Tissue-marking agent containing fluorescent dye-labelled particles

Abstract

Provided is a tissue-marking agent that improves the dispersion stability of fine particles labelled with a fluorescent dye in an aqueous medium.　The present invention involves dispersing fine particles labelled with a fluorescent dye in an aqueous medium containing a water-soluble viscous substance.

Description

Tissue marking agent containing fluorescent dye-labeled particles

[0001] The present invention relates to a tissue marking agent containing fluorescent dye-labeled particles, and more particularly to a tissue marking agent containing fluorescent dye-labeled particles in a specific aqueous medium.

[0002] In surgery to remove cancerous tissue, the amount of normal tissue that can be preserved is important for maintaining normal organ function after surgery. For this reason, tumor sites are marked before surgery to avoid removing unnecessary tissue. In particular, gastrointestinal cancers are usually located inside the gastrointestinal tract, so there is a need for a marking method that allows tumor sites to be identified from outside the gastrointestinal tract.

[0003] In the past, when performing surgery for gastrointestinal cancer, the tumor site was marked by injecting ink into it using an endoscope (ink injecting method) or by placing a clip.

[0004] However, marking with ink is difficult to see because it does not use a fluorescent agent, and the diffusion of the ink makes it difficult to determine the boundary between the tumor site and normal tissue. Furthermore, clips cannot be used in laparoscopic surgery, and even in open surgery, they can be difficult to palpate from the serosal surface (outside the digestive tract).

[0005] In recent years, endoscopic clips using fluorescent materials have been developed, but there is a problem that the clip gets caught in the stapler when trying to cut tissue with the stapler (Non-Patent Document 1).

[0006] To address these problems with the ink-drop and clipping methods, the use of indocyanine green as a tissue marking agent has been investigated. However, because indocyanine green is a low-molecular-weight compound, it rapidly diffuses even when administered locally, and does not function adequately as a tissue marking agent (Non-Patent Documents 2 and 3).

[0007] It has also been reported that tissues were marked using hollow silica nanoparticles with indocyanine green (ICG) electrostatically adsorbed onto their surfaces (Non-Patent Document 4). However, this marking agent simply disperses ICG-adsorbed silica nanoparticles in water, and no consideration is given to dispersion stability. Furthermore, this document states that marking by silica nanoparticles with ICG electrostatically adsorbed was confirmed for up to 12 days, and there is a demand for longer-term marking.

[0008] Narihiro et al., International Journal of Surgery Yoshiaki Ozawa, Masahiko Murakami, (2020). Examination of preoperative marking methods in laparoscopic rectal cancer surgery - Comparison of ink marking method and ICG fluorescence method. Showa Gakushikai Zasshi, 80.1:1-6. Tetsuta Satoyoshi et al., Surgical Endoscopy (2021) 35:763-769 Adrian Garcia Badaracco et al., Indocyanine green modified silica shells for colon tumor marking, Appl Surf Sci. 2020 January 1; 499: doi:10.1016 / j. apsusc. 2019.143885

[0009] The inventors have investigated biological tissue marking agents containing microparticles labeled with fluorescent dyes and have discovered a new problem: simply dispersing the microparticles labeled with fluorescent dyes in an aqueous medium results in low dispersion stability, leading to a concentration gradient within the injection needle, making it difficult to administer the appropriate amount of tissue marking agent and, in some cases, causing the needle to become clogged.

[0010] The present invention aims to solve this problem, that is, to provide a tissue marking agent in which the dispersion stability of fluorescent dye-labeled microparticles in an aqueous medium is improved.

[0011] The present inventors have discovered that dispersing fine particles labeled with a fluorescent dye in an aqueous medium containing a water-soluble viscous substance improves the dispersion stability of the particles, leading to the development of the present invention.

[0012] That is, embodiments of the present invention are as follows. [1] A tissue marking agent (tissue marking composition) comprising fluorescent dye-labeled microparticles in an aqueous medium, the fluorescent dye-labeled microparticles being dispersed in an aqueous medium in which a water-soluble viscous substance is dissolved. [2] The tissue marking agent according to [1], wherein the water-soluble viscous substance comprises a polyhydric alcohol, a polyether or a salt thereof, a water-soluble protein, or a polysaccharide or a salt thereof. [3] The tissue marking agent according to [2], wherein the water-soluble viscous substance comprises a polysaccharide or a salt thereof. [4] The tissue marking agent according to any of [1] to [3], wherein the fluorescent dye is covalently bonded to the microparticles. [5] The tissue marking agent according to any of [1] to [4], wherein the pH of the tissue marking agent is in the range of 5.0 to 8.0. [6] The tissue marking agent according to any one of [3] to [5], wherein the polysaccharide is at least one selected from the group consisting of hyaluronic acid, alginic acid, guar gum, xanthan gum, acacia gum, pullulan, dextran, and agarose, carrageenan, pectin, galactomannan, xanthan gum, gellan gum, curdlan, carboxymethylcellulose, chitin / chitosan, and carrageenan. [7] The tissue marking agent according to [6], wherein the polysaccharide includes or is alginic acid, dextran, and / or hyaluronic acid. [8] The tissue marking agent according to any one of [1] to [7], wherein the fluorescent dye is selected from the group consisting of indocyanine green, coumarin, rhodamine, xanthene, porphyrin, fluorescamine, fluorescein, and derivatives thereof. [9] The tissue marking agent according to [8], wherein the fluorescent dye is indocyanine green or a derivative thereof.

[10] The tissue marking agent according to any one of [1] to [9], wherein the microparticles are selected from the group consisting of silica particles, polyester particles, polyurethane particles, silicon particles, polyethylene particles, polypropylene particles, polystyrene particles, polymethacrylate particles, titanium alloy particles, hydroxyapatite particles, cellulose particles, silk particles, alumina particles, zirconia particles, and titania particles.

[11] The tissue marking agent according to any one of [1] to

[10] , wherein the microparticles have an average particle size in the range of 1 nm to 2 μm.

[12] The tissue marking agent according to any one of [1] to

[11] , wherein the dispersion medium is at least one selected from the group consisting of physiological saline, phosphate buffer, acetate buffer, citrate buffer, prolamin buffer, carbon buffer, and purified water.

[13] The tissue marking agent according to any one of [1] to

[12] , wherein the concentration of the microparticles in the dispersion medium is in the range of 0.01 mg / mL to 1000 mg / mL.

[14] The tissue marking agent according to any one of [1] to

[13] , wherein the concentration of the water-soluble viscous substance in the dispersion medium is in the range of 0.001% by mass to 20% by mass.

[15] The tissue marking agent according to any one of [1] to

[14] , for marking a local area of ​​the digestive tract.

[16] Use for preparing a tissue marking agent, which is an aqueous composition comprising fluorescent dye-labeled microparticles dispersed in an aqueous medium containing a water-soluble viscous substance dissolved therein.

[17] The use according to

[16] , wherein the water-soluble viscous substance comprises a polyhydric alcohol, a polyether or a salt thereof, a water-soluble protein, or a polysaccharide or a salt thereof.

[18] The use according to

[16] , wherein the water-soluble viscous substance comprises a polysaccharide or a salt thereof.

[19] The use according to any of

[16] to

[18] , wherein the fluorescent dye is covalently bonded to the microparticles.

[20] The use according to any of

[16] to

[19] , wherein the pH of the tissue marking agent is in the range of 5.0 to 8.0.

[21] The use according to any one of

[18] to

[20] , wherein the polysaccharide is at least one selected from the group consisting of hyaluronic acid, alginic acid, guar gum, xanthan gum, acacia gum, pullulan, dextran, and agarose, carrageenan, pectin, galactomannan, xanthan gum, gellan gum, curdlan, carboxymethylcellulose, chitin / chitosan, and carrageenan.

[22] The use according to

[21] , wherein the polysaccharide comprises or is alginic acid, dextran, and / or hyaluronic acid.

[23] The use according to any one of

[16] to

[22] , wherein the fluorescent dye is selected from the group consisting of indocyanine green, coumarin, rhodamine, xanthene, porphyrin, fluorescamine, and derivatives thereof.

[24] The use according to

[23] , wherein the fluorescent dye is indocyanine green or a derivative thereof.

[25] The use according to any one of

[16] to

[24] , wherein the microparticles are selected from the group consisting of silica particles, polyester particles, polyurethane particles, silicon particles, polyethylene particles, polypropylene particles, polystyrene particles, polymethacrylate particles, titanium alloy particles, hydroxyapatite particles, cellulose particles, silk particles, alumina particles, zirconia particles, and titania particles.

[26] The use according to any one of

[16] to

[25] , wherein the microparticles have an average particle size in the range of 1 nm to 2 μm.

[27] The use according to any one of

[16] to

[26] , wherein the dispersion medium is at least one selected from the group consisting of physiological saline, phosphate buffer, acetate buffer, citrate buffer, prolamin buffer, carbon buffer, and purified water.

[28] The use according to any one of

[16] to

[27] , wherein the concentration of the microparticles in the dispersion medium is in the range of 0.01 mg / mL to 1000 mg / mL.

[29] The use according to any one of

[16] to

[28] , wherein the concentration of the water-soluble viscous substance in the dispersion medium is in the range of 0.001 mass% to 20 mass%.

[30] The use according to any one of

[16] to

[29] , wherein the tissue marking agent is used to mark a localized area of ​​the digestive tract.

[31] An aqueous composition used for tissue marking, wherein microparticles labeled with a fluorescent dye are dispersed in an aqueous medium in which a water-soluble viscous substance is dissolved.

[32] The aqueous composition for use according to

[31] , wherein the water-soluble viscous substance comprises a polyhydric alcohol, a polyether or a salt thereof, a water-soluble protein, or a polysaccharide or a salt thereof.

[33] The aqueous composition for use according to

[31] , wherein the water-soluble viscous substance comprises a polysaccharide or a salt thereof.

[34] The aqueous composition for use according to any of

[31] to

[33] , wherein the fluorescent dye is covalently bound to the microparticles.

[35] The aqueous composition for use according to any one of

[31] to

[34] , wherein the pH of the aqueous composition is in the range of 5.0 to 8.0.

[36] The aqueous composition for use according to any one of

[31] to

[35] , wherein the polysaccharide is at least one selected from the group consisting of hyaluronic acid, alginic acid, guar gum, xanthan gum, acacia gum, pullulan, dextran, and agarose, carrageenan, pectin, galactomannan, xanthan gum, gellan gum, curdlan, carboxymethylcellulose, chitin / chitosan, and carrageenan.

[37] The aqueous composition for use according to

[36] , wherein the polysaccharide comprises or is alginic acid, dextran, and / or hyaluronic acid.

[38] The aqueous composition for use according to any one of

[31] to

[37] , wherein the fluorescent dye is selected from the group consisting of indocyanine green, coumarin, rhodamine, xanthene, porphyrin, fluorescamine, and derivatives thereof.

[39] The aqueous composition for use according to

[38] , wherein the fluorescent dye is indocyanine green or a derivative thereof.

[40] The aqueous composition for use according to any one of

[31] to

[39] , wherein the microparticles are selected from the group consisting of silica particles, polyester particles, polyurethane particles, silicon particles, polyethylene particles, polypropylene particles, polystyrene particles, polymethacrylate particles, titanium alloy particles, hydroxyapatite particles, cellulose particles, silk particles, alumina particles, zirconia particles, and titania particles.

[41] The aqueous composition for use according to any one of

[31] to

[40] , wherein the microparticles have an average particle size in the range of 1 nm to 2 μm.

[42] The aqueous composition for use according to any one of

[31] to

[41] , wherein the dispersion medium is at least one selected from the group consisting of physiological saline, phosphate buffer, acetate buffer, citrate buffer, prolamine buffer, carbon buffer, and purified water.

[43] The aqueous composition for use according to any one of

[31] to

[42] , wherein the concentration of the microparticles in the dispersion medium is in the range of 0.01 mg / mL to 1000 mg / mL.

[44] The aqueous composition for use according to any one of

[31] to

[43] , wherein the concentration of the water-soluble viscous substance in the dispersion medium is in the range of 0.001% by mass to 20% by mass.

[45] The aqueous composition for use according to any one of

[31] to

[44] , wherein the tissue marking agent is used to mark a localized area of ​​the digestive tract.

[46] A tissue marking method, comprising locally administering, for example, injecting, the tissue marking agent according to any one of [1] to

[15] or the aqueous composition according to any one of

[31] to

[45] to a target tissue site.

[47] The tissue marking agent according to any one of [1] to

[15] or the aqueous composition according to any one of

[31] to

[45] , which contains an antioxidant.

[48] The tissue marking agent or aqueous composition according to

[47] , which contains 0.02 mg / mL to 10 mg / mL of the antioxidant.

[49] The use according to any one of

[16] to

[30] , wherein the tissue marking agent contains an antioxidant.

[50] The use according to

[49] , wherein the tissue marking agent contains the antioxidant at 0.02 mg / mL to 10 mg / mL.

[0013] Dispersing fluorescent dye-labeled microparticles in an aqueous medium containing a water-soluble viscous substance improves the dispersion stability of the particles, thereby enabling the administration of an appropriate amount of tissue marking agent and preventing clogging of the injection needle.

[0014] Figure 1 shows electron microscope photographs of silica nanoparticles or indocyanine green-conjugated silica nanoparticles (siICG) with particle sizes of 20, 65, or 200 nm. Figure 2 shows the dispersion stability of siICG when siICG with a particle size of 200 nm is dispersed at a concentration of 5 mg / mL in phosphate buffer containing 0, 0.2, or 0.4% by mass sodium hyaluronate. Figure 3 shows the change over time in the transmittance of light at a wavelength of 500 nm when siICG with a particle size of 20, 65, or 200 nm is dispersed at a concentration of 5 mg / mL in phosphate buffer containing 0, 0.1, 0.2, or 0.4% by mass sodium hyaluronate. Figure 4 shows the change over time in the transmittance of light at a wavelength of 500 nm when siICG with a particle size of 20, 65, or 200 nm is dispersed at a concentration of 5 mg / mL in phosphate buffer containing 0 or 0.5% by mass sodium alginate. Figure 5 shows the time course of the transmittance of light at a wavelength of 500 nm when siICG particles with a particle size of 20, 65, or 200 nm were dispersed at a concentration of 5 mg / mL in phosphate buffer containing 0, 2, or 10% by mass of dextran. Figure 6 shows the results of subcutaneous administration of low molecular weight indocyanine green to the back of a mouse. Even one day after administration, no low molecular weight indocyanine green remained at the administration site. Figure 7 shows photographs taken with a near-infrared camera 28 days after subcutaneous administration of siICG particles with a particle size of 20, 65, or 200 nm dispersed at a concentration of 10 mg / mL in phosphate buffer containing 0, 0.2, or 0.4% by mass of sodium hyaluronate. Even 28 days after administration, siICG remained at the administration site and exhibited strong near-infrared emission. 8 shows photographs taken with a near-infrared camera 56 days after subcutaneous administration of siICG solutions prepared by dispersing siICG particles with diameters of 20, 65, or 200 nm in phosphate buffer containing 0, 0.2, or 0.4% by mass of sodium hyaluronate at a concentration of 10 mg / mL. Even 56 days after administration, the siICG remained at the administration site and exhibited strong near-infrared emission.Figure 9 shows photographs of siICG particles with diameters of 20, 65, or 200 nm dispersed at a concentration of 10 mg / mL in phosphate buffer containing 0.2% by mass sodium hyaluronate, administered to the gastric submucosa of a rabbit, and then taken with a near-infrared camera 21 days after administration. An excitation light intensity of 2 was used. All siICG particles with diameters of 20, 65, or 200 nm remained at the administration site even 21 days after administration, exhibiting strong near-infrared emission. Figure 10 shows photographs of siICG particles with diameters of 200, 450, 700, 1000, or 1500 nm dispersed at a concentration of 10 mg / mL in phosphate buffer containing 0.2% by mass sodium hyaluronate, administered subcutaneously to a mouse, and then taken with a near-infrared camera 28 days after administration. An excitation light intensity of 2 was used. The photograph on the far right of the top row was taken to confirm the location where the mouse was placed. For all samples, mice were placed in approximately the same position. 200, 450, 700, 1000, or 1500 nm siICG remained at the administration site and exhibited near-infrared emission even 28 days after administration. Figure 11 shows photographs taken with a near-infrared camera 28 days after subcutaneous administration of siICG particles with a particle size of 200 nm dispersed in phosphate buffer containing 0.2% sodium hyaluronate at concentrations of 1, 10, and 300 mg / mL. The siICG solutions at each concentration were administered subcutaneously to mice. The photograph on the far left of the top row was taken to confirm the location of the mouse placement. For all samples, mice were placed in approximately the same position. Photographs were generally taken at an excitation light intensity of 2, except for the bottom photograph of 1 mg / mL siICG, which was taken at an excitation light intensity increased from 2 to 10. siICG at concentrations of 1, 10, and 300 mg / mL remained at the administration site even 28 days after administration and exhibited near-infrared emission. Figure 12 shows a graph showing the fluorescence intensity of siICG solutions prepared by dispersing siICG with a particle size of 200 nm in a phosphate buffer containing 0.2% by mass sodium hyaluronate at a concentration of 10 mg / mL and adding ascorbic acid as an antioxidant at concentrations of 0.04, 0.4, or 4 mg / mL, stored in the dark at 4°C for 3 days, and then irradiated with near-infrared light. The fluorescence intensity is shown as a ratio of the fluorescence intensity immediately before storage in the dark to 100%.The addition of ascorbic acid suppressed the decrease in the fluorescence intensity of indocyanine green.

[0015]

[0023] Embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments. <Tissue marking agent of the present invention> In this specification, the term "tissue marking agent" means an agent used to mark tissue during surgery or the like. The tissue marking agent of the present invention comprises fine particles labeled with a fluorescent dye, a water-soluble viscous substance, and a dispersion medium containing at least water, and the fine particles labeled with a fluorescent dye are dispersed in an aqueous viscous medium containing the water-soluble viscous substance, typically in which the water-soluble viscous substance is dissolved.

[0016] The fluorescent dye is not particularly limited, but examples include indocyanine green, coumarin, rhodamine, xanthene, porphyrin, fluorescamine, fluorescein, and derivatives thereof. Two or more fluorescent dyes may be combined, for example, a mixture of particles in which two or more fluorescent dyes are adsorbed or bound to particles of different particle sizes or different materials may be used. Because near-infrared cameras are used in surgical procedures and the near-infrared region allows for highly sensitive observation with no background signal, fluorescent dyes that emit light in the near-infrared region, such as indocyanine green or its derivatives, are preferred.

[0017] The microparticles are not particularly limited, but are preferably made of biocompatible materials. Furthermore, the microparticles may be biodegradable or non-biodegradable, but non-biodegradable particles are more preferred from the viewpoint of maintaining the tissue marking agent in the body for a long period of time. Examples of microparticles are not particularly limited, but include, for example, silica particles, polyester particles, polyurethane particles, silicon particles, polyethylene particles, polypropylene particles, polystyrene particles, polymethacrylate particles, titanium alloy particles, hydroxyapatite particles, cellulose particles, silk particles, alumina particles, zirconia particles, and titania particles. Two or more of these particles may be combined, for example, a mixture of two or more types of particles may be used.

[0018] In microparticles labeled with fluorescent dyes, the fluorescent dye is incorporated into the microparticles or bound or adsorbed to the microparticles on their surface or interior, thereby suppressing the rapid diffusion that occurs when the fluorescent dye is administered locally alone. As used herein, "bound" refers to a chemically bound state, and "adsorption" refers to a state of physical proximity due to physical interaction, chemical interaction, or both. Examples of "bound" include covalent bonding, ionic bonding, and hydrogen bonding. Examples of "adsorption" include a state of physical proximity due to electrostatic interaction, affinity, metal coordination, physical adsorption, host-guest interaction, hydrophobic interaction, π-stacking interaction, van der Waals forces, and dipole-dipole interaction. Depending on the type of interaction between the fluorescent dye and the microparticles, the diffusibility of the fluorescent dye after injection of the tissue marking agent into tissue may vary. To prevent diffusion of the fluorescent dye and enable clear and accurate marking of the target site over a long period of time, it is preferable for the fluorescent dye to be covalently bound to the microparticles.

[0019] The average particle size of the microparticles is not particularly limited, but can typically be in the range of 1 nm to 2 μm. However, as demonstrated in the Examples described below, from the viewpoint of dispersion stability and fluorescent signal intensity, the average particle size of the microparticles is preferably in the range of 10 nm to 1.8 μm, more preferably in the range of 15 nm to 1.5 μm, more preferably in the range of 30 nm to 1 μm, even more preferably in the range of 40 nm to 700 nm, and even more preferably in the range of 50 nm to 500 nm. A particularly preferred range is 65 nm to 300 nm. In the present specification, the term "average particle size" refers to a value determined by randomly selecting photographs of particles observed with a transmission electron microscope (e.g., JEM-2100F, manufactured by JEOL Ltd.) and measuring and averaging the particle sizes (major diameters) of 100 particles using Image J (ver. 1.53k; open-source, public domain image processing software).

[0020] The amount of fluorescent dye in the fluorescent dye-labeled microparticles can be in the range of 0.01 to 5.0 μg per 1 mg of microparticles, preferably in the range of 0.1 to 2.5 μg, more preferably in the range of 0.25 to 1.2 μg, and particularly preferably in the range of 0.5 to 0.6 μg.

[0021] In the present invention, the fluorescent dye-labeled microparticles described above are dispersed in an aqueous medium containing a water-soluble viscous substance. Dispersing the fluorescent dye-labeled microparticles described above in such a dispersion medium improves the dispersion stability of the microparticles, resulting in a uniform dispersion liquid that remains uniform for a long period of time. The dispersion medium is typically an aqueous dispersion medium, and an aqueous dispersion medium whose safety has been confirmed when applied to living organisms is preferred. Examples of aqueous dispersion mediums include physiological saline, phosphate buffer, acetate buffer, citrate buffer, prolamin buffer, carbon buffer, and purified water. The dispersion medium can be used alone or in combination with multiple types. Furthermore, various buffers may be included, but it is preferable that the dispersion medium does not include basic substances.

[0022] A water-soluble viscous substance is a water-soluble substance that can increase the viscosity of a dispersion medium and is typically soluble in the dispersion medium. Such substances are typically polymeric compounds containing multiple hydrophilic functional groups, such as hydroxyl groups. To provide the dispersion medium with an appropriate viscosity, they preferably have a molecular weight (Mw) of 10,000 to 1,000,000, more preferably 10,000 to 1,000,000. Preferred water-soluble viscous substances include polyhydric alcohols, polyethers or their salts, water-soluble proteins, and polysaccharides or their salts. Of these, biocompatible water-soluble viscous substances are more preferred. In particular, polysaccharides or their salts exhibit excellent dispersion stability of microparticles, as demonstrated in the Examples below, making them particularly preferred water-soluble viscous substances from the standpoints of microparticle dispersion stability and biocompatibility.

[0023] Examples of polyhydric alcohols include ethylene glycol and glycerin. Furthermore, from the viewpoint of imparting an appropriate viscosity to the dispersion medium, polyhydric alcohols having a molecular weight (Mw) of 50 to 1,000 are preferred. Examples of polyethers include polyethylene glycol. Furthermore, from the viewpoint of imparting an appropriate viscosity to the dispersion medium, polyethers having a molecular weight (Mw) of 1,000 to 1,000,000 are preferred. Furthermore, examples of water-soluble proteins include albumin (particularly human serum albumin), fibrin, globulin (particularly human immunoglobulin), and the like. In this specification, unless otherwise specified, "molecular weight" refers to the weight-average molecular weight, and this weight-average molecular weight refers to the weight-average molecular weight determined by gel permeation chromatography (GPC).

[0024] The polysaccharide is not particularly limited, and examples thereof include hyaluronic acid, alginic acid, guar gum, xanthan gum, acacia gum, pullulan, dextran, agarose, carrageenan, pectin, galactomannan, xanthan gum, gellan gum, curdlan, carboxymethylcellulose, chitin / chitosan, and carrageenan. Polysaccharides whose safety when injected into the body has been established are preferred, and from the viewpoint of dispersion stability and biocompatibility of the microparticles, hyaluronic acid, dextran, and alginic acid are more preferred, with hyaluronic acid and alginic acid being particularly preferred. The polysaccharide may be in the form of a salt, and is not particularly limited as long as it is biocompatible. Examples of the salt include inorganic base salts. Examples of the inorganic base salt include salts derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum. Polysaccharides or salts thereof can be used singly or in combination. The polysaccharide or its salt is usually dissolved in an aqueous medium and is not adsorbed or bound to the fine particles.

[0025] The concentration of the microparticles in the dispersion medium is not particularly limited and can usually be 0.01 mg / mL or more and 1000 mg / mL or less, preferably in the range of 0.1 mg / mL or more and 500 mg / mL or less, more preferably in the range of 1 mg / mL or more and 400 mg / mL or less, even more preferably in the range of 10 mg / mL or more and 300 mg / mL or less, and particularly preferably in the range of 50 mg / mL or more and 300 mg / mL or less.

[0026] The concentration of the water-soluble viscous substance in the dispersion medium varies depending on the water-soluble viscous substance used, but can usually be adjusted within a range of 0.001% by mass to 20% by mass to prepare a dispersion medium with an appropriate viscosity.In the case of a polyhydric alcohol-containing medium, the concentration of the polyhydric alcohol in the dispersion medium varies depending on the molecular weight and hydrophilicity of the polyhydric alcohol used, and it is preferable to select an appropriate range of concentration from the viewpoint of solubility and particle dispersion stability.Usually, it can be selected from a range of 0.01% by mass to 20% by mass, preferably from a range of 0.1% by mass to 15% by mass, and particularly preferably from 0.2% by mass to 10% by mass.In the case of a polyether-containing medium, the concentration of the polyether in the dispersion medium varies depending on the molecular weight and hydrophilicity of the polyether used, and it is preferable to select an appropriate range of concentration from the viewpoint of solubility and particle dispersion stability. Typically, the concentration can be selected from the range of 0.01% by mass to 20% by mass, preferably from the range of 0.1% by mass to 15% by mass, and particularly preferably from 0.2% by mass to 10% by mass. In the case of a water-soluble protein-containing medium, the protein concentration in the dispersion medium varies depending on the molecular weight and hydrophilicity of the protein used, and it is preferable to select a concentration within an appropriate range from the viewpoints of solubility and particle dispersion stability. Typically, the concentration can be selected from the range of 0.01% by mass to 20% by mass, preferably from the range of 0.1% by mass to 15% by mass, and particularly preferably from the range of 0.2% by mass to 10% by mass. In the case of a medium containing a polysaccharide or a salt thereof, the concentration of the polysaccharide or a salt thereof in the dispersion medium varies depending on the molecular weight and hydrophilicity of the polysaccharide or a salt thereof used, and it is preferable to select a concentration within an appropriate range from the viewpoints of solubility and particle dispersion stability. Typically, the concentration can be selected from the range of 0.001% by mass to 20% by mass, and preferably from the range of 0.01% by mass to 10% by mass. In the case of a medium containing hyaluronic acid or its salt, the concentration of hyaluronic acid or its salt in the dispersion medium can usually be selected from the range of 0.01% by mass to 10% by mass, preferably 0.1% by mass to 5% by mass, more preferably 0.15% by mass to 1% by mass, and particularly preferably 0.15% by mass to 0.5% by mass.In the case of a medium containing alginic acid or a salt thereof, the concentration of alginic acid or a salt thereof in the dispersion medium is usually selected from the range of 0.01% by mass to 10% by mass, preferably 0.1% by mass to 5% by mass, more preferably 0.15% by mass to 1% by mass, and particularly preferably 0.2% by mass to 0.8% by mass. In the case of a medium containing dextran or a salt thereof, the concentration of dextran or a salt thereof in the dispersion medium is usually selected from the range of 2.0% by mass to 20% by mass, preferably 3.0% by mass to 17% by mass, more preferably 5.0% by mass to 15% by mass, and particularly preferably 8.0% by mass to 12.0% by mass.

[0027] The pH of the tissue marking agent is preferably in the range of about 5.0 to about 8.0, more preferably about 5.5 to about 8.0, about 6.0 to about 8.0, about 6.5 to about 8.0, about 5.0 to about 7.5, about 5.5 to about 7.5, about 6.0 to about 7.5, or about 6.5 to about 7.5. A pH within this range not only ensures high safety by preventing inflammation upon localized injection into the body, but also maintains good solubility of the water-soluble viscous substance in the dispersion medium, making it less likely to be adsorbed or bound to microparticles. The tissue marking agent may optionally contain various additives. For example, a dye that does not require a near-infrared camera to confirm successful administration at the time of administration, typically a dye that can be confirmed visually, or an antioxidant may be added to prevent a decrease in the fluorescence intensity of the above-mentioned fluorescent dyes (e.g., indocyanine green) due to oxidation during storage. Examples of such dyes include indigo carmine, cardio blue, brilliant blue, methylene blue, toliidine blue, trypan blue, etc., and examples of antioxidants include ascorbic acid, sodium ascorbate, vitamin E, etc. The amount of antioxidant such as ascorbic acid or sodium ascorbate added to the tissue marking agent is usually 0.01 mg / mL or more, preferably 0.02 mg / mL to 10 mg / mL, more preferably 0.04 mg / mL to 7 mg / mL, and particularly preferably 2 mg / mL to 6 mg / mL.

[0028] <Method for Producing the Tissue Marking Agent of the Present Invention> The tissue marking agent of the present invention is produced by adding fluorescent dye-labeled particles to a dispersion medium containing a water-soluble viscous substance, and stirring the mixture.

[0029] Fluorescent dye-labeled microparticles are produced by labeling the interior or surface of the microparticle with a fluorescent dye through physical interaction, chemical interaction, or both. Methods for labeling microparticles with fluorescent dyes are well known in the art to which the present invention pertains. Typically, functional groups that induce physical and / or chemical interaction between the microparticle and the fluorescent dye are introduced into one or both of the microparticle and the fluorescent dye, and the two are then bound or adsorbed through the functional groups. For example, depending on the fluorescent dye used, the surface of the microparticle may be appropriately modified using a silane coupling agent or the like to change the surface charge, hydrophobicity, hydrophilicity, etc. of the microparticle, or to introduce reactive functional groups into the microparticle. Similarly, depending on the modification introduced into the surface of the microparticle, if necessary, appropriate modifications, such as the introduction of corresponding functional groups, may also be introduced into the fluorescent dye. After introducing appropriate modifications into one or both of the microparticle and the fluorescent dye, the two are brought into contact (reacted) to generate the desired physical and / or chemical interaction, resulting in binding or adsorption of the two. Examples of functional groups that can be introduced into the microparticles and / or fluorescent dyes include amino groups, carboxyl groups, thiol groups, vinyl groups, epoxy groups, styryl groups, methacryl groups, acrylic groups, ureido groups, mercapto groups, and isocyanate groups.

[0030] Methods for modifying fine particles with silane coupling agents are well known in the technical field to which the present invention pertains. A method for modifying alumina particles with a silane coupling agent is described, for example, in Composites Science and Technology 68 (2008) 2965-2975. A method for modifying zirconia particles with a silane coupling agent is described, for example, in Langmuir 2008, 24, 11497-11505. A method for modifying cellulose particles with a silane coupling agent is described, for example, in Journal of Colloid and Interface Science 289 (2005) 249-261. A method for modifying titanium alloy particles and titania particles with a silane coupling agent is described, for example, in Colloids and Surfaces A: Physicochem. Eng. Aspects 413 (2012) 273-279. A method for modifying hydroxyapatite particles with a silane coupling agent is described, for example, in Materials Science and Engineering C 58 (2016) 675-681. A method for modifying polypropylene particles with a silane coupling agent is described, for example, in Polymer Composites 32.10 (2011): 1568-1583. A method for modifying polystyrene particles with a silane coupling agent is described, for example, in Chem. Mater. 2002, 14, 1325-1331 and Applied Surface Science 499 (2020) 143885, and polyester particles, polyurethane particles, silicone particles, polyethylene particles, silk particles, and polymethacrylate particles can also be modified by the methods described therein, the contents of which are incorporated herein by reference.

[0031] <Uses of the Tissue Marking Agent of the Present Invention> The tissue marking agent of the present invention can be used to mark localized areas of biological tissue. There are no particular limitations on the tissues to be marked, and examples include cancer, lymph nodes, digestive tract, pancreas, mammary glands, skin, lungs, kidneys, bladder, thyroid gland, prostate, head and neck, liver, bile duct, peripheral nerves, brain, skeletal muscle, smooth muscle, adipose tissue, and uterus. The tissue marking agent of the present invention can also mark at the cellular level, and examples of targets to be marked include tumor cells, tumor cell masses, solid tumors, areas near solid tumors, and precancerous lesions. In one embodiment of the present invention, the tissue marking agent can be injected subcutaneously for body decoration, taking advantage of its ability to be retained locally for a long period of time. The method of administration of the tissue marking agent of the present invention is not particularly limited as long as it can be administered to the target site, and examples include submucosal injection, intramuscular injection, intratumoral injection, subcutaneous injection, and intraperitoneal injection.

[0032] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0033] 1. Production of Indocyanine Green-Bound Silica Nanoparticles 1-1. Synthesis of Amino-Modified Silica Nanoparticles Commercially available silica nanoparticles (SNOWTEX, manufactured by Nissan Chemical Industries, Ltd., product names: 22 nm; ST-50-T, 65 nm; ST-YL, 200 nm; ST MP-2040) with three different particle sizes were used. 0.05 mL of (3-aminopropyl)triethoxysilane, 1.5 mL of aqueous ammonia (28% by mass), and 15 mL of ultrapure water were added to 5 mL (480 mg / mL) of the silica nanoparticles with the three different particle sizes, and the mixture was vigorously stirred to obtain a mixture. The mixture was then heated at 75°C for 3 hours, placed in a dialysis membrane with a molecular weight cutoff of 3500, and dialyzed against water for 1 day to purify the amino-modified silica nanoparticles.

[0034] 1-2. Synthesis of indocyanine green-conjugated silica nanoparticles (siICG) To 2 mL of the amino group-modified silica nanoparticles obtained after dialysis, 1 μL of phosphate buffer (200 mM, pH 7.0) was added to adjust the pH, yielding an aqueous solution containing dispersed silica nanoparticles. Meanwhile, in a separate container, 0.3 mg of ICG NHS ester (catalog no. POS1604, manufactured by Funakoshi Co., Ltd.), 30 mg of WSCD-HCl (manufactured by Peptide Institute, Inc.), and 23 mg of N-hydroxysuccinimide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were dissolved in 5 mL of pure water. The entire volume of this solution was added to the aqueous solution containing dispersed silica nanoparticles, and the mixture was allowed to react at 25°C for 2 hours. The reacted aqueous solution was then placed in a dialysis membrane with a molecular weight cutoff of 3500 and dialyzed first against methanol for 3 hours, and then against pure water for 1 day to purify the indocyanine green-conjugated silica nanoparticles (siICG). Indocyanine green not bound to silica nanoparticles can be removed by adding methanol or ethanol to the prepared particles, as it is released from the nanoparticles. On the other hand, the bound indocyanine green does not detach even when methanol is added, confirming that indocyanine green is bound to the silica nanoparticles. After dialysis, the siICG particles were lyophilized and recovered. The amount of indocyanine green bound per 1 mg of the recovered siICG particles was 0.5-0.6 μg. Ethylene oxide gas sterilization was also performed for sterilization. Electron micrographs of silica nanoparticles before and after ICG labeling are shown in Figure 1.

[0035] 2. Preparation of tissue marking agent (1) Sodium hyaluronate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to 1 mL of phosphate buffer solution (PBS, manufactured by Nacalai Tesque, Inc.) to prepare phosphate buffer solutions containing sodium hyaluronate at concentrations of 0.1% by mass, 0.2% by mass, and 0.4% by mass. 5 mg of recovered siICG powder or 10 mg of recovered siICG powder was added and stirred to the phosphate buffer solutions containing sodium hyaluronate at each concentration to prepare tissue marking agents. As a comparative example, 5 mg of recovered siICG powder or 10 mg of recovered siICG powder was added and stirred to a phosphate buffer solution without sodium hyaluronate to prepare tissue marking agents. The list of tissue marking agents obtained is as follows:

[0036] 3. Preparation of tissue marking agent (2) Sodium alginate (Fujifilm Wako Pure Chemical Industries, Ltd.) or dextran (Fujifilm Wako Pure Chemical Industries, Ltd., 40 kDa) was added to a phosphate buffer solution (PBS, Nacalai Tesque, Inc.) to prepare a phosphate buffer solution containing 0.5% by mass of sodium alginate, or 2% by mass or 10% by mass of dextran. 5 mg of the recovered siICG particles was added to 1 mL of each solution and stirred to prepare a tissue marking agent. The tissue marking agents obtained were as follows:

[0037] 4. Evaluation of the Dispersion State of siICG in Dispersion Medium Figure 2 shows photographs of the tissue marking agents of Comparative Example 1 and Examples 2 and 3, which were prepared by adding 5 mg of siICG (particle size 200 nm) to 1 mL of phosphate buffer solution (PBS) containing 0.0, 0.2, or 0.4% by mass of sodium hyaluronate and stirring, taken immediately after the addition of siICG and stirring, and taken after 30 minutes of standing time after stirring. In the tissue marking agent of Comparative Example 1, in which siICG was dispersed in PBS containing no sodium hyaluronate, siICG precipitated within 30 minutes. On the other hand, in the tissue marking agents of Examples 2 and 3, in which siICG was dispersed in PBS containing 0.2 or 0.4% by mass of sodium hyaluronate, the siICG remained dispersed for more than 30 minutes.

[0038] To evaluate the dispersion state of siICG in the dispersion medium, the transmittance of the tissue marking agents of Examples 1 to 9 and Comparative Examples 1 to 3 was measured using a UV-vis spectrophotometer (UV-2600, Shimadzu Corp.) ( Figure 3 ). Comparing the sedimentation rates of solutions containing siICG with particle sizes of 20, 65, or 200 nm, the smaller the siICG particle size, the faster the sedimentation rate tended to be. Comparing the tissue marking agents of Comparative Examples 1 to 3, in which siICG was dispersed in PBS without sodium hyaluronate, approximately 60% of the siICG settled after 30 minutes in the tissue marking agent of Comparative Example 1, which contained siICG with a particle size of 20 nm, and approximately 30% of the siICG settled after 30 minutes in the tissue marking agents of Comparative Examples 2 and 3, which contained siICG with a particle size of 65 or 200 nm. In the tissue marking agents of Examples 1 to 3, in which siICG with a particle size of 20 nm was dispersed in PBS containing 0.1% by mass or more of sodium hyaluronate, approximately 60% of the siICG remained dispersed even after 30 minutes. In the tissue marking agents of Examples 2 and 3, in which siICG was dispersed in PBS containing 0.2% by mass or more of sodium hyaluronate, almost all of the siICG remained dispersed even after 30 minutes. In the tissue marking agents of Examples 4 to 9, in which siICG with a particle size of 65 nm or 200 nm was dispersed in PBS containing 0.1% by mass or more of sodium hyaluronate, almost all of the siICG remained dispersed even after 30 minutes. Although not shown in the graph, the tissue marking agents of Examples 10 to 18 and Comparative Examples 4 to 6, which contained 10 mg / mL of siICG, also obtained results similar to those of the tissue marking agents of Examples 1 to 9 and Comparative Examples 1 to 3, respectively.

[0039] Next, the tissue marking agents of Examples 19 to 21 and Examples 22 to 27, in which siICG was dispersed in PBS containing sodium alginate or dextran, were compared with the tissue marking agent of Comparative Example 1, in which siICG was dispersed in PBS not containing these water-soluble viscous substances, to evaluate the dispersion state of siICG. As shown in Figure 4, in the tissue marking agents of Examples 19 to 21, in which siICG with particle sizes of 20, 65, or 200 nm was dispersed in PBS containing 0.5% by mass of sodium alginate, most of the siICG remained dispersed even after 30 minutes. Furthermore, as shown in Figure 5, the tissue marking agents of Examples 22 to 27, in which siICG with particle sizes of 20, 65, or 200 nm was dispersed in PBS containing 0, 2, or 10% by mass of dextran, showed improved dispersion stability compared to the tissue marking agent of Comparative Example 1, in which siICG was dispersed in PBS containing no dextran. In the tissue marking agents of Examples 25 to 27, in which siICG was dispersed in PBS containing 10% by mass of dextran, most of the siICG remained dispersed even after 30 minutes.

[0040] 5. Local Subcutaneous Administration of a Solution Containing Indocyanine Green or siICG to Mice The hair on the backs of ICR mice (female, 5 weeks old) was removed using clippers. 100 μL of low molecular weight indocyanine green (product code: I0535, Tokyo Chemical Industry Co., Ltd.) was injected subcutaneously into the removed backs. One day later, under isoflurane anesthesia, the injection site was irradiated with a 780 nm LED light, and the injection site was photographed using a near-infrared digital camera (ORCAspark, Hamamatsu Photonics) (Figure 6). One day after administration, no low molecular weight indocyanine green remained at the injection site.

[0041] 50 μL of each of the tissue marking agents of Examples 10 to 18 and Comparative Examples 4 to 6 was injected subcutaneously into both sides of the back of hair-shaved ICR mice (female, 5 weeks old). 28 and 56 days after injection, the injection site was irradiated with a 780 nm LED light under isoflurane anesthesia, and the injection site was photographed using a near-infrared digital camera (ORCAspark, Hamamatsu Photonics). As shown in Figures 7 and 8, regardless of the tissue marking agent administered, strong near-infrared emission was detected from the injection site 28 and 56 days after administration, confirming that siICG remained at the injection site.

[0042] 6. Local Administration to the Gastric Submucosa of Rabbits The sedative medetomidine was injected intramuscularly. Under inhalation anesthesia with isoflurane, the surgical field was shaved with clippers and disinfected with isodine. A midline abdominal incision (approximately 10 cm) was made, and the stomach was exposed to the outside of the abdominal cavity through the incision. 100 μL of the tissue marking agent of Examples 11, 14, or 17, containing siICG particles with a particle diameter of 20 nm, 65 nm, or 200 nm, was locally injected into the stomach wall (near the submucosa). After 21 days, the animals were euthanized. The excised stomach was opened, and a 780 nm LED light was irradiated onto the injection site. The injection site was photographed using a near-infrared digital camera (ORCAspark, Hamamatsu Photonics). An excitation light intensity of 2 was used. Even 21 days after administration, fluorescent signals were detected from the tissue marking agent injection site, confirming that siICG remained at the injection site (Figure 9). Furthermore, the sites where tissue marking agents containing siICG with particle diameters of 65 nm and 200 nm were administered showed fluorescent signals that were approximately 2.6 times and 2.2 times stronger, respectively, than the fluorescent intensity at the site where tissue marking agents containing siICG with particle diameters of 20 nm were administered. This suggests that larger siICG particle diameters may improve the retention of tissue marking agents at the administration site, and this is thought to be because diffusion due to gastrointestinal motility and diffusion into the mucosal layer is more easily suppressed as the particle diameter increases.

[0043] 7. Evaluation of particle size Silica particles with a particle size of 200 nm (manufactured by Nissan Chemical Industries, Ltd., product name: SNOWTEX ST MP-2040), silica particles with a particle size of 450 nm (manufactured by Nissan Chemical Industries, Ltd., product name: SNOWTEX ST Indocyanine green-conjugated silica nanoparticles (siICG) were produced according to the description in "1. Production of indocyanine green-conjugated silica nanoparticles," except that silica particles having a particle diameter of 700 nm (manufactured by Tokuyama Corporation, product name: SS-07), silica particles having a particle diameter of 1000 nm (manufactured by Tokuyama Corporation, product name: SS-10), or silica particles having a particle diameter of 1500 nm (manufactured by Tokuyama Corporation, product name: SS-15) were used, and a tissue marking agent was prepared according to the description in "2. Preparation of tissue marking agent (1)," except that 10 mg of the recovered siICG was added to 1 mL of phosphate buffer containing 0.2% by mass sodium hyaluronate and stirred. 50 μL of each prepared tissue marking agent was injected subcutaneously into both sides of the back of hairless ICR mice (female, 5 weeks old) as described in "5. Local subcutaneous administration of a solution containing indocyanine green or siICG into mice." Immediately after administration (day 0) and 28 days after administration, the injection site was irradiated with a 780 nm LED light under isoflurane anesthesia, and the injection site was photographed using a near-infrared digital camera (pde-neo, Hamamatsu Photonics). An excitation light intensity of 2 was used.

[0044] Photographs of the administration site of mice administered with siICG of each particle size, taken with a digital camera (pde-neo, Hamamatsu Photonics) immediately after administration (day 0) and 28 days after administration, are shown in Figure 10. An excitation light intensity of 2 was used. As can be seen from Figure 10, there was a tendency for the fluorescence intensity per weight of siICG to decrease as the particle size of siICG increased, but even with siICG with a particle size of 1500 nm, near-infrared emission was detected after 28 days, confirming that siICG remained at the administration site.

[0045] 8. Evaluation of siICG Concentration Indocyanine green-conjugated silica nanoparticles (siICG) were produced according to the description in "1. Production of indocyanine green-conjugated silica nanoparticles," except that silica particles with a particle size of 200 nm (manufactured by Nissan Chemical Industries, Ltd., product name: SNOWTEX, ST MP-2040) were used, and tissue marking agents were prepared according to the description in "2. Preparation of tissue marking agent (1)," except that 1, 10, or 300 mg of the recovered siICG was added to 1 mL of phosphate buffer containing 0.2 mass% sodium hyaluronate and stirred. 50 μL of the prepared tissue marking agent of each concentration was injected subcutaneously into both sides of the back of hairless ICR mice (female, 5 weeks old) as described in "5. Local subcutaneous administration of a solution containing indocyanine green or siICG into mice." Immediately after administration (day 0) and 28 days after administration, the injection site was irradiated with a 780 nm LED light under isoflurane anesthesia, and the injection site was photographed using a near-infrared digital camera. An excitation light intensity of 2 was generally used, but when a 1 mg / mL tissue marking agent was used, excitation light intensities of 2 and 10 were used.

[0046] Photographs of the injection site of mice administered with various concentrations of tissue marking agent taken with a digital camera immediately after administration (day 0) and 28 days after administration are shown in Figure 11. As can be seen from Figure 11, there was a tendency for the fluorescence intensity to increase concentration-dependently, and near-infrared emission was detected after 28 days in the concentration range of 1 to 300 mg / mL, confirming that siICG remained at the injection site.

[0047] 9. Effect of Inhibiting Decrease in Fluorescence Intensity Due to Addition of Antioxidants Indocyanine green-conjugated silica nanoparticles (siICG) were produced as described in "1. Production of Indocyanine Green-Conjugated Silica Nanoparticles," except that silica particles with a particle size of 200 nm (Nissan Chemical Industries, Ltd., product name: SNOWTEX, ST MP-2040) were used. 10 mg of the recovered siICG was added to 1 mL of phosphate buffer containing 0.2% by mass sodium hyaluronate, and tissue marking agents were prepared as described in "2. Preparation of Tissue Marking Agent (1)," except that no ascorbic acid, or 0.04, 0.4, or 4 mg of ascorbic acid was added as an antioxidant and stirred. Each prepared tissue marking agent was stored at 4°C in a light-proof room for 3 days, after which 100 μL was transferred to a black 96-well plate and fluorescence intensity was measured using a plate reader (Tecan, product name: Trading AG, TECAN).

[0048] The fluorescence intensity of the tissue marking agents containing no or various concentrations of ascorbic acid after storage in the dark is shown in Figure 12. As can be seen from Figure 12, it was confirmed that the addition of ascorbic acid suppressed the decrease in the fluorescence intensity of indocyanine green, and the decrease in fluorescence intensity was significantly suppressed in the tissue marking agent containing 4 mg of ascorbic acid.

Claims

1. A tissue marking agent comprising fluorescent dye-labeled microparticles in an aqueous medium, the microparticles are selected from the group consisting of silica particles, polyester particles, polyurethane particles, silicon particles, polyethylene particles, polypropylene particles, polystyrene particles, polymethacrylate particles, hydroxyapatite particles, cellulose particles, and silk particles; The tissue marking agent comprises the fluorescent dye-labeled microparticles dispersed in an aqueous medium in which a water-soluble viscous substance is dissolved.

2. The tissue marking agent according to claim 1 , wherein the water-soluble viscous substance comprises a polyhydric alcohol, a polyether or a salt thereof, a water-soluble protein, or a polysaccharide or a salt thereof.

3. The tissue marking agent of claim 2 , wherein the water-soluble viscous substance comprises a polysaccharide or a salt thereof.

4. 4. The tissue marking agent according to claim 1, wherein the fluorescent dye is covalently bound to the microparticles.

5. The tissue marking agent according to any one of claims 1 to 3, wherein the pH of the tissue marking agent is in the range of 5.0 to 8.

0.

6. 4. The tissue marking agent according to claim 3, wherein the polysaccharide is at least one selected from the group consisting of hyaluronic acid, alginic acid, guar gum, xanthan gum, acacia gum, pullulan, dextran, agarose, carrageenan, pectin, galactomannan, gellan gum, curdlan, carboxymethylcellulose, and chitin / chitosan.

7. The tissue marking agent of claim 6 , wherein the polysaccharide comprises alginate, dextran, and / or hyaluronic acid.

8. 4. The tissue marking agent according to claim 1, wherein the fluorescent dye is selected from the group consisting of indocyanine green, coumarin, rhodamine, xanthene, porphyrin, fluorescamine, fluorescein, and derivatives thereof.

9. The tissue marking agent of claim 8 , wherein the fluorescent dye is indocyanine green or a derivative thereof.

10. The tissue marking agent according to any one of claims 1 to 3, wherein the microparticles are silica particles.

11. The tissue marking agent according to any one of claims 1 to 3, wherein the microparticles have an average particle size in the range of 1 nm to 2 µm.

12. The tissue marking agent according to any one of claims 1 to 3, wherein the aqueous medium is at least one selected from the group consisting of physiological saline, phosphate buffer, acetate buffer, citrate buffer, prolamine buffer, carbon buffer, and purified water.

13. The tissue marking agent according to any one of claims 1 to 3, wherein the concentration of the microparticles in the aqueous medium is in the range of 0.01 mg / mL to 1000 mg / mL.

14. The tissue marking agent according to any one of claims 1 to 3, wherein the concentration of the water-soluble viscous substance in the aqueous medium is in the range of 0.001% by mass to 20% by mass.

15. The tissue marking agent according to any one of claims 1 to 3, for marking a localized area of ​​the digestive tract.

16. Use of an aqueous composition containing microparticles labeled with a fluorescent dye for preparing a tissue marking agent, comprising: the microparticles are selected from the group consisting of silica particles, polyester particles, polyurethane particles, silicon particles, polyethylene particles, polypropylene particles, polystyrene particles, polymethacrylate particles, hydroxyapatite particles, cellulose particles, and silk particles; The fluorescent dye-labeled microparticles are dispersed in an aqueous medium in which a water-soluble viscous substance is dissolved.

17. The use according to claim 16, wherein the water-soluble viscous substance comprises a polyhydric alcohol, a polyether or a salt thereof, a water-soluble protein, or a polysaccharide or a salt thereof.

18. The use according to claim 16, wherein the water-soluble viscous substance comprises a polysaccharide or a salt thereof.

19. The use according to any one of claims 16 to 18, wherein the fluorescent dye is covalently bound to the microparticles.

20. The use according to any one of claims 16 to 18, wherein the pH of the tissue marking agent is in the range of 5.0 to 8.

0.

21. The use according to claim 18, wherein the polysaccharide is at least one selected from the group consisting of hyaluronic acid, alginic acid, guar gum, xanthan gum, acacia gum, pullulan, dextran, agarose, carrageenan, pectin, galactomannan, gellan gum, curdlan, carboxymethylcellulose, and chitin / chitosan.

22. 22. The use according to claim 21, wherein the polysaccharide comprises or is alginic acid, dextran and / or hyaluronic acid.

23. The use according to any one of claims 16 to 18, wherein the fluorescent dye is selected from the group consisting of indocyanine green, coumarin, rhodamine, xanthene, porphyrin, fluorescamine, and derivatives thereof.

24. 24. The use according to claim 23, wherein the fluorescent dye is indocyanine green or a derivative thereof.

25. The use according to any one of claims 16 to 18, wherein the microparticles are silica particles.

26. The use according to any one of claims 16 to 18, wherein the microparticles have an average particle size in the range of 1 nm to 2 µm.

27. The use according to any one of claims 16 to 18, wherein the aqueous medium is at least one selected from the group consisting of physiological saline, phosphate buffer, acetate buffer, citrate buffer, prolamine buffer, carbon buffer, and purified water.

28. The use according to any one of claims 16 to 18, wherein the concentration of the microparticles in the aqueous medium is in the range of 0.01 mg / mL to 1000 mg / mL.

29. The use according to any one of claims 16 to 18, wherein the concentration of the water-soluble viscous substance in the aqueous medium is in the range of 0.001% by mass or more and 20% by mass or less.

30. The use according to any one of claims 16 to 18, wherein the tissue marking agent is used to mark a localized area of ​​the digestive tract.

31. The tissue marking agent according to any one of claims 1 to 3, which comprises an antioxidant.

32. 32. The tissue marking agent of claim 31, comprising 0.02 mg / mL to 10 mg / mL of the antioxidant.

33. The use according to any one of claims 16 to 18, wherein the tissue marking agent comprises an antioxidant.

34. 34. The use of claim 33, wherein the tissue marking agent comprises 0.02 mg / mL to 10 mg / mL of the antioxidant.