WO2012128162A1 - 画像診断用シリカナノ粒子、その製造方法、及び生体物質標識剤 - Google Patents
画像診断用シリカナノ粒子、その製造方法、及び生体物質標識剤 Download PDFInfo
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- WO2012128162A1 WO2012128162A1 PCT/JP2012/056623 JP2012056623W WO2012128162A1 WO 2012128162 A1 WO2012128162 A1 WO 2012128162A1 JP 2012056623 W JP2012056623 W JP 2012056623W WO 2012128162 A1 WO2012128162 A1 WO 2012128162A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B67/00—Influencing the physical, e.g. the dyeing or printing properties of dyestuffs without chemical reactions, e.g. by treating with solvents grinding or grinding assistants, coating of pigments or dyes; Process features in the making of dyestuff preparations; Dyestuff preparations of a special physical nature, e.g. tablets, films
- C09B67/0001—Post-treatment of organic pigments or dyes
- C09B67/0004—Coated particulate pigments or dyes
- C09B67/0005—Coated particulate pigments or dyes the pigments being nanoparticles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
- A61K49/0089—Particulate, powder, adsorbate, bead, sphere
- A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
- A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/04—X-ray contrast preparations
- A61K49/0409—Physical forms of mixtures of two different X-ray contrast-enhancing agents, containing at least one X-ray contrast-enhancing agent which is not a halogenated organic compound
- A61K49/0414—Particles, beads, capsules or spheres
- A61K49/0423—Nanoparticles, nanobeads, nanospheres, nanocapsules, i.e. having a size or diameter smaller than 1 micrometer
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B67/00—Influencing the physical, e.g. the dyeing or printing properties of dyestuffs without chemical reactions, e.g. by treating with solvents grinding or grinding assistants, coating of pigments or dyes; Process features in the making of dyestuff preparations; Dyestuff preparations of a special physical nature, e.g. tablets, films
- C09B67/0071—Process features in the making of dyestuff preparations; Dehydrating agents; Dispersing agents; Dustfree compositions
- C09B67/0083—Solutions of dyes
Definitions
- the present invention relates to an imaging diagnostic silica nanoparticle encapsulating an organic fluorescent dye and a metal complex that can be used in X-ray CT, fluorescence imaging, and the like, a method for producing the same, and a biological material labeling agent using the imaging imaging silica nanoparticle .
- X-ray CT X-ray computed tomography
- MRI Magnetic resonance imaging
- the tissue of the lesioned part is surgically collected while confirming the lesion. At that time, since the modality is different between the X-ray CT or MRI and the ultrasonic diagnostic apparatus, it may be difficult to determine the ablation range.
- the accuracy of biopsy can be dramatically improved by making it possible to observe the lesion detected by one method even with imaging that can be used for biopsy at the same time, and it is possible to prevent misdiagnosis due to false negatives. .
- X-ray CT and MRI can obtain anatomical information.
- X-ray CT is based on the detection of differences in X-ray absorption between normal tissue and tumor tissue.
- a labeling agent for X-ray CT a molecule containing an iodine atom, a gold atom, a gadolinium atom or the like is used.
- MRI is based on the detection of the difference between T 1 (spin-lattice relaxation time) and T 2 (spin-spin relaxation time) in tissues close to the MRI contrast agent.
- Paramagnetic ions including gadolinium ions are used as MRI contrast agents.
- Patent Document 1 discloses an MRI contrast agent in which a gadolinium complex is bonded to the surface of silica nanoparticles.
- Fluorescence imaging does not expose patients to ionizing radiation and is therefore always highly acceptable as a diagnostic modality. Fluorescence imaging is based on detecting differences in fluorescence from normal and tumor tissues.
- the fluorescent labeling agent an organic fluorescent dye substance that emits fluorescence having a wavelength different from that of excitation light is excellent.
- the present invention has been made in view of the above problems and situations, and a solution to the problem is to provide silica nanoparticles for diagnostic imaging that can be used in X-ray CT, fluorescence imaging, and the like, and have high emission intensity, and a method for producing the same. It is to be.
- Another object of the present invention is to provide a biological substance labeling agent using the silica nanoparticles for diagnostic imaging.
- a silica nanoparticle for diagnostic imaging that includes an organic fluorescent dye and a metal complex, wherein each of the organic fluorescent dye and the metal complex is linked to the skeleton of the silica nanoparticle through an organic molecule.
- silica nanoparticle for diagnostic imaging according to the item 1, wherein the organic molecule is an organic molecule having a silyl group.
- silica nanoparticle for diagnostic imaging according to item 4, wherein the covalent bond includes an amide bond.
- An imaging diagnostic silica nanoparticle comprising an organic fluorescent dye and a metal complex encapsulated at least through the following steps (a), (b) and (c): Production method.
- Step (b) reacting an organic molecule having an amino group and a silyl group substituted with a hydrolyzable substituent in the same molecule with a gadolinium complex having a functional group that reacts with the amino group
- a biological substance labeling agent wherein the diagnostic imaging silica nanoparticles according to any one of items 1 to 6 and a molecular labeling substance are bonded via an organic molecule.
- silica nanoparticle for image diagnosis that can be used in X-ray CT, fluorescence imaging, and the like and has high emission intensity and a method for producing the same.
- a biological substance labeling agent using the silica nanoparticles for diagnostic imaging can be provided.
- the diagnostic imaging silica nanoparticles of the present invention are diagnostic imaging silica nanoparticles containing an organic fluorescent dye and a metal complex, and each of the organic fluorescent dye and the metal complex has a skeleton of silica nanoparticles via an organic molecule. It is connected to.
- This feature is a technical feature common to the inventions according to claims 1 to 8.
- the organic molecule is an organic molecule having a silyl group from the viewpoint of manifesting the effect of the present invention. Further, the organic molecule is preferably a molecule having an amino group and a silyl group substituted with a hydrolyzable substituent in the same molecule.
- the organic molecule and the metal complex ligand are preferably linked via a covalent bond.
- the covalent bond preferably includes an amide bond.
- the metal complex is preferably a gadolinium complex.
- a method for producing a silica nanoparticle for diagnostic imaging of the present invention a method for producing a silica nanoparticle for diagnostic imaging containing an organic fluorescent dye and a metal complex through at least the steps (a) to (c). It is preferable that
- the silica nanoparticles for diagnostic imaging of the present invention can be suitably used for a biological substance labeling agent in which the diagnostic imaging silica nanoparticles and a molecular labeling substance are bound via organic molecules.
- ⁇ is used to mean that the numerical values described before and after it are included as a lower limit value and an upper limit value.
- the diagnostic imaging silica nanoparticles of the present invention are diagnostic imaging silica nanoparticles containing an organic fluorescent dye and a metal complex, and each of the organic fluorescent dye and the metal complex has a skeleton of silica nanoparticles via an organic molecule. It is connected to.
- organic molecule refers to an organic molecule having a plurality of sites capable of binding to other substances, and particularly means an organic molecule that can function as a linker.
- skeleton of silica nanoparticles refers to a framework composed of bonds of oxygen atoms and silicon atoms constituting nanosized silicon dioxide crystal particles (average particle size of 30 to 800 nm).
- the diagnostic imaging silica nanoparticles of the present invention are diagnostic imaging silica nanoparticles containing an organic fluorescent dye, characterized in that the organic fluorescent dye is linked to the skeleton of the silica nanoparticles via an organic molecule. To do.
- Examples of the method of encapsulating the organic fluorescent dye in the silica nanoparticles include a method in which the organic molecules and the organic fluorescent dye are linked through a covalent bond, an ionic bond, a hydrogen bond, or the like in the silica nanoparticle.
- the organic molecule and the organic fluorescent dye are preferably covalently bonded from the viewpoint of chemical stability. If the organic molecule is not bonded to the organic fluorescent dye, the organic fluorescent dye may gradually leak while stored as an aqueous dispersion, which is not preferable for application to a biological substance labeling agent.
- the organic fluorescent dye used in the silica nanoparticles for diagnostic imaging of the present invention when excited by ultraviolet to near infrared light having a wavelength in the range of 200 to 700 nm, visible light having a wavelength in the range of 400 to 900 nm can be used. It is preferable that the organic fluorescent dye has an aspect that emits near-infrared light.
- Organic fluorescent dyes include fluorescein dye molecules, rhodamine dye molecules, Alexa Fluor (Invitrogen) dye molecules, BODIPY (Invitrogen) dye molecules, cascade dye molecules, coumarin dye molecules, and eosin dyes. Examples include molecules, NBD dye molecules, pyrene dye molecules, Texas Red dye molecules, cyanine dye molecules, and the like.
- organic compounds having a silyl group can be used as the organic molecules for encapsulating the organic fluorescent dye in the silica nanoparticles.
- Organic molecules having a silyl group can be used.
- the “silyl group” is a group provided for bonding to the “skeleton of silica nanoparticles” described later.
- a silyl group substituted with a hydrolyzable substituent is used as the silyl group.
- a silyl group substituted with a hydrolyzable substituent is used as the silyl group.
- the “silyl group substituted with a hydrolyzable substituent” means a silyl group having a substituent that hydrolyzes in the presence of moisture to generate silanol, such as a trimethoxysilyl group, triethoxy Examples thereof include alkoxysilyl groups such as silyl group and methyldimethoxysilyl group.
- the method of encapsulating the organic fluorescent dye in the silica nanoparticles that is, the method of combining the organic fluorescent dye with the “skeleton of the silica nanoparticles” described later is not particularly limited.
- a reaction between an organic molecule having a silyl group substituted with a hydrolyzable substituent and an organic fluorescent dye having a functional group capable of reacting with a reactive functional group can be used. More specifically, this reaction includes an organic fluorescent molecule having a reactive functional group and an organic molecule having a silyl group substituted with at least one hydrolyzable substituent, and a functional group capable of reacting with the reactive functional group. It can be carried out as a reaction with a dye.
- the “reactive functional group” is a functional group capable of forming a bond with another substance, and in the normal case, the above “silyl group” capable of forming a bond with another substance. Is a different functional group.
- the reactive functional group constituting the “organic molecule for encapsulating the organic fluorescent dye in the silica nanoparticles” is a condition that the above-mentioned “silyl group” does not lose the ability to bind to the “skeleton of the silica nanoparticles” described later.
- reactive functional groups include active ester groups such as amino groups, mercapto groups, maleimide groups, isocyanate groups, isothiocyanate groups, carboxyl groups, and N-hydroxysuccinimide groups. Of these, amino groups are preferred from the standpoint of stability and reactivity.
- the molecule having a silyl group substituted with an amino group and at least one hydrolyzable substituent is not particularly limited, and can include various compounds generally called silane coupling agents. Specific examples include 3-aminopropylmethyldimethoxysilane, 3-aminopropyltriethoxysilane, and 3-aminopropyltrimethoxysilane.
- the organic fluorescent dye having a functional group capable of reacting with the reactive functional group includes amino group, mercapto group, maleimide group, isocyanate group, isothiocyanate group, carboxyl group, and N-hydroxysuccinimide ester group. There are those having an ester group.
- the organic fluorescent dye is also preferably an active ester group such as a functional group isocyanate group, isothiocyanate group, carboxyl group, and N-hydroxysuccinimide ester group that reacts with the amino group. is there.
- the “active ester group” means an ester group having a highly acidic electron-attracting group on the alcohol side of the ester group and activating a nucleophilic reaction, that is, an ester having a high reaction activity. Refers to the group. Actually, the “active ester group” is an ester group having an electron withdrawing group on the alcohol side of the ester group and activated more than the alkyl ester. The active ester group is an amino group. , Reactive to groups such as thiol groups or hydroxyls.
- such a reaction between the organic molecule and the organic fluorescent dye may be performed based on the reactivity of the “reactive functional group” and the “functional group capable of reacting with the reactive functional group” itself.
- additives, catalysts, and the like that promote the reaction may be used as necessary.
- EDC 1-Ethyl A condensing agent such as carbodiimide such as -3- [3-Dimethylaminopropyl] carbohydride (produced by Pierce) may be used.
- the organic solvent used is not particularly limited as long as it has no reactivity with the hydrolyzable group on the silyl group, and examples thereof include tetrahydrofuran, dimethyl sulfoxide, dimethylformamide and the like.
- the reaction temperature is not particularly limited, but it can be carried out between -20 to 50 ° C.
- the reaction time is preferably 1 hour or more and 50 hours or less, preferably 1 hour or more to complete the reaction, and the yield is improved.
- the reaction time is preferably 1 hour or more and 50 hours or less, preferably 1 hour or more to complete the reaction, and the yield is improved.
- the diagnostic imaging silica nanoparticles of the present invention are diagnostic imaging silica nanoparticles encapsulating a metal complex, wherein each of the metallic complexes is linked to the skeleton of the silica nanoparticles via an organic molecule. To do.
- Examples of the method of encapsulating the metal complex in the silica nanoparticles include a method in which the organic molecules and the organic fluorescent dye are bound to each other via a covalent bond, an ionic bond, a hydrogen bond, or the like in the silica nanoparticle. That is, in the silica nanoparticles for diagnostic imaging according to the present invention, examples of the bond that links the metal complex and the organic molecule include a covalent bond, an ionic bond, and a hydrogen bond.
- the metal complex When the metal complex is not covalently bonded to an organic molecule, the metal complex may gradually leak while being stored as an aqueous dispersion, which may be undesirable for application to a biosubstance labeling agent. . Therefore, in the present invention, it is preferable that the metal complex and the organic molecule are bound by a covalent bond. At this time, the bond between the metal complex and the organic molecule is usually performed in such a manner that the ligand of the metal complex and the organic molecule are linked via a covalent bond.
- Examples of the covalent bond include a ketone bond, an ester bond, a thioester bond, an amide bond, an ether bond, and an imino bond, and an amide bond is preferable from the viewpoint of high hydrolysis stability.
- metal complexes include, but are not limited to, various metal complexes such as lanthanoid complexes, iron complexes, and gold complexes.
- lanthanoid complex which is a paramagnetic ion that can be used for MRI, and particularly a gadolinium complex is preferable because of its high X-ray absorption ability.
- organic molecules for encapsulating the metal complex in the silica nanoparticles the same “organic molecules used for encapsulating the organic fluorescent dye in the silica nanoparticles” described above can be used. That is, various organic compounds having a silyl group can be used. For example, an organic molecule having a silyl group substituted with a reactive functional group and at least one hydrolyzable substituent can be used.
- silica nanoparticles As the “silyl group substituted with a hydrolyzable substituent”, “substituted with a hydrolyzable substituent” that can be used in the above-mentioned “organic molecule used to encapsulate an organic fluorescent dye in silica nanoparticles” Those similar to the “silyl group” can be used.
- the organic molecule for encapsulating the metal complex in the silica nanoparticles may be the same as or different from the organic molecule used for encapsulating the organic fluorescent dye in the silica nanoparticles.
- the method for covalently encapsulating the metal complex with the organic molecule that is, the method for linking the metal complex to the “skeleton of silica nanoparticles” described later is not particularly limited.
- a reaction between an organic molecule having a reactive functional group and a silyl group substituted with at least one hydrolyzable substituent and a metal complex having a functional group capable of reacting with the reactive functional group is performed.
- a reaction between an organic molecule having a reactive functional group and a silyl group substituted with at least one hydrolyzable substituent and a metal complex having a functional group capable of reacting with the reactive functional group is performed.
- the reactive functional group constituting the “organic molecule for encapsulating the metal complex in the silica nanoparticles” is a condition in which the above-mentioned “silyl group” does not lose the ability to bind to the “skeleton of the silica nanoparticles” described later.
- reactive functional groups include active ester groups such as amino groups, mercapto groups, maleimide groups, isocyanate groups, isothiocyanate groups, carboxyl groups, and N-hydroxysuccinimide groups.
- an amino group that forms an amide bond is preferable from the viewpoint of stability and reactivity.
- the molecule having a silyl group substituted with an amino group and at least one hydrolyzable substituent is not particularly limited, and can include various compounds generally called silane coupling agents. Specific examples include 3-aminopropylmethyldimethoxysilane, 3-aminopropyltriethoxysilane, and 3-aminopropyltrimethoxysilane.
- the metal complex having a functional group capable of reacting with the reactive functional group has an active ester group such as an amino group, a mercapto group, a maleimide group, an isocyanate group, an isothiocyanate group, a carboxyl group, or an N-hydroxysuccinimide group. Things can be raised.
- the metal complex also includes an active ester group such as a functional group that reacts with the amino group, an isothiocyanate group, a carboxyl group, and an N-hydroxysuccinimide ester group.
- carboxyl groups that form amide groups and active ester groups such as N-hydroxysuccinimide ester groups are preferred.
- a complex having a carboxyl group that is, a metal complex having a carboxyl group
- a metal complex having a carboxyl group examples include 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid gadolinium complex (hereinafter referred to as Gd-DOTA).
- Gd-DTPA diethylenetriaminepentaacetic acid gadolinium complex
- such a reaction between an organic molecule and a metal complex may be performed based on the reactivity of the “reactive functional group” and the “functional group capable of reacting with a reactive functional group” itself.
- additives, catalysts, and the like that promote the reaction may be used as necessary.
- EDC 1-Ethyl- A condensing agent such as carbodiimide such as 3- [3-Dimethylaminopropyl] carbodiimide (produced by Pierce) may be used.
- the organic solvent used is not particularly limited as long as it has no reactivity with the hydrolyzable group on the silyl group, and examples thereof include tetrahydrofuran, dimethyl sulfoxide, dimethylformamide and the like.
- the reaction temperature is not particularly limited, but it can be carried out between -20 to 50 ° C.
- the reaction time is preferably 1 hour or more and 50 hours or less, and preferably 1 hour or more in that the reaction is completed.
- the reaction time is preferably 1 hour or more and 50 hours or less, and preferably 1 hour or more in that the reaction is completed.
- the diagnostic imaging silica nanoparticles of the present invention are diagnostic imaging silica nanoparticles containing an organic fluorescent dye and a metal complex, and each of the organic fluorescent dye and the metal complex has a skeleton of silica nanoparticles via an organic molecule. It is connected to.
- the “skeleton of silica nanoparticles” has a role of supporting the structure of silica nanoparticles for diagnostic imaging as a matrix portion.
- Silica nanoparticles not containing an organic fluorescent dye or the like that is, the “skeleton of silica nanoparticles” itself can be produced by various conventionally known production methods. For example, Journal of Colloid Science Vol. 26, 62 It is preferably produced by a method called “Stober method” described in page (1968), in which a silicon-containing alkoxide compound such as tetraethoxysilane is hydrolyzed under alkaline conditions using aqueous ammonia or the like.
- silica nanoparticles for diagnostic imaging hydrolysis of such a silicon-containing alkoxide compound in the presence of an organic fluorescent dye and a metal complex into which the organic molecule has been introduced is also performed.
- a skeleton of silica nanoparticles can be suitably formed.
- formation of the “skeleton of the silica nanoparticles” and the inclusion of the organic fluorescent dye and the metal complex in the “skeleton of the silica nanoparticles” can be advantageously performed simultaneously.
- the organic fluorescent dye and the metal complex are present in an encapsulated state on the surface or inside of the “skeleton of the silica nanoparticles” via organic molecules.
- the particle size of the diagnostic imaging silica nanoparticles of the present invention can be freely adjusted by applying known reaction conditions with respect to the water, ethanol, alkali amount, etc. to be added, and can be about 30 to 800 nm in average particle size. Further, the coefficient of variation indicating the variation in particle diameter can be 20% or less.
- the average particle diameter is an electron micrograph taken with a scanning electron microscope (SEM), the cross-sectional area is measured for a sufficient number of particles, and the measured value is the area of a corresponding circle.
- the diameter was determined as the particle size.
- the arithmetic average of the particle diameters of 1000 particles is defined as the average particle diameter.
- the coefficient of variation was also a value calculated from the particle size distribution of 1000 particles.
- the method for producing diagnostic imaging silica nanoparticles according to the present invention comprises imaging diagnostic silica nanoparticles encapsulating an organic fluorescent dye and a metal complex through at least the following steps (a), (b) and (c). It is characterized by manufacturing.
- the silica nanoparticles for diagnostic imaging co-encapsulating the organic fluorescent dye and metal complex of the present invention can be obtained by referring to a known method, for example, a method described in a non-patent document (Langmuir 8, Vol. 2921 (1992)). can do.
- the method for producing the diagnostic imaging silica nanoparticles of the present invention will be described in more detail.
- the diagnostic imaging silica nanoparticles containing the organic fluorescent dye and the metal complex are produced through the following steps (1) to (5). It is preferable to do.
- Step (1) A molecule having an amino group, a silyl group substituted with at least one hydrolyzable substituent in the same molecule, and an organic fluorescent dye having a functional group that reacts with the amino group are reacted.
- Step (2) A molecule having an amino group, a silyl group substituted with at least one hydrolyzable substituent in the same molecule, and a gadolinium complex having a functional group that reacts with the amino group are reacted.
- Step (3) A silicon-containing alkoxide compound such as tetraethoxysilane is mixed with the product obtained in step (1) and step (2).
- Step (4) An organic solvent such as ethanol, water and a base are mixed and the reaction proceeds.
- Step (5) Silica nanoparticles for diagnostic imaging encapsulating an organic fluorescent dye and a metal complex generated from the reaction mixture are collected by filtration or centrifugation.
- step (1) corresponds to step (a)
- step (2) corresponds to step (b)
- steps (3) to (5) correspond to step (c).
- the biological substance labeling agent can also be produced by further performing the following step (6) on the silica nanoparticles for diagnostic imaging of the present invention obtained through the above steps (1) to (5):
- the details of the biological substance labeling agent and the method for producing the same are described in the section “Biological substance labeling agent” described later.
- Examples of the silicon-containing alkoxide compound used in the step (3) include tetraalkoxide silanes such as tetraethoxysilane and tetramethoxysilane, trialkoxysilanes such as methyltrimethoxysilane, methylethoxysilane, and phenyltriethoxysilane. be able to.
- the silicon-containing alkoxide compound which has an organic functional group can be mentioned. Specific examples include mercaptopropyltriethoxysilane and aminopropyltriethoxysilane.
- the silicon-containing alkoxide compound can be used alone or in combination of two or more.
- the mixing ratio of the silicon-containing alkoxide compound and the organic fluorescent dye-binding molecule obtained in the step (1) is not limited, but 1 ⁇ 10 ⁇ 6 mol / L or more and 1 ⁇ 10 ⁇ 2 mol in the finally obtained silica nanoparticles. It is preferable to mix so that it may become / L or less. Sufficient fluorescence can be obtained by setting the concentration to 1 ⁇ 10 ⁇ 6 mol / L or more. Moreover, it is preferable at 1 * 10 ⁇ -2 > mol / L or less at the point which can disperse
- the mixing ratio of the silicon-containing alkoxide compound and the gadolinium complex-bonded molecule obtained in the step (2) is not limited, but it is 1 ⁇ 10 ⁇ 6 mol / L or more and 1 ⁇ 10 ⁇ 2 mol / L in the finally obtained silica nanoparticles. It is preferable to mix so that it may become L or less. Sufficient X-ray CT or MRI contrast can be obtained by setting the concentration to 1 ⁇ 10 ⁇ 6 mol / L or more. Moreover, it is preferable at 1 * 10 ⁇ -2 > mol / L or less at the point which can disperse
- any organic solvent may be used as long as it is used in the usual hydrolysis reaction of a silicon-containing alkoxide compound, and methanol, ethanol, tetrahydrofuran, dimethylformamide, dimethyl sulfoxide and the like are used. It is good also as a 1 type, or 2 or more types of mixture.
- a base used at a process (4) what is necessary is just to be used by the hydrolysis reaction of a normal silicon-containing alkoxide compound, Ammonia, sodium hydroxide, potassium hydroxide etc. can be used, respectively, It may be used as
- tetraethoxysilane When tetraethoxysilane is 1 mol, a is 20 to 400, r is 10 to 200, and b is 10 to 40, where ethanol is a mol, water is r mol, and ammonia is b mol.
- a 20 to 400
- r 10 to 200
- b 10 to 40
- ethanol is a mol
- water is r mol
- ammonia is b mol.
- the reaction temperature may be a condition applied in a normal hydrolysis reaction of a silicon-containing alkoxide compound, and can be performed between room temperature and 50 ° C.
- the reaction time in the step (4) may be a condition applied in a normal hydrolysis reaction of a silicon-containing alkoxide compound, and is preferably 1 hour or more and 50 hours or less. By setting it for 1 hour or more, it is preferable at the point which reaction is completed, and setting at 50 hours or less is preferable at the point which can prevent reaction progressing and insoluble matter formation.
- the method for recovering the silica nanoparticles for diagnostic imaging encapsulating the organic fluorescent dye and the metal complex generated from the reaction mixture may be filtration or centrifugation that is usually performed for recovering the nanoparticles.
- the silica nanoparticles for diagnostic imaging encapsulating the collected organic fluorescent dye and metal complex may be washed with an organic solvent or water in order to remove unreacted raw materials as necessary.
- the biological material labeling agent according to the present invention includes the above-described silica nanoparticles for diagnostic imaging encapsulating an organic fluorescent dye modified with an organic molecule and a metal complex, and a molecular labeling substance bonded to the diagnostic diagnostic silica nanoparticles.
- the biological substance labeling agent of the present invention does not preclude that the molecular labeling substance is a biological substance labeling agent in an embodiment in which the molecular labeling substance is directly bonded to the diagnostic imaging silica nanoparticles without intervening other molecules.
- the biological material labeling agent of the present invention has a mode in which the above-described silica nanoparticles for diagnostic imaging and the molecular labeling material are bound by organic molecules.
- the silica nanoparticles for diagnostic imaging and the molecular labeling substance are bound via an organic molecule.
- the form of the bond is not particularly limited, and examples thereof include a covalent bond, an ionic bond, a hydrogen bond, a coordinate bond, physical adsorption, and chemical adsorption.
- a bond having a strong bonding force such as a covalent bond is preferable from the viewpoint of bond stability.
- the organic molecule bound to the imaging diagnostic silica nanoparticles and the molecular labeling substance may be directly bound or bound via a second linker compound. You may do it.
- the biological substance labeling agent according to the present invention can label a biological substance by specifically binding and / or reacting with the target biological substance. That is, the biological material labeling agent according to the present invention can be suitably used for the purpose of labeling biological materials.
- the biological substance labeling agent according to the present invention enables labeling with both fluorescence and X-ray absorption modalities, and particularly enables highly sensitive detection in various assay methods such as immunoassay.
- the molecular labeling substance is not particularly limited as long as it is a substance that can specifically bind and / or react with a biological substance to be labeled.
- Examples of molecular labeling substances that can be used in the present invention include nucleotide chains, proteins, and antibodies.
- Organic molecules that bind the diagnostic imaging silica nanoparticles and the molecular labeling substance are sites that can bind to the diagnostic imaging silica nanoparticles.
- an organic molecule having a site capable of binding directly or indirectly to the molecular labeling substance is not particularly limited. However, in the present invention, it is widely used as an organic molecule that binds to the surface of an imaging diagnostic silica nanoparticle encapsulating an organic fluorescent dye and a metal complex and can also bind to a molecular labeling substance, for example, to bind an inorganic substance to an organic substance.
- a silane coupling agent that is a compound that is present can be used.
- This silane coupling agent is a compound having an alkoxysilyl group that gives a silanol group by hydrolysis at one end of the molecule and a functional group such as a carboxyl group, an amino group, an epoxy group, an aldehyde group at the other end, Bonding with an inorganic substance through an oxygen atom of the silanol group.
- Specific examples include mercaptopropyltriethoxysilane, glycidoxypropyltriethoxysilane, and aminopropyltriethoxysilane.
- a silane coupling agent having a polyethylene glycol chain for example, PEG-silane no. SIM6492.7 manufactured by Gelest
- PEG-silane no. SIM6492.7 manufactured by Gelest
- a second linker compound may be further interposed between the organic molecule and the molecular target substance, if necessary.
- a second linker compound may function as a spacer that secures a certain distance between the diagnostic imaging silica nanoparticles and the molecular target substance, or is introduced into the diagnostic imaging silica nanoparticles. It may also function as an adapter for binding organic molecules and molecular target substances.
- the second linker compound is not particularly limited as long as it has a portion capable of directly binding to the organic molecule introduced into the diagnostic imaging silica nanoparticles and a portion capable of binding to the molecular target substance. Absent. For example, when an organic molecule introduced into the silica nanoparticles for diagnostic imaging has an amino group, a carboxyl group or the like can be cited as a site that can directly bind to the organic molecule in the second linker compound, and binds to a molecular target substance. Examples of such sites include functional groups that can selectively react with mercapto groups, such as maleimide groups. Specific examples of the second linker compound include sulfo-SMCC (Sulfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate: manufactured by Pierce).
- the biological material labeling agent according to the present invention is obtained by binding the above-described silica nanoparticles for diagnostic imaging, a molecular labeling substance and an organic molecule.
- the method for producing a biological material labeling agent according to the present invention may be a method of passing a step of introducing an organic molecule into the silica nanoparticles for diagnostic imaging and then a step of binding the molecular labeling substance to the organic molecule.
- the molecular labeling substance may be modified in advance with an organic molecule to form an organic molecule-modified molecular labeling substance, followed by a process of introducing the organic molecule-modifying molecular labeling substance into the image diagnostic silica nanoparticles. May be.
- the biological material according to the present invention is passed through a step of introducing an organic molecule into the silica nanoparticles for diagnostic imaging and then a step of binding a molecular labeling substance to the organic molecule. It is preferable to produce a labeling agent.
- diagnostic imaging silica nanoparticles encapsulating an organic fluorescent dye and a metal complex
- a silane coupling agent having an appropriate reactive functional group for example, aminopropyl group
- the reactive functional group is introduced into the silica nanoparticles for diagnostic imaging, and the functional group-modified silica nanoparticles for diagnostic imaging are obtained.
- step (ii) functional group-modified silica nanoparticles for diagnostic imaging obtained by the above step (i),
- a molecular labeling substance for example, an antibody
- a functional group for example, a carboxyl group
- bonding is reacted to form a functional group-modified diagnostic imaging silica nanoparticle and the molecular labeling substance.
- the second linker compound for example, sulfo having a functional group capable of forming a bond with the reactive functional group (for example, a carboxyl group and an active ester thereof) and a second reactive functional group (for example, a maleimide group).
- a known method can be used as a reaction procedure between the diagnostic imaging silica nanoparticles encapsulating the organic fluorescent dye and the metal complex and the silane coupling agent.
- the obtained diagnostic imaging silica nanoparticles containing the organic fluorescent dye and the metal complex are dispersed in pure water, aminopropyltriethoxysilane is added, and the reaction is performed at room temperature for 12 hours.
- silica nanoparticles for diagnostic imaging whose surface is modified with an aminopropyl group can be obtained by centrifugation or filtration.
- step (ii) by reacting an amino group of a silica nanoparticle for image diagnosis modified with aminopropyltriethoxysilane with a carboxyl group in the antibody, the antibody is used for image diagnosis via an amide bond. It can be combined with silica nanoparticles. If necessary, a condensing agent such as EDC (1-Ethyl-3- [3-Dimethylaminopropyl] carbohydrate, Hydrochloride: Pierce) may be used.
- EDC 1-Ethyl-3- [3-Dimethylaminopropyl] carbohydrate, Hydrochloride: Pierce
- a site capable of directly binding to the organic molecule-modified silica nanoparticles for diagnostic imaging and a site capable of binding to the molecular target substance if necessary, a site capable of directly binding to the organic molecule-modified silica nanoparticles for diagnostic imaging and a site capable of binding to the molecular target substance. It is possible to use a linker compound having the second linker compound.
- sulfo-SMCC Sulfosuccinimidyl 4- [N-maleidomethyl] cyclohexane-1-carboxylate: manufactured by Pierce
- sulfo-SMCC Sulfosuccinimidyl 4- [N-maleidomethyl] cyclohexane-1-carboxylate: manufactured by Pierce
- the antibody-coupled diagnostic silica nanoparticle can be produced.
- Step (1) 2.6 mg (0.0048 mmol) of N-hydroxysuccinimide ester derivative (5 (6) -TAMRA-NHS, SE, manufactured by Invitrogen) of an organic fluorescent dye TAMRA (hereinafter referred to as “TAMRA”) is added to dimethyl
- TAMRA organic fluorescent dye
- Step (2) When 2.6 mg (0.0048 mmol) of diethylenetriaminepentaacetic acid gadolinium complex (manufactured by Aldrich) was dissolved in 0.8 ml of dimethylformamide (hereinafter abbreviated as “DMF”), 1-Ethyl-3 -[3-Dimethylaminopropyl] carbohydride Hydrochloride (manufactured by Pierce) 0.7 mg (0.0048 mmol), then 3-aminopropyltriethoxysilane (manufactured by Gelest) 1 ⁇ l (0.0048 mmol) was added and stirred for 30 minutes.
- DMF dimethylformamide
- Step (3) The DMF solution obtained in Step (1) and Step (2) and 40 ⁇ l of tetraethoxysilane were mixed.
- Step (5) The mixture prepared in Step (3) was added to the mixture prepared in Step (4) while stirring at room temperature. Stirring was performed for 12 hours from the start of addition.
- Step (6) The reaction mixture was centrifuged at 10,000 g for 60 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. In the same procedure, washing with ethanol and pure water was performed once.
- the obtained silica nanoparticles 1 for diagnostic imaging were observed with a scanning electron microscope (SEM; Model S-800 manufactured by Hitachi, Ltd.). As a result, the average particle size was 110 nm and the coefficient of variation was 12%. In this observation, the cross-sectional area is measured for 1000 particles, the diameter when the measured value is the area of the corresponding circle is obtained as the particle size, and the arithmetic average of the particle size of 1000 particles is the average particle size. The diameter. The coefficient of variation was also a value calculated from the particle size distribution of 1000 particles.
- Synthesis Example 2 Synthesis of diagnostic imaging silica nanoparticles 2 containing Cy5 and Gd-DOTA Cy5N-hydroxysuccinimide ester derivative (manufactured by GE Healthcare) as an organic fluorescent dye, Gd-DOTA (trademark of DOTAREM, manufactured by Guerbet) as a gadolinium complex
- the diagnostic imaging silica nanoparticles 2 were synthesized by the same procedure as in Synthesis Example 1 except that was used.
- the average particle size was 100 nm and the coefficient of variation was 10%.
- Synthesis Example 3 Synthesis of TAMRA / Gd-DTPA-encapsulating silica nanoparticles 3 for diagnostic imaging Diagnostic imaging was performed by the method of the following steps (1) to (6) without performing the step of covalently bonding Gd-DTPA to silica nanoparticles. Silica nanoparticles 3 were prepared.
- Step (1) N-hydroxysuccinimide ester derivative of TAMRA (Invitrogen 5 (6) -TAMRA-NHS, SE) 2.6 mg (0.0048 mmol) was dissolved in dimethylformamide 0.8 ml, ice Under cooling, 1 ⁇ l (0.0048 mmol) of 3-aminopropyltriethoxysilane (manufactured by Gelest) was added and stirred for 30 minutes.
- TAMRA Invitrogen 5 (6) -TAMRA-NHS, SE
- Step (2) 2.6 mg (0.0048 mmol) of diethylenetriaminepentaacetic acid gadolinium complex (manufactured by Aldrich) was dissolved in 0.8 ml of dimethylformamide.
- Step (3) The DMF solution obtained in Step (1) and Step (2) and 40 ⁇ l of tetraethoxysilane were mixed.
- Step (5) The mixture prepared in Step (3) was added to the mixture prepared in Step (4) while stirring at room temperature. Stirring was performed for 12 hours from the start of addition.
- Step (6) The reaction mixture was centrifuged at 10,000 g for 60 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. In the same procedure, washing with ethanol and pure water was performed once.
- silica nanoparticles 3 for diagnostic imaging were observed with a scanning electron microscope (SEM; model S-800 manufactured by Hitachi, Ltd.). As a result, the average particle size was 105 nm and the coefficient of variation was 12%.
- Synthesis Example 4 Synthesis of Silica Nanoparticle 4 for Image Diagnosis Encapsulating TAMRA Silica nanoparticles 4 for image diagnosis encapsulating only an organic fluorescent dye were prepared by the methods of the following steps (1) to (5).
- Step (1) N-hydroxysuccinimide ester derivative of TAMRA (Invitrogen 5 (6) -TAMRA-NHS, SE) 2.6 mg (0.0048 mmol) was dissolved in dimethylformamide 0.8 ml, ice Under cooling, 1 ⁇ l (0.0048 mmol) of 3-aminopropyltriethoxysilane (manufactured by Gelest) was added and stirred for 30 minutes.
- TAMRA Invitrogen 5 (6) -TAMRA-NHS, SE
- Step (2) The DMF solution obtained in step (1) and 40 ⁇ l of tetraethoxysilane were mixed.
- Step (3) Ethanol 40 ml and 14% ammonia water 10 ml were mixed.
- Step (4) The mixture prepared in step (2) was added to the mixture prepared in step (4) while stirring at room temperature. Stirring was performed for 12 hours from the start of addition.
- Step (5) The reaction mixture was centrifuged at 10,000 g for 60 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. In the same procedure, washing with ethanol and pure water was performed once.
- silica nanoparticles 4 for diagnostic imaging were observed with a scanning electron microscope (SEM; Model S-800, manufactured by Hitachi, Ltd.). As a result, the average particle size was 110 nm and the coefficient of variation was 10%.
- Synthesis Example 5 Synthesis of diagnostic imaging silica nanoparticles 5 encapsulating Gd-DTPA By the method of the following steps (1) to (5), the diagnostic imaging silica nanoparticles 5 encapsulating only gadolinium complexes in silica particles by covalent bonds. Produced.
- Step (1) When 2.6 mg (0.0048 mmol) of diethylenetriaminepentaacetic acid gadolinium complex (manufactured by Aldrich) was dissolved in 0.8 ml of dimethylformamide, 1-Ethyl-3- [3-Dimethylaminopropyl] hydrochloride ( (Pierce) 0.7 mg (0.0048 mmol) and 3-aminopropyltriethoxysilane (Gelest) 1 ⁇ l (0.0048 mmol) were added and stirred for 30 minutes.
- 1-Ethyl-3- [3-Dimethylaminopropyl] hydrochloride ( (Pierce) 0.7 mg (0.0048 mmol)
- 3-aminopropyltriethoxysilane (Gelest) 1 ⁇ l (0.0048 mmol) were added and stirred for 30 minutes.
- Step (2) The DMF solution obtained in step (1) and 40 ⁇ l of tetraethoxysilane were mixed.
- Step (3) Ethanol 40 ml and 14% ammonia water 10 ml were mixed.
- Step (4) The mixture prepared in step (2) was added to the mixture prepared in step (4) while stirring at room temperature. Stirring was performed for 12 hours from the start of addition.
- Step (5) The reaction mixture was centrifuged at 10,000 g for 60 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. In the same procedure, washing with ethanol and pure water was performed once.
- silica nanoparticles 5 for diagnostic imaging were observed with a scanning electron microscope (SEM; Model S-800, manufactured by Hitachi, Ltd.). As a result, the average particle size was 102 nm and the coefficient of variation was 11%.
- the X-ray absorption capacity is obtained by placing the obtained silica nanoparticle 1 nM PBS dispersion in a plastic tube, irradiating X-rays with an acceleration voltage of 70 kV, and transmitting the transmitted X-ray dose using a flat panel detector PaxScan registered trademark 1313R (trade name). : Manufactured by Varian). Evaluation was made as a relative value when the measured value of the part without the sample was taken as 100. The evaluation results are shown in Table 1. In Table 1, the smaller the value shown in the “X-ray absorption” column, the smaller the transmitted X-ray dose and the better the X-ray absorption ability.
- the diagnostic imaging silica nanoparticles 1 and 2 of the present invention are detected by both the fluorescence and X-ray absorption modalities.
- the particle 3 in which the gadolinium complex is not encapsulated by a covalent bond has a reduced X-ray absorption rate. This is presumably because the gadolinium complex was not covalently bonded to the particle skeleton, so it easily leaked from the silica particles and was removed by washing.
- the particles 4 containing only the organic fluorescent dye could not be detected by X-ray absorption, but could only be detected by fluorescence.
- grains 5 which included only the gadolinium complex were not able to be detected by fluorescence, only the detection by X-ray absorption was possible.
- the particles containing only one of the organic fluorescent dye and the gadolinium complex cannot be detected by plural modalities, and the effects of the present invention cannot be obtained.
- silica nanoparticles A (Molecular Modification Silica Nanoparticle A: Amino Group Modification to Silica Nanoparticle for Image Diagnosis Encapsulating TAMRA / Gd-DTPA) 1 mg of silica nanoparticles 1 for diagnostic imaging was dispersed in 5 ml of pure water. 100 ⁇ l of aminopropyltriethoxysilane aqueous dispersion was added and stirred at room temperature for 12 hours.
- reaction mixture was centrifuged at 10,000 g for 60 minutes, and the supernatant was removed. Ethanol was added to disperse the sediment and centrifuged again. Further washing with ethanol and pure water was performed in the same procedure. As a result, molecular modified silica nanoparticles A were obtained.
- Silica nanoparticles for diagnostic imaging containing TAMRA / Gd-DTPA treated with sulfo-SMCC and anti-hCG antibody treated with DTT were mixed and reacted for 1 hour. 10 mM mercaptoethanol was added to stop the reaction. Unreacted substances were removed by a gel filtration column to obtain diagnostic imaging silica nanoparticles (biological substance labeling agent 1) encapsulating TAMRA / Gd-DTPA bound with anti-hCG antibody.
- [Preparation of biological material labeling agent 2] (Biological substance labeling agent 2: antibody conjugate to silica nanoparticles for diagnostic imaging encapsulating amino group-modified Cy5 / Gd-DOTA) Similar to the preparation of the biological material labeling agent 1 for the diagnostic imaging silica nanoparticles encapsulating the amino group-modified Cy5 / Gd-DOTA obtained in “Preparation of molecularly modified silica nanoparticles B”, that is, the molecularly modified silica nanoparticles B. Through the procedure, silica nanoparticles for diagnostic imaging (biological substance labeling agent 2) encapsulating Cy5 / Gd-DOTA bound with anti-hCG antibody were obtained.
- An immunoassay using biological substance labeling agents 1 and 2 was carried out according to the following procedure. 1) Anti-h ⁇ subunit was immobilized in the well on the microplate. 2) The antigen hCG was added to each well at different concentrations. 3) After removing excess hCG by washing, a biological material labeling agent dispersion was added to each well. 4) Excess biological material labeling agent was removed by washing. 5) The fluorescence intensity of each well was measured with a microplate reader Fluoroscan Ascent FL (trade name: manufactured by Thermo Fisher). 6) The microplate was irradiated with X-rays, and the X-ray absorption rate of each well was measured.
- the fluorescence intensity measured at each hCG antibody concentration when the fluorescence intensity when the hCG antibody concentration is 1 ng / ml is 100 is shown in Table 2, and X when the hCG antibody concentration is 1 ng / ml.
- Table 3 shows the X-ray absorption rate measured at each hCG antibody concentration when the linear absorption rate is 10.
- the biological substance labeling agent obtained in the present invention does not impair the antigen recognition ability. That is, this result shows that the present invention can provide a biological substance labeling agent capable of highly sensitive detection by a plurality of modalities.
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Abstract
Description
工程(a):同一分子内にアミノ基と、加水分解性置換基で置換されたシリル基とを有する有機分子と、当該アミノ基と反応する官能基を有する有機蛍光色素とを反応させる工程、
工程(b)同一分子内にアミノ基と、加水分解性置換基で置換されたシリル基とを有する有機分子と、当該アミノ基と反応する官能基を有するガドリニウム錯体とを反応させる工程、
工程(c):前記工程(a)及び工程(b)で得られた反応生成物を含ケイ素アルコキシドと混合し、塩基性条件下で加水分解反応を行う工程。
本発明の画像診断用シリカナノ粒子は、有機蛍光色素と金属錯体とを内包した画像診断用シリカナノ粒子であって、当該有機蛍光色素と金属錯体のそれぞれが、有機分子を介して、シリカナノ粒子の骨格に連結していることを特徴とする。
なお、「シリカナノ粒子の骨格」とは、ナノサイズ(平均粒径30~800nm)の二酸化ケイ素結晶粒子を構成する酸素原子とケイ素原子との結合からなる骨組みをいう。
本発明の画像診断用シリカナノ粒子は、有機蛍光色素を内包した画像診断用シリカナノ粒子であって、当該有機蛍光色素が、有機分子を介して、シリカナノ粒子の骨格に連結していることを特徴とする。
ここで、「シリル基」は、後述する「シリカナノ粒子の骨格」との結合に供される基であり、本発明においては、このシリル基として、加水分解性置換基で置換されたシリル基が好ましく用いられる。ここで、「加水分解性置換基で置換されたシリル基」は、水分の存在により加水分解してシラノールを生じさせるような置換基を有するシリル基をいい、例えば、トリメトキシシリル基、トリエトキシシリル基、メチルジメトキシシリル基などのアルコキシシリル基が挙げられる。シリル基として「加水分解性置換基で置換されたシリル基」を用いると、有機分子と、後述する「シリカナノ粒子の骨格」との結合を容易に形成することが可能になるとともに、そのような結合の形成反応を制御しやすくもなるので、有利である。
本発明において「有機蛍光色素をシリカナノ粒子に内包させるための有機分子」を構成する反応性官能基は、上述の「シリル基」が後述する「シリカナノ粒子の骨格」と結合する能力を失わない条件下で、有機蛍光色素との結合を可能とするものであることが好ましく、特に、加水分解を生じさせる条件を用いることなく、有機蛍光色素との結合を可能とするものであることがより好ましい。このような反応性官能基としては、アミノ基、メルカプト基、マレイミド基、イソシアネート基、イソチオシアネート基、カルボキシル基、N-ヒドロキシスクシンイミド基など活性エステル基があげられる。中でも安定性や反応性からアミノ基が好ましい。
本発明の画像診断用シリカナノ粒子は、金属錯体を内包した画像診断用シリカナノ粒子であって、当該金属錯体のそれぞれが、有機分子を介して、シリカナノ粒子の骨格に連結していることを特徴とする。
なお、金属錯体をシリカナノ粒子に内包させるための有機分子は、有機蛍光色素をシリカナノ粒子に内包させるために用いられる有機分子と同一のものであっても良く、あるいは、異なっていても良い。
金属錯体を有機分子と共有結合させ内包させる方法、すなわち、金属錯体を、後述する「シリカナノ粒子の骨格」と結びつける方法は、特に限定されるものではない。ただ、好ましい方法として、反応性官能基と少なくとも一種の加水分解性置換基で置換されたシリル基を有する有機分子と、当該反応性官能基と反応しうる官能基をもつ金属錯体との反応を用いることができる。
本発明の画像診断用シリカナノ粒子は、有機蛍光色素と金属錯体とを内包した画像診断用シリカナノ粒子であって、当該有機蛍光色素と金属錯体のそれぞれが、有機分子を介して、シリカナノ粒子の骨格に連結していることを特徴とする。
ここで、本発明において、「シリカナノ粒子の骨格」は、マトリクス部分として、画像診断用シリカナノ粒子の構造を支持する役割を有する。
あるいは、「画像診断用シリカナノ粒子の製造方法」の項で後述するように、上記有機分子を導入した有機蛍光色素および金属錯体存在下でこのような含ケイ素アルコキシド化合物の加水分解を行うことによっても、「シリカナノ粒子の骨格」を好適に形成することができる。この場合、「シリカナノ粒子の骨格」の形成と、「シリカナノ粒子の骨格」への有機蛍光色素および金属錯体の内包を同時に行うことができるので有利である。
本発明の画像診断用シリカナノ粒子において、上記有機蛍光色素および金属錯体は、有機分子を介して、上記「シリカナノ粒子の骨格」の表面または内部に内包された状態で存在している。
本発明の画像診断用シリカナノ粒子の製造方法は、少なくとも、下記工程(a)、工程(b)及び工程(c)を経由して有機蛍光色素と金属錯体とを内包した画像診断用シリカナノ粒子を製造することを特徴とする。
工程(a):同一分子内にアミノ基と、加水分解性置換基で置換されたシリル基とを有する有機分子と、当該アミノ基と反応する官能基を有する有機蛍光色素とを反応させる工程、
工程(b)同一分子内にアミノ基と、加水分解性置換基で置換されたシリル基とを有する有機分子と、当該アミノ基と反応する官能基を有するガドリニウム錯体とを反応させる工程、
工程(c):前記工程(a)及び工程(b)で得られた反応生成物を含ケイ素アルコキシドと混合し、塩基性条件下で加水分解反応を行う工程。
そして、上記工程(1)~(5)を経ることによって得られる本願発明の画像診断用シリカナノ粒子に対して、下記工程(6)をさらに行い、生体物質標識剤を製造することもできる:
工程(6):工程(5)で得られた有機蛍光色素及び金属錯体を内包した画像診断用シリカナノ粒子を分子標識物質と結合させ、生体物質標識剤を得る。
なお、生体物質標識剤およびその製造方法についての詳細は、後述する「生体物質標識剤」の項で述べる。
本発明に係る生体物質標識剤は、有機分子修飾された有機蛍光色素及び金属錯体を内包した上述の画像診断用シリカナノ粒子と、当該画像診断用シリカナノ粒子に結合した分子標識物質とを含む。
本発明の生体物質標識剤は、分子標識物質が他の分子を介することなく直接画像診断用シリカナノ粒子に結合した態様の生体物質標識剤であることを妨げるものではない。ただ、本発明の生体物質標識剤は、上記画像診断用シリカナノ粒子と分子標識物質とが有機分子により結合されている態様であることが好ましい。すなわち、本発明の生体物質標識剤の好適な態様において、上記画像診断用シリカナノ粒子と分子標識物質とが、有機分子を介して結合されている。上記結合の態様としては、特に限定されず、共有結合、イオン結合、水素結合、配位結合、物理吸着及び化学吸着等が挙げられる。結合の安定性から共有結合などの結合力の強い結合が好ましい。
なお、本発明の生体物質標識剤において、上記画像診断用シリカナノ粒子と結合した有機分子と、分子標識物質とは、直接結合していても良いし、あるいは、第2のリンカー化合物を介して結合していても良い。
本発明において、分子標識物質は、標識対象とする生体物質と特異的に結合及び/又は反応することのできる物質である限り特に限定されるものではない。本発明で用いうる分子標識物質の例として、ヌクレオチド鎖、タンパク質、抗体等が挙げられる。
本発明において、画像診断用シリカナノ粒子と分子標識物質とを結合するために用いうる有機分子は、画像診断用シリカナノ粒子と結合可能な部位と、分子標識物質と直接的にまたは間接的に結合可能な部位とを有する有機分子である限り特に限定されるものではない。ただ、本発明では、有機蛍光色素及び金属錯体を内包した画像診断用シリカナノ粒子の表面に結合し、分子標識物質とも結合しうる有機分子として、例えば、無機物と有機物を結合させるために広く用いられている化合物であるシランカップリング剤を用いることができる。このシランカップリング剤は、分子の一端に加水分解でシラノール基を与えるアルコキシシリル基を有し、他端に、カルボキシル基、アミノ基、エポキシ基、アルデヒド基などの官能基を有する化合物であり、上記シラノール基の酸素原子を介して無機物と結合する。具体的には、メルカプトプロピルトリエトキシシラン、グリシドキシプロピルトリエトキシシラン、アミノプロピルトリエトキシシランなどがあげられる。
また、本発明に係る画像診断用シリカナノ粒子と分子標的物質との間の結合として、必要により、上記有機分子と分子標的物質との間に、第2のリンカー化合物をさらに介在させることもできる。このような第2のリンカー化合物は、画像診断用シリカナノ粒子と分子標的物質との間に一定の距離を確保するスペーサーとして機能するものであっても良く、あるいは、画像診断用シリカナノ粒子に導入された有機分子と分子標的物質とが結合するためのアダプターとして機能するものであっても良い。
この第2のリンカー化合物は、画像診断用シリカナノ粒子に導入された上記有機分子と直接結合しうる部位と、分子標的物質と結合しうる部位とを有するものであれば、特に限定されるものではない。
例えば、画像診断用シリカナノ粒子に導入された有機分子がアミノ基を有する場合、この第2のリンカー化合物における上記有機分子と直接結合しうる部位として、カルボキシル基などが挙げられ、分子標的物質と結合しうる部位として、マレイミド基など、メルカプト基と選択的に反応しうる官能基が挙げられる。この第2のリンカー化合物の具体例として、sulfo-SMCC(Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate:Pierce社製)などが挙げられる。
本発明に係る生体物質標識剤は、上述した画像診断用シリカナノ粒子と、分子標識物質と有機分子を介して結合させて得られる。
ここで、本発明に係る生体物質標識剤の製造方法は、画像診断用シリカナノ粒子に有機分子を導入する工程を経てから、分子標識物質を当該有機分子と結合させる工程を経るものであっても良いし、分子標識物質を予め有機分子で修飾して有機分子修飾分子標識物質を形成する工程を経てから、画像診断用シリカナノ粒子に当該有機分子修飾分子標識物質を導入する工程を経るものであっても良い。ただ、後処理の容易さ等の観点から、画像診断用シリカナノ粒子に有機分子を導入する工程を経てから、分子標識物質を当該有機分子と結合させる工程を経ることによって、本発明に係る生体物質標識剤を製造することが好ましい。
本発明に係る生体物質標識剤の好適な製造方法として、具体的には、
(i) まず、有機蛍光色素及び金属錯体を内包した画像診断用シリカナノ粒子と、
適当な反応性官能基(例えば、アミノプロピル基)を有するシランカップリング剤と
を反応させることにより、反応性官能基を画像診断用シリカナノ粒子に導入して、官能基修飾画像診断用シリカナノ粒子を得る工程と、
(ii) 上記工程 (i) により得られた官能基修飾画像診断用シリカナノ粒子と、
当該反応性官能基と結合形成可能な官能基(例えば、カルボキシル基)を有する分子標識物質(例えば、抗体)と
を反応させて、官能基修飾画像診断用シリカナノ粒子と分子標識物質との間に結合を生じさせることにより、生体物質標識剤を得る工程
を含むものが挙げられる。
ここで、上記工程 (ii) に代えて、
(ii') 上記工程 (i) により得られた官能基修飾画像診断用シリカナノ粒子と、
当該反応性官能基と結合形成可能な官能基(例えば、カルボキシル基およびその活性エステル)と、第2の反応性官能基(例えば、マレイミド基)とを有する上記第2のリンカー化合物(例えば、sulfo-SMMC)と
を反応させて、当該第2の反応性官能基を画像診断用シリカナノ粒子に導入して、第2の官能基修飾画像診断用シリカナノ粒子を得る工程と、
(iii') 上記工程 (ii') により得られた第2の官能基修飾画像診断用シリカナノ粒子と、
当該第2の反応性官能基と結合形成可能な官能基(例えば、チオール基)を有する分子標識物質(例えば、抗体)と
を反応させて、当該第2の官能基修飾画像診断用シリカナノ粒子と分子標識物質との間に結合を生じさせることにより、生体物質標識剤を得る工程
からなる2つの工程を行っても良い。
下記工程(1)~(6)の方法により、画像診断用シリカナノ粒子1を作製した。
有機蛍光色素としてCy5N-ヒドロキシスクシンイミドエステル誘導体(GE Healthcare社製)ガドリニウム錯体としてGd-DOTA(Guerbet社製DOTAREM登録商標)を用いた他は合成例1と同様の手順により画像診断用シリカナノ粒子2を合成した。
Gd-DTPAをシリカナノ粒子と共有結合する工程を行わずに下記工程(1)~(6)の方法により、画像診断用シリカナノ粒子3を作製した。
下記工程(1)~(5)の方法により、有機蛍光色素のみ内包した画像診断用シリカナノ粒子4を作製した。
下記工程(1)~(5)の方法により、ガドリニウム錯体のみをシリカ粒子に共有結合により内包した画像診断用シリカナノ粒子5を作製した。
有機蛍光色素及び金属錯体の両方を内包した画像診断用シリカナノ粒子1及び2を用いて、下記に示す方法により分子修飾シリカナノ粒子A及びBを各々調製し、更にこれを用いて生体物質標識剤1及び2を調製して、生体物質標識剤の長期保存性を評価した。
(分子修飾シリカナノ粒子A:TAMRA/Gd-DTPAを内包した画像診断用シリカナノ粒子へのアミノ基修飾)
画像診断用シリカナノ粒子1 1mgを純水5mlに分散させた。アミノプロピルトリエトキシシラン水分散液100μlを添加し、室温で12時間撹拌した。
(分子修飾シリカナノ粒子B:Cy5/Gd-DOTAを内包した画像診断用シリカナノ粒子へのアミノ基修飾)
画像診断用シリカナノ粒子2について、分子修飾シリカナノ粒子Aの調製と同様の手順によりアミノ基修飾を行った。その結果、分子修飾シリカナノ粒子Bが得られた。
(生体物質標識剤1:アミノ基修飾TAMRA/Gd-DTPAを内包した画像診断用シリカナノ粒子への抗体結合体)
上記「分子修飾シリカナノ粒子Aの調製」で得られたアミノ基修飾TAMRA/Gd-DTPAを内包した画像診断用シリカナノ粒子、すなわち、上記分子修飾シリカナノ粒子A0.5mgを純水0.5mlに分散させたもの0.1mlをDMSO2mlに添加した。そこへ、sulfo-SMCC(Pierce社製)をいれ1時間反応させた。過剰のsulfo-SMCCなどを遠心分離により除去する、一方で、抗hCG抗体を1Mジチオスレイトール(DTT)で還元処理を行い、ゲルろ過カラムにより過剰のDTTを除去した。
(生体物質標識剤2:アミノ基修飾Cy5/Gd-DOTAを内包した画像診断用シリカナノ粒子への抗体結合体)
上記「分子修飾シリカナノ粒子Bの調製」で得られたアミノ基修飾Cy5/Gd-DOTAを内包した画像診断用シリカナノ粒子、すなわち、上記分子修飾シリカナノ粒子Bについて、生体物質標識剤1の調製と同様の手順で、抗hCG抗体が結合したCy5/Gd-DOTAを内包した画像診断用シリカナノ粒子(生体物質標識剤2)を得た。
1)マイクロプレート上ウェル内にアンチ-hαサブニットを固定化した。
2)抗原であるhCGを各ウェルに濃度を変えて入れた。
3)過剰のhCGを洗浄により除去後、各ウェルに生体物質標識剤分散液を入れた。
4)過剰の生体物質標識剤を洗浄により除去した。
5)マイクロプレートリーダー フルオロスキャンアセントFL(商品名:サーモフィッシャー社製)により各ウェルの蛍光強度を測定した。
6)マイクロプレートにX線を照射し、各ウェルのX線吸収率を測定した。
Claims (8)
- 有機蛍光色素と金属錯体とを内包した画像診断用シリカナノ粒子であって、当該有機蛍光色素と金属錯体のそれぞれが、有機分子を介して、シリカナノ粒子の骨格に連結していることを特徴とする画像診断用シリカナノ粒子。
- 前記有機分子が、シリル基を有する有機分子であることを特徴とする請求項1に記載の画像診断用シリカナノ粒子。
- 前記有機分子が、同一分子内にアミノ基と、加水分解性置換基で置換されたシリル基を有する分子であることを特徴とする請求項1または2に記載の画像診断用シリカナノ粒子。
- 前記有機分子と前記金属錯体の配位子が共有結合を介して連結していることを特徴とする請求項1~3のいずれか一項に記載の画像診断用シリカナノ粒子。
- 前記共有結合として、アミド結合を含むことを特徴とする請求項4に記載の画像診断用シリカナノ粒子。
- 前記金属錯体が、ガドリニウム錯体であることを特徴とする請求項1~5のいずれか一項に記載の画像診断用シリカナノ粒子。
- 少なくとも、下記工程(a)、工程(b)及び工程(c)を経由して有機蛍光色素と金属錯体とを内包した画像診断用シリカナノ粒子を製造することを特徴とする画像診断用シリカナノ粒子の製造方法。
工程(a):同一分子内にアミノ基と、加水分解性置換基で置換されたシリル基とを有する有機分子と、当該アミノ基と反応する官能基を有する有機蛍光色素とを反応させる工程、
工程(b)同一分子内にアミノ基と、加水分解性置換基で置換されたシリル基とを有する有機分子と、当該アミノ基と反応する官能基を有するガドリニウム錯体とを反応させる工程、
工程(c):前記工程(a)及び工程(b)で得られた反応生成物を含ケイ素アルコキシドと混合し、塩基性条件下で加水分解反応を行う工程。 - 請求項1~6のいずれか一項に記載の画像診断用シリカナノ粒子と分子標識物質とが、有機分子を介して結合されていることを特徴とする生体物質標識剤。
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