Composition and kit for gastrointestinal tumor marking

Abstract

To provide a gastrointestinal tumor-marking composition that finds the optimum material and composition of a marking material, causes no deviation when marking the tumor site, holds the marker for a long time, and enable precise surgery.SOLUTION: Provided is a gastrointestinal tumor-marking composition containing chitosan carrying a near-infrared fluorescent dye, the chitosan being crosslinked with a crosslinker. The marking composition has: a fluid state that allows injection into the tumor from the inside of the gastrointestinal tract using an endoscope within a predetermined time after cross-linking; and a non-fluid state that gels after a predetermined time and stays at the injected position for at least 24 hours.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present disclosure relates to a composition for gastrointestinal tumor marking, and more particularly to a near-infrared marking composition and kit for visualizing tumors during laparoscopic gastrectomy. [Background technology]

[0002] Laparoscopic gastrectomy is a surgical treatment method for early-stage gastric cancer. This is a surgical procedure for early-stage gastric cancer that cannot be completely removed with an endoscope (commonly known as a gastroscope), meaning that the cancer has penetrated deep into the submucosa and it is deemed necessary to remove lymph nodes near the stomach. Most early-stage gastric cancer is classified as stage I, and it is known that the chances of treatment are very high. Laparoscopic surgery is a treatment method that allows for such highly treatable surgery to be performed with as little strain on the body as possible and allows for faster postoperative recovery.

[0003] Laparoscopic surgery is performed under the same general anesthesia as open surgery. First, carbon dioxide gas is injected into the abdominal cavity to inflate it, and then a thin, high-performance camera (laparoscope) developed for this surgery is inserted through the belly button. At the same time, four or five small holes measuring 5 to 10 mm are made on both sides of the abdomen to insert surgical instruments, and the image of the inside of the abdomen taken by the laparoscope is displayed on a monitor while the tumor is removed and the surrounding lymph nodes are removed.

[0004] In laparoscopic surgery, endoscopic marking from inside the digestive tract is essential for accurately identifying tumor sites. Traditionally, India ink has been used as a marker. When injected into tissue, India ink often diffuses widely into the surrounding area. This makes the marking location unclear and inaccurate, and ink leakage can cause local inflammation. Furthermore, with conventional tissue markers that use dyes instead of India ink, the dye (staining agent) diffuses, meaning that even if the marking is made immediately before surgery, the dye spreads widely during surgery, resulting in a misalignment between the tumor site and the resection site. Furthermore, this method is only suitable for tumors with shallow penetration, and there are other issues, such as the marker coming off during the time leading up to surgery and the marker's borders becoming blurred, resulting in inaccuracies.

[0005] To address these issues, marker compositions using chitosan have been developed. For example, Patent Document 1 discloses a tissue marker containing partially deacetylated chitin or chitosan, carbon powder, or a dye applicable to the human body. Patent Document 2 also discloses a chitosan-based pharmaceutical composition, specifically a method for transferring a therapeutically active substance into host cells using a chitosan compound containing 5 to 300 glucosamine repeating units. Patent Document 3 further describes a drug carrier that contains a first component, which is a biocompatible polymer having amino groups, a saccharide, and a second component, which is a biocompatible material, and that protects the drug from gastric acid and swells or disintegrates under specific pH conditions to release the drug. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2007-262062 [Patent Document 2] Special Publication No. 2001-502736 [Patent Document 3] Japanese Patent Application Laid-Open No. 2014-074005 Summary of the Invention [Problem to be solved by the invention]

[0007] However, the tissue marker described in Patent Document 1 is produced by simply mixing chitosan and a dye in an aqueous solution and adjusting the pH, so the dye diffuses and is thought to have little ability to remain at the labeled site. Furthermore, Patent Document 2 describes the modification of chitosan compounds and their application to living organisms, but does not describe the use of chitosan as a tissue marker by labeling it with a fluorescent dye. Patent Document 3 describes a drug carrier in which chitosan gel used in the stomach is crosslinked with genipin, but does not disclose the use of chitosan as a tissue marker by labeling it with a fluorescent dye.

[0008] Therefore, the problem that the present invention aims to solve is to find the optimal material and composition for a marking composition, and to provide a composition for gastrointestinal tumor marking that will not shift when marking the tumor site, will retain the marker for a long period of time, and will enable accurate surgery. [Means for solving the problem]

[0009] The present disclosure has been made to solve the above-mentioned problems and relates to a composition containing chitosan carrying a near-infrared fluorescent dye. By crosslinking this chitosan with a crosslinking agent, it is possible to obtain a fluid state that allows it to be injected into a tumor from the inside of the digestive tract using an endoscope within a predetermined time after crosslinking, and a non-fluid state that gels after a predetermined time and remains at the injected site for a predetermined period of time. Specifically, the present disclosure includes the following embodiments.

[0010] (1) A composition for gastrointestinal tumor marking, comprising chitosan carrying a near-infrared fluorescent dye, the chitosan being crosslinked with a crosslinking agent, the composition having a fluid state that allows it to be injected into a tumor from the inside of the gastrointestinal tract using an endoscope within a predetermined time after crosslinking, and a non-fluid state that gels after the predetermined time has elapsed and remains at the injected location for at least 24 hours. (2) A marking composition according to (1) or (2) for detecting the location of a tumor from outside the digestive tract using a laparoscopic microscope equipped with a near-infrared camera. (3) The marking composition according to (1) or (2), wherein the predetermined time during which the composition remains in a fluid state after crosslinking is within 60 minutes. (4) The marking composition according to any one of (1) to (3), which remains at the injected site for at least 14 days. (5) The marking composition according to (4), which has a retention time of 30 days. (6) The marking composition according to any one of (1) to (5), wherein the gelled composition has a storage modulus of 500 to 100,000 Pa. (7) The marking composition according to any one of (1) to (6), wherein the swelling ratio of the gelled composition is 0.5 to 1.5. (8) A digestive tract tumor marking composition kit in which a single dose of chitosan carrying a near-infrared fluorescent dye and a single dose of a chitosan crosslinker are separately filled into individual containers, and the contents are mixed at the time of use to form an injectable preparation, in which the chitosan is dissolved in a solvent, the crosslinker includes genipin or a derivative thereof, and the kit is pre-filled so that the final concentrations of each component of the injectable preparation are 5 to 40 g / L of chitosan and 0.0001 to 1 mol / L of crosslinker. (9) The kit according to (8), wherein the pH of the injection formulation is within the range of 1 to 7. (10) The kit according to (8) or (9), wherein the near-infrared fluorescent dye is indocyanine green (ICG). [Effects of the Invention]

[0011] By injecting the marking composition of the present disclosure into the mucosal surface of the stomach or the like, its position can be accurately conveyed to the serosal surface, allowing a clear image to be read out with a single touch using a near-infrared camera during surgery. [Brief explanation of the drawings]

[0012] [Figure 1]Figure 1 is a graph showing the absorption wavelength of ICG-chitosan gel. The ICG concentrations at the time of preparation were ◯: 0.43×10 mol / L, □: 1.08×10 mol / L, and ▲: 2.15×10 mol / L. [Figure 2] Figure 2 shows the gelation state of the ICG-chitosan gel. (A) shows the state before gelation, and (B) shows the state after gelation. [Figure 3] Figure 3 is a graph showing the elastic modulus of ICG-chitosan gel. ●: storage modulus G' (Pa), □: loss modulus G'' (Pa). [Figure 4] Figure 4 shows the results of the ex vivo experiment conducted in Example 1, showing fluorescence observations of the ICG-chitosan gel (top) and ICG solution (bottom). (C) and (I) show the injection site. (M) is a photograph after gelation, and (N) shows the time course of the fluorescence intensity of the ICG-modified chitosan gel and the ICG-PBS solution. [Figure 5] Figure 5 shows the results of the ex vivo experiment in which five injection points were performed in Example 1, showing fluorescence observation of the ICG-chitosan gel (top row) and the ICG solution (bottom row). The injection points of the solution are indicated by white circles in (C) and (H). [Figure 6] Figure 6 is a graph showing the results of the swelling experiment conducted in Example 2 (◯ and ●: pH 1, △ and ▲: pH 3, ◇ and ◆: pH 5, □ and ■: pH 7, solid line indicates the presence of lysozyme, dashed line indicates the absence of lysozyme). [Figure 7] FIG. 7 is a graph showing the elastic modulus measured in Example 2 (pH 1 to 7). DETAILED DESCRIPTION OF THE INVENTION

[0013] Next, each embodiment of the present disclosure will be described with reference to the drawings. Note that each embodiment described below does not limit the invention according to the claims, and not all of the elements and combinations thereof described in each embodiment are necessarily essential to the solution of the present invention.

[0014] The present disclosure relates to a composition for gastrointestinal tumor marking, which comprises chitosan carrying a near-infrared fluorescent dye, the chitosan being crosslinked with a crosslinking agent. The following describes the chitosan, crosslinking agent, and near-infrared fluorescent dye that make up the composition, followed by a method for producing the marking composition using these components, the physical properties of the produced marking composition, and a method and kit for marking gastrointestinal tumors using the composition.

[0015] (Chitosan) Chitosan is derived from chitin, a type of aminopolysaccharide found in the exoskeletons of crustaceans such as crabs and shrimp, and is a naturally occurring polymer with a chemical structure consisting of repeating units of glucosamine and a small amount of N-acetylglucosamine. Generally, chitosan is obtained by deproteinizing the exoskeletons of crustaceans with an alkali such as caustic soda, decalcifying them 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.

[0016] In this case, the degree of deacetylation (also called 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 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.

[0017] 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. Chitosan in the present disclosure can be any commercially available chitosan used 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 any weak acid such as acetic acid or a strong acid such as hydrochloric acid.

[0018] (Crosslinking agent) In the present disclosure, the cross-linking agent used for cross-linking chitosan is not particularly limited, and may be, for example, commonly used glutaraldehyde, or cross-linking agents such as genipin, procyanidin, carbodiimide, or a mixture of the above cross-linking agents.

[0019] In a preferred embodiment, the crosslinker used is genipin. The term "genipin" refers to the non-sugar portion of geniposide, a natural component of the iridoid glycoside family, which is the main pharmacologically active component of gardenia fruit and can be represented by the following chemical formula:

[0020] [ka]

[0021] The term "derivatives of genipin" in the present disclosure includes, without limitation, compounds that exhibit the same action as genipin and can crosslink chitosan, such as geniposide, geniposidic acid, penta-acetylgeniposide, 6a-hydroxygeniposide, 6b-hydroxygeniposide, 6a-methoxygeniposide, or 6b-methoxygeniposide (Journal of Health Science, 52(6), 743-747, 2006).

[0022] (near-infrared fluorescent dye) In the present disclosure, the term "near-infrared fluorescent dye" refers to an organic compound that absorbs light and emits light in the near-infrared region. The term may be an organic compound that emits fluorescence with a near-infrared wavelength of preferably 700 nm to 1500 nm, more preferably 750 nm to 900 nm. The near-infrared fluorescence emitted from the near-infrared fluorescent dye can be photographed or monitored in real time using a device such as a fluorescence camera or a fluorescence sensing probe. For the purposes of the present disclosure, near-infrared fluorescent dyes can be injected into the site of a gastrointestinal tumor and used to accurately identify the tumor lesion before surgical removal, thereby improving the success rate of the surgical procedure. In particular, unlike dyes visible to the naked eye, near-infrared fluorescent dyes can detect the location of the tumor outside the body before incision and direct identification of the lesion, allowing for rapid and accurate surgery. Examples of such near-infrared fluorescent dyes include indocyanine green, rhodamine 800, oxazine 750, IR780, IR813, and IR1048. Among these, indocyanine green (ICG) represented by the following formula is preferred.

[0023] [ka]

[0024] Indocyanine green is a fluorescent dye in the near-infrared region that is widely used in the fields of biology and medicine. It decomposes or is excreted within about an hour after injection into the human body, making it suitable for clinical applications as a fluorescent dye that can be used in the human body. Numerous papers have reported on the use of indocyanine green in human applications. For example, its safe clinical use in 18 breast cancer patients has been reported (T. Kitai, et al., Breast Cancer, 12:211-215, 2005).

[0025] (Method for producing marking composition) The method for preparing the marking composition of the present disclosure is as follows: Pre-deacetylated chitosan is modified to carry a near-infrared fluorescent dye. The near-infrared fluorescent dye is attached to the chitosan polymer chain via amidation of the primary amine groups present in the glucosamine units of chitosan using a chemical reaction such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or N-hydroxysuccinimide (NHS). Chitosan is dissolved in acetic acid at room temperature. NHS or EDC is then added to the aqueous solution under strong magnetic stirring. Finally, the near-infrared fluorescent dye is added to the mixture, and the attachment reaction is carried out for 24 hours. The chitosan carrying the near-infrared fluorescent dye is then purified by a dialysis process using deionized water for 5 days. The purified chitosan is then recovered through a freeze-drying process. The near-infrared fluorescent dye may be derivatized with NHS or EDC in advance to be attached to the chitosan polymer chain.

[0026] In the marking composition of the present disclosure, the mixing ratio of chitosan to near-infrared fluorescent dye is not particularly limited and can be 100:1 to 1:100, preferably 50:1 to 1:50, and more preferably 20:1 to 1:20.

[0027] The chitosan loaded with the near-infrared fluorescent dye is then crosslinked with a crosslinking agent. Suitable crosslinking agents for use in this disclosure contain at least two electrophilic reactive sites designed to readily react with amines. When a crosslinking agent has two reactive sites, it is bifunctional and can therefore react with two amino groups, for example, two glucosamine units in different chitosan chains. The distance between these reactive groups can be increased by a spacer component. This spacer is often an aliphatic chain or a polyether structure such as poly- or oligoethylene glycol. For high-yield reactions, it is preferable to use a bifunctional crosslinking agent that reacts readily at a pH close to or above the pKa of the glucosamine in the polymer chain (approximately 6.8) and consumes a significant amount of crosslinking molecules. Typical examples of such crosslinking functionalities are reactive esters, Michael acceptors, and epoxides. Suitable crosslinking agents are well known and include glutaraldehyde, glyoxal, diethyl squarate, diepoxides such as diglycyl ether, tripolyphosphate, genipin, and formaldehyde.

[0028] The structure of the marking composition of the present disclosure is affected by the concentration of chitosan and the amount of cross-linking agent used. Therefore, marking compositions with appropriate viscoelasticity can be produced by using a high concentration of chitosan or by increasing the number of cross-linkers. The molar ratio of cross-linker to chitosan, based on the number of functional groups in the cross-linker and the number of accessible amino groups in chitosan, will depend on the reactivity of the cross-linker and the accessibility to the amino groups of chitosan (only deacetylated amino groups will be reactive). Obviously, the number of available amino groups will be determined by the degree of deacetylation of chitosan.

[0029] (cross-linking with genipin) Genipin is known to react with free amino groups of lysine, hydroxylysine, or arginine residues in biological tissues (Biomaterials, 1999;20:1759-72). Prior art documents the structure of an intermediate blue pigment produced from genipin and methylamine (the simplest primary amine). The proposed mechanism involves nucleophilic attack of methylamine on the olefinic carbon at the C-3 position of genipin, liberating the dihydropyran ring, followed by attack of a second amino group on the resulting aldehyde group, resulting in the formation of genipin-methylamine monomer. The blue pigment is believed to be produced by oxygen radical-induced polymerization and dehydrogenation of several intermediate pigments. In this study, the amino groups of chitosan are linked via genipin as a crosslinker.

[0030] (Physical Properties of Marking Composition) The marking composition of the present disclosure can be easily injected into tissues via a syringe needle. Therefore, it remains fluid for a predetermined period of time after crosslinking the chitosan. The predetermined period of time refers to any period between immediately after mixing the chitosan and the crosslinker (or within a few tens of seconds) and within 60 minutes, preferably 5 minutes, more preferably 15 minutes, and even more preferably 30 minutes, and it can be injected into a tumor site from inside the digestive tract using an endoscope. The fluid state may be viscous or viscoelastic enough to be injected into tissues via a syringe needle.

[0031] On the other hand, after the chitosan gels after a predetermined time has passed, it becomes non-fluid and can remain at the injected site for a certain period of time (about 1 to 30 days), at least until the end of surgery (preferably 14 days or more). Furthermore, there is only a slight tissue reaction at the injection site. The non-fluid state is achieved when the storage modulus of the gelled composition is 500 Pa or more. This storage modulus is preferably 800 Pa or more, and more preferably approximately 1000 Pa. There is no particular upper limit to the storage modulus, but it is sufficient if it is approximately 100,000 Pa or less, and preferably 10,000 Pa or less.

[0032] The storage modulus expresses the rheological elasticity of chitosan gelled by crosslinking, and indicates the degree of crosslinking between chitosan and a crosslinking agent. The higher the storage modulus, the more accelerated the crosslinking between chitosan and a crosslinking agent, and the more rigid the network structure in the chitosan gel. The more rigid the network structure, the more stable the chitosan gel. The storage modulus is a value obtained, for example, by measuring using a dynamic viscoelasticity device under conditions of a heating rate of 5°C / min, tension mode, and frequency of 10 Hz.

[0033] The marking composition of the present disclosure preferably has a swelling ratio of 0.5 to 1.5 within a predetermined period of time after gelation, preferably within one month. This is because a stable presence in an in vivo environment is desirable to increase the accuracy of marking. The swelling ratio refers to the rate at which the volume of a marking composition administered to a living body changes after a predetermined period of time. A decrease in the swelling ratio also indicates degradation in the in vivo environment. For example, when administered between the gastric mucosa and serous membrane, the pH environment is considered to be near neutral, similar to that found in the general living body, so a stable presence at near neutral pH is preferred. On the other hand, if cancer infiltration or ulceration occurs at the marking site, the pH may be lowered, so stability under low pH conditions is also important. Therefore, in a more preferred embodiment, the marking composition of the present disclosure has a swelling ratio of 0.5 to 1.5 within one month under conditions of pH 1 to 7.

[0034] (Method of using the marking composition) The marking composition of the present disclosure can be used to mark gastrointestinal tumors. Gastrointestinal tumors refer to benign or malignant tumors or polyps originating in the stomach, esophagus, or colon of mammals, particularly humans. By injecting the composition into the gastrointestinal wall directly under an endoscope and leaving a small marker, the composition can be used to determine the treatment area, etc. For example, the composition can be injected into the tumor site with 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 tumor tissue through laparoscopic surgery.

[0035] (kit) The gastrointestinal tumor marking composition kit of the present disclosure is prepared by separately filling containers with a single dose of chitosan carrying a near-infrared fluorescent dye and a chitosan crosslinker. The contents can then be mixed at the time of use to prepare an injectable formulation. The chitosan is dissolved in an appropriate solvent, such as a 0.1 mol / L aqueous acetic acid solution, and the crosslinker contains genipin or a derivative thereof. The kit is pre-filled so that the final chitosan concentration in the final formulation upon mixing is 5-40 g / L, preferably 10-30 g / L, and the final crosslinker concentration is 0.0001-1 mol / L, preferably 0.001-0.1 mol / L. Each formulation for separate filling is filled separately in 2.5-30 mL, preferably approximately 5-20 mL, and the two formulations are mixed at the time of use.

[0036] The containers used to fill each of the separately filled formulations in the gastrointestinal tumor 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 formulations filled in the pre-filled syringe containers, each or any of the components constituting each of the separately filled formulations 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 only a limited amount that does not adversely affect the resulting composition). Examples of the inert gas used include nitrogen gas.

[0037] The gastrointestinal tumor marking composition of the present disclosure thus obtained is filtered and sterilized using conventional methods, filled into a container, and then sterilized by high-pressure steam as necessary and stored at room temperature. This dispensing process is also preferably carried out under an inert gas atmosphere such as nitrogen gas. Usually, dissolved oxygen in the injectable formulation is degassed using conventional methods, and then nitrogen gas is injected to replace the atmosphere.

[0038] Furthermore, the gastrointestinal tumor marking composition of the present disclosure can be freeze-dried by conventional methods and prepared as a kit together with a solubilizer so that it can be dissolved immediately before use. As the solubilizer for the formulation to be used immediately, a solution of polyhydric alcohol and / or sugar can be used.

[0039] The digestive tract tumor marking composition kit of the present disclosure is composed of an integrated two-compartment container containing a container filled with a near-infrared fluorescent dye-carrying chitosan and a crosslinking agent, and may be a container that can be mixed by opening the partition between the two compartments when used, or may be separate containers that can be mixed when used, with the former being more convenient. Integrated two-compartment containers are widely used when mixing substances that require separate storage, and these can be used.

[0040] The present invention will now be described in more detail with reference to the following examples, but the present invention is not limited to these examples. In the following examples, the unit % used to indicate the amount of each component added means % by mass. [Example]

[0041] [Example 1] Marking gel visualization experiment under tissue (Synthesis of ICG-chitosan) Chitosan (Fujifilm Wako Pure Chemical Industries, Ltd., 100 mg, 0.66 × 10 -5 mol) was dissolved in hydrochloric acid (5 mL, 0.1 mol / L) and then adjusted to pH 5.0. The ICG-NHS powder was reacted with 2, 5, or 10 μL of a 1.3 mmol / L ICG solution dissolved in DMSO, and the resulting mixture was purified and recovered. Depending on the amount of ICG-NHS solution added during the reaction, 2, 5, or 10 μL of ICG-chitosan was used. The amount of ICG incorporated into chitosan was evaluated using a UV-Vis-NIR spectrophotometer (V-670, JASCO Corporation).

[0042] (Preparation of ICG-chitosan gel) 10 μL of ICG-chitosan (20 mg, 1.3 × 10 -6mol) was dissolved in acetic acid (1 mL, 0.1 mol / L), and genipin solution (25 μL, 4 mol / L) was added, stirred, and then allowed to stand at 40°C to gel. The chitosan concentration of the prepared gel and the genipin solution were 1.3 × 10 -3 The concentrations were 0.1 mol / L and 0.1 mol / L. Gelation was confirmed by tilting and color change. The elastic modulus of the gel was measured using a rheometer (MCR301; Anton Paar).

[0043] (Ex vivo experiment) 50 μL of the pregel solution (viscous solution before gelation) mixed under the above conditions was injected subcutaneously into chicken meat (thickness: 2-3 mm) using a syringe, and fluorescence observation was performed through the skin. Fluorescence at 860 nm was taken, and the same region of interest (ROI) was selected to compare the fluorescence intensity. A 2.4 μmol ICG solution was prepared and used as a control.

[0044] (Results and Discussion) (Synthesis of ICG-chitosan) To confirm the incorporation of ICG into chitosan, UV-vis measurement was performed. Peaks derived from chitosan and ICG were observed, confirming the incorporation of ICG into chitosan. In addition, the amount / concentration of ICG at the time of preparation (2 μL / 0.43 × 10 -6 mol / L, 5 μL / 1.08 × 10 -6 mol / L, or 10 μL / 2.15 × 10 -6 The effect of the amount of ICG introduced into chitosan was investigated by changing the ICG concentration (mol / L). As a result, the absorption intensity at 800 nm increased with increasing ICG amount / concentration during the preparation (Figure 1). Furthermore, chitosan was produced with high yields regardless of the amount of ICG introduced, confirming that the amount of ICG supported on chitosan can be controlled by controlling the ICG concentration during preparation.

[0045] [Table 1]

[0046] (Preparation of ICG-chitosan gel) Gelation was confirmed using the tilting method. Before gelation (Figure 2(A)), the solution was in a liquid state, whereas after gelation (Figure 2(B)), it was confirmed that the solution did not flow even when tilted. It has also been reported that the genipin and chitosan system changes from colorless and transparent to deep blue upon gelation, and this was confirmed in this experiment.

[0047] (Elastic modulus measurement of ICG-chitosan gel) Using a rheometer (MCR301; Anton Paar), the frequency dependence of the storage modulus and loss modulus was investigated. The results are shown in Figure 3. The storage modulus was almost constant with respect to frequency, suggesting the presence of chemical bonds. The value was 1000 Pa (the elastic modulus of the human stomach wall is approximately 1900 kPa).

[0048] (Ex vivo experiment) We investigated whether the ICG-chitosan gel prepared above could be observed through tissue. In this experiment, we simulated a stomach membrane (<2 mm thick) and placed the prepared gel between chicken meat (2 cm thick) and skin (2 mm thick). Fluorescence observation was performed through the skin. A 0.002 mg / mL ICG-PBS solution was used as a control. Figure 4 shows fluorescence images of chicken subcutaneously injected with ICG solution or ICG-chitosan gel. The fluorescence images and white light images are shown before injection and 1 and 20 minutes after injection (Figure 4(B, E, F, H, K, L)). ICG fluorescence was observed immediately after injection. Figure 4(N) also shows the increase in fluorescence intensity over time. The preparation conditions for the ICG solution and ICG-chitosan gel were adjusted to achieve comparable fluorescence intensities. However, due to diffusion, the fluorescence intensity of the ICG solution was lower than that of the ICG-chitosan gel. Furthermore, after gelation, the blue gelation could be confirmed even from the subcutaneous area (Figure 4(M)).

[0049] Next, to evaluate it as a marking material, five injections were made, just like an actual marker, and fluorescence observation was performed (Figure 5). The thicknesses of the chicken meat and chicken skin used were 2 cm and 2 mm, respectively, as before. As in the previous experiment, we confirmed that the fluorescence intensity increased over time and reached a plateau after about 20 minutes. With the ICG solution, the fluorescence spread over the entire area, making it impossible to distinguish the injection site, whereas with the ICG-chitosan gel, it was possible to distinguish the five injection sites. With the ICG solution, there were localized areas of high fluorescence intensity, and it was confirmed that the ICG solution had accumulated due to the unevenness of the chicken meat, but the fluorescence intensity in the gel was almost constant and homogeneous.

[0050] [Example 2] Surface-degrading chitosan gel (Preparation of chitosan gel) Chitosan was dissolved in acetic acid (0.1 M), and genipin solution was added. The mixture was left to stand at 40°C to prepare a gel (cylindrical shape with a diameter of 15 mm). The chitosan concentration of the prepared gel and the genipin solution were 2.0 × 10 -6 The molar ratio of the two was approximately 1:10.

[0051] (Evaluation of decomposition behavior) The prepared gels were immersed in a 1.5 mg / L lysozyme solution at 37°C, and their diameters were measured at regular intervals. The external solution was changed every two days. The pH of the external solution was also varied from 1 to 7, and the effect on degradation behavior was examined. Surface or bulk degradation was determined by compression tests. The results are shown in Figures 6 and 7. The vertical axis of Figure 6 shows the ratio of the gel diameter measured after each time period to the gel diameter at the start of the test (swelling ratio). Figure 7 shows the compressive modulus measured using gels immersed for 30 days in the presence or absence of lysozyme.

[0052] (Results and Discussion) (Evaluation of decomposition behavior) Changes were investigated over a one-month period, and it was confirmed that the gel remained intact under all conditions. At pH 1-3, the volume increased slightly (remained nearly constant) over time, while at pH 5-7, the volume decreased over time. It was also suggested that decomposition accelerated in the presence of lysozyme. Bulk decomposition, as seen in typical gels, involves swelling over time and eventually explosive disappearance. However, the results showed that the size remained nearly constant or shrunk over time, and that there was no significant decrease in the elastic modulus, confirming that surface decomposition, rather than bulk decomposition, was occurring. [Industrial Applicability]

[0053] The gastrointestinal tumor marking composition disclosed herein does not damage other organs, can maintain the injected position for at least several weeks, does not shift the positional information marked in the mucosal layer and serous layer, and can be removed when desired, making it suitable for use in surgical treatment methods such as laparoscopic resection.

Claims

1. A composition for gastrointestinal tumor marking, comprising chitosan carrying a near-infrared fluorescent dye, the chitosan being crosslinked with a crosslinking agent, A marking composition having a fluid state that allows it to be injected into the tumor from inside the digestive tract using an endoscope within a predetermined time after crosslinking, and a non-fluid state that gels after the predetermined time has passed and remains at the injected location for at least 24 hours.

2. The marking composition according to claim 1, for detecting the location of the tumor from outside the digestive tract using a laparoscopic microscope equipped with a near-infrared camera.

3. 3. The marking composition according to claim 1, wherein the predetermined time is 60 minutes or less.

4. 4. The marking composition according to claim 1, wherein the marking composition remains in a non-fluid state at the injected position for at least 14 days.

5. 5. The marking composition of claim 4, wherein the residence time is 30 days.

6. 6. The marking composition according to claim 1, wherein the gelled composition has a storage modulus of 500 to 100,000 Pa.

7. 7. The marking composition according to claim 1, wherein the swelling ratio of the gelled composition is 0.5 to 1.

5.

8. A digestive tract tumor marking composition kit, comprising: a near-infrared fluorescent dye-carrying chitosan; and a crosslinking agent for the chitosan, each of which is separately filled in a single dose into each container, and the contents are mixed together at the time of use to prepare an injectable preparation, The chitosan is dissolved in a solvent, and the cross-linking agent comprises genipin or a derivative thereof; The injectable preparation is filled in advance so that the final concentrations of the components thereof are 5 to 40 g / L of chitosan and 0.0001 to 1 mol / L of crosslinking agent, The above kit, wherein the genipin derivative is geniposide, geniposidic acid, penta-acetylgeniposide, 6a-hydroxygeniposide, 6b-hydroxygeniposide, 6a-methoxygeniposide, or 6b-methoxygeniposide.

9. The kit according to claim 8, wherein the pH of the injection formulation is within the range of 1 to 7.

10. 10. The kit according to claim 8, wherein the near-infrared fluorescent dye is indocyanine green.

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