Patent Application
20200124521
The present invention relates to a fluorescent probe for cytometry and a method for screening fluorescence-labeled cells.
In recent years, in vivo fluorescence imaging analysis using animals such as mice has been widely performed in the fields of cancer research, embryological research, and the like. The fluorescence imaging analysis is a major technique for analyzing information about positions at which cells such as stem cells including iPS cells, ES cells, and the like and disease-related cells including cancer cells, cirrhotic cells, and the like differentiate, grow, and metastasize after being transplanted into mice. Under current circumstances, only methods using the fluorescence imaging analysis can be used to observe the progression of transplanted cells, particularly with animals such as mice that are still alive, and such methods are regarded as particularly important analysis methods.
In general, fluorescence imaging analysis on transplanted cells in vivo is performed as follows: cells are labeled with a fluorescent probe in vitro in advance before the cells are transplanted, the cells are transplanted into a living organism such as a mouse, and the cells are observed using an in vivo fluorescence imaging apparatus. For example, Patent Document 1 discloses use of a porphyrin-containing complex obtained by binding anionic porphyrin, cationic organoalkoxysilane, and non-cationic silane together as a near-infrared fluorescent probe for observation of a living organism.
However, under current circumstances, the fluorescence of the near-infrared fluorescent probe for observation of a living organism cannot be detected using an in vitro fluorescence imaging apparatus such as a fluorescence microscope or flow cytometer, and it is difficult to observe fluorescence-labeled cells bound to the near-infrared fluorescent probe in vitro.
In order to solve the foregoing conventional problems, the present invention provides a fluorescent probe for flow cytometry that can be used in flow cytometry analysis using a flow cytometer, and a method for screening fluorescence-labeled cells.
The present invention relates to a fluorescent probe for flow cytometry including a carrier molecule, and porphyrin bound to the carrier molecule, wherein an excitation wavelength of the fluorescent probe for flow cytometry is in a range of 350 to 650 nm.
The present invention also relates to a method for screening fluorescence-labeled cells using a flow cytometer, and the method includes a step of fluorescently labeling cells with a fluorescent probe for flow cytometry, and a step of screening fluorescence-labeled cells labeled with the fluorescent probe for flow cytometry using a flow cytometer, wherein the fluorescent probe for flow cytometry includes a carrier molecule and porphyrin bound to the carrier molecule, an excitation wavelength of the fluorescent probe for flow cytometry is in a range of 350 to 650 nm, and the screening of the fluorescence-labeled cells using a flow cytometer is performed by irradiating the fluorescence-labeled cells with an excitation light with a wavelength of 350 to 650 nm and detecting fluorescence.
It is preferable that the carrier molecule is polysiloxane.
A configuration may be employed in which the fluorescent probe for flow cytometry is specifically bound to a surface of a cell so that the cell is labeled, or a configuration may be employed in which the fluorescent probe for flow cytometry is taken up by a cell so that the cell is labeled.
It is preferable that a fluorescence wavelength of the fluorescent probe for flow cytometry is in a range of 400 to 800 nm.
The present invention can provide a fluorescent probe for flow cytometry that can be used in flow cytometry analysis using a flow cytometer. Moreover, when cells are labeled with the fluorescent probe for flow cytometry, the fluorescence-labeled cells can be screened using a flow cytometer.
The inventors of the present invention found that a fluorescent probe that includes a carrier molecule and porphyrin bound to the carrier molecule and in which an excitation wavelength of the fluorescent probe for flow cytometry was in a range of 350 to 650 nm can be used to detect and screen fluorescence-labeled cells in fluorescence analysis using flow cytometry, and the present invention was thus achieved.
Fluorescent Probe for Flow Cytometry
A fluorescent probe for flow cytometry includes a carrier molecule and porphyrin bound to the carrier molecule.
The excitation wavelength of the fluorescent probe for flow cytometry is 350 to 650 nm, and preferably 380 to 580 nm from the viewpoint of suitability for flow cytometry analysis using a general-purpose flow cytometer. Moreover, the fluorescence wavelength of the fluorescent probe for flow cytometry is preferably in a range of 400 to 800 nm, and more preferably 450 to 680 nm, from the viewpoint of suitability for flow cytometry analysis using a general-purpose flow cytometer.
There is no particular limitation on the type of porphyrin, but porphyrin having a carboxyl group can be used from the viewpoint of suitability for fluorescence analysis using a flow cytometer. It should be noted that the term “porphyrin” is used to collectively refer to a ring compound in which four pyrrole rings are alternately bound to four methine groups at a positions, and derivatives thereof. The excitation wavelength of porphyrin having a carboxyl group is generally in a range of 400 to 650 nm, and its fluorescence wavelength is in a range of 600 to 740 nm.
For example, a compound represented by General Formula (I) below can be used as the porphyrin having a carboxyl group.
In General Formula (I) above, R1b, R2b, R3b, and R4b are optionally the same or different and represent a carboxyl group (COOH), a sulfo group (SO3H), or a hydrogen atom (H) (it should be noted that a case where all of these are hydrogen atoms and a case where all of these are sulfo groups are excluded). It is preferable that, in General Formula (I) above, all of R1b, R2b, R3b, and R4b are carboxyl groups, or R1b is a carboxyl group and R2b, R3b, and R4b are hydrogen atoms. It is more preferable to use, as the porphyrin having a carboxyl group, a compound that is represented by General Formula (I) above and in which all of R1b, R2b, R3b, and R4b are carboxyl groups. The compound in which all of R1b, R2b, Ra, and R4b are carboxyl groups is called tetrakis(4-carboxyphenyl)porphyrin (TCPP).
In addition, bilirubin, hemin, protoporphyrin, and the like can also be used as the porphyrin having a carboxyl group, for example.
The porphyrin having a carboxyl group used in the present invention is a known compound or can be easily manufactured using a known method. For example, commercially available compounds can be obtained from Tokyo Chemical Industry Co., Ltd., and the like.
The fluorescent probe of the present invention may also contain another fluorescent dye in addition to porphyrin. When another fluorescent dye is contained, fluorescence resonance energy transfer (FRET) should not occur between porphyrin and the other fluorescent dye. There is no particular limitation on the excitation wavelength of the other fluorescent dye (also referred to as “fluorescent dye b” hereinafter) as long as it is different from the excitation wavelength of porphyrin and FRET does not occur.
There is no limitation on the excitation wavelength of the fluorescent dye b as long as there is no influence on flow cytometry analysis using porphyrin. The excitation wavelength of the fluorescent dye b is preferably in a range of 600 to 850 nm, and more preferably in a range of 630 to 790 nm.
Fluorescent dyes for in vivo fluorescence imaging such as indocyanine compounds, coumarin, rhodamine, xanthene, hematoporphyrin, and fluorescamine may be used as the fluorescent dye b, for example.
It is preferable that the carrier molecule can bind to porphyrin and allows the original excitation wavelength of porphyrin to be maintained. Examples thereof include inorganic polymer such as polysilane, polygermane, polystannane, polysiloxane, polysilsesquioxane, polysilazane, polyborazirene, and polyphosphazene, and organic polymers such as polypyrrole, polyethylene glycol, and polysaccharides. For example, a silica polymer is formed through hydrolysis and polycondensation of silane as described later.
The fluorescent probe for flow cytometry may be specifically bound to the surface of a cell so that the cell is labeled. In this case, it is preferable that the fluorescent probe for flow cytometry includes a cell surface binding substance capable of binding to a substance for specific recognition of the cell surface. The fluorescent probe for flow cytometry specifically binds to the cell surface via a cell surface binding substance, and the cell can be thus labeled therewith. The “substance for specific recognition of the cell surface” refers to a protein, a lipid, a sugar chain, and/or a nucleic acid that is present on the surface of a specific cell. For example, a folic acid receptor, a transferrin receptor, an antigen, and the like that are specific to cancer cells are present on the surfaces of cancer cells. Cancer cells can be specifically labeled with a fluorescent probe including folic acid, transferrin, an antibody, or the like. A cell surface marker (membrane protein) and the like are present on the surfaces of stem cells such as iPS cells and ES cells. Stem cells such as iPS cells and ES cells can be specifically labeled with a fluorescent probe to which a molecule or the like that specifically binds to the cell surface marker of iPS cells or ES cells is bound.
The fluorescent probe for flow cytometry may be taken up by a cell so that the cell is labeled. Examples of such a cell include a macrophage, a dendritic cell, an immune cell, a cancer cell, and an iPS cell. Macropharges, dendritic cells, immune cells, cancer cells, iPS cells, or the like that have taken up the fluorescent probe can be used to confirm the dynamic behavior of macropharges, dendritic cells, or the like inside a body in an immune cell therapy.
The fluorescent probe for flow cytometry of the present invention can be produced by binding, preferably covalently binding, a carrier molecule to porphyrin to form a complex of the carrier molecule and porphyrin. A cell surface binding substance may be bound to the carrier molecule in the complex of the carrier molecule and porphyrin as needed. It is preferable that the excitation wavelength and the fluorescence wavelength of porphyrin in the fluorescent probe for flow cytometry of the present invention barely change before and after the carrier molecule is bound to porphyrin.
When the carrier molecule is polysiloxane, the fluorescent probe for flow cytometry of the present invention can be produced as described below, for example.
First, silane having an amino group and porphyrin having a carboxyl group are reacted to obtain silane including porphyrin in its molecule (also referred to as “porphyrin-silane” hereinafter). Specifically, silane having an amino group and porphyrin having a carboxyl group are dissolved in a solvent, and a condensing agent is added thereto to initiate an amidation reaction. An example of the solvent is N,N-dimethylformamide (DMF). An example of the condensing agent is carbodiimide. An example of carbodiimide is N,N′-dipropylcarbodiimide, but there is no particular limitation thereto. Succinimide or the like may be added in order to reduce by-products. An example of succinimide is N-hydroxysuccinimide, but there is no particular limitation thereto. The reaction temperature is preferably 20 to 150° C., and more preferably 20 to 80° C., for example, from the viewpoint of synthesis cost, but there is no particular limitation thereto. The reaction time is preferably 1 to 24 hours, and more preferably 3 to 15 hours, for example, but there is no particular limitation thereto. After the reaction, the product is collected as a precipitate through centrifugation, and porphyrin-silane thus can be obtained.
In the above-mentioned reaction, the molar ratio of the silane having an amino group to the porphyrin having a carboxyl group (silane having an amino group:porphyrin having a carboxyl group) is preferably 4:1 to 1:1, more preferably 4:1 to 2:1, and even more preferably 4:1.
Next, a complex of polysiloxane and porphyrin is obtained through hydrolysis and polycondensation reaction between the porphyrin-silane obtained as described above and silane having one or more functional groups (also referred to as “functional silane” hereinafter). Specifically, the porphyrin-silane and the functional silane are dissolved in a solvent, and then an alkali solution is added thereto to initiate a reaction. An example of the solvent is DMF. Examples of the alkali solution include an aqueous solution of ammonia and an aqueous solution of sodium hydroxide whose pH is 8 or higher. The reaction temperature is preferably 20 to 200° C., and more preferably 60 to 80° C., for example, from the viewpoint of synthesis cost, but there is no particular limitation thereto. The reaction time is preferably 1 to 72 hours, and more preferably 3 to 24 hours, for example, but there is no particular limitation thereto. After the reaction, the product is collected as a precipitate through centrifugation, and the complex of polysiloxane and porphyrin in which porphyrin is covalently bound to polysiloxane thus can be obtained.
In the above-mentioned reaction, the molar ratio of the porphyrin-silane to the functional silane (porphyrin-silane:functional silane) is preferably 1:2 to 1:100, more preferably 1:21 to 1:50, and even more preferably 1:30 to 1:40.
There is no particular limitation on the silane having an amino group as long as an amino group is included. For example, a compound represented by General Formula (II) below can be favorably used.
Xi—Si(R2a)j(OR1a)(4-i-j) (I)
In General Formula (II), X represents a group represented by H2NCmH2m—, H2NCnH2n—HNCmH2m—, or Ph-NHCmH2m— (where Ph represents a phenyl group). In particular, a group represented by H2NCmH2m— or H2NCnH2n—HNCmH2m— is favorable. m and n are the same or different and represent an integer of 1 to 6. m is preferably 1, 2, 3, or 4, and more preferably 1, 2, or 3. n is preferably 1, 2, 3, or 4, and more preferably 1, 2, or 3. H2NCmH2m—, H2NCnH2n—HNCmH2m—, and Ph-NHCmH2m— are preferably H2N(CH2)m—, H2N(CH2)n—HN(CH2)m—, and Ph-NH(CH2)m—, respectively.
R1a and R2a are the same or different and represent an alkyl group having 1 to 6 carbon atoms. The alkyl group may be a linear chain or a branched chain, and is preferably a linear chain. It is preferable that the alkyl group has 1, 2, 3, or 4 carbon atoms. Specific examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, a sec-butyl group, an n-pentyl group, a 1-ethylpropyl group, an isopentyl group, a neopentyl group, an n-hexyl group, a 1,2,2-trimethylpropyl group, a 3,3-dimethylbutyl group, a 2-ethylbutyl group, an isohexyl group, and a 3-methylpentyl group. In particular, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, and an isobutyl group are preferable.
i represents 1 or 2, and j represents 0 or 1 (it should be noted that a relationship (4-i-j)≥2 is satisfied). That is, (i,j) represents (1,0), (1,1) or (2,0).
Examples of the compound represented by General Formula (II) include
For example, commercially available compounds manufactured by Tokyo Chemical Industry Co., Ltd. can be used as the above-described silane having an amino group. The silanes having an amino group can be used alone or in combination of two or more.
There is no particular limitation on the functional silane as long as one or more functional groups are included. The functional silane may be monofunctional silane having one functional group or polyfunctional silane having two or more functional groups. A compound represented by General Formula (III):
Yp—Si(R2c)q(OR1c)(4-p-q) (III)
can be favorably used as the silane having a functional group, for example.
In General Formula (III) above, Y represents a group represented by CH2═CH—, a group represented by CH2═CHCH2—, a group including alkene, a group including thiol, a group including disulfide, a group including amine, a group including ester, a group including amide, a group including carboxylic acid, a group including urea, a group including thiourea, a group represented by OCNCH2CH2—, a group represented by ClCαH2α—, a group represented by HSCßH2ß—, a group represented by CF3CγF2γ—CδH2δ—, a group represented by CH2═C(CH3)COOCεH2ε—, a group represented by CH2═CHCOOCζH2ζ—, a group represented by HN—CONH—CηH2η—, a group represented by Chemical Formula (a) below, a group represented by Chemical Formula (b) below, an alkyl group having 1 to 18 carbon atoms, or a phenyl group.
In the description above, α, ß, δ, ε, ζ, η, θ, and ι independently represent an integer of 1 to 6, and preferably an integer of 1, 2, 3, or 4. γ represents an integer of 0 to 8, and preferably an integer of 0, 1, 2, 3, 4, 5, 6, or 7. The alkyl group having 1 to 18 carbon atoms preferably has 1 to 12 carbon atoms, and more preferably 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. The alkyl group having 1 to 18 carbon atoms may be a linear chain or a branched chain, and is preferably a linear chain. ClCαH2α—, HSCßH2ß—, CF3CγF2γ—CδH2δ—, CH2═C(CH3)COOCεH2ε—, CH2═CHCOOCζH2ζ—, and HN—CONH—CηH2η— above are preferably Cl(CH2)α—, HS(CH2)ß—, CF3 (CF2)γ—(CH2)δ—, CH2═C(CH3)COO(CH2)ε—, CH2═CHCOO(CH2)ζ—, and HN—CONH—(CH2)η—, respectively.
R1c represents an alkyl group having 1 to 6 carbon atoms or —(CH2)τ—OCH3, and R2c represents an alkyl group having 1 to 6 carbon atoms or a phenyl group. R1c and R2c may be the same or different. In both R1c and R2c, the alkyl group having 1 to 6 carbon atoms may be a linear chain or a branched chain, and is preferably a linear chain. An alkyl group having 1, 2, 3, or 4 carbon atoms is preferable. Specific examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, a sec-butyl group, an n-pentyl group, a 1-ethylpropyl group, an isopentyl group, a neopentyl group, an n-hexyl group, a 1,2,2-trimethylpropyl group, a 3,3-dimethylbutyl group, a 2-ethylbutyl group, an isohexyl group, and a 3-methylpentyl group. In particular, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, and an isobutyl group are preferable. i represents an integer of 1 to 4 (preferably 1, 2, or 3).
p represents 1, 2, or 3, and q represents 0, 1, or 2 (it should be noted that a relationship (4-p-q)≥1 is satisfied). That is, (p,q) represents (1,0), (1,1), (1,2), (2,0), (2,1) or (3,0).
A compound represented by General Formula (IV) can also be favorably used as the functional silane, for example.
Z—SiCl3 (IV)
In General Formula (IV) above, Z represents a group represented by CH2═CH—, a group represented by CH2═CHCH2—, a group including alkene, a group including thiol, a group including disulfide, a group including amine, a group including ester, a group including amide, a group including carboxylic acid, a group including urea, a group including thiourea, a group represented by ClCκH2κ—, a group represented by CF3CλF2λ—CζH2ζ—, an alkyl group having 1 to 18 carbon atoms, a phenyl group, or a cyclohexyl group. κ and ξ independently represent an integer of 1 to 6, and preferably an integer of 1, 2, 3, or 4. λ represents an integer of 0 to 8, and preferably an integer of 0, 1, 2, 3, 4, 5, 6, or 7. The alkyl group having 1 to 18 carbon atoms preferably has 1 to 12 carbon atoms, and more preferably 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. The alkyl group having 1 to 18 carbon atoms may be a linear chain or a branched chain, and is preferably a linear chain. ClCκH2κ— and CF3CλF2λ—CξH2ξ— above are preferably Cl(CH2)κ— and CF3 (CF2)λ—(CH2)ξ—, respectively.
An N-[2-(N-vinylbenzylamino)ethyl]-3-aminopropyltrimethoxysilane hydrochloride may also be used as the polyfunctional silane.
Specific examples of the compound represented by General Formula (III) include tetramethoxysilane, tetraethoxysilane, tetrapropoxysilane, tetrabutoxysilane, tetraisopropoxysilane, allyltriethoxysilane, allyltrimethoxysilane, diethoxymethylvinylsilane, dimethoxymethylvinylsilane, triethoxyvinylsilane, vinyltrimethoxysilane, vinyltris(2-methoxyethoxy)silane, (chloromethyl)triethoxysilane, 3-chloropropyldimethoxymethylsilane, 3-chloropropyltriethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, 3-mercaptopropyl(dimethoxy)methylsilane, (3-mercaptopropyl)triethoxysilane, (3-mercaptopropyl)trimethoxysilane, 3-(triethoxysilyl)propyl isocyanate, 3-(trimethoxysilyl)propyl methacrylate, triethoxy-1H,1H,2H,2H-tridecafluoro-n-octylsilane, 3-(trimethoxysilyl)propyl acrylate, 3-trimethoxysilylpropylchloride, 1-[3-(trimethoxysilyl)propyl] urea, trimethoxy(3,3,3-trifluoropropyl)silane, 3-ureidepropyltriethoxysilane, diethoxy(3-glycidyloxypropyl)methylsilane, 3-glycidyloxypropyl(dimethoxy)methylsilane, 3-glycidyloxypropyltrimethoxysilane, 2-cyanoethyltriethoxysilane, diacetoxydimethylsilane, diethoxydimethylsilane, dimethoxydimethylsilane, dimethoxydiphenylsilane, dimethoxymethylphenylsilane, dodecyltriethoxysilane, hexyltrimethoxysilane, octadecyltriethoxysilane, octadecyltrimethoxysilane, n-octyltriethoxysilane, pentyltriethoxysilane, triacetoxymethylsilane, triethoxyethylsilane, trimethoxy(methyl)silane, trimethoxyphenylsilane, and trimethoxy(propyl)silane. In particular, 3-mercaptopropyl(dimethoxy)methylsilane, (3-mercaptopropyl)triethoxysilane, (3-mercaptopropyl)trimethoxysilane, and the like are preferable.
Specific examples of the compound represented by General Formula (IV) include allyltrichlorosilane, trichlorovinylsilane, 3-chloropropyltrichlorosilane, trichloro(1H,1H,2H,2H-heptadecafluorodecyl)silane, trichloro(1H,1H,2H,2H-tridecafluoro-n-octyl)silane, butyltrichlorosilane, cyclohexyltrichlorosilane, decyltrichlorosilane, dodecyltrichlorosilane, ethyltrichlorosilane, n-octyltrichlorosilane, phenyltrichlorosilane, trichloro-2-cyanoethylsilane, trichlorohexylsilane, trichloro(methyl)silane, trichlorooctadecylsilane, trichloro(propyl)silane, and trichlorotetradecylsilane.
For example, commercially available compounds manufactured by Tokyo Chemical Industry Co., Ltd. can be used as the above-described functional silane. The functional silanes can be used alone or in combination of two or more.
Next, a cell surface binding substance is bound to a nanoparticle made of the complex of polysiloxane and porphyrin as needed. Specifically, an aqueous solution of the cell surface binding substance is added to an aqueous solution of the nanoparticles made of the complex of polysiloxane and porphyrin, and the resultant mixture is stirred at a temperature of 4 to 50° C. for 1 to 24 hours and reacted. For example, folic acid serving as the cell surface binding substance may be bound to the nanoparticle made of the complex of polysiloxane and porphyrin. A cell surface binding substance modified with a functional group capable of binding to the functional group present on the surface of the nanoparticle made of the complex of polysiloxane and porphyrin is used. For example, when the functional silane used to form the complex of polysiloxane and porphyrin is a silane having a thiol group such as 3-mercaptopropyl(dimethoxy)methylsilane, (3-mercaptopropyl)triethoxysilane, or (3-mercaptopropyl)trimethoxysilane, a compound obtained by respectively binding a maleimide group and folic acid to both of the termini of polyethylene glycol (PEG) can be used as the folic acid.
In the above-mentioned reaction, the molar ratio of the functional group of the nanoparticle made of the complex of polysiloxane and porphyrin that is covalently bound to folic acid to the functional group with which the folic acid is modified and that is covalently bound to the complex of polysiloxane and porphyrin (the functional group of the nanoparticle made of the complex of polysiloxane and porphyrin that is covalently bound to folic acid:the functional group with which the folic acid is modified) is preferably 1:1 to 3:1, more preferably 1:1 to 2:1, and even more preferably 1:1. It should be noted that when the fluorescent probe for flow cytometry of the present invention includes the fluorescent dye b in addition to porphyrin, the fluorescent dye b may be bound to the complex of polysiloxane and porphyrin together with the folic acid.
When TCPP is used as the porphyrin, it is preferable that the carboxyl group of TCPP forms an amide bond in the complex of polysiloxane and porphyrin, and it is more preferable that TCPP is incorporated into a siloxane network (main chain of polysiloxane) by an amide bond. In the present invention, the structure of the fluorescent probe such as the complex of polysiloxane and porphyrin can be analyzed using Fourier transform infrared spectrophotometry as described later.
The fluorescent probe for flow cytometry preferably includes four pyrrole ring structure moieties (porphine) in porphyrin in an amount of 1 mass % or more, and more preferably 5 to 90 mass %, from the viewpoint of suitability for flow cytometry analysis using a flow cytometer, but there is no particular limitation thereto. As described later, in the present invention, analyzing a fluorescent probe such as the nanoparticle made of siloxane and porphyrin through thermogravimetric-differential thermal analysis makes it possible to measure the content of a fluorescent dye such as porphyrin and a carrier molecule such as silica polymer.
The fluorescent probe for flow cytometry preferably has an average particle diameter of 3 to 250 nm, and more preferably 10 to 80 nm, from the viewpoint of the cells being easy to label, but there is no particular limitation thereto. In the present invention, the average particle diameter is measured through dynamic light scattering.
Method for Screening Fluorescence-Labeled Cells
A method for screening fluorescence-labeled cells of the present invention includes a step of fluorescently labeling cells with the above-mentioned fluorescent probe for flow cytometry, and a step of screening the fluorescence-labeled cells labeled with the fluorescent probe for flow cytometry using a flow cytometer.
First, cells are fluorescently labeled with the fluorescent probe. Cells can be labeled by binding the fluorescent probe for flow cytometry to the cell surface or causing cells to take up the fluorescent probe for flow cytometry. When the fluorescent probe is bound to the cell surface, the fluorescent probe includes a cell surface binding substance. For example, when the fluorescent probe includes folic acid, cancer cells such as HeLa S3 cells derived from human cervical cancer and HCT116 cells derived from human large intestine cancer can be labeled by binding the fluorescent probe to the surfaces of these cancer cells. Specifically, culturing cells in a cell culture medium containing the fluorescent probe makes it possible to label the cells with the fluorescent probe. Examples of the cells include stem cells such as iPS cells and ES cells, and disease-related cells such as cancer cells and cirrhotic cells.
Next, the fluorescence-labeled cells labeled with the fluorescent probe for flow cytometry are screened using a flow cytometer. Screening of the fluorescence-labeled cells using a flow cytometer is performed as follows: cells that have been subjected to a fluorescence labeling operation are irradiated with an excitation light with an excitation wavelength of 350 to 650 nm and then fluorescence is detected. Specifically, cells that have been subjected to a fluorescence labeling operation using a fluorescent probe for flow cytometry are supplied to a flow cytometer and analyzed through flow cytometry analysis using a predetermined excitation wavelength and a predetermined fluorescence wavelength, such as an excitation wavelength of 350 to 650 nm and a fluorescence wavelength of 400 to 800 nm, thus making it possible to screen the fluorescence-labeled cells that have been fluorescently labeled with the fluorescent probe for flow cytometry. There is no particular limitation on the flow cytometer, and any flow cytometer can be used.
Hereinafter, the present invention will be specifically described by way of examples. It should be noted that the present invention is not limited to the following examples.
Reagents
All of tetrakis(4-carboxyphenyl)porphyrin (TCPP), (3-mercaptopropyl)trimethoxysilane (MPTMS), N,N-dimethylformamide (DMF), polyethylene glycol (PEG) in which folic acid and a maleimide group are respectively bound to both of the termini (molecular weight: 3400; also referred to simply as “FA-PEG-Mal” hereinafter), and indocyanine green to which a maleimide group is bound (molecular weight: 853; also referred to simply as “ICG-Mal” hereinafter) were obtained from Tokyo Chemical Industry Co., Ltd. The following are the structural formulae of MPTMS, FA-PEG-Mal, and ICG-Mal.
Production of Fluorescent Probe
Production of complex of carrier molecule and porphyrin
(1) APTES (462 μmol) was added to a DMF solution of TCPP (3.8 mM: 32 mL) obtained by dissolving TCPP in DMF, and then N,N′-dipropylcarbodiimide (480 μmol) and N-hydroxysuccinimide (480 μmol) were added thereto. The resultant mixture was stirred at 50° C. for 24 hours.
(2) Next, 200 μL (porphyrin-silane: 0.75 μmol) of the DMF solution of porphyrin-silane obtained as described above was mixed with 5 μl (0.027 mmol) of MPTMS. To the obtained mixed solution, 500 μL of DMF and 300 μL of an aqueous solution of ammonia (concentration: 28 mass %, pH 11) were added, and a reaction was carried out at 80° C. for 24 hours.
(3) After 24 hours, the product was collected as a precipitate through centrifugation (15000 rpm for 20 minutes). Then, the product was washed with water and ethanol several times, and was finally dispersed in 1 mL of water.
(4) The obtained substance was a complex of polysiloxane and porphyrin in which the polysiloxane serves as a carrier molecule and the porphyrin serves as a fluorescent dye, and the porphyrin is covalently bound to the polysiloxane. From the results of the measurement performed through dynamic light scattering (“DelsaMax Pro” manufactured by Beckman Coulter), it was found that the obtained substance was a fluorescent hybrid nanoparticle (also referred to simply as “PPS HNPs” hereinafter) with an average particle diameter of about 40 nm.
Step of Binding Fluorescent Dye b to Complex of Carrier Molecule and Porphyrin
(1) To 1 mL of the aqueous dispersion of the PPS HNPs obtained as described above, 63 μl of an aqueous solution of ICG-Mal (concentration: 2 mg/mL, ICG-Mal: 1.48×10−4 mmol) was added. The resultant mixture was stirred at 30° C. for 3 hours to carry out a reaction.
(2) After the reaction, the product was collected as a precipitate through centrifugation (15000 rpm for 20 minutes). Then, the product was washed with water several times, and was finally dispersed in 1 mL of water.
(3) The obtained substance was a complex (fluorescent probe) in which the porphyrin and the indocyanine green (fluorescent dye b) were covalently bound to the polysiloxane. From the results of the measurement performed through dynamic light scattering (“DelsaMax Pro” manufactured by Beckman Coulter), it was found that the obtained substance was a fluorescent hybrid nanoparticle (also referred to simply as “ICG-PPS HNPs” hereinafter) with an average particle diameter of about 50 nm.
Production of Fluorescent Probe
Production of Complex of Carrier Molecule and Porphyrin
PPS HNPs was obtained in the same manner as in Example 1.
Step of binding fluorescent dye b and cell surface binding substance to complex of carrier molecule and porphyrin
(1) To 1 mL of the aqueous dispersion of the PPS HNPs obtained as described above, 251 μl of an aqueous solution of FA-PEG-Mal (concentration: 2 mg/mL, FA-PEG-Mal: 1.48×10−4 mmol) and 63 μl of an aqueous solution of ICG-Mal (concentration: 2 mg/mL, ICG-Mal: 1.48×10−4 mmol) were added. The resultant mixture was stirred at 30° C. for 3 hours.
(2) After the reaction, the product was collected as a precipitate through centrifugation (15000 rpm for 20 minutes). Then, the product was washed with water several times, and was finally dispersed in 1 mL of water.
(3) The obtained substance was a complex (fluorescent probe) in which the porphyrin, the indocyanin green (fluorescent dye b), and the folic acid (cell surface binding substance) are covalently bound to the polysiloxane. From the results of the measurement performed through dynamic light scattering (“DelsaMax Pro” manufactured by Beckman Coulter), it was found that the obtained substance was a fluorescent hybrid nanoparticle (also referred to simply as “FA-PEG/ICG-PPS HNPs” hereinafter) with an average particle diameter of about 65 nm.
Confirmation Through Fourier Transform Infrared Spectrophotometry
The PPS HNPs obtained in Examples 1 and 2 and the TCPP used as a raw material were analyzed using a Fourier transform infrared spectrophotometer (“NEXUS470” manufactured by Nicolet).
The structure of the PPS HNPs obtained in Examples 1 and 2 was analyzed through 29Si solid-state NMR.
Thermogravimetric-Differential Thermal Analysis
The PPS HNPs was analyzed through thermogravimetric-differential thermal analysis (TG-DTA) using a thermal analyzer (TG8120, manufactured by Rigaku Corporation).
Quantification of FA-PEG and ICG Bound to PPS HNPs
In order to determine the amounts of the FA-PEG and the ICG bound to the PPS HNPs, an absorption spectrum of the supernatant (1 mL) after the reaction was measured using a spectrophotometer (“V-570” manufactured by JASCO Corporation), and the amounts of unreacted FA-PEG-Mal and ICG-Mal were calculated.
Confirmation of Absorption Spectrum Through Spectroscopy
The absorption spectra of the aqueous dispersion (also referred to as “aqueous solution”) of the PPS HNPs obtained in Examples 1 and 2 and the aqueous solution of the FA-PEG/ICG-PPS HNPs obtained in Example 2 were measured using a spectrophotometer (“V-570” manufactured by JASCO Corporation).
Confirmation of Wavelength Characteristics of Fluorescent Probe
The aqueous solution of the FA-PEG/ICG-PPS HNPs obtained in Example 2 was used to analyze the excitation wavelength spectrum and the fluorescence wavelength spectrum of the FA-PEG/ICG-PPS HNPs using a fluorescence spectrophotometer FP-6600 manufactured by JASCO Corporation.
Analysis Using Fluorescence Microscope
(1) Cells
RAW264.7 cells derived from a mouse macrophage, which are homologous to mouse cells, and HeLa S3 cells derived from human cervical cancer and HCT116 cells derived from human large intestine cancer, which are not homologous to mouse cells, were used. In the examples, cells and cell culture media were obtained from Sigma-Aldrich. Cell culture dishes manufactured by Thermo Fisher Scientific were used.
(2) Fluorescent Probe Labeling of Cells
(a) Cells (0.3×106 cells/mL) were seeded into a cell culture medium (DMEM medium containing 10% FBS) in a cell culture dish (with a diameter of 35 mm) and cultured overnight.
(b) The cultured cells were transferred into a cell culture medium (DMEM medium containing 10% FBS) containing the fluorescent probe (50 μg/mL), and cultured for 24 hours. At this time, the RAW264.7 cells were transferred into a cell culture medium containing the fluorescent probe ICG-PPS HNPs, and the HeLa S3 cells and the HCT116 cells were transferred into a cell culture medium containing the fluorescent probe FA-PEG/ICG-PPS HNPs.
(c) The supernatant was removed, and the cultured cells were washed with 2 mL of phosphate buffered saline (1×PBS, pH 7.4: “D-PBS(−) (045-29795)” manufactured by Wako Pure Chemical Corporation).
(d) The supernatant was removed, and 2 mL of 4% paraformaldehyde (PFA) was added to the cultured cells. The cells were left to stand at room temperature (20±5° C.) for 30 minutes.
(e) The supernatant was removed, and 1 mL of 1×PBS was added to the cultured cells. This step was repeated three times.
(3) Observation Using Fluorescence Microscope
The fluorescence-labeled cells labeled with the fluorescent probe were observed using a fluorescence microscope EVOS (registered trademark) FL Cell Imaging System manufactured by Life Technologies. All the cells were observed using an excitation wavelength of 422 nm and a fluorescence wavelength of 655 nm.
Analysis Using Flow Cytometer
(1) Cells
RAW264.7 cells derived from a mouse macrophage, which are homologous to mouse cells, and HeLa S3 cells derived from human cervical cancer and HCT116 cells derived from human large intestine cancer, which are not homologous to mouse cells, were used.
(2) Fluorescent Probe Labeling of Cells
(a) Cells were seeded into a cell culture medium (DMEM medium containing 10% FBS) in a cell culture dish (with a diameter of 35 mm) in a seeding amount shown in Table 1 below and cultured overnight.
(b) The cultured cells were transferred into a cell culture medium (DMEM medium containing 10% FBS) containing the fluorescent probe (50 μg/m) shown in Table 1 below, and cultured for another 48 hours.
(c) The cells were collected from the cell culture dish. At this time, the HeLa S3 cells and the HCT116 cells were collected using a 0.25% trypsin/EDTA solution.
(d) After the collected solution was centrifuged at 1000 revolutions for 5 minutes, the supernatant was removed, and the cells were suspended in 400 μL of a cell culture medium (DMEM medium containing 10% FBS).
(e) After the suspension was centrifuged at 1000 revolutions for 5 minutes, the supernatant was removed, and 1 mL of 4% PFA was added to the cells. The cells were left to stand at room temperature (20±5° C.) for 30 minutes.
(f) After the resuspension was centrifuged at 1000 revolutions for 5 minutes, the supernatant was removed, and 1 mL of 1×PBS was added to the cells. This step was repeated three times.
Observation Using Flow Cytometer
The cells labeled with the fluorescent probe were observed using a flow cytometer LSR Fortessa X-20 manufactured by BD.
As is clear from
| Number | Date | Country | Kind |
|---|---|---|---|
| 2016-091356 | Apr 2016 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2017/004979 | 2/10/2017 | WO | 00 |
