Marking composition, marking composition kit, and method for forming marking composition

Abstract

Provided are: a marking composition which is a gelled composition that contains a structural unit derived from at least one polymer (A) having an amino group and carrying no near-infrared fluorescent dye and a structural unit derived from at least one crosslinking agent (B) selected from the group consisting of genipin and glutaraldehyde, and which emits fluorescence when irradiated with near-infrared light; a marking composition kit; and a method for forming a marking composition.

Description

Marking composition, marking composition kit, and method for forming a marking composition

[0001] FIELD OF THE DISCLOSURE The present disclosure relates to marking compositions, marking composition kits, and methods of forming marking compositions.

[0002] Near-infrared light is known as the optimal wavelength for bioimaging because it is gentle on the body and its wavelength (700-1400 nm) is in the region known as the biological window, which does not overlap with biological components. For this reason, near-infrared cameras have been introduced in many hospitals in recent years. However, there are very few fluorescent molecules that absorb near-infrared light, and in the case of organic compounds, the synthesis process is complex and therefore expensive.

[0003] Indocyanine green (ICG) is a widely known fluorescent dye that responds to near-infrared light. Because ICG is a low-molecular-weight drug used to identify the location of blood vessels and tumors, it is unsuitable for use in areas where it must remain for a certain period of time. For example, if it is used as an imaging material that must remain in a specific area of ​​the body for a certain period of time during surgery, the low-molecular-weight reagent will leak out, preventing its function from being expected. Therefore, Patent Document 1 proposes a composition for gastrointestinal tumor marking that retains ICG in a specific area by crosslinking ICG-loaded chitosan with a crosslinking agent to form a gel.

[0004] Patent Document 1: Japanese Patent Application Laid-Open No. 2023-90180

[0005] By fixing ICG to a gel skeleton, as in the ICG-loaded gel disclosed in Patent Document 1, it is possible to retain the tissue marking material locally for a long period of time. However, retaining ICG in the body for a long period of time is difficult from the standpoint of safety and functionality for the living body. In addition, fluorescent dyes that respond to near-infrared light, such as ICG, are expensive, making it difficult to use them in large quantities for tissue marking, substance detection, etc.

[0006] In view of the above circumstances, the present disclosure aims to provide a marking material and related technology that emits fluorescence when irradiated with near-infrared light without using a fluorescent dye that responds to near-infrared light, such as ICG, and that can be manufactured relatively inexpensively.

[0007] The above-mentioned problems are solved by the following means. <1> A marking composition that emits fluorescence when irradiated with near-infrared light, the marking composition being a gelling composition containing constitutional units derived from at least one polymer (A) that has an amino group and does not carry a near-infrared fluorescent dye, and constitutional units derived from at least one crosslinking agent (B) selected from the group consisting of genipin and glutaraldehyde. <2> The marking composition according to <1>, in which the polymer (A) is chitosan. <3> The marking composition according to <1> or <2>, in which the crosslinking agent (B) is genipin. <4> The marking composition according to any one of <1> to <3>, which contains a thickener (C). <5> The marking composition according to <4>, in which the thickener (C) is at least one selected from the group consisting of glycerin, polyalkylene glycol, polyalkylene oxide, and carboxyvinyl polymer. <6> A marking composition kit comprising: a container A containing at least one polymer (A) having an amino group and not carrying a near-infrared fluorescent dye; and a container B containing at least one crosslinking agent (B) selected from the group consisting of genipin and glutaraldehyde, wherein the contents of container A and container B are mixed to gel the polymer (A) and the crosslinking agent (B), thereby forming a marking composition that emits fluorescence when irradiated with near-infrared light. <7> The marking composition kit according to <6>, wherein the polymer (A) is chitosan. <8> The marking composition kit according to <6> or <7>, wherein the crosslinking agent (B) is genipin. <9> The marking composition kit according to any one of <6> to <8>, wherein a thickener (C) is contained in at least one of container A and container B. <10> The marking composition kit according to <9>, wherein the thickener (C) is at least one selected from the group consisting of glycerin, polyalkylene glycol, polyalkylene oxide, and carboxyvinyl polymer. <11> The marking composition kit according to any one of <6> to <8>, further comprising a container C containing a thickener (C), and wherein the contents of the containers A, B, and C are mixed together to gel the polymer (A) and the crosslinking agent (B).<12> The marking composition kit according to <11>, wherein the thickener (C) is at least one selected from the group consisting of glycerin, polyalkylene glycol, polyalkylene oxide, and carboxyvinyl polymer. <13> A method for forming a marking composition, comprising injecting at least one polymer (A) having an amino group and not carrying a near-infrared fluorescent dye, and at least one crosslinker (B) selected from the group consisting of genipin and glutaraldehyde, into a specific site in an animal's body and allowing them to gel, thereby forming a marking composition that emits fluorescence when irradiated with near-infrared light. <14> The method for forming a marking composition according to <13>, wherein the polymer (A) is chitosan. <15> The method for forming a marking composition according to <13> or <14>, wherein the crosslinker (B) is genipin. <16> The method for forming a marking composition according to any one of <13> to <15>, wherein a thickener (C) is injected into the specific site together with the polymer (A) and the crosslinking agent (B). <17> The method for forming a marking composition according to <16>, wherein the thickener (C) is at least one selected from the group consisting of glycerin, polyalkylene glycol, polyalkylene oxide, and carboxyvinyl polymer.

[0008] According to the present disclosure, there is provided a marking material and related technology that exhibits fluorescence when irradiated with near-infrared light without using a fluorescent dye that responds to near-infrared light, such as ICG, and that can be manufactured relatively inexpensively.

[0009] 1 is an image showing the results of observing the gelling compositions of Example 1, Comparative Example 1, and Comparative Example 2 using white light and near-infrared fluorescence. 2 is an image showing the results of observing a composition gelled by subcutaneous injection of a chitosan-genipin solution under visible light. 3 is an image showing the results of observing a composition gelled by subcutaneous injection of a chitosan-genipin solution under chicken skin under near-infrared fluorescence. 4 is an image showing the results of observing a composition gelled by subcutaneous injection of a chitosan-genipin-PEG solution under chicken skin under visible light. 5 is an image showing the results of observing a composition gelled by subcutaneous injection of a chitosan-genipin-PEG solution under chicken skin under near-infrared fluorescence. 6 is a graph showing the fluorescence intensity when the gelling compositions prepared in the examples are excited at a wavelength of 700 nm. 7 is a graph showing the fluorescence intensity over time for three types of gelling compositions prepared using chitosan-genipin.

[0010]

[0013] The present inventors have conducted extensive research to develop a technology for relatively inexpensively producing a marking material that exhibits fluorescence when irradiated with near-infrared light without using indocyanine green (ICG), and have found that a gelling composition formed by mixing a specific polymer with a specific crosslinker component can be used as a marking composition that exhibits fluorescence when irradiated with near-infrared light, even if it does not contain a fluorescent dye such as ICG.

[0011] [Marking Composition] The marking composition according to the present disclosure is a composition obtained by mixing and gelling at least one polymer (A) (sometimes referred to simply as "polymer (A)" in this disclosure) that has an amino group and does not carry a near-infrared fluorescent dye with at least one crosslinker (B) selected from the group consisting of genipin and glutaraldehyde. The marking composition contains structural units derived from the polymer (A) and structural units derived from the crosslinker (B), and emits fluorescence when irradiated with near-infrared light. The marking composition according to the present disclosure does not contain (does not carry) a near-infrared fluorescent dye such as ICG, and the gel itself is the fluorescent molecule, making it a marking material with a new concept. Furthermore, the marking composition according to the present disclosure can increase its fluorescence intensity by further containing a specific thickener.

[0012] Conventional materials that respond to near-infrared light are mainly low-molecular-weight compounds and nanoparticles, but the near-infrared photogel that utilizes the gel skeleton in this disclosure, and the technology that enables the increase in fluorescence intensity by introducing substances, will lead to the creation of a new concept in near-infrared materials. They will also contribute to the creation of a new gel property, namely, conversion to fluorescent molecules through gelation.

[0013] Hereinafter, each component constituting the marking composition according to the present disclosure will be described.

[0014] <Polymer (A)> The polymer (A) is a polymer that has amino groups but does not carry a near-infrared fluorescent dye. It forms the main skeleton of the marking composition according to the present disclosure through crosslinking with a crosslinking agent (B). Note that "not carrying a near-infrared fluorescent dye" means that an organic compound that absorbs light and emits light in the near-infrared region of 700 to 1500 nm is not immobilized in the polymer. The polymer (A) is not particularly limited as long as it has amino groups but does not carry a near-infrared fluorescent dye, and emits fluorescence in response to near-infrared light upon crosslinking with a crosslinking agent (B) to form a gel. The polymer (A) used to form the marking composition according to the present disclosure can be any polymer that does not carry a near-infrared fluorescent dye and has two or more tertiary amino groups in its molecule. Examples of such polymers include chitosan, a basic polysaccharide; water-soluble collagen (collagen peptide) derived from a biopolymer; synthetic polymers such as polyalkylene glycols with amine-modified terminals; polyallylamine; branched polyethyleneimine; and aminoethylated acrylic polymers. When the marking composition according to the present disclosure is used as a bioimaging material, from the viewpoint of safety for the living body, at least one selected from the group consisting of chitosan, water-soluble collagen, and polyethylene glycol having an amine-modified end is preferred, and among these, chitosan is more preferred.

[0015] (Chitosan) Chitosan is derived from chitin, a type of aminopolysaccharide found in the exoskeletons of crustaceans such as crabs and shrimp, and has the following chemical structure: it is a naturally occurring polymer consisting of a repeating structure of glucosamine and a small amount of N-acetylglucosamine.

[0016]

[0017] Chitosan is generally obtained by deproteinizing the exoskeleton of crustaceans with an alkali such as caustic soda, decalcifying it with an acid solution such as hydrochloric acid, and then partially deacetylating the resulting chitin with a highly concentrated aqueous alkali solution such as caustic soda.

[0018] In this case, the degree of deacetylation (also referred to as DAC degree) can be adjusted by appropriately changing the alkali concentration, temperature, and treatment time used. Generally, the DAC degree is 60% or higher, and these have the property of being insoluble in water but soluble in aqueous acid solutions such as acetic acid. The average molecular weight of chitosan that can be used in the present disclosure is generally a weight-average molecular weight (calculated by GPC molecular weight measurement using pullulan as a standard) of approximately 10,000 to 4,000,000, preferably 15,000 to 3,000,000. The degree of deacetylation is preferably 65% ​​or higher, and more preferably 80% or higher.

[0019] The molecular weight of chitosan can be measured by GPC using an aqueous GPC column. Furthermore, unless otherwise specified, the degree of deacetylation can be measured by colloid titration using a polyvinyl potassium sulfate solution. The chitosan used in this disclosure may be commercially available as a food additive. For example, chitosan manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. can be used. Chitosan can be dissolved in a certain acid solution to prepare a salt. The acid used in the acid aqueous solution of chitosan can be a weak acid such as acetic acid or a strong acid such as hydrochloric acid.

[0020] (Water-soluble collagen (collagen peptide)) Collagen is a major protein extracted from the connective tissues of animals and fish, and is found in large amounts in animal skin, bones, and cartilage, as well as fish scales and skin. Among these, water-soluble collagen, also known as collagen peptide, is a type of collagen that, unlike regular collagen, is easily soluble in water, and its properties vary significantly depending on the processing method. Specifically, water-insoluble collagen can be processed by acid / alkali treatment, enzyme treatment, heat treatment, or other methods, as appropriate. By using a method that specifically decomposes collagen using an enzyme, it is possible to produce water-soluble collagen with an adjusted molecular weight and low viscosity. Such water-soluble collagen generally has a molecular weight ranging from several hundred to several tens of thousands.

[0021] (Polyalkylene glycol with amine-modified terminals) Polyalkylene glycol is a polymer obtained by polymerizing ethylene glycol or propylene glycol, and has a molecular weight of up to about 40,000. In the present disclosure, in addition to the linear polymer in which both terminals of the alkylene glycol are amine-modified as described above, multi-arm polyalkylene glycols branched into three or more branches can also be selected and used as appropriate. Such polyalkylene glycols branched into three or more branches can be preferably used in the present disclosure because they increase the number of crosslinking points with the crosslinking agent (B) described below and make it easier to form a gel structure.

[0022] Specific examples of amine-terminated polyalkylene glycols include poly(ethylene glycol)bis(amine), poly(propylene glycol)bis(2-aminopropyl ether), O,O'-bis(2-aminopropyl)polyethylene glycol-block-polyethylene glycol-block-polyethylene glycol, multi-arm poly(ethylene glycol)amines (e.g., 3-arm poly(ethylene glycol)amine, 4-arm poly(ethylene glycol)amine, 6-arm poly(ethylene glycol)amine, 8-arm poly(ethylene glycol)amine), trimethylolpropane tris[poly(propylene glycol), amine terminated]ether, and the like.

[0023] The molecular weight of the polymer (A) that forms the main skeleton of the marking composition by gelation with the crosslinking agent (B) is preferably about 1,500 to 375,000, more preferably about 1,500 to 200,000, from the viewpoint of viscosity.

[0024] <Crosslinking agent (B)> The crosslinking agent (B) is at least one selected from the group consisting of genipin and glutaraldehyde, and crosslinks the polymer (A) to form a gel composition. When the marking composition according to the present disclosure is used as a bioimaging material, genipin is preferred as the crosslinking agent (B) from the viewpoint of safety to living organisms.

[0025] Genipin is a compound having the structure represented by the following formula, and is a plant-derived cross-linking agent with extremely low cytotoxicity that is present in the extract of the fruit of Genipa americana.

[0026]

[0027] (Glutaraldehyde) Glutaraldehyde is a compound having a structure represented by the following formula, and reacts with the amino groups of the polymer (A) to form intramolecular or intermolecular crosslinks.

[0028]

[0029] <Thickener (C)> The marking composition according to the present disclosure may further contain a thickener (C), and as the thickener (C), any thickener (C) used in food, cosmetics, pharmaceuticals, or industrial applications may be appropriately selected and used, as long as it is a water-soluble compound or polymer that does not have an amino group. Specific examples of the thickener (C) include starch, starch derivatives (carboxymethyl starch, hydroxyethyl starch, cationic starch, etc.), cellulose derivatives (carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, cationic cellulose, etc.), polysaccharides (xanthan gum, tragacanth gum, guar gum, gum arabic, pectin, dextran, etc.), polyacrylates, polyvinyl alcohol, polyalkylene glycols (polyethylene glycol, polypropylene glycol, etc.), polyalkylene oxides (polyethylene oxide, polypropylene oxide, etc.), carboxyvinyl polymers, alginate esters, glycerin, erythritol, sorbitol, maltitol, etc. These thickeners (C) can be used alone or in combination of two or more. When the above-mentioned thickener (C) is further added to the marking composition, it is more preferable to include one thickener (C) selected from the group consisting of glycerin, polyalkylene glycols, polyalkylene oxides, and carboxyvinyl polymers. By including a thickener (C) in the marking composition according to the present disclosure, the fluorescence intensity can be increased; for example, by adding a thickener (C) to a chitosan-genipin gelling composition to form a marking composition, the fluorescence intensity when irradiated with near-infrared light can be increased.

[0030] <Method for Producing Marking Composition> The marking composition according to the present disclosure can be produced by mixing the aforementioned polymer (A) and crosslinker (B) to form a gel, and optionally adding a thickener (C). For example, the amino groups of chitosan are bonded via genipin as a crosslinker to form a gel composition. The structure of the marking composition according to the present disclosure is influenced by the types and blend ratios of the polymer (A), crosslinker (B), and optionally added thickener (C). The blend ratio of the polymer (A) to the crosslinker (B) can be selected depending on the type and intended use of the marking composition. For example, when chitosan is crosslinked with genipin to form a gel composition, the blend ratio (molar ratio) of chitosan to genipin can be in the range of 1:3 to 1:150. When preparing a gel from chitosan and genipin, the concentration required for gelation is preferably 0.001 M or more for a chitosan concentration of 30 g / L. Furthermore, when genipin is 0.01 M, gelation occurs if chitosan is 10 g / L, but gelation is difficult if chitosan is 5 g / L or less.

[0031] When the marking composition according to the present disclosure is used as a bioimaging material, a gel composition (marking composition) obtained by adding and mixing a crosslinking agent (B) to a solution obtained by dissolving a polymer (A) in water and heating the resulting mixture may be injected into a specific site inside the body of an animal, or a marking composition that emits fluorescence when irradiated with near-infrared light may be formed by injecting the polymer (A) and the crosslinking agent (B) separately into a specific site inside the body of an animal and allowing them to gel.

[0032] <Marking Composition Kit> The present disclosure also provides a marking composition kit that includes a container A containing at least one polymer (A) that has an amino group and does not carry a near-infrared fluorescent dye, and a container B containing at least one crosslinking agent (B) selected from the group consisting of genipin and glutaraldehyde, and that is used to form a marking composition that emits fluorescence when irradiated with near-infrared light by mixing the contents of container A with the contents of container B to gel the polymer (A) and the crosslinking agent (B).

[0033] For example, a marking composition kit may be prepared by separately filling a single dose of chitosan and genipin into separate containers. The contents (containers) may then be mixed at the time of use to produce an injectable formulation. Chitosan is dissolved in an appropriate solvent, such as a 0.1 mol / L aqueous acetic acid solution, and the final formulation is pre-filled with a final chitosan concentration of 5-40 g / L, preferably 10-30 g / L, and a genipin concentration of 0.0001-1 mol / L, preferably 0.001-0.1 mol / L, upon mixing. Each formulation for fractional filling is filled separately in 2.5-30 mL, preferably approximately 5-20 mL, and mixed at the time of use.

[0034] Containers for filling each of the fractional fill formulations of the marking composition kit of the present disclosure include bags, plastic containers, ampoules, vials, and glass containers. The containers may be for storage purposes only or may be pre-filled syringe containers for storage. In the method for producing the fractional fill formulations in a storage syringe, each or any of the components constituting each fractional fill formulation is produced under an inert gas atmosphere (under conditions where oxygen in the atmosphere or in the water used is substantially absent or present in a limited amount that does not adversely affect the resulting composition). Examples of the inert gas used include nitrogen gas.

[0035] The marking composition of the present disclosure thus obtained is filtered and sterilized by conventional methods, filled into a container, and then sterilized by high-pressure steam as necessary and stored at room temperature. This dispensing step is also preferably carried out under an inert gas atmosphere such as nitrogen gas. Usually, dissolved oxygen in the injectable preparation is degassed according to conventional methods, and then nitrogen gas is injected to replace the atmosphere.

[0036] The marking composition of the present disclosure can also be freeze-dried by a conventional method to prepare a kit together with a solubilizer so that the marking composition can be dissolved immediately before use. As a dissolving solution for the preparation to be used immediately, a solution of a polyhydric alcohol and / or a sugar can be used.

[0037] Furthermore, when the marking composition kit of the present disclosure also uses the aforementioned thickener (C), the thickener (C) may be contained in at least one of the container A containing the polymer (A) and the container B containing the crosslinking agent (B), or a container C containing the thickener (C) may be provided separately from containers A and B. When the marking composition kit of the present disclosure includes container C containing the thickener (C), the contents of container A, container B, and container C may be mixed to gel the polymer (A) and crosslinking agent (B).

[0038] <Method of Using the Marking Composition> The use of the marking composition according to the present disclosure is not limited, but the marking composition according to the present disclosure is suitable as a bioimaging material and can be used, for example, to mark gastrointestinal tumors. A gastrointestinal tumor refers to a benign or malignant tumor or polyp derived from the stomach, esophagus, or large intestine of a mammal, particularly a human.

[0039] The marking composition according to the present disclosure can be injected into tissue, for example, via a syringe needle. Alternatively, the marking composition can be formed by injecting the polymer (A) and crosslinking agent (B) into specific tissue sites via a syringe needle and allowing them to crosslink (gel) within the tissue. For example, after mixing chitosan and genipin, the composition remains in a fluid state for a predetermined period of time (e.g., within 60 minutes) while the crosslinking reaction is in progress. This fluid composition can be injected into the tumor site from inside the digestive tract using an endoscope via a syringe needle. Alternatively, a thickener (C) can be injected into a specific site together with the polymer (A) and crosslinking agent (B).

[0040] The marking composition according to the present disclosure can be injected into the gastrointestinal wall directly under an endoscope to leave a small mark, allowing it to be used to determine the treatment area. For example, the marking composition can be injected into the tumor site using an endoscopic injection needle to mark the area for surgical resection. The location of the marked tumor can then be detected from outside the gastrointestinal tract using a laparoscopic microscope equipped with a near-infrared camera, allowing for reliable resection of the tumor tissue during laparoscopic surgery.

[0041] Examples of the marking composition according to the present disclosure will be described below, but the present disclosure is not limited to the following examples.

[0042] In the examples and comparative examples, the following reagents and equipment were used: (Reagents) Chitosan (CS) (Chitosan, Purified Powder MW -15,000: manufactured by Olysciences) Four-branched amine-modified polyethylene glycol (referred to as modified PEG) (4-arm PEG-NH ２ , molecular weight 20k: manufactured by SINOPEG Co., Ltd.) 25% glutaraldehyde solution (GLA) (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) Genipin (Ge) (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) 3-(3-dimethylaminopropyl)carbodiimide (manufactured by Dojindo Laboratories, Inc.) N-hydroxysuccinimide (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) L(+)-tartaric acid, special grade (Kanto Chemical Co., Ltd.) CHO-PEG-CHO, 6K (Biopharma PEG Scientific Inc.) Glycerin (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) Polyethylene glycol (polyethylene glycol 1,000: manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) Polyethylene oxide (600K g / mol, manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) Carboxyvinyl polymer (Hibiswako 104, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (Apparatus) Near-infrared fluorescence intensity measuring device (Maestro, manufactured by Cambridge Research & Instrumentation (CRi)) Multiplate reader (Spectra Maxmini, manufactured by Molecular Devices) Spectrofluorometer (RF-6000, manufactured by Shimadzu Corporation)

[0043] [Fluorescence Intensity Measurement] Example 1 (Preparation of Gelling Composition) Chitosan (30 g / L, 90 mg) was added to 1 mL of acetic acid (0.1 mol / L) and stirred, followed by the addition of 2 mL of aqueous sodium hydroxide solution (0.2 mol / L). 7.5 μL of genipin solution (4 mol / L) was added to the prepared chitosan solution and mixed, then placed in a cylindrical mold (inner diameter 15 mm, height 7 mm) and allowed to stand at 37°C for 1 day to prepare a gelling composition (CS-Ge gel).

[0044] (Near-infrared fluorescence observation using Maestro) Near-infrared fluorescence observation was performed on the cylindrical gel (diameter 15 mm, height 7 mm) prepared as described above using the following procedure. The gel was removed from the gel frame and placed on a petri dish. The gel and petri dish were then placed in a fluorescence intensity measurement device, and images were taken using excitation light of 704 nm and fluorescence of 804 nm. The fluorescence observation resulted in a fluorescence intensity of 0.0599. Near-infrared fluorescence was observed in the gel prepared using chitosan and genipin.

[0045] (Fluorescence Intensity Measurement Using a Spectrofluorometer) Chitosan (30 g / L, 90 mg) was added to 1 mL of acetic acid (0.1 mol / L) and stirred, followed by the addition of 2 mL of aqueous sodium hydroxide solution (0.2 mol / L). 7.5 μL of genipin solution (4 mol / L) was added to the prepared chitosan solution, mixed, and then placed in a 1 cm square cuvette and allowed to stand at 37°C for 1 day to prepare a gelling composition (CS-Ge gel). The prepared gelling composition was excited at a wavelength of 700 nm using a spectrofluorometer, and the fluorescence intensity was observed. The results of the fluorescence intensity versus wavelength are shown in Figure 4.

[0046] Comparative Example 1: A gelling composition was prepared using tartaric acid (Comparative Example 1) as a crosslinker for chitosan. Preparation method: (i) Chitosan (106.33 mg) was dissolved in acetic acid (0.1 mol / L, 1 mL), and then aqueous sodium hydroxide solution (0.2 mol / L, 2 mL) was added. (ii) 3-(3-dimethylaminopropyl)carbodiimide (346.66 mg) and N-hydroxysuccinimide (203.33 mg) were added to acetic acid (0.1 mol / L, 0.07 mL) and aqueous sodium hydroxide solution (0.2 mol / L, 0.14 mL) and stirred. (iii) Tartaric acid (8.33 mg) was dissolved in acetic acid (0.1 mol / L, 0.0235 mL) and aqueous sodium hydroxide solution (0.2 mol / L, 0.047 mL). The above three solutions were mixed, placed in a cylindrical mold (inner diameter 15 mm), and left to stand at 37°C for one day to prepare a gelled composition (chitosan-tartaric acid gel). The near-infrared fluorescence intensity of the gelled composition was measured in the same manner as in Example 1. However, no near-infrared fluorescence was observed in this gelled composition.

[0047] Comparative Example 2: Chitosan (90 mg) was dissolved in acetic acid (0.1 mol / L, 1 mL), and then aqueous sodium hydroxide solution (0.2 mol / L, 2 mL) was added. A CHO-PEG-CHO solution (6K, 0.04 mol / L, 750 μL) was added to the prepared chitosan solution, and the mixture was allowed to stand at 37°C for one day to prepare a gelled composition (chitosan-PEG gel). The near-infrared fluorescence intensity of the gelled composition was measured in the same manner as in Example 1. However, no near-infrared fluorescence was observed in this gelled composition. The results of observing the gelled compositions of Example 1, Comparative Example 1, and Comparative Example 2 under white light and near-infrared fluorescence are shown in Figure 1.

[0048] Example 2 (Preparation of Marking Composition) A gelling composition was prepared using the following combination of polymers and crosslinkers. (1) Preparation of Chitosan-Genipin Gel (CS-Ge) Chitosan (15 g / L, 45 mg) was dissolved in 1 mL of acetic acid (0.1 mol / L), and 2 mL of aqueous sodium hydroxide solution (0.2 mol / L) was added. 7.5 μL of genipin solution (4 mol / L) was added to the prepared chitosan solution and mixed, then the mixture was placed in a cylindrical mold (inner diameter 15 mm, height 7 mm) and left to stand at 37°C for 1 day to prepare a gelling composition (CS-Ge gel).

[0049] (2) Preparation of chitosan-glutaraldehyde gel (CS-GLA) Chitosan (45 mg in the case of 15 g / L) was dissolved in acetic acid (0.1 mol / L, 1 mL), and sodium hydroxide solution (0.2 mol / L, 2 mL) was added. The solution was then mixed with GLA solution (5 wt%, 0.45 mL), allowed to stand at 37°C for 1 day, and placed in a cylindrical mold (inner diameter 15 mm, height 7 mm) to prepare a gelling composition (CS-GLA).

[0050] (3) Preparation of four-arm amine-modified polyethylene glycol-genipin gel (modified PEG-Ge) ２ A PEG solution (70 g / L) prepared by dissolving -20k (210 mg) in phosphate buffer (pH 7, 3 mL) was placed in a cylindrical mold (inner diameter 15 mm, height 7 mm), and genipin (4 mol / L, 7.5 μL) was added. The mixture was left to stand at 37°C for one day to prepare modified PEG-Ge.

[0051] (4) Preparation of four-arm amine-modified polyethylene glycol-glutaraldehyde gel (modified PEG-GLA) 4arm PEG-NH ２ A PEG solution (70 g / L) prepared by dissolving -20k (210 mg) in phosphate buffer (pH 7, 3 mL) was mixed with a GLA solution (5 wt %, 0.45 mL), placed in a cylindrical mold (inner diameter 15 mm, height 7 mm) and left to stand at 37°C for 1 day to prepare a gelling composition (modified PEG-GLA).

[0052] (Fluorescence observation using Maestro) Near-infrared fluorescence observation was carried out in the same manner as in Example 1. The results are shown in Table 1.

[0053]

[0054] [Effect of Substances on Increase in Near-Infrared Fluorescence Intensity] The effect of thickeners on fluorescence intensity was investigated. Glycerin, a low molecular weight compound, and polyethylene glycol, a high molecular weight compound, were used as thickeners.

[0055] Example 3 (Glycerin) An experiment was conducted on a gel prepared by crosslinking 15 g / L chitosan and 0.01 M genipin in Example 2. Chitosan (45 mg) was dissolved in acetic acid (0.1 mol / L, 1 mL), and then aqueous sodium hydroxide solution (0.2 mol / L, 2 mL) was added. Glycerin (0.6 mL) was added to the prepared chitosan solution and stirred, followed by the addition of genipin (4 mol / L, 7.5 μL). The mixture was left standing at 37°C for one day to prepare a gelling composition containing glycerin. Fluorescence observation revealed a fluorescence intensity of 0.151. Compared to Example 2, it was confirmed that the addition of glycerin increased the fluorescence intensity.

[0056] Example 4 (Polyethylene Glycol) An experiment was conducted on a gel prepared using 15 g / L chitosan and 0.01 M genipin as in Example 2. Chitosan (45 mg) was dissolved in acetic acid (0.1 mol / L, 1 mL), and then aqueous sodium hydroxide solution (0.2 mol / L, 2 mL) was added. PEG (1K, 0.03 g) was added to the prepared chitosan solution and stirred, followed by genipin (4 mol / L, 7.5 μL). The mixture was allowed to stand at 37°C for one day to prepare a gelation composition containing polyethylene glycol. Fluorescence observation revealed a value of 0.0999. Compared to Example 2, it was confirmed that the addition of polyethylene glycol increased the fluorescence intensity.

[0057] Example 5 (Polyethylene Oxide) Experiments were conducted on gels prepared using 15 g / L chitosan and 0.01 M genipin as in Example 2. Chitosan (45 mg) was dissolved in acetic acid (0.1 mol / L, 1 mL), followed by the addition of aqueous sodium hydroxide solution (0.2 mol / L, 2 mL). PEO (600K, 0.03 g for 1 wt% and 0.06 g for 2 wt%) was added to the prepared chitosan solution and stirred. Genipin (4 mol / L, 7.5 μL) was then added and the mixture was allowed to stand at 37°C for one day to prepare a gelling composition containing polyethylene oxide. Fluorescence observation revealed that the fluorescence intensities for the 1 wt% and 2 wt% polyethylene oxide solutions were 0.0883 and 0.0925, respectively. Compared to Example 2, it was confirmed that the fluorescence intensity increased with increasing polyethylene oxide content.

[0058] Example 6 (Polyethylene Glycol) An experiment was conducted on a gel prepared using 15 g / L chitosan and 0.35 wt% glutaraldehyde as in Example 2. Chitosan (45 mg) was dissolved in acetic acid (0.1 mol / L, 1 mL), followed by the addition of aqueous sodium hydroxide solution (0.2 mol / L, 2 mL). PEG (1K, 0.03 g) was added to the prepared chitosan solution and dissolved. The solution was then mixed with GLA solution (5 wt%, 0.45 mL) and allowed to stand at 37°C for one day to prepare a gel composition containing polyethylene glycol. Fluorescence observation revealed a fluorescence intensity of 0.3080. Compared to Example 2, the addition of polyethylene glycol confirmed an increase in fluorescence intensity.

[0059] The fluorescence intensity increased or decreased depending on the presence or absence and type of thickener, and the fluorescence intensity of both the chitosan-genipin gel composition (CS-Ge) and the chitosan-glutaraldehyde gel composition (CS-GLA) increased with the addition of polyethylene glycol.

[0060] Example 7 (Fluorescence observation using a multiplate reader) A cylindrical gel (diameter 15 mm, height 7 mm) prepared by the following procedure was removed from the gel frame and placed in a 12-well well, and the fluorescence intensity was measured at an excitation light of 710 nm and a fluorescence wavelength of 810 nm.

[0061] (Preparation of Gelling Composition from Chitosan-Genipin-Glycerin) An experiment was performed on a gel prepared by crosslinking 15 g / L chitosan and 0.003 M genipin. Chitosan (210 mg) was dissolved in acetic acid (0.1 mol / L, 466 mL), and then an aqueous sodium hydroxide solution (0.2 mol / L, 932 mL) was added to prepare a chitosan solution. Genipin (4 mol / L, 10.5 μL) was then added to this chitosan solution. Glycerin was then added to a concentration of 10 wt %, and the mixture was stirred and then allowed to stand at 37°C for one day to prepare a gelling composition containing glycerin. Fluorescence observation of this gelling composition using a multiplate reader revealed that the fluorescence intensity was 2.8 × 10 ６ It was.

[0062] (Preparation of Gelling Composition from Chitosan-Genipin-Polyethylene Glycol) An experiment was performed on a gel prepared by crosslinking 15 g / L chitosan and 0.003 M genipin. Chitosan (210 mg) was dissolved in acetic acid (0.1 mol / L, 466 mL), and then an aqueous sodium hydroxide solution (0.2 mol / L, 932 mL) was added to prepare a chitosan solution. Genipin (4 mol / L, 10.5 μL) was then added to this chitosan solution. Polyethylene glycol was then added to the solution to give a concentration of 1 wt %, and the mixture was stirred and then allowed to stand at 37°C for one day to prepare a gelling composition containing polyethylene glycol. Fluorescence observation of this gelling composition using a multiplate reader revealed that the fluorescence intensity was 3.6 × 10 ６ It was.

[0063] (Preparation of Gelling Composition from Chitosan-Genipin-Carboxyvinyl Polymer) An experiment was performed on a gel prepared by crosslinking 15 g / L chitosan and 0.003 M genipin. Chitosan (210 mg) was dissolved in acetic acid (0.1 mol / L, 466 mL), and then aqueous sodium hydroxide solution (0.2 mol / L, 932 mL) was added to prepare a chitosan solution. Genipin (4 mol / L, 10.5 μL) was then added to this chitosan solution. After that, carboxyvinyl polymer was added so that the concentration was 1 wt %, and the mixture was stirred. The mixture was then left to stand at 37°C for 1 day to prepare a gelling composition containing carboxyvinyl polymer. Fluorescence observation of this gelling composition using a multiplate reader revealed that the fluorescence intensity was 2.4 × 10 ６ It was.

[0064] (Fluorescence observation of a gel prepared from chitosan-genipin over time) An experiment was conducted on a gel prepared by crosslinking 15 g / L chitosan and 0.003 M genipin. Chitosan (210 mg) was dissolved in acetic acid (0.1 mol / L, 466 mL), and then aqueous sodium hydroxide solution (0.2 mol / L, 932 mL) was added to prepare a chitosan solution. Genipin (4 mol / L, 10.5 μL) was added to this chitosan solution. After stirring, the mixture was left to stand at 37°C for one day to prepare a gelling composition. The prepared gel was subjected to fluorescence observation using a multiplate reader after a predetermined time had elapsed. The prepared gel was immersed in a citrate-phosphate buffer solution at pH 4.5 at 37°C, except for fluorescence observation. The results are shown in Figure 5.

[0065] (Fluorescence Observation of Gels Prepared from Chitosan-Genipin-Glycerin Over Time) An experiment was conducted on a gel prepared by crosslinking 15 g / L chitosan and 0.003 M genipin. Chitosan (210 mg) was dissolved in acetic acid (0.1 mol / L, 466 mL), and then aqueous sodium hydroxide solution (0.2 mol / L, 932 mL) was added to prepare a chitosan solution. Genipin (4 mol / L, 10.5 μL) was then added to this chitosan solution. Glycerin was then added to a concentration of 20 wt%, and the mixture was stirred and then allowed to stand at 37°C for one day to prepare a gelling composition. The prepared gel was subjected to fluorescence observation using a multiplate reader after a predetermined time had elapsed. The prepared gel was immersed in a citrate-phosphate buffer solution at pH 4.5 at 37°C, except for fluorescence observation. The results are shown in Figure 5.

[0066] (Fluorescence Observation of Gels Prepared from Chitosan-Genipin-Polyethylene Glycol Over Time) An experiment was conducted on a gel prepared by crosslinking 15 g / L chitosan and 0.003 M genipin. Chitosan (210 mg) was dissolved in acetic acid (0.1 mol / L, 466 mL), and then aqueous sodium hydroxide solution (0.2 mol / L, 932 mL) was added to prepare a chitosan solution. Genipin (4 mol / L, 10.5 μL) was then added to this chitosan solution. Polyethylene glycol was then added to the solution to a concentration of 10 wt%, and the mixture was stirred and then allowed to stand at 37°C for one day to prepare a gelling composition. The prepared gel was subjected to fluorescence observation using a multiplate reader after a predetermined time had elapsed. The prepared gel was immersed in a citrate-phosphate buffer solution at pH 4.5 at 37°C, except for fluorescence observation. The results are shown in Figure 5.

[0067] The results of fluorescence observation over time in Figure 5 show that the fluorescence intensity 28 days after gel preparation was almost the same as that on day 1. It was also found that the addition of a thickener prevented the decrease in fluorescence intensity over time.

[0068] (Fluorescence observation of gel prepared from chitosan-genipin-carboxyvinyl polymer over time) The gel prepared in Example 7 was subjected to fluorescence observation using a multiplate reader after 7 days. As a result, the fluorescence intensity was 10.7 × 10 ６ The prepared gel was immersed in a citrate-phosphate buffer solution of pH 4.5 at 37°C except for the fluorescence observation.

[0069] Example 8 (Preparation of Chitosan-Genipin Gel-Forming Solution 1) Chitosan (90 mg) was added to acetic acid (0.1 mol / L, 1 mL) and stirred, and then an aqueous sodium hydroxide solution (0.2 mol / L, 2 mL) was added to prepare a chitosan solution. Genipin (4 mol / L, 7.5 μL) was added to this chitosan solution to prepare Gel-Forming Solution 1.

[0070] (Preparation of Chitosan-Genipin-PEG Gel-Forming Solution 2) Chitosan (90 mg) was added to acetic acid (0.1 mol / L, 1 mL) and stirred, and then aqueous sodium hydroxide solution (0.2 mol / L, 2 mL) was added to prepare a chitosan solution. Genipin (4 mol / L, 7.5 μL) and polyethylene glycol were added to this chitosan solution so that the concentration was 1 wt %, thereby preparing Gel-Forming Solution 2.

[0071] (Fluorescence Observation with Maestro) Approximately 5 mm of chicken meat was placed in a 36 mm diameter Petri dish, with approximately 2 mm of chicken skin placed on top. Gelling composition-forming solutions 1 and 2 prepared as described above were each injected subcutaneously with a syringe and left for 150 minutes. After gelation was confirmed, near-infrared fluorescence observation was performed with Maestro. Figure 2A shows the results of visible light observation of the gelling composition gelled under chicken skin using gelling composition-forming solution 1, and Figure 2B shows the results of near-infrared fluorescence observation of the same composition. Figure 3A shows the results of visible light observation of the gelling composition gelled under chicken skin using gelling composition-forming solution 2, and Figure 3B shows the results of near-infrared fluorescence observation of the same composition. Fluorescence was observed in both cases.

[0072] The marking composition according to the present disclosure does not require a fluorescent dye that responds to near-infrared light, can be produced relatively inexpensively in large volumes (sizes), and has potential applications in the fields of engineering, agriculture, and medicine as a material that responds to near-infrared light. Furthermore, because the fluorescence intensity increases or decreases depending on the introduction of a substance, it can also be used for substance detection. By investigating the factors related to the increase or decrease in fluorescence intensity, it is possible to detect substances, and new industrial developments are expected.

[0073] The disclosures of Japanese Patent Application No. 2024-058054, filed on March 29, 2024, and Japanese Patent Application No. 2024-117196, filed on July 22, 2024, are incorporated herein by reference in their entirety. All publications, patent applications, and technical standards mentioned herein are incorporated herein by reference to the same extent as if each individual publication, patent application, and technical standard was specifically and individually indicated to be incorporated herein by reference.

Claims

1. A marking composition that is a gelling composition comprising a constituent unit derived from at least one polymer (A) that has an amino group and does not carry a near-infrared fluorescent dye, and a constituent unit derived from at least one crosslinking agent (B) selected from the group consisting of genipin and glutaraldehyde, and that emits fluorescence when irradiated with near-infrared light.

2. The marking composition according to claim 1, wherein the polymer (A) is chitosan.

3. The marking composition according to claim 2, wherein the crosslinking agent (B) is genipin.

4. The marking composition according to any one of claims 1 to 3, which contains a thickener (C).

5. The marking composition according to claim 4, wherein the thickener (C) is at least one selected from the group consisting of glycerin, polyalkylene glycol, polyalkylene oxide, and carboxyvinyl polymer.

6. A marking composition kit comprising: a container A containing at least one polymer (A) that has an amino group and does not carry a near-infrared fluorescent dye; and a container B containing at least one crosslinking agent (B) selected from the group consisting of genipin and glutaraldehyde; the kit being used to form a marking composition that emits fluorescence when irradiated with near-infrared light by mixing the contents of container A with the contents of container B to gel the polymer (A) and the crosslinking agent (B).

7. The marking composition kit according to claim 6, wherein the polymer (A) is chitosan.

8. The marking composition kit according to claim 7, wherein the crosslinking agent (B) is genipin.

9. A marking composition kit according to any one of claims 6 to 8, wherein at least one of said container A and said container B contains a thickener (C).

10. The marking composition kit according to claim 9, wherein the thickener (C) is at least one selected from the group consisting of glycerin, polyalkylene glycol, polyalkylene oxide, and carboxyvinyl polymer.

11. A marking composition kit according to any one of claims 6 to 8, which includes a container C containing a thickener (C), and which gels the polymer (A) and the crosslinking agent (B) by mixing the contents of said container A, the contents of said container B, and the contents of said container C.

12. The marking composition kit according to claim 11, wherein the thickener (C) is at least one selected from the group consisting of glycerin, polyalkylene glycol, polyalkylene oxide, and carboxyvinyl polymer.

13. A method for forming a marking composition, comprising injecting at least one polymer (A) having an amino group and not carrying a near-infrared fluorescent dye, and at least one crosslinking agent (B) selected from the group consisting of genipin and glutaraldehyde, into a specific site in an animal's body and causing them to gel, thereby forming a marking composition that emits fluorescence when irradiated with near-infrared light.

14. The method for forming a marking composition according to claim 13, wherein the polymer (A) is chitosan.

15. The method for forming a marking composition according to claim 14, wherein the crosslinking agent (B) is genipin.

16. A method for forming a marking composition according to any one of claims 13 to 15, wherein a thickener (C) is injected into the specific area together with the polymer (A) and the crosslinking agent (B).

17. The method for forming a marking composition according to claim 16, wherein the thickener (C) is at least one selected from the group consisting of glycerin, polyalkylene glycol, polyalkylene oxide, and carboxyvinyl polymer.

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