WO2019142913A1 - Microparticules fluorescentes permettant la cartographie de la concentration de glucose à l'intérieur d'un tissu tridimensionnel - Google Patents

Microparticules fluorescentes permettant la cartographie de la concentration de glucose à l'intérieur d'un tissu tridimensionnel Download PDF

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WO2019142913A1
WO2019142913A1 PCT/JP2019/001475 JP2019001475W WO2019142913A1 WO 2019142913 A1 WO2019142913 A1 WO 2019142913A1 JP 2019001475 W JP2019001475 W JP 2019001475W WO 2019142913 A1 WO2019142913 A1 WO 2019142913A1
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glucose
fluorescent
group
fluorescent microparticles
unit
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PCT/JP2019/001475
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English (en)
Japanese (ja)
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昌治 竹内
淳 澤山
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国立大学法人東京大学
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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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • the present invention relates to fluorescent microparticles capable of continuously measuring glucose present in three-dimensional cell tissue, and continuous glucose monitoring using the fluorescent microparticles.
  • Biological tissues such as organs have a three-dimensional structure formed by cells. Biological functions of living tissue are known to appear inside tissue through interactions between cells and the surrounding environment.
  • three-dimensional tissue construction techniques have been developed in various fields, such as regenerative medicine research aimed at replacing organs and tissues, and “Organ on a chip” research for drug evaluation using human cell chips instead of animal experiments. Has attracted attention.
  • cell aggregates such as spheroids are used, and monitoring of the glucose concentration inside the spheroids etc. not only controls the environment of the culture system, but also reveals the mechanism of cell metabolism etc. are also useful for the purpose (for example, Non-Patent Document 1).
  • an object of the present invention is to provide a novel detection material and detection method capable of continuously measuring glucose in a three-dimensional cell tissue such as a spheroid in a simple, highly accurate and non-invasive manner. It is.
  • the inventors of the present invention have found, on the surface of fine particles having an average particle size on the order of micrometers, a fluorescent dye molecule exhibiting a fluorescence response by binding to glucose, and a target By covalently modifying a cell adhesion molecule capable of adhering to three-dimensional cell tissue, it is possible to produce glucose-responsive fluorescent microparticles that change fluorescence intensity according to increase or decrease of glucose concentration, By using the said fluorescent microparticles, it discovers that glucose in the inside of three-dimensional cell tissue, such as a spheroid, can be continuously monitored simply, with high accuracy and non-invasiveness, and the present invention has been completed based on such findings. It is
  • the present invention ⁇ 1> A fluorescent microparticle for measuring glucose in a three-dimensional cell tissue, wherein the core particle has an average particle size of the order of micrometers, and a fluorochrome unit exhibiting a fluorescence response by binding with glucose, A cell adhesion unit having a cell adhesion molecule; the fluorochrome unit and the cell adhesion unit having a structure covalently immobilized on the surface of the core particle, Said fluorescent microparticles; ⁇ 2> The fluorescent microparticles according to ⁇ 1>, wherein the core particle is selected from the group consisting of a silicon-containing material, a polymer, and a metal fine particle each having a functional group for modification on the surface; ⁇ 3> The fluorescent microparticles according to ⁇ 1>, wherein the core particle is a porous material having a modifying functional group on the surface; ⁇ 4> One or more functional groups selected from the group consisting of a carboxyl group, a thiol group,
  • Fluorescent microparticles according to claim 2 or 3, which are groups. ⁇ 5> The fluorescent microparticles according to any one of ⁇ 1> to ⁇ 4>, wherein the core particle has an average particle diameter of 100 nm to 100 ⁇ m.
  • ⁇ 6> The fluorescent microparticles according to any one of the above ⁇ 1> to ⁇ 5>, wherein the cell adhesion unit comprises a polypeptide or an oligopeptide consisting of four or more amino acid residues; ⁇ 7> The ⁇ 1>, wherein the cell adhesion unit comprises one or more selected from the group consisting of collagen, gelatin, proteoglycan, hyaluronic acid, fibronectin, laminin, tenascin, entactin, elastin, and compounds derived therefrom.
  • the fluorescent microparticles according to any one of ⁇ 5>; ⁇ 8>
  • the fluorochrome unit has a fluorophore and a quencher; the fluorophore is quenched by the quencher in the absence of glucose, but the fluorophore is bound to glucose by the fluorophore.
  • the fluorescent microparticles according to any one of the above; and ⁇ 11> the fluorescent dye unit is represented by the following formula (I) or (II): (In the formula (II), “PEG” represents a polyethylene glycol chain.)
  • the fluorescent micro particle according to any one of the above ⁇ 1> to ⁇ 10> is provided.
  • the present invention ⁇ 12> A glucose monitoring sensor comprising the fluorescent microparticles according to any one of ⁇ 1> to ⁇ 11>above; ⁇ 13> A glucose detection method comprising the step of detecting the presence of glucose in a three-dimensional cell tissue as a fluorescence response using the fluorescent microparticles according to any one of ⁇ 1> to ⁇ 11>above;> A continuous glucose monitoring method characterized by continuously monitoring the presence of glucose in a three-dimensional cell tissue by the glucose detection method described in ⁇ 13> above.
  • the size of the microparticles can be adjusted to a size suitable for three-dimensional cell tissue such as spheroid to be measured, and measurement can be performed without being affected by changes in the surrounding environment, The presence and concentration of glucose can be measured with high accuracy.
  • noncontact measurement is performed without consuming glucose at the time of measurement, it is possible to minimize the invasion in three-dimensional cell tissue, and to map the glucose concentration in three-dimensional cell tissue in a more natural state. become.
  • FIG. 1 is an electron microscope image and a fluorescence image of the fluorescent microparticles of the present invention.
  • A GF-particles,
  • B GF-RGD-particles,
  • c GF-Col-particles.
  • FIG. 2 is a SEM image of the fluorescent microparticles of the present invention.
  • A GF-particles,
  • B GF-RGD-particles,
  • c GF-Col-particles.
  • FIG. 3 is a graph showing (a) change in fluorescence spectrum when 0 to 1,000 mg / dL of glucose is added to the fluorescent microparticles of the present invention, and (b) a graph plotting change in fluorescence intensity at 470 nm. .
  • FIG. 4 is a fluorescence image when the fluorescent microparticles of the present invention are introduced into HepG2 (hepatic liver cells) spheroids.
  • A GF-particles
  • B GF-RGD-particles
  • c GF-Col-particles.
  • FIG. 5 is an image obtained by monitoring the glucose concentration inside the spheroid using the fluorescent microparticles of the present invention.
  • the fluorescent microparticles of the present invention are fluorescent microparticles for measuring glucose in three-dimensional cell tissue, 1) Core particles having an average particle size on the order of micrometers, 2) A fluorochrome unit that exhibits a fluorescence response upon binding to glucose, 3) It is characterized by being comprised by the cell adhesion unit which has a cell adhesion molecule. Furthermore, the fluorescent dye unit and the cell adhesion unit are characterized in that they have a structure immobilized by covalent bonding on the surface of the core particle. In general, "microparticles" are particles having a particle size of submicron to submillimeter.
  • the fluorescent microparticles of the present invention have a size capable of entering into the interior of three-dimensional cell tissue such as spheroid to be measured, and stably adhere to the three-dimensional cell tissue by the cell adhesion unit. be able to. And, since the surface of the core particle is provided with a fluorescent dye unit that changes the fluorescence intensity according to the increase or decrease of the glucose concentration, the inside of the three-dimensional cell tissue without invading the three-dimensional cell tissue by observing the fluorescence response The presence and concentration of glucose can be measured and monitored continuously and accurately.
  • “three-dimensional cell tissue” is a three-dimensional (typically spherical) cell aggregate in which a plurality of cells are aggregated and aggregated, and is also called a cell aggregate or spheroid.
  • Core particle is a microparticle (microparticulate) having an average particle size on the order of micrometers, and is a base of the fluorescent microparticle of the present invention. It is possible to adjust fluorescent microparticles to a desired size by appropriately changing the size of core particles used.
  • the average particle size of the core particles is 100 nm to 100 ⁇ m, preferably 1 to 20 ⁇ m, and more preferably 2 to 10 ⁇ m.
  • the material of the core particle is not particularly limited as long as it is a material that can be used for a living body such as cellular tissue, and it is processed into a material having a particle shape having a uniform particle diameter to some extent or such particle shape It can be a possible material.
  • silicon-containing materials, polymer polymers, and metal microparticles can be used as the core material.
  • silica gel, zeolite, etc. can be mentioned.
  • polystyrene, nylon, etc. can be mentioned as an example of a high molecular weight polymer
  • Gold (Au) microparticles can be mentioned as an example of metal particulates.
  • polylactic acid, polyethylene, polyphenol, polyurethane, polyacryl, benzoamine melamine, polycarbonate, polyolefin, polyester and the like can be used.
  • a core particle is a porous material from another viewpoint.
  • the fluorescent dye unit is immobilized on the surface of the core particle, but by using a porous material, fluorescent molecules can be immobilized also inside the particle, whereby fluorescence It offers the advantage of being less susceptible to the surrounding environment in binding molecules with glucose.
  • porous materials include inorganic porous materials such as mesoporous silica and zeolite, and porous polymer materials such as polystyrene beads.
  • porous polymer materials such as polylactic acid, polyethylene, polyphenol, polyurethane, polyacrylic, benzoamine melamine, polycarbonate, polyolefin and polyester can be used.
  • the core particle is a porous material, for example, the bulk density can be 3.0 to 4.5 g / cm 3 .
  • these core particles have a functional group for modification for modifying and immobilizing the fluorochrome unit and the cell adhesion unit on the surface thereof.
  • a functional group on the surface of the core particle is reacted with a functional group present at the end of the fluorescent dye unit or the like to form a covalent bond and chemically fix the functional unit to the surface of the core particle.
  • Such functional groups for modification include one or more functional groups selected from the group consisting of a carboxyl group, a thiol group, an isocyano group, a thioisocyano group, an epoxy group, an activated carboxylic acid ester, a maleimide group, an acetyl group, and an azide group. Groups can be mentioned.
  • the functional group for modification is a carboxyl group and the fluorochrome unit and the cell adhesion unit have an amino group
  • these units are immobilized on the surface of the core particle by forming an amide bond.
  • Techniques known in the art can be used to provide the functional group for modification on the surface of the core particle.
  • core particles having a functional group for modification on the surface examples include silica gel with a carboxyl group modified on the surface, polymer particles with a carboxyl group in the side chain, and gold fine particles with a thiol group on the surface. . However, it is not limited to these.
  • glucose which is a measurement object is what has a hydroxyl group (OH group)
  • a core particle does not have OH group on the surface.
  • surface treatment can be performed by silane coupling.
  • the cell adhesion unit is to impart cell adhesion to the fluorescent microparticles of the present invention. Thereby, when the fluorescent microparticles are in proximity to a three-dimensional cell tissue, they can adhere to the cells and can be stably present inside (or on the surface), and the fluorescence response change due to the presence of glucose can be detected more accurately can do.
  • the cell adhesion unit can comprise, as a cell adhesion factor, a polypeptide or oligopeptide consisting of four or more amino acid residues.
  • the cell adhesion unit can include one or more selected from the group consisting of collagen, gelatin, proteoglycan, hyaluronic acid, fibronectin, laminin, tenascin, entactin, elastin, and compounds derived therefrom. Mixtures of these can also be used.
  • Collagen is one of the proteins constituting the dermis, ligaments, tendons, bones, cartilage and the like, and is an element constituting the extracellular matrix.
  • the peptide chain of collagen protein has a primary structure in which "-(glycine)-(amino acid X)-(amino acid Y)-" and glycine repeat every three residues. In many types of collagen, three peptide chains gather to form a helical structure and are called tropocollagen.
  • one peptide chain of type I collagen has a sequence repeating 1014 amino acid residues and has a molecular weight of about 100,000. It is known that there are 30 or more types of human collagen.
  • type I collagen is mainly used for dermis, ligaments, tendons, bones and the like
  • type II collagen is mainly used for articular cartilage
  • the basement membrane which is the lining structure of all epithelial tissues, mainly contains type IV collagen.
  • the most abundant in the body is type I collagen.
  • water soluble collagen can be used.
  • gelatin means what was extracted in water by heating with water for a long time among collagens. The gelatin may be peptided.
  • the cell adhesion unit can use mammalian or fish-derived collagen, atelocollagen, gelatin, their derivatives and mixtures thereof.
  • "Atelocollagen” is collagen obtained by enzymatic treatment of removing telopeptides present at both ends of a collagen molecule, and is collagen that has become soluble in water. Atelocollagen may be peptided.
  • the cell adhesion unit forms a covalent bond on the surface of the core particle by the reaction with the functional group for modification by the functional group inherently contained in them or the functional group introduced by modification, and the surface of the core particle is formed. It can be immobilized on top.
  • An amino group etc. are illustrated as a functional group of a cell adhesion unit.
  • the fluorescent microparticles of the present invention at least include a fluorescent dye unit that exhibits a fluorescent response upon binding to glucose. Thereby, the presence of glucose in the three-dimensional cell tissue can be detected as a fluorescence response.
  • the fluorochrome unit has a fluorophore and a quencher.
  • a "fluorophore” is a site that contains a molecule that fluoresces upon being excited at a particular wavelength.
  • the “quencher” is a site containing an electron acceptor capable of reducing the emission of light from the fluorophore by interaction with the fluorophore, and such quenching action is eliminated by binding to glucose, thereby making the fluorophore The light emission is caused again.
  • the fluorophore is quenched by the quencher in the absence of glucose, the presence of glucose is ON-OFF due to the fluorophore emitting light due to the binding of the quencher to glucose. It becomes possible to detect as
  • said fluorophore comprises anthracene and said quencher comprises arylboronic acid.
  • said quencher comprises arylboronic acid.
  • a fluorophore and a quencher known in the art can also be used.
  • the fluorescent dye unit is immobilized by covalent bonding on the surface of the core particle. Therefore, preferably, the fluorescent dye unit has a functional group capable of forming a covalent bond by a chemical reaction with the modifying functional group on the surface of the core particle.
  • the fluorochrome unit has one or more amino groups at its end, and is immobilized on the surface of the fluorescent microparticles by covalent bonding of the amino group and the functional group for modification.
  • Preferred examples of the fluorescent dye unit used in the fluorescent microparticles of the present invention include compounds represented by the following formula (I).
  • the central atracene is a fluorophore and the two arylboronic acids on either side of it is a quencher.
  • the fluorescence of athracene is quenched by the arylboronic acid site in the absence of glucose, but when the arylboronic acid site is bound to glucose, the electrostatics of the nitrogen and boron atoms in the molecule are generated.
  • the interaction suppresses the electron transfer to the anthracene site, and the quenching action is eliminated, resulting in the emission of athracene fluorescence.
  • Such recognition mechanism allows the presence of glucose to be detected as an ON-OFF fluorescence response.
  • the compound of the formula (I) has two amino groups at the end, and the amino group reacts with the modifying functional group on the surface of the core particle to fluoresce the fluorescent dye unit. It can be immobilized on the surface of the microparticles by covalent bonding.
  • the terminal functional group of the compound of Formula (I) is an amino group was shown here, it is not limited to this, The functional group which can be covalently couple
  • Another preferable example of the fluorescent dye unit used in the fluorescent microparticles of the present invention includes a compound represented by the following formula (II).
  • PEG represents a polyethylene glycol chain.
  • the polyethylene glycol chain preferably has 2 to 60 repeating units. However, the length of the polyethylene glycol chain can be appropriately changed and used.
  • the compound represented by the formula (II) is different from the compound of the above formula (I) and has two maleimide groups at the terminal.
  • the fluorescent dye unit can be covalently immobilized on the surface of the fluorescent micro particle.
  • the fluorescent microparticles of the present invention can be prepared by gel production methods known in the art. For example, since a microparticulate material having a modifying functional group such as a carboxyl group on the surface is commercially available, such a microparticulate material is used as a core particle and a known amide condensing agent is used as shown in the examples described later.
  • the fluorescent microparticles of the present invention can be obtained by modifying and immobilizing the cell adhesion unit and the fluorescent dye unit sequentially on the surface of the core particle. The surface modification ratio of the fluorochrome unit and the cell adhesion unit can be appropriately adjusted.
  • an amide condensing agent DMT-MM can be mentioned.
  • the present invention further provides a method for detecting and monitoring glucose in three-dimensional cell tissue such as spheroids using the above-mentioned fluorescent microparticles, and a sensor including the fluorescent microparticles. It also relates to Specifically, the fluorescent microparticles of the present invention are brought into contact with the three-dimensional cell tissue to be measured, and the presence of glucose in the three-dimensional cell tissue is detected as a fluorescence response by irradiating light of a specific wavelength. be able to.
  • the fluorescent microparticles of the present invention can be introduced into three-dimensional cell tissue, for example, by forming them in a culture solution in three-dimensional cell culture to form spheroids.
  • a compact sensor device provided with a light source such as an LED, a detector such as a photomultiplier tube, a wireless data transfer system and the like can be manufactured and applied to a culture system to be used as a continuous glucose monitoring sensor.
  • the detection of a fluorescence signal can use apparatuses, systems, etc. well-known in the said technical field, such as a fluorescence measurement apparatus and a microscope.
  • microparticles immobilized on the surface of glucose responsive fluorochrome only
  • microparticles GF-RGD-particle
  • fluorochrome and RGD peptide are immobilized on the surface
  • fluorochrome and atelocollagen immobilized on the surface Microparticle
  • Both RGD peptide and atelocollagen are molecules having cell adhesion.
  • each fluorescent microparticle was prepared by the procedure of adding a fluorescent dye.
  • the glucose-responsive dye contains anthracene which acts as a specific glucose recognition site and as a fluorogenic site, respectively.
  • the fluorescence is quenched in the absence of the glucose molecule, but when the glucose molecule is attached to the diboronic acid moiety, it exhibits fluorescence.
  • FIG. 1 An electron microscope image and a fluorescence image of fluorescent microparticles in a state before introduction into a spheroid are shown in FIG. 1, and their SEM images are shown in FIG. All the figures are (a) GF-particles, (b) GF-RGD-particles, and (c) GF-Col-particles. From these images, it can be seen that the fine particles can be modified with protein and that homogeneous particles are supported.
  • FIG. 3 (a) (excitation wavelength: 405 nm). It is FIG. 3 (b) which plotted the fluorescence intensity change in 470 nm. As a result, it was confirmed that the glucose responsive dye increased in fluorescence intensity in response to an increase in glucose concentration.
  • these fluorescent microparticles were introduced into the HepG2 (hepatic liver cells) spheroids using an ultra-low adhesion multilayer plate, and a Lucose responsiveness test was performed. Time-lapse video was used to observe the fluorescence intensity of the fluorescent microparticles to monitor glucose concentration.
  • FIG. 5 shows the result of monitoring the glucose concentration inside the spheroid using “GF-Col-particle” (after 0 to 2 hours).
  • GF-Col-particles can be used to suitably measure the glucose concentration inside the spheroid.

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Abstract

Le problème décrit par la présente invention est de fournir un nouveau matériau de détection et une méthode de détection qui permettent de réaliser de manière pratique, précise et non invasive une mesure continue du glucose à l'intérieur de tissus cellulaires tridimensionnels tels que des sphéroïdes. La solution de l'invention porte sur des microparticules fluorescentes qui mesurent le glucose à l'intérieur d'un tissu cellulaire tridimensionnel, et comprennent des particules à noyau, dont le diamètre de particule moyen est de l'ordre de micromètres, une unité de pigment fluorescent qui présente une réponse de fluorescence par liaison au glucose, et une unité d'adhésion cellulaire possédant des molécules adhérant aux cellules, l'unité de pigment fluorescent et l'unité d'adhésion cellulaire étant fixées par des liaisons covalentes à la surface des particules à noyau.
PCT/JP2019/001475 2018-01-18 2019-01-18 Microparticules fluorescentes permettant la cartographie de la concentration de glucose à l'intérieur d'un tissu tridimensionnel WO2019142913A1 (fr)

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