Tissue marking agent

Abstract

To provide a tissue marking agent having improved dispersion stability of fine particles labeled with a fluorescent dye in an aqueous medium.SOLUTION: Silica particles in which a plurality of particles are linked are labeled with a fluorescent dye and dispersed in an aqueous medium.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a tissue marking agent comprising silica particles of a specific structure labeled with a fluorescent dye. [Background technology]

[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 not only difficult to see because it does not use a fluorescent agent, but also has the problem of making it difficult to determine the boundary between the tumor site and normal tissue due to the diffusion of the ink.In addition, clips cannot be used as markings in laparoscopic surgery because they cannot directly touch the tissue, and even in open surgery, they can be difficult to palpate from the serosal surface (outside the digestive tract).

[0005] Although endoscopic clips using fluorescent materials have been developed in recent years, 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] In addition, it has been reported that tissues can be marked using hollow silica nanoparticles with indocyanine green (ICG) electrostatically adsorbed onto their surfaces (Non-Patent Document 4). As described in this document, this marking agent is made by adsorbing ICG onto spherical silica particles that exist independently, and according to the findings of the present inventors, it is assumed that there are problems with dispersion stability. Furthermore, this document states that marking by silica nanoparticles with electrostatically adsorbed ICG was confirmed for up to about 12 days, and there is a demand for marking that lasts for a longer period of time.

[0008] On the other hand, it has been taught that the surface of metal oxide particles or metal particles containing indocyanine green (ICG) can be modified with a dispersant such as dextran (Patent Document 1). However, as a result of modifying the surface of these particles with a dispersant such as dextran, they do not remain at the administration site but diffuse throughout the body, making them unsuitable as marking agents that are administered locally to the site to be marked and retained there. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Patent No. 7164133 [Patent Document 2] Patent No. 4328935 [Patent Document 3] Patent Publication No. 2022-106093 [Patent Document 4] Patent Publication No. 2014-001175 [Non-patent literature]

[0010] [Non-Patent Document 1] Narihiro et al., International Journal of Surgery [Non-patent document 2] Ozawa, Yoshiaki, Murakami, Masahiko, (2020). Examination of preoperative marking methods in laparoscopic rectal cancer surgery: Comparison of ink marking and ICG fluorescence. Showa Gakushikai Zasshi, 80.1:1-6. [Non-patent document 3] Tetsuta Satoyoshi et al., Surgical Endoscopy (2021) 35:763-769 [Non-patent document 4] 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 Summary of the Invention [Problem to be solved by the invention]

[0011] An object of the present invention is to provide a tissue marking agent in which the dispersion stability of silica particles labeled with a fluorescent dye in an aqueous medium is improved, and another object is to provide a tissue marking agent that, when administered locally to a target tissue site, is retained in place for a long period of time. [Means for solving the problem]

[0012] While investigating the suitability of various silica particles for tissue marking agents, the present inventors unexpectedly found that when silica particles in which multiple particles are linked, typically via covalent bonds, are labeled with a fluorescent dye and dispersed in an aqueous medium, the dispersion stability of the particles is improved. Furthermore, they confirmed that when such labeled silica particles are administered locally to a target tissue site, they are retained in place for a long period of time. The present invention was made based on these findings.

[0013] That is, the embodiments of the present invention are as follows. [1] A tissue marking agent (tissue marking composition) comprising silica particles labeled with a fluorescent dye in an aqueous medium, wherein a plurality of the silica particles are linked together. [2] The tissue marking agent described in [1], wherein the linked individual silica particles have an average particle size in the range of 1 nm to 40 nm. [3] The tissue marking agent according to [1] or [2], wherein the number of linked silica particles is 5 to 1,000. [4] The tissue marking agent according to any one of [1] to [3], wherein the total length of the linked silica particles is 30 nm to 1 μm. [5] The tissue marking agent according to any one of [1] to [4], wherein the plurality of silica particles are linked in a linear or branched chain configuration. [6] The tissue marking agent according to any one of [1] to [5], wherein the fluorescent dye is covalently bonded to the silica particles. [7] The tissue marking agent according to any one of [1] to [6], wherein the aqueous medium contains a water-soluble viscous substance. [8] The tissue marking agent according to [7], 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. [9] The tissue marking agent according to [8], wherein the water-soluble viscous substance comprises a polysaccharide or a salt thereof.

[10] The tissue marking agent according to [9], 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, xanthan gum, gellan gum, curdlan, carboxymethylcellulose, chitin / chitosan, and carrageenan.

[11] The tissue marking agent according to

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

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

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

[13] The tissue marking agent according to

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

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

[13] , wherein the aqueous 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.

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

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

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

[15] , wherein the concentration of the silica particles in the aqueous medium is in the range of 0.01 mg / mL or more and 1000 mg / mL or less.

[17] The tissue marking agent according to any one of [7] to

[16] , 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.

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

[17] , for marking a localized area of ​​the digestive tract.

[19] Use of an aqueous composition in which silica particles labeled with a fluorescent dye are dispersed in an aqueous medium for preparing a tissue marking agent, wherein a plurality of the silica particles are linked together.

[20] The use according to

[19] , wherein the linked individual silica particles have an average particle size in the range of 1 nm to 40 nm.

[21] The use according to

[19] or

[20] , wherein the number of linked silica particles is 5 to 1,000.

[22] The use according to any one of

[19] to

[21] , wherein the total length of the linked silica particles is 5 nm to 1 μm.

[23] The use according to any one of

[19] to

[22] , wherein the plurality of silica particles are linked in a linear or branched chain configuration.

[24] The use according to any one of

[19] to

[23] , wherein the fluorescent dye is covalently bonded to the silica particles.

[25] The tissue marking agent according to any one of

[19] to

[24] , wherein the aqueous medium contains a water-soluble viscous substance.

[26] The use according to

[25] , 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.

[27] The use according to

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

[28] The use according to

[27] , 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, xanthan gum, gellan gum, curdlan, carboxymethylcellulose, chitin / chitosan, and carrageenan.

[29] The use according to

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

[30] The use according to any one of

[19] to

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

[31] The use according to

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

[32] The use according to any one of

[19] to

[31] , wherein the aqueous 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.

[33] The use according to any one of

[19] to

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

[34] The use according to any one of

[19] to

[33] , wherein the concentration of the silica particles in the aqueous medium is in the range of 0.01 mg / mL or more and 1000 mg / mL or less.

[35] The use according to any one of

[25] to

[34] , wherein the concentration of the water-soluble viscous substance in the dispersion medium is in the range of 0.001% by mass or more and 20% by mass or less.

[36] The use according to any one of

[19] to

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

[37] An aqueous composition used for tissue marking, comprising silica particles labeled with a fluorescent dye dispersed in an aqueous medium, wherein the silica particles are linked together.

[38] The aqueous composition described in

[37] , wherein the linked individual silica particles have an average particle size in the range of 1 nm to 40 nm.

[39] The aqueous composition according to

[37] or

[38] , wherein the number of linked silica particles is 5 to 1,000.

[40] The aqueous composition according to any one of

[37] to

[39] , wherein the total length of the linked silica particles is 30 nm to 1 μm.

[41] The aqueous composition according to any one of

[37] to

[40] , wherein the plurality of silica particles are linked in a linear or branched chain configuration.

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

[37] to

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

[43] The aqueous composition according to any one of

[37] to

[42] , wherein the aqueous medium contains a water-soluble viscous substance.

[44] The aqueous composition for use according to

[43] , 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.

[45] The aqueous composition for use according to

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

[46] The aqueous composition for use according to

[45] , 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, xanthan gum, gellan gum, curdlan, carboxymethylcellulose, chitin / chitosan, and carrageenan.

[47] The aqueous composition for use according to

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

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

[37] to

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

[49] The aqueous composition for use according to

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

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

[37] to

[49] , 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.

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

[37] to

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

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

[37] to

[51] , wherein the concentration of the silica particles in the aqueous medium is in the range of 0.01 mg / mL or more and 1000 mg / mL or less.

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

[43] to

[52] , 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.

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

[37] to

[53] , which is used to mark a localized area of ​​the digestive tract.

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

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

[37] to

[54] to the site of the tissue to be marked.

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

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

[37] to

[54] , which contains an antioxidant.

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

[56] , containing the antioxidant at a concentration of 0.02 mg / mL to 10 mg / mL.

[58] The use according to any one of

[19] to

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

[59] The use according to

[58] , wherein the tissue marking agent contains the antioxidant at 0.02 mg / mL to 10 mg / mL. [Effects of the Invention]

[0014] The dispersion stability of the fluorescent dye-labeled silica particles in an aqueous medium is improved by linking the particles together, allowing for the administration of an appropriate amount of tissue marking agent and preventing clogging of the injection needle. Furthermore, when administered locally to the target tissue site, the agent is retained in place for a long period of time. [Brief explanation of the drawings]

[0015] [Figure 1] Figure 1(a) is an electron microscope photograph of indocyanine green-linked silica nanoparticles (siICG) in the tissue marking agent of Comparative Example 1. Figure 1(b) is an electron microscope photograph of indocyanine green-linked silica nanoparticles (siICG) in the tissue marking agent of Example 1. Figure 1(c) is an electron microscope photograph of indocyanine green-linked silica nanoparticles (siICG) in the tissue marking agent of Example 2. [Figure 2] 2 is a graph showing the fluorescence intensity of the tissue marking agents of Examples 1 to 4 and Comparative Examples 1 and 2. The excitation wavelength used was 760 nm, and the fluorescence wavelength used was 820 nm. [Figure 3]3 is a graph showing the change over time in transmittance of the tissue marking agents of Example 1, Example 2, and Comparative Example 1. After stirring to thoroughly disperse the tissue marking agent, the change over time in transmittance was measured. [Figure 4] 4 is a graph showing the change over time in transmittance of the tissue marking agents of Examples 3 and 4, and Comparative Example 2. After stirring to thoroughly disperse the tissue marking agents, the change over time in transmittance was measured. [Figure 5] FIG. 5 is a graph showing the change over time in transmittance of the tissue marking agents of Comparative Examples 1 and 2. [Figure 6] FIG. 6 is a graph showing the change over time in transmittance of the tissue marking agents of Examples 1 to 4. [Figure 7] Figures 7(A) and (B) show photographs of the area around the administration site 32 days after the tissue marking agent of Comparative Example 3 was administered directly and locally to the submucosa of a pig's stomach. Figure 7(A) is a photograph taken with a conventional camera, and Figure 7(B) is a photograph taken with a near-infrared camera. The arrows indicate the location where the tissue marking agent was administered. [Figure 8] 8 is a photograph taken with a near-infrared camera 32 days after administration of the tissue marking agent of Example 5 into the submucosa of a pig stomach. The arrow indicates the location where the tissue marking agent was administered. [Figure 9] 9 is a photograph taken with a near-infrared camera 32 days after administration of the tissue marking agent of Example 6 into the submucosa of a pig stomach. The arrow indicates the location where the tissue marking agent was administered. The arrow indicates the location where the tissue marking agent was administered. DETAILED DESCRIPTION OF THE INVENTION

[0016] The embodiments of the present invention will be described in detail below, but the present invention is not limited to the following embodiments. <Tissue marking agent of the present invention> As used herein, the term "tissue marking agent" refers to a drug used to mark any site of tissue that is the target of treatment, such as excision or administration, during surgery, etc., and is a labeling substance that can be administered directly to any target site and retained there, making it possible to identify the site. The tissue marking agent of the present invention comprises silica particles labeled with a fluorescent dye and an aqueous dispersion medium, and the fluorescent dye-labeled silica particles have a specific structure and are dispersed in the aqueous medium.

[0017] The fluorescent dye is not particularly limited, and examples thereof include indocyanine green, coumarin, rhodamine, xanthene, porphyrin, fluorescamine, fluorescein, and derivatives thereof. Two or more types of fluorescent dyes may be combined, and for example, a mixture of particles in which two or more types of fluorescent dyes are adsorbed or bound to particles of different particle sizes or different materials may be used. Since near-infrared cameras are used in surgical procedures and the near-infrared region allows 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.

[0018] In the tissue marking agent of the present invention, a plurality of silica particles labeled with a fluorescent dye are dispersed in a medium in a linked state. As will be demonstrated in the examples described below, the marking agent in which a plurality of silica particles labeled with a fluorescent dye are dispersed in a medium in a linked state has significantly improved dispersion stability compared to a marking agent in which spherical silica particles labeled with a fluorescent dye are dispersed in a medium in an independent state. Here, in this specification, "linked" refers to a chemically bonded state. Examples of "linked" silica particles include multiple silica particles linked to each other by covalent bonds, ionic bonds, or hydrogen bonds, and typically multiple silica particles linked to each other by covalent bonds. These "linked" silica particles are clearly distinguished from a state in which multiple silica particles are physically close to each other due to physical interactions, chemical interactions, or both. Therefore, aggregates in which silica particles are gathered together due to hydrophobic interactions, etc., are not included in the "linked" silica particles in this specification and are clearly distinguished from the "linked" silica particles in this specification. A simple method for distinguishing "linked" silica particles from silica particle aggregates in this specification is to observe the silica particle dispersion before and after ultrasonic treatment using an electron microscope. While aggregates are broken down by ultrasonic treatment and individual particles are observed, "linked" silica particles maintain their linked state even after ultrasonic treatment.

[0019] The individual linked silica particles are usually silica nanoparticles, i.e., silica particles with a size of 1 nm or more and less than 1 μm, and preferably have an average particle size in the range of 1 nm to 40 nm, more preferably 5 nm to 30 nm. Here, the "average particle size" in this specification refers to a value determined by randomly selecting particles from a photograph observed with a transmission electron microscope (e.g., JEM-2100F, manufactured by JEOL Ltd.), measuring the particle size (major axis) of seven particles using Image J (ver. 1.53k; open-source, public domain image processing software), and averaging the measured values.

[0020] Furthermore, a structure composed of multiple linked silica particles preferably comprises 5 to 1,000 silica particles, more preferably 5 to 500 silica particles, and even more preferably 5 to 200 silica particles, in one structural unit.

[0021] The structure composed of multiple linked silica particles preferably has an overall length of 30 nm to 1 μm, more preferably an overall length of 40 nm to 700 nm, and even more preferably an overall length of 50 nm to 600 nm.

[0022] The connected silica particles can form various shapes or structures, for example, the silica particles may be connected in a linear or branched chain, the silica particles may be fused together to form an earthworm-like shape, or the silica particles may be connected in a state where the individual particle shapes can be confirmed, forming a rosary or pearl necklace-like shape.Furthermore, the silica particles may be shaped like an earthworm, a rosary or a pearl necklace, and may have a complex branched structure, and such silica particles with a complex structure with many branches are preferred from the viewpoint of dispersion stability.

[0023] In microparticles labeled with a fluorescent dye, the fluorescent dye may be incorporated into the interior of the silica particle, or may be bound or adsorbed to the particle on the surface or interior of the silica particle. As used herein, "binding" is synonymous with the aforementioned "linking," and "adsorption" refers to a state of physical proximity due to physical interaction, chemical interaction, or both. 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 force, dipole-dipole interaction, etc. 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 that the fluorescent dye be covalently bound to the microparticles. Alternatively, the fluorescent dye may be bound or adsorbed to silica particles in a state where individual molecules are crosslinked and associated, which is also beneficial for enabling clear and accurate marking of the target site over a long period of time. This embodiment is described in detail in U.S. Patent No. 6,299,499, which is incorporated herein by reference.

[0024] The amount of fluorescent dye in the fluorescent dye-labeled silica particles can be in the range of 0.01 to 5.0 μg per 1 mg of silica particles, preferably 0.1 to 2.5 μg, more preferably 0.25 to 1.2 μg, and particularly preferably 0.5 to 0.6 μg, in order to improve dispersion stability while exhibiting the fluorescence intensity required for marking at the administration site.

[0025] The dispersion medium is not particularly limited, but is typically an aqueous dispersion medium, and an aqueous dispersion medium whose safety when applied to a living body is preferred. Examples of aqueous dispersion media include physiological saline, phosphate buffer, acetate buffer, citrate buffer, prolamin buffer, carbon buffer, and purified water. One or more dispersion media can be used in combination. Furthermore, various buffers may be contained, but it is preferable that they do not contain basic substances.

[0026] In a preferred embodiment of the present invention, the dispersion medium contains a water-soluble viscous substance. As demonstrated in the examples below, dispersing the above-mentioned fluorescent dye-labeled silica particles in a dispersion medium containing a water-soluble viscous substance improves the dispersion stability of the labeled silica particles, resulting in a uniform dispersion for a long period of time. This suppresses the reduction in fluorescence intensity caused by aggregation of the labeled silica particles, thereby achieving the desired fluorescence intensity upon administration to the target tissue site. A water-soluble viscous substance is a water-soluble substance that can increase the viscosity of the dispersion medium and is typically a substance that dissolves 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, among which 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 described below, and are particularly preferred water-soluble viscous substances from the viewpoints of dispersion stability and biocompatibility of microparticles.

[0027] Examples of polyhydric alcohols include ethylene glycol, glycerin, etc. Furthermore, the polyhydric alcohol preferably has a molecular weight (Mw) of 50 to 1,000, from the viewpoint of imparting an appropriate viscosity to the dispersion medium. Examples of polyethers include polyethylene glycol, etc. 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. Examples of water-soluble proteins include albumin (particularly human serum albumin), fibrin, and globulin (particularly human immunoglobulin). In this specification, unless otherwise specified, the term "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).

[0028] 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 viewpoints 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 there is no particular limitation as long as the salt 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. The polysaccharides or salts thereof can be used singly or in combination of two or more. The polysaccharides or salts thereof are usually dissolved in an aqueous medium and are not adsorbed or bound to the fine particles.

[0029] 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.

[0030] The concentration of the water-soluble viscous substance in the dispersion medium varies depending on the water-soluble viscous substance used, but it is usually adjusted to 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 a concentration within an appropriate range from the viewpoint of solubility and particle dispersion stability. Usually, it is sufficient to select 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 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 a concentration within an appropriate range from the viewpoint of solubility and particle dispersion stability. Usually, the concentration is 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 is 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 the medium containing polysaccharide or its salt, the concentration of polysaccharide or its salt in dispersion medium varies depending on the molecular weight and hydrophilicity of the polysaccharide or its salt used, and it is preferable to select a suitable range of concentration from the viewpoint of solubility and particle dispersion stability.Usually, it can be selected from the range of 0.001% by mass or more and 20% by mass or less, and it is preferable to select from the range of 0.01% by mass or more and 10% by mass or less.In the case of the medium containing hyaluronic acid or its salt, the concentration of hyaluronic acid or its salt in dispersion medium can be selected from the range of 0.01% by mass or more and 10% by mass or less, it is preferably 0.1% by mass or more and 5% by mass or less, more preferably 0.15% by mass or more and 1% by mass or less, and it is particularly preferable to select from 0.15% by mass or more and 0.5% by mass or less. 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% to 10% by mass, preferably 0.1% to 5% by mass, more preferably 0.15% to 1% by mass, and particularly preferably 0.2% 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% to 20% by mass, preferably 3.0% to 17% by mass, more preferably 5.0% to 15% by mass, and particularly preferably 8.0% to 12.0% by mass. The concentration of water-soluble viscous substances in dispersion media is discussed in detail in International Application PCT / JP2023 / 044604, the contents of which are incorporated herein by reference.

[0031] 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. When the pH is set within this range, not only is it highly safe and does not cause inflammation when locally injected into the body, but the water-soluble viscous substance maintains good solubility in the dispersion medium, making it less likely to be adsorbed or bound to silica particles. 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, typically a dye that can be visually confirmed, or an antioxidant can 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, cardioblue, brilliant blue, methylene blue, toliidine blue, and trypan blue. Examples of antioxidants include ascorbic acid, sodium ascorbate, and vitamin E. The amount of antioxidants, such as ascorbic acid and 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. The addition of antioxidants is discussed in detail in International Application PCT / JP2023 / 044604, the contents of which are incorporated herein by reference.

[0032] <Method for producing the tissue marking agent of the present invention> The tissue marking agent of the present invention is produced by adding a plurality of linked silica particles labeled with a fluorescent dye to a dispersion medium optionally containing a water-soluble viscous substance, and stirring the mixture.

[0033] Fluorescent dye-labeled silica particles are produced by labeling the interior or surface of silica particles with a fluorescent dye through physical interaction, chemical interaction, or both. Methods for labeling silica particles with a fluorescent dye are well known in the art. Typically, functional groups that induce physical and / or chemical interaction between the silica particles and / or the fluorescent dye are introduced into one or both of the silica particles 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 particles may be appropriately modified using a silane coupling agent or the like to change the surface charge, hydrophobicity, hydrophilicity, etc. of the particles, or to introduce reactive functional groups into the particles. Similarly, depending on the modifications introduced into the surface of the particles, appropriate modifications, such as the introduction of corresponding functional groups, may also be introduced into the fluorescent dye, if necessary. After introducing appropriate modifications into one or both of the particles 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.

[0034] Silica in which multiple silica particles are linked together can be synthesized by known methods, for example, by a sol-gel method under the conditions described in Patent Documents 1 to 4. The contents of these documents are incorporated herein by reference. Furthermore, such silica particles are commercially available, so commercially available products can be used. Examples of commercially available silica particles include SNOWTEX UP, SNOWTEX ST-UP, SNOWTEX OUP, IPA-ST-UP, MEK-ST-UP, SNOWTEX PS-S, SNOWTEX PS-SO, SNOWTEX PS-M, SNOWTEX PS-MO, SNOWTEX ST-PS-S, and SNOWTEX ST-PS-M, manufactured by Nissan Chemical Industries, Ltd.

[0035] <Uses of the tissue marking agent of the present invention> The tissue marking agent of the present invention remains at the site of administration for a long period of time, and can therefore be used to mark localized biological tissues. There are no particular limitations on the tissues to be marked, and examples include cancer, lymph nodes, digestive tract, pancreas, mammary gland, skin, lung, kidney, bladder, thyroid, prostate, head and neck, liver, bile duct, peripheral nerve, 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. Furthermore, in one embodiment of the present invention, the tissue marking agent can be used for body decoration by subcutaneous injection, taking advantage of its ability to remain localized for a long period of time. The method of administration of the tissue marking agent of the present invention is local administration to the target site, and examples include submucosal injection, intramuscular injection, intratumoral injection, subcutaneous injection, intraperitoneal injection, etc. The marked site can be confirmed by irradiating the administration site with light of an appropriate wavelength depending on the fluorescent dye used. [Example]

[0036] 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.

[0037] Example 1: Tissue marking agent containing siICG particles with an average particle size of 12 nm, to which silica nanoparticles are covalently linked, at a concentration of 25 mg / ml in a solution containing dextran (1) Silica nanoparticles used Commercially available silica nanoparticles (SNOWTEX ST-UP, manufactured by Nissan Chemical Co., Ltd.) were used. These silica nanoparticles consist of multiple fused particles, forming an elongated, earthworm-like shape, with a complex branched chain structure (see the particle structure shown in Figure 1(b) after ICG labeling). According to the product catalog, the average particle diameter of each particle is 12 nm. Observation with a transmission electron microscope revealed that the connected silica nanoparticle clusters were 100–550 nm long (judging by the linear distance between the longest points) and consisted of 10–160 silica nanoparticles. The "average particle size" was determined by randomly selecting particles from photographs taken with a transmission electron microscope (JEM-2100F, manufactured by JEOL Ltd.) and measuring and averaging the particle sizes (longer diameters) of seven particles using Image J (ver. 1.53k; open-source, public domain image processing software). The size of the connected silica nanoparticle clusters and the number of constituent silica nanoparticles were also measured by observation with a transmission electron microscope (JEM-2100F, manufactured by JEOL Ltd.) (the same applies below). Note that multiple particles fused together to form an earthworm-like shape, making it difficult to grasp the overall shape of each particle. Therefore, the particle size was determined by measuring the diameter in the direction perpendicular to the stretching direction. Observation by transmission electron microscope was performed as follows: a silica nanoparticle product (SNOWTEX ST-UP) was diluted 10,000 times with saline containing 10% dextran (molecular weight: 40,000), and 20 μL of the diluted solution was applied to an electron microscope grid (Microgrid NC-15, Oken Shoji) and allowed to dry. After that, observation was performed using a transmission electron microscope JEM-2100F (manufactured by JEOL Ltd.) (the same applies below).

[0038] (2) Preparation of siICG particles To 25 mL of an aqueous dispersion of silica nanoparticles (200-300 mg / mL), 1.8 mL of (3-aminopropyl)triethoxysilane, 1 mL of aqueous ammonia (28% by mass), and 75 mL of purified water were added, vigorously stirred, and heated at 75°C for 18 hours. Then, 2.2 mL of 10 mM HCl was added and the pH was adjusted to 1.6. The dispersion was then centrifuged (10,000 rpm, 18,800 × g, 20°C, 15 minutes) and the supernatant was removed. 300 mL of 10 mM HCl was added to adjust the pH to approximately 2.0, and the mixture was centrifuged again and the supernatant was removed. The precipitate was collected using a spoon, and 50 mL of 10 mM HCl was added. The mixture was stirred using a magnetic stirrer to redisperse the amino-modified silica nanoparticles. Then, 200 μL of 8 N NaOH was added in 200 μL increments to adjust the pH to 7.4. Next, 12.5 mg of ICG-NHS (15.0 μmol, Catalog No. POS16 04, Funakoshi Co., Ltd.) in DMF (1 mL), 121 mg of Water-Soluble Carbodiimide Hydrochloride-HCl (WSCD-HCl) (81 μmol, Peptide Institute, Inc.), and 93 mg of N-hydroxysuccinimide (81 μmol, Fujifilm Wako Pure Chemical Industries, Ltd.) were dissolved in 100 mL of ethanol and added to amino-functionalized silica nanoparticles (100 mL, pH 7.4). The mixture was incubated at room temperature (25°C) for 18 hours in the dark. The reaction mixture was then centrifuged (10,000 rpm, 18,800 × g, 4°C, 15 minutes) and the supernatant was removed. 300 mL of ethanol was added, the mixture was centrifuged again, and the supernatant was removed. The precipitate was then collected using a spoonful, and 400 mL of ethanol was added. The mixture was stirred using a magnetic stirrer and redispersed. This centrifugation was repeated twice to remove unreacted ICG-NHS. Finally, the particles were dispersed in 400 mL of ethanol and dried using a spray dryer to recover siICG particles. As shown in Figure 1(b), the resulting siICG particles were elongated and fused together, forming an earthworm-like shape with a complex branched chain structure. The average particle diameter of each particle was 12 nm, and the length of the connected silica nanoparticle clusters ranged from 100 to 550 nm, consisting of 10 to 160 silica nanoparticles.Because multiple particles fuse together to form a worm-like shape, making it difficult to grasp the overall shape of each particle, the particle size was determined by measuring the diameter perpendicular to the stretching direction. The length of the silica nanoparticle cluster was determined by the linear distance at its longest point.

[0039] Transmission electron microscopy was performed as follows: The recovered siICG particles were dispersed in saline containing 10% dextran (molecular weight: 40,000) to prepare a 0.01 mg / mL siICG particle dispersion. To facilitate the distinction between "linked" silica nanoparticles and silica nanoparticle aggregates, the dispersion was sonicated for 30 seconds at 10% amplitude using an ultrasonic homogenizer (BRANSON Digital Sonifer, Model 102C) to disrupt silica nanoparticle aggregates. 20 μL of the solution was then applied to an electron microscopy grid (Microgrid NC-15, Oken Shoji) and allowed to dry. The specimens were then observed using a JEM-2100F transmission electron microscope (JEOL Ltd.).

[0040] (3) Preparation of tissue marking agent An aqueous solution was prepared by dissolving dextran (40 kDa, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) at a concentration of 10% by mass and NaCl (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) at a concentration of 0.9% by mass in purified water. The recovered siICG particles were added to 1 mL of the prepared solution at a concentration of 25 mg / mL and stirred to prepare a tissue marking agent.

[0041] Example 2: Tissue marking agent containing siICG with an average particle size of 15 nm and covalently linked silica nanoparticles at a concentration of 25 mg / ml in a solution containing dextran (1) Silica nanoparticles used The silica nanoparticles used were commercially available silica nanoparticles (SNOWTEX ST-PS-S, manufactured by Nissan Chemical Co., Ltd.). These silica nanoparticles have a complex, branched chain structure, with multiple particles linked together to form a rosary or pearl necklace-like shape (see the particle structure in Figure 1(c) after ICG labeling). According to the product catalog, the average particle diameter of each particle is 15 nm. Observation with a transmission electron microscope revealed that the clusters of linked silica nanoparticles were 50–500 nm long (judging by the linear distance at their longest point) and consisted of 5–120 silica nanoparticles.

[0042] (2) Preparation of siICG particles To 25 mL of an aqueous dispersion of silica nanoparticles (200-300 mg / mL), 1.8 mL of (3-aminopropyl)triethoxysilane, 1 mL of aqueous ammonia (28% by mass), and 75 mL of purified water were added, vigorously stirred, and heated at 75°C for 18 hours. Then, 2.2 mL of 10 mM HCl was added and the pH was adjusted to 1.6. The dispersion was then centrifuged (10,000 rpm, 18,800 × g, 20°C, 15 minutes) and the supernatant was removed. 300 mL of 10 mM HCl was added to adjust the pH to approximately 2.0, and the mixture was centrifuged again and the supernatant was removed. The precipitate was collected using a spoon, and 50 mL of 10 mM HCl was added. The mixture was stirred using a magnetic stirrer to redisperse the amino-modified silica nanoparticles. The pH was then adjusted to 7.4 by adding 200 μL of 8 N NaOH in 200 μL increments. Next, 12.5 mg of ICG-NHS (15.0 μmol, Catalog No. POS16 04, Funakoshi Co., Ltd.) in DMF (1 mL), 121 mg of Water-Soluble Carbodiimide Hydrochloride-HCl (WSCD-HCl) (81 μmol, Peptide Institute, Inc.), and 93 mg of N-hydroxysuccinimide (81 μmol, Fujifilm Wako Pure Chemical Industries, Ltd.) were dissolved in 100 mL of ethanol and added to amino-functionalized silica nanoparticles (100 mL, pH 7.4). The mixture was incubated at room temperature (25°C) for 18 hours in the dark. The reaction mixture was then centrifuged (10,000 rpm, 18,800 × g, 4°C, 15 minutes) and the supernatant was removed. 300 mL of ethanol was added, the mixture was centrifuged again, and the supernatant was removed. The precipitate was then collected using a medicine spoon, 400 mL of ethanol was added, and the mixture was redispersed by stirring using a magnetic stirrer. This centrifugation procedure was repeated twice to remove unreacted ICG-NHS. Finally, the precipitate was dispersed in 400 mL of ethanol and dried using a spray dryer to recover the powder. As shown in Figure 1(c), the resulting powder had a complex, branched chain structure in which multiple particles were linked together, forming a rosary-like or pearl necklace-like shape. The average particle diameter of each particle was 15.0 nm, and the size of the linked silica nanoparticle clusters ranged from 50 nm to 500 nm, consisting of 5 to 120 silica nanoparticles.

[0043] (3) Preparation of tissue marking agent An aqueous solution was prepared by dissolving dextran (40 kDa, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) at a concentration of 10% by mass and NaCl (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) at a concentration of 0.9% by mass in purified water. The recovered siICG particles were added to 1 mL of the prepared solution at a concentration of 25 mg / mL and stirred to prepare a tissue marking agent.

[0044] Comparative Example 1: Tissue marking agent containing siICG, in which individual silica nanoparticles are spherical and independent, with an average particle size of 65 nm, at a concentration of 25 mg / ml in a solution containing dextran. (1) Silica nanoparticles used The silica nanoparticles used were commercially available silica nanoparticles (SNOWTEX ST-YL, manufactured by Nissan Chemical Co., Ltd., 65 nm) with an average particle diameter of 65 nm. These silica nanoparticles consisted of individual spherical silica nanoparticles (after ICG labeling, see the particle structure in Figure 1(a)). The average particle diameter was 65 nm. Note that in Figure 1(a), some particles aggregate, resulting in a strong black contrast near the periphery of each particle. This is due to the re-aggregation of multiple particles, resulting in overlapping particles. As can be seen from Figures 1(b) and 1(c), the areas of strong black contrast near the periphery of each particle are almost nonexistent, suggesting that the particles in the aggregate shown in Figure 1(a) are not bonded together. (2) Preparation of siICG particles To 75 mL of silica nanoparticles (480 mg / mL), 5.625 mL of (3-aminopropyl)triethoxysilane, 3 mL of aqueous ammonia (28% by mass), and 225 mL of purified water were added, vigorously stirred, and heated at 75°C for 18 hours. Then, 2.8 mL of 10 mM HCl was added to adjust the pH to 1.6. The dispersion was then centrifuged (10,000 rpm, 18,800 × g, 20°C, 15 minutes) and the supernatant was removed. 300 mL of 10 mM HCl was added to adjust the pH to approximately 2.0, and the dispersion was centrifuged again and the supernatant was removed. The precipitate was collected using a spoon, and 100 mL of 10 mM HCl was added. The mixture was stirred using a magnetic stirrer to redisperse the amino-modified silica nanoparticles. Then, 400 μL of 8 N NaOH was added in aliquots to adjust the pH to 7.4. Next, 25 mg of ICG-NHS (30.0 μmol, Catalog No. POS16 04, Funakoshi Co., Ltd.) in DMF (1 mL), 243 mg of Water-Soluble Carbodiimide Hydrochloride-HCl (WSCD-HCl) (162 μmol, Peptide Institute, Inc.), and 186 mg of N-hydroxysuccinimide (162 μmol, Fujifilm Wako Pure Chemical Industries, Ltd.) were dissolved in 100 mL of ethanol and added to amino-functionalized silica nanoparticles (100 mL, pH 7.4). The mixture was incubated at room temperature (25°C) for 18 hours in the dark. The reaction mixture was then centrifuged (10,000 rpm, 18,800 × g, 4°C, 15 minutes) and the supernatant was removed. 400 mL of ethanol was added, the mixture was centrifuged again, and the supernatant was removed. The precipitate was then collected using a medicine spoon, 400 mL of ethanol was added, and the mixture was redispersed by stirring using a magnetic stirrer. This centrifugation procedure was repeated twice to remove unreacted ICG-NHS. Finally, the mixture was dispersed in 400 mL of ethanol and dried using a spray dryer (B-290, Nippon Buchi Co., Ltd.) to recover the powder. As shown in Figure 1(a), the resulting powder contained individual, spherical silica nanoparticles, with an average particle diameter of 65 nm. As mentioned above, overlapping particles in Figure 1(a) are observed as a strong black contrast, and the particle clusters shown in Figure 1(a) are not bonded together.

[0045] (3) Preparation of tissue marking agent An aqueous solution was prepared by dissolving dextran (40 kDa, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) at a concentration of 10% by mass and NaCl (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) at a concentration of 0.9% by mass in purified water. The recovered siICG particles were added to 1 mL of the prepared solution at a concentration of 25 mg / mL and stirred to prepare a tissue marking agent.

[0046] Example 3: Tissue marking agent containing siICG with covalently linked silica nanoparticles and an average particle size of 12 nm at a concentration of 25 mg / ml in a solution containing no dextran.

[0047] NaCl (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in purified water to prepare a 0.9% NaCl aqueous solution, to which the same siICG as in the tissue marking agent of Example 1 was added at a concentration of 25 mg / mL and stirred to prepare the tissue marking agent.

[0048] Example 4: Tissue marking agent containing siICG with covalently linked silica nanoparticles having an average particle size of 15 nm at a concentration of 25 mg / ml in a solution containing no dextran. NaCl (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in purified water to prepare a 0.9% NaCl aqueous solution, to which the same siICG as used in the tissue marking agent in Example 2 was added at a concentration of 25 mg / mL and stirred to prepare the tissue marking agent.

[0049] Comparative Example 2: Tissue marking agent containing siICG, in which individual silica nanoparticles are spherical and independent, with an average particle size of 65 nm, at a concentration of 25 mg / ml in a solution that does not contain dextran. NaCl (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in purified water to prepare a 0.9% NaCl aqueous solution, to which the same siICG as used in the tissue marking agent of Comparative Example 1 was added at a concentration of 25 mg / mL and stirred to prepare the tissue marking agent.

[0050] Example 5: Tissue marking agent containing siICG with an average particle size of 12 nm and covalently linked silica nanoparticles at a concentration of 100 mg / ml in a solution containing dextran The same siICG as used in the tissue marking agent of Example 1 was added to an aqueous solution containing 10% dextran and 0.9% NaCl in an amount to give a concentration of 100 mg / mL, and the mixture was stirred to prepare a tissue marking agent.

[0051] Example 6: Tissue marking agent containing siICG with an average particle size of 15 nm and covalently linked silica nanoparticles at a concentration of 100 mg / ml in a solution containing dextran The same siICG as used in the tissue marking agent of Example 2 was added to an aqueous solution containing 10% dextran and 0.9% NaCl in an amount to give a concentration of 100 mg / mL, followed by stirring, to prepare a tissue marking agent.

[0052] Comparative Example 3: Tissue marking agent containing siICG, in which individual silica nanoparticles are spherical and independent and have an average particle size of 65 nm, at a concentration of 200 mg / ml in a solution containing dextran. The same siICG as used in the tissue marking agent of Comparative Example 1 was added to an aqueous solution containing 10% dextran and 0.9% NaCl in an amount to give a concentration of 200 mg / mL, followed by stirring, to prepare a tissue marking agent.

[0053] The tissue marking agents of each example and comparative example are summarized in the table below. [Table 1]

[0054] 1. Fluorescence Intensity Measurement To evaluate the fluorescence intensity of siICG in the dispersion medium of the tissue marking agents of Examples 1 to 4 and Comparative Examples 1 and 2, physiological saline containing 10% dextran was added to each tissue marking agent, stirred, and then 100 μL was added to a 96-well black plate, and the fluorescence intensity was measured using a plate reader (Spark multimode microplate reader (Tecan Trading AG, Switzerland)). The excitation wavelength was 760 nm, and the fluorescence wavelength was 820 nm. Figure 2 shows the fluorescence intensity of each tissue marking agent. The tissue marking agents of Examples 1, 2, and Comparative Example 1, which contained siICG in a solution containing dextran, exhibited similar fluorescence intensities, and no difference in fluorescence intensity was observed due to differences in silica particle structure. On the other hand, the tissue marking agents of Examples 3, 4, and Comparative Example 2, which contained siICG in a solution not containing dextran, exhibited slightly reduced fluorescence intensities compared to the tissue marking agents of Examples 1, 2, and Comparative Example 1. This is presumably because the absence of a dispersant caused the hydrophobic indocyanine green on the silica surface to aggregate, resulting in quenching.

[0055] 2. Evaluation of dispersibility To evaluate the dispersion state of siICG in the dispersion medium for the tissue marking agents of Examples 1 to 4 and Comparative Examples 1 and 2, the transmittance of each tissue marking agent was measured over time using a UV-vis spectrophotometer (UV-2600, Shimadzu Corp.). The transmittance of each tissue marking agent is shown in Figures 3 to 6. Comparing the tissue marking agents of Examples 1, 2, and Comparative Example 1, which contain siICG particles at a concentration of 25 mg / ml in a solution containing dextran, as shown in Figure 3, the tissue marking agents of Examples 1 and 2, which contain siICG particles in which silica nanoparticles are covalently linked, had a stably low absorbance of around 0.1% for 30 minutes, and had significantly improved dispersion stability compared to the tissue marking agent of Comparative Example 1, which contains siICG particles in which each silica nanoparticle is spherical and independent. Similarly, when comparing the tissue marking agents of Examples 3, 4, and Comparative Example 2, which contain siICG particles at a concentration of 25 mg / ml in a solution that does not contain dextran, as shown in Figure 4, the tissue marking agents of Examples 3 and 4, which contain siICG particles in which silica nanoparticles are covalently linked, have significantly improved dispersion stability compared to the tissue marking agent of Comparative Example 2, which contains siICG particles in which each silica nanoparticle is spherical and independent. Furthermore, when comparing the tissue marking agents of Comparative Example 1 and Comparative Example 2, as shown in Figure 5, the tissue marking agent of Comparative Example 1 showed a greater increase in transmittance over time, indicating that the presence or absence of dextran had a significant impact on dispersion stability, whereas when comparing the tissue marking agents of Examples 1 to 4, as shown in Figure 6, there was no difference in dispersion stability depending on the presence or absence of dextran, indicating that the particle structure contributes significantly to dispersion stability. Note that the transmittance of the tissue marking agent of Example 3 increased to 1.2% after 30 minutes, but the rate of increase to 1.2% was extremely low compared to the comparative examples, indicating that the covalently linked structure contributes to maintaining dispersion stability.

[0056] 3. Administration test into the porcine gastric submucosa using an endoscope that can be extrapolated to humans A pig (40 kg) was deeply anesthetized and moved to the operating table and fixed in the left lateral position. Then, using an endoscope (GIF-Q260J, manufactured by Olympus Corporation) and an endoscope unit (VISERA ELIT II, ​​manufactured by Olympus Corporation), 200 μL of the tissue marking agents of Examples 5, 6, and Comparative Example 3 were administered locally to several sites in the gastric submucosa. After 32 days of feeding, an abdominal midline incision was made to expose the stomach to the outside of the abdominal cavity, and the abdominal aorta and inferior vena cava were incised and the pig was euthanized. The stomach was then excised, and the tissue administered with the tissue marking agent was collected. Near-infrared fluorescence was observed using an infrared observation camera system (pde-neo, manufactured by Hamamatsu Photonics K.K.). An excitation light of 2.0 was used. Figure 7(A) shows a photograph of the area around the site where the tissue marking agent of Comparative Example 3 was administered, taken with a conventional camera, and Figure 7(B) shows a photograph of the same area taken with a near-infrared camera. Photographs taken with a near-infrared camera of the areas around the sites where the tissue marking agents of Examples 5 and 6 were administered are shown in Figures 8 and 9, respectively. In Figure 7(B), Figures 8 and 9, the arrows indicate the locations where the tissue marking agents were administered. As shown in Figures 7(B), 8 and 9, it was confirmed that the tissue marking agents of Comparative Example 3, Example 5 and Example 6 remained without diffusing even one month after administration to the gastric submucosa.

Claims

1. A tissue marking agent comprising silica particles labeled with a fluorescent dye in an aqueous medium, wherein a plurality of said silica particles are linked together.

2. 10. The tissue marking agent of claim 1, wherein the linked individual silica particles have an average particle size in the range of 1 nm to 40 nm.

3. 2. The tissue marking agent of claim 1, wherein the number of linked silica particles is 5 to 1,000.

4. 2. The tissue marking agent of claim 1, wherein the total length of the linked silica particles is between 30 nm and 1 μm.

5. The tissue marking agent according to claim 1 , wherein the plurality of silica particles are linked in a linear or branched chain.

6. The tissue marking agent of claim 1 , wherein the fluorescent dye is covalently bound to the silica particles.

7. The tissue marking agent of claim 1 , wherein the aqueous medium comprises a water-soluble viscous substance.

8. The tissue marking agent according to claim 7 , 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.

9. The tissue marking agent of claim 8 , wherein the water-soluble viscous substance comprises a polysaccharide or a salt thereof.

10. The tissue marking agent according to claim 9, 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, xanthan gum, gellan gum, curdlan, carboxymethylcellulose, chitin / chitosan, and carrageenan.

11. The tissue marking agent of claim 10 , wherein the polysaccharide comprises alginate, dextran, and / or hyaluronic acid.

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

13. The tissue marking agent of claim 12 , wherein the fluorescent dye is indocyanine green or a derivative thereof.

14. 2. The tissue marking agent of claim 1, 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.

15. 10. The tissue marking agent of claim 1, wherein the pH of the tissue marking agent is in the range of 5.0 to 8.

0.

16. The tissue marking agent of claim 1 , wherein the concentration of the silica particles in the aqueous medium is in the range of 0.01 mg / mL to 1000 mg / mL.

17. The tissue marking agent according to claim 7 , 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.

18. The tissue marking agent of claim 1 for marking a localized area of ​​the digestive tract.

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