Patent Application
20230142721
The present invention relates to a composite of a silicate-based base material and a rare-earth compound, a method for producing a composite of a silicate-based base material and a rare-earth compound, light-emitting nanoparticles, a cell detection method, an animal treating method, and a medical device.
In recent years, there has been a demand for a technique of detecting tumors such as cancer cells or the like with high sensitivity at a cellular level by a marker material having high biocompatibility.
Patent Document 1 discloses, as a bioimaging material, a two-photon absorbing material consisting of a water soluble dendrimer in which a dye having a two-photon absorbing property and a fluorescence property and a dendron are bonded.
Patent Document 2 discloses use of a water-dispersible quantum dot in which a surface of the quantum dot is coated with a surfactant-type polymerization initiator including 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 including 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 having 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 as a phenomenon of obtaining visible light fluorescence with the use of a low energy light such as an infrared light or the like as an excitation light, 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+, and Yb3+.
Patent Document 5 discloses a semiconductor nanoparticle to be used for molecule/cell imaging, which is a semiconductor nanoparticle having an average particle size of 1 to 20 nm, containing an atom pair of a main component atom constituting the same, and an atom pair of the corresponding differing atom or a differing atom having an equivalent valence electron arrangement, and further the dopant distributed on the semiconductor nanoparticle surface or in the vicinity thereof.
Patent Document 6 discloses a fluorescent labeling agent for pathological diagnosis including fluorescent material encapsulated nanoparticles including a first fluorescent substance, and a second fluorescent substance having a distinguishable excitation/light-emitting property from the first fluorescent substance.
Patent Document 7 discloses a fluorescent labeling agent including a rare-earth fluorescent complex-containing silica particle containing rare-earth fluorescent complex, and a target molecule measurement kit.
Furthermore, Non-Patent Documents 1 to 4 also disclose use of quantum dots, fluorescent dyes, or the like, for bioimaging.
As described above, in recent years, there has been a demand for a technique of detecting tumors such as cancer cells with high sensitivity at the cellular level by a marker material having high biocompatibility. For example, in the cancerization of a cell, before a morphological change occurs, an activity change at the molecular level occurs. For example, a cancer cell tends to consume a large amount of glucose compared with a normal cell. At the same time, folate receptors are overexpressed on the cell membrane, resulting in tendency of specifically binding to / taking up folic acid molecules. High sensitivity imaging of such changes at the molecular level of a cell would make it possible to realize very early stage detection of cancer cells and the like.
However, in the case of using an organic molecule as an imaging material for imaging, 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. Furthermore, in the case of using an inorganic material such as quantum dots as a bioimaging material, highly toxic elements such as cadmium may be included, and there have been problems with biocompatibility and the like.
Thus, the present inventors created a composite particle wherein a light-emitting molecule or ion is included in an inorganic material, and developed a light-emitting nanoparticle provided with light emitting stability and light resistance, and having low biological toxicity (Patent Document 8).
In order to achieve bioimaging with high sensitivity, light-emitting intensity of light-emitting particles is desirably high. However, in Patent Document 8, light-emitting molecules/ions may agglomerate, or the amount of light-emitting molecules is small and the light-emitting intensity may be degraded. Note here that high light-emitting intensity is similarly demanded for applications other than bioimaging, and the problem that when light-emitting molecules/ions agglomerate or the amount of light-emitting substance is small, the light-emitting intensity may be degraded similarly occurs in applications other than bioimaging.
The present invention has been made in view of the above-mentioned problem, and has an object to provide a composite of a silicate-based base material and a rare-earth compound, having high light-emitting intensity and capable of being used as light-emitting particles, and light-emitting nanoparticle including the same, a cell detection method, a method for treating an animal, a medical device, and a method for producing the composite of a silicate-based base material and a rare-earth compound.
The present inventors have completed the present invention based on the finding that when a composite of a silicate-based base material and a rare-earth compound includes the silicate-based base material containing elemental silicon (Si) and elemental oxygen (O) and the rare-earth compound, the rare-earth compound includes at least one selected from a chloride of a rare-earth element and a fluoride of a rare-earth element, and the silicate-based base material has a solid 29Si-NMR spectrum satisfying Q4/Q3 of 1.6 to 3.9 where Q4 represents a peak area derived from Si(OSi)4 and Q3 represents a peak area derived from HO—Si(OSi)3, an amount of a light-emitting rare-earth element such as Eu is sufficient, agglomeration of light-emitting rare-earth ions is suppressed, and the light-emitting intensity is high. In other words, the present invention includes the following configurations.
[1] A composite of a silicate-based base material and a rare-earth compound, the silicate-based base material containing elemental silicon (Si) and elemental oxygen (O), in which the rare-earth compound includes at least one selected from a chloride of a rare-earth element and a fluoride of a rare-earth element, and
the silicate-based base material has a solid 29Si-NMR spectrum satisfying Q4/Q3 of 1.6 to 3.9 where Q4 represents a peak area derived from Si(OSi)4 and Q3 represents a peak area derived from HO—Si(OSi)3.
[2] The composite of a silicate-based base material and a rare-earth compound as described in the above [1], in which the rare-earth element is at least one selected from europium (Eu) and terbium (Tb).
[3] The composite of a silicate-based base material and a rare-earth compound as described in the above [2], in which the rare-earth compound includes at least one compound selected from compounds represented by the following chemical formulae (1) to (4):
(in the formula (1), x is 0.05 or more and 5 or less).
[4] The composite of a silicate-based base material and a rare-earth compound as described in the above [3], in which the rare-earth compound has a powder X-ray diffraction pattern having a first diffraction peak in a diffraction angle (2θ) range of 34.3° to 36.1° in which a half value width of the first diffraction peak is 1.8° or less, and/or having a second diffraction peak in a diffraction angle (2θ) range of 28.6° to 29.6° and a third diffraction peak in a diffraction angle (2θ) range of 36.8° to 38.4° in which a half value width of the second diffraction peak is 1.0° or less and a half value width of the third diffraction peak is 1.6° or less.
[5] The composite of a silicate-based base material and a rare-earth compound as described in the above [2], in which the rare-earth compound includes a compound represented by the following chemical formula (5) or a compound represented by the following chemical formula (6), and a mixed crystal of at least one compound selected from the compound represented by the chemical formula (5) and the compound represented by the chemical formula (6) and amorphous silica.
[6] The composite of a silicate-based base material and a rare-earth compound as described in the above [5], in which the rare-earth compound has a powder X-ray diffraction pattern having a fourth diffraction peak in a diffraction angle (2θ) range of 26.6° to 28.6°, a fifth diffraction peak in a diffraction angle (2θ) range of 44.8° to 46.8°, and a sixth diffraction peak in a diffraction angle (2θ) range of 24.3° to 26.3° in which a half value width of the fourth diffraction peak is 0.3° or less, a half value width of the fifth diffraction peak is 0.57° or less, and a half value width of the sixth diffraction peak is 0.22° or less, and/or having the fourth diffraction peak, the fifth diffraction peak, and a seventh diffraction peak in a diffraction angle (2θ) range of 30.8° to 32.8° in which a half value width of the fourth diffraction peak is 0.3° or less, a half value width of the fifth diffraction peak is 0.57° or less, and a half value width of the seventh diffraction peak is 0.38° or less.
[7] A composite of a silicate-based base material and a rare-earth compound as described in any one of the above [1] to [6], in which the rare-earth element is on a surface of the silicate-based base material.
[8] A composite of a silicate-based base material and a rare-earth compound as described in any one of the above [1] to [7], including a molecule containing elemental carbon on a surface thereof.
[9] The composite of a silicate-based base material and a rare-earth compound as described in any one of the above [1] to [8], in which a percentage of a number of moles of the rare-earth element with respect to a total number of moles of the elemental silicon and the rare-earth element is 0.1 mol% or more and 7 mol% or less.
[10] The composite of a silicate-based base material and a rare-earth compound as described in any one of the above [1] to [9], in which the silicate-based base material is a base material including silica or a silicate.
[11] The composite of a silicate-based base material and a rare-earth compound as described in any one of the above [1] to [10], in which the silicate-based base material is an amorphous.
[12] The composite of a silicate-based base material and a rare-earth compound as described in any one of the above [1] to [11], in which the silicate-based base material is powder having an average particle diameter of 50 nm or more and 470 nm or less.
[13] A light-emitting nanoparticle including a composite of the silicate-based base material and the rare-earth compound as described in the above [12], the rare-earth compound being a light-emitting substance.
[14] The light-emitting nanoparticle as described in the above [13] being used for bioimaging.
[15] A cell detection method, including allowing the light-emitting nanoparticle as described in the above [13] to be taken into a cell, irradiating the light-emitting nanoparticle with light, and observing the cell.
[16] A treating method for treating a non-human animal, the method including administering the light-emitting nanoparticle as described in the above [13] to the animal, irradiating the light-emitting nanoparticle with light, and treating the animal.
[17] A medical device including a testing portion for carrying out testing of an internal cell, a diagnosis portion for carrying out diagnosis of the internal cell, and/or a treatment portion
[18] A method for producing the composite of a silicate-based base material and a rare-earth compound as described in any one of the above [1] to [12], the method including:
[19] A method for producing the composite of a silicate-based base material and a rare-earth compound as described in any one of the above [1] to [12], the method including:
[20] A mixed crystal including at least one compound selected from a compound represented by the following chemical formula (5) and a compound represented by the following chemical formula (6), and amorphous silica, the mixed crystal having a powder X-ray diffraction pattern having a fourth diffraction peak in a diffraction angle (2θ) range of 26.6° to 28.6°, a fifth diffraction peak in a diffraction angle (2θ) range of 44.8° to 46.8°, and a sixth diffraction peak in a diffraction angle (2θ) range of 24.3° to 26.3°, in which a half value width of the fourth diffraction peak is 0.3° or less, a half value width of the fifth diffraction peak is 0.57° or less, and a half value width of the sixth diffraction peak is 0.22° or less, and/or having the fourth diffraction peak, the fifth diffraction peak, and a seventh diffraction peak in a diffraction angle (2θ) range of 30.8° to 32.8°, in which a half value width of the fourth diffraction peak is 0.3° or less, a half value width of the fifth diffraction peak is 0.57° or less, and a half value width of the seventh diffraction peak is 0.38° or less.
The present invention can provide a composite of a silicate-based base material and a rare-earth compound having high light-emitting intensity and being usable as a light-emitting particle, a light-emitting nanoparticle using the same, a cell detection method, a method for treating an animal, a medical device, and a method for producing a composite of a silicate-based base material and a rare-earth compound.
Hereinafter, the present invention will be described in more detail.
A composite of a silicate-based base material and a rare-earth compound of the present invention includes a silicate-based base material containing elemental silicon (Si) and elemental oxygen (O), and a rare-earth compound. The rare-earth compound includes at least one selected from chloride of a rare-earth element and fluoride of a rare-earth element. Furthermore, the silicate-based base material has a solid 29Si-NMR spectrum satisfying Q4/Q3 of 1.6 to 3.9 where Q4 represents a peak area derived from Si(OSi)4 and Q3 represents a peak area derived from HO—Si(OSi)3.
In this way, a composite of a specific silicate-based base material and a rare-earth compound including chloride of the rare-earth element or fluoride of the rare-earth element, as shown in the below-mentioned Examples, agglomeration of rare-earth element ions as light-emitting substance is suppressed and a supporting amount of the rare-earth element into the silicate-based base material becomes appropriate, fluorescence intensity (light-emitting intensity) becomes high. On the other hand, when Q4/Q3 is less than 1.6, since the supporting amount of the rare-earth element is large, agglomeration occurs, and fluorescence intensity is deteriorated. Furthermore, when Q4/Q3 is more than 3.9, since the supporting amount of the compound of the rare-earth element is small, the fluorescence intensity is deteriorated.
The “composite of a silicate-based base material and a rare-earth compound” is not a simple mixture obtained by mixing a silicate-based base material and a rare-earth compound, but is a structure body having a rare-earth compound, for example, on a surface of the silicate-based base material, and a structure body in which a silanol group of a silicate-based base material and an oxygen atom of a siloxane bond, and rare-earth ions constituting the rare-earth compound are chemically bonded one another by, for example, a coordination bond.
The rare-earth ions constituting the rare-earth compound are not particularly limited but are 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, trivalent Lu, and trivalent Tb. Any of these are a light-emitting substance. Among these, Eu3+ that is trivalent Eu is preferable. This is preferable because Eu3+ is excited and emits light in the visible light region. For example, a crystal of a europium compound excites and emits light in a visible light region, and the excitation wavelength λex is, for example, 395 nm and 464 nm, and the fluorescence wavelength λem is, for example, 615 nm.
The rare-earth compound may be an amorphous but is preferably a crystal. When the rare-earth compound is a crystal, as compared with an amorphous, elusion of ions such as a chloride ion and a fluoride ion does not easily occur, so that an adverse effect by the elusion of ions is suppressed. Therefore, cytotoxicity is low, and can be preferably used for bioimaging.
The silicate-based base material contains elemental silicon (Si) and elemental oxygen (O). The molar ratio (O/Si) that is a ratio of O to Si in the silicate-based base material, is preferably 2.0 to 2.2. The silicate-based base material is a material as a skeleton of the composite of the silicate-based base material and the rare-earth compound. Examples of the silicate-based base material include silicon oxide such as silica, a base material including silicate. These may be a crystal but is preferably an amorphous from the viewpoint of biotoxicity.
Furthermore, the silicate-based base material has a solid 29Si-NMR spectrum satisfying Q4/Q3 of 1.6 to 3.9 where Q4 represents a peak area derived from Si(OSi)4 and Q3 represents a peak area derived from HO—Si(OSi)3. The Q4/Q3 is preferably 2.0 to 3.9, and more preferably 2.2 to 2.6.
Such a silicate-based base material containing elemental silicon (Si) and elemental oxygen (O) and satisfying Q4/Q3 of 1.6 to 3.9 can be obtained as a particulate substance (a soot body or a by-product) generated in a method for producing silica glass by a conventionally known soot method (for example, a production method of silica glass by Vapor phase Axial Deposition (VAD)). For example, when silica glass is produced as porous synthetic quartz glass (soot body) by hydrolyzing silicon tetrachloride in an oxygen-hydrogen flame using silicon tetrachloride as a raw material, particles which are hydrolyzed in the oxygen-hydrogen flame and then desorbed from the flame and do not become porous synthetic quartz glass can be the silicate-based base material. By adjusting the above conditions, the above silicate-based base material is obtained. For example, silicon tetrachloride as a raw material is introduced into the central portion of the oxygen-hydrogen flame burner to adjust a flame temperature zone length and gas balance of oxygen-hydrogen gas. Specifically, nucleation and grain growth are adjusted, for example, in a range of the flame temperature zone length at 1000° C. or more: 100 mm or more and 800 mm or less, and volume ratio of hydrogen to oxygen (H2/O2): 1.0 or more and 2.5 or less. As the flame temperature zone length is increased, Q4/Q3 tends to increase, and as H2/O2 increases, Q4/Q3 tends to decrease.
The shape of the silicate-based base material is not particularly limited. The shape may be, for example, a spherical shape (powdery) or may be a plate shape, but the shape is preferably spherical shape because the cytotoxicity is low. Also, the size of the silicate-based base material is not particularly limited. When the composite of the silicate-based base material and the rare-earth compound is used for bioimaging, from the viewpoint of cytotoxicity, it is preferable that the silicate-based base material has a spherical shape and an average particle size of 50 nm or more and 470 nm or less and is amorphous.
When the silicate-based base material has a spherical shape, as shown in a schematic sectional view of
In the composite, a percentage of the number of moles of the rare-earth element with respect to a total number of moles of the elemental silicon and the rare-earth element (number of moles of rare-earth element / (number of moles of rare-earth element + number of moles of Si)) is preferably 0.1 mol% or more and 7 mol% or less, and more preferably 4 mol% or more and 7 mol% or less.
When the rare-earth compound includes the chloride of the rare-earth element, the rare-earth compound includes, for example, compounds represented by the following chemical formulae (1) to (4).
(In the formula (1), x is 0.05 or more and 5 or less.)
Furthermore, it is preferable that the compounds represented by the above chemical formulae (1) to (4) have a powder X-ray diffraction pattern having a first diffraction peak in a diffraction angle (2θ) range of 34.3° to 36.1° in which a half value width (half width) of the first diffraction peak is 1.8° or less, and/or having a second diffraction peak in a diffraction angle (2θ) range of 28.6° to 29.6° and a third diffraction peak in a diffraction angle (2θ) range of 36.8° to 38.4° in which a half value width of the second diffraction peak is 1.0° or less and a half value width of the third diffraction peak is 1.6° or less. The compounds represented by the chemical formulae (1) to (4) may have a first diffraction peak in a diffraction angle (2θ) range of 34.3° to 36.1° in which a half value width of the first diffraction peak is 1.8° or less; may have a second diffraction peak in a diffraction angle (2θ) range of 28.6° to 29.6°and a third diffraction peak in a diffraction angle (2θ) range of 36.8° to 38.4° in which a half value width of the second diffraction peak is 1.0° or less and a half value width of the third diffraction peak is 1.6° or less; and also may have a first diffraction peak in a diffraction angle (2θ) range of 34.3° to 36.1° in which a half value width of the first diffraction peak is 1.8° or less, a second diffraction peak in a diffraction angle (2θ) range of 28.6° to 29.6° and a third diffraction peak in a diffraction angle (2θ) range of 36.8° to 38.4° in which a half value width of the second diffraction peak is 1.0° or less and a half value width of the third diffraction peak is 1.6° or less. The half value width of the first diffraction peak is preferably 1.1° or less. The half value width of the second diffraction peak is preferably 0.6° or less. The half value width of the third diffraction peak is preferably 1.0° or less. The first diffraction peak is derived from a crystal of the compound represented by the chemical formula (1) or (2). The second diffraction peak and the third diffraction peak are derived from a crystal of the compound represented by the chemical formula (3) or (4). In the chemical formula (1), x is preferably 0.2 or more and 0.6 or less.
The description “having a diffraction peak in a diffraction angle (2θ) range of a° to b°” means that a peak top position of the diffraction peak (a diffraction peak top position) is within a range of a° to b°. Therefore, for example, in a broad peak, all of the peaks from an end portion to another end portion are not necessarily required to be included within a range of a° to b°.
Furthermore, the crystal bodies of the compounds represented by the chemical formulae (1) to (4) may have a different peak other than the above specific diffraction peak. For example, the compound represented by the chemical formula (1) or (2) may also have a diffraction peak in a diffraction angle (2θ) range of 25.9° to 26.5° and a diffraction peak in a diffraction angle (2θ) range of 31.6° to 32.2°. The diffraction peak in a diffraction angle (2θ) range of 25.9° to 26.5° and the diffraction peak in a diffraction angle (2θ) range of 31.6° to 32.2°respectively have preferably a half value width of 0.6° or less, and further preferably 0.4° or less. Furthermore, the crystal bodies of the compound represented by the chemical formula (3) or (4) may also have a diffraction peak in a diffraction angle (2θ) range of 39.0° to 40.2°. The half value width of the diffraction peak in a diffraction angle (2θ) range of 39.0° to 40.2° is preferably 1.2° or less, and further preferably 0.8° or less.
Conventionally, crystal bodies of the compounds represented by the above chemical formulae (1) to (4) have not been known. However, as will be described in detail later, when a specific silicate-based base material and europium chloride (III) hexahydrate are subjected to a solid-phase mechanochemical reaction under specific conditions, crystals of compounds represented by chemical formulae (1) to (4) having the specific diffraction peaks can be obtained. Note here that the “crystal” means a substance not being amorphous, and a substance having crystallinity represented by the following formula (7) of more than 0. The crystallinity is preferably more than 0.10.
Crystallinity = {crystal diffraction peak area / amorphous halo diffraction peak area} (7) (In the formula (7), the “crystal diffraction peak area” is a sum of the areas of the diffraction peaks derived from the crystal in 2θ = 20° to 55°; the “amorphous halo diffraction peak area” is a value obtained by subtracting the crystal diffraction peak area from the sum of the areas of all the diffraction peaks observed in 2θ = 20° to 55°.)
When the rare-earth compound includes a fluoride of a rare-earth element, the rare-earth compound includes, for example, a compound represented by chemical formula (5) or (6). A mixed crystal of at least one compound selected from compounds represented by chemical formulae (5) and (6) and silica derived from a silicate-based base material may be used.
Furthermore, it is preferable that the compound represented by the above chemical formula (5) or (6) has a powder X-ray diffraction pattern having a fourth diffraction peak in a diffraction angle (2θ) range of 26.6° to 28.6°, a fifth diffraction peak in a diffraction angle (2θ) range of 44.8° to 46.8°, and sixth diffraction peak in a diffraction angle (2θ) range of 24.3° to 26.3, in which a half value width of the fourth diffraction peak is 0.3° or less, a half value width of the fifth diffraction peak is 0.57° or less, a half value width of the sixth diffraction peak is 0.22° or less, and/or and having the fourth diffraction peak, the fifth diffraction peak, and a seventh diffraction peak in a the diffraction angle (2θ) range of 30.8° to 32.8°, in which a half value width of the fourth diffraction peak is 0.3° or less, a half value width of the fifth diffraction peak is 0.57° or less, and a half value width of the seventh diffraction peak is 0.38° or less. The fourth diffraction peak, the fifth diffraction peak, and the seventh diffraction peak are derived from the crystal of the compound represented by the formula (5). Furthermore, the fourth diffraction peak, the fifth diffraction peak, and the sixth diffraction peak are derived from the crystal of the compound represented by the formula (6).
The rare-earth compound may be, for example, terbium chloride (TbCl3), and terbium fluoride (TbF3).
Note here that in the present invention, the rare-earth compound may have a different peak other than the above-specified diffraction peak.
The composite of the silicate-based base material and the rare-earth compound of the present invention preferably includes a molecule containing a carbon element on a surface thereof. The amount of molecule containing a carbon element is not particularly limited, but, for example, the percentage of the number of moles of carbon atoms with respect to the number of moles of silicon atoms included in the composite of the silicate-based base material and the rare-earth compound (number of moles of C / number of moles of Si) is preferably 0.05 mol% to 160 mol%, and more preferably 6 mol% to 9 mol%.
A composite of the silicate-based base material and the rare-earth compound of the present invention can be produced by a production method including mixing a silicate-based base material and a raw material of a rare-earth compound with each other to carry out a solid-phase mechanochemical reaction, in which the silicate-based base material has a solid 29Si-NMR spectrum satisfying Q4/Q3 of 1.6 to 3.9 where Q4 represents a peak area derived from Si(OSi)4 and Q3 represents a peak area derived from HO—Si(OSi)3.
The silicate-based base material is described in the above-mentioned <Composite of the silicate-based base material and rare-earth compound>.
Examples of the raw material of the rare-earth compound include europium chloride (III) hexahydrate, europium chloride (III) anhydride in a case where the rare-earth compound is chloride of the rare-earth element and include europium fluoride (III) hexahydrate and europium fluoride (III) anhydride in a case where the rare-earth compound is fluoride of the rare-earth element.
Such a silicate-based base material and a raw material of a rare-earth compound are mixed and subjected to a solid-phase mechanochemical reaction under a load (for example, under a load of 2 N or more and 24 N or less, and preferably 2 N or more and 12 N or less). A method for subjecting a silicate-based base material and a raw material of a rare-earth compound to a solid-phase mechanochemical reaction is not particularly limited. For example, a powdery silicate-based base material and a raw material of a rare-earth compound may be placed in a mortar and pulverized by rotating a pestle while applying a load of 2 N or more and 24 N or less to the pestle. When the above operation is carried out with the mortar mounted on the electronic balance (electronic scale), the load value can be read by the metric indicator of the electronic balance. By increasing the load to be loaded, minute particulate composite is produced. The silicate-based base material and the rare-earth compound are composed (bonded) at a load of 2 N or more, and the rare-earth compound starts to be crystalized at a load of 4 N or more. By increasing the load, crystallinity can be enhanced. Note here that when the load exceeds 12 N, peeling of the rare-earth compound (light-emitting layer) accompanying pulverization tends to progress to cause phase separation and refinement (less than 50 nm). Therefore, the load is preferably 12 N or less. Furthermore, pulverization may be carried out by mounting europium chloride (III) on a surface of the plate-shaped silicate-based base material and moving the pestle while applying a load of 4 N or more and 24 N or less, and preferably 2 N or more and 12 N or less. In this case, by carrying out the above-mentioned operation while a plate-shaped silicate-based base material is mounted on an electronic balance, the load value can be read with a metric indicator of the electronic balance.
The percentage of the silicate-based base material and the raw material of the earth compound is not particularly limited, but the percentage of the number of moles of the rare-earth element with respect to the total number of moles of Si and the rare-earth element (number of moles of rare-earth element) / (number of moles of rare-earth element + number of moles of Si)) is preferably 0.1 mol% or more and 7.0 mol% or less. When europium chloride (III) hexahydrate is used as the raw material of the rare-earth compound, when the amount of Eu with respect to Si is increased, the compound represented by the chemical formula (3) tends to be generated through the compound represented by the chemical formula (4). Furthermore, when europium fluoride (III) hexahydrate is used as the raw material of the rare-earth compound, the compound represented by the chemical formula (5) is generated when the amount of Eu with respect to Si is small, and when the amount of Eu with respect to Si is increased, the compound represented by the chemical formula (5) is generated and at the same time, the compound represented by the chemical formula (6) tends to remain.
In the solid-phase mechanochemical reaction using the silicate-based base material satisfying Q4/Q3 of 2.0 to 3.9, europium chloride (III) hexahydrate is added such that the percentage of the number of moles of the europium element with respect to the total number of moles of elemental silicon and a europium element is 1.0 mol% or more and, and the above reaction is carried out under a load at 4 N or more and 24 N or less. Thus, a composite of the crystal of a europium compound and the silicate-based base material can be produced. The percentage of the number of moles of the europium element with respect to the total number of moles of the elemental silicon and a europium element is preferably 1.0 mol% or more and 7.0 mol% or less.
The europium compound is conventionally difficult to be made separately, but according to the above-mentioned production method, the compounds represented by chemical formulae (1) to (4) can be separated based on the amount of Eu in the solid-phase mechanochemical reaction. Specifically, in a state in which the concentration of europium during a solid-phase mechanochemical reaction is low (in a state in which OH during the reaction is small in amount), the compound represented by the chemical formulae (1) and (2) coexist, and when a multilayer structure starts to be formed according to increase in concentration of europium during reaction, the compound represented by the chemical formula (1) and the compound represented by the chemical formula (2) interact with each other, and tend to be changed into the compounds represented by the chemical formulae (3) and (4). Therefore, the compounds represented by the chemical formulae (1) to (4) can be made separately based on the amount of Eu in the solid-phase mechanochemical reaction.
Furthermore, in the solid-phase mechanochemical reaction using the silicate-based base material satisfying Q4/Q3 of 2.0 to 3.9, europium fluoride is added such that the percentage of the number of moles of the europium element with respect to the total number of moles of elemental silicon and a europium element is 1.0 mol% or more and, and the above reaction is carried out under a load at 4 N or more and 24 N or less. Thus, a composite of the crystal of a europium compound and the silicate-based base material can be produced.
A solid-phase mechanochemical reaction is carried out, and then baking is carried out, and then washing with organic solvent or water is carried out as necessary.
With this production method, the rare-earth compound is formed on a surface of the silicate-based base material. For example, when the spherical-shaped silicate-based base material is used, a composite having a rare-earth compound covering at least a part of the surface of the spherical-shaped silicate-based base material is obtained. It is inferred that the rare-earth compound strongly binds to the silicate-based base material by some kind of chemical bonds such as a coordination bond between oxygen atoms existing on the surface of the silicate-based base material and the rare-earth atoms contained in the rare-earth compound.
The above-mentioned composite of the silicate-based base material and the rare-earth compound can be produced also by the gas phase method. Specifically, the composite of the silicate-based base material and the rare-earth compound can be produced by a production method including a step of dissolving the surface of the silicate-based base material in an basic atmosphere, and a step of reacting the silicate-based base material having a surface dissolved with the raw material of the rare-earth compound, in which the silicate-based base material has a solid 29Si-NMR spectrum satisfying Q4/Q3 of 1.6 to 3.9 where Q4 represents a peak area derived from Si(OSi)4 and Q3 represents a peak area derived from HO—Si(OSi)3.
Firstly, the surface of a silicate-based base material is dissolved in a basic atmosphere. The reactivity with the raw material of the rare-earth compound can be enhanced by dissolving the surface of the silicate-based base material in a basic atmosphere. Examples of the basic atmosphere include an atmosphere including ammonia vapor. The silicate-based base material is similar to that described for the solid phase mechanical method. In the case where the silicate-based base material is spherical (powdery), the surface of the silicate-based base material may be dissolved by mixing and pulverizing the silicate-based base material and the raw material of the rare-earth compound and placing the obtained mixed and pulverized product in a basic atmosphere.
Next, a silicate-based base material having a surface that has been dissolved is allowed to react with the raw material of the rare-earth compound. Thus, the silicate-based base material such as silica that has been dissolved is deposited again and Eu3+ coprecipitates. Thereby, a crystal of the rare-earth compound and NH4Cl is formed on the surface of the silicate-based base material. The raw material of the rare-earth compound is the same as those described for the solid phase mechanical method. After the reaction is carried out by a gas phase method, baking is carried out, and washing with organic solvent or water may be carried out as necessary.
Note here that as described above, without using a surfactant, a composite in which agglomeration of the rare-earth ions is suppressed can be produced.
A composite of a silicate-based base material and a rare-earth compound of the present invention has low cytotoxicity, and has the rare-earth compound formed on the surface of the silicate-based base material functioning as a light-emitting substance, so that the composite can be preferably used for bioimaging technique. For example, the composite of the silicate-based base material and the rare-earth compound of the present invention can be used as the light-emitting nanoparticle for the bioimaging technique.
The average particle size of the light-emitting nanoparticle is preferably 50 nm to 470 nm. By making the average particle size within this range, it becomes easy for the particles to be taken up by the target cells, and also tends to be suitable in observing the cells. On the other hand, it is not preferable that the average particle size is small because there is a tendency to cause toxicity problems by acting on the active function of the cell. The average particle size of the light-emitting nanoparticle is preferably 100 nm to 400 nm. Note here that in the case where the light-emitting nanoparticles are not used, for example, for cell imaging, the average particle size may be larger than 470 nm.
The light-emitting nanoparticle to be used for bioimaging technique preferably includes a hydroxyl group (OH group) on the surface thereof. Also preferably, the surface of the light-emitting nanoparticle is modified by an amino group, and, for example, may also be formed using a silane coupling agent containing an amino group. When silicate-based base material has pores, the OH group and the amino group may be on the inner surface of the pores, but preferably on the outer surface of the pores. When the OH group or the amino group is fixed by a hydrogen bond or a covalent bond by a condensation polymerization to the cell binding molecule, and the surface of the light-emitting nanoparticle is modified by the cell binding molecule, the cell binding molecule can specifically bind to a cancer cell or a normal cell. When the cell binding molecule specifically binds to a cell, the light-emitting nanoparticle is taken up into the cell. Thus, the light-emitting nanoparticle is made to emit light inside the cell, and a cancer cell or the like can be detected.
Examples of the cell binding molecule include an HER2 antibody, antibodies specifically binding to human epidermal growth factor receptor, cancer-specific antibodies, phosphorylation protein antibodies, folic acid, antibodies specifically binding to folic acid receptor β, vascular endothelial cell-specific antibodies, tissue-specific antibodies, transferrin, transferrin-bonding peptide, proteins having affinity to sugar chains, and the like. Among these, folic acid, having a tendency to be taken up by cancer cells, is preferably used as the cell binding molecule. Folic acid receptors are over-expressed on cell membranes for cancer cells, resulting in a tendency of selectively binding to and taking up folic acid molecules.
Furthermore, the surface of the light-emitting nanoparticle may be modified by an anticancer agent molecule. When the anticancer agent molecule specifically binds to the cancer cell, the light-emitting nanoparticle will be taken up into the cell. In this way, the light-emitting nanoparticle inside the cell is allowed to emit light and can detect cancer cells. Further, the anticancer agent is also taken up by the cell, and the anticancer agent molecule can act, and the proliferation of cancer cells can be suppressed.
The cell binding molecule or anticancer agent molecule are preferably modified and fixed to the surface of the light-emitting nanoparticle by a chemical bond. Examples of the chemical bond include a peptide bond (—CO—NH—), a hydrogen bond, or the like.
The excitation wavelength and the light-emission wavelength of the light-emitting nanoparticles are preferably in the visible light region. When 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 due to irradiating with light. Furthermore, light scattering of a sample surface can be reduced, and the observation sensitivity can be increased. Note here that in an application using the light-emitting nanoparticles, when it is not necessary to consider damage to biological tissue and labeling material, the excitation wavelength and the light-emission wavelength are not necessarily in the visible light region.
Use of the light-emitting nanoparticles permits detection of cells, and treatment of animals. Specifically, the cell detection method of the present invention includes introducing the light-emitting nanoparticle into a cell, irradiating the light-emitting nanoparticle with light, and observing the cell. According to this detection method, because the light-emitting nanoparticle of the present embodiment has high fluorescence intensity and high sensitivity, observation of cells can be observed more easily.
The treatment method of an animal of the present invention is a method for treating a non-human animal. The method includes steps of administering the light-emitting nanoparticle to a non-human animal, irradiating light onto the light-emitting nanoparticle, and treating the animal. According to this treatment method, the light-emitting nanoparticle of the present embodiment has high fluorescence intensity and high sensitivity, and further, high biocompatibility, a condition of disease in an animal body can be detected with high sensitivity, and safely. Thus, it becomes possible to suitably treat a disease of a non-human animal.
Furthermore, the medical device of the present invention includes a testing portion for carrying out testing of an internal cell, a diagnosis portion for carrying out diagnosis of the internal cell, and/or a treatment portion for carrying out treatment of the internal cell, and further include a light irradiation portion which introduces the light-emitting nanoparticles into the internal cell, and irradiates the light-emitting nanoparticles with light in carrying out the testing, diagnosis, and/or treatment. Herein, examples of the testing portion for carrying out testing of an internal cell include a fluorescence endoscope for carrying out precision image diagnosis. Furthermore, examples of the diagnosis portion for carrying out diagnosing of an internal cell include a device for carrying out tissue biopsies. Furthermore, examples of the treatment portion for carrying out treatment of an internal cell include a device for extracting a tumor site by endoscopy. Furthermore, examples of the internal cell include cancer cells pertaining to oral cavity cancer, pharyngeal cancer, esophageal cancer, large intestinal cancer, small intestinal cancer, lung cancer, breast cancer, and bladder cancer. According to this medical device, the light-emitting nanoparticle of the present embodiment have high fluorescence intensity and high sensitivity, and high biocompatibility, whereby it becomes possible to carry out testing, diagnosis, and treatment of an internal cell with high sensitivity, and safely.
Furthermore, the silicate-based base material and the rare-earth compound of the present invention can be used for applications other than the bioimaging application, and, for example, applications of light emitting devices such as a light emitting diode are expected.
Hereinafter, the present invention will be described with reference to Examples in more detail, but the present invention is not necessarily limited by these examples.
Silicon tetrachloride as a raw material was subjected to hydrolysis reaction sufficiently in an oxygen-hydrogen flame to produce silica glass as porous synthetic quartz glass (soot body). At this time, particles that did not become porous synthetic quartz glass after hydrolysis in an oxygen-hydrogen flame and desorbed from the flame were used as the silicate-based base material 1. Note here that silicon tetrachloride was introduced from the center portion of the oxygen-hydrogen flame burner, and the length of the flame temperature zone of 1000° C. or more was 400 mm, and the volume ratio (H2/O2) of hydrogen to oxygen was 1.5.
Ultrapure water (22.8 mL), hexadecylammonium chloride (0.10 g), and 2N-sodium hydroxide aqueous solution (0.35 mL) were added to a screw tube, then 50 mg of silicate-based base material 1 was added thereto, and the obtained product was subjected to dispersion treatment by carrying out perform ultrasonic irradiation for 5 minutes. Tetraethoxysilane (0.55 mL) was then added and stirred at 1500 rpm for 48 hours at room temperature. Then, centrifugation (3000 rpm, 20 min) was carried out, washing was carried out twice with ultrapure water, dried at 120° C. for two hours, and baked at 550° C. for six hours. Thereafter, the obtained product was washed with 40 mL of ethanol, and subjected to solid-liquid separation by centrifugation, and the solid phase was dried at 120° C. for two hours to obtain a silicate-based base material 2.
A silicate-based base material 3 was obtained in the same manner as described above (production of the silicate-based base material 2) except that hexadecylammonium chloride and the silicate-based base material 1 were not added and the baking temperature was set at 750° C.
A silicate-based base material 4 was obtained in the same manner as described above (production of the silicate-based base material 2) except that the silicate-based base material 1 was not added. Note here that all of the obtained silicate-based base materials 1 to 4 were spherical-shaped powders.
(Q4/Q3 of silicate-based base material in solid 29Si-NMR spectrum)
For the obtained silicate-based base materials 1 to 4, the solid 29Si-NMR spectrum was measured in the following conditions. The results are shown in
Name of device: Bruker Advance 300 wbs spectrometer (manufactured by BRUKER) Measurement method: DD (Dipolar Decoupling) method
Separation method: Solver function of Microsoft Office 2016 Excel (registered trademark) Used function: Gaussian function (in the formula, A denotes a peak height, B denotes a peak position, and C denotes a half value width)
Peak assignment:
From
For the silicate-based base materials 1 to 4, the powder X-ray diffraction (XRD) patterns were measured. The measurement was carried out using a sample horizontal type X-ray diffractometer (XRD) (Smart Lab manufactured by Rigaku Corporation), under the conditions of X radiation source: CuKα radiation source (λ: 1.5418 Å), output: 40 kV/30 mA, scanning speed: 3.0°/min, sampling width: 0.01°, and measurement mode: continuous. As an example, the result of the silicate-based base material 1 is shown in
The silicate-based base materials 1 to 4 (particle powder) were fixed to a sample stand for observation with carbon paste, and dried. Next, particles were observed under a field emission scanning electron microscope (FE-SEM) (SU8230 manufactured by Hitachi High-Technologies, Ltd.), 100 or more of particle diameters were measured, and the average particle size was calculated. Specifically, for 300 particles, the longer diameter and the shorter diameter of each particle were measured, and “(longer diameter + shorter diameter) / 2” was defined as a particle diameter (particle size) of each particle. An average value of the particle diameter of each particle (a value obtained by dividing the total values of the particle diameter of each particle by number of particles (300)) was defined as an average particle size (Ave.), and coefficient of variance (Cv.) was calculated. As an example, the result of silicate-based base material 1 is shown in
Particles of Example 2 were obtained by carrying out the same operation as in Example 1 except that europium chloride hexahydrate was added such that the percentage of the number of moles of Eu with respect to the total number of moles of Si and Eu was 2.5 mol%.
Particles of Example 3 were obtained by carrying out the same operation as in Example 1 except that europium chloride hexahydrate was added such that the percentage of the number of moles of Eu with respect to the total number of moles of Si and Eu was 1.25 mol%.
Particles of Example 4 were obtained by carrying out the same operation as in Example 1 except that the europium chloride hexahydrate was added such that the percentage of the number of moles of Eu with respect to the total number of moles of Si and Eu was 0.625 mol%.
Particles of Example 5 were obtained by carrying out the same operation as in Example 1 except that the silicate-based base material 2 was used instead of the silicate-based base material 1.
Europium chloride hexahydrate was used as particles for Comparative Example 1.
Particles of Comparative Example 2 were obtained by carrying out the same operation as in Example 1 except that the silicate-based base material 3 was used instead of the silicate-based base material 1.
Particles of Comparative Example 3 were obtained by carrying out the same operation as in Example 1 except that the silicate-based base material 4 was used instead of the silicate-based base material 1.
Particles of Examples 1 to 5 and Comparative Examples 1 to 3 were subjected to measurement of powder X-ray diffraction (XRD) pattern in the following conditions. Measurement was carried out using the sample horizontal type X-ray diffractometer (XRD, Smart Lab manufactured by Rigaku Corporation) under conditions of X radiation source: CuKα radiation source (λ: 1.5418 Å), output: 40 kV/30 mA, scanning speed: 3.0°/min, sampling width: 0.01°, and measurement mode: continuous. The diffraction peak position, diffraction angle, and half-value width were obtained by software (manufactured by Rigaku Corporation, software name: PDXL) attached to the device. The diffraction peaks were detected by the automatic profiling process of PDXL, by the second-order differential method (a method of detecting a region in which the peak second-order differential is negative (convex upward) as a peak), by removing the background, removing the Kα2 line, and smoothing in this order, and fitting with the B-spline function (the split pseudo-Voigt function). The threshold (cut value of the standard deviation) in detecting the diffraction peak was 3.0. The threshold means that the diffraction peak is not regarded as a diffraction peak when the intensity of the diffraction peak is 3.0 times or less of its error. Three points were selected in descending order of the diffraction peak intensity (the height of the diffraction peak) and used for identification of the crystal. Furthermore, the crystallinity was calculated by the above formula (7). The results are shown in Table 1 and
As shown in Table 1 and
Furthermore, Example 1 included a crystal of a compound represented by the chemical formula (3) or (4) having a powder X-ray diffraction pattern including diffraction peaks (second diffraction peak and third diffraction peak) in the diffraction angle (2θ) range of 28.6° to 29.6° and a diffraction angle (2θ) range of 36.8° to 38.4° in which the half-value width of the diffraction peak in the diffraction angle (2θ) range of 28.6° to 29.6° was 1.0° or less and the half-value width of the diffraction peak in the diffraction angle (2θ) range of 36.8° to 38.4° was 1.6° or less. Furthermore, Example 1 had diffraction peaks in the diffraction angle (2θ) range of 39.0° to 40.2° in which the half-value width of the diffraction peak in the diffraction angle (2θ) range of 39.0° to 40.2° was 1.2° or less. Furthermore, as shown in Table 1 and
An excitation spectrum and a fluorescence spectrum of particles of Examples 1 to 5 and Comparative Examples 1 to 3 were measured in the following conditions. As an example, the results of Examples 1 to 3 are shown in
The excitation spectrum was obtained by fixing the detection wavelength (615 nm) with a spectrophotometer (PL, FP-8500 manufactured by JASCO Corporation). The measurement conditions were atmosphere: air, excitation/detection slit size: 2.5 nm/2.5 nm, step width: 1.0 nm, sample weight: 20 mg, shape: pellet.
The sample was irradiated with excitation light from the Xe lamp using a spectrophotometer (PL, FP-8500 manufactured by JASCO Corporation) at room temperature (excitation wavelength: 395 nm), and a PL spectrum (fluorescence spectrum) was obtained. The measurement conditions were atmosphere: air, excitation/detection slit size: 2.5 nm / 2.5 nm, step width: 1.0 nm, sample weight: 20 mg, shape: pellet.
As a result, as shown in
Particles of Examples 1 to 5 and Comparative Examples 1 to 3 were subjected to the X-ray fluorescence analysis (XRF) in the following conditions. A sample pellet obtained by pressurizing about 100 mg of sample powder which had not been diluted was subjected to XRF analysis using the fluorescent X-ray analyzer (XRF, manufactured by Rigaku Corporation, device name: ZSX PrimusII) and software attached to the device (manufactured by Rigaku Corporation, name of the software: EZ scan program). In measurement, without using a primary filter and a calibration curve, semi-quantitative analysis was carried out using the measurement program attached to the device is used. Analysis results and results converted in element ratios are shown in Tables 2 to 5.
Particles of Examples 3 to 4 were observed under transmission electron microscope (TEM). Firstly, the particles were dispersed in ethanol at a concentration of 0.1 mass%, subjected to ultrasonication for 15 minutes, and dried on a carbon microgrid. Then, the center portion of the particle membrane was evaluated and analyzed under transmission electron microscopy (TEM) (HT 7700, manufactured by Hitachi High-Technologies, Ltd.). As an example of the results, the result of Example 1 is shown in
For the particles of Examples 1 to 4, 100 or more particle diameters were measured using the field emission-type scanning electron microscope (FE-SEM), and an average particle size was calculated. Note here that for 300 particles, the longer diameter and the shorter diameter of each particle were measured, respectively, and “(longer diameter + shorter diameter) / 2” was defined as a particle diameter of each particle. An average value of the particle diameters of each particle (a value obtained by dividing the total values of the particle diameters of each particle by the number of particles (300)) was defined as an average particle size (Ave.). As an example, Example 1 is shown in
Particles of Example 6 were obtained by the same operation as in Example 1 except that the load was made to be 8.0 N.
Particles of Example 7 were obtained by the same operation as in Example 1 except that the load was made to be 12.0 N.
For the particles of Examples 6 to 7, results obtained in the same manner as in <Powder X-ray diffraction (XRD)> mentioned above are shown in Table 6 and
Particles of Examples 1 to 4 or Comparative Example 1 were subjected to toxicity evaluation by cancer cell imaging and a fluorescence intensity measurement by the following methods.
To 250 mg of the particles of Examples 1 to 4 or Comparative Example 1, 12 ml of an HCl aqueous solution (pH = 2) was added, and ultrasonic treatment 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-treated solution to obtain a mixed solution. The mixed solution was stirred for 20 hours at 40° C. (pH < 6.5). After stirring was completed, the mixed solution was centrifuged and washed with ethanol. After washing, drying was carried out under reduced pressure to obtain 150 mg of particles having a surface modified with APTES. To 150 mg of these particles having a surface modified with APTES, 25 mL of a 50 mM phosphate buffer solution (pH = 7.0) was added, and ultrasonic treatment was carried out. Next, a solution containing 430 mg (0.8 mmol) of FA-NHS (folic acid derivative) in 12 mL of dimethylsulfoxide (DMSO) was prepared, and the prepared solution was added to the ultrasonic-treated solution to obtain a mixed solution. The mixed solution was stirred for 3 hours at room temperature. After stirring was completed, the mixed solution was centrifuged and washed with water. After washing, drying was carried out under reduced pressure to obtain FA (folic acid)-modified particles of Examples 1 to 4 or Comparative Example 1.
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 peeled off and separated. The Hela concentration was (0.99 ± 0.07) × 105 cells/mL. Concentration of the cells was adjusted, and 10 vol% FBS (fetal bovine serum) was cultured in DMEM (Dulbecco modification nutrient medium). The obtained cells were 7.5 × 104 cells per 1 mL. An amount of 2.25 mL/TCPS was disseminated to polystyrene dishes (TCPS) (cultivation area: 9.6 cm2), and the dissemination density was 1.8 × 104 cells/cm2. (Microscope observation). Thereafter, culturing was carried out (temperature: 37° C., CO2 concentration: 5%, humidity: 100%). After 12 hours, FA (folic acid)-modified particles of Examples 1 to 4 or Comparative Example 1 were added to 10 vol% DMEM, and dispersed, and the concentration was adjusted to 100 mg/mL.
For living cell imaging, at 3 hours, 12 hours, 24 hours, 36 hours and 48 hours after spraying FA (folic acid)-modified particles on the cell surface, the medium was removed.
After the above cells were cultured, a polystyrene dish (TCPS) including cells was washed twice with 1 ml of phosphate buffered saline (PBS), then FA (folic acid)-modified particles which had not taken up by the cells were removed, 0.1 ml of 0.05% Trypsin-EDTA was placed in a TCPS including cells, followed by allowing to stand still in a CO2 incubator for 12 minutes, and cells were peeled off from TCPS. Peeling was observed, a suspension including cells was taken into a 50-ml conical tube and subjected to centrifugation (2000 rpm, 2 min). After centrifugation, a supernatant was discarded, 7 ml of culture medium was added to the tube, overturning stirring was carried out about 10 times, and then centrifugation (2000 rpm,2 min) was carried out. Thereafter, the supernatant was discarded, 20 ml of medium was added to the tube, pipetting was carried out about 20 times, 1 ml was aliquoted into 15 ml-tubes, the cell suspension was placed into a disposable hemocytometer using a micropipette outside a clean bench, the cell number was confirmed under a microscope, and the average value (cell/cm2) was calculated. At 3 hours, 12 hours, 24 hours, 36 hours, and 48 hours after spraying FA (folic acid)-modified particles on the cell surface, the average value of cell density was calculated, and the results are shown in
Using an integrating sphere (ISF834 manufactured by JASCO Corporation), PL spectrum measurement was carried out by a spectrophotometer (PL; FP-8500 manufactured by JASCO Corporation), under the conditions: atmosphere: air, excitation/detection slit size: 10 nm/10 nm, a step width: 1.0 nm. Specifically, TCPS including cells after cell culture and FA (folic acid)-modified particles was washed with 1 ml of phosphate buffered saline twice, FA (folic acid)-modified particles which had not been taken up by the cells were removed, and further washing with 1 ml of ultrapure water twice. TCPS after washing (i.e., TCPS including cells which had taken up FA (folic acid)-modified particles) were freeze-dried. After drying, the cell layer present in the TCPS was peeled off to form a powder, and the powder was placed into a powder cell holder. Then, the PL spectrum was measured. The excitation wavelength herein was 395 nm, and integrated light-emission intensity centered on the peak top of5D0→7F2 transition was calculated. Specifically, the PL spectrum area integrated light-emission intensity in the wavelength region between 600 to 635 nm was obtained. Note here that light emission from TCPS was not observed in this wavelength region. The results are shown in
As shown in
As shown in
PL spectra of particles of Examples 1 and 5 and Comparative Examples 2 and 3 were measured. A PL spectrum (fluorescence spectrum) was obtained by irradiating a sample with excitation light (excitation wavelength: 394 nm) from the Xe lamp using a spectrophotometer (PL, FP-8500 manufactured by JASCO Corporation) at room temperature under the conditions: atmosphere: air, excitation/detection slit size: 2.5 nm / 2.5 nm, a step width: 1.0 nm, sample mass: 20 mg, shape: pellet. Thereafter, the integrated light-emission intensity centered on the peak top of the 5D0→7F2 transition (left vertical axis in
As shown in
As shown in
A mixed dispersion was obtained by mixing 10 mg of silicate-based base material (silicate-based base material 1), and 1.0 mL of europium chloride (III) aqueous solution having a concentration of 10 g/L, followed by ultrasonication for 2 minutes. Particles of Example 9 were obtained by carrying out the same operation as in Example 8 except that 0.1 mL of the obtained mixed dispersion was used instead of the mixed and pulverized product.
Particles of Example 10 were obtained by carrying out the same operation as in Example 9 except that the amount of mixed dispersion was made to be 1.0 mL.
For particles of Examples 8 to 10, results obtained in the same manner as in the above <Powder X-ray diffraction (XRD)> are shown in Table 7 and
As shown in Table 7 and
For particles of Examples 8 to 10, an X-ray fluorescence analysis (XRF) was carried out as in Example 1. Results are shown in Tables 8 and 9.
Particles of Example 11 were produced by using a production device of
Particles of Example 12 were obtained by carrying out the same operation as in Example 11 except that europium fluoride was added such that the percentage of the number of moles of Eu with respect to the total number of moles of Si and Eu was 2.5 mol%.
Particles of Example 13 were obtained by carrying out the same operation as in Example 11 except that europium fluoride was added such that the percentage of the number of moles of Eu with respect to the total number of moles of Si and Eu was 5.0 mol%.
Europium fluoride was used as a particle of Comparative Example 4.
For the particles of Examples 11 to 13 and Comparative Example 4, results obtained in the same manner as in the <Powder X-ray diffraction (XRD)> mentioned above are shown in Table 10 and
As shown in Table 10 and
For particles of Examples 11 to 13, an excitation spectrum and a fluorescence spectrum were measured in the same manner as in Example 1. As an example of the results, fluorescence spectrum is shown in
As a result, in all of Examples 11 to 13, an excitation peak (395 nm) and a light-emitting peak (613 nm) derived from an Eu(III) ion were observed. Furthermore, for particles of Examples 11 to 13, when internal quantum yields of the particles were obtained where excitation wavelength λex was 395 nm and light-emission wavelength λem was 613 nm, the yield was 1.47 in Example 11, 5.39 in Example 12, and 6.70% in Example 13.
For the particles of Examples 11 to 13 and Comparative Example 4, the X-ray fluorescence analysis (XRF) was carried out in the same manner as in Example 1. Results are shown in Tables 11 and 12. [0149]
[0150]
For particles of Examples 11 to 13, 100 or more particle diameters were measured using the field emission-type scanning electron microscope (FE-SEM), and an average particle size was calculated. Note here that for 300 particles, the longer diameter and the shorter diameter of each particle were measured, respectively, and “(longer diameter + shorter diameter) / 2” was defined as a particle diameter of each particle. An average value of the particle diameter of each particle (a value obtained by dividing the total values of the particle diameter of each particle by number of particles (300)) was defined as an average particle size. As a result, an average particle size of the particles of Example 11 was 140 nm, an average particle size of the particles of Example 12 was 149 nm, and an average particle size of the particles of Example 13 was 151 nm.
Toxicity evaluation was carried out using the particles of Examples 11 to 13 and Comparative Example 4 by the same method as in Example 1. As a result, Examples 11 to 13, in which crystal of a europium compound were formed, exhibited normal cell proliferation behavior and no cytotoxicity. On the other hand, in Comparative Example 4, since fluoride was unstable, this eluted into a cell culture solution, and was formed into Eu ion or Eu fluoride ion and reacted with cells directly, resulting in showing substantially the same level of toxicity as in Comparative Example 1. Note here that for the fluorescence property of the particles which have taken up by the cells, since the crystal did not elute into the cell culture solution, finally, after 48 hours of culture, high fluorescence intensity which was higher than the result of Comparative Example 4 was shown.
As mentioned above, it is shown that when composite particles having a chloride of the rare-earth element or a fluoride of the rare-earth element on a surface of a specific silicate-based base material is used, cells grow well, and the particles can be taken up by the cells and visualized. Since agglomeration of the rare-earth element as a light-emitting substance is suppressed, and the support amount of the rare-earth element is appropriate, the fluorescence intensity is high, and detection can be carried out with high sensitivity.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2020-046605 | Mar 2020 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2021/010645 | 3/16/2021 | WO |
