescent particle according to any one of items 1 to 11 or the luminescent particle for labeling according to item 12 is used. An evaluation method characterized by:

[0025] 16. An oxygen concentration evaluation method for evaluating the oxygen concentration of a cell, comprising: The luminescent particle according to any one of items 1 to 11 or the luminescent particle for labeling according to item 12 is used. 1. A method for evaluating oxygen concentration.

[0026] 17. A method for evaluating the activity of a cell, comprising: Use the oxygen concentration evaluation method described in paragraph 16. A method for evaluating activity. [Effects of the Invention]

[0027] By the above-mentioned means of the present invention, it is possible to provide luminescent particles, luminescent particles for labeling, methods for producing luminescent particles, evaluation methods, oxygen concentration evaluation methods, and activity evaluation methods that can be introduced into cells without using organic solvents and that do not impair the original functions of the luminescent dyes contained therein.

[0028] The mechanism by which the effects of the present invention are manifested or the mechanism of action is not clear, but is speculated as follows.

[0029] The luminescent particles of the present invention contain a luminescent dye and a polymer with biomembrane affinity. This allows the entire luminescent particle to have affinity with biomembranes without changing the structure of the luminescent dye. Furthermore, the luminescent particles can be introduced into cells without using organic solvents. As a result, it is believed that the effects of organic solvents on cells can be eliminated and the original functions of the luminescent dye can be exhibited. [Brief explanation of the drawings]

[0030] [Figure 1] Schematic cross-sectional view of an example of a luminescent particle [Figure 2] Emission spectrum of luminescent particles when the ratio C of luminescent pigments is changed in six patterns [Figure 3] A graph showing the change in maximum luminescence intensity IC when the mass ratio C of the luminescent dye is changed [Figure 4] Optical imaging results for luminescent particles with different luminescent dye ratios C [Figure 5] Energy diagrams for molecular fluorescence and phosphorescence [Figure 6] Absorption spectrum of luminescent particles 1-3 [Figure 7] DLS measurement results for luminescent particles 1-3 [Figure 8] Emission spectra of luminescent particles 1 to 3 under normal air and low-oxygen conditions [Figure 9] Optical imaging results of breast cancer cell KPL4 using Luminescent Particle 2 [Figure 10] Optical imaging results of NK cells using Luminescent Particle 2 [Figure 11] Optical imaging results of luminescent particle 2 in mouse killer T cells (CTLL-2 cells) cultured at different glucose concentrations [Figure 12]Optical imaging results of human NK92-CD16 cells cultured at different glucose concentrations using Luminescent Particle 2. DETAILED DESCRIPTION OF THE INVENTION

[0031] The luminescent particle of the present invention is a luminescent particle containing a luminescent dye and a polymer, characterized in that the polymer is a polymer with affinity for biomembranes. This feature is a technical feature common to or corresponding to the following embodiments.

[0032] In an embodiment of the present invention, the biomembrane-compatible polymer is preferably water-soluble from the viewpoint of ease of introduction into cells.

[0033] In an embodiment of the present invention, from the viewpoint of obtaining the effects of the present invention remarkably, it is preferable that the luminescent dye is poorly soluble in water.

[0034] In an embodiment of the present invention, the luminescent particles are preferably cell membrane permeable, from the viewpoint of ease of introduction into cells.

[0035] In an embodiment of the present invention, the luminescent dye is preferably a phosphorescent dye, from the viewpoint of applicability to intracellular oxygen concentration evaluation.

[0036] In an embodiment of the present invention, the phosphorescent dye is preferably an iridium complex from the viewpoint of high oxygen responsiveness.

[0037] In an embodiment of the present invention, from the viewpoint of performance as a luminescent particle, the mass ratio C of the luminescent dye to the biomembrane-compatible polymer in the luminescent particle is preferably within the range of C1 to C2 above.

[0038] In an embodiment of the present invention, the biomembrane-compatible polymer preferably has a structure derived from a phospholipid, from the viewpoint of ease of introduction into cells.

[0039] In an embodiment of the present invention, the biomembrane-compatible polymer is preferably a polymer of 2-methacryloyloxyethyl phosphorylcholine, from the viewpoint of ease of introduction into cells.

[0040] In an embodiment of the present invention, in order to facilitate the formation of particles containing iridium complexes, it is preferable that the biomembrane-compatible polymer has a site capable of forming at least one of dipole-dipole interactions, CH-π interactions, and π-π interactions with molecules contained in the luminescent particles.

[0041] In an embodiment of the present invention, from the viewpoint of ease of introduction into cells, the average primary particle size of the luminescent particles measured by dynamic light scattering is preferably within the range of 10 to 1000 nm.

[0042] The labeling luminescent particles of the present invention are characterized by containing the above-mentioned luminescent particles.

[0043] The method for producing the luminescent particles of the present invention is characterized by comprising the steps of preparing an organic solvent solution of the luminescent dye, preparing an aqueous solution of the biomembrane-compatible polymer, mixing and homogenizing the organic solvent solution of the luminescent dye and the aqueous solution of the biomembrane-compatible polymer, and removing the organic solvent to form particles containing the luminescent dye and the biomembrane-compatible polymer. As an embodiment of the present invention, from the viewpoint of performance as a luminescent particle, C The value of I max It is preferable to adjust the concentration of the luminescent dye in the organic solvent solution so that the concentration is 80% or more of the value of (1).

[0044] The evaluation method of the present invention is a method for evaluating cells or microorganisms, and is characterized by using the above-mentioned luminescent particles or the above-mentioned luminescent particles for labeling.

[0045] The oxygen concentration evaluation method of the present invention is a method for evaluating the oxygen concentration of a cell, and is characterized by using the above-mentioned light-emitting particles or the above-mentioned light-emitting particles for labeling.

[0046] The activity evaluation method of the present invention is a method for evaluating the activity of cells, characterized in that it uses the oxygen concentration evaluation method described above.

[0047] The present invention, its components, and embodiments for carrying out the present invention will be described in detail below. In this application, the symbol "to" is used to mean that the numerical values ​​before and after it are included as lower and upper limits.

[0048] 1. Overview of Luminescent Particles The luminescent particle of the present invention is a luminescent particle containing a luminescent dye and a polymer, characterized in that the polymer is a polymer with affinity for biomembranes.

[0049] In the present invention, the term "luminescent dye" refers to a dye that is excited by external energy and then emits fluorescence, phosphorescence, or the like in the process of returning from the excited state to the ground state. The luminescent dye is preferably poorly soluble in water. In the present invention, "poorly soluble in water" means that the solubility of a solute (for example, a luminescent dye) in 100 g of water at 20°C is less than 5 g.

[0050] The term "biomembrane-compatible polymer" refers to a polymer that has a relatively high affinity with components that make up biomembranes. Details will be described later.

[0051] In the present invention, the luminescent particles may contain a luminescent dye and a polymer with affinity for biomembranes, and the particle structure is not limited. Figure 1 is a cross-sectional schematic diagram of an example of a luminescent particle. The luminescent particle 1 is formed by entanglement of a luminescent dye 2 and a biomembrane-compatible polymer 3. Although it is not clear, it is thought that the hydrophobic portion of the luminescent dye 2 and the hydrophobic portion 5 of the biomembrane-compatible polymer probably interact with each other. Furthermore, since the biomembrane-compatible polymer hydrophilic portion 4 is located on the outside of the luminescent particle, it is thought that the particle exhibits high dispersibility in water.

[0052] In the present invention, whether or not luminescent particles are formed can be determined by measuring the UV-Vis absorption of a dispersion containing luminescent particles and measuring the particle size by dynamic light scattering (DLS).

[0053] Specifically, for example, UV-Vis absorption measurement of the sample solution is performed using an ultraviolet-visible spectrophotometer "U-3300" (manufactured by Hitachi High-Tech Science Corporation). If a clear absorption spectrum is obtained by the measurement, it is understood that the luminescent dye is dispersed or dissolved in the sample solution.

[0054] Furthermore, for example, the particle size of the sample liquid is measured by dynamic light scattering (DLS) using a zeta potential measurement device "Zetasizer Nano S" (manufactured by Malvern). If the particle size can be measured, it is clear that particles are present in the sample liquid.

[0055] In other words, if a clear absorption spectrum can be obtained by UV-Vis absorption measurement and the particle size can be measured, it can be determined that the luminescent dye is in the form of particles and dispersed in the sample solution.In theory, it is also thought that if single particles of the luminescent dye are dispersed in the sample solution, a clear absorption spectrum can be obtained by UV-Vis absorption measurement and the particle size can be measured.

[0056] However, in reality, it is difficult to disperse a luminescent dye, which is poorly soluble in water, in water. Even if the luminescent dye can be dispersed, it will lose its dispersed state over time and float or settle in the water. When single particles of the luminescent dye float or settle in water, a clear absorption spectrum cannot be obtained in UV-Vis absorption measurement. Therefore, if a clear absorption spectrum can be obtained by UV-Vis absorption measurement and the particle size can be measured, it can be identified that the luminescent dye is dispersed in the sample solution in a form different from that of single particles.

[0057] The luminescent particles of the present invention contain a water-soluble biomembrane-compatible polymer, and therefore, although in the form of particles, have excellent dispersion stability in water.

[0058] 2. Components of luminescent particles The luminescent particles of the present invention contain a luminescent dye and a polymer with affinity for biomembranes.

[0059] (1) Luminescent dye The luminescent dye according to the present invention is not particularly limited, and examples thereof include phosphorescent dyes and fluorescent dyes. Hereinafter, the terms "phosphorescent dye" and "fluorescent dye" will also be simply referred to as "phosphorescent dye" and "fluorescent dye." The luminescent dye according to the present invention may be composed of, for example, only a phosphorescent dye or only a fluorescent dye, or may contain both a phosphorescent dye and a fluorescent dye.

[0060] (1.1) Phosphorescent dyes In the present invention, the term "phosphorescent dye" refers to a compound in which luminescence associated with a transition from an excited triplet state to a ground singlet state is observed. Specifically, the term is defined as a compound that emits phosphorescence at room temperature (25°C) and has a phosphorescence quantum yield of 0.01 or more at 25°C. In particular, a phosphorescence quantum yield of 0.1 or more is preferred.

[0061] The phosphorescence quantum yield can be measured by the method described in Spectroscopy II, 4th Edition, Experimental Chemistry Lectures 7, p. 398 (1992, Maruzen). The phosphorescence quantum yield in solution can be measured using various solvents. The phosphorescent dye used in the present invention may have a phosphorescence quantum yield of 0.01 or more in any solvent.

[0062] The phosphorescent dye is not particularly limited, and known dyes can be used, such as iridium complexes, ruthenium complexes, osmium complexes, platinum complexes, rhenium complexes, molybdenum complexes, strontium aluminate, and lanthanoid ions (complex salts).

[0063] Specific examples include compounds described in the following documents: Nature 395,151(1998), Appl.Phys.Lett.78,1622(2001), Adv.Mater.19,739(2007), Chem.Mater.17,3532(2005), Adv.Mater.17,1059(2005), WO 2009 / 100991, WO 2008 / 101842, WO 2003 / 040257, U.S. Patent Application Publication No. 2006 / 835469, U.S. Patent Application Publication No. 2006 / 0202194, U.S. Patent Application Publication No. 2007 / 008732 No. 1, U.S. Patent Application Publication No. 2005 / 0244673, Inorg.Chem.40,1704(2001), Chem.Mater.16,2480(2004), Adv.Mater.16,2003(2004), Angew.Chem.lnt.Ed.2006,45,7800, Appl.Phys.Lett.86,153505(2005), Chem.Lett.34,592(2005), Chem.Commun.2906(2005), Inorg.Chem.42,1248(2003), International Publication No. 2009 / 050290, International Publication No. 2002 / 015645, International Publication No. 2009 / 000673, U.S. Patent Application Publication No. 2002 / 0034656, U.S. Patent No. 7,332,232, U.S. Patent Application Publication No. 2009 / 0108737, U.S. Patent Application Publication No. 2009 / 0039776, U.S. Patent No. 6,921,915, U.S. Patent No. 6,687,266, U.S. Patent Application Publication No. 2007 / 0190359, U.S. Patent Application Publication No. 2006 / 0008670, U.S. Patent Application Publication No. 2009 / 0165846, U.S. Patent Application Publication No. 2008 / 0015355, U.S. Patent No. 7,250,226, U.S. Patent No. 7,396,598, U.S. Patent Application Publication No. 2006 / 0263635, U.S. Patent Application Publication No. 2003 / 0138657, U.S. Patent Application Publication No. 2003 / 0152802, U.S. Patent No. 7,090,928, Angew. Chem. lnt. Ed. 47, 1 (2008), Chem. Mater. 18, 5119 (2006), Inorg. Chem.46,4308(2007), Organometallics 23,3745(2004), Appl.Phys.Lett.74,1361(1999), WO 2002 / 002714, WO 2006 / 009024, WO 2006 / 056418, WO 2005 / 019373, WO 2005 / 123873, WO 2005 / 123873, WO 2007 / 004380, WO 2006 / 082742, U.S. Patent Application Publication No. 2006 / 0251923, U.S. Patent Application Publication No. 2005 / 0260441, U.S. Patent No. 7,393,599, U.S. Patent No. 7,534,505, U.S. Patent No. 7,445,855, U.S. Patent Application Publication No. 2007 / 0190359, U.S. Patent Application Publication No. 2008 / 0297,033, U.S. Patent No. 7,338,722, U.S. Patent Application Publication No. 2002 / 0134,984, U.S. Patent No. 7,279,704, U.S. Patent Application Publication No. 2006 / 098,120, U.S. Patent Application Publication No. 2006 / 1. No. 03874, International Publication No. 2005 / 076380, International Publication No. 2010 / 032663, International Publication No. 2008 / 140115, International Publication No. 2007 / 052431, International Publication No. 2011 / 134013, International Publication No. 2011 / 157339, International Publication No. 2010 / 086089, International Publication No. 2009 / 113646, International Publication No. 2012 / 020327, International Publication No. 2011 / 051404, These include International Publication No. 2011 / 004639, International Publication No. 2011 / 073149, U.S. Patent Application Publication No. 2012 / 228583, U.S. Patent Application Publication No. 2012 / 212126, Japanese Patent Application Laid-Open No. 2012-069737, Japanese Patent Application No. 2011-181303, Japanese Patent Application Laid-Open No. 2009-114086, Japanese Patent Application Laid-Open No. 2003-81988, Japanese Patent Application Laid-Open No. 2002-302671, and Japanese Patent Application Laid-Open No. 2002-363552.

[0064] In the present invention, among these known phosphorescent dyes, those that are poorly soluble in water can be more preferably used. These may be contained alone or in combination of two or more.

[0065] Among these, the phosphorescent dye is preferably an iridium complex having iridium as the central metal, and more preferably a complex having at least one of the coordination modes of a metal-carbon bond, a metal-nitrogen bond, a metal-oxygen bond, and a metal-sulfur bond.

[0066] Specific examples of phosphorescent dyes are shown below, but the present invention is not limited thereto.

[0067] [ka]

[0068] [ka]

[0069] [ka]

[0070] [ka]

[0071] [ka]

[0072] [ka]

[0073] [ka]

[0074] [ka]

[0075] (1.2) Fluorescent dyes The fluorescent dye is not particularly limited, and examples thereof include rhodamine-based dye molecules, squarylium-based dye molecules, cyanine-based dye molecules, perylene-based (perylene diimide-based) dye molecules, pyrene-based dye molecules, oxazine-based (benzophenoxazine-based) dye molecules, azobenzene-based (azonaphthalene-based) dye molecules, carbopyronine-based dye molecules, pyrromethene-based dye molecules, Alexa Fluor (registered trademark, manufactured by Invitrogen)-based dye molecules, BODIPY (registered trademark, manufactured by Invitrogen)-based dye molecules, Cy (registered trademark, manufactured by GE Healthcare)-based dye molecules, DY (registered trademark, manufactured by DYOMICS)-based dye molecules, HiLyte (registered trademark, manufactured by Anaspec)-based dye molecules, DyLight (registered trademark, manufactured by Thermo Scientific)-based dye molecules, ATTO (registered trademark, manufactured by ATTO-TEC)-based dye molecules, MFP (registered trademark, manufactured by Mobitec)-based dye molecules, and FM-based dye molecules. Fluorescent dyes also include thermally activated delayed fluorescence (TADF) compounds.

[0076] In the present invention, among these known fluorescent dyes, those that are poorly soluble in water can be more preferably used. These may be contained alone or in combination of two or more.

[0077] Specific examples of BODIPY dye molecules include "BODIPY493 / 503," "BODIPY FL," "BODIPY R6G," "BODIPY TMR," "BODIPY 581 / 591," "BODIPY TR," "BODIPY 630 / 650," and "BODIPY 650 / 665."

[0078] Examples of FM dye molecules include "FM 1-43," "FM 1-43FX," "FM 4-64," and "FM 4-64FX." Examples of oxazine (benzophenoxazine) dye molecules include Nile Red and Nile Blue. Examples of azobenzene (azonaphthalene) dye molecules include Oil Red O.

[0079] These fluorescent dyes have aromatic rings in the basic skeleton, which is the main part of their molecular structure, and tend to be poorly soluble in water. The basic skeleton of fluorescent dyes is shown below.

[0080] [ka]

[0081] (2) Biomembrane-compatible polymers In the present invention, the term "biomembrane" is a general term for the membrane structure that constitutes a cell, and is also called a "cell membrane." Biological membranes are selectively permeable membranes that separate cells from the external environment and form intracellular compartments. Biological membranes are mainly composed of proteins and lipids.

[0082] In the present invention, the term "biomembrane-compatible polymer" refers to a polymer having functional groups with relatively high affinity for proteins and lipids that mainly constitute biomembranes. "Biomembrane-compatible" means that the polymer does not induce significant adverse reactions, such as long-term and chronic inflammatory reactions, when it comes into contact with a biomembrane. The biomembrane-compatible polymer is preferably water-soluble, specifically, preferably has a solubility of 0.1 g or more in 100 g of water at 20°C.

[0083] The biomembrane-compatible polymer is not particularly limited as long as it has affinity for biomembranes, and there is no particular limitation as to whether it is degradable in vivo. Examples of non-biodegradable materials include polytetrafluoroethylene (PTFE), polyurethane, polypropylene, polyester, vinyl chloride, polycarbonate, acrylic, stainless steel, titanium, silicone, and MPC (2-methacryloyloxyethyl phosphorylcholine) polymer.

[0084] Examples of biodegradable materials include polypeptides such as recombinant peptides or chemically synthesized peptides. Specific examples include gelatin, which will be described later. Other examples of biodegradable materials include polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer (PLGA), hyaluronic acid, glycosaminoglycans, proteoglycans, chondroitin, cellulose, agarose, carboxymethylcellulose, chitin, and chitosan.

[0085] The biomembrane-compatible polymer may be crosslinked or not, but is preferably crosslinked, which can prevent the biomembrane-compatible polymer from being instantly decomposed when the luminescent particles are introduced into cells.

[0086] Common crosslinking methods include crosslinking with aldehydes such as formaldehyde and glutaraldehyde. Other crosslinking methods include crosslinking with condensing agents such as carbodiimide and cyanamide. Other crosslinking methods include thermal crosslinking, enzymatic crosslinking, photocrosslinking, ultraviolet crosslinking, hydrophobic interaction, hydrogen bonding, and ionic interaction. Among these, thermal crosslinking, ultraviolet crosslinking, and enzymatic crosslinking are preferred, with thermal crosslinking being more preferred.

[0087] When crosslinking is performed using an enzyme, the enzyme is not particularly limited as long as it has the ability to crosslink between polymers. The enzyme is preferably transglutaminase or laccase, and more preferably transglutaminase. Specific examples of proteins that can be enzymatically crosslinked using transglutaminase include proteins having lysine residues and glutamine residues.

[0088] Transglutaminase may be derived from mammals or microorganisms. Specific examples include the "Activa Series" (manufactured by Ajinomoto Co., Inc.), mammalian transglutaminases available as reagents, such as guinea pig liver transglutaminase, goat transglutaminase, and rabbit transglutaminase, available from Oriental Yeast Co., Ltd., Upstate USA Inc., and Biodesign International, as well as human blood coagulation factor (Factor XIIIa, Haematologic Technologies, Inc.).

[0089] For example, the reaction temperature when thermal crosslinking is carried out is not particularly limited as long as crosslinking is possible. The reaction temperature is preferably within the range of -100 to 500°C, more preferably within the range of 0 to 300°C, even more preferably within the range of 50 to 300°C, particularly preferably within the range of 100 to 250°C, and most preferably within the range of 120 to 200°C. However, T (Kelvin: K) = t (Celsius degree: °C) + 273.15.

[0090] The biomembrane-compatible polymer preferably has a site capable of forming at least one of dipole-dipole interaction, CH-π interaction, and π-π interaction with the molecules contained in the luminescent particles, particularly the luminescent dye molecules. By having a site capable of forming an interaction, the biomembrane-compatible polymer can uniformly incorporate the molecules contained in the luminescent particles, particularly the luminescent dye molecules, into the particles. Furthermore, the luminescent particles can be easily produced using the method described below.

[0091] (2.1) Phosphorylcholine group-containing polymer The biomembrane-compatible polymer preferably contains a phosphorylcholine group. The phosphorylcholine group is a polar group having a structure similar to that of the polar group of phospholipids (phosphatidylcholine), the main component of biomembranes. Therefore, the polymer containing the phosphorylcholine group exhibits extremely good affinity for the surface of biomembranes.

[0092] The polymer structure of the biomembrane-compatible polymer is not particularly limited, but it preferably has a chemical structure in which a monomer component having an acrylic skeleton is polymerized.

[0093] Examples of monomer components having an acrylic skeleton and a phosphorylcholine group include 2-methacryloyloxyethyl phosphorylcholine, 2-acryloyloxyethyl phosphorylcholine, N-(2-methacrylamido)ethyl phosphorylcholine, 4-methacryloyloxybutyl phosphorylcholine, 6-methacryloyloxyhexyl phosphorylcholine, 10-methacryloyloxydecyl phosphorylcholine, ω-methacryloyldioxyethylene phosphorylcholine, 4-styryloxybutyl phosphorylcholine, etc. Among these, 2-methacryloyloxyethyl phosphorylcholine is preferred.

[0094] The monomer component may be contained alone or in combination of two or more. A polymer containing 2-methacryloyloxyethyl phosphorylcholine as a monomer component is also called an "MPC polymer."

[0095] The phosphorylcholine group-containing polymer preferably has a structure represented by the following formula (1).

[0096] [ka]

[0097] In formula (1), m and n each independently represent an integer of 1 or greater. m is preferably an integer in the range of 1 to 10, more preferably an integer in the range of 1 to 5, and even more preferably an integer in the range of 1 to 3. In the MPC polymer, m is 1.

[0098] The phosphorylcholine group-containing polymer may have a structure represented by the following formula (2) in addition to the structure represented by the above formula (1).

[0099] [ka]

[0100] In formula (2), n represents an integer of 1 or greater, and R represents a hydrogen atom or a hydrocarbon group, which may further have a substituent.

[0101] Examples of the hydrocarbon group include aliphatic hydrocarbon groups such as methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, and n-hexyl groups, as well as aromatic hydrocarbon groups such as phenyl, tolyl, xylyl, mesityl, benzyl, naphthyl, and anthracenyl groups.

[0102] Examples of the substituent include halogeno groups such as a fluoro group, a chloro group, a bromo group, and an iodo group, as well as hydroxyl, carboxyl, aldehyde, ketone, ether, ester, nitro, amino, and sulfo groups.

[0103] When the phosphorylcholine group-containing polymer is a polymer containing multiple types of monomer components, the phosphorylcholine group-containing polymer may be, for example, any of an alternating copolymer, a random copolymer, a block copolymer, and a graft copolymer.

[0104] The content of the monomer component having a phosphorylcholine group relative to all the monomer components constituting the phosphorylcholine group-containing polymer is preferably within a range of 10 to 80 mol %, more preferably within a range of 20 to 70 mol %, and even more preferably within a range of 25 to 60 mol %.

[0105] The weight-average molecular weight of the phosphorylcholine group-containing polymer is not particularly limited, and is preferably within the range of 8,000 to 160,000, more preferably within the range of 30,000 to 150,000, and even more preferably within the range of 50,000 to 120,000. The weight-average molecular weight can be measured by gel permeation chromatography (GPC).

[0106] The method for synthesizing the phosphorylcholine group-containing polymer is not particularly limited. For example, a method of radical polymerization using a polymerization initiator can be used. Specifically, a method of radical polymerization in an organic solvent can be used.

[0107] The radical polymerization initiator used in the polymerization is not particularly limited, and examples thereof include azo-based initiators such as 2,2'-azobisisobutyronitrile (AIBN), 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(2-amidinopropane) dihydrochloride, 4,4'-azobis(4-cyanopentanoic acid), and 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile).Other examples include peroxide-based initiators such as benzoyl peroxide, t-butyl hydroperoxide, and potassium persulfate.

[0108] The amount of radical polymerization initiator used is preferably adjusted depending on the polymerizability of the monomer and the molecular weight of the required polymer, and is, for example, preferably within a range of 0.001 to 3 mass %, more preferably within a range of 0.01 to 1 mass %, based on the total mass of all monomers used in the polymerization.

[0109] Examples of solvents used in radical polymerization include ester solvents such as ethyl acetate, alcohol solvents such as methanol, ethanol, 1-propanol, 2-propanol, and 1-butanol, ketone solvents such as acetone, ether solvents such as dioxane, amide solvents such as N,N-dimethylformamide and N,N-dimethylacetamide, aromatic solvents such as benzene and toluene, nitrile solvents such as acetonitrile, and halogenated solvents such as methylene chloride, chloroform, and dichloroethane. Among these, alcohol solvents such as methanol, ethanol, 1-propanol, 2-propanol, and 1-butanol are preferred. These may be used alone or in combination of two or more.

[0110] The reaction temperature during the polymerization reaction can be appropriately set depending on the molecular weight of the required polymer, the type of polymerization initiator, etc. For example, the reaction temperature is preferably within the range of 30 to 100°C.

[0111] After the polymerization reaction, the resulting solution of the phosphorylcholine group-containing polymer may be purified as needed.

[0112] The purification method is not particularly limited, and examples thereof include reprecipitation. In the reprecipitation method, examples of poor solvents include ether solvents such as diethyl ether, dioxane, and tetrahydrofuran; ester solvents such as 3-methoxybutyl acetate, ethyl acetate, isopropyl acetate, and butyl acetate; ketone solvents such as methyl ethyl ketone, acetone, methyl isobutyl ketone, cyclohexanone, and isophorone; and halogenated hydrocarbon solvents such as chloromethane, dichloromethane, and chloroform. These may be used alone or in combination of two or more.

[0113] Furthermore, the phosphorylcholine group-containing polymer can be purified, for example, by ultrafiltration.

[0114] (3) Mass ratio C of luminescent pigment In the present invention, the "mass ratio C of the luminescent dye" refers to the ratio of the mass of the luminescent dye to the mass of the biomembrane-compatible polymer contained in the luminescent particle. Hereinafter, the mass ratio C of the luminescent dye will also be simply referred to as "ratio C."

[0115] The luminescent particles are irradiated with excitation light of the maximum absorption wavelength of the luminescent dye, and the luminescence spectrum is measured. The maximum luminescence intensity of this luminescence spectrum is defined as I. The maximum luminescence intensity of the luminescent particle with a luminescent dye content mass ratio of C is defined as I. C Let's say.

[0116] Ratio C and luminescence intensity I C The following study was conducted regarding the relationship between Luminescent particles containing Ir-1 as the luminescent dye and MPC polymer as the biomembrane-compatible polymer were prepared using the method described below. The luminescent dye ratio C was varied in six patterns. Each luminescent particle was irradiated with excitation light at the absorption maximum wavelength of the luminescent dye, and the emission spectrum was measured.

[0117] Figure 2 shows the emission spectra of luminescent particles when the ratio C of the luminescent dye is changed in six patterns. As shown by emission spectra (1) to (4) in Figure 2, the luminescence intensity increases as the ratio C increases. However, as shown by emission spectra (4) to (6) in Figure 2, it was found that the luminescence intensity decreases when the ratio C exceeds a certain value.

[0118] In other words, the maximum luminous intensity I C is maximized when the ratio C is a certain value. In the present invention, the maximum emission intensity in the emission spectrum when the ratio C is changed is defined as I max The ratio C at which the maximum luminescence intensity peak appears is defined as C max Let's say.

[0119] Figure 3 shows the maximum luminescence intensity I when the mass ratio C of the luminescent dye is changed. C This is a graph showing the change in I C The maximum value of I maxThe mass ratio of the luminescent dye at this time is C max As shown in Figure 3, the maximum luminescence intensity I C But, I max There are two values ​​of the ratio C of the luminescent pigment that will be 80% of the maximum luminescence intensity I, and these are designated C1 and C2, starting from the smallest one. C I max It is 80% or more of C max exists between C1 and C2. From the viewpoint of performance as a luminescent particle, the ratio C of the luminescent dye is preferably within the range of C1 to C2. Maximum luminous intensity of luminous particles I C I max It is preferable that the ratio is 20% or more of I max It is more preferable that the ratio is 50% or more of I max It is more preferable that the ratio is 80% or more.

[0120] Figure 4 shows the results of optical imaging of luminescent particles with different luminescent dye ratios C. (a) is a graph showing the luminescence intensity (I) under normal air and hypoxia conditions for luminescent particles with different luminescent dye ratios C (Concentration of Dye). (b) shows the results of optical imaging in normal air when the luminescent dye ratios C are (I) to (III). (c) shows the results of optical imaging under hypoxia conditions when the luminescent dye ratios C are (I) to (III). Note that (I) to (III) in (b) and (c) correspond to (I) to (III) in graph (a). Details of cell evaluation by optical imaging will be described later.

[0121] Graph (II) in Figure 4(a) shows that the ratio C is C max The emission intensity I when max In addition, the optical imaging results (II) in Figure 4 (b) and (c) show that the ratio C is C maxThe graph (I) in (a) of FIG. 4 and the optical imaging results (I) in (b) and (c) of FIG. 4 show the optical imaging results when the ratio C is C max Graph (III) and the optical imaging results (b) and (c) show an example where the ratio C is smaller than C. max Here is an example where the ratio C is greater than C max When the light-emitting particle has the highest light-emitting intensity, the highest quality light-emitting image can be obtained in optical imaging.

[0122] 3. Physical properties of luminescent particles (Average primary particle size) The average primary particle size of the luminescent particles measured by dynamic light scattering is preferably within the range of 10 to 1000 nm, more preferably within the range of 30 to 300 nm. When the average primary particle size is 10 nm or more, the particle shape can be stably maintained. When the average primary particle size is 1000 nm or less, excellent cell membrane permeability can be achieved.

[0123] The average primary particle size can be measured using, for example, "Microtrac UPA-150" (manufactured by Nikkiso Co., Ltd.).

[0124] 4. Method for producing luminescent particles The method for producing luminescent particles of the present invention is characterized by comprising the steps of preparing an organic solvent solution of the luminescent dye, preparing an aqueous solution of the biomembrane-compatible polymer, mixing and homogenizing the organic solvent solution of the luminescent dye and the aqueous solution of the biomembrane-compatible polymer, and forming particles containing the luminescent dye and the biomembrane-compatible polymer by removing the organic solvent.

[0125] An organic solvent solution of a luminescent dye and an aqueous solution of a polymer are prepared, and these solutions are mixed and homogenized, whereby the luminescent dye molecules change from interacting with the organic solvent molecules to interacting with the polymer molecules in an entropically favorable manner. The organic solvent is then removed, and the interaction between the luminescent dye molecules and the polymer molecules becomes stronger in an enthalpic favorable manner. In this manner, particles containing a luminescent dye and a polymer are formed. In the description of the production method, the "biomembrane-compatible polymer" is also simply referred to as the "polymer."

[0126] For luminescent dyes that are poorly soluble in water, an organic solvent is used as the solvent for the luminescent dye solution. On the other hand, biomembrane-compatible polymers have many hydrophilic sites and are relatively soluble in water, so water is used as the solvent for the polymer solution. By mixing and homogenizing these solutions, the hydrophobic sites of the luminescent dye and the hydrophobic sites of the polymer approach each other, resulting in entanglement of the luminescent dye and the polymer. This is because a state in which polymer molecules, capable of forming multiple interactions with a single molecule, interact with the luminescent dye molecules is more entropically advantageous than a state in which many organic solvent molecules interact with the luminescent dye molecules. The solvent for this dispersion is a mixture of organic solvent and water. Removing the organic solvent from this dispersion results in the release of organic solvent molecules outside the system, which is entropically disadvantageous. To compensate for this, strengthening the interaction between the luminescent dye molecules and the polymer molecules is enthalpiically advantageous. As a result, particle formation progresses, resulting in a dispersion of particles in which the dispersion solvent is water.

[0127] (1) A step of preparing an organic solvent solution of a luminescent dye In this step, a solution of a luminescent dye in an organic solvent is prepared by a known method.

[0128] If the organic solvent remains in the final luminescent particles and dispersion, the organic solvent will also be introduced into the cells when the luminescent particles are introduced into the cells. Therefore, it is preferable to select an organic solvent that is less likely to remain in the luminescent particles and dispersion. Also, from the viewpoint of the particle formation process, it is preferable to select an organic solvent that dissolves the luminescent dye and whose solvent molecules are easily removed. In other words, it is preferable that the organic solvent can dissolve the luminescent dye, be present in the mixed liquid when forming the luminescent particles, and be ultimately removable from the dispersion.

[0129] Examples of the organic solvent include dichloromethane, methyl ethyl ketone, ethyl acetate, butyl acetate, tetrahydrofuran, and 1,3-dioxolane.

[0130] (2) Prepare an aqueous solution of a biomembrane-compatible polymer In this step, an aqueous solution of a biomembrane-compatible polymer is prepared by a known method.

[0131] (3) A step of mixing and homogenizing the organic solvent solution of the luminescent dye and the aqueous solution of the biomembrane-compatible polymer. In this step, the solutions obtained above are mixed and homogenized by a known method. The concentration and mixing ratio of each solution are not particularly limited, but it is preferable that the amount of organic solvent is smaller.

[0132] The dispersing machine used for homogenization is not particularly limited, and examples thereof include a stirring device equipped with a high-speed rotating rotor, "Clearmix" (manufactured by M Technique Co., Ltd.), an ultrasonic dispersing machine, a mechanical homogenizer, a Manton-Gaulin homogenizer, and a pressure homogenizer.

[0133] It is preferable that the organic solvent used in preparing the luminescent dye solution remains in the mixed solution during this process. If the organic solvent is removed before the luminescent dye molecules and the polymer molecules sufficiently interact with each other, precipitation of the luminescent dye is likely to occur. In this process, it is preferable to adjust the temperature, pressure, etc. so that the organic solvent is not removed by evaporation, etc. However, it is preferable that the temperature and pressure here are within a range that does not affect the luminescent dye and the polymer.

[0134] The average primary particle size of the luminescent particles can be adjusted by adjusting the homogenization conditions, such as the ratio of the dye to the biomembrane-compatible polymer added, the dispersion strength, speed, temperature, and the like.

[0135] (4) Removal of organic solvent from particles In this step, the organic solvent in the particles obtained above is removed. Specifically, the organic solvent is removed from the dispersion obtained above. As a result, a dispersion of particles in which the dispersion solvent is water is obtained.

[0136] The method for removing the organic solvent is not particularly limited, but from the viewpoint of ease of operation, removal by evaporation is preferred. Temperature, pressure, etc. may be adjusted as necessary. However, from the viewpoint of ease of operation and less influence on the luminescent dye and polymer, the temperature during evaporation is preferably room temperature.

[0137] (5) An example of a method for producing luminescent particles An example of a method for producing luminescent particles will be described below, although the present invention is not limited thereto. In the following example, the phosphorescent dye Ir-1 is used as the luminescent dye, dichloromethane is used as the organic solvent, and MPC polymer is used as the biomembrane-compatible polymer.

[0138] The phosphorescent dye Ir-1 is dissolved in dichloromethane to prepare a dichloromethane solution of the phosphorescent dye Ir-1 with a concentration of 1 mM or less. Furthermore, an MPC polymer is dissolved in water to prepare a 0.5% by mass MPC polymer aqueous solution. These solutions are mixed so that the mass ratio of the luminescent dye solution to the polymer solution is 1:9. The MPC polymer used is "LIPIDURE (registered trademark)-BL206" (manufactured by NOF Corporation).

[0139] The resulting mixture is homogenized for 30 minutes with a homogenizer while being cooled with 4°C water to obtain a dispersion. The resulting dispersion is then left at room temperature for at least 1 hour to evaporate the dichloromethane, yielding a dispersion of particles in which the only dispersion solvent is water.

[0140] Dichloromethane evaporates easily at room temperature. Therefore, by homogenizing the mixture while cooling it with water at 4°C, the evaporation of dichloromethane before homogenization can be suppressed. Furthermore, cooling the mixture with water at 4°C has only a minimal effect on the luminescent dye and MPC polymer. If the mixture is homogenized at room temperature without cooling, the dichloromethane will evaporate quickly, and the phosphorescent dye Ir-1 will precipitate in water before it can entangle with the MPC polymer, failing to form the luminescent particles of the present invention.

[0141] When using an organic solvent that has a higher boiling point than dichloromethane and is difficult to evaporate at room temperature, the mixture may be homogenized at room temperature, and then the dispersion may be heated to evaporate the organic solvent.

[0142] (6) Evaluation of the validity of the particle formation process by calculation To demonstrate that the process in which the organic solvent molecules are replaced by polymer molecules around the luminescent dye molecules is valid, an example of calculation will be described. In the following example, Ir-1 and Ir-15 were used as the luminescent dyes, the structure represented by the following formula (3) was used as the polymer, and dichloromethane was used as the organic solvent.

[0143] [ka]

[0144] The stabilization energy when x molecules of dichloromethane as a solvent or y molecules of polymers of formula (3) are placed around Ir-1 and Ir-15 as solutes is expressed as Dmol 3 The results are shown in Table I below. The stabilization energy was defined as the sum of the energy when a solute molecule exists alone and the energy when a solvent molecule exists alone, minus the energy when a solvent molecule is placed around the solute molecule. Therefore, when calculating the stabilization energy, the stabilization energies for the cases when Ir-1 and Ir-15 exist alone, when x dichloromethane molecules exist independently, and when y polymers of formula (3) exist independently were calculated as Dmol 3 The results were calculated using DFT calculations.

[0145] [Table 1]

[0146] Table I shows that Ir-1 and Ir-15 have a higher stabilization energy when polymer (3) is present than when dichloromethane is present around them. This calculation result indicates that Ir-1 and Ir-15 are more stable when interacting with a smaller number of polymer (3) molecules than when interacting with a larger number of dichloromethane molecules. In other words, the process in which organic solvent molecules are replaced by polymer molecules around Ir-1 and Ir-15 is plausible.

[0147] 5. Application of Luminescent Particles to Cell Evaluation The evaluation method of the present invention is a method for evaluating cells or microorganisms, and is characterized by using the above-mentioned light-emitting particles. In the present invention, the luminescent particles used for labeling purposes are also referred to as "luminescent particles for labeling" or "probes."

[0148] The luminescent particles of the present invention can be introduced into cells by containing a polymer that is compatible with biomembranes, and can be used as an optical imaging agent in optical imaging methods.

[0149] Optical imaging is a method of imaging the internal details of a subject by irradiating the subject with light and measuring the intensity of the signal generated by an optical imaging agent introduced into the subject. By imaging the internal details of the subject, specific components present in the subject or the internal state of the subject can be labeled.

[0150] Examples of optical imaging methods include photoultrasound imaging and fluorescence imaging. The term "fluorescence imaging" as used herein includes detection of not only fluorescence but also phosphorescence. Optical imaging methods are capable of imaging the internal details of a subject without destroying it, and are therefore used in applications such as in vivo diagnostic imaging.

[0151] Objects to be evaluated using optical imaging include biological tissues, which are single cells or cell aggregates, and microorganisms. Optical imaging can be used to evaluate the characteristics, state, and function of cells and biological tissues, as well as the environment within the cells and biological tissues. Optical imaging can also be used to evaluate the characteristics, state, function, and environment within microorganisms. Examples of microorganisms include prokaryotes such as eubacteria and archaea; and eukaryotes such as microalgae, protists, fungi, and slime molds.

[0152] The cells to be evaluated may be any of plate-cultured cells, spheroids, and suspension cells. They may also be established cultured cells or primary cultured cells. Furthermore, the luminescent particles of the present invention may be administered to laboratory animals such as mice and rats to detect areas with reduced oxygen concentration.

[0153] Examples of cells include cancer cells, oocytes, fertilized egg cells, sperm cells, embryonic stem cells, iPS cells, adult stem cells, hematopoietic stem cells, tissue stem cells, fibroblasts, feeder cells, vascular endothelial cells, bone marrow (stem) cells, dental pulp (stem) cells, immune cells, hepatocytes, kidney cells, nerve cells, pancreatic cells, smooth muscle cells, cardiac muscle cells, myoblasts, corneal cells, retinal cells, osteocytes, osteoclasts, chondrocytes, chondrocyte precursor cells, synovial membrane-derived cells, synovial stem cells, osteoblasts, odontoblasts, periodontal ligament cells, oral mucosal cells, nasal mucosal cells, mesenchymal stem cells, adipocytes, adipose stem cells, epithelial cells, endothelial cells, muscle cells, epidermal cells, ovaries, red blood cells, white blood cells, platelets, and plant cells.

[0154] For example, by using a phosphorescent dye as the luminescent dye in the luminescent particles, the oxygen concentration of cells can be evaluated. Furthermore, by using a dye that stains a specific component as the luminescent dye in the luminescent particles, the specific component can be detected. Specifically, by using Oil Red O as a fluorescent dye, lipid droplets can be detected. Furthermore, by using the luminescent particles, specific components can be detected by fluorescent immunostaining.

[0155] (1) Oxygen concentration evaluation method The oxygen concentration evaluation method of the present invention is characterized by using the above-mentioned luminescent particles.

[0156] Figure 5 shows the energy state diagram for molecular fluorescence and phosphorescence. As shown in Figure 5, fluorescence and phosphorescence are emissions that occur due to transitions from an excited singlet state or an excited triplet state to the ground state, respectively. Since the ground state of many molecules is a singlet, fluorescence is an allowed transition, while phosphorescence is a forbidden transition. Phosphorescence has a significantly longer emission lifetime than fluorescence. Therefore, some molecules in the excited triplet state collide with oxygen molecules by diffusion during their excitation lifetime. The molecules then transfer their excitation energy to oxygen and deactivate to the ground state without exhibiting phosphorescence. In other words, they are quenched by oxygen. Taking advantage of this property, intracellular oxygen concentrations can be evaluated using luminescent particles containing phosphorescent dyes.

[0157] When the oxygen concentration in the cell is relatively low, the phosphorescent pigment is not quenched by oxygen. The resulting phosphorescence is high in intensity and has a long lifetime. On the other hand, when the oxygen concentration in the cell is relatively high, the phosphorescent pigment is quenched by oxygen. The resulting phosphorescence is low in intensity and has a short lifetime.

[0158] Luminescent particles containing phosphorescent dyes can be introduced into cells, such as planar cultured cells and spheroids, or into biological tissues, and emit strong phosphorescence in hypoxic regions within these cells. Therefore, luminescent particles containing phosphorescent dyes are useful as reagents for assessing oxygen concentration and can be used in oxygen concentration assessment methods. Furthermore, by using phosphorescent dyes that emit phosphorescence in a color tone that can be observed using commonly used filters and have low cytotoxicity, they can be widely used as reagents for assessing oxygen concentration.

[0159] The method for evaluating oxygen concentration of the present invention can be carried out in the same manner as known methods, except that the luminescent particles of the present invention are used as reagents. The luminescent particles of the present invention are preferably introduced into cells in the form of a dispersion.

[0160] Specific examples of the oxygen concentration evaluation method of the present invention include a method comprising a step of contacting a measurement target with the luminescent particles of the present invention. After this contact, the cells are cultured, as needed, for a period ranging from immediately after contact to approximately 14 days. The phosphorescence intensity or phosphorescence lifetime is then observed. This allows for the assessment that the intracellular oxygen concentration is low when the phosphorescence intensity is high or the phosphorescence lifetime is long. Furthermore, by determining the relationship between the oxygen concentration and the phosphorescence intensity, the oxygen concentration can be quantitatively measured.

[0161] The phosphorescence intensity or phosphorescence lifetime can be observed using a fluorescence microscope, a fluorescence measuring device, a phosphorescence lifetime imaging device, or the like.

[0162] The method for evaluating oxygen concentration of the present invention is useful for monitoring the oxygen concentration in cells or biological tissues, screening for compounds that affect the oxygen concentration in cells or biological tissues, etc. It can also be used for co-staining, multicolor imaging, etc.

[0163] Evaluating oxygen concentration can be used to diagnose and treat conditions involving hypoxia, such as cancer, stroke, and myocardial infarction. Highly active cells consume more oxygen than less active cells, making them more likely to exhibit hypoxic conditions. Therefore, evaluating oxygen concentration can be used to evaluate the activity of cells.

[0164] (1.1) Activity evaluation method The activity evaluation method of the present invention is characterized by using the above-described oxygen concentration evaluation method.

[0165] In the present invention, "activity" refers to a state in which a cell maintains its life and exerts its characteristics and functions. Furthermore, "activity" is an index that indicates the degree to which a cell exerts its characteristics and functions. Cells consume oxygen to maintain life. Therefore, the shorter the time it takes for a cell to consume a certain amount of oxygen, the higher the activity of the cell can be evaluated.

[0166] An example of a method for evaluating activity will be described below, although the present invention is not limited thereto. The oxygen supply is cut off to the cells to be evaluated. Immediately after cutting off, the amount of oxygen is relatively high, so quenching is easy and the phosphorescence intensity is low. In addition, the phosphorescence lifetime is short. However, as time passes, oxygen is consumed and the amount of oxygen present decreases, making quenching more difficult and increasing the phosphorescence intensity. In addition, the phosphorescence lifetime becomes longer. The time from when the oxygen supply is cut off until the phosphorescence intensity or lifetime reaches a specific reference value is measured. Cells with a short time can be evaluated as having rapid oxygen consumption and high activity.

[0167] (2) Fluorescent immunostaining In "fluorescent immunostaining," specific components within cells, etc., are labeled with a probe containing a fluorescent dye using an antibody to prepare a sample. The sample is then irradiated with excitation light. The image of the fluorescence emitted by the molecules of the labeled specific component is observed, for example, with a fluorescence microscope, and the expression state of the specific component within the cell, etc. is observed.

[0168] In the fluorescent immunostaining method, "fluorescence" refers to fluorescence in a broad sense, and includes not only fluorescence in the narrow sense but also phosphorescence. Hereinafter, the probe used in the fluorescent immunostaining method will also be referred to as a "fluorescently labeled probe." The luminescent dye used in the fluorescently labeled probe may be a fluorescent dye or a phosphorescent dye.

[0169] The fluorescent immunostaining method may be a direct method or an indirect method. In the direct method, a primary antibody is specifically bound or immobilized to a particular component (antigen), and then labeled with a probe containing a fluorescent dye. On the other hand, in the indirect method, a primary antibody is specifically bound or immobilized to a specific component (antigen). Next, a secondary antibody is specifically bound to the bound or immobilized primary antibody. The secondary antibody is then labeled with a probe containing a fluorescent dye.

[0170] From the viewpoint of easy availability of antibodies, it is preferable to use the indirect method. The specific component (antigen) to be detected is not particularly limited as long as it is a component to which fluorescent immunostaining can be applied.

[0171] (2.1) Primary antibody A primary antibody is an antibody that recognizes an epitope specific to an antigen. An "epitope" is a specific structural unit of an antigen that an antibody recognizes and binds to. The primary antibody may be a free primary antibody.

[0172] When a polyclonal antibody is used as the primary antibody, a typical polyclonal antibody can be used, which is generated by using a specific sequence portion containing the epitope of an antigen as the immunizing antigen. Alternatively, a mixture of two or more monoclonal antibodies can be used as a substitute (equivalent) for a polyclonal antibody. In this case, it is preferable to combine monoclonal antibodies that specifically bind to different epitopes for each antibody.

[0173] As long as the secondary antibody can bind to the primary antibody when the primary antigen is bound to the antigen, the primary antibody does not need to be a full-length antibody like a natural antibody. The primary antibody may be an antibody fragment or an antibody derivative. In other words, as used herein, the term "antibody" encompasses not only full-length antibodies but also derivatives such as antibody fragments, chimeric antibodies (humanized antibodies, etc.), and multifunctional antibodies.

[0174] The type of animal that produces the primary antibody (immunized animal) is not particularly limited, and can be selected from mice, rats, guinea pigs, rabbits, goats, sheep, and the like.

[0175] The primary antibody is preferably an IgG antibody. For example, when PD-L1 is used as the antigen, the IgG antibody used is preferably an anti-PD-L1 antibody. Furthermore, when HER2 is used as the antigen, the IgG antibody used is preferably an anti-HER2 antibody.

[0176] (2.2) Secondary antibody The secondary antibody specifically recognizes the unreacted portion of the antigen-immobilized primary antibody (Fc, F(ab), or F(ab')). The secondary antibody then binds to some or all of the primary antibody. The secondary antibody does not bind to the antigen. The secondary antibody is or will be conjugated to a fluorescently labeled probe.

[0177] The secondary antibody is preferably an IgG antibody. Typically, the secondary antibody is produced in an animal immunized different from the primary antibody, and is produced so as to recognize the antibody (Fc region, etc.) of the animal species of the primary antibody. The type of animal (immunized animal) producing the primary or secondary antibody is not particularly limited and can be selected from mouse, rat, guinea pig, rabbit, goat, sheep, etc.

[0178] (2.3) Binding of secondary antibodies to fluorescently labeled probes The secondary antibody and the fluorescently labeled probe can be directly linked via their functional groups, or they can be indirectly linked using a linker and first and second binding group substances.

[0179] (Indirect binding of secondary antibodies to fluorescently labeled probes) In a method for indirectly linking the secondary antibody and the fluorescently labeled probe, for example, a linker is attached to each of the secondary antibody and the fluorescently labeled probe, and the secondary antibody and the fluorescently labeled probe are linked by a specific binding reaction between the first and second binding group substances attached to the ends of the linkers, respectively.

[0180] Examples of the first and second binding group substances include a combination of biotin and avidin biomolecules. Examples of the first and second binding group substances include a combination of known substances other than biotin and avidin (e.g., a hapten and an anti-hapten antibody). The first and second binding group substances do not include those that react with primary and secondary antibodies or antigens. As the linker, a known linker can be used.

[0181] The secondary antibody is modified with a first binding group substance via a linker. The fluorescently labeled probe is modified with a second binding group substance via a linker. The first and second binding group substances are then bound to each other, whereby the fluorescently labeled probe binds to the secondary antibody and the secondary antibody is fluorescently labeled. The binding of the first and second binding group substances to each other can be carried out at a desired timing before or after the step of binding the secondary antibody to the primary antibody.

[0182] (Direct binding of secondary antibodies to fluorescently labeled probes) Instead of binding the first and second binding group substances to each other, the secondary antibody and the fluorescently labeled probe may be linked via a linker. In this case, it is preferable to bind the fluorescently labeled probe to the secondary antibody before binding the primary antibody to the antigen. As the linker, a known linker can be used.

[0183] (Linker length, material) In a complex of a secondary antibody and a fluorescently labeled probe bound via a linker, the length of the linker-derived linking portion is preferably within the range of 15 to 1000 angstroms, and more preferably within the range of 30 to 65 angstroms. Usable linkers are not particularly limited, and examples thereof include hydrophilic polymers such as polyethylene glycol, polypropylene glycol, ficoll, and polyvinyl alcohol.

[0184] (2.4) Fluorescently labeled probe The fluorescently labeled probe contains the luminescent particle of the present invention and has the property of being able to bind to a primary antibody or a secondary antibody. Preferably, the fluorescently labeled probe can emit fluorescence of sufficient intensity to represent each antigen molecule as a bright spot.

[0185] (2.4.1) Method for preparing fluorescently labeled probes It is preferable that the fluorescently labeled probe be linked to a biologically relevant binding substance by further surface modification of the luminescent particle of the present invention.

[0186] By carrying out surface modification, it is possible to link biologically relevant binding substances to the surface of the fluorescently labeled probe. As a method for linking a biologically relevant binding substance to the surface of a fluorescently labeled probe, a known method for linking a fluorescent label to a biologically relevant binding substance can be used.

[0187] For example, the reaction between reactive functional groups such as carboxyl groups, amino groups, aldehyde groups, thiol groups, and maleimide groups is utilized. In other words, one reactive functional group present on the surface of a fluorescent label is bonded to another reactive functional group present in the molecule of a biologically relevant binding substance. If the functional groups cannot be bonded directly to each other, they can also be bonded via a "linker molecule" that has a specific functional group at each end of the molecule. This reaction can be carried out by adding the necessary reagents and allowing the specified time to elapse.

[0188] As a specific example, a fluorescently labeled probe having a hydroxyl group on its surface is reacted with a silane coupling agent, such as aminopropyltrimethoxysilane, to introduce an amino group. Meanwhile, a thiol group-introducing reagent, such as N-succimidyl S-acetylthioacetate, is reacted with streptavidin to introduce a thiol group. Finally, a PEG (polyethylene glycol) linker molecule having maleimide groups at both ends is reacted to link the fluorescently labeled probe to streptavidin. The maleimide group is reactive with both amino and thiol groups.

[0189] In addition, when acrylic resins are synthesized using glycidyl methacrylate as a raw material monomer, epoxy groups derived from the monomer are present on the surface of the fluorescently labeled probe. By adding ammonia water to the fluorescently labeled probe, the epoxy groups are converted into amino groups. Furthermore, desired biologically relevant binding substances can be linked to the amino groups.

[0190] (2.5) Analysis method An analytical method for detecting specific components using fluorescently labeled probes will be described. However, the analytical method can be any known method and is not limited to this. Hereinafter, "incubating cells" refers to allowing a reaction acting on the cells to proceed.

[0191] (fixation treatment) The cultured cell samples from the first sample were placed in each 24-well cell culture plate. Then, tissue fixative "FB002" (4% formaldehyde, manufactured by Thermo Fisher Scientific) was added to the cultured cell samples. The cells were incubated for 15 minutes at room temperature (25°C). Then, the cells were washed three times with phosphate-buffered saline (PBS).

[0192] (Transparency) Next, 0.5% Triton X-100 included in the permeabilization kit "R37602" (Thermo Fisher Scientific) is added to the cells. The cells are incubated for 20 minutes at room temperature (25°C). The cells are then washed three times with phosphate-buffered saline (PBS). For blocking, 3% bovine serum albumin (BSA) is added, and the cells are incubated for 30 minutes at room temperature (25°C).

[0193] (dyeing process) The anti-HER3 antibody "Anti-ErbB3, Intracellular, Mouse-Mono (5A12)" (NTA) was added to the cells as a primary antibody and allowed to react overnight at 4°C. The cells were then washed with phosphate-buffered saline (PBS).

[0194] Next, the anti-mouse IgG antibody is biotinylated using the antibody / protein labeling kit "Biotin Labeling Kit-NH2" (Dojindo Laboratories, Ltd.), and the biotinylated anti-mouse IgG antibody is then added to the cells as a secondary antibody and allowed to react.

[0195] The cells are then fluorescently labeled with two types of fluorescent probes: a fluorescently labeled probe and streptavidin-Alexa Fluor 488 conjugate (Thermo Fisher Scientific). The cells are then washed three times with phosphate-buffered saline (PBS). Hoechst 33342 (Invitrogen) (Thermo Fisher Scientific), diluted to 10 μg / mL in phosphate-buffered saline (PBS), is added to the cells as a nuclear staining dye. The cells are then incubated for five minutes at room temperature (25°C). The cells are then washed three times with phosphate-buffered saline (PBS). After all staining steps are complete, the cells are mounted using the anti-fading mounting medium Prolong Gold (Invitrogen) (Thermo Fisher Scientific) to obtain a second sample.

[0196] (Measurement of fluorescent spot number and brightness) An example of the measurement method will be described. The second sample was observed using, for example, a point-scanning confocal microscope, "A1R + The images are taken using a 4x objective lens (NA = 0.45) with a pinhole diameter set to 31.9 μm. The images are digitized using, for example, high-definition image analysis software "Imaris (ver. 9.1.1)" (Bitplane). The number and brightness of fluorescent foci from the fluorescently labeled probes are measured using, for example, a scanning electron microscope (SEM) "S4500" (Hitachi). Then, using a separately prepared calibration curve, the number of fluorescently labeled probes per cell is calculated, and fluorescent staining measurement data is obtained.

[0197] The calibration curve can be prepared by the following method. A dilute solution of the fluorescently labeled probe is dropped onto a glass slide and allowed to dry. Then, images are taken under the same conditions as for cells, and the images are processed. As with cells, the same field of view is observed using an SEM. The number and brightness of fluorescent spots produced by the fluorescently labeled probe are measured, and a calibration curve for the number and brightness of fluorescent spots is created. [Example]

[0198] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these. In the examples, the terms "parts" and "%" are used, but unless otherwise specified, they represent "parts by mass" or "% by mass." In the following examples, unless otherwise specified, all operations were carried out at room temperature (25°C).

[0199] 1. Preparation of Luminescent Particles 1 to 6 (1) Preparation of luminescent particles 1 The blue phosphorescent dye Ir-15 was dissolved in dichloromethane to prepare a dichloromethane solution of the blue phosphorescent dye Ir-15 with a concentration of 10 mM or less. This solution was mixed with a 0.5% by mass aqueous solution of MPC polymer at a ratio of 1: 9. The MPC polymer used was "LIPIDURE (registered trademark)-BL206" (manufactured by NOF Corporation). The concentration of the blue phosphorescent dye Ir-15 was measured by the above-mentioned method, specifically, by changing the amount of blue phosphorescent dye Ir-15 added as shown in FIG. max The ratio C of blue phosphorescent dyes max The ratio of blue phosphorescent pigment was calculated as C max The concentration of the blue phosphorescent dye Ir-15 was adjusted so that

[0200] The resulting mixture was homogenized for 30 minutes using a homogenizer while being cooled with water at 4°C. The mixture was left to stand at room temperature for at least one hour to evaporate the dichloromethane, and a dispersion of luminescent particles 1 was obtained.

[0201] The luminescent particles 1 were uniformly dispersed in the dispersion with extremely small particle diameters, so the particles could not be confirmed visually. However, the formation of particles was confirmed by DLS measurement, which will be described later.

[0202] When the same amount of blue phosphorescent dye Ir-15 used in producing luminescent particles 1 was mixed with the same amount of water as the dispersion of luminescent particles 1, the luminescent dye alone did not dissolve in water. Furthermore, even when the luminescent dye alone was stirred in water, it was confirmed that visually visible particles of the luminescent dye were floating in the water when the stirring was stopped.

[0203] (2) Preparation of luminescent particles 2 to 6 Luminescent particles 2 to 6 were produced in the same manner as in the production of luminescent particle 1, except that the luminescent dye was changed as shown in Table I. For luminescent particle 4, the surfactant "Brij (registered trademark) 58" was used instead of the polymer. The concentration of each dichloromethane solution was set to a concentration at which the luminescent dye was completely dissolved or less.

[0204] The luminescent dyes Ir-15, Ir-1, and Hex-Ir(piq)3 listed in Table II are blue, green, and red phosphorescent dyes, respectively. These are iridium complexes that are poorly soluble in water. Their luminescence intensities, whether in solid form or in soluble organic solvents, vary depending on the oxygen concentration.

[0205] Compound (1) as a luminescent dye listed in Table II is a compound in which a highly hydrophilic sulfo group is introduced into Ir-1, and is a phosphorescent dye. Compound (1) is water-soluble by itself. Oil Red O, listed in Table II, is a fluorescent dye.

[0206] The chemical structures of the luminescent dye compound (1) and Oil Red O are shown below.

[0207] [ka]

[0208] [ka]

[0209] The luminescent particles 2 were uniformly dispersed in the dispersion with extremely small particle diameters, so the particles could not be confirmed visually. However, the formation of particles was confirmed by DLS measurement, which will be described later.

[0210] When the same amount of green phosphorescent dye Ir-1 used in producing luminescent particles 2 was mixed with the same amount of water as the dispersion of luminescent particles 2, the luminescent dye alone did not dissolve in water. Furthermore, even when the luminescent dye alone was stirred in water, it was confirmed that visually visible particles of the luminescent dye were floating in the water when the stirring was stopped. The same phenomenon was confirmed for luminescent particles 3 and 6.

[0211] 2. Confirmation of luminescent particles 1 to 6 The obtained dispersions of luminescent particles 1 to 6 were subjected to UV-Vis absorption measurement using an ultraviolet-visible spectrophotometer "U-3300" (manufactured by Hitachi High-Tech Science Corporation). Clear absorption spectra were obtained for all dispersions of luminescent particles 1 to 6. This indicates that the luminescent dye was dispersed or dissolved in water. FIG. 6 shows the absorption spectra of luminescent particles 1 to 3. (a) corresponds to luminescent particle 1, (b) to luminescent particle 2, and (c) to luminescent particle 3.

[0212] For the obtained luminescent particles 1 to 6, the average primary particle diameter of the luminescent particles was measured by dynamic light scattering (DLS) using a zeta potential measurement device "Zetasizer Nano S" (manufactured by Malvern). For luminescent particle 5, the particle diameter could not be measured and the particles could not be confirmed. Figure 7 shows the results of DLS measurement for luminescent particles 1 to 3. (a) corresponds to luminescent particle 1, (b) to luminescent particle 2, and (c) to luminescent particle 3. In (a) to (c), the peaks appearing around 100 nm are all thought to be due to the luminescent particles.

[0213] 3. Confirmation of extracellular luminescence function of bioluminescent particles 1-3 It was confirmed by the emission spectrum whether the luminescent particles have the same luminescent function as the luminescent dye alone. The dispersion of luminescent particles 1 to 6 was maintained in the atmosphere (normal air) for 2 hours. Then, the emission spectrum of this dispersion was measured using a spectrofluorometer "F-7000" (manufactured by Hitachi High-Tech Science Corporation) by exciting it with light at the absorption maximum wavelength of the luminescent dye alone.

[0214] Separately, a dispersion of luminescent particles 1 to 6 was maintained for 2 hours under hypoxic conditions with an oxygen concentration of less than 0.1% using a hypoxic culture kit "BIONIX" (manufactured by Sugiyamagen Co., Ltd.) Then, the emission spectrum of this dispersion was measured using a spectrofluorometer "F-7000" (manufactured by Hitachi, Ltd.) by exciting it with light at the absorption maximum wavelength of the luminescent dye alone.

[0215] FIG. 8 shows the emission spectra of light-emitting particles 1 to 3 under normal air and low-oxygen conditions. (a) corresponds to light-emitting particle 1, (b) to light-emitting particle 2, and (c) to light-emitting particle 3. It was found that the phosphorescent intensity increased in all dispersions of light-emitting particles 1 to 3 when maintained under low-oxygen conditions. In other words, it was found that light-emitting particles 1 to 3 function as oxygen-responsive dyes.

[0216] 4. Confirmation of the intracellular luminescence function of luminescent particles 1-6 For light-emitting particles 1 to 5, optical imaging of breast cancer cells KPL4 was carried out using a confocal microscope "LSM780" (manufactured by Zeiss).

[0217] The dispersion of luminescent particles 2 obtained in the above step (1) of preparing luminescent particles 2 was diluted 20-fold with phosphate-buffered saline (PBS). This diluted solution was then added to breast cancer cells KPL4 from which the medium had been removed, and the breast cancer cells were incubated in a CO2 incubator at 37°C for 30 minutes. Luminescence images of the breast cancer cells were obtained using a confocal microscope "LSM780" (manufactured by Zeiss). A cover glass was then placed on the breast cancer cells, and the cells were incubated in a CO2 incubator at 37°C for 15 minutes. Luminescence images of the breast cancer cells were obtained using a confocal microscope "LSM780" (manufactured by Zeiss). Luminescence images were captured using 405 nm excitation light and detecting light with wavelengths in the range of 480 to 570 nm.

[0218] FIG. 9 shows the results of optical imaging of breast cancer cells KPL4 using light-emitting particles 2. The luminescence image under normal air (open air) in Figure 9 shows that a weak signal was successfully detected throughout the breast cancer cells before the cover glass was placed. This result demonstrates that luminescent particles 2 were successfully introduced into breast cancer cells without using organic solvents. Furthermore, the luminescence image under hypoxic conditions (hypoxia) in Figure 9 shows that a stronger signal was successfully detected throughout the breast cancer cells 15 minutes after the cover glass was placed. Note that the placement of a cover glass limits the amount of oxygen supplied to the breast cancer cells. This result demonstrates that the luminescence intensity of luminescent particles 2 increased in response to a hypoxic environment.

[0219] FIG. 10 shows the results of optical imaging of NK cells using luminescent particles 2. Using the same procedure as for the breast cancer cells KPL4, luminescence images were obtained for NK cells under normal air conditions (open air) and hypoxia conditions (hypoxia), and the same results were obtained as for the breast cancer cells. In other words, it can be seen that the luminescence intensity of luminescent particles 2 increased in response to a hypoxic environment not only for the breast cancer cells KPL4, which are adherent cells, but also for the NK cells, which are suspended cells.

[0220] This demonstrates that by forming a phosphorescent dye into the luminescent particles of the present invention, it can function as an oxygen-responsive dye even within cells.

[0221] When optical imaging was performed on light-emitting particles 1 and 3 in the same manner as for light-emitting particle 2, similar results were obtained.

[0222] Light imaging was also performed on luminescent particle 4 in the same way as for luminescent particle 2. However, in the luminescent image taken under normal air, no signal derived from the luminescent dye could be detected from within the cells. Furthermore, in the luminescent image taken under hypoxic conditions, no signal derived from the luminescent dye could be detected from within the cells. From these results, it is believed that luminescent particle 4 could not be introduced into the breast cancer cells.

[0223] Light imaging was also performed on light-emitting particle 5 in the same manner as light-emitting particle 2. However, no difference in signal intensity was observed between the light-emitting images under normal air and those under hypoxic conditions. Furthermore, the signal in the light-emitting image under normal air was weaker than that of light-emitting particle 2.

[0224] For luminescent particle 6, optical imaging of breast cancer cells KPL4 was performed using a confocal microscope "LSM780" (manufactured by Zeiss) using the same procedure as for luminescent particle 2. When a luminescent image was acquired, red-stained lipid droplets were detected. However, the function as an oxygen-responsive dye was not confirmed.

[0225] The structures of the luminescent particles 1 to 6 and the results of the above measurements are shown in Table II. In the table, "-" indicates that no measurement was performed.

[0226] [Table 2]

[0227] In confirming the luminescent function within cells, all of luminescent particles 1 to 6 were introduced into cells without using an organic solvent. The oxygen response function was confirmed for luminescent particles 1 to 3. Furthermore, the lipid droplet staining function was confirmed for luminescent particle 6. This demonstrates that the luminescent particles of the present invention can be introduced into cells without using an organic solvent, and that the original function of the luminescent dye contained therein is not impaired. Furthermore, by changing the amount of each luminescent dye added, luminescent particles with average primary particle sizes of 10 nm and 1000 nm were confirmed. Furthermore, the amount of each luminescent dye added was adjusted. C The value of I max The concentration of the luminescent dye in the organic solvent solution was adjusted so that it was 80% or more of the value of 1. It was confirmed that this allowed the luminescent particles to exhibit sufficient performance.

[0228] 5. Application of Luminescent Particles 2 to Evaluating Immune Cell Activity For luminescent particles 2, optical imaging of mouse killer T cells CTLL-2 and human NK cells NK92-CD16 cultured in a hyperglycemic model medium was performed using a confocal microscope "LSM780" (Zeiss).

[0229] CTLL-2 cells were cultured for one week in media with different glucose concentrations. The media with higher glucose concentrations than the normal culture medium were used as a hyperglycemic model medium.

[0230] The dispersion of luminescent particles 2 obtained in the above step (1) of preparing luminescent particles 2 was diluted 20-fold with phosphate-buffered saline (PBS). CTLL-2 cells, which had been cultured in each medium for one week, were then mixed with this diluted solution and incubated in a CO2 incubator at 37°C for 30 minutes. A cover glass was then placed on the CTLL-2 cells, and the cells were incubated in a CO2 incubator at 37°C for 15 minutes. Luminescent images of the CTLL-2 cells were obtained using a confocal microscope "LSM780" (manufactured by Zeiss). Luminescent images were captured using an excitation light of 405 nm and detecting light with wavelengths ranging from 480 to 570 nm.

[0231] Figure 11 shows the results of optical imaging of CTLL-2 cells cultured at different glucose concentrations using light-emitting particles 2. (a) is an optical imaging image, and (b) is a graph showing the intracellular signal intensity (Mean intensity / cell) in the optical imaging image. Note that "**" in the graph indicates that the P value in Tukey's test was below the significance level of 0.01, indicating a statistically significant difference. As shown in Figure 11, CTLL-2 cells cultured in a hyperglycemic model medium tend to show a decrease in luminescence intensity. Note that because the oxygen supply to the breast cancer cells is limited by the cover glass, this result indicates that oxygen consumption is weaker in CTLL-2 cells cultured in a medium with a high glucose concentration. This response corresponds to the exhaustion and reduced activity of CTLL-2 cells exposed to a hyperglycemic model medium.

[0232] Figure 12 shows the results of optical imaging of human NK92-CD16 cells cultured at different glucose concentrations using luminescent particles 2. (a) is an optical imaging image, and (b) is a graph showing the intracellular signal intensity (Mean intensity / cell) in the optical imaging image. Note that "**" in the graph indicates that the P value in Tukey's test was below the significance level of 0.01, indicating a statistically significant difference. Using the same procedure as for the CTLL-2 cells described above, NK92-CD16 cells were cultured in media with different glucose concentrations, and luminescence images were taken with a cover glass placed on top. The same results were obtained as for the CTLL-2 cells described above. In other words, it was found that luminescent particle 2 was able to evaluate the effect of the glucose concentration of the medium on cell activity in different types of immune cells. [Explanation of symbols]

[0233] 1. Luminescent particles 2. Luminescent dyes 3. Biomembrane-compatible polymers 4. Biomembrane-compatible polymer hydrophilic part 5. Biomembrane-compatible polymer hydrophobic part

Claims

1. A luminescent particle comprising a luminescent dye and a polymer, The polymer is a biomembrane-compatible polymer. A luminescent particle characterized by:

2. The biomembrane-compatible polymer is water-soluble.

2. The luminescent particle according to claim 1.

3. The luminescent dye is poorly soluble in water.

2. The luminescent particle according to claim 1.

4. The luminescent particles are cell membrane permeable.

2. The luminescent particle according to claim 1.

5. The luminescent dye is a phosphorescent dye.

2. The luminescent particle according to claim 1.

6. The phosphorescent dye is an iridium complex 6. The luminescent particle according to claim 5.

7. In the emission spectrum measured when irradiated with excitation light having the absorption maximum wavelength of the luminescent dye, the maximum luminescence intensity when the mass ratio of the luminescent dye to the biomembrane-compatible polymer is C is defined as I. Ｃ , the I when the content mass ratio C of the luminescent dye is changed Ｃ The maximum value of I ｍａｘ , the above I Ｃ The value of I ｍａｘ The smaller of the two values ​​of the content mass ratio of the luminescent dye when the value is 80% of the value of １ , the larger value is C ２ When In the luminescent particle, the content mass ratio C of the luminescent dye to the biomembrane-compatible polymer is C １ ~C ２ is within the range 2. The luminescent particle according to claim 1.

8. The biomembrane-compatible polymer has a structure derived from a phospholipid.

2. The luminescent particle according to claim 1.

9. The biomembrane-compatible polymer is a polymer of 2-methacryloyloxyethyl phosphorylcholine.

2. The luminescent particle according to claim 1.

10. The biomembrane-compatible polymer has a site capable of forming at least one of a dipole-dipole interaction, a CH-π interaction, and a π-π interaction with a molecule contained in the luminescent particle.

2. The luminescent particle according to claim 1.

11. The average primary particle diameter of the luminescent particles measured by dynamic light scattering is within the range of 10 to 1000 nm.

2. The luminescent particle according to claim 1.

12. A material containing the luminescent particles according to claim 1 A luminescent particle for labeling characterized in that:

13. A method for producing luminescent particles, comprising: The luminescent particles are the luminescent particles according to any one of claims 1 to 11, preparing a solution of the luminescent dye in an organic solvent; preparing an aqueous solution of the biomembrane-compatible polymer; a step of mixing and homogenizing the organic solvent solution of the luminescent dye and the aqueous solution of the biomembrane-compatible polymer; removing the organic solvent to form particles containing the luminescent dye and the biomembrane-compatible polymer. A method for producing luminescent particles, comprising:

14. In the emission spectrum measured when the luminescent particle is irradiated with excitation light having the maximum absorption wavelength of the luminescent dye, the maximum luminescence intensity when the mass ratio of the luminescent dye to the biomembrane-compatible polymer is C is defined as I. Ｃ , the I when the content mass ratio C of the luminescent dye is changed Ｃ The maximum value of I ｍａｘ When the above I Ｃ The value of I ｍａｘ The concentration of the luminescent dye in the organic solvent solution is adjusted so that the value of The method for producing luminescent particles according to claim 13 .

15. An evaluation method for evaluating a cell or a microorganism, comprising: The luminescent particle according to any one of claims 1 to 11 or the luminescent particle for labeling according to claim 12 is used. An evaluation method characterized by:

16. An oxygen concentration evaluation method for evaluating a cellular oxygen concentration, comprising: The luminescent particle according to any one of claims 1 to 11 or the luminescent particle for labeling according to claim 12 is used.

1. A method for evaluating oxygen concentration.

17. An activity evaluation method for evaluating the activity of a cell, comprising: The oxygen concentration evaluation method according to claim 16 is used. A method for evaluating activity.

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