Patent Application
20230114986
The present invention relates to a light emitting nanoparticle. Particularly preferably, the present invention relates to a light emitting nanoparticle used for bioimaging, a cell detection method using the same, a method of treating animals, medical devices, visualization methods of cells, and damage reduction methods for cells.
Recently, there has been demand for a technique of detecting with high sensitivity at the cell level, tumors such as cancer cells and the like, by a marker material having a high biocompatibility.
Patent Document 1 discloses, as a bioimaging material, a two-photo absorbing material consisting of a water soluble dendrimer wherein a dye having a two photon absorbing-property and a dendron are bonded.
Patent Document 2 discloses the use of a water-dispersible quantum dot wherein a surface of the quantum dot is coated with a surfactant-type polymerization initiator comprising a hydrophobic group and a polar group, as a particle for in-vivo bioimaging.
Patent Document 3 discloses a production method of a bioimaging nanoparticle comprising the step of producing a hydrophobic nanoparticle which maintains individual dispersibility in a nonpolar organic solvent, wherein in a hydrophobic inorganic nanoparticle having a core or core/shell structure protected by a surfactant, the surfactant is partially substituted by adding 1 to 30 equivalents of an organic ligand wherein a thiol group and a hydrophilic group are bonded by a hydrocarbon chain with a carbon number of 8 to 20, and the surface of the nanoparticles is surface-modified so that only one part is hydrophilic, by forming a metal thiolate (M-S) bond, and the like.
Patent Document 4 discloses a fluorescent particle used for bioimaging, which is a fluorescent particle having upconversion characteristics which is a phenomenon which uses a low energy light such as an infrared light or the like as an excitation light, to obtain visible light fluorescence, and the material of the fluorescent particles is one material, or a combination of two or more materials among Y2O3:Er3+, Yb3+, Y2O3:Er3+, NaYF4:Er3+, Yb3+.
Patent Document 5 discloses a semiconductor nanoparticle used for molecule/cell imaging, which is a semiconductor nanoparticle with an average particle diameter of 1 to 20 nm, comprising an atom pair a main component element constituting the same, and an atom pair of the corresponding differing atom or a differing atom which has an equivalent valence electron arrangement, and further the dopant is distributed on the semiconductor nanoparticle surface or the vicinity thereof.
Patent Document 6 discloses a fluorescent labeling agent for pathological diagnosis comprising fluorescent material encapsulated nanoparticles comprising a first fluorescent material, and a second fluorescent material having a distinguishable excitation/luminescence property from the first fluorescent material.
Patent Document 7 discloses a fluorescent labeling agent, and a kit for target molecule measurement, consisting of a rare earth fluorescent complex-comprising silica particle comprising a rare earth fluorescent complex.
Further, Non-Patent Documents 1 to 4 also disclose the use of quantum dots and fluorescent dyes for bioimaging.
As described above, in recent years, there has been demand for a technique for high sensitivity detection of tumors such as cancer cells or the like at the cellular level by a marker having high biocompatibility. For example, in the canceration of a cell, before a morphological change occurs, an operational change at the molecular level occurs. For example, a cancer cell tends to consume a large amount of dextrose compared to a normal cell. At the same time, because folate receptors are overexpressed on the cell membrane, there is a tendency to selectively bond/acquire folic acid molecules. If high sensitivity imaging of such changes at the molecular level of a cell were possible, it would become possible to realize very early stage detection of cancer cells and the like.
However, as an imaging material for imaging, in the case of using an organic molecule, the rate of degradation/fading is high, for example, there has been the problem that light-emitting particles become quenched or the like by photoirradiation after several tens of minutes under fluorescence observation. Further, as a bioimaging material, in the case of using an inorganic material such as quantum dots or the like, in some cases, highly poisonous elements such as cadmium or the like are included, and there have been problems with biocompatibility and the like.
The present invention has the objective of providing light-emitting nanoparticles, which is provided with light-emitting stability and light resistance, and is low in biological toxicity, and a cell detection method, treatment method of animals, medical device, a cell visualization method, and a damage reduction method of cells using the same.
The present inventors, in order to achieve the above objective, as a result of diligent research, created a composite particle wherein a light-emitting molecule or ion is included in an inorganic material, and invented a light-emitting nanoparticle which is provided with light emitting stability and light resistance, and is low in biological toxicity, and a cell detection method, treatment method of animals, medical device, cells visualization method, and damage reduction method of cells using the same.
Namely, the present invention is the following (1) to (23).
(1) Alight-emitting nanoparticle comprising a matrix material, and a light-emitting substance included in the matrix material, wherein the matrix material comprises at least one cationic element selected from the group consisting of Ti, Si, Ca, Al and Zr, and at least one anionic element selected from the group consisting of O and P, and a concentration of the light-emitting substance in the matrix material is a concentration whereby an average distance between the light-emitting substance is 1.2 nm or more.
(2) Alight-emitting nanoparticle as disclosed in (1), wherein the matrix material comprises at least one selected from the group consisting of TiO2, SiO2, Ca10(PO4)6(OH)2, Al2O3, and ZrO2.
(3) Alight-emitting nanoparticle comprising a matrix material and a light-emitting substance included in the matrix material, wherein the matrix material comprises at least one cationic element selected from the group consisting of Ti, Ca, Al and Zr, and at least one anionic element selected from the group consisting of O and P.
(4) Alight-emitting nanoparticle as disclosed in (3), wherein the matrix material comprises at least one selected from the group consisting of TiO2, Ca10(PO4)6(OH)2, Al2O3, and ZrO2.
(5) A light-emitting nanoparticle as disclosed in (3) or (4), wherein a concentration of the light-emitting substance in the matrix material is a concentration whereby an average distance between the light-emitting substance is 1.2 nm or more.
(6) Alight-emitting nanoparticle as disclosed in any one of (1) to (5), wherein the light-emitting substance is at least one selected from the group consisting of an organic light-emitting dye, and a rare earth ion.
(7) A light-emitting nanoparticle as disclosed in (6), wherein the organic light-emitting dye is a fluorescein-based dye molecule.
(8) A light-emitting nanoparticle as disclosed in (6) or (7), wherein a contained concentration of the organic light-emitting dye is 1 mmol % to 6 mol % with respect to the cationic element.
(9) A light-emitting nanoparticle as disclosed in (6), wherein the rare earth ion is trivalent Eu.
(10) Alight-emitting nanoparticle as disclosed in (6) or (9), wherein a contained concentration of the rare earth ion is 1 mmol % to 10 mol % with respect to the cationic element.
(11) Alight-emitting nanoparticle as disclosed in any one of (1) to (10), wherein the matrix material comprises a surfactant molecule.
(12) Alight-emitting nanoparticle as disclosed in any one of (1) to (11), wherein an average particle diameter of the light-emitting nanoparticles is 10 nm to 500 nm.
(13) Alight-emitting nanoparticle as disclosed in any one of (1) to (12), wherein a surface is provided with a micropore with a pore diameter of 0.1 to 10 nm.
(14) Alight-emitting nanoparticle as disclosed in any one of (1) to (13), wherein a hydroxyl group and/or amino group bonded to the cationic element is formed at the surface.
(15) Alight-emitting nanoparticle as disclosed in any one of (1) to (14), wherein a surface is modified by a cell bonding molecule.
(16) Alight-emitting nanoparticle as disclosed in any one of (1) to (15), wherein an excitation wavelength and a light emission wavelength are in the visible light region.
(17) Alight-emitting nanoparticle as disclosed in any one of (1) to (16), used for bioimaging.
(18) Alight-emitting nanoparticle as disclosed in any one of (1) to (17), used as a therapeutic agent and which supports a drug at a pore of a surface.
(19) A cell detection method, comprising a step of inserting the light-emitting nanoparticle as disclosed in any one of (1) to (17) into a cell, irradiating the light-emitting nanoparticle with light, and observing the cell.
(20) A method of treating an animal, comprising a step of administering the light-emitting nanoparticle as disclosed in any one of (1) to (18) to an animal, irradiating the light-emitting nanoparticle with light, and treating the animal.
(21) A medical device provided with an examination portion which carries out examination of an internal cell, a diagnosis portion which carries out diagnosis of the internal cell, and/or a treatment portion which carries out treatment of the internal cell, wherein when the examination, diagnosis, and/or treatment are carried out, the light-emitting nanoparticle as disclosed in any one of (1) to (18) is inserted into the internal cell, and which is further provided with a light irradiating portion which irradiates light on the light-emitting nanoparticle.
(22) A cell visualization method, comprising a step of inserting into a cell a light-emitting nanoparticle comprising a matrix material, and a light-emitting substance included in the matrix material, wherein the matrix material comprises at least one cationic element selected from the group consisting of Ti, Si, Ca, Al, and Zr, and at least one anionic element selected from the group consisting of O and P, irradiating the light-emitting nanoparticle with light, and visualizing the cell.
(23) A damage reduction method cells of inserting into a cell a light-emitting nanoparticle comprising a matrix material, and a light-emitting substance included in the matrix material, wherein the matrix material comprises at least one cationic element selected from the group consisting of Ti, Si, Ca, Al, and Zr, and at least one anionic element selected from the group consisting of O and P, and exciting the light-emitting nanoparticle with light of a wavelength in the visible light region.
According to the present invention, it is possible to provide a light-emitting nanoparticle which is provided with light-emitting stability and light resistance, and is low in biological toxicity, and a cell detection method, treatment method of animals, medical device, a cell visualization method, and a damage reduction method of cells using the same.
Below, examples of the invention are described. Further, the present invention is not to be interpreted as being limited by these examples.
The first light-emitting nanoparticle of the present examples comprise a matrix material, and a light-emitting substance included in the matrix material. The matrix material comprises at least one type of cationic element selected from the group consisting of Ti, Si, Ca, Al and Zr, and at least one type of anionic element selected from the group consisting of O and P.
In the first light-emitting nanoparticle 1, the concentration of the light-emitting substance in the matrix material is a concentration whereby the average distance between the light-emitting substance becomes 1.2 nm or more. Herein, the “average distance between the light-emitting substance” is a theoretical value derived by calculating as below, from the average particle diameter of the light-emitting particles, the density, the molecular weight of the matrix material, the concentration of the light-emitting substance and the like. Accordingly, “the concentration of the light-emitting substance in the matrix material is a concentration whereby the average distance between the light-emitting substance becomes 1.2 nm or more” is a concentration set such that the theoretical average distance of the light-emitting substance, derived by calculations using formulas (1) to (6), becomes 1.2 nm or more.
Furthermore, when calculating the average distance of the light-emitting substance, it is confirmed that the light-emitting substance is in a state of approximately uniformly distribution in the light-emitting nanoparticle. The confirmation of the distribution state of the light-emitting substance can be carried out by transmission electron microscopy (TEM). Further, it can also be confirmed that the light-emitting substance is in a state of approximately uniformly distribution according to the fluorescence life measurement method, from the relationship between the light-emitting substance concentration and the fluorescence life. In a plurality of light-emitting nanoparticles where the concentrations of the light-emitting substance differ, when the light-emitting substance in the respective light-emitting nanoparticles is approximately uniform, a plot of the fluorescence life vs each concentration of the light-emitting substance displays a correlation with a negative linearity. This is because, as the concentration of the light-emitting substance increases, the occupied volume contributed by the light-emitting substance linearly decreases, the distance between the light-emitting substance becomes shorter, and the rate of the cross-relaxation process becomes higher. If the light-emitting substance aggregates, such a correlation does not occur. Accordingly, according to the fluorescence life measurement method, in order to confirm whether or not the light-emitting substance in the light-emitting nanoparticle is approximately uniform without agglomerating, the following procedure may be used. First, the concentration of the constituent components of these light-emitting nanoparticles and the light-emitting substance are analyzed, and a plurality of samples having differing concentrations of the light-emitting substance are prepared, consisting of the same constituent components as these light-emitting nanoparticles. Next, the fluorescence life of the plurality of samples is measured, and it is confirmed that the relationship between the concentration and the fluorescence life is a linear correlation. For the plurality of samples, if the relationship between the concentration of the light-emitting substance and the fluorescence life can be confirmed to be a linear correlation, after this, the fluorescence life of the light-emitting nanoparticle which is the subject of analysis is measured, and it is confirmed whether or not in the relationship between the concentration of the light-emitting substance and the fluorescence life, the plot of the sample and analysis is a linear correlation. If the plot of the sample and analysis is a linear correlation, it can be confirmed that the light-emitting substance in the light-emitting nanoparticle is approximately uniformly distributed.
The average particle diameter of the light-emitting nanoparticle is the averaged value of a plurality of particles obtained by determining the particle diameter of the light-emitting nanoparticle as {(long diameter+short diameter)/2}. For example, it is preferable to calculate the average particle diameter for 100 arbitrary particles in a predetermined region, using a scanning electron microscope (FE-SEM).
Specifically, the average distance between the centers of the light-emitting substances can be calculated from the following formulas (1) to (6). In this regard, as for the light-emitting substance number B with respect to the inorganic molecule number, the concentration of the light-emitting substance with respect to the metal element of the inorganic phase which is the matrix material, can be calculated from a result obtained using fluorescence X-ray (XRF) analysis.
De=A·ρn/M Formula(1)
De: inorganic molecular number density (number of molecules/nm3)
A: Avogadro's number (6.02×1023)
ρn: mass of matrix material per nm3 (g/nm3)
M: molecular weight of matrix material (silica (S): 60.1, titania (T) 79.866, hydroxyapatite
V
1=(4π/3)·(R/2)3 Formula (2)
V1: average one particle volume (nm3/1 particle)
R: average particle diameter (nm)
X
1=De·V1 Formula (3)
X1: inorganic molecule number contained in the light-emitting nanoparticle (molecule number/1 particle)
X
2
=X
1
·B Formula (4)
X2: light-emitting substance number contained in the light-emitting nanoparticle (molecule number/1 particle)
B: inorganic molecule number per light-emitting substance number (molecule number/substance number)
V
2
=V
1
/X
2 Formula (5)
V2: occupied volume of 1 molecule of light-emitting substance (nm3/1 molecule of light-emitting substance)
D={V
2·3/(4π)}1/3 Formula (6)
D: average distance between centers of light emitting substances (nm)
When the average distance between the light-emitting substance is 1.2 nm or more, it is possible to prevent density quenching caused by agglomerates of the light-emitting substance 4, and the light-emitting nanoparticles have excellent light emission stability. On the other hand, the present inventors surmise that if the average distance between the light-emitting substance is short, because of behavior as an excited complex, the energy occurring when excited is transferred and does not become light-emission energy, and light-emission rate reduction and the like occurs. The average distance between the light-emitting substance is preferably 1.5 nm or more, and more preferably 2 nm or more. Further, the upper limit of the average distance between the light-emitting substance is preferably 10 nm or less from the viewpoint of the sensitivity of general purpose fluorescence detection equipment. The concentration of the light-emitting substance in the matrix material corresponds to the light-emitting substance number B with respect to the inorganic molecule number. Therefore, a concentration where the average distance between the light-emitting substance is 1.2 nm or more can be determined depending on the used each molecular weight M and average particle diameter R according to the above equations (1) to (6).
The matrix material comprises at least one type of cationic element selected from the group consisting of Ti, Si, Ca, Al, and Zr, and at least one type of anionic element selected from the group consisting of O and P. As a suitable matrix material, titanium oxide comprising Ti which is a cationic element and O which is an anionic element, or silicon oxide comprising Si which is a cationic element, and O which is an anionic element, or aluminum oxide comprising Al which is a cationic element and O which is an anionic element, or zirconium oxide comprising Zr which is a cationic element and O which is an anionic element, may be mentioned.
The titanium oxide and silicon oxide may be particles of metal alkoxides shown by the general formula M(OR)n(M is Si or Ti, R is an alkyl group with a carbon number of 1 to 5) are agglomerated by hydrogen bonds, or may be particles wherein metal alkoxides are dehydration condensed with each other, to form a skeleton structure (-(M-O-M)n-).
As the matrix material, a calcium phosphate comprising Ca which is a cationic element and P which is an anionic element may also be mentioned.
The calcium phosphate compound is preferably a mixed compound of a phosphoric oxide source (one or more types of salt selected from the group of phosphoric oxide, monosodium phosphate, disodium phosphate, monocalcium phosphate, dicalcium phosphate, monoantimony phosphate, diantimony phosphate, and the like), and a calcium source (one or more types salt selected from the group of calcium nitrate, calcium carbonate, calcium chloride, calcium hydroxide, calcium acetate, and the like), or mixed reactants. As the calcium phosphate compound, calcium monohydrogen phosphate anhydride (CaHPO4), calcium monohydrogen phosphate dihydride (CaHPO4.2H2O), tricalcium phosphate (Ca3(PO4)2), dihydrogen calcium phosphate anhydride (Ca(H2PO4)2), calcium dihydrogen phosphate hydride (Ca(H2PO4)2.H2O), tetracalcium phosphate (Ca4O(PO4)2), hydroxyapatite (Ca10(PO4)6(OH)2), octacalcium phosphate (Ca8H2(PO4)6.5H2O), amorphous calcium phosphate (Ca3(PO4)2.nH2O) may be mentioned.
In the first light-emitting nanoparticle, the matrix material preferably comprises at least one type selected from the group consisting of TiO2, SiO2, Ca10(PO4)6(OH)2, Al2O3, and ZrO2. These matrix materials have low toxicity towards living bodies, and are excellent in biocompatibility. Among these, TiO2, SiO2, and Ca10(PO4)6(OH)2 are preferable as the matrix material of the first light-emitting nanoparticle.
The second light-emitting nanoparticle of the present embodiment is a light-emitting nanoparticle comprising a matrix material, and a light-emitting substance included in the matrix material, and the matrix material comprises at least one type of cationic element selected from the group consisting of Ti, Ca, Al, and Zr, and at least one type of anionic element selected from the group consisting of O and P.
In the second light-emitting nanoparticle, the matrix material preferably comprises at least one type selected from the group consisting of TiO2, Ca10(PO4)6(OH)2, Al2O3, and ZrO2. These matrix materials have low toxicity towards living bodies, and are excellent in biocompatibility. Among these, TiO2 and Ca10(PO4)6(OH)2 are more preferable as the matrix material of the second light-emitting nanoparticle.
Also in the second light-emitting nanoparticle, it is preferable if the average distance between the light-emitting substance is 1.2 nm or more, because it is possible to prevent concentration quenching of the light-emitting substance, and it becomes possible to obtain light-emitting nanoparticle with excellent light-emission stability. The average distance between the light-emitting substance is preferably 1.5 nm or more, and more preferably 2 nm or more. Further, the upper limit of the average distance between the light-emitting substance is preferably 10 nm or less from the viewpoint of the sensitivity of general purpose fluorescence detection equipment.
In the first light-emitting nanoparticle or the second light-emitting nanoparticle (below, when the first light-emitting nanoparticle as well as the second light-emitting nanoparticle are the subject, these may be referred to simply as “light-emitting nanoparticle”), the light-emitting substance preferably comprises at least one type selected from the group consisting of an organic light-emitting dye and a rare earth ion.
The organic light-emitting dye is not particularly limited, but is preferably at least one type selected from the group consisting of fluoresceine-based dye molecules, rhodamine-based dye molecules, cascade system dye molecules, coumalin-based dye molecules, eosin-based dye molecules, pyrene-based dye molecules, and cyanine-based dye molecules. Among these, fluoresceine-based dye molecules are preferable, and for example, fluorescein isothiocyanate (FITC) is excited and emits in the visible light region, and therefore is suitably used as the light-emitting substance in the present examples.
The rare earth ion is preferably at least one type selected from the group consisting of trivalent Ce, tetravalent Ce, trivalent Pr, trivalent Nd, trivalent Pm, trivalent Sm, divalent Eu, trivalent Eu, trivalent Gd, trivalent Tb, trivalent Dy, trivalent Ho, trivalent Er, trivalent Tm, trivalent Yb, and trivalent Lu. Among these, Eu3+ which is trivalent Eu is excited and emits in the visible light region, and therefore is suitably used as the light-emitting substance in the light-emitting nanoparticles of the present examples.
The contained concentration of the organic light-emitting dye is preferably 1 mmol % to 6 mol % with respect to the cationic element of the matrix material. When the contained concentration of the organic light-emitting dye is within this range, there is a tendency to readily maintain an average distance between the light-emitting substance of 1.2 nm or more. The contained concentration of the organic light-emitting dye is more preferably 100 mmol % to 5.5 mol %, and even more preferably 1 mol % to 5 mol %, with respect to the cationic element. The contained concentration of the rare earth ion is preferably 1 mmol % to 10 mol % with respect to the cationic element of the matrix material. When the contained concentration of the rare earth ion is within this range, there is a tendency to readily maintain an average distance between the light-emitting substance of 1.2 nm or more. The contained concentration of the rare earth ion is more preferably 100 mmol % to 10 mol %, and even more preferably 1 mol % to 5 mol %, with respect to the cationic element.
The matrix material preferably comprises a surfactant molecule. By comprising a surfactant molecule, there is a tendency to readily maintain a suitable average distance between the light-emitting substances. The surfactant molecule is not particularly limited, and for example, hexadecyltrimethylammonium bromide (tyltrimethylammonium bromide), hexadecyl trimethyl ammonium chloride, octadecyl trimethyl ammonium bromide, decyltrimethylammonium bromide, dodecyltrimethylammonium bromide, hexadecyl dimethyl ethyl ammonium bromide, hexadecylamine, sodium dodecyl sulfate, hexadecylamine, octadecylamine, octylphenol ethoxylate, polyoxyethylene cetyl ether, polyoxyethylene lauryl ether, polyoxyethylene(EO)-polyoxypropylene glycol (PO) copolymer (EO20PO30), polyoxyethylene (EO)-polyoxypropylene glycol (PO)-polyoxyethylene (EO) amphiphilic triblock copolymer (EO5PO6sEO5, EO20PO70PEO20, EO97PO67EO97) and the like, may be used. Further, in the case that it is not required to maintain a suitable average distance between the light-emitting substance, it is not necessary for the matrix material to comprise a surfactant molecule.
The content of the surfactant molecule in the matrix material is preferably a mol ratio of 0.01 or more with respect to the metal element of the matrix material. By making the mol ratio 0.01 more with respect to the metal element of the matrix material, there is a tendency to improve the dispersibility of the light-emitting substance in the matrix material, and to maintain the average distance between the light-emitting substance in a suitable range. The mol ratio is more preferably 0.05 or more, and even more preferably 0.1 or more. Further, the upper limit of the above mole ratio is preferably 1.5 or less, more preferably 0.2 or less, from the viewpoint of preventing segregation of only a liquid crystal phase to the outside of the particle or the particle surface of the surfactant.
In the case that the matrix material is prepared by a condensation reaction, it is preferable to use a cationic surfactant. By using a cationic surfactant, it is possible to prevent phase separation by electrostatic interaction, and a state with excellent dispersibility can be formed. Further, it is thought that if the surfactant is dispersed in advance in the solution before adding the matrix material, mycelles are formed with the hydrophilic groups facing outwards, and amorphous cluster structures of silica or titania or the like, and the light-emitting substance will agglomerate in a conjugated state on a nanoscale. In this way, there is a tendency to improve the dispersibility of the light-emitting substance in the matrix material, and maintain the average distance of the light-emitting substance in a suitable range, which is a preferable condition. Further, depending for example on the matrix material, a nonionic surfactant or an anionic surfactant may be used instead of the cationic surfactant.
The average particle diameter of the light-emitting nanoparticle is preferably 10 nm to 500 nm. By making the average particle diameter within this range, it becomes easy for the particles to be taken up by the target cells, and is also suitable in the context of observing the cells. On the other hand, if the average particle diameter is small, there is a tendency to have an effect on the active functions of the cell, and for the problem of toxicity to arise, which is not preferable. The average particle diameter of the light-emitting nanoparticle is more preferably 50 nm to 450 nm, and even more preferably 100 to 400 nm. Further, in the case that the present light-emitting nanoparticles are not used, for example, for cell imaging, the average particle diameter may be larger than 500 nm.
The light-emitting nanoparticle is preferably provided at its surface with pores with a pore diameter of 0.1 to 10 nm. By firing or solvent extracting the light-emitting nanoparticle surface, there is a tendency to form pores with a pore diameter of 0.1 to 10 nm on the light-emitting nanoparticle surface. By providing pores with a pore diameter of 0.1 to 10 nm, it is possible, for example, to support small molecule drugs on the light-emitting nanoparticle, and the use as a therapeutic agent also become possible. The pore diameter of the pores is more preferably 1 nm to 10 nm, and even more preferably 2 nm to 6 nm. Further, the light-emitting nanoparticle, depending for example on the application, may also not be provided with pores with a pore diameter of 0.1 to 10 nm on its surface. When the matrix material is silica, there is a tendency for pores to be formed. This can be thought to be because the interactions between the surfactant and the silica which is the matrix material comprising the light-emitting substance are relatively weak, and the surfactant is eliminated by the firing or solvent extraction process, whereby pores are formed. On the other hand, in the case that the matrix material is titania or a calcium phosphate compound, there is a tendency that pores are difficult to form. This is conjectured to be because the interaction between the surfactant, and the titania or calcium phosphate compound is strong, and it is difficult to eliminate the surfactant.
At the surface of the light-emitting nanoparticle, a hydroxide group (OH) bonded to the cationic element of the matrix material is preferably formed. Further, the surface of the matrix material is also preferably modified by an amino group, and for example, may also be formed using a silane coupling agent comprising an amino group. The OH group and amino group are not limited to the surface inside the pores, and may be on the particle surface such as the surface outside of the pores, and the surface outside the pores is preferable. The OH group or amino group is fixed by covalent bonding by hydrogen bonding or condensation polymerization to the cell bonding molecule, and if the surface of the light-emitting nanoparticle is modified by the cell bonding molecule, the cell bonding molecule can selectively bond to a cancer cell or a normal cell. If the cell bonding molecule selectively binds to a cell, the light-emitting nanoparticle is taken up into the cell. In this way, the light-emitting nanoparticle is made to emit light inside the cell, and it becomes possible to detect cancer or the like inside a cell. Further, according to the application of the light-emitting nanoparticle, the OH group and amino group do not have to be formed at the surface.
As the cell bonding molecule, HER2 antibody, antibodies selectively binding to human epidermal growth factor receptor, cancer-specific antibodies, phosphorylation protein antibodies, folic acid, antibodies selectively binding to folic acid receptor p, vascular endothelial cell-specific antibodies, tissue-specific antibodies, transferrin, transferring-bonding peptide, proteins having affinity to sugar chains, and the like may be mentioned. Among these, folic acid, which has tendency to be taken up by cancer cells, is preferably used as a cell bonding molecule. Because folic acid receptors are over-expressed on cell membranes for cancer cells, there is a tendency for folic acid molecules to be selectively bonded and taken up.
Further, the surface of the light-emitting nanoparticle may be modified with an anticancer agent molecule. If the anticancer agent molecule selectively binds to the cancer cell, the light-emitting nanoparticle will be taken up into the cell. In this way, the light-emitting nanoparticle is made to emit light inside the cell, and the cancer cells can be detected, and further, the anticancer agent is also taken up by the cell, and the anticancer agent molecule can operate, and can suppress the proliferation of cancer cells. Further, the light-emitting nanoparticle of the present embodiment has a wide range of applications other than cancer cells, and therefore the surface does not have to be modified with an anticancer agent molecule.
The cell bonding molecule or anticancer agent molecule are preferably modified and fixed to the surface of the light-emitting nanoparticle by a chemical bond. As the chemical bond, a peptide bond (—CO—NH—), hydrogen bond or the like may be mentioned.
For the light-emitting nanoparticles of the present embodiment, the excitation wavelength and the light-emission wavelength are preferably in the visible light region. If the excitation wavelength and the light-emission wavelength are in the visible light wavelength or higher, degradation of biological tissue and labeling material can be reduced. Further, optical scattering of the test surface can be reduced, and the observation sensitivity can be increased. Moreover, in an application using the light-emitting nanoparticles, in cases where it is not necessary to consider damage to biological tissue and labeling material, the excitation wavelength and the light-emission wavelength are preferably do not have to be in the visible light region.
The light-emitting nanoparticles of the present embodiment are preferably used for biological imaging. In the case that an organic molecule is used as the matrix material, the degradation and fading speed are fast, and there has been the problem that by exciting with ultraviolet rays, normal biological tissue is damaged. Further, in the case of using an inorganic material such as a quantum dot or the like, there are problems of biocompatibility such as the inclusion of highly toxic elements and the like, and the wavelengths of the excitation light include the ultraviolet region, and therefore, there has been concern of imparting damage to biological tissue. In contrast, the light-emitting nanoparticle of the present embodiment is provided with light-emitting stability and light resistance, and damage to biological tissue can be reduced, and living body toxicity is also low, whereby it can be suitably applied to biological imaging. Further, the light-emitting nanoparticle of the present embodiment is in a state which is preferable for use in applications other than biological imaging.
Further, the cell detection method of the present embodiment has the step of inserting the light-emitting nanoparticle into a cell, irradiating light onto the light-emitting nanoparticle, and observing the cell. According to the present detection method, because the light-emitting nanoparticle of the present embodiment has high sensitivity, it becomes possible to easily carry out observation of cells.
The treatment method of animals of the present embodiment includes the steps of administering the light-emitting nanoparticle to an animal, irradiating light onto the light-emitting nanoparticle, and treating the animal. According to the present treatment method, the light-emitting nanoparticle of the present embodiment has high sensitivity, and further, high biocompatibility, a condition of internal disease of an animal can be detected with good sensitivity, and further safely, and it becomes possible to suitably treat a disease of an animal.
The medical device of the present embodiment is provided with a testing portion which carries out testing of an internal cell, a diagnosis portion which carries out diagnosis of an internal cell, and/or a treatment portion which carries out treatment of an internal cell, and when the testing, diagnosis, and/or treatment is carried out, the light-emitting nanoparticles disclosed in any of (1) to (18) are inserted into an internal cell, and the medical device is further provided with a light irradiation portion which irradiates light on the light-emitting nanoparticles. Herein, as the testing portion which carries out testing of internal cells, for example a fluorescence endoscope which carries out precision image diagnosis may be mentioned. Further, as the diagnosis portion which carries out diagnosing of internal cells, for example, a device which carries out tissue biopsies may be mentioned. Further, as the treatment portion which carries out treatment of internal cells, a device for tumor portion extraction by endoscopy may be mentioned. Furthermore, as examples of internal cells, cancer cells pertaining to oral cavity cancer, pharyngeal cancer, esophageal cancer, colorectal cancer, small intestinal cancer, pulmonary cancer, breast cancer, and bladder cancer may be mentioned as examples. According to the present medical device, the light-emitting nanoparticle of the present embodiment have high sensitivity, and further, high biocompatibility, whereby it becomes possible to carry out testing, diagnosis, and treatment of internal cells with good sensitivity, and further, safely.
The visualization method of cells of the present embodiment is a method having the steps of inserting into a cell a light-emitting nanoparticle comprising a matrix material, and a light-emitting substance included in the matrix material, wherein the matrix material comprises at least one cationic element selected from the group consisting of Ti, Si, Ca, Al, and Zr, and at least one anionic element selected from the group consisting of O and P; irradiating the light-emitting nanoparticles with light, and visualizing the cell. According to the present visualization method of cells, the light-emitting nanoparticles of the present embodiment have high sensitivity, whereby it becomes possible to easily carry out visualization of the cells.
The damage reduction method cells of the present embodiment is a method having the steps of inserting into a cell a light-emitting nanoparticle comprising a matrix material, and a light-emitting substance included in the matrix material, wherein the matrix material comprises at least one cationic element selected from the group consisting of Ti, Si, Ca, Al, and Zr, and at least one anionic element selected from the group consisting of O and P; and exciting the light-emitting nanoparticles with light of a wavelength in the visible light region. According to the present method, the light-emitting nanoparticles of the present embodiment have high sensitivity, and the wavelength of light exciting the light-emitting nanoparticles is in the visible light region, whereby it becomes possible to reduce damage to cells compared to cell imaging methods of the prior art.
Below, specific examples of the present invention are explained. Further, the present invention is not to be interpreted as being limited by these examples.
To 225 mL of deionized water, 1.0 g of cetyltrimethylammonium bromide (CTAB) was added, and further, 3.5 mL of 2.0 M NaOH was added, and stirring was carried out at 353 K for 30 min. To the stirred solution, 5.515 mL of tetraethoxysilane (TEOS), and 15 mL of deionized water containing EuCl3 were added (when the EuCl3 was 0 g, the charge-in quantity at the synthesis starting time of Eu was 0 mol %, and is shown as “Eu0 mol %-S” (Comparative Example 1). When the EuCl3 was 0.452 g, the charge-in quantity at the synthesis starting time of Eu was 5 mol %, and is shown as “Eu5 mol %-S” (Example 1). When the EuCl3 was 0.904 g, the charge-in quantity at the synthesis starting time of Eu was 10 mol %, and is shown as “Eu10 mol %-S” (Example 2)), stirring was carried out at 353 K for 2 hours, and filtered. The residue was washed 4 times with 20 mL deionized water, and 1 time with 10 mL ethanol. After this, it was dried for 1 day at room temperature, and fired at 550° C. for 6 hours. The concentrations and the like of the elements constituting the particles of Examples 1 and 2, and Comparative Example 1 are as shown in Table 1.
To 0.011 mL (4.63×10−5 mol) of 3-aminopropyltriethoxysilane (APTES: C9H23NO3Si), 0 mg (0 mol, Comparative Example 2), or 91.1 mg (Example 3), or 192 mg (4.68×10−4 mol, Comparative Example 3) of fluorescein isothiocyanate (FITC: C21H11NO5S), and 36.1 mL (0.471 mol) of 2-propanol (IPA) were mixed, and stirring was carried out for 24 hours using a magnetic stirrer. To this solution, 1.37 mL (4.68×10−3 mol) of titanium tetraisopropoxide (TTTP: C12H28O4Ti) was added so that the Ti/APTES mol ratio became=100, whereby the solution A was prepared. The charge-in quantity at the synthesis starting time of the FITC of Comparative Example 2 was shown as 0 mol %, “FITCOmol %-T”, the charge-in quantity at the synthesis starting time of the FITC of Example 3 was 5 mol %, “FITC5 mol %-T”, and the charge-in quantity at the synthesis starting time of the FITC of Comparative Example 3 was 10 mol %, “FITC10 mol %-T”. 37.3 mL (0.487 mol) ofTIPA and 0.231 mL (1.28×10−2 mol) of ion-exchanged water were mixed, whereby solution B was prepared. 205 mg (7.61×10−4 mol) of octadecylamine (ODA: C18H39N), 189 mL (2.47 mol) ofTIPA, and 0.900 mL (4.99×10−2 mol) of ion-exchanged water were mixed, whereby solution C was prepared in a polypropylene vessel.
Herein, APTES was expected to express an interaction accompanying the formation of hydrogen bonds or the like of ODA and FITC. The solutions A and B were liquid-fed at respective flow rates of 30 mL·min−1 and mixed. Using IPA as the good solvent for TTIP, APTES, FITC and ODA, and using ion exchanged water as the reactant for TTIP and APTES hydrolysis, ODA was used for control of the form, size and nanostructure of the product. The reaction liquid thereof was delivered at a flow rate of 60 mL·min−1 to the vessel of the solution C, and until the end of delivery, stirring was carried out with a magnetic stirrer, and after this, was allowed to stand at room temperature for 24 hours, and a particle dispersion was obtained. Solid-liquid separation was done by centrifuge (9000 rpm, 10 min), and after discarding the supernatant liquid, the precipitate was dried overnight at 60° C., and a sample powder was obtained. The concentrations and the like of the elements constituting the particles of Example 3, and Comparative Examples 2 and 3 are as in Table 2.
To a solution consisting of 100 mL H2O (80° C.), and 8.75 g cetyltrimethylammonium bromide (CTAB, molecular weight 364.45) (0.024 mol), 2.09 g (0.012 mol) K2HPO4, and 1N NaOH were added by dropwise, so that the solution pH became 13, and cooled to 40° C. or less. Next, 60 mL H2O, CaCl2.2H2O was 2.87 g (0.0195 mol), and EuCl3.6H2O was 0 g (0 mmol), 0.357 g (0.9 mmol), or 0.714 g (1.9 mmol). The charge-in quantity at the synthesis starting time of the Eu of Comparative Example 4 was shown as 0 mol %, “Eu0 mol %-CP”, the charge-in quantity at the synthesis starting time of the Eu of Example 4 was 5 mol %, “Eu5 mol %-CP”, and the charge-in quantity at the synthesis starting time of the Eu of Example 5 was 10 mol %, “Eu10 mol %-CP”. These Eu-containing solutions were added dropwise to the solution cooled to 40° C. or less, at a dropping flow rate of 6 mL/min. After the dropping, while stirring, this was reflux heated for 24 hours at 40° C. The obtained white precipitate was washed two times with pure water, and washed two times with ethanol. After washing, and centrifuging (10000 G, 15 min, 4° C.), drying was carried out for 24 hours at 100° C. The concentrations and the like of the elements constituting Examples 4 and 5, and Comparative Example 4, as as in Table 3.
For the light-emitting nanoparticles of Examples 2 and 5, and Comparative Example 3, observation was carried out for a light-emitting substance distribution by a transmission electron microscope (TEM). Specifically, each type of particle powder was dispersed at a concentration of 1 wt % in ethanol, an ultrasound wave treatment was applied for 15 min, and the particle suspension liquid was cast on a glass substrate with a concentration of 0.01 mL/cm2. Vacuum drying was applied for 1 day, carbon deposition (film thickness: 10 nm) was applied to the substrate surface, a cross section of the particle film was cut out with a focused ion beam (surface area 8 μm×6 μm), and mounted on a carbon micro grid. Next, the central portion of the particle film was evaluated and analyzed by the transmission electron microscope (TEM) (HT 7700 by Hitachi High Technologies K.K.), and attached EDS (energy dispersion type X-ray spectroscopy). The observation results are shown in
It could be confirmed by TEM that the light-emitting substance was distributed in the matrix material, and therefore, the average distance between the light-emitting substance was calculated from the average particle diameter and concentration of the light-emitting substance. Specifically, the distance between the light-emitting substance was calculated by a density computation of the light-emitting substance with respect to the metal element of the inorganic phase which is the matrix material by fluorescence X-ray (XRF) analysis and scanning electron microscope (FE-SEM) observation.
As shown below in Table 4, by the density (known value) of the inorganic phase, the molecular number density of the inorganic phase was calculated.
For the light-emitting nanoparticles of Examples 1 to 5, and Comparative Example 3, the particle diameters of 100 or more light-emitting nanoparticles was measured using FE-SEM, and the average particle diameter was calculated. Further, the inorganic molecule number of the inorganic phase included per 1 molecule was calculated (refer to Table 5).
From the density of the light-emitting substance corresponding to the inorganic metal element obtained by XRF, the distance between light-emitting substance was calculated. As shown in Table 6, for the light-emitting nanoparticle produced in Examples 1 to 5 and Comparative Example 3, the average distance between the light-emitting substance included in the matrix material was 1.2 nm or more except for FITC10 mol %-T, which is Comparative Example 3.
By fluorescence life measurement, the dispersibility of the light-emitting substance was tested. The sample of the light-emitting substance, in addition of the 5 mol %, 10 mol % of the light-emitting substance synthesized when producing the below Examples, a sample of 2.5 mol % was also prepared. For the Eu light-emitting substance, an FP-8500 fluorospectrophotometer made by JASCO Corp. was used. For the FITC light-emitting substance, a DeltaPro fluorescence life photometer made by Horiba, Ltd. was used. For a light source, a xenon flash tube was used, for the excitation wavelength, the same wavelength as the fluorescence spectra was used, and as the test wavelength, the largest wavelength of the fluorescence spectra was used. The slit bandwidth at the excitation side and the receiving side was 2 nm. From immediately after the lighting of the flash lamp, for the light-emitting substance Eu, the florescence intensity changes were measured for an interval of 50 ms, and the light-emitting substance FITC, the florescence intensity changes were measured for an interval of 200 ns, and these decay curves of fluorescence intensity were measured with 10 repetitions. These 10 batches of decay curves were fit to the below Formula (7), and the fluorescence life T was calculated.
I(t)=I(0)exp(−t/τ) Formula (7)
Herein, I(t) is the fluorescence intensity at the time t, I(0) is the fluorescence intensity immediately after the lighting of the flash lamp. As a result, the fluorescence life T was as shown below in Table 7. Then a plot with the light-emitting substance density on the horizontal axis, and the fluorescence life T on the vertical axis was prepared. The results are shown in
The aspect ratio of the particles of Examples 1 and 2 and Comparative Example 1 were as shown below in Table 8. The aspect ratio is determined by dividing the long axis size of the particle by the short axis size. As the concentration of Eu becomes higher, the aspect ratio is reduced, and the particles show a change in shape from needle shape to spherical shape.
The average distance between the light-emitting substance in the Eu3+-containing silica particles of Examples 1 and 2 were respectively 1.5 nm and 1.2 nm (refer to the above Table 6). Further, concerning the Eu3+-containing silica particles of Examples 1 and 2, by solvent extraction or firing (oxidative decomposition) of the surfactant, pores with diameters in a range of 1 to 10 nm were observed. The analysis results of the specific surface area and pore diameter are shown in Table 9. Further,
The aspect ratios of the particles of Example 3 and Comparative Examples 2 and 3 were as shown in the following Table 10. The aspect ratio was determined by dividing the long axis size of the particle by the short axis size.
The average distance between the light-emitting substance in the FITC-containing titania particles of Example 3 and Comparative Example 3 were respectively 1.2 nm and 0.9 nm (refer to Table 6) and showed a tendency to decrease along with a density increase of the light-emitting substance. Further, concerning the FITC-containing titania particles of Example 3, by solvent extraction or firing (oxidative decomposition) of the surfactant, pores were observed. This is conjectured to be because the interaction between the surfactant and the FITC-containing titania particles of Example 3 is stronger than the interaction between the surfactant and the Eu3+-containing silica particles of Examples 1 and 2, whereby it was difficult to eliminate the surfactant.
For the aspect ratios of the particles of Examples 4 and 5, and Comparative Example 4, Table 11 below shows that, as the concentration of Eu increases, the aspect ratio decreases, and the particles change from a needle shape to a spherical form.
The average distances between the light-emitting substance in the Eu3+-containing calcium phosphate compound particles of Examples 4 and 5 were respectively 1.6 nm and 1.3 nm (refer to Table 6). For the Eu3+-containing calcium phosphate compound particles of Examples 4 and 5, pores were not observed due to the solvent extracting processes or firing (oxidative decomposition) of the surfactant. This is surmised to be because the interaction between the surfactant and the Eu3+-containing calcium phosphate compound particles is stronger than the interaction between the surfactant and the Eu3+-containing silica particles of Examples 1 and 2, whereby surfactant in the Eu3+-containing calcium phosphate compound particles was difficult to eliminate.
(Modification of Cancer Cell Binding Molecule (Folic Acid Derivative FA-NHS) with Light-Emitting Substance 5 Mol %-Containing Particles)
To 250 mg of the particles containing 5 mol % of each light-emitting substance of Examples 1, 3, and 4, 12 ml of an HCl aqueous solution (pH=2) was added, and treatment with ultrasonic waves was carried out. Next, a solution containing 0.78 ml (3.3 mmol) of 3-aminopropyltriethoxysilane (APTES) in 5 mL of ethanol was prepared, and added to the ultrasonic wave-treated solution, and a mixed solution was obtained. This mixed solution was stirred for 20 hours at 40° c. (pH<6.5). After completion of the stirring, the mixed solution was separated by centrifuge, and washed with ethanol. After washing, it was vacuum dried, and 150 mg of particles containing 5 mol % light-emitting substance with a surface modified by APTES was obtained. To 150 mg of these APTES/light-emitting substance 5 mol %-containing particles, 25 mL of a 50 mM phosphate buffer solution (pH=7.0) was added, and treatment with ultrasonic waves was carried out. Next, a solution comprising 430 mg (0.8 mmol) of FA-NHS in 12 mL of dimethylsulfoxide (DMSO) was prepared, and added to the ultrasonic wave-treated solution, and a mixed solution was obtained. This mixed solution was stirred for 3 hours at room temperature. After completion of the stirring, the mixed solution was separated by centrifuge, and washed with water. After washing, it was vacuum dried, and the particles containing 5 FA (folic acid)/light-emitting substance 5 mol % of Examples 6 to 8 were obtained.
For 5 mol % samples and 10 mol % samples of each light-emitting substance when synthesized and prepared, particles were synthesized using the surfactant, and in the case that the light-emitting substance was approximately uniformly distributed, particles were synthesized by the same experimental method as the present example completely without using surfactant, and the quantum yield was measured in the case that the particles were agglomerated in the light-emitting substance. The quantum yield was determined by a fluorescence spectrum measurement device. Measurements were carried out using an ISF-834 integrating sphere with p 60 mm, and in the measurement of the excitation scattered light, the standard white plate was set and measured in a state with the quartz window plate attached at the reflection position of the integrating sphere. The spectra of the incident light, scattered light, and fluorescence light were measured, their integrated peak intensities were calculated, respectively abbreviated as I0, I1, and I2, and the quantum yield (internal quantum efficiency)<Φint was calculated by Formula (8). Further, concerning the excitation/fluorescence spectra, the maximum wavelength seen in the excitation/fluorescence spectra of each sample was used.
Φint=I2/(I0−I1)×100 Formula (8)
Eu5 mol %-S-synthesis using surfactant: 11.5%
Eu10 mol %-S-synthesis using surfactant: 8.3%
Eu5 mol %-S-synthesis not using surfactant: 2.5%
Eu10 mol %-S-synthesis not using surfactant: 1.3%
FITC5 mol %-T-synthesis using surfactant: 19.4%
FITC5 mol %-T-synthesis not using surfactant: 13.1%
Eu5 mol %-CP-synthesis using surfactant: 7.1%
Eu10 mol %-CP-synthesis using surfactant: 4.8%
Eu5 mol %-CP-synthesis not using surfactant: 3.6%
Eu10 mol %-CP-synthesis not using surfactant: 1.9%
Normal cells (NIH3T3 cells) were cultured in a PS flask (dissemination density: 100×104 cells/37 cm2) After this, thawing and dissemination were carried out for 7 days, and cells were ablated and separated. The density of the NIH3T3 cells was (1.97±0.15)×105 cells/mL. Concentration adjustment of the cells was carried out, and 10 vol % FBS (fetal bovine serum) was cultured in DMEM (Dulbecco modification nutrient medium). There were 7.5×104 cells per 1 mL. An amount of 0.9 mL/well was disseminated to a 12 well plate (culture area: 3.8 cm2/well). The disseminated concentration was 1.8×104 cells/cm2. After this, it was cultured (temperature: 37° C., CO2 concentration: 5%, humidity 100%) After 12 hours, FA-Ef:NPS particles were added to 10 vol % DMEM, dispersed, and a concentration of 100 mg/mL was prepared.
Cell proliferation examination was executed by an MTT assay. The MTT assay is a method wherein formazan arising from the reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] by mitochondrial dehydrogenase inside a cell is extracted by an organic solvent, the absorbance at 570 nm is measured, and the viable cell rate is measured. At 24 hours, 48 hours, and 72 hours after dissemination, 100 μL of MTT reagent (Cat. No. 10009591) was added, and cultured for 3 hours (temperature: 37° C., CO2 concentration: 5%, humidity: 100%). After this, the culture medium was removed, 1 mL of a crystal dissolving solution (Cat. No. 10009593) was added, and stirred (variable mode, 1 min.). The absorbency at 570 nm was measured. The viable cell rate (%) was calculated by the formula below. Viable cell rate (%)=(absorbency of cell which is targeted for an evaluation−absorbency of blank)/(absorbency of cells without added particles−absorbency of blank)
Hela cancer cells were cultured in a PS flask (dissemination density: 100×104 cells/37 cm2). Thawing and dissemination were carried out for 7 days. The cells were ablated and separated. The Hela concentration was (0.99+0.07)×105 cells/mL. Concentration adjustment of the cells was carried out, and cultured in DMEM (Dulbecco modification nutrient medium) 10 vol % FBS (fetal bovine serum). There were 7.5×104 cells per 1 mL. An amount of 2.25 mL/PS was disseminated to PS dishes (cultivation area: 9.6 cm2), and the dissemination density was 1.8×104 cells/cm2.
After this, culturing was performed (temperature: 37° C., CO2 concentration: 5%, humidity 100%). After 12 hours, FA-Eu:NPS particles were added to 10 vol % DMEM, dispersed, and the concentration was adjusted to 100 mg/mL.
For living cell imaging, after 3 hours, 24 hours and 48 hours from spraying the particles on the cell surface, the nutrient medium was removed. After this, 1 mL PBS (phosphate buffered saline) was added, and removed (one time). Further, 1 mL of distilled water was added, and removed (one time). Fluorescence intensity measurements were carried out.
Eu3+ content: Ex filter: 485 nm±40 nm
Em filter: 590 nm±35 nm
FITC content: Ex filter: 485 nm±40 nm
Em filter: 540 nm±35 nm
Fluorescence microscope observation was carried out only after 24 hours. Further, after cultivation the culture medium was removed and the fluorescence intensity (PL) was measured at specified excitation wavelengths and detection wavelengths, after eliminating the “particles which are not bonded to cells”, or “particles which are not taken up into cells” with PBS and distilled water. Therefore, the obtained fluorescence intensities are for light emission caused only by “particles which are bonded to cells”, or “particles which are taken up into cells”.
The main measurement device used in the present examples was as follows.
1 . . . light-emitting nanoparticle, 2 . . . matrix material, 3 . . . light-emitting substance, 4 . . . OH group or amino group, 5 . . . peptide bond, 6 . . . cell bonding molecule, 7 . . . cell bonding molecule-modified light-emitting nanoparticle, 10 . . . cancer cell, 11 . . . receptor.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2016-064240 | Mar 2016 | JP | national |
The present application is a continuation application of U.S. patent application Ser. No. 16/089,758, filed on Sep. 28, 2018, the entire contents of which are incorporated herein by reference and priority to which is hereby claimed. Application 16/089,758 is a U.S. national stage of application No. PCT/JP2017/012605, filed on Mar. 28, 2017. Priority under 35 U.S.C. § 119(a) and 35 U.S.C. § 365(b) is hereby claimed from Japanese Application No. 2016-064240, filed on Mar. 28, 2016, the disclosures all of which are both incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16089758 | Sep 2018 | US |
| Child | 17962198 | US |
