Patent Description:
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 <NUM> 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 <NUM> 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 <NUM> 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 <NUM> to <NUM> equivalents of an organic ligand wherein a thiol group and a hydrophilic group are bonded by a hydrocarbon chain with a carbon number of <NUM> to <NUM>, 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 <NUM> 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 Y<NUM>O<NUM>:Er<NUM>+, Yb<NUM>+, Y<NUM>O<NUM>:Er<NUM>+, NaYF<NUM>:Er<NUM>+, Yb<NUM>+.

Patent Document <NUM> discloses a semiconductor nanoparticle used for molecule/cell imaging, which is a semiconductor nanoparticle with an average particle diameter of <NUM> to <NUM>, 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 <NUM> 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 <NUM> 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 <NUM> to <NUM> also disclose the use of quantum dots and fluorescent dyes for bioimaging. Non-Patent Document <NUM> discloses luminescent europium(III)-doped nanoporous silica nanospheres. Non-Patent Document <NUM> discloses luminescent Eu<NUM>+-doped hydroxyapatite nanorods for the preparation of fluorescent inks. Non-Patent Document <NUM> discloses the synthesis of cytocompatible luminescent titania/fluorescin hybrid nanoparticles. Non-Patent Document <NUM> discloses the synthesis and luminescence properties of Eu(III)-doped nanoporous silica spheres. Non-Patent Document <NUM> relates to Eu(III)-containing nanoporous silica, which was synthesized by two different loadings utilizing adsorption and doping. Non-Patent Document <NUM> describes luminescent Eu(III)-doped nanoporous silica spheres by a supramolecular templating method. Non-Patent Document <NUM> relates to calcium phosphate nanoparticles doped with Eu<NUM>+, Mn<NUM>+ and Ln<NUM>+ ions synthesized by a hydrothermal route. Non-patent document <NUM> describes strontium hydroxyapatite nanorods with luminescent and mesoporous properties synthesized by a hydrothermal method.

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.

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 surfactant molecule, and at least one type of anionic element selected from the group consisting of O and P.

<FIG> is a schematic diagram schematically showing the outer appearance of the first light-emitting nanoparticle <NUM> of the present example. The first light-emitting nanoparticle <NUM> is provided with the matrix material <NUM>, and the light-emitting substance <NUM> is included in the matrix material <NUM>.

In the first light-emitting nanoparticle <NUM>, the concentration of the light-emitting substance in the matrix material is a concentration whereby the average distance between the light-emitting substance becomes <NUM> 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 <NUM> or more" is a concentration set such that the theoretical average distance of the light-emitting substance, derived by calculations using formulas (<NUM>) to (<NUM>), becomes <NUM> 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)/<NUM>}. For example, it is preferable to calculate the average particle diameter for <NUM> arbitrary particles in a predetermined region, using a scanning electron microscope (FE-SEM).

Specifically, the average distance between the light-emitting substance can be calculated from the following formulas (<NUM>) to (<NUM>).

<MAT> X<NUM>: inorganic molecule number contained in the light-emitting nanoparticle (molecule number/<NUM> particle).

<MAT> V<NUM>: occupied volume of <NUM> molecule of light-emitting substance (nm<NUM>/<NUM> molecule of light-emitting substance).

<MAT> D: distance between centers of light emitting substance (nm).

When the average distance between the light-emitting substance is <NUM> or more, it is possible to prevent density quenching caused by agglomerates of the light-emitting substance <NUM>, 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 <NUM> or more, and more preferably <NUM> or more. Further, the upper limit of the average distance between the light-emitting substance is preferably <NUM> 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 <NUM> or more can be determined depending on the used each molecular weight M and average particle diameter R according to the above equations (<NUM>) to (<NUM>).

The matrix material comprises at least one type of cationic element selected from the group consisting of Ti, Ca, Al, and Zr. 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 <NUM> to <NUM>) 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 (CaHPO<NUM>), calcium monohydrogen phosphate dihydride (CaHPO<NUM>•<NUM><NUM>O), tricalcium phosphate (Ca<NUM>(PO<NUM>)<NUM>), dihydrogen calcium phosphate anhydride (Ca(H<NUM>PO<NUM>)<NUM>), calcium dihydrogen phosphate hydride (Ca(H<NUM>PO<NUM>)<NUM>•H<NUM>O), tetracalcium phosphate (Ca<NUM>O(PO<NUM>)<NUM>), hydroxyapatite (Ca<NUM>(PO<NUM>)<NUM>(OH)<NUM>), octacalcium phosphate (CaBH<NUM>(PO<NUM>)<NUM>•SH<NUM>O), amorphous calcium phosphate (Ca<NUM>(PO<NUM>)<NUM>•nH<NUM>O) may be mentioned.

In the first light-emitting nanoparticle, the matrix material preferably comprises at least one type selected from the group consisting of TiO<NUM>, SiO<NUM>, Ca<NUM>(PO<NUM>)<NUM>(OH)<NUM>, Al<NUM>O<NUM>, and ZrO<NUM>. These matrix materials have low toxicity towards living bodies, and are excellent in biocompatibility. Among these, TiO<NUM>, SiO<NUM>, and Ca<NUM>(PO<NUM>)<NUM>(OH)<NUM> 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 TiO<NUM>, Ca<NUM>(PO<NUM>)<NUM>(OH)<NUM>, Al<NUM>O<NUM>, and ZrO<NUM>. These matrix materials have low toxicity towards living bodies, and are excellent in biocompatibility. Among these, TiO<NUM> and Ca<NUM>(PO<NUM>)<NUM>(OH)<NUM> 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 <NUM> 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 <NUM> or more, and more preferably <NUM> or more. Further, the upper limit of the average distance between the light-emitting substance is preferably <NUM> 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, Eu<NUM>+ 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 <NUM> mmol% to <NUM> 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 <NUM> or more. The contained concentration of the organic light-emitting dye is more preferably <NUM> mmol% to <NUM> mol%, and even more preferably <NUM> mol% to <NUM> mol%, with respect to the cationic element. The contained concentration of the rare earth ion is preferably <NUM> mmol% to <NUM> 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 <NUM> or more. The contained concentration of the rare earth ion is more preferably <NUM> mmol% to <NUM> mol%, and even more preferably <NUM> mol% to <NUM> 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 (EO<NUM>PO<NUM>), polyoxyethylene (EO)-polyoxypropylene glycol (PO)-polyoxyethylene (EO) amphiphilic triblock copolymer (EO<NUM>PO<NUM>EO<NUM>, EO<NUM>PO<NUM>PEO<NUM>, EO<NUM>PO<NUM>EO<NUM>) 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 <NUM> or more with respect to the metal element of the matrix material. By making the mol ratio <NUM> 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 <NUM> or more, and even more preferably <NUM> or more. Further, the upper limit of the above mole ratio is preferably <NUM> or less, more preferably <NUM> 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 <NUM> to <NUM>. 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 <NUM> to <NUM>, and even more preferably <NUM> to <NUM>. 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 <NUM>.

The light-emitting nanoparticle is preferably provided at its surface with pores with a pore diameter of <NUM> to <NUM>. By firing or solvent extracting the light-emitting nanoparticle surface, there is a tendency to form pores with a pore diameter of <NUM> to <NUM> on the light-emitting nanoparticle surface. By providing pores with a pore diameter of <NUM> to <NUM>, 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 <NUM> to <NUM>, and even more preferably <NUM> to <NUM>. Further, the light-emitting nanoparticle, depending for example on the application, may also not be provided with pores with a pore diameter of <NUM> to <NUM> 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 β, 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.

<FIG> is a diagram schematically showing the mechanism by which the light-emitting nanoparticle which one embodiment of the present invention is taken up in a cancer cell. As shown in <FIG>, the light-emitting nanoparticle <NUM> provided with the matrix material <NUM> comprising the light-emitting substance <NUM>, may form a cell binding molecule-modified light-emitting nanoparticle <NUM> having a bonding means <NUM> of an OH group or an amino group at the surface of the matrix material <NUM>, which is bonded to a cell bonding molecule <NUM> by a peptide bond <NUM>. The cell bonding molecule-modified light-emitting nanoparticle may bond to a receptor <NUM> of the cancer cell <NUM>, and be taken up into the cancer cell <NUM>.

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.

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 <NUM> of deionized water, <NUM> of cetyltrimethylammonium bromide (CTAB) was added, and further, <NUM> of <NUM> NaOH was added, and stirring was carried out at <NUM> for <NUM>. To the stirred solution, <NUM> of tetraethoxysilane (TEOS), and <NUM> of deionized water containing EuCl<NUM> were added (when the EuCl<NUM> was <NUM>, the charge-in quantity at the synthesis starting time of Eu was <NUM> mol%, and is shown as "Eu0mol%-S" (Comparative Example <NUM>). When the EuCl<NUM> was <NUM>, the charge-in quantity at the synthesis starting time of Eu was <NUM> mol%, and is shown as "Eu5mol%-S" (Example <NUM>). When the EuCl<NUM> was <NUM>, the charge-in quantity at the synthesis starting time of Eu was <NUM> mol%, and is shown as "Eu10mol%-S" (Example <NUM>)), stirring was carried out at <NUM> for <NUM> hours, and filtered. The residue was washed <NUM> times with <NUM> deionized water, and <NUM> time with <NUM> ethanol. After this, it was dried for <NUM> day at room temperature, and fired at <NUM> for <NUM> hours. The concentrations and the like of the elements constituting the particles of Examples <NUM> and <NUM>, and Comparative Example <NUM> are as shown in Table <NUM>.

To <NUM> (<NUM> x <NUM>-<NUM> mol) of <NUM>-aminopropyltriethoxysilane (APTES: C<NUM>H<NUM>NO<NUM>Si), <NUM> (<NUM> mol, Comparative Example <NUM>), or <NUM> (Example <NUM>), or <NUM> (<NUM> × <NUM>-<NUM> mol, Comparative Example <NUM>) of fluorescein isothiocyanate (FITC: C<NUM>H<NUM>NO<NUM>S), and <NUM> (<NUM> mol) of <NUM>-propanol (IPA) were mixed, and stirring was carried out for <NUM> hours using a magnetic stirrer. To this solution, <NUM> (<NUM> × <NUM>-<NUM> mol) of titanium tetraisopropoxide (TTIP: C<NUM>H<NUM>O<NUM>Ti) was added so that the Ti/APTES mol ratio became = <NUM>, whereby the solution A was prepared. The charge-in quantity at the synthesis starting time of the FITC of Comparative Example <NUM> was shown as <NUM> mol%, "FITC0mol%-T", the charge-in quantity at the synthesis starting time of the FITC of Example <NUM> was <NUM> mol%, "FITC5mol%-T", and the charge-in quantity at the synthesis starting time of the FITC of Comparative Example <NUM> was <NUM> mol%, "FITC10mol%-T". <NUM> (<NUM> mol) of IPA and <NUM> (<NUM> × <NUM>-<NUM> mol) of ion-exchanged water were mixed, whereby solution B was prepared. <NUM> (<NUM> × <NUM>-<NUM> mol) of octadecylamine (ODA: C<NUM>H<NUM>N), <NUM> (<NUM> mol) of IPA, and <NUM> (<NUM> × <NUM>-<NUM> 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 <NUM>•min-<NUM> 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 <NUM>•min-<NUM> 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 <NUM> hours, and a particle dispersion was obtained. Solid-liquid separation was done by centrifuge (<NUM> rpm, <NUM>), and after discarding the supernatant liquid, the precipitate was dried overnight at <NUM>, and a sample powder was obtained. The concentrations and the like of the elements constituting the particles of Example <NUM>, and Comparative Examples <NUM> and <NUM> are as in Table <NUM>.

To a solution consisting of <NUM> H<NUM>O (<NUM>), and <NUM> cetyltrimethylammonium bromide (CTAB, molecular weight <NUM>) (<NUM> mol), <NUM> (<NUM> mol) K<NUM>HPO<NUM>, and 1N NaOH were added by dropwise, so that the solution pH became <NUM>, and cooled to <NUM> or less. Next, <NUM> H<NUM>O, CaCl<NUM>•<NUM><NUM>O was <NUM> (<NUM> mol), and EuCl<NUM>•<NUM><NUM>O was <NUM> (<NUM> mmol), <NUM> (<NUM> mmol), or <NUM> (<NUM> mmol). The charge-in quantity at the synthesis starting time of the Eu of Comparative Example <NUM> was shown as <NUM> mol%, "Eu0mol%-CP", the charge-in quantity at the synthesis starting time of the Eu of Example <NUM> was <NUM> mol%, "Eu5mol%-CP", and the charge-in quantity at the synthesis starting time of the Eu of Example <NUM> was <NUM> mol%, "Eu10mol%-CP". These Eu-containing solutions were added dropwise to the solution cooled to <NUM> or less, at a dropping flow rate of <NUM>/min. After the dropping, while stirring, this was reflux heated for <NUM> hours at <NUM>. The obtained white precipitate was washed two times with pure water, and washed two times with ethanol. After washing, and centrifuging (<NUM>, <NUM>, <NUM>), drying was carried out for <NUM> hours at <NUM>. The concentrations and the like of the elements constituting Examples <NUM> and <NUM>, and Comparative Example <NUM>, as as in Table <NUM>.

For the light-emitting nanoparticles of Examples <NUM> and <NUM>, and Comparative Example <NUM>, 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 <NUM> wt% in ethanol, an ultrasound wave treatment was applied for <NUM>, and the particle suspension liquid was cast on a glass substrate with a concentration of <NUM>/cm<NUM>. Vacuum drying was applied for <NUM> day, carbon deposition (film thickness: <NUM>) was applied to the substrate surface, a cross section of the particle film was cut out with a focused ion beam (surface area <NUM> × <NUM> pm), 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 <NUM> by Hitachi High Technologies K. ), and attached EDS (energy dispersion type X-ray spectroscopy). The observation results are shown in <FIG>. In <FIG>, the light-emitting substance is present as single molecules or ions with a white, approximately circular shape, and it could be confirmed that they are present distributed inside the light-emitting nanoparticles.

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 <NUM>, 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 <NUM> to <NUM>, and Comparative Example <NUM>, the particle diameters of <NUM> 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 <NUM> molecule was calculated (refer to Table <NUM>).

In the case of a silica phase, the correspondence of one Si per one inorganic silica molecule unit, and in the case of a titania phase, the correspondence of one Ti per one inorganic silica molecule unit, and in the case of a hydroxyapatite phase the correspondence of six Ca per one inorganic silica molecule unit was applied.

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 <NUM>, for the light-emitting nanoparticle produced in Examples <NUM> to <NUM> and Comparative Example <NUM>, the average distance between the light-emitting substance included in the matrix material was <NUM> or more except for FITC10mol%-T, which is Comparative Example <NUM>.

By fluorescence life measurement, the dispersibility of the light-emitting substance was tested. The sample of the light-emitting substance, in addition of the <NUM> mol%, 10mol% of the light-emitting substance synthesized when producing the below Examples, a sample of <NUM> mol% was also prepared. For the Eu light-emitting substance, an FP-<NUM> fluorospectrophotometer made by JASCO Corp. For the FITC light-emitting substance, a DeltaPro fluorescence life photometer made by Horiba, Ltd. 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 <NUM>. 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 <NUM>, and the light-emitting substance FITC, the florescence intensity changes were measured for an interval of <NUM> ns, and these decay curves of fluorescence intensity were measured with <NUM> repetitions. These <NUM> batches of decay curves were fit to the below Formula (<NUM>), and the fluorescence life τ was calculated. <MAT> Herein, I(t) is the fluorescence intensity at the time t, I(<NUM>) is the fluorescence intensity immediately after the lighting of the flash lamp. As a result, the fluorescence life τ was as shown below in Table <NUM>. Then a plot with the light-emitting substance density on the horizontal axis, and the fluorescence life τ on the vertical axis was prepared. The results are shown in <FIG>.

<FIG> shows the relationship between the light-emitting substance concentration and the fluorescence life for a silica phase (S), <NUM>(b) for a titania phase (T), and <NUM>(c) for hydroxyapatite (CP), and as shown in <FIG>, the plot of fluorescence life with respect to each concentration shows a negative linear correlation. This correlation is a monotonic decrease, therefore, it is surmised that this is an equivalent matrix environment with respect to the light-emitting substance. Namely, along with a concentration increase of the light-emitting substance, the occupied volume contributed to the light-emitting substance is linearly reduced, the distance between the light-emitting substance becomes short, and the probability of cross relaxation process becomes high (the excitation energy is partially transferred to proximal ions, and the resulting two low excitation state ions display the phenomenon of rapid relaxation to the base state). The correlation between the light-emitting substance concentration and fluorescence life shows a high correlation of <NUM> or more, and it can be considered that the light-emitting substance itself is present approximately uniformly distributed as individual molecules or ions which do not relax each other, and the distance between the light-emitting substance is small. As above, it was confirmed that in the light-emitting nanoparticles used in the Examples, the light-emitting substance was present approximately uniformly distributed.

<FIG> is an electron microscope observed image (TEM image) by concentration of Eu<NUM>+-containing silica particles. <FIG> is Comparative Example <NUM> (Eu0mol%-S), <FIG> is Example <NUM> (Eu5mol%-S), and <FIG> is Example <NUM> (Eu10mol%-S). <FIG>, show the trend that as the concentration of Eu becomes higher, the average particle diameter (D) of the Eu<NUM>+-containing silica particles becomes smaller. Further, the coefficient of variation (CV), which is a relative standard deviation, was <NUM> to <NUM>%.

The aspect ratio of the particles of Examples <NUM> and <NUM> and Comparative Example <NUM> were as shown below in Table <NUM>. 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 Eu<NUM>+-containing silica particles of Examples <NUM> and <NUM> were respectively <NUM> and <NUM> (refer to the above Table <NUM>). Further, concerning the Eu<NUM>+-containing silica particles of Examples <NUM> and <NUM>, by solvent extraction or firing (oxidative decomposition) of the surfactant, pores with diameters in a range of <NUM> to <NUM> were observed. The analysis results of the specific surface area and pore diameter are shown in Table <NUM>. Further, <FIG> shows the nitrogen absorption/desorption isotherm and pore diameter distribution. <FIG> relate to EuOmol%-S, <NUM>(b) and (e) relate to Eu5mol%-S, and <FIG> relate to Eu10mol%-S. Concerning the measurement method, the BET specific surface area and (determined by the BJH method) BJH pore diameter distribution were measured by the nitrogen absorption/desorption isotherm (BELSORP-mini manufactured by MicrotracBEL Corp. The sample was degassed for a whole day and night, dried for <NUM> hours at <NUM>, and measured at an adsorption temperature of -<NUM> and maximum equilibrium pressure of <NUM> Torr. As a result, as shown in Table <NUM>, it was confirmed that the mesopores were enlarged along with an increase in the doping amount of Eu. The distribution center of the included mesopore diameter was approximately <NUM> to <NUM>.

<FIG> is an electron microscope observed image (FE-SEM) by density of the FITC-containing titania particles, and a particle diameter distribution. <FIG> is Comparative Example <NUM> (FITC0mol%-T), <FIG> is Example <NUM> (FITC5mol%-T), and <FIG> is Comparative Example <NUM> (FITC10mol%-T). <FIG> show a tendency that as the density of the FITC becomes higher, the average particle diameter (D) of the FITC-containing titania particles becomes greater. Further, the coefficient of variation (CV) was <NUM> to <NUM>%.

The aspect ratios of the particles of Example <NUM> and Comparative Examples <NUM> and <NUM> were as shown in the following Table <NUM>. 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 <NUM> and Comparative Example <NUM> were respectively <NUM> and <NUM> (refer to Table <NUM>), 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 <NUM>, 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 <NUM> is stronger than the interaction between the surfactant and the Eu<NUM>+-containing silica particles of Examples <NUM> and <NUM>, whereby it was difficult to eliminate the surfactant.

<FIG> is an electron microscope observed image (TEM image) by concentration of Eu<NUM>+-containing calcium phosphate compound particles. <FIG> is Comparative Example <NUM> (Eu0mol%-CP), <FIG> is Example <NUM> (Eu5mol%-CP), and <FIG> is Example <NUM> (Eu10mol%-CP), <FIG> show a trend that the trend that as the concentration of Eu becomes higher, the average particle diameter (D) of the Eu<NUM>+-containing calcium phosphate compound particles becomes smaller. Further, the coefficient of variation (CV) was <NUM> to <NUM>%.

For the aspect ratios of the particles of Examples <NUM> and <NUM>, and Comparative Example <NUM>, Table <NUM> 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.

<FIG> is a graph showing X-ray diffraction patterns, (a) Eu<NUM>+-containing silica particles, (b) FITC-containing titania particles, and (c) Eu<NUM>+-containing calcium phosphate compound particles. <FIG> shows an amorphous structure, wherein the peaks deriving from the precipitate of Eu and the silica crystals were not observed, and shows a tendency wherein the greater the content of Eu, the lower the peak intensity. <FIG> shows a tendency that the greater the FITC content, the higher the peak intensity of the left end side of the graph. In <FIG>, from the assignment of the Miller indices, the crystal structure is a hydroxyapatite single phase, and it is seen that as the Eu content becomes greater, portions where the peak width at half height becomes lower were seen.

<FIG> is a graph showing the infrared absorption spectrum before surfactant removal for the Eu<NUM>+-containing silica particles. Characteristic absorption bands of an Si-OH stretching vibration of a hydrogen bond type at <NUM>-<NUM>, a C-H stretching vibration at <NUM>-<NUM> (-CH<NUM>), a C-H stretching vibration at <NUM>-<NUM> (-CH<NUM>-), a bending vibration at <NUM>-<NUM> (-CH<NUM>-), an Si-O-Si asymmetric stretching vibration at <NUM>-<NUM> ((Si-O-Si)n derived), an Si-O-Si symmetric stretching vibration at <NUM>-<NUM> ((Si-O-Si)n derived), an Si-OH stretching vibration at <NUM>-<NUM>, and an Si-OH stretching vibration at <NUM>-<NUM>, and the like were observed. The presence of a surfactant was confirmed by the presence of absorption bands of a C-H stretching vibration at <NUM>-<NUM> (-CH<NUM>), a C-H stretching vibration at <NUM>-<NUM> (-CH<NUM>-), and a bending vibration at <NUM>-<NUM> (-CH<NUM>-).

<FIG> is a graph showing the infrared absorption spectrum, (a) Eu<NUM>+-containing silica particles, (b) FITC-containing titania particles, and (c) Eu<NUM>+-containing calcium phosphate compound particles. In <FIG>, characteristic absorption bands of an Si-OH stretching vibration of a hydrogen bond type at <NUM>-<NUM>, an Si-O-Si asymmetric stretching vibration at <NUM>-<NUM> ((Si-O-Si)n derived), an Si-O-Si symmetric stretching vibration at <NUM>-<NUM> ((Si-O-Si)n derived), an Si-OH stretching vibration at <NUM>-<NUM>, and an Si-OH stretching vibration at <NUM>-<NUM>, and the like were observed. As a result of the firing or solvent extracting processes, the absorption bands of a C-H stretching vibration at <NUM>-<NUM> (-CH<NUM>), a C-H stretching vibration at <NUM>-<NUM> (-CH<NUM>-), a bending vibration at <NUM>-<NUM> (-CH<NUM>-) disappear, whereby it was judged that the surfactant was eliminated. In <FIG> characteristic absorption bands of a stretching vibration of an OH group in a titania structure at <NUM>-<NUM>, a stretching vibration of a Ti-OH and H<NUM>O of a particle surface at <NUM>-<NUM>-<NUM>, a stretching vibration of a -CH<NUM> and -CH<NUM>- caused by surfactant ODA (octadecylamine) and the light-emitting substance FITC at <NUM>-<NUM> and <NUM>-<NUM>, a bending vibration of -CH<NUM>- at <NUM>-<NUM>, and a stretching vibration of C=O at <NUM>-<NUM> were observed. Ultimately, the surfactant survived the washing process by IPA. From this, it was surmised that the surfactant survived due to the interaction between the surfactant and titania/FITC. In <FIG> characteristic absorption bands of a stretching vibration of an OH group in a crystal structure of hydroxyapatite at <NUM>-<NUM>, P-O stretching vibrations of phosphate groups at <NUM>-<NUM>, <NUM>-<NUM>, and <NUM>-<NUM>, stretching vibrations of OH groups of H<NUM>O of a particle surface at <NUM>-<NUM>-<NUM> and <NUM>-<NUM>, and the like were observed. The P-O and -OH stretching vibrations which are characteristic peaks of calcium phosphate (especially, hydroxyapatite) were observed. Ultimately, the surfactant was not observed. This is because the surfactant was sufficiently eliminated by the washing. It was confirmed by the XRF results that the surfactant was ultimately eliminated by the washing process, and pores were formed for CP.

The average distances between the light-emitting substance in the Eu<NUM>+-containing calcium phosphate compound particles of Examples <NUM> and <NUM> were respectively <NUM> and <NUM> (refer to Table <NUM>). For the Eu<NUM>+-containing calcium phosphate compound particles of Examples <NUM> and <NUM>, 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 Eu<NUM>+-containing calcium phosphate compound particles is stronger than the interaction between the surfactant and the Eu<NUM>+-containing silica particles of Examples <NUM> and <NUM>, whereby surfactant in the Eu<NUM>+-containing calcium phosphate compound particles was difficult to eliminate.

To <NUM> of the particles containing <NUM> mol% of each light-emitting substance of Examples <NUM>, <NUM>, and <NUM>, <NUM> of an HCl aqueous solution (pH = <NUM>) was added, and treatment with ultrasonic waves was carried out. Next, a solution containing <NUM> (<NUM> mmol) of <NUM>-aminopropyltriethoxysilane (APTES) in <NUM> of ethanol was prepared, and added to the ultrasonic wave-treated solution, and a mixed solution was obtained. This mixed solution was stirred for <NUM> hours at <NUM> (pH < <NUM>). After completion of the stirring, the mixed solution was separated by centrifuge, and washed with ethanol. After washing, it was vacuum dried, and <NUM> of particles containing <NUM> mol% light-emitting substance with a surface modified by APTES was obtained. To <NUM> of these APTES/light-emitting substance <NUM> mol%-containing particles, <NUM> of a <NUM> phosphate buffer solution (pH = <NUM>) was added, and treatment with ultrasonic waves was carried out. Next, a solution comprising <NUM> (<NUM> mmol) of FA-NHS in <NUM> 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 <NUM> 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 <NUM> FA (folic acid)/light-emitting substance <NUM> mol% of Examples <NUM> to <NUM> were obtained.

<FIG> is a graph showing an excitation spectrum, (a) Eu<NUM>+-containing silica particles, (b) FITC-containing titania particles, and (c) Eu<NUM>+-containing calcium phosphate compound particles. In <FIG> a peak caused by an f-f transition at <NUM> was observed. In <FIG>, at <NUM>, <NUM>, and <NUM>, peaks caused by minus ionized single molecules of the FITC light-emitting substance were observed (the FITC which interacts with the cationic agent ODA (octadecylamine) was introduced individually distributed inside the particles). In <FIG>, a peak caused by an f-f transition at <NUM> was observed.

<FIG> is a graph showing an emission spectrum, (a) Eu<NUM>+-containing silica particles, (b) FITC-containing titania particles, and (c) Eu<NUM>+-containing calcium phosphate compound particles. In <FIG>, peaks caused by a transition from <NUM>D<NUM> to <NUM>F<NUM> of <NUM>, a transition from <NUM>D<NUM> to <NUM>F<NUM> of <NUM>, <NUM>, and <NUM>, a transition from D<NUM> to <NUM>F<NUM> of <NUM>, a transition from <NUM>D<NUM> to <NUM>F<NUM> of <NUM>, and a transition from <NUM>D<NUM> to <NUM>F<NUM> of <NUM> are observed. Ultimately, there was no change in the form and intensity of the light emission spectrum due to the firing or solvent extracting processes (elimination process of the surfactant). From this result, it was thought that the surfactant plays an important role in the approximately uniform dispersion and fixing of the light-emitting substance during the process wherein the particles are nucleated and the crystal growth. In <FIG>, a peak caused by an individually dispersed molecule or a two molecule associated state of the FITC light-emitting substance in the vicinity of <NUM> was observed. Because a peak caused by aggregates was not observed, it was thought to interact with the surfactant and to be present in an approximately uniform distribution. In <FIG>, a fluorescence peak caused by a 4f-4f transition of a light-emitting substance Eu(III) ion; and peaks caused by a transition from <NUM>D<NUM> to <NUM>F<NUM> of <NUM>, a transition from <NUM>D<NUM> to <NUM>F<NUM> of <NUM>, a transition from <NUM>D<NUM> to <NUM>F<NUM> of <NUM>, and a transition from <NUM>D<NUM> to <NUM>F<NUM> of <NUM> were observed. There was no change in light-emission spectral shape or intensity due to the final washing process (surfactant elimination process). From this result, it was thought that the surfactant plays an important role in the approximately uniform dispersion and fixing of the light-emitting substance during the process wherein the particles are nucleated and the crystal growth.

For <NUM> mol% samples and <NUM> 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-<NUM> integrating sphere with ϕ <NUM>, 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 I<NUM>, I<NUM>, and I<NUM>, and the quantum yield (internal quantum efficiency) Φint was calculated by Formula (<NUM>). Further, concerning the excitation/fluorescence spectra, the maximum wavelength seen in the excitation/fluorescence spectra of each sample was used.

<FIG> is a graph showing the intensity spectra of the incident light, scattered light, and fluorescent light. Further, the results of the measured quantum yield as below. The quantum yield in the case of synthesis using the surfactant was higher than the case of not using the surfactant, and it was shown that the surfactant is important in the approximately uniform dispersion of the light-emitting substance (high efficiency light emission).

Normal cells (NIH3T3 cells) were cultured in a PS flask (dissemination density: <NUM> x <NUM><NUM> cells/<NUM><NUM>) After this, thawing and dissemination were carried out for <NUM> days, and cells were ablated and separated. The density of the NIH3T3 cells was (<NUM> ± <NUM>) × <NUM><NUM> cells/mL. Concentration adjustment of the cells was carried out, and <NUM> vol% FBS (fetal bovine serum) was cultured in DMEM (Dulbecco modification nutrient medium). There were <NUM> × <NUM><NUM> cells per <NUM>. An amount of <NUM>/well was disseminated to a <NUM> well plate (culture area: <NUM><NUM>/well). The disseminated concentration was <NUM> × <NUM><NUM> cells/cm<NUM>. After this, it was cultured (temperature: <NUM>, CO<NUM> concentration: <NUM>%, humidity <NUM>%) After <NUM> hours, FA-Ef:NPS particles were added to <NUM> vol% DMEM, dispersed, and a concentration of <NUM>/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 [<NUM>-(<NUM>,<NUM>-dimethylthiazol-<NUM>-yl)-<NUM>,<NUM>-diphenyltetrazolium bromide] by mitochondrial dehydrogenase inside a cell is extracted by an organic solvent, the absorbance at <NUM> is measured, and the viable cell rate is measured. At <NUM> hours, <NUM> hours, and <NUM> hours after dissemination, <NUM>µL of MTT reagent (Cat. No. <NUM>) was added, and cultured for <NUM> hours (temperature: <NUM>, CO<NUM> concentration: <NUM>%, humidity: <NUM>%). After this, the culture medium was removed, <NUM> of a crystal dissolving solution (Cat. No. <NUM>) was added, and stirred (variable mode, <NUM>. The absorbency at <NUM> 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).

<FIG> is a graph showing the results of cytotoxic quantification, (a) light-emitting nanoparticles which are not modified by folic acid bonding to cells, (b) light-emitting nanoparticles which are modified by folic acid bonding to cells. As shown in <FIG>, in all of the folic acid nonmodified (before folic acid modification) particles, the particle additive-free samples, namely, normal cell growth characteristics induced tissue culture polystyrene only same growth characteristics were seen. As shown in <FIG>, in all of the particles after folic acid modification, growth characteristics similar to those of folic acid (FA) only, which does not harm the cell proliferation properties, were seen. As above, the particles of the present embodiment show normal growth characteristics without giving toxicity to the cells.

Hela cancer cells were cultured in a PS flask (dissemination density: <NUM> × <NUM><NUM> cells/<NUM><NUM>). Thawing and dissemination were carried out for <NUM> days. The cells were ablated and separated. The Hela concentration was (<NUM> ± <NUM>) × <NUM><NUM> cells/mL. Concentration adjustment of the cells was carried out, and cultured in DMEM (Dulbecco modification nutrient medium) <NUM> vol% FBS (fetal bovine serum). There were <NUM> × <NUM><NUM> cells per <NUM>. An amount of <NUM>/PS was disseminated to PS dishes (cultivation area: <NUM><NUM>), and the dissemination density was <NUM> × <NUM><NUM> cells/cm<NUM>.

After this, culturing was performed (temperature: <NUM>, CO<NUM> concentration: <NUM>%, humidity <NUM>%). After <NUM> hours, FA-Eu:NPS particles were added to <NUM> vol% DMEM, dispersed, and the concentration was adjusted to <NUM>/mL.

For living cell imaging, after <NUM> hours, <NUM> hours and <NUM> hours from spraying the particles on the cell surface, the nutrient medium was removed. After this, <NUM> PBS (phosphate buffered saline) was added, and removed (one time). Further, <NUM> of distilled water was added, and removed (one time). Fluorescence intensity measurements were carried out.

Fluorescence microscope observation was carried out only after <NUM> 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".

<FIG> is, for Eu<NUM>+-containing silica particles differing by having or not having modification of cell bonding molecules, and (a) is a graph showing the relationship between fluorescent light intensity and culture time, and (b) and (c) are fluorescence imaging images of cells which have taken up the particles. As shown in <FIG>, Eu<NUM>+-containing silica particles having modification of cell bonding molecules showed a larger increase of fluorescence intensity with respect to culture time than Eu<NUM>+-containing silica particles not having modification of cell bonding molecules, and after <NUM> hours showed approximately five times the fluorescence intensity. As shown in <FIG>, in the case of Eu<NUM>+-containing silica particles not having modification of cell bonding molecules live cell imaging was not possible, but for Eu<NUM>+-containing silica particles having modification of cell bonding molecules, live cell imaging became possible (<FIG>). Further, these results show that Eu<NUM>+-containing silica particles are provided with excellent light-emitting stability and light resistance.

<FIG> is, for FITC-containing titania particles differing by having or not having modification of cell bonding molecules, and (a) is a graph showing the relationship between fluorescent light intensity and culture time, and (b) and (c) are fluorescence imaging images of cells which have taken up the particles. As shown in <FIG>, FITC-containing silica particles having modification of cell bonding molecules showed a larger increase of fluorescence intensity with respect to culture time than FITC-containing silica particles not having modification of cell bonding molecules, and after <NUM> hours showed approximately five times the fluorescence intensity. As shown in <FIG>, in the case of FITC-containing silica particles not having modification of cell bonding molecules live cell imaging was not possible, but for FITC-containing silica particles having modification of cell bonding molecules, live cell imaging became possible (<FIG>). Further, these results show that FITC-containing silica particles are provided with excellent light-emitting stability and light resistance.

<FIG> is, for Eu<NUM>+-containing calcium phosphate compound particles differing in having or not having modification of cell bonding molecules, and (a) is a graph showing the relationship between fluorescent light intensity and culture time, and (b) and (c) are fluorescence imaging images of cells which have taken up the particles. As shown in <FIG>, Eu<NUM>+-containing calcium phosphate compound particles having modification of cell bonding molecules showed a larger increase of fluorescence intensity with respect to culture time than Eu<NUM>+-containing calcium phosphate compound particles not having modification of cell bonding molecules, and after <NUM> hours showed approximately four times the fluorescence intensity. As shown in <FIG>, in the case of Eu<NUM>+-containing calcium phosphate compound particles not having modification of cell bonding molecules live cell imaging was not possible, but for Eu<NUM>+-containing calcium phosphate compound particles having modification of cell bonding molecules, live cell imaging became possible (<FIG>). Further, these results show that Eu<NUM>+-containing calcium phosphate compound particles are provided with excellent light-emitting stability and light resistance.

The main measurement device used in the present examples was as follows.

Claim 1:
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 surfactant molecule, and at least one anionic element selected from the group consisting of O and P, preferably comprising at least one selected from the group consisting of TiO<NUM>, Ca<NUM>(PO<NUM>)<NUM>(OH)<NUM>, Al<NUM>O<NUM>, and ZrO<NUM>.