Applications of polysaccharide derivatives

Abstract

[Problem] To provide an additional use of a polysaccharide derivative such as a tissue adhesive material. [Solution] According to some embodiments, a tissue adhesive material is provided, which is intended to be used for a hard and thick tissue and / or a connective tissue and comprises a polysaccharide derivative having such a structure that a group represented by formula (A) is introduced in an acidic, basic or amphoteric polysaccharide.　According to some embodiments, a crosslinked structure and a tissue adhesive material comprising the crosslinked structure are provided, in which the crosslinked structure comprises (a) a polysaccharide derivative having such a structure that a group represented by formula (A) is introduced into an acidic, basic or amphoteric polysaccharide and (b) at least one of an amino-group-containing polymer and an amino-group-containing low-molecular-weight compound each containing at least two primary amino groups, hydrazide groups or aminooxy groups and a group represented by formula (x1), and the crosslinking is achieved by the covalent bonding between a primary amino group, a hydrazide group or an aminooxy group contained in the amino-group-containing polymer and the amino-group-containing low-molecular-weight compound and the group represented by formula (A) contained in the polysaccharide derivative through a Schiff base.　According to some embodiments, a tissue adhesive material having a gel-like form is provided, which comprises a polysaccharide derivative having such a structure that a group represented by formula (A-2) is introduced into a polysaccharide containing an amino group, in which the group represented by formula (A-2) is introduced into the polysaccharide by substituting for a hydrogen atom in the amino group in the polysaccharide to form an amide bond.

Description

[Technical Field]

[0001] This invention relates to the applications of polysaccharide derivatives, such as tissue adhesive materials. [Background technology]

[0002] Polysaccharides such as hyaluronic acid and alginic acid are generally widely used as medical materials, food additives, cosmetic and daily necessities additives, and thickeners due to their excellent water solubility, water retention, moderate viscosity, adhesiveness, and biocompatibility. For example, hyaluronic acid (HA) is a biodegradable and biocompatible natural polymer that is abundant in the body and is widely used as a material for healthcare products such as pharmaceuticals in orthopedics and ophthalmology, medical devices, cosmetics, and contact lenses. Alginic acid (AL) is a natural polysaccharide found in brown algae and other organisms, and due to its high biocompatibility and properties such as crosslinking with polyvalent metal ions such as Ca, it is used in pharmaceuticals, medical devices, pharmaceutical additives, foods, supplements, and food additives such as hemostatic agents and wound dressings.

[0003] In recent years, research has been progressing on the use of these polysaccharides as drug carrier materials in drug delivery systems (DDS). HA specifically binds to the CD44 receptor, is overexpressed on the surface of various tumor cells, and can be easily derivatized using its hydroxyl and carboxyl groups, making it a promising drug carrier material. For example, several HA drug conjugates utilizing Schiff bases have been reported (Non-Patent Literature 1: Materials Science and Engineering: C, 2014, 36: 287-293; Non-Patent Literature 2: Carbohydrate Polymers 134 (2015) 293-299; Non-Patent Literature 3: Carbohydrate Polymers 189 (2018) 273-279; Non-Patent Literature 4: Carbohydrate Polymers 216 (2019) 63-71). Schiff bases are imine group-containing compounds formed by the condensation reaction of a primary amine with a reactive carbonyl compound. Schiff bases are expected to selectively release drugs because they exhibit reversible pH-responsive dissociation. However, Schiff bases formed between typical aldehydes or ketones and amines are not sufficiently stable in water because the reverse reaction occurs rapidly. Therefore, drug conjugates using Schiff bases often have a fast sustained drug release rate and do not provide sufficient performance. On the other hand, by using hydrazide groups instead of amino groups to slow down the hydrolysis rate, they have been used as biocompatible in situ crosslinked hydrogels with hyaluronic acid or alginic acid as the backbone.

[0004] It has been reported that benzaldehyde, due to its aromatic structure, can form stable Schiff bases with amine groups contained in various drugs (Non-Patent Literature 5: Materials Chemistry Frontiers, 2018, 2(10): 1765-1778). Non-Patent Literature 6 (J. Biomed. Nanotechnol. 2017, 13, 1647-1659) discloses a conjugate of carboxymethyl chitosan and daunorubicin utilizing Schiff base formation between the aldehyde group of triazolebenzylaldehyde and the amino group of daunorubicin. The conjugate formation in Non-Patent Literature 6 involves a complex and multi-step reaction process in which the drug is reacted with azidobenzylaldehyde, and this is then reacted with alkyne-modified carboxymethyl chitosan to form a triazole ring, thereby linking the drug and chitosan. In addition, it required the drug to be reacted with azidobenzylaldehyde beforehand for conjugate formation. Furthermore, this reaction involves the use of a copper catalyst, which presented challenges in terms of manufacturing and safety. In addition, Non-Patent Document 7 (Polymer Bulletin 31 (1993) 145-149) discloses dextran derivatives in which benzaldehyde is introduced via an ester bond at the position of the hydroxyl group of dextran. Because these polysaccharide derivatives use dextran, a neutral polysaccharide, it was difficult to introduce hydrophobic benzaldehyde with a high modification rate. Furthermore, the resulting polysaccharide derivatives also had poor water solubility, posing challenges in terms of application. The same document also discloses derivatives in which benzaldehyde is introduced into dextran that has been derivatized by reacting it with epihydrochlorin or epoxy. While these derivatives are expected to improve hydrophilicity due to the introduction of hydroxyl groups derived from epihydrochlorin or epoxy into the side chains, the synthesis of these derivatives requires multiple reaction steps, and there is room for improvement in terms of industrial production and the modification rate of benzaldehyde.

[0005] Numerous reports exist regarding conjugates of hyaluronic acid without Schiff bases with drugs. For example, HA protein conjugates (Non-Patent Literature 8: Carbohydrate Polymers 92 (2013) 2163-2170), HA-IFNα2a conjugates (Non-Patent Literature 9: Journal of Controlled Release 236 (2016) 79-89), and HA-pemetrexide conjugates (Non-Patent Literature 10: European Journal of Pharmaceutical Sciences 138 (2019) 105008) have been reported. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Materials Science and Engineering: C, 2014, 36: 287-293 [Non-Patent Document 2] Carbohydrate Polymers 134 (2015) 293-299 [Non-Patent Document 3] Carbohydrate Polymers 189 (2018) 273-279 [Non-Patent Document 4] Carbohydrate Polymers 216 (2019) 63-71 [Non-Patent Document 5] Materials Chemistry Frontiers 2 (2018) 1765-1778 [Non-Patent Document 6] J.Biomed.Nanotechnol. 13 (2017) 1647-1659 [Non-Patent Document 7] Polymer Bulletin 31 (1993) 145-149 [Non-Patent Document 8] Carbohydrate Polymers 92 (2013) 2163-2170 [Non-Patent Document 9] Journal of Controlled Release 236 (2016) 79-89 [Non-Patent Document 10] European Journal of Pharmaceutical Sciences 138 (2019) 105008 [Overview of the Initiative]

[0007] In PCT / JP2021 / 029573 (filing date: August 10, 2021), the inventors provided a novel polysaccharide derivative having a modifying group capable of forming a Schiff base with an amino group, such as benzaldehyde. The present invention relates to further improvements of the polysaccharide derivative, and in particular to further applications of the polysaccharide derivative such as tissue adhesive materials, and applications to improved crosslinked structures using the polysaccharide derivative.

[0008] In a first embodiment of the present invention, a tissue adhesive material is provided that exhibits adhesion not only to soft tissues in living organisms, but also to relatively hard and thick tissues such as the dura mater, perichondrium, synovial membrane, and / or connective tissue. In a second embodiment of the present invention, a crosslinked structure of a polysaccharide derivative and an amino group-containing material having a polyhydroxyphenyl group, and a tissue-adhesive material containing the same are provided. A third embodiment of the present invention provides a tissue-adhesive material comprising a polysaccharide derivative containing an amino group. In some forms, tissue adhesive materials are used for repairing / restoring biological tissues.

[0009] The present invention is, for example, as follows: [1] A tissue adhesive material used for rigid, thick tissues and / or connective tissue, A tissue-adhesive material comprising a polysaccharide derivative in which a group represented by the following formula (A) is introduced into an acidic, basic, or amphoteric polysaccharide. [ka] (In the formula, R 1 is a hydrogen atom or C1-4 Represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or pyridine ring may be substituted with one or more substituents independently selected from a halogen atom, -CF3, -NO2, a carboxyl group, and -SO3H. Y is -NH-, -C(=O)-, -S-, -O-, C 1-6 Alkylene, -(CH2CH2O) n - represents a divalent base selected from the group consisting of , and any combination thereof, where n is an integer from 1 to 9. * indicates a linkage with a polysaccharide. [2] The polysaccharide is selected from alginic acid, its derivatives or salts thereof, hyaluronic acid, its derivatives or salts thereof, carboxymethylcellulose, its derivatives or salts thereof, carboxymethyl dextran, its derivatives or salts thereof, carboxymethyl starch, its derivatives or salts thereof, heparin, its derivatives or salts thereof, heparan sulfate, its derivatives or salts thereof, chondroitin sulfate, its derivatives or salts thereof, dermantan sulfate, its derivatives or salts thereof, chitosan, its derivatives or salts thereof, regenerated oxidized cellulose, its derivatives or salts thereof, and pectinic acid, its derivatives or salts thereof, and the tissue adhesive material according to [1].

[0010] [3] The polysaccharide is a polysaccharide containing a carboxyl group, wherein the group represented by formula (A) is the group represented by formula (A-1) below, and the group represented by formula (A-1) is introduced into the polysaccharide by substituting the -OH of the carboxyl group of the polysaccharide to form an amide bond; or; The tissue-adhering material according to [1] or [2], wherein the polysaccharide is a polysaccharide containing an amino group, the group represented by formula (A) is the group represented by the following formula (A-2), and the group represented by formula (A-2) is introduced into the polysaccharide by substituting a hydrogen atom of the amino group of the polysaccharide to form an amide bond. [ka] (In formulas (A-1) and (A-2), R 1 represents a hydrogen atom or C 1-4 alkyl, ring P is a phenyl ring or a pyridine ring, and the phenyl ring or pyridine ring may be substituted with one or more substituents independently selected from a halogen atom, -CF3, -NO2, a carboxyl group, and -SO3H, L 1 is a single bond or represents a divalent group selected from the group consisting of -C(=O)-, -S-, -O-, C 1―6 alkylene, -(CH2CH2O) n -, and any combination thereof, L 2 is a single bond or represents a divalent group selected from the group consisting of -NH-, -S-, -O-, C 1―6 alkylene, -(CH2CH2O) n -, and any combination thereof, n is an integer from 1 to 9 * represents the linking portion with the polysaccharide.)

[0011] [4] The polysaccharide is alginic acid, a derivative thereof, or a salt thereof, The polysaccharide derivative contains a structural unit represented by the following formula (c11) and / or (c12), and is the tissue adhesive material according to any one of [1] to [3]. [Chemical formula] (In the formula, R 11 , R 12 , R 13 , and R 14 are each independently selected from a hydrogen atom, C 1-6 alkyl, and -C(=O)-C 1-6 alkyl, R 1 represents a hydrogen atom or C 1-4 alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or pyridine ring may be substituted with one or more substituents independently selected from a halogen atom, -CF3, -NO2, a carboxyl group, and -SO3H. Y is -L 1 -NH- represents, and in this case L 1 It is bonded to ring P, L 1 C is a single bond. 1-6 Alkylene and -(CH2CH2O) n - Selected from, n is an integer between 1 and 9.

[0012] [5] The group represented by formula (A) is [ka] (In the formula, * represents the linkage with the polysaccharide.) A tissue adhesive material selected from the group consisting of [1] to [4], according to any one of [1] to [4]. [5-1] The hard, thick tissue and / or connective tissue is selected from the group consisting of dura mater tissue, perichondrium tissue, synovial tissue and periodontal ligament tissue, and is a tissue adhesive material according to any one of [1] to [4]. [5-2] The tissue-adhesive material according to any one of [1] to [4], wherein the hard, thick tissue and / or connective tissue is dura mater tissue. [5-3] The tissue-adhesive material according to any one of [1] to [4], wherein the rigid, thick tissue and / or connective tissue is a rigid, thick tissue. [5-4] The tissue-adhesive material according to any one of [1] to [4], wherein the rigid, thick tissue and / or connective tissue is connective tissue.

[0013] [6] A tissue-adhesive material used for rigid, thick tissues and / or connective tissues, A tissue-adhesive material comprising a crosslinked structure in which at least a portion of a polysaccharide derivative is crosslinked, in which a group represented by the following formula (A) is introduced into an acidic, basic, or amphoteric polysaccharide. [ka] (In the formula, R 1 is a hydrogen atom or C 1-4 Represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or pyridine ring may be substituted with one or more substituents independently selected from a halogen atom, -CF3, -NO2, a carboxyl group, and -SO3H. Y is -NH-, -C(=O)-, -S-, -O-, C 1-6 Alkylene, -(CH2CH2O) n - represents a divalent base selected from the group consisting of , and any combination thereof, where n is an integer from 1 to 9. * indicates a linkage with a polysaccharide. [6-1] The tissue-adhesive material according to [6], wherein the crosslinked structure is a polysaccharide derivative crosslinked with a crosslinking agent selected from a divalent metal ion; an amino group-containing polymer and an amino group-containing low molecular weight compound containing two or more primary amino groups, hydrazide groups, or aminooxy groups; and a compound containing two or more aldehyde groups such as glutaraldehyde. [6-2] The polysaccharide is selected from alginic acid, its derivatives or salts thereof, hyaluronic acid, its derivatives or salts thereof, carboxymethylcellulose, its derivatives or salts thereof, carboxymethyl dextran, its derivatives or salts thereof, carboxymethyl starch, its derivatives or salts thereof, heparin, its derivatives or salts thereof, heparan sulfate, its derivatives or salts thereof, chondroitin sulfate, its derivatives or salts thereof, dermantane sulfate, its derivatives or salts thereof, chitosan, its derivatives or salts thereof, regenerated oxidized cellulose, its derivatives or salts thereof, and pectinic acid, its derivatives or salts thereof, as described in [6] or [6-1]. [6-3] The polysaccharide is a polysaccharide containing a carboxyl group, the group represented by formula (A) is the group represented by formula (A-1) below, and the group represented by formula (A-1) is introduced into the polysaccharide by substituting the -OH of the carboxyl group of the polysaccharide to form an amide bond; or; The tissue-adhering material according to [6], [6-1], or [6-2], wherein the polysaccharide is a polysaccharide containing an amino group, the group represented by formula (A) is the group represented by formula (A-2) below, and the group represented by formula (A-2) is introduced into the polysaccharide by substituting a hydrogen atom of the amino group of the polysaccharide to form an amide bond. [ka] (In equations (A-1) and (A-2), R 1 is a hydrogen atom or C 1-4 Represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or pyridine ring may be substituted with one or more substituents independently selected from a halogen atom, -CF3, -NO2, a carboxyl group, and -SO3H. L 1 It is either a single bond or -C(=O)-, -S-, -O-, C 1―6 Alkylene, -(CH2CH2O) n - represents a divalent group selected from the group consisting of any combination thereof, L 2 It is either a single bond or -NH-, -S-, -O-, C 1―6 Alkylene, -(CH2CH2O) n - Represents a divalent group selected from the group consisting of any combination thereof. n is an integer from 1 to 9. * indicates a linkage with a polysaccharide. [6-4] The polysaccharide is alginic acid, its derivatives, or salts thereof. The polysaccharide derivative is the tissue adhesive material according to any one of [6], [6-1] to [6-3], which contains a structural unit represented by the following formula (c11) and / or (c12). [Chemical formula] (In the formula, R 11 , R 12 , R 13 , and R 14 are each independently selected from a hydrogen atom, C 1-6 alkyl, and -C(=O)-C 1-6 alkyl, R 1 represents a hydrogen atom or C 1-4 alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or pyridine ring may be substituted with one or more substituents independently selected from a halogen atom, -CF3, -NO2, a carboxyl group, and -SO3H, Y represents -L 1 -NH-, where L 1 is bonded to ring P, L 1 is selected from a single bond, C 1-6 alkylene, and -(CH2CH2O) n -, n is an integer from 1 to 9.) [6-5] The group represented by the formula (A) is [Chemical formula] (where * represents the linking part with the polysaccharide.) The tissue adhesive material according to any one of [6], [6-1] to [6-4], which is selected from the group consisting of [7] The tissue adhesive material according to any one of [1] to [6], which is in the form of a gel, sponge, sheet, or film. [8] The tissue adhesive material according to any one of [1] to [7], which further contains a non-woven fabric, sheet, or film.

[0014] [9] (a) Polysaccharide derivatives obtained by introducing a group represented by the following formula (A) into an acidic, basic, or amphoteric polysaccharide, (b) comprising at least one amino group-containing polymer and an amino group-containing low molecular weight compound, which contain two or more primary amino groups, hydrazide groups, or aminooxy groups and a group represented by the following formula (x1), A crosslinked structure in which a primary amino group, hydrazide group, or aminooxy group contained in the amino group-containing polymer and amino group-containing low molecular weight compound is covalently bonded via a Schiff base to a group represented by formula (A) contained in the polysaccharide derivative. [ka] (In formula (A), R 1 is a hydrogen atom or C 1-4 Represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or pyridine ring may be substituted with one or more substituents independently selected from a halogen atom, -CF3, -NO2, a carboxyl group, and -SO3H. Y is -NH-, -C(=O)-, -S-, -O-, C 1-6 Alkylene, -(CH2CH2O) n - represents a divalent base selected from the group consisting of , and any combination thereof, where n is an integer from 1 to 9. * indicates a linkage with a polysaccharide. [ka] (In equation (x1), k is an integer between 2 and 5, L 5 It is either a single bond or -C(=O)-, -S-, -O-, alkylene, -(CH2CH2O) n - and a divalent group selected from the group consisting of any combination thereof, n is an integer from 1 to 9. * indicates a linkage with an amino group-containing polymer or amino group-containing low-molecular-weight compound.

[0015]

[10] The crosslinked structure according to [9], wherein the amino group-containing polymer is at least one selected from linear, branched or dendritic polyamines; polyalkylene glycols substituted with amino groups, hydrazide groups, or aminooxy groups; polyallylamines; polyvinylamines; polyacrylamines; amino group-containing polysaccharides; amino group-containing proteins; and polyamino acids.

[0016]

[11] The polysaccharide is alginic acid, its derivatives, or salts thereof. The aforementioned polysaccharide derivative comprises a structural unit represented by the following formula (c11) and / or (c12) as described in [9] or

[10] , the crosslinked structure as described in [9] or

[10] . [ka] (In the formula, R 11 , R 12 , R 13 , and R 14 These are, independently, hydrogen atoms and C 1-6 Alkyl and -C(=O)-C 1-6 Selected from alkyl groups, R 1 is a hydrogen atom or C 1-4 Represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or pyridine ring may be substituted with one or more substituents independently selected from a halogen atom, -CF3, -NO2, a carboxyl group, and -SO3H. Y is -L 1 -NH- represents, and in this case L 1 It is bonded to ring P, L 1 C is a single bond. 1-6 Alkylene and -(CH2CH2O) n - Selected from, n is an integer between 1 and 9. A tissue-adhesive material comprising a crosslinked structure as described in any of

[12] [9] to

[11] .

[0017]

[13] A tissue-adhesive material The polysaccharide derivative includes a polysaccharide containing an amino group into which a group represented by the following formula (A-2) has been introduced. The group represented by formula (A-2) is introduced into the polysaccharide by substituting a hydrogen atom of the amino group of the polysaccharide, thereby forming an amide bond. A tissue-adhering material in gel form. [ka] (In equation (A-2), R 1 is a hydrogen atom or C 1-4 Represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or pyridine ring may be substituted with one or more substituents independently selected from a halogen atom, -CF3, -NO2, a carboxyl group, and -SO3H. L 2 It is either a single bond or -NH-, -S-, -O-, C 1―6 Alkylene, -(CH2CH2O) n - Represents a divalent group selected from the group consisting of any combination thereof. n is an integer from 1 to 9. * indicates a linkage with a polysaccharide.

[0018]

[14] Shear rate of 0.001 s, measured at 25°C -1 viscosity (η) 0.001 ) and shear rate 1000s -1 viscosity (η) 1000 ) ratio (η 0.001 / η 1000 ) is 10-10 9 The composition described in

[13] .

[15] The tissue-adhering material according to

[13] or

[14] , wherein the polysaccharide derivative is crosslinked by forming a covalent bond between the amino group contained in the polysaccharide derivative and the group represented by formula (A-2) via a Schiff base, thereby forming a crosslinked structure.

[0019]

[16] The polysaccharide is chitosan, its derivative or their salt, The polysaccharide derivative is the tissue adhesive material according to any one of

[13] to

[15] , which contains a structural unit represented by the following formula (c16).

Chemical formula

[0020] According to a first embodiment of the present invention, a tissue adhesive material is provided that is used for repairing / restoring biological tissue and has adhesive properties not only to soft tissues in the body but also to relatively hard and thick tissues such as the dura mater, perichondrium, and synovial membrane, and / or connective tissue, and contains a polysaccharide derivative. The tissue adhesive material has good adhesion to tissue by forming Schiff bases between the amino groups of the tissue and the aldehydes of the polysaccharide derivative. According to some embodiments, adhesion to tissue can be improved by forming hydrophobic interactions and hydrogen bonds between the tissue proteins constituting the tissue and the polysaccharide derivative. The material adhered to the tissue acts to protect and repair the tissue, and in the case of the dura mater, for example, it can prevent cerebrospinal fluid leakage. According to a second embodiment of the present invention, a crosslinked structure is provided between a polysaccharide derivative and an amino group-containing material having a polyhydroxyphenyl group. This crosslinked structure has good adhesion to tissues and can be used in applications such as tissue adhesion materials. According to a third embodiment of the present invention, a gel-type tissue adhesive is provided, comprising a polysaccharide derivative containing an amino group. The tissue adhesive material comprises an aldehyde group or a ketone group (-C(=O)R) of the modifying group (A-2) of the polysaccharide derivative. 1 The polysaccharide derivative exhibits good adhesion to tissues by forming a Schiff base between the amino group of the biological tissue and the aldehyde of the polysaccharide derivative. In addition, in some forms, interactions occur between the amino group of the polysaccharide derivative and the carboxyl group of the biological tissue, which can improve adhesion to tissues. In some forms, the polysaccharide derivative containing the amino group has pH-responsive self-crosslinking properties. [Brief explanation of the drawing]

[0021] [Figure 1] Figure 1A shows the 1H NMR spectra of benzaldehyde-modified alginate (AL-ABA) and alginate (AL) synthesized in Example I-1, and Figure 1B shows the regions corresponding to peaks b to d shown in Figure 1A. [Figure 2]Figure 2 shows the ultraviolet-visible light absorption spectra (UV-vis) of benzaldehyde-modified alginic acid (AL-ABA), alginic acid (AL), and 4-aminobenzaldehyde (ABA) synthesized in Example I-1. [Figure 3] Figure 3 shows the FT-IR spectra of benzaldehyde-modified alginic acid (AL-ABA) and alginic acid (AL) synthesized in Example I-1. [Figure 4] Figure 4 shows the cytotoxicity test results (WST assay results) of benzaldehyde-modified alginate (AL-ABA) and alginate (AL) synthesized in Example I-1 at different concentrations (0.01 mg / mL, 0.1 mg / mL, and 1 mg / mL) against MeT-5A (human mesothelial cell line), NIH / 3T3 (mouse embryonic fibroblasts), HUVEC (human umbilical vein endothelial cells), and RAW264.7 cells (mouse macrophage-like cell line). [Figure 5] Figure 5 shows the 1H NMR spectrum of AAP-modified alginate (AL-AAP) synthesized in Example I-3. [Figure 6] Figure 6 shows the FT-IR spectrum of AAP-modified alginate (AL-AAP) synthesized in Example I-3. [Figure 7] Figure 7 shows the 1H NMR spectrum of ADFBA-modified alginate (AL-ADFBA) synthesized in Example I-4. [Figure 8] Figure 8 shows the FT-IR spectrum of ADFBA-modified alginate (AL-ADFBA) synthesized in Example I-4. [Figure 9] Figure 9 shows the 1H NMR spectrum of AAP-modified alginate (AL-APCA) synthesized in Example I-5. [Figure 10] Figure 10 shows the FT-IR spectrum of AAP-modified alginate (AL-APCA) synthesized in Example I-5. [Figure 11] Figure 11 shows the 1H NMR spectrum of ANA-modified alginate (AL-ANA) synthesized in Example I-6. [Figure 12]Figure 12 shows the FT-IR spectrum of ANA-modified alginate (AL-ANA) synthesized in Example I-6. [Figure 13] Figure 13 shows the 1H NMR spectra of the benzaldehyde-modified alginate-vancomycin conjugate (AL-ABA-Van) synthesized in Example I-7, the benzaldehyde-modified alginate (AL-ABA), vancomycin (Van), and alginate (AL) synthesized in Example I-1. [Figure 14] Figure 14 shows the ultraviolet-visible light absorption spectra (UV-vis) of benzaldehyde-modified alginate-vancomycin conjugate (AL-ABA-Van) synthesized in Example I-7, benzaldehyde-modified alginate (AL-ABA) synthesized in Example I-1, and vancomycin (Van). [Figure 15] Figure 15 shows the FT-IR spectra of benzaldehyde-modified alginate-vancomycin conjugate (AL-ABA-Van) synthesized in Example I-7, benzaldehyde-modified alginate (AL-ABA), vancomycin (Van), and alginate (AL) synthesized in Example I-1. [Figure 16A] Figure 16A shows the release behavior of vancomycin (Van) from the benzaldehyde-modified alginate-vancomycin conjugate (AL-ABA-Van) solution synthesized in Example I-7 under different pH levels (pH = 5.0, 6.0, 7.4). The Cumulative Release %) shown on the vertical axis represents the cumulative release rate of Van, calculated from the absorbance measured by UV-vis spectroscopy. [Figure 16B] Figure 16B shows the release behavior of vancomycin from a mixed solution of sodium alginate and vancomycin (AL+Van) under different pH levels (pH=5.0, 6.0, 7.4) (control experiment). The Cumulative Release %) shown on the vertical axis represents the cumulative release rate of vancomycin, calculated from absorbance measured by UV-vis spectroscopy. [Figure 16C]Figure 16C shows the release behavior of vancomycin from a vancomycin solution (van only) under different pH levels (pH = 5.0, 6.0, 7.4) (control experiment). The Cumulative Release %) shown on the vertical axis represents the cumulative release rate of van, calculated from the absorbance measured by UV-vis spectroscopy. [Figure 17] Figure 17 shows confocal microscope images of FTSC-supported AL-ABA capsules and FTSC-supported AL capsules prepared in Example I-8. The top row shows confocal microscope images of AL-capsules (Alg microcapsules) in FTSC-containing saline, the middle row shows confocal microscope images of AL-ABA capsules (Alg-ABA microcapsules) in FTSC-containing saline, and the bottom row shows confocal microscope images of AL-ABA capsules (Alg-ABA microcapsules) in FTSC-containing DMEM. In each image, the left side shows the transmitted image, the middle shows the fluorescence image (Amine-Fluorescein), and the right side shows the merged image of both. [Figure 18] Figure 18 shows the release behavior of vancomycin (Van) from van-supported AL-ABA microcapsules (AL-ABA-Van capsules) prepared in Example I-9 and van-supported AL microcapsules (AL-Van capsules) as a control. Figure 18A shows the cumulative release rate (%) of van on the vertical axis, and Figure 18B shows the release rate (Vancomycin Release %) of van at each time point on the vertical axis. The release rate (Vancomycin Release %) of van was calculated from the absorbance measured by UV-vis spectroscopy. [Figure 19] Figure 19 shows photographs illustrating the growth of Staphylococcus aureus around filter paper impregnated with sustained-release solution from vancomycin-loaded capsules. (a) shows the results when using sustained-release solution from vancomycin-loaded AL capsules (AL-Van), and (b) shows the results when using sustained-release solution from vancomycin-loaded AL-ABA capsules (AL-ABA-Van). The upper panel shows the results immediately after adding the sustained-release solution, and the lower panel shows the results 24 hours after addition. [Figure 20] Figure 20 shows a comparison of the area of ​​Staphylococcus aureus inhibition by the sustained-release solution of vancomycin-loaded AL capsules (AL-Van) and vancomycin-loaded AL-ABA capsules (AL-ABA-Van) at various sustained-release times (n=3). [Figure 21] Figure 21 shows the ultraviolet-visible light absorption spectra (UV-vis) of the benzaldehyde-modified alginate-bacitracin conjugate (AL-ABA-Bac) synthesized in Example I-10, the benzaldehyde-modified alginate (AL-ABA) synthesized in Example 1, and bacitracin (Bac). [Figure 22] Figure 22 shows the FT-IR spectra of the benzaldehyde-modified alginate-bacitracin conjugate (AL-ABA-Bac), bacitracin (Bac) synthesized in Example I-10, the benzaldehyde-modified alginate (AL-ABA), and alginate (AL) synthesized in Example 1. [Figure 23] Figure 23 shows the 1H NMR spectrum of the benzaldehyde-modified alginate-dopamine conjugate (AL-ABA-DOPA) synthesized in Example I-11. [Figure 24] Figure 24 shows the FT-IR spectrum of the benzaldehyde-modified alginate-dopamine conjugate (AL-ABA-DOPA) synthesized in Example I-11. [Figure 25] Figure 25 shows the 1H NMR spectrum of the benzaldehyde-modified alginate-serotonin conjugate (AL-ABA-Serotonin) synthesized in Example I-13. [Figure 26] Figure 26 shows the FT-IR spectrum of the benzaldehyde-modified alginate-serotonin conjugate (AL-ABA-Serotonin) synthesized in Example I-13. [Figure 27] Figure 27 shows the 1H NMR spectrum of the benzaldehyde-modified alginate-celecoxib conjugate (AL-ABA-Celecoxib) synthesized in Example I-14. [Figure 28]Figure 28 shows the preparation method of AL-ABA-Apt and the experimental procedure for the sustained-release experiment of AL-ABA-Apt in Example I-15. [Figure 29] Figure 29 shows the release behavior of HGF aptamers (Apt) synthesized in Example I-15 from AL-ABA-HGF aptamer (AL-ABA-Apt), a mixture of alginic acid and HGF aptamer (ALG-Apt), and HGF aptamer alone (Apt) under physiological pH conditions (pH 7.4). The vertical axis represents the cumulative release rate (%) of HGF aptamers, calculated from the absorbance measured by UV-vis spectroscopy. [Figure 30] Figure 30 shows an optical photograph of the benzaldehyde-modified alginate sponge prepared in Example I-16. The Control is an AL sponge prepared as a control, while A, B, and C are AL / AL-ABA mixed sponges with AL to AL-ABA mixing ratios (by weight) of 75:25, 50:50, and 25:75, respectively. [Figure 31] Figure 31 shows SEM images of the benzaldehyde-modified alginate sponge prepared in Example I-16. The Control is an AL sponge prepared as a control, while A, B, and C are AL / AL-ABA mixed sponges with AL and AL-ABA mixing ratios (by weight) of 75:25, 50:50, and 25:75, respectively. [Figure 32] Figure 32 shows the swelling and decomposition profile (change in hydrogel weight) of Ca-crosslinked AL-ABA hydrogel. [Figure 33] Figure 33 is an SEM image (magnification X500; 24 hours after the start of swelling) of a sponge obtained by freeze-drying Ca-crosslinked AL-ABA hydrogel. The porous structure of the dried hydrogel is confirmed. [Figure 34] Figure 34 shows a schematic diagram of the crosslinked structure of AL-ABA and amino group-containing polymer (DPI; dendritic polyethyleneimine) prepared in Example I-18. [Figure 35] Figure 35 shows a photograph of a hydrogel consisting of a cross-linked structure of AL-ABA and DPI. [Figure 36A] Figure 36A shows the 1H NMR spectrum of PEG-COOH prepared in Example I-19. [Figure 36B] Figure 36B shows the 1H NMR spectrum of PEGDH prepared in Example I-19. [Figure 36C] Figure 36C shows a photograph of the hydrogel made of a cross-linked structure of AL-ABA and PEGDH prepared in Example I-19. [Figure 37] Figure 37 shows the results of dynamic viscoelasticity measurements of a hydrogel consisting of a crosslinked structure of AL-ABA and an amino group-containing polymer (PEGDH) prepared in Example I-19. [Figure 38] Figure 38 shows a photograph of the hydrogel of AL-ABA and polyallylamine prepared in Example I-21. [Figure 39] Figure 39 shows the change in appearance over time when benzaldehyde-modified alginate (AL-ABA) and alginate (AL) gels (AL-ABA gel and AL gel), respectively, were administered onto the esophagus of a pig and immersed in physiological saline in Example I-22. In Figure 39, ALG (IL-6G) represents AL gel, and ALG-ABA represents AL-ABA gel. [Figure 40] Figure 40 shows the change in gel weight (adhesion rate,%) over time when gels of benzaldehyde-modified alginate (AL-ABA) and alginate (AL), respectively, cross-linked with calcium (AL-ABA gel and AL gel), were administered onto the esophagus of a pig and immersed in physiological saline in Example I-22. In Figure 40, ALG (IL-6G) represents AL gel, and ALG-ABA represents AL-ABA gel. [Figure 41] Figure 41 is a schematic diagram showing the binding of AL-ABA and AL to the esophageal mucosa and submucosa. [Figure 42] Figure 42 shows the procedure for evaluating the adhesion of AL-ABA and AL to the esophageal mucosa and submucosa in Example I-23. [Figure 43]Figure 43 shows the changes in appearance over time when gels (AL-ABA, AL) formed by calcium crosslinking of benzaldehyde-modified alginate (AL-ABA) and alginate (AL) at Ca2+ concentrations of 50 mM and 100 mM, respectively, were applied to the mucosa (Control) and submucosa (ESD) of a pig's esophagus and immersed in physiological saline. The circles (dotted lines) in the figure represent areas where the material remains. [Figure 44] Figure 44 shows the change over time in gel weight (adhesion rate, %) when gels (AL-ABA, AL) formed by calcium crosslinking of benzaldehyde-modified alginate (AL-ABA) and alginate (AL) at Ca2+ concentrations of 50 mM and 100 mM, respectively, were applied to the mucosa (Con) and submucosa (ESD) of a pig esophagus and immersed in physiological saline. [Figure 45] Figure 45 is a schematic diagram showing the tensile test method in Example I-23. [Figure 46] Figure 46 shows the tensile test results of AL-ABA and AL Ca-crosslinked hydrogels adhered to the mucosa (Control) and submucosa (ESD) in Example I-23. [Figure 47] Figures 47(A) and (B) are schematic diagrams showing the experimental procedure for the lap shear method in Example I-24. [Figure 48] Figure 48 shows the results of measuring the adhesion strength (Pa) of AL-ABA and AL Ca-crosslinked hydrogels, as well as conventional tissue adhesives, which were adhered to submucosa and skin tissue in Example I-24, using the overlapping shear method. [Figure 49] Figure 49(A) is a schematic diagram showing the apparatus used for the burst test in Example I-25. Figure 49(B) is a schematic diagram showing the method of the burst test. [Figure 50]Figure 50 shows the results of measuring the burst pressure (mmHg) by burst tests for AL-ABA and AL Ca-crosslinked hydrogels, as well as conventional tissue adhesives, that were adhered to submucosa and skin tissue in Example I-25. [Figure 51] Figure 51 shows the 1H NMR spectrum of benzaldehyde-modified hyaluronic acid (HA-ABA) synthesized in Example II-1. [Figure 52] Figure 52 shows the ultraviolet-visible light absorption spectra (UV-vis) of benzaldehyde-modified hyaluronic acid (HA-ABA), hyaluronic acid (HA), and 4-aminobenzaldehyde (ABA) synthesized in Example II-1. [Figure 53] Figure 53 shows the FT-IR spectra of benzaldehyde-modified hyaluronic acid (HA-ABA), hyaluronic acid (HA), and 4-aminobenzaldehyde (ABA) synthesized in Example II-1. [Figure 54] Figure 54 shows the results of cytotoxicity tests (WST assay results) of benzaldehyde-modified hyaluronic acid (HA-ABA) and hyaluronic acid (HA) synthesized in Example II-1 at different concentrations (0.01 mg / mL, 0.1 mg / mL, and 1 mg / mL) against MeT-5A (human mesothelial cell line), HUVEC (human umbilical vein endothelial cells), RAW264.7 cells (mouse macrophage-like cell line), NIH / 3T3 (mouse embryonic fibroblasts), and AB22 cells (mouse mesothelioma cells). [Figure 55] Figure 55 shows the 1H NMR spectra of benzaldehyde-modified hyaluronic acid-pemetrexed conjugate (HA-ABA-PMX), benzaldehyde-modified hyaluronic acid (HA-ABA), hyaluronic acid (HA), and pemetrexed (PMX) synthesized in Example II-2. [Figure 56]Figure 56 shows the ultraviolet-visible light absorption spectra (UV-vis) of benzaldehyde-modified hyaluronic acid-pemetrexed conjugate (HA-ABA-PMX) and benzaldehyde-modified hyaluronic acid (HA-ABA) synthesized in Example II-2. [Figure 57] Figure 57 shows the release behavior of pemetrexed (PMX) from benzaldehyde-modified hyaluronic acid-pemetrexed conjugate solution (HA-ABA-PMX) under different pH levels (pH = 5.0, 6.0, 7.4), the release behavior of PMX from PMX solution (Free PMX), and the release behavior of PMX from a mixed solution of HA and PMX (Free PMX mixed with HA). The vertical axis represents the cumulative drug release rate (%) of PMX, calculated from absorbance measured by UV-vis spectroscopy. [Figure 58] Figures 58A and 58B show the cell proliferation inhibitory effects of benzaldehyde-modified hyaluronic acid-pemetrexed conjugate (HA-ABA-PMX), free pemetrexed (PMX), and HA-ADH-PMX (where PMX is bound to HA via an irreversible amide bond) synthesized in Example II-2 with different PMX concentrations (10⁻⁴ μg / mL, 10⁻³ μg / mL, 10⁻² μg / mL, 10⁻¹ μg / mL, 1 μg / mL, and 10 μg / mL), respectively, on AB22 cells (mouse mesothelioma cells) and MeT-5A (human mesothelial cell line). The vertical axis represents cell viability (%). [Figure 59] Figure 59 shows the ultraviolet-visible light absorption spectra (UV-vis) of benzaldehyde-modified hyaluronic acid-doxorubicin conjugate (HA-ABA-DOX) synthesized in Example II-3, benzaldehyde-modified hyaluronic acid (HA-ABA) synthesized in Example II-1, and doxorubicin (DOX). [Figure 60]Figure 60 shows the release behavior of doxorubicin (DOX) from a benzaldehyde-modified hyaluronic acid-doxorubicin conjugate solution (HA-ABA-DOX) under different pH levels (pH = 5.0, 6.0, and 7.4). The vertical axis represents the cumulative drug release rate (%) of DOX, calculated from absorbance measured by UV-vis spectroscopy. [Figure 61] Figure 61 shows the cell proliferation inhibitory effects of benzaldehyde-modified hyaluronic acid-doxorubicin conjugate (HA-ABA-DOX) synthesized in Example II-3 and free doxorubicin (DOX) on AB22 cells (mouse mesothelioma cells) at different DOX concentrations (10⁻³ μg / mL, 10⁻² μg / mL, 10⁻¹ μg / mL, 1 μg / mL, 10 μg / mL, 100 μg / mL). The vertical axis represents cell viability (%). [Figure 62] Figure 62 shows the ultraviolet-visible light absorption spectra (UV-vis) of benzaldehyde-modified hyaluronic acid-gemcitabine conjugate (HA-ABA-GEM) synthesized in Example II-4, benzaldehyde-modified hyaluronic acid (HA-ABA) synthesized in Example II-1, and gemcitabine (GEM). [Figure 63] Figure 63 shows the 1H NMR spectrum of the benzaldehyde-modified hyaluronic acid-adenine conjugate (HA-ABA-Adenine) synthesized in Example II-5. [Figure 64] Figure 64 shows the 1H NMR spectrum of the benzaldehyde-modified hyaluronic acid-cytosine conjugate (HA-ABA-cytosine) synthesized in Example II-5. [Figure 65] Figure 65 shows the 1H NMR spectrum of the benzaldehyde-modified hyaluronic acid-guanine conjugate (HA-ABA-guanine) synthesized in Example II-5. [Figure 66]Figure 66 shows the FT-IR spectra of benzaldehyde-modified hyaluronic acid-DNA nucleotide conjugates (HA-ABA-Adenine, HA-ABA-cytosine, and HA-ABA-guanine) synthesized in Example II-5 and benzaldehyde-modified hyaluronic acid (HA-ABA) synthesized in Example II-1. [Figure 67] Figure 67 shows the 1H NMR spectrum of benzaldehyde-modified carboxymethylcellulose (CMC-ABA) synthesized in Example III-1. [Figure 68] Figure 68 shows the FT-IR spectra of benzaldehyde-modified carboxymethylcellulose (CMC-ABA) and carboxymethylcellulose (CMC) synthesized in Example III-1. [Figure 69] Figure 69 shows the 1H NMR spectrum of benzaldehyde-modified carboxymethyl dextran (CMDX-ABA) synthesized in Example IV-1. [Figure 70] Figure 70 shows the FT-IR spectra of benzaldehyde-modified carboxymethyl dextran (CMD-ABA) and carboxymethyl dextran (CMD) synthesized in Example IV-1. [Figure 71] Figure 71 shows the FT-IR spectra of benzaldehyde-modified carboxymethyl dextran-DNA nucleotide conjugates synthesized in Example IV-2 (CMD-ABA-Adenine, CMD-ABA-Cytosine, and CMD-ABA-Guanine), and benzaldehyde-modified carboxymethyl dextran (CMD-ABA) and carboxymethyl dextran (CMD) synthesized in Example IV-1. [Figure 72] Figure 72 shows the 1H NMR spectrum of benzaldehyde-modified chitosan (Chitosan-CBA) synthesized in Example V-1. [Figure 73] Figure 73 shows the FT-IR spectrum of benzaldehyde-modified chitosan (Chitosan-CBA) synthesized in Example V-1. [Figure 74]Figure 74(A) is a schematic diagram showing the experimental procedure for evaluating adhesion to dura mater tissue using the overlapping shear method in Example I-26. Figure 74(B) shows the results of measuring the adhesion strength (Pa) to dura mater tissue of various alginate derivatives (AL-ABA, AL-AFA, AL-AAP, AL-APA, AL-ANA) and conventional tissue adhesives using the overlapping shear method. [Figure 75A] Figure 75(A) is a schematic diagram showing the method of the burst test in Example I-27. Figure 75(B) shows the results of measuring the burst pressure (mmHg) by burst test for various alginic acid derivatives (AL-ABA, AL-AFA, AL-AAP, AL-APA, AL-ANA) and conventional tissue adhesives adhered to dura mater tissue in Example I-27. Samples ending in "Neovil" are samples using Neovil® as the nonwoven fabric, while samples ending in "Double Vicryl mesh" and "Single Vicryl mesh" are samples using two and one sheets of "Ethicon Vicryl Mesh" as the nonwoven fabric, respectively. [Figure 75B] Figure 75(C) shows the results of burst pressure (mmHg) measurements by burst test for various alginate derivatives (AL-ABA, AL-AFA, AL-AAP, AL-APA, AL-ANA) and conventional tissue adhesives adhered to dura mater tissue in Example I-27. Samples ending in "Neovil" are samples using Neovil® as the nonwoven fabric, while samples ending in "Double Vicryl mesh" and "Single Vicryl mesh" are samples using two and one sheets of "Ethicon Vicryl Mesh" as the nonwoven fabric, respectively. [Figure 76] Figure 76 shows the results of the viscoelastic analysis (flow curve) in Example I-28. [Figure 77] Figure 77 shows the results of the frequency sweep test in Example I-28. [Figure 78] Figure 78 shows the results of the hemolysis test in Example I-29. [Figure 79] Figure 79 shows the 1H NMR spectrum of dendritic poly(ethyleneimine) (DPIGA) conjugated with gallic acid synthesized in Example I-30. The spectra of gallic acid (GA) and dendritic poly(ethyleneimine) (DPI) are also shown. [Figure 80] Figure 80 shows the FT-IR spectrum of DPIGA synthesized in Example I-30. The spectra of GA and DPI are also shown. [Figure 81] Figure 81 shows the ultraviolet-visible absorption spectrum (UV-vis) of DPIGA synthesized in Example I-30. The spectra of GA and DPI are also shown. [Figure 82] Figure 82(A) shows the adhesion strength (Pa) of the conjugate (cross-linked structure) (AL-ABA / DPI-GA) of AL-ABA and GA-modified amino group-containing polymer prepared in Example I-30 to skin tissue or submucosal tissue, measured by the overlapping shear method. AL-ABA and DPI-GA were used as controls. Figure 82(B) is a schematic diagram showing the binding morphology of the conjugate (cross-linked structure) of AL-ABA and GA-modified amino group-containing polymer to tissue. [Figure 83] Figure 83(A) shows the 1H NMR spectrum of benzaldehyde-modified chitosan (CH-CBA) synthesized in Example V-2. Figure 83(B) shows the FT-IR spectrum of CH-CBA prepared in Example V-2. Figure 83(C) shows the ultraviolet-visible absorption spectrum (UV-vis) of CH-CBA prepared in Example V-2. [Figure 84] Figure 84 shows the results of gelation tests at different pH values ​​measured by the vial inversion method for CH-CBA prepared in Example V-2. [Figure 85]Figure 85 shows the results of viscoelastic analysis for CH-CBA at different pH levels prepared in Example V-2. Figure 85(A) shows the storage modulus (G') and loss modulus (G") of CH-CBA hydrogels at different pH levels, Figure 85(B) plots the loss coefficient (tanδ) of CH-CBA hydrogels at different pH levels, and Figure 85(C) plots the viscosity (η) of CH-CBA hydrogels at different pH levels as the shear rate is varied. [Figure 86] Figure 86 shows the relationship between the pH of CH-CBA prepared in Example V-2 and the viscosity ratio (η0.001 / η1000) between the viscosity at a shear rate of 10⁻³ s⁻¹ (η0.001) and the viscosity at a shear rate of 10³ s⁻¹ (η1000). [Figure 87] Figure 87 shows SEM images of CH-CBA hydrogels gelled at different pH levels prepared in Example V-2. [Figure 88] Figure 88(A) is a schematic diagram showing the pH-responsive self-crosslinking properties of CH-CBA hydrogel. Figure 88(B) is a schematic diagram showing the adhesion of CH-CBA hydrogel to tissue. [Figure 89] Figure 89(A) shows the cytotoxicity test results (WST-8 assay results) of CH-CBA prepared in Example V-2. Figure 89(B) shows the results of the Live / Dead cell viability assay of CH-CBA prepared in Example V-2. The results for the control groups, Fibrin Glue and Albumin / Glutaraldehyde (Albumin / GTA), are also shown. [Figure 90] Figure 90 is a schematic diagram illustrating the method of a superposition shear test using CH-CBA hydrogel. [Figure 91A] Figures 91(A) and (B) show the results of tack tests on CH-CBA hydrogels at different pH levels prepared in Example V-2. Figure 91(C) shows the results of overlap shear tests on mucous membranes using CH-CBA hydrogels at different pH levels prepared in Example V-2. [Figure 91B] Figures 91(D) to (E) show the results of overlapping shear tests of CH-CBA hydrogels at different pH levels prepared in Example V-2 on submucosal tissue and skin, respectively. [Modes for carrying out the invention]

[0022] In this specification, "halogen atom" refers to a fluorine atom (F), a chlorine atom (Cl), a bromine atom (Br), or an iodine atom (I). In this specification, "C 1-k "Alkyl" refers to a linear or branched alkyl group with 1 to k carbon atoms. In this specification, "C 1-k "Alkylene" refers to a linear or branched alkylene group with 1 to k carbon atoms. In this specification, "polysaccharide" means a compound comprising two or more monosaccharides linked to each other by glycosidic bonds, its derivatives (for example, polysaccharides modified by esterification, maleimide modification, thiol modification, acrylate modification, aldehyde modification, disulfide modification (e.g., pyridyl disulfide), alkyne modification including cyclic alkynes, tetrazine modification, furan ring modification, etc.), its salts, and crosslinked products thereof. While compounds composed of 2 to 10 linked monosaccharides are sometimes called oligosaccharides, in this specification, "polysaccharide" includes not only the narrow definition of "polysaccharide" consisting of 10 or more linked monosaccharides, but also "oligosaccharides." In this specification, "polysaccharide derivative-drug conjugate" means a complex formed by linking a drug and a polysaccharide derivative via a bond.

[0023] I. First Form A first embodiment of the present invention relates to a tissue adhesive material for use with tissues that are adhesive not only to soft tissues in living organisms but also to relatively hard and thick tissues such as the dura mater and / or connective tissue. The tissue adhesive material of this embodiment is a tissue adhesive material for use with hard and thick tissues and / or connective tissue and comprises a polysaccharide derivative (hereinafter also simply referred to as "polysaccharide derivative") in which a group represented by the following formula (A) is introduced into an acidic, basic, or amphoteric polysaccharide. In some embodiments, the tissue adhesive material is used for biological tissue repair / restoration. [ka] (In the formula, R 1 is a hydrogen atom or C 1-4 Represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or pyridine ring may be substituted with one or more substituents independently selected from a halogen atom, -CF3, -NO2, a carboxyl group, and -SO3H. Y is -NH-, -C(=O)-, -S-, -O-, C 1-6 Alkylene, -(CH2CH2O) n - represents a divalent base selected from the group consisting of , and any combination thereof, where n is an integer from 1 to 9. * indicates a linkage with a polysaccharide.

[0024] In this specification, "hard, thick tissue" refers to tissues other than solid biological components containing calcium and phosphate, such as bone and teeth, that contain collagen and are in the form of membranes, tubes, or plates, or tissues present at the ends of bones, such as the dura mater, perichondrium, periodontal ligament, and cartilage. The thickness of such "hard, thick tissue" is, for example, in the range of 0.1 to 10 mm. Connective tissue, derived from mesenchyme (mesenchyma), is a tissue that connects and supports various organs and tissues by filling the spaces between them. Connective tissue consists of fibers such as collagen fibers and elastic fibers, and connective tissue cells such as fibrous cells, lymphocytes, plasma cells, and leukocytes. Depending on the types of fibers and cells that make up the tissue, it is classified into gelatinous tissue, reticular tissue, fibrous connective tissue, elastic tissue, adipose tissue, pigment tissue, etc., and the shape and composition of connective tissue vary. Examples of connective tissue include fascia, tendons, ligaments, bursae, cartilage, periosteum, dura mater, sclera, and dermis. Although there is some overlap between "hard, thick tissue" and "connective tissue," if the tissue falls into either category, a tissue-adhesive material can be preferably applied in the first form. The hard, thick tissues and connective tissues that are the target of this study are present throughout the entire body. In certain embodiments, the hard, thick tissue and / or connective tissue is selected from the dura mater, perichondrium, synovium, periodontal ligament, and elastocartilage. In some embodiments, the hard, thick tissue and / or connective tissue is selected from the dura mater, perichondrium, synovial membrane, and periodontal ligament.

[0025] One of the most representative hard, thick tissues and / or connective tissues is the dura mater. The dura mater is a hard membrane that surrounds the brain and spinal cord, and is divided into the cerebral dura mater, which surrounds the brain, and the spinal dura mater, which surrounds the spinal cord. Beneath the dura mater are the arachnoid mater, pia mater, and brain / spinal cord parenchyma. The dura mater, arachnoid mater, and pia mater together are called the meninges, and the dura mater plays a role in protecting the brain and spinal cord from trauma and infection. The dura mater consists of two layers: an inner layer (also called the inner lobe or inner plate) and an outer layer (also called the outer lobe or outer plate). In the spinal cord, there is a space called the epidural space (supradural space) between the outer and inner layers, but in the brain, they are tightly attached in most parts except for the dural venous sinuses. There is a space called the subdural space between the outer and inner layers and the inner arachnoid mater. In this specification, the dura mater may refer to either the cerebral dura mater or the spinal dura mater. The perichondrium is the membrane that covers the cartilage of the knee, elbow, hip, and fingers, while the synovial membrane is the membrane that covers the joints, especially the knee, elbow, and fingers. Both are membranes that enclose synovial fluid within the joint cavity. The dura mater tissue has primary amino groups on its surface. Specifically, the main component of the dura mater is collagen, and the cross-linking sites of collagen contain numerous lysine residues (Lys) with amino groups. According to this form of tissue adhesive material, the amino group of the lysine residue (Lys), an amino acid abundant in collagen, and the modifying group (A)(aldehyde group or ketone group (-C(=O)R) of the polysaccharide derivative are combined. 1 By forming Schiff bases between them, good adhesion to the dura mater tissue is achieved. In a preferred embodiment, the tissue adhesive material is used for dura mater tissue. Collagen is a major component of the so-called membrane structure in living organisms, and the fact that this form of tissue adhesive material adheres to the dura mater indicates that this form of tissue adhesive material also adheres to other membrane components besides the dura mater. In the examples described later, the dura mater was used to demonstrate adhesion to hard, thick tissues and / or connective tissue. The reason for focusing on the dura mater is that it is a relatively hard and thick material among the soft tissues and membrane components of living organisms, and a stronger adhesion is required that can withstand the pressure of cerebrospinal fluid leakage from beneath the dura mater. In addition to good adhesion, this form of tissue adhesive material has appropriate flexibility, resulting in excellent resistance to the pressure of cerebrospinal fluid leakage. Furthermore, polysaccharide derivatives such as alginate derivatives, hyaluronic acid derivatives, and carboxymethyl dextran derivatives can form hydrophobic interactions and hydrogen bonds (for example, hydrogen bonds between the hydroxyl groups of hydroxyproline residues in collagen and the hydroxyl or carboxyl groups of polysaccharide derivatives) with tissue proteins, potentially improving adhesion to tissues.

[0026] Other typical examples of hard, thick tissues and / or connective tissues include the perichondrium and periodontal ligament. The perichondrium is connective tissue that covers the surface of cartilage. For example, when transplanting cartilage fragments to fill cartilage defects, this tissue adhesive material can be used as a substitute for the perichondrium, or to cover the defect in the perichondrium. Not only donor cartilage, but also cartilage grafts cultured in vitro, stem cells, etc., can be mixed with the tissue adhesive and bonded to the defect at the same time as filling it, and it has the advantage of not requiring a covering material in addition to the filler. The periodontal ligament is a collagen-rich membrane, approximately 0.3 mm thick, that connects the alveolar bone to the tooth root. This type of tissue adhesive material can also be used in dentistry for bonding the alveolar bone to the periodontal ligament, or for bonding the gums to the periodontal ligament.

[0027] In its first form, the tissue adhesive material is a material used for repairing / restoring biological tissue, and it adheres not only to soft tissues in the body but also to relatively hard and thick tissues such as the dura mater and / or connective tissue. It is used for repairing / reinforcing tissue and its damaged areas, preventing the spread of tissue fracture / damage and increasing the physical strength of the reinforced area. While its use as a tissue adhesive is not particularly limited, in neurosurgery, for example, it is used in surgery for brain injury, brain tumors, and intracerebral hemorrhage to repair the dura mater, the outermost layer of the meninges. This means repairing and restoring the dura mater after an incision, or as an adhesive when filling defects with artificial dura mater (dura mater repair and protection). In orthopedics, it is used during spinal and spinal cord surgery to repair and protect the dura mater in particular. In otolaryngology, it is also used for bonding when transplanting or repairing elastic cartilages such as auricular cartilage, nasal cartilage, and epiglottic cartilage.

[0028] There are no particular restrictions on the specific areas of biological tissue to which the material can be applied; areas where tissue adhesive materials have conventionally been used can be suitably adopted. Examples of such application areas include injury sites, surgical suture sites, and bleeding sites. In some embodiments, tissue adhesive materials are used for the purpose of repairing and regenerating dural damage and preventing cerebrospinal fluid fistulas. In some embodiments, tissue adhesive materials are used to prevent the repair and regeneration of perichondrium damage, and to fill cartilage defects when transplanting cartilage fragments. In some embodiments, tissue adhesive materials are also used on synovial damage in joints such as the knee, elbow, and fingers for the purpose of joint repair and protection. In some embodiments, tissue adhesive materials are used for the purpose of preventing the repair and regeneration of periodontal ligament damage, for bonding the alveolar bone to the periodontal ligament in dentistry, or for bonding the gingiva to the periodontal ligament.

[0029] Conventional tissue adhesives include: 1) cyanoacrylate adhesives (Dermabond, Liquiband), 2) gelatin-aldehyde adhesives (Bioglue), 3) fibrin glue adhesives (Beriplast, Volheel), and 4) other materials (Hydrofit (Mazdaite)). 1) Cyanoacrylate adhesives utilize the polymerization reaction of cyanoacrylate monomers and offer high adhesive strength. They also have the advantage of a short bonding time. However, after the material has set, the polymer undergoes hydrolysis in the body, generating formaldehyde, which is highly toxic to living organisms. It can also inhibit the healing of the bonded area. Therefore, there have been problems with using these adhesives in areas that come into direct contact with the central nervous system or blood vessels within the body. 2) Gelatin-aldehyde adhesives are adhesives that utilize the cross-linking reaction between the biopolymer gelatin and formaldehyde or glutaraldehyde. While these adhesives have high adhesive strength and polymer strength after curing, they exhibit biotoxicity because they use toxic formaldehyde compounds as cross-linking agents. 3) Fibrin glue adhesives are adhesives that utilize the biological component of blood coagulation. They utilize the formation of fibrin in blood, have no toxicity to biological tissues, and are the most widely used tissue adhesives for use in vivo. Because they are derived from biological components, they have high affinity with biological tissues and do not inhibit tissue regeneration at the application site. However, as an adhesive, they are soft and have weak adhesive strength. Furthermore, although they are mixed immediately before use, there are challenges in terms of the amount of preparation time and effort required beforehand, such as the need to prepare two types of solutions at the time of use and assemble a special filling device. In addition, once dissolved, the adhesive has a short usage time and cannot be left standing, which is another challenge. Moreover, since the raw material for fibrin is human blood, it is difficult to secure the raw material blood and avoid the risk of contamination by pathogenic viruses, etc. 4) Other materials include, for example, urethane adhesives. Urethane is non-absorbent and is mainly used for local hemostasis. It is a viscous liquid composed of polyether-based fluorine-containing urethane prepolymers that react with the moisture in blood and biological tissue at the vascular anastomosis site to polymerize, forming a gel-like polymer that adheres closely to the vascular anastomosis site and stops bleeding.

[0030] On the other hand, this form of tissue adhesive material uses polysaccharide components such as alginic acid, which has the advantages of avoiding the use of toxic formaldehyde (1) and 2), avoiding the risk of bloodborne infection (3), and avoiding retention in the body due to its non-absorbable nature (4). Furthermore, in contrast to cyanoacrylate-based adhesives (1) which bond and fix strongly and quickly, this form of tissue adhesive material retains appropriate flexibility (viscosity) even after bonding. As a result, this form of tissue adhesive material has the advantage of preventing leakage from the bonding site (gaps) while also having excellent resistance to leakage pressure. Thus, this form of tissue adhesive material is superior in terms of biocompatibility and safety when considered for use in vivo. Furthermore, depending on the site of application, it may be necessary to adhere to tissues that are very wet or rich in blood and bodily fluids, but this form of tissue adhesive material is thought to be able to function even under such wet conditions by directly adhering to the proteins that make up the tissue. In addition, conventional tissue adhesives are used to adhere soft tissues to soft tissues in the body (for example, to adhere bioabsorbable materials to vascular anastomoses or to repair tissues excised by surgery), but the discovery that it can also adhere to hard tissues such as the dura mater, which is the outermost layer of the meninges, represents an improvement as a new tissue adhesive material and will broaden its range of use in medical settings. The excellent tissue adhesion of this embodiment of tissue adhesive material to dura mater tissue is demonstrated, for example, in Examples I-26 and I-27 described later. In particular, as shown in Example I-26 (Figure 74(B)) described later, many conventional tissue adhesives (DuraSeal® (a gel of activated ester-terminated PEG and trilysine using NHS) and fibrin glue) have poor dura mater adhesion, and this embodiment of tissue adhesive material, which has excellent adhesion to dura mater tissue, is highly useful. It is presumed that in this embodiment of tissue adhesive material, the benzaldehyde group of the polysaccharide derivative has appropriate hydrophobicity, while the hydrophilicity due to the abundant hydroxyl groups of the polysaccharide contributes to the excellent properties such as adhesion.

[0031] 1. Polysaccharide derivative In the present invention, the polysaccharide derivative is an acidic, basic, or amphoteric polysaccharide to which a group represented by the following formula (A) (hereinafter also simply referred to as "modifying group (A)") has been introduced. [ka]

[0032] Polysaccharide derivatives can react with primary amino groups to form Schiff bases under neutral to basic pH conditions. The Schiff bases formed between the polysaccharide derivative and the primary amino group are stable under neutral to basic pH conditions, while they can dissociate under low pH conditions. This property allows for their use as drug delivery carriers, bioabsorbable materials, medical devices, and separation material devices.

[0033] The polysaccharide derivative in this form has at least one of the following advantages. (1) Polysaccharide derivatives can form stable conjugates with various drugs having primary amino groups under neutral to basic pH conditions, while also releasing drugs having primary amino groups. In particular, due to its aromatic structure, the above-mentioned modifying group (A) can form stable Schiff bases with amine groups contained in various drugs, even in water. (2) Since polysaccharide derivatives are based on acidic, basic, or amphoteric polysaccharides, even if the modifying group (A) has a hydrophobic benzene ring or pyridyl ring, the modifying group (A) can be introduced with a good modification rate. Furthermore, even when the modifying group (A) is introduced with a high modification rate, the resulting polysaccharide derivative exhibits excellent water solubility due to the presence of unmodified acidic or basic functional groups (anionic or cationic functional groups). In particular, polysaccharides having anionic functional groups (such as carboxyl groups) yield polysaccharide derivatives with especially excellent water solubility, even when the modification rate of the modifying group (A) is high. (3) Because the polysaccharide has acidic, basic, or both (amphoteric) functional groups (i.e., charged functional groups), it can react with highly hydrophobic phenylaldehyde or pyridylaldehyde compounds in a single step under mild and safe conditions, and a modifying group (A) can be easily introduced to the polysaccharide. (4) In some embodiments, a stable polysaccharide derivative-drug conjugate can be easily obtained in one pot using mild and safe reaction conditions in a single step. Since there is no need for modification of the drug before conjugate formation, and no complex experimental equipment or catalysts are required for conjugate formation, the polysaccharide derivative-drug conjugate can be prepared in situ from the polysaccharide derivative and the drug. (5) In some embodiments, polysaccharide derivatives and conjugates of polysaccharide derivatives and drugs can be crosslinked using a crosslinking agent to form crosslinked structures of various shapes (e.g., tubular structures, fibrous structures, fibers, beads, gels, approximately spherical gels, capsules, sponges, sheets). In addition, in some embodiments, polysaccharide derivatives and conjugates of polysaccharide derivatives and drugs can form the above-mentioned various forms of crosslinked structures with amino group-containing polymers.

[0034] (Modifying group (A): The group represented by formula (A)) In the above formula (A), R 1 is a hydrogen atom or C 1-4 Represents alkyl. 1Preferably, is a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a tert-butyl group, a sec-butyl group, an iso-butyl group, or an n-butyl group, and more preferably a hydrogen atom or a methyl group. In one embodiment, R 1 This is a hydrogen atom. In the above formula (A), * represents the linkage with the polysaccharide.

[0035] In formula (A) above, ring P is a phenyl ring or a pyridine ring. The phenyl ring or pyridine ring may be substituted with one or more substituents independently selected from halogen atoms (F, Cl, Br, and / or I), -CF3 (trifluoromethyl group), -NO2, carboxyl group, and -SO3H. In one embodiment, ring P is a phenyl ring, and the phenyl ring may be substituted with 1 to 4 substituents independently selected from halogen atoms (F, Cl, Br, and / or I), -CF3, -NO2, carboxyl groups, and -SO3H. In one embodiment, ring P is an unsubstituted phenyl ring. In one embodiment, ring P is a pyridine ring, which may be substituted with 1 to 3 substituents independently selected from halogen atoms (F, Cl, Br, and / or I), -CF3, -NO2, carboxyl groups, and -SO3H. In one embodiment, ring P is an unsubstituted pyridine ring.

[0036] In the above formula (A), Y is -NH-, -C(=O)-, -S-, -O-, alkylene, -(CH2CH2O) n - represents a divalent group selected from the group consisting of and any combination thereof, where n is an integer from 1 to 9 (preferably 1 to 4, more preferably 1 to 2). The number of carbon atoms in the alkylene group in Y is 1 to 6, preferably 1 to 4, more preferably 1 to 2, and even more preferably 1, from the viewpoint of hydrophilicity.

[0037] In some embodiments, Y is -L 1 -NH- or -L 2-C(=O)- is selected, and in this case, L 1 and L 2 It is bonded to ring P. That is, the modifying group (A) is selected from the following formulas (A-1) or (A-2). [ka] In the above formulas (A-1) and (A-2), * represents the linkage with the polysaccharide. Also, in the above formulas (A-1) and (A-2), R 1 The definition and preferred embodiment of ring P is R in formula (A) above. 1 And it is the same as the definition and preferred embodiment of ring P.

[0038] In equation (A-1), L 1 It is either a single bond or -C(=O)-, -S-, -O-, alkylene, -(CH2CH2O) n - represents a divalent base selected from the group consisting of and any combination thereof, where n is an integer between 1 and 9 (preferably 1 to 4, more preferably 1 to 2). 1 The number of carbon atoms in the alkylene group is 1 to 6, preferably 1 to 4, more preferably 1 to 2, and even more preferably 1, from the viewpoint of hydrophilicity. Among them, L 1 C is a single bond. 1―6 Alkylene, -(CH2CH2O) n -,-(CH2) m1 -(CH2CH2O) n -(CH2) m2 -, and -(CH2) m1 -O-(CH2CH2O) n -(CH2) m2 - Selected from. n is an integer between 1 and 9 (preferably 1 to 4, more preferably 1 to 2). m1 and m2 are each independently integers between 1 and 9 (preferably 1 to 4, more preferably 1 to 2). L 1 C in 1―6 From the viewpoint of hydrophilicity, alkylene is preferably C 1―4 It is an alkylene, and farC 1―2It is an alkylene, and more preferably a methylene. In one embodiment, L 1 is a single bond or C 1―2 It is alkylene. 1 It is a single bond.

[0039] In equation (A-2), L 2 It is either a single bond or -NH-, -S-, -O-, alkylene, -(CH2CH2O) n - represents a divalent base selected from the group consisting of and any combination thereof, where n is an integer between 1 and 9 (preferably 1 to 4, more preferably 1 to 2). 2 The number of carbon atoms in the alkylene group is 1 to 6, preferably 1 to 4, more preferably 1 to 2, and even more preferably 1, from the viewpoint of hydrophilicity. Among them, L 2 C is a single bond. 1―6 Alkylene, -(CH2CH2O) n -,-(CH2) m1 -(CH2CH2O) n -(CH2) m2 -, and -(CH2) m1 -O-(CH2CH2O) n -(CH2) m2 - Selected from. n is an integer between 1 and 9 (preferably 1 to 4, more preferably 1 to 2). m1 and m2 are each independently integers between 1 and 9 (preferably 1 to 4, more preferably 1 to 2). L 2 C in 1―6 From the viewpoint of hydrophilicity, alkylene is preferably C 1―4 It is an alkylene, and farC 1―2 It is an alkylene, and more preferably a methylene. In one embodiment, L 2 is a single bond or C 1―2 It is alkylene. 2 It is a single bond.

[0040] In some embodiments, the modifying group (A) is a group represented by formula (A-1) above, ring P is a phenyl ring, and the phenyl ring may be substituted with 1 to 4 substituents independently selected from halogen atoms (F, Cl, Br, and / or I), -CF3, -NO2, carboxyl groups, and -SO3H.

[0041] For example, the modifying group (A) is selected from the following formulas (1), (2), and (3) (which may hereafter be referred to as "formulas (1) to (3)"). [ka] In the above formulas (1) to (3), * represents the linkage with the polysaccharide. Also, in the above formulas (1) to (3), R 1 The definition and preferred embodiment of R in formula (A) above is 1 The definition and preferred embodiment are identical, L 1 The definition and preferred embodiment of L in formula (A-1) above is 1 This is identical to the definition and preferred embodiment of [the term]. In the above equations (1) to (3), R 51 , R 52 , R 53 , and R 54 Each of these is independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), -CF3, -NO2, a carboxyl group, and -SO3H. In one embodiment, R 51 , R 52 , R 53 , and R 54 In one embodiment, R 51 , R 52 , R 53 , and R 54 Each of these is independently selected from hydrogen atoms and halogen atoms (F, Cl, Br, I; preferably F). That is the case.

[0042] In a particular embodiment, in formulas (1) to (3) above, R 1 L is a hydrogen atom. 1 It is a single bond, R 51, R 52 , R 53 , and R 54 Each of these is independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), -CF3, -NO2, a carboxyl group, and -SO3H. In one embodiment, in formulas (1) to (3) above, R 1 L is a hydrogen atom. 1 It is a single bond, R 51 , R 52 , R 53 , and R 54 These are all hydrogen atoms. In one embodiment, in formulas (1) to (3) above, R 1 L is a hydrogen atom. 1 It is a single bond, R 51 , R 52 , R 53 , and R 54 Each of these is independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0043] In a particular embodiment, in formulas (1) to (3) above, R 1 It is methyl, L 1 It is a single bond, R 51 , R 52 , R 53 , and R 54 Each of these is independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), -CF3, -NO2, a carboxyl group, and -SO3H. In one embodiment, in formulas (1) to (3) above, R 1 It is methyl, L 1 It is a single bond, R 51 , R 52 , R 53 , and R 54 These are all hydrogen atoms. In one embodiment, in formulas (1) to (3) above, R 1 It is methyl, L 1 It is a single bond, R 51 , R 52 , R 53 , and R 54 Each of these is independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0044] In a particular embodiment, in formulas (1) to (3) above, R 1 L is a hydrogen atom. 1 R is methylene (-CH2-) or ethylene (-C2H4-), 51 , R 52 , R 53 , and R 54 Each of these is independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), -CF3, -NO2, a carboxyl group, and -SO3H. In one embodiment, in formulas (1) to (3) above, R 1 L is a hydrogen atom. 1 R is methylene (-CH2-) or ethylene (-C2H4-), 51 , R 52 , R 53 , and R 54 These are all hydrogen atoms. In one embodiment, in formulas (1) to (3) above, R 1 L is a hydrogen atom. 1 R is methylene (-CH2-) or ethylene (-C2H4-), 51 , R 52 , R 53 , and R 54 Each of these is independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0045] In a particular embodiment, in formulas (1) to (3) above, R 1 It is methyl, L 1 R is methylene (-CH2-) or ethylene (-C2H4-), 51 , R 52 , R 53 , and R 54 Each of these is independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), -CF3, -NO2, a carboxyl group, and -SO3H. In one embodiment, in formulas (1) to (3) above, R 1 It is methyl, L 1 R is methylene (-CH2-) or ethylene (-C2H4-), 51 , R 52 , R53 , and R 54 These are all hydrogen atoms. In one embodiment, in formulas (1) to (3) above, R 1 It is methyl, L 1 R is methylene (-CH2-) or ethylene (-C2H4-), 51 , R 52 , R 53 , and R 54 Each of these is independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0046] In one embodiment, the modifying group (A) is selected from the groups represented by the following formula. [ka] (In the formula, * represents the linkage with the polysaccharide.) In one embodiment, the modifying group (A) is a group represented by the following formula. [ka] (In the formula, * represents the linkage with the polysaccharide.)

[0047] In some embodiments, the modifying group (A) is a group represented by formula (A-1) above, the ring P is a pyridine ring, and the pyridine ring may be substituted with 1 to 3 substituents independently selected from halogen atoms (F, Cl, Br, I), -CF3, -NO2, carboxyl groups, and -SO3H. For example, the modifying group (A) is selected from the following formulas (4), (5), (6), (7), (8), (9), (10), (11), (12), and (13) (hereinafter sometimes referred to as "formulas (4) to (13)"). [ka] In the above formulas (4) to (13), * represents the linkage with the polysaccharide. Also, in the above formulas (4) to (13), R 1 The definition and preferred embodiment of R in formula (A) above is 1The definition and preferred embodiment are identical, L 1 The definition and preferred embodiment of L in formula (A-1) above is 1 This is identical to the definition and preferred embodiment of [the term]. In the above equations (4) to (13), R 61 , R 62 , and R 63 Each of these is independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), -CF3, -NO2, a carboxyl group, and -SO3H. In one embodiment, R 61 , R 62 , and R 63 In one embodiment, R 61 , R 62 , and R 63 Each of these is independently selected from hydrogen atoms and halogen atoms (F, Cl, Br, I).

[0048] In a particular embodiment, in formulas (4) to (13) above, R 1 L is a hydrogen atom. 1 It is a single bond, R 61 , R 62 , and R 63 Each of these is independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), -CF3, -NO2, a carboxyl group, and -SO3H. In one embodiment, in formulas (4) to (13) above, R 1 L is a hydrogen atom. 1 It is a single bond, R 61 , R 62 , and R 63 These are all hydrogen atoms. In one embodiment, in formulas (4) to (13) above, R 1 L is a hydrogen atom. 1 It is a single bond, R 61 , R 62 , and R 63 Each of these is independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0049] In a particular embodiment, in formulas (4) to (13) above, R 1It is methyl, L 1 It is a single bond, R 61 , R 62 , and R 63 Each of these is independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), -CF3, -NO2, a carboxyl group, and -SO3H. In one embodiment, in formulas (4) to (13) above, R 1 It is methyl, L 1 It is a single bond, R 61 , R 62 , and R 63 These are all hydrogen atoms. In one embodiment, in formulas (4) to (13) above, R 1 It is methyl, L 1 It is a single bond, and R 61 , R 62 , and R 63 Each of these is independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0050] In a particular embodiment, in formulas (4) to (13) above, R 1 L is a hydrogen atom. 1 R is methylene (-CH2-) or ethylene (-C2H4-), 61 , R 62 , and R 63 Each of these is independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), -CF3, -NO2, a carboxyl group, and -SO3H. In one embodiment, in formulas (4) to (13) above, R 1 L is a hydrogen atom. 1 R is methylene (-CH2-) or ethylene (-C2H4-), 61 , R 62 , and R 63 These are all hydrogen atoms. In one embodiment, in formulas (4) to (13) above, R 1 L is a hydrogen atom. 1 It is methylene (-CH2-) or ethylene (-C2H4-), and R 61 , R 62 , and R 63Each of these is independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0051] In a particular embodiment, in formulas (4) to (13) above, R 1 It is methyl, L 1 R is methylene (-CH2-) or ethylene (-C2H4-), 61 , R 62 , and R 63 Each of these is independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), -CF3, -NO2, a carboxyl group, and -SO3H. In one embodiment, in formulas (1) to (3) above, R 1 It is methyl, L 1 R is methylene (-CH2-) or ethylene (-C2H4-), 61 , R 62 , and R 63 These are all hydrogen atoms. In one embodiment, in formulas (1) to (3) above, R 1 It is methyl, L 1 R is methylene (-CH2-) or ethylene (-C2H4-), 61 , R 62 , and R 63 Each of these is independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0052] In one embodiment, the modifying group (A) is selected from the groups represented by the following formula. [ka] (In the formula, * represents the linkage with the polysaccharide.)

[0053] In one embodiment, the modifying group (A) is selected from the groups represented by the following formula. [ka] (In the formula, * represents the linkage with the polysaccharide.)

[0054] In one embodiment, the modifying group (A) is a group represented by the following formula. [ka] This configuration may improve the adhesive strength of tissue-adhering materials.

[0055] In some embodiments, the modifying group (A) is a group represented by formula (A-2) above, ring P is a phenyl ring, and the phenyl ring may be substituted with 1 to 4 substituents independently selected from halogen atoms (F, Cl, Br, and / or I), -CF3, -NO2, carboxyl groups, and -SO3H.

[0056] For example, the modifying group (A) is selected from the following formulas (14), (15), and (16) (hereinafter sometimes referred to as "formulas (14) to (16)"). [ka] In equations (14) to (16) above, * represents the linkage with the polysaccharide. Also, in equations (14) to (16) above, R 1 The definition and preferred embodiment of R in formula (A) above is 1 The definition and preferred embodiment are identical, L 2 The definition and preferred embodiment of L in formula (A-2) above is 2 This is identical to the definition and preferred embodiment of [the term]. In the above equations (1) to (3), R 71 , R 72 , R 73 , and R 74 Each of these is independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), -CF3, -NO2, a carboxyl group, and -SO3H. In one embodiment, R 71 , R 72 , R 73 , and R 74 In one embodiment, R 71 , R 72 , R 73 , and R 74Each of these is independently selected from hydrogen atoms and halogen atoms (F, Cl, Br, I; preferably F). That is the case.

[0057] In a particular embodiment, in formulas (14) to (16) above, R 1 L is a hydrogen atom. 2 It is a single bond, R 71 , R 72 , R 73 , and R 74 Each of these is independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), -CF3, -NO2, a carboxyl group, and -SO3H. In one embodiment, in formulas (14) to (16) above, R 1 L is a hydrogen atom. 2 It is a single bond, R 71 , R 72 , R 73 , and R 744 These are all hydrogen atoms. In one embodiment, in formulas (14) to (16) above, R 1 L is a hydrogen atom. 2 It is a single bond, R 71 , R 72 , R 73 , and R 74 Each of these is independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0058] In a particular embodiment, in formulas (14) to (16) above, R 1 It is methyl, L 2 It is a single bond, R 71 , R 72 , R 73 , and R 74 Each of these is independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), -CF3, -NO2, a carboxyl group, and -SO3H. In one embodiment, in formulas (14) to (16) above, R 1 It is methyl, L 2 It is a single bond, R 71 , R 72 , R 73 , and R 74These are all hydrogen atoms. In one embodiment, in formulas (14) to (16) above, R 1 It is methyl, L 2 It is a single bond, R 71 , R 72 , R 73 , and R 74 Each of these is independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0059] In a particular embodiment, in formulas (14) to (16) above, R 1 L is a hydrogen atom. 2 R is methylene (-CH2-) or ethylene (-C2H4-), 71 , R 72 , R 73 , and R 74 Each of these is independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), -CF3, -NO2, a carboxyl group, and -SO3H. In one embodiment, in formulas (14) to (16) above, R 1 L is a hydrogen atom. 2 R is methylene (-CH2-) or ethylene (-C2H4-), 71 , R 72 , R 73 , and R 74 These are all hydrogen atoms. In one embodiment, in formulas (14) to (16) above, R 1 L is a hydrogen atom. 2 R is methylene (-CH2-) or ethylene (-C2H4-), 71 , R 72 , R 73 , and R 74 Each of these is independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0060] In a particular embodiment, in formulas (14) to (16) above, R 1 It is methyl, L 2 R is methylene (-CH2-) or ethylene (-C2H4-), 71 , R 72 , R 73, and R 74 Each of these is independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), -CF3, -NO2, a carboxyl group, and -SO3H. In one embodiment, in formulas (14) to (16) above, R 1 It is methyl, L 2 R is methylene (-CH2-) or ethylene (-C2H4-), 71 , R 72 , R 73 , and R 74 These are all hydrogen atoms. In one embodiment, in formulas (14) to (16) above, R 1 It is methyl, L 2 R is methylene (-CH2-) or ethylene (-C2H4-), 71 , R 72 , R 73 , and R 74 Each of these is independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0061] In one embodiment, the modifying group (A) is a group represented by the following formula. [ka] (In the formula, * represents the linkage with the polysaccharide.)

[0062] In some embodiments, Y is -L 3 -S- or -L 4 -Selected from, in this case L 3 and L 4 It is bonded to ring P. That is, the modifying group (A) is selected from the following formulas (A-3) or (A-4). [ka] In the above formulas (A-3) and (A-4), * represents the linkage with the polysaccharide. Also, in the above formulas (A-3) and (A-4), R 1 The definition and preferred embodiment of ring P is R in formula (A) above. 1 And it is the same as the definition and preferred embodiment of ring P.

[0063] In equation (A-3), L 3 It is either a single bond or -NH-, -C(=O)-, -O-, alkylene, -(CH2CH2O) n - represents a divalent base selected from the group consisting of and any combination thereof, where n is an integer between 1 and 9 (preferably 1 to 4, more preferably 1 to 2). 3 The number of carbon atoms in the alkylene group is 1 to 6, preferably 1 to 4, more preferably 1 to 2, and even more preferably 1, from the viewpoint of hydrophilicity. Among them, L 3 C is a single bond. 1―6 Alkylene, -(CH2CH2O) n -,-(CH2) m1 -(CH2CH2O) n -(CH2) m2 -, and -(CH2) m1 -O-(CH2CH2O) n -(CH2) m2 - Selected from. n is an integer between 1 and 9 (preferably 1 to 4, more preferably 1 to 2). m1 and m2 are each independently integers between 1 and 9 (preferably 1 to 4, more preferably 1 to 2). L 3 C in 1―6 From the viewpoint of hydrophilicity, alkylene is preferably C 1―4 It is an alkylene, and farC 1―2 It is an alkylene, and more preferably a methylene. In one embodiment, L 3 is a single bond or C 1―2 It is alkylene. 3 It is a single bond.

[0064] In equation (A-4), L 4 It is either a single bond or -NH-, -C(=O)-, -S-, -O-, alkylene, -(CH2CH2O) n - represents a divalent base selected from the group consisting of and any combination thereof, where n is an integer between 1 and 9 (preferably 1 to 4, more preferably 1 to 2).4 The number of carbon atoms in the alkylene group is 1 to 6, preferably 1 to 4, more preferably 1 to 2, and even more preferably 1, from the viewpoint of hydrophilicity. Among them, L 4 C is a single bond. 1―6 Alkylene, -(CH2CH2O) n -,-(CH2) m1 -(CH2CH2O) n -(CH2) m2 -, and -(CH2) m1 -O-(CH2CH2O) n -(CH2) m2 - Selected from. n is an integer between 1 and 9 (preferably 1 to 4, more preferably 1 to 2). m1 and m2 are each independently integers between 1 and 9 (preferably 1 to 4, more preferably 1 to 2). L 4 C in 1―6 From the viewpoint of hydrophilicity, alkylene is preferably C 1―4 It is an alkylene, and farC 1―2 It is an alkylene, and more preferably a methylene. In one embodiment, L 4 C 1―2 It is alkylene.

[0065] The modifying group (A) has -C(=O)R in the head portion. 1 It has an (aldehyde group or ketone group), and this -C(=O)R 1 It can react with a primary amino group to form a Schiff base. In this invention, -C(=O)R 1 This can form a hyperconjugated structure with an adjacent phenyl or pyridine ring, stabilizing the resulting Schiff base and potentially improving its binding stability with the amino group.

[0066] (polysaccharide) The polysaccharide is acidic, basic, or amphoteric, and is not particularly limited as long as it can be modified with the above-mentioned modifying group (A). It may be a polysaccharide extracted or isolated from natural plants or animals, a polysaccharide produced by genetically modified microorganisms, or a chemically synthesized polysaccharide.

[0067] Acidic polysaccharides are polysaccharides that have anionic functional groups (e.g., carboxyl groups, sulfate groups, phosphate groups, etc.) in their structure. Examples of acidic polysaccharides include polysaccharides containing uronic acid (e.g., guluronic acid, mannuronic acid, glucuronic acid, iduronic acid, galacturonic acid, etc.), polysaccharides having sulfate groups or phosphate groups in part of their structure, or polysaccharides having both structures. Acidic polysaccharides include not only polysaccharides that inherently have anionic functional groups in their structure, but also polysaccharides that do not inherently have anionic functional groups (e.g., neutral polysaccharides) in which anionic functional groups have been introduced, such as by substituting some or all of the hydrogen atoms of the hydroxyl groups with anionic functional groups such as carboxyalkyl groups. Specific examples of acidic polysaccharides include alginic acid, hyaluronic acid, carboxymethylcellulose, carboxymethyl dextran, carboxymethyl starch, heparin, heparan sulfate, chondroitin sulfate, dermantan sulfate, regenerated oxidized cellulose, pectic acid, gellan gum, gum arabic, xanthan gum, agar, agaropectin, carrageenan, and their derivatives or salts.

[0068] Basic polysaccharides are polysaccharides that have a cationic functional group (such as an amino group) in their structure. Basic polysaccharides include not only polysaccharides that inherently have a cationic functional group in their structure, but also polysaccharides that do not inherently have a cationic functional group (for example, neutral polysaccharides) into which a cationic functional group has been introduced. Examples of basic polysaccharides include chitosan, its derivatives, or salts thereof.

[0069] Amphoteric polysaccharides are polysaccharides that have both anionic functional groups (e.g., carboxyl groups, sulfate groups, phosphate groups, etc.) and cationic functional groups (e.g., amino groups, etc.) in their structure. Specific examples of amphoteric polysaccharides include anionic modified basic polysaccharides such as succinic acid-modified chitosan; deacetylated polysaccharides such as deacetylated hyaluronic acid; amine-modified acidic polysaccharides such as cationic-modified alginic acid and cationic-modified hyaluronic acid; phosphocholine-modified polysaccharides such as phosphocholine-modified hyaluronic acid; carbobetaine-modified polysaccharides; and sulfobetaine-modified polysaccharides.

[0070] By using acidic, basic, or amphoteric polysaccharides, even modifying groups (A) having hydrophobic benzene or pyridyl rings can be introduced with a good modification rate. Furthermore, even when hydrophobic modifying groups (A) are introduced with a high modification rate, the presence of unmodified acidic or basic functional groups (anionic or cationic functional groups) results in polysaccharide derivatives with excellent water solubility.

[0071] In some forms, the polysaccharide is selected from alginic acid, its derivatives or salts thereof; hyaluronic acid, its derivatives or salts thereof; carboxymethylcellulose, its derivatives or salts thereof; carboxymethyl dextran, its derivatives or salts thereof; carboxymethyl starch, its derivatives or salts thereof; heparin, its derivatives or salts thereof; heparan sulfate, its derivatives or salts thereof; chondroitin sulfate, its derivatives or salts thereof; dermantan sulfate, its derivatives or salts thereof; chitosan, its derivatives or salts thereof; regenerated oxidized cellulose, its derivatives or salts thereof; and pectinic acid, its derivatives or salts thereof.

[0072] In the present invention, the polysaccharide derivative may be a polysaccharide that has been subjected to any modification. Examples include polysaccharides that have been modified by esterification, maleimide modification, thiol modification, acrylate modification, aldehyde modification, disulfide modification (e.g., pyridyl disulfide), alkyne modification including cyclic alkynes, tetrazine modification, furan ring modification, etc. Methods for these modifications are known to those skilled in the art, and polysaccharides can be modified using conventionally known methods such as the carbodiimide reaction. For example, the method for maleimide modification is described in International Publication WO2019 / 189330. As a method for alkyne modification including cyclic alkynes, for example, Akira Takahashi et al., Biomacromolecules, 2013, 14(10), 3581-3588 can be used. For tetrazine modification, for example, the method described in VianneyDelplace et al., Nonswelling, Ultralow Content Inverse Electron‐Demand Diels-Alder Hyaluronan Hydrogels with Tunable Gelation Time: Synthesis and In Vitro Evaluation, Advanced Functional Materials, 2020, 30(14)) can be used. For furan ring modification, for example, the method described in Nimmo, Chelsea M. et al., BIOMACROMOLECULES, 2011, 12(3) 824-830; RSC Adv., 2018, 8, 11036-11042 can be used. By applying these modifications, it is possible to introduce a second drug (an additional drug of a different type) or to crosslink polysaccharide derivatives, by combining them with copper-free click reactions (e.g., reactions between cyclic alkynes and azides), reverse electron-demanded Diels-Alder reactions (e.g., reactions between tetrazine and cyclic alkenes), and Diels-Alder reactions (e.g., reactions between furan and maleimide).

[0073] In some forms, polysaccharides are acidic. Polysaccharides having anionic functional groups yield polysaccharide derivatives that exhibit particularly excellent water solubility, even when the modification rate of the modifying group (A) is high.

[0074] In some embodiments, the polysaccharide is a polysaccharide containing a carboxyl group. The carboxyl group-containing polysaccharide can form an amide bond through a reaction between the carboxyl group and an amino group, thereby introducing the above-mentioned modifying group (A) into the polysaccharide. In certain embodiments, the polysaccharide is a polysaccharide containing a carboxyl group, and the group represented by formula (A) is the group represented by formula (A-1), and the group represented by formula (A-1) is introduced into the polysaccharide by substituting the -OH of the carboxyl group of the polysaccharide, thereby forming an amide bond. By forming an amide bond, a polysaccharide derivative with superior stability can be obtained.

[0075] A polysaccharide containing a carboxyl group is acceptable as long as it has at least one unmodified carboxyl group. Polysaccharides having a carboxyl group include polysaccharides having a carboxyl group in their structure, or derivatives thereof or salts thereof (for example, alginic acid, its derivatives or salts thereof, hyaluronic acid, its derivatives or salts thereof, carboxymethylcellulose, its derivatives or salts thereof, carboxymethyl dextran, its derivatives or salts thereof, carboxymethyl starch, its derivatives or salts thereof, heparin, its derivatives or salts thereof, heparan sulfate, its derivatives or salts thereof, chondroitin sulfate, its derivatives or salts thereof, dermantane sulfate, its derivatives or salts thereof, pectinic acid, its derivatives or salts thereof, regenerated oxidized cellulose, its derivatives or salts thereof, gellan gum, its derivatives or salts thereof, gum arabic, its derivatives or salts thereof, xanthan gum, its derivatives or salts thereof, agarose, its derivatives or salts thereof, agaropectin, its derivatives or salts thereof, etc.; preferably alginic acid, its derivatives or salts thereof, hyaluronic acid, its derivatives or salts thereof, carboxymethylcellulose In addition to rose, its derivatives or salts thereof, carboxymethyl dextran, its derivatives or salts thereof, carboxymethyl starch, its derivatives or salts thereof, heparin, its derivatives or salts thereof, heparan sulfate, its derivatives or salts thereof, chondroitin sulfate, its derivatives or salts thereof, dermantan sulfate, its derivatives or salts thereof, pectin acid, its derivatives or salts thereof, regenerated oxidized cellulose, its derivatives or salts thereof), polysaccharides that do not have carboxyl groups (for example, chitosan, its derivatives or salts thereof, curdlan, its derivatives or salts thereof, agar, its derivatives or salts thereof, carrageenan, its derivatives or salts thereof, guar gum, its derivatives or salts thereof, roasted gum bean gum, its derivatives or salts thereof, tamarind seed gum, its derivatives or salts thereof; preferably chitosan, its derivatives or salts thereof, etc.), the term also includes polysaccharides that do not have carboxyl groups, in which carboxyl groups have been introduced by substituting some or all of the hydrogen atoms of the hydroxyl groups with carboxyalkyl groups.

[0076] In certain embodiments, the polysaccharide having a carboxyl group is selected from alginic acid or a salt thereof, hyaluronic acid or a salt thereof, carboxymethylcellulose or a salt thereof, carboxymethyl dextran or a salt thereof, carboxymethyl starch or a salt thereof, heparin or a salt thereof, heparan sulfate or a salt thereof, chondroitin sulfate or a salt thereof, dermantan sulfate or a salt thereof, pectinic acid or a salt thereof, regenerated oxidized cellulose or a salt thereof. In certain embodiments, the polysaccharide is selected from alginic acid, its derivatives or salts thereof, hyaluronic acid, its derivatives or salts thereof, carboxymethylcellulose, its derivatives or salts thereof, and carboxymethyl dextran, its derivatives or salts thereof. In certain embodiments, the polysaccharide is selected from alginic acid or its salts, hyaluronic acid or its salts, carboxymethylcellulose or its salts, and carboxymethyl dextran or its salts. In certain embodiments, the polysaccharide is alginic acid, its derivatives or salts thereof, or hyaluronic acid, its derivatives or salts thereof. In certain embodiments, the polysaccharide is alginic acid or a salt thereof, or hyaluronic acid or a salt thereof.

[0077] (Alginic acid derivatives) In one embodiment, the polysaccharide is alginic acid, its derivatives, or salts thereof (hereinafter also referred to as "alginic acid derivatives"). Alginic acid is a biodegradable and biocompatible polymer, a linear polymer formed by the polymerization of two types of uronic acids, D-mannuronic acid (M) and L-guluronic acid (G). More specifically, it is a block copolymer in which a homopolymer fraction of D-mannuronic acid (MM fraction), a homopolymer fraction of L-guluronic acid (GG fraction), and a fraction in which D-mannuronic acid and L-guluronic acid are randomly arranged (MG fraction) are arbitrarily linked. The composition ratio of D-mannuronic acid to L-guluronic acid (M / G ratio) of alginic acid varies mainly depending on the type of organism from which it is derived, such as seaweed, and is also influenced by the habitat and season of the organism, resulting in a wide range from a high-G type with an M / G ratio of approximately 0.2 to a high-M type with an M / G ratio of approximately 5.

[0078] Derivatives of alginic acid are not particularly limited and are alginic acid with any modifications or salts thereof. Any modifications to alginic acid include, for example, esterification, sulfation, maleimide modification, thiol modification, acrylate modification, aldehyde modification, and disulfide modification (e.g., pyridyl disulfide), and cationic modification (e.g., heparin-conjugated alginic acid). In one embodiment, derivatives of alginic acid include alginic acid esters, propylene glycol alginate, sulfated alginic acid with sulfated hydroxyl groups, maleimide-modified alginic acid, thiol-modified alginic acid, acrylate-modified alginic acid, and cationic-modified alginic acid (e.g., heparin-conjugated alginic acid as described in Japanese Patent Application Publication No. 2001-233786). These modifications to alginic acid can be carried out by known methods or similar methods.

[0079] Examples of alginic acid or its derivatives include metal salts of alginic acid or its derivatives. For example, the hydrogen atom (hydrogen ion) of the carboxylic acid at position 6 of alginic acid or its derivative may be replaced with a monovalent metal ion (e.g., Na). + Ya K +Examples include water-soluble salts produced by ion exchange with alkali metal ions (such as Mg). Specifically, examples include sodium alginate, potassium alginate, sodium salts of alginate derivatives, or potassium salts of alginate derivatives. In certain embodiments, the salt of alginic acid or its derivatives is sodium alginate or a sodium salt of an alginate derivative. A solution of a monovalent metal salt of alginic acid or its derivatives forms a gel when mixed with a crosslinking agent. Alternatively, the metal salt of alginic acid or its derivatives may exchange the hydrogen atom (hydrogen ion) of the carboxylic acid at the 6th position of alginic acid or its derivatives with a divalent metal ion (e.g., Mg 2+ Ca 2+ It may also be a salt (bridged material) produced by ion exchange with alkaline earth metal ions such as [other alkaline earth metal ions].

[0080] Alginic acid, initially extracted from brown algae, has a high molecular weight and relatively high viscosity. However, during processes such as heat drying, freeze-drying, and purification, its molecular weight decreases and its viscosity declines. Therefore, by appropriately controlling the temperature at each stage of production, alginic acid compounds with different molecular weights can be manufactured. Controlling the temperature at each stage of production to a lower level yields alginic acid compounds with higher molecular weights, while higher temperatures yield alginic acid compounds with lower molecular weights. Furthermore, alginic acid compounds with different molecular weights can also be manufactured by appropriately selecting the brown algae used as raw materials, or by performing molecular weight fractionation during the manufacturing process. In addition, after measuring the molecular weight or viscosity of the alginic acid compounds manufactured by each method, it is possible to obtain alginic acid compounds with the desired molecular weight by mixing them with alginic acid compounds from a different batch that have a different molecular weight or viscosity.

[0081] The viscosity of the alginic acid compounds used is not particularly limited, but when the viscosity is measured as an aqueous solution of 1 w / w% alginic acid compounds, it is preferably 10 mPa·s to 1000 mPa·s, and more preferably 50 mPa·s to 800 mPa·s.

[0082] The viscosity of aqueous solutions of alginates can be measured according to conventional methods. For example, it can be measured using a rotational viscometer, such as a coaxial double-cylinder rotational viscometer, a single-cylinder rotational viscometer (Brookfield type viscometer), or a cone-plate rotational viscometer. Preferably, it is desirable to follow the viscosity measurement method of the Japanese Pharmacopoeia (16th edition). More preferably, a cone-plate viscometer is used.

[0083] The position of the modifying group (A) introduced into the alginic acid derivatives is not particularly limited, but it is preferable that it be introduced at the position of the carboxyl group at position 6 of D-mannuronic acid (M) and L-guluronic acid (G) (uronic acid) that constitute the alginic acid derivatives. In certain embodiments, the group represented by formula (A-1) above is introduced into the polysaccharide by substituting the -OH group of the carboxyl group of alginic acid.

[0084] In one embodiment, the polysaccharide derivative includes constituent units represented by the following formulas (c11) and / or (c12). [ka] In equations (c11) and (c12), R 1 The definition and preferred embodiment of ring P is R in formula (A) above. 1 And it is the same as the definition and preferred embodiment of ring P. In equations (c11) and (c12), Y is -L 1 -NH- represents, and in this case L 1 It is bonded to ring P. L 1 The definition and preferred embodiment of L in formula (A-1) 1 This is identical to the definition and preferred embodiment of [the term]. In equations (c11) and (c12), R 11 , R 12 , R 13 , and R 14 These are, independently, hydrogen atoms and C 1-6 Alkyl and -C(=O)-C 1-6 Selected from alkyl. In one embodiment, R11 , R 12 , R 13 , and R 14 These are all hydrogen atoms.

[0085] (Hyaluronic acid derivatives) In one embodiment, the polysaccharide is hyaluronic acid, its derivatives, or salts thereof (hereinafter also referred to as "hyaluronic acid derivatives"). Hyaluronic acid is a biodegradable and biocompatible polymer having a repeating structure in which the disaccharides D-glucuronic acid and N-acetyl-D-glucosamine are linked in a linear fashion.

[0086] Derivatives of hyaluronic acid are not particularly limited and are hyaluronic acid with any modifications or salts thereof. Any modifications to hyaluronic acid include, for example, esterification, sulfation, maleimide modification, thiol modification, acrylate modification, aldehyde modification, and disulfide modification (e.g., pyridyl disulfide). In one embodiment, the derivative of hyaluronic acid is hyaluronic acid ester, propylene glycol hyaluronate, sulfated hyaluronic acid with sulfated hydroxyl groups, maleimide-modified hyaluronic acid, thiol-modified hyaluronic acid, or acrylate-modified hyaluronic acid. These modifications to hyaluronic acid can be carried out by known or similar methods.

[0087] Examples of salts of hyaluronic acid or its derivatives include metal salts of hyaluronic acid or its derivatives. For example, the hydrogen atom (hydrogen ion) of the carboxylic acid at position 6 of hyaluronic acid or its derivative may be replaced with a monovalent metal ion (e.g., Na). + Ya K +Examples include water-soluble salts produced by ion exchange with alkali metal ions (such as Mg). Specifically, examples include sodium hyaluronate, potassium hyaluronate, sodium salts of hyaluronic acid derivatives, or potassium salts of hyaluronic acid derivatives. In certain embodiments, the salt of hyaluronic acid or its derivatives is sodium hyaluronate or a sodium salt of a hyaluronic acid derivative. A solution of a monovalent metal salt of hyaluronic acid or its derivatives forms a gel when mixed with a crosslinking agent. Alternatively, the metal salt of hyaluronic acid or its derivatives may exchange the hydrogen atom (hydrogen ion) of the carboxylic acid at position 6 of hyaluronic acid or its derivatives with a divalent metal ion (e.g., Mg 2+ Ca 2+ It may also be a salt (bridged material) produced by ion exchange with alkaline earth metal ions such as [examples of alkaline earth metal ions].

[0088] The position of the modifying group (A) introduced in hyaluronic acid derivatives is not particularly limited, but it is preferable that it be introduced at the position of the carboxyl group of D-glucuronic acid, which constitutes hyaluronic acid. In certain embodiments, the group represented by formula (A-1) above is introduced into the polysaccharide by substituting the -OH group of the carboxyl group of hyaluronic acid.

[0089] In one embodiment, the polysaccharide derivative includes a constituent unit represented by the following formula (c13). [ka] In equation (c13), R 1 The definition and preferred embodiment of ring P is R in formula (A) above. 1 And it is the same as the definition and preferred embodiment of ring P. In equation (c13), Y is -L 1 -NH- represents, and in this case L 1 It is bonded to ring P. L 1 The definition and preferred embodiment of L in formula (A-1) 1 This is identical to the definition and preferred embodiment of [the term]. In equation (c13), R 21 , R22 , R 23 , and R 24 These are, independently, hydrogen atoms and C 1-6 Alkyl and -C(=O)-C 1-6 Selected from alkyl. In one embodiment, R 21 , R 22 , R 23 , and R 24 These are all hydrogen atoms.

[0090] (Carboxymethylcellulose) In one embodiment, the polysaccharide is carboxymethylcellulose, its derivatives, or salts thereof (hereinafter also referred to as "carboxymethylcelluloses"). Carboxymethylcellulose is a cellulose derivative that has been solubilized by introducing carboxymethyl groups into cellulose, and it possesses excellent thickening, water absorption, and water retention properties.

[0091] Derivatives of carboxymethylcellulose are not particularly limited and are carboxymethylcellulose with any modifications or salts thereof. Any modifications to carboxymethylcellulose include, for example, esterification, sulfation, maleimide modification, thiol modification, acrylate modification, aldehyde modification, and disulfide modification (e.g., pyridyl disulfide). These modifications to carboxymethylcellulose can be carried out by known or equivalent methods.

[0092] Examples of salts of carboxymethylcellulose or its derivatives include metal salts of carboxymethylcellulose or its derivatives. For example, the hydrogen atoms (hydrogen ions) of the carboxylic acid contained in carboxymethylcellulose or its derivatives are replaced with monovalent metal ions (e.g., Na). + Ya K +Examples include water-soluble salts produced by ion exchange with alkali metal ions (such as Mg). Specifically, examples include sodium carboxymethylcellulose, potassium carboxymethylcellulose, sodium salts of carboxymethylcellulose derivatives, or potassium salts of carboxymethylcellulose derivatives. In certain embodiments, the salt of carboxymethylcellulose or its derivatives is sodium carboxymethylcellulose or sodium salt of a carboxymethylcellulose derivative. A solution of a monovalent metal salt of carboxymethylcellulose or its derivatives forms a gel when mixed with a crosslinking agent. Alternatively, the metal salt of carboxymethylcellulose or its derivatives can exchange the hydrogen atoms (hydrogen ions) of the carboxylic acid contained in carboxymethylcellulose or its derivatives with divalent metal ions (e.g., Mg 2+ Ca 2+ It may also be a salt (bridged material) produced by ion exchange with alkaline earth metal ions such as [examples of alkaline earth metal ions].

[0093] The position of the modifying group (A) introduced in carboxymethylcellulose is not particularly limited, but it is preferable that it be introduced at the position of a carboxyl group contained in carboxymethylcellulose. In certain embodiments, the group represented by formula (A-1) is introduced into the polysaccharide by substituting the -OH group of the carboxyl group of carboxymethylcellulose.

[0094] In one embodiment, the polysaccharide derivative includes a constituent unit represented by the following formula (c14). [ka] In equation (c14), R 31 , R 32 , and R 33 One to three of these represent a group represented by the following formula (i). [ka] In the above equation (i), R 1The definition and preferred embodiment of ring P is R in formula (A) above. 1 And it is the same as the definition and preferred embodiment of ring P. Furthermore, in equation (i) above, Y is -L 1 -NH- represents, and in this case L 1 It is bonded to ring P. L 1 The definition and preferred embodiment of L in formula (A-1) 1 This is identical to the definition and preferred embodiment of [the term]. R 31 , R 32 , and R 33 The remaining ones (substituents other than the group represented by formula (i)) are, independently, a hydrogen atom and a carbon atom. 1-6 Alkyl, -C(=O)-C 1-6 Selected from alkyl and -CH2COOH, preferably a hydrogen atom and -CH2COOH, more preferably a hydrogen atom. In one embodiment, R 31 The group is represented by the above formula (i), and R 32 and R 33 It is a hydrogen atom.

[0095] (Carboxymethyl dextran compounds) In one embodiment, the polysaccharide is carboxymethyl dextran, its derivatives, or salts thereof (hereinafter also referred to as "carboxymethyl dextrans"). Carboxymethyl dextran is the carboxymethyl ether of dextran.

[0096] Derivatives of carboxymethyl dextran are not particularly limited and are carboxymethyl dextran with any modifications, or salts thereof. Any modifications to carboxymethyl dextran include, for example, esterification, sulfation, maleimide modification, thiol modification, acrylate modification, aldehyde modification, and disulfide modification (e.g., pyridyl disulfide). These modifications to carboxymethyl dextran can be carried out by known or equivalent methods.

[0097] Examples of salts of carboxymethyl dextran or its derivatives include metal salts of carboxymethyl dextran or its derivatives. For example, the hydrogen atoms (hydrogen ions) of the carboxylic acid contained in carboxymethyl dextran or its derivatives are replaced with monovalent metal ions (e.g., Na). + Ya K + Examples include water-soluble salts produced by ion exchange with alkali metal ions (such as Mg). Specifically, examples include sodium carboxymethyl dextran, potassium carboxymethyl dextran, sodium salts of carboxymethyl dextran derivatives, or potassium salts of carboxymethyl dextran derivatives. In certain embodiments, the salt of carboxymethyl dextran or its derivatives is the sodium salt of carboxymethyl dextran or a carboxymethyl dextran derivative. A solution of a monovalent metal salt of carboxymethyl dextran or its derivatives forms a gel when mixed with a crosslinking agent. Alternatively, the metal salt of carboxymethyl dextran or its derivatives exchanges the hydrogen atoms (hydrogen ions) of the carboxylic acid contained in carboxymethyl dextran or its derivatives with divalent metal ions (e.g., Mg 2+ Ca 2+ It may also be a salt (bridged material) produced by ion exchange with alkaline earth metal ions such as [examples of alkaline earth metal ions].

[0098] The position of the modifying group (A) introduced in carboxymethyl dextran is not particularly limited, but it is preferable that it be introduced at the position of the carboxyl group contained in carboxymethyl dextran. In certain embodiments, the group represented by formula (A-1) is introduced into the polysaccharide by substituting the -OH group of the carboxyl group of carboxymethyl dextran.

[0099] In one embodiment, the polysaccharide derivative includes a constituent unit represented by the following formula (c15). [ka] In equation (c15), R 41, R 42 , and R 43 One to three of these represent a group represented by the following formula (i). [ka] In the above equation (i), R 1 The definition and preferred embodiment of ring P is R in formula (A) above. 1 And it is the same as the definition and preferred embodiment of ring P. Furthermore, in equation (i) above, Y is -L 1 -NH- represents, and in this case L 1 It is bonded to ring P. L 1 The definition and preferred embodiment of L in formula (A-1) 1 This is identical to the definition and preferred embodiment of [the term]. R 41 , R 42 , and R 43 The remaining ones (substituents other than the group represented by formula (i)) are, independently, a hydrogen atom and a carbon atom. 1-6 Alkyl, -C(=O)-C 1-6 Selected from alkyl and -CH2COOH, preferably a hydrogen atom and -CH2COOH, more preferably a hydrogen atom. In one embodiment, R 41 The group is represented by the above formula (i), and R 42 and R 43 It is a hydrogen atom.

[0100] The polysaccharide may be heparin, its derivatives or salts thereof, heparan sulfate, its derivatives or salts thereof, chondroitin sulfate, its derivatives or salts thereof, dermantane sulfate, its derivatives or salts thereof, regenerated oxidized cellulose, its derivatives or salts thereof, and pectinic acid, or its derivatives or salts thereof. In one embodiment of the polysaccharide derivative, the polysaccharide is heparin, its derivatives or salts thereof, heparan sulfate, its derivatives or salts thereof, chondroitin sulfate, its derivatives or salts thereof, dermantane sulfate, its derivatives or salts thereof, regenerated oxidized cellulose, its derivatives or salts thereof, and pectinic acid, or its derivatives or salts thereof, and the group represented by the above formula (A-1) is introduced by substituting the -OH of the carboxyl group contained in the polysaccharide.

[0101] In some embodiments, the polysaccharide is a polysaccharide containing an amino group. The amino group-containing polysaccharide can form an amide bond through a reaction between the amino group and a carboxyl group, thereby introducing the above-mentioned modifying group (A) into the polysaccharide. In a particular embodiment, the polysaccharide is a polysaccharide containing an amino group, and the group represented by formula (A) is the group represented by formula (A-2), and the group represented by formula (A-2) is introduced into the polysaccharide by substituting a hydrogen atom of the amino group of the polysaccharide to form an amide bond. By forming an amide bond, a polysaccharide with superior stability can be obtained. The term "polysaccharides containing amino groups" includes not only polysaccharides that inherently contain amino groups in their structure, their derivatives, or salts thereof (for example, chitosan, its derivatives, or salts thereof), but also polysaccharides that do not contain amino groups but have had amino groups introduced into a portion of them.

[0102] For example, the following are examples of polysaccharide derivatives in which a group represented by the above formula (A-2) is introduced into a polysaccharide having an amino group. [ka] In the above formula, R 1The definition and preferred embodiment of ring P is R in formula (A) above. 1 And it is the same as the definition and preferred embodiment of ring P. Furthermore, in the above formula, L 2 The definition and preferred embodiment of L in formula (A-2) 2 This is identical to the definition and preferred embodiment of [the term].

[0103] (Chitosan derivatives) The polysaccharide may be chitosan, its derivatives, or salts thereof (also called "chitosans"). Chitosan is a polysaccharide in which D-glucosamine units and N-acetyl-D-glucosamine units are linked by β-(1-4) glycosidic bonds. Chitosan can be obtained by partial deacetylation of the polysaccharide chitin. The degree of deacetylation is not particularly limited. In some embodiments, the chitosans are deacetylated to a degree greater than about 50% (more typically greater than about 75%). Derivatives of chitosan are not particularly limited and include chitosan with any modifications or salts thereof. Any modifications to chitosan include, for example, esterification, sulfation, maleimide modification, thiol modification, acrylate modification, aldehyde modification, and disulfide modification (e.g., pyridyl disulfide). Examples of chitosan derivatives include carboxymethyl chitosan, hydroxylbutyl chitin, N-acyl chitosan, O-acyl chitosan, N-alkyl chitosan, O-alkyl chitosan, N-alkylidene chitosan, O-sulfonyl chitosan, sulfated chitosan, phosphorylated chitosan, nitrated chitosan, alkali chitosan, or metal chelates with chitosan. These modifications to chitosan can be carried out by known or similar methods. Commercially available products can also be used.

[0104] Chitosan has a primary amino group. In one embodiment of the polysaccharide derivative, the polysaccharide is chitosan, a derivative thereof, or a salt thereof, and the group represented by formula (A-2) above is introduced by substituting a hydrogen atom of the amino group contained in the polysaccharide. The position of the modifying group (A) introduced into the chitosan is not particularly limited, but it is preferable that it be introduced at the position of the amino group of the D-glucosamine unit constituting the chitosan. In a particular embodiment, the group represented by formula (A-2) above is introduced into the polysaccharide by substituting a hydrogen atom of the amino group of the chitosan.

[0105] In one embodiment, the polysaccharide derivative includes a constituent unit represented by the following formula (c16). [ka] In equation (c16), R 1 The definition and preferred embodiment of ring P is R in formula (A) above. 1 And it is the same as the definition and preferred embodiment of ring P. In equation (c16), Y is -L 2 -C(=O)- represents, and in this case L 2 It is bonded to ring P. L 2 The definition and preferred embodiment of L in formula (A-2) 2 This is identical to the definition and preferred embodiment of [the term]. In equation (c16), R 81 and R 82 These are, independently, hydrogen atoms and C 1-6 Alkyl and -C(=O)-C 1-6 Selected from alkyl. In one embodiment, R 81 and R 82 These are all hydrogen atoms.

[0106] In a particular embodiment, the polysaccharide is a polysaccharide having an ethylenically double bond group. A polysaccharide containing an ethylenically double bond group can form a sulfide bond (-S-) through a reaction between the ethylenically double bond group and a thiol group (-SH), thereby introducing the modifying group (A) into the polysaccharide. In a particular embodiment, the polysaccharide is a polysaccharide containing an ethylenically double bond group, the group represented by formula (A) is the group represented by formula (A-3), and the group represented by formula (A) is introduced into the polysaccharide by reacting with the ethylenically double bond group of the polysaccharide.

[0107] Polysaccharides having an ethylenically double bond are not particularly limited, but examples include polysaccharides (e.g., alginic acid, hyaluronic acid, carboxymethylcellulose, carboxymethyl dextran, carboxymethyl starch, pectin, regenerated oxidized cellulose, chitosan) that have been modified with maleimide or acrylate, or salts thereof. For example, the following are examples of polysaccharide derivatives in which a group represented by the above formula (A-3) is introduced into a maleimide-modified polysaccharide. [ka] In the above formula, R 1 The definition and preferred embodiment of ring P is R in formula (A) above. 1 And it is the same as the definition and preferred embodiment of ring P. Furthermore, in the above formula, L 3 The definition and preferred embodiment of L in formula (A-3) 3 This is identical to the definition and preferred embodiment of [the term].

[0108] The polysaccharide derivative may have a structure in which a modifying group (A) is introduced at the position of the hydroxyl group contained in the polysaccharide. In certain embodiments, the group represented by formula (A-4) above is introduced by substituting a hydrogen atom of a hydroxyl group contained in the polysaccharide. In this embodiment, as shown below, the polysaccharide derivative has an ether bond formed by an oxygen atom derived from the hydroxyl group contained in the polysaccharide. [ka]

[0109] (Molecular weight and viscosity of polysaccharide derivatives) While it is generally difficult to precisely determine the molecular weight of high molecular weight polysaccharides, they typically have a weight-average molecular weight in the range of 1,000 to 10,000,000.

[0110] For example, the molecular weight of the polysaccharide derivative is preferably 1,000 to 5,000,000, and more preferably 1,000 to 3,000,000, as measured by gel filtration chromatography (GFC) with a weight-average molecular weight (Mw). The preferred molecular weight range for polysaccharide derivatives varies depending on the type of polysaccharides that make up the derivative (such as the presence or absence of degrading enzymes and inflammatory responses) and the intended use of the polysaccharide derivative. For example, when used in DDS applications, although it depends on the charge of the polysaccharide derivative, generally, the kidney's excretion limit is said to be 40,000 to 50,000. Therefore, polysaccharide derivatives composed of non-biodegradable polysaccharides (dextran, cellulose derivatives, alginic acid, their salts, or derivatives thereof) for which there are no enzymes to break them down in the body are generally preferred to have a weight-average molecular weight (Mw) in the range of 1,000 to 50,000 or 1,000 to 40,000. However, although alginate does not have a biodegrading enzyme in the body, it has very little immunogenicity, so it is possible to use alginate with a larger weight-average molecular weight (e.g., 1,000 to 5,000,000 or 1,000 to 2,000,000 Mw). Because hyaluronic acid has abundant differentiation enzymes in the body, it is possible to use hyaluronic acid with a large weight-average molecular weight (for example, less than 2,000,000 or less, or less than 5,000,000 Mw). However, hyaluronic acid with a low molecular weight (around 100,000) is known to induce inflammation, so hyaluronic acid with a molecular weight of 500,000 or more is preferred. Typically, when calculating the molecular weight of high-molecular-weight polysaccharides by gel filtration chromatography, a measurement error of 10-20% or more can occur. For example, a value of 400,000 may fluctuate within a range of 320,000-480,000, 500,000 within a range of 400,000-600,000, and 1,000,000 within a range of 800,000-1,200,000. Therefore, a suitable weight-average molecular weight range for polysaccharides is at least 1,000. Polysaccharides with excessively high molecular weights are difficult to manufacture and can cause problems such as excessive viscosity when dissolved in aqueous solution, reduced solubility, and difficulty in maintaining physical properties during long-term storage. For this reason, a weight-average molecular weight of 5,000,000 or less is preferable, and more preferably 3,000,000 or less. The weight-average molecular weight of a polysaccharide derivative to which modifying group (A) has been introduced will be higher than that of the polysaccharide before the introduction of modifying group (A). By appropriately selecting the molecular weight of the starting polysaccharide, a polysaccharide derivative with the desired molecular weight can be obtained.

[0111] Generally, polymeric substances derived from natural products do not have a single molecular weight, but rather are aggregates of molecules with various molecular weights. Therefore, they are measured as a molecular weight distribution with a certain range. A typical measurement method is gel filtration chromatography. Typical information about the molecular weight distribution obtained by gel filtration chromatography includes weight-average molecular weight (Mw), number-average molecular weight (Mn), and dispersion ratio (Mw / Mn). Weight-average molecular weight, which emphasizes the contribution of large molecular weight polymers to the average molecular weight, is expressed by the following formula. Mw = Σ(WiMi) / W = Σ(HiMi) / Σ(Hi) The number-average molecular weight is calculated by dividing the total weight of the polymer by the total number of polymers. Mn=W / ΣNi=Σ(MiNi) / ΣNi=Σ(Hi) / Σ(Hi / Mi) Here, W is the total weight of the polymer, Wi is the weight of the i-th polymer, Mi is the molecular weight at the i-th elution time, Ni is the number of molecules with molecular weight Mi, and Hi is the height at the i-th elution time.

[0112] It is known that the molecular weight of naturally derived polymers can vary depending on the measurement method (for example, hyaluronic acid: Chikako YOMOTA et.al., Bull. Natl. Health Sci., Vol.117, pp135-139 (1999), Chikako YOMOTA et.al., Bull. Natl. Inst. Health Sci., Vol.121, pp30-33 (2003)). Regarding the molecular weight measurement of alginates, there are literatures describing methods for calculation from intrinsic viscosity and methods using SEC-MALLS (Size Exclusion Chromatography with Multiple Angle Laser Light Scattering Detection) (ASTM F2064-00 (2006), published by ASTM International). Furthermore, the document recommends that when measuring molecular weight by size exclusion chromatography (=gel filtration chromatography), a multi-angle light scattering detector (MALS) should be used in conjunction with a calibration curve using pullulan as a standard substance (=measurement by SEC-MALS). SEC-MALS is described in ASTM F2065-16. There are also examples where molecular weight obtained by SEC-MALS is used as the standard value in alginate catalogs (FMC Biopolymer, PRONOVA). TM sodium alginates catalog). In this invention, when determining the molecular weight of a polysaccharide derivative, unless otherwise specified, the weight-average molecular weight calculated by gel filtration chromatography is used.

[0113] Typical conditions for gel filtration chromatography include using a calibration curve with pullulan as the standard substance. Preferably, pullulan molecules with molecular weights of at least 1.6 million, 788,000, 404,000, 212,000, and 112,000 are used as the standard substance. Other conditions such as the eluent (200 mM sodium nitrate solution) and column conditions can also be specified. For column conditions, it is preferable to use a polymethacrylate resin-based packing material and at least one to three columns with an exclusion limit molecular weight of 10 million or more. A typical column is TSKgel GMPW. x1 (Diameter 7.8mm x 300mm) and G2500PW XL (Diameter 7.8mm x 300mm) (Manufactured by Tosoh Corporation).

[0114] (qualification rate) The modification rate refers to the number of modifying groups (A) per monosaccharide unit contained in a polysaccharide derivative.

number

[0115] The modification rate of the modifying group (A) in polysaccharide derivatives can be in the range of, for example, 0.01 to 1, 0.02 to 0.6, 0.05 to 0.2, or 0.1 to 0.2. Such ranges of modification rates may be used when polysaccharide derivatives are used as drug transport carriers. In another embodiment, the modification rate of the modifying group (A) in the polysaccharide derivative can be in the range of, for example, 0.1 to 1, 0.1 to 0.8, or 0.15 to 0.6. Such a range of modification rates may be used when the polysaccharide derivative is used in hydrogel form, for example. In another embodiment, the modification rate of the modifying group (A) in the polysaccharide derivative can be in the range of, for example, 0.00001 to 0.01.

[0116] For example, if the polysaccharide is alginic acid, each monosaccharide unit has one carboxyl group. Therefore, when introducing a modifying group (A) to the carboxyl group, the modification rate of the modifying group (A) in the polysaccharide derivative is at most 1, and can be in the range of, for example, 0.01 to 1, 0.01 to 0.6, 0.05 to 0.2, or 0.00001 to 0.01. For example, if the polysaccharide is hyaluronic acid, since hyaluronic acid is composed of monosaccharide units having one carboxyl group (D-glucuronic acid) and monosaccharide units not having a carboxyl group (N-acetyl-D-glucosamine), the modification rate of the modifying group (A) in the polysaccharide derivative when introducing a modifying group (A) to the carboxyl group is a maximum of 0.5, and can be in the range of, for example, 0.01 to 0.5, 0.05 to 0.2, or 0.00001 to 0.01. For example, when the polysaccharide is a carboxymethylcellulose, it has 1 to 3 carboxyl groups in the monosaccharide constituent unit, and when a modifying group (A) is introduced to the carboxyl group, the modification rate of the modifying group (A) in the polysaccharide derivative is a maximum of 3 (when carboxymethyl substitution is made at all three positions), and can be in the range of, for example, 0.01 to 3, 0.05 to 1.5, 0.05 to 1, or 0.00001 to 0.01. For example, if the polysaccharide is a carboxymethyl dextran, it has 1 to 3 carboxyl groups in the monosaccharide constituent unit, and when a modifying group (A) is introduced to the carboxyl group, the modification rate of the modifying group (A) in the polysaccharide derivative is a maximum of 3 (when carboxymethyl substitution is made at all three positions), and can be in the range of, for example, 0.01 to 3, 0.05 to 1.5, or 0.05 to 1, or 0.00001 to 0.01. Modification rate 1 It can be calculated by 1H NMR measurement (D2O). Furthermore, the modification rate can be calculated using a colorimetric aldehyde assay. 1 Repair rates calculated by 1H NMR measurement and colorimetric aldehyde assay do not always perfectly match, and discrepancies may occur between the two. However, in this invention, 1 The repair rate calculated by either 1H NMR measurement or a colorimetric aldehyde assay must fall within the above range.

[0117] A spacer may be used when introducing the modifying group (A). Suitable spacers can be those commonly used in the field (e.g., spacers described in Greg T. Hermanson, Bioconjugate Techniques, Third Edition (2013)), and more specifically, include polyylene glycols, polyamides such as peptides, and hydrocarbons.

[0118] 2. Method for producing polysaccharide derivatives The method for producing polysaccharide derivatives is not particularly limited, but for example, a polysaccharide derivative can be produced by a method that includes reacting a compound (a) represented by the following formula (a) with a polysaccharide having a functional group that can react with the -X group of compound (a). This yields a polysaccharide derivative (C) in which a modifying group (A) (the group represented by formula (A) described above) corresponding to compound (a) is introduced into the polysaccharide. [ka]

[0119] In equation (a) above, X is selected according to Y in equation (C). Y is -L 1 -NH-(L at this point 1 If X is bonded to ring P, then X is -L 1 It is -NH2. Y is -L 2 -C(=O)-(L in this case) 2 If X is bonded to ring P, then X is -L 2 It is -C(=O)OH. Y is -L 3 -S-(L at this point) 3If X is bonded to ring P, then X is -L 3 -SH Y is -L 4 -(L at this point 4 If X is bonded to ring P, then X is -L 4 -R L And R L The halogen atom is selected from F, Cl, Br, or I. In the above formulas (a) and (C), rings P and R 1 Furthermore, the definition and preferred embodiment of Y in formula (C) are the same as the definition and preferred embodiment in formula (A) and formulas (A-1), (A-2), (A-3), and (A-4) described above.

[0120] In this method of production, the polysaccharide has acidic, basic, or both (amphoteric) functional groups (i.e., charged functional groups), which allows it to react with a compound (a) represented by formula (a) having a highly hydrophobic phenyl ring or pyridyl ring in a single step under mild and safe conditions to introduce a modifying group (A) into the polysaccharide.

[0121] In a particular embodiment, a method for producing a polysaccharide derivative represented by formula (C) includes reacting an acidic, basic, or amphoteric polysaccharide with compound (a) represented by formula (a) by adding compound (a) in an aqueous solvent, a polar solvent, or a higher alcohol to a solution containing the polysaccharide. This reaction allows for the introduction of a modifying group (A) into the polysaccharide with a high degree of modification.

[0122] Below, Y is -L 1 -NH-(L at this point 1 (is bonded to ring P), and X is -L 1 The method for producing polysaccharide derivatives will be explained using the case where Y is -NH2 as an example. 1 Even in cases other than -NH-, the polysaccharide derivative represented by formula (C) above can be produced by methods known in the literature.

[0123] One embodiment of the present invention relates to a method for producing a polysaccharide derivative represented by the following formula (C1). The polysaccharide derivative represented by the following formula (C1) can be obtained by condensing the carboxyl group of the polysaccharide with the amino group of compound (a1) represented by the following formula (a1). In this reaction, an amide bond is formed between the amino group of compound (a1) and the carboxyl group of the polysaccharide, and the polysaccharide derivative represented by the following formula (C1) is obtained in which a modifying group (A) (the group represented by formula (A-1) described above) corresponding to compound (a1) is introduced into the polysaccharide. [ka]

[0124] In the above formulas (a1) and (C1), rings P and R 1 , and also L in formula (C1) 1 The definition and preferred embodiment are the same as the definition and preferred embodiment in formulas (A) and (A-1) above.

[0125] The condensation reaction method is not particularly limited and can be carried out according to methods known from the literature, for example, according to methods described in "Experimental Chemistry Course, 5th Edition, Vol. 16, Synthesis of Organic Compounds IV, Carboxylic Acids and Derivatives, Esters, pp. 35-70, Acid Amides and Acid Imides, pp. 118-154, Amino Acids and Peptides, pp. 258-283, 2007, Maruzen." The reaction is usually carried out in a solvent.

[0126] In a particular embodiment, a method for producing a polysaccharide derivative represented by formula (C1) includes a condensation reaction between a polysaccharide containing a carboxyl group and a compound (a1) represented by the following formula (a1) in a solvent (e.g., an aqueous solvent). In one embodiment, the compound (a1) is dissolved or dispersed in an aqueous solvent, a polar solvent, or a higher alcohol solvent and added to a solution containing a polysaccharide to react with the polysaccharide. This allows the modifying group (A) to be introduced into the polysaccharide with a high modification rate. Compound (a1), represented by formula (a1), generally has low solubility in aqueous solvents. Therefore, by adding compound (a1) to an aqueous solvent containing a polysaccharide and reacting it with the polysaccharide under stirring, the reaction can be carried out with compound (a1) suspended. In one embodiment, compound (a1) is dissolved in a polar solvent (preferably DMSO) or a higher alcohol solvent, and the solution of compound (a1) is added to an aqueous solvent containing a polysaccharide. This increases the modification rate of compound (a1). It was surprising that the modification rate improved dramatically by a simple method of dissolving or dispersing compound (a1) in a specific solvent and adding it to the polysaccharide. Although the details of the factors contributing to the improved modification rate are unclear, the inventors speculate that an excess of carboxyl groups was present relative to compound (a1), and that the less soluble (hydrophobic) compound (a1) was consumed in the reaction under these conditions, thereby promoting the dissolution of the next compound (a1) and leading to an improved modification rate. In addition, they speculate that the reaction between the carboxyl group of the polysaccharide and the amino group of compound (a) proceeded more efficiently than the reaction between the amino group of compound (a) and the aldehyde group of compound (a) (Schiff base formation reaction), which also contributed to the improved modification rate. However, the manufacturing method of this embodiment is not limited to this mechanism.

[0127] Examples of polar solvents include N,N-dimethylformamide, dimethyl sulfoxide (DMSO), dioxane, and N-methyl-2-pyrrolidone (NMP). Examples of aqueous solvents include water, ether-based solvents such as tetrahydrofuran (THF) and 1,4-dioxane (to the extent that water and polysaccharides do not precipitate), alcohol-based solvents such as methanol, ethanol, and 2-propanol, and mixed solvents with solvents selected from polar solvents such as N,N-dimethylformamide, dimethyl sulfoxide (DMSO), dioxane, and N-methyl-2-pyrrolidone (NMP).

[0128] The condensation reaction between compound (a1) and the polysaccharide is preferably carried out under conditions of pH 5 to 10 (more preferably pH 5.5 to 8.5, and even more preferably pH 7.5 to 8.0). By adjusting the pH within this range while the reaction proceeds, the modification rate of the modifying group (A) can be further improved. For adjusting the pH range, it is preferable to use an aqueous sodium hydroxide solution or an aqueous hydrochloric acid solution. Alternatively, the reaction can be carried out using a buffer solution.

[0129] The condensation reaction between compound (a1) and polysaccharide is preferably carried out in the presence or absence of an inorganic base such as sodium bicarbonate or sodium carbonate, or an organic base such as triethylamine or pyridine, in the presence or absence of a condensing agent selected from 1,3-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC·HCl), benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent), bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl), 2-chloro-1,3-dimethylimidazolinium hexafluorophosphate (CIP), or 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMT-MM), etc. Such condensation reactions can be carried out, for example, in the range of 0°C to 50°C. The preferred temperature range is 15°C to 40°C. By carrying out the reaction at a temperature near room temperature, the reaction time can be shortened while suppressing the decrease in the molecular weight of the polysaccharide. Furthermore, by using such a temperature range, side reactions (Schiff base formation reaction) between the amino group of compound (a) and the aldehyde group of compound (a) can also be suppressed. To suppress the formation of by-products in the condensation reaction, additives such as 1-hydroxy-1H-benzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt) can be added.

[0130] After the reaction, the product may be purified by filtration and / or dialysis.

[0131] One embodiment of the present invention relates to a method for producing a polysaccharide derivative represented by the following formula (C2). In some embodiments, the method for producing the polysaccharide derivative represented by formula (C2) includes a condensation reaction between a polysaccharide containing an amino group and a compound (a2) represented by the following formula (a2). The polysaccharide derivative represented by the following formula (C2) can be obtained by condensing the amino group of the polysaccharide with the carboxyl group of compound (a2) represented by the following formula (a2). In this reaction, an amide bond is formed between the carboxyl group of compound (a2) and the amino group of the polysaccharide, and the polysaccharide derivative represented by the following formula (C2) is obtained in which a modifying group (A) (the group represented by formula (A-2) described above) corresponding to compound (a2) is introduced into the polysaccharide. [ka]

[0132] Rings P and R in the above formulas (a2) and (C2) 1 , and also L in equation (C2) 2 The definition and preferred embodiment are the same as the definition and preferred embodiment in formulas (A) and (A-2) above.

[0133] The method of the condensation reaction is not particularly limited and can be carried out using conventionally known methods, such as those using carbodiimide-based condensing agents. The condensation reaction between compound (a2) and polysaccharide is preferably carried out under conditions of pH 4 to 7 (more preferably pH 5 to 6). By adjusting the pH within this range while the reaction proceeds, precipitation of the reaction product can be suppressed and the reaction can be carried out efficiently. For adjusting the pH range, it is preferable to use an aqueous sodium hydroxide solution or an aqueous hydrochloric acid solution. Alternatively, the reaction can be carried out using a buffer solution. Furthermore, it is preferable to react compound (a2) with the polysaccharide in a molar ratio of carboxyl groups of compound (a2) to amino groups of the polysaccharide within the range of 0.8:1 to 1.2:1. In this case, precipitation of the reaction product can be suppressed, and the reaction can proceed efficiently.

[0134] 3. Polysaccharide derivative-drug conjugates The polysaccharide derivative may form a polysaccharide derivative-drug conjugate with a drug containing a primary amino group and the polysaccharide derivative in the form described above. Therefore, in some embodiments, the tissue adhesive material comprises a polysaccharide derivative-drug conjugate. The polysaccharide derivative can form a conjugate by forming a Schiff base with a drug having a primary amino group. Specifically, the aldehyde or ketone group (-CR) of the polysaccharide derivative 1 (=O) reacts with the primary amino group of the drug to form a Schiff base. Therefore, in this form of polysaccharide derivative-drug conjugate, a covalent bond is formed between the drug and the group represented by formula (A) (modifying group (A)) contained in the polysaccharide via a Schiff base, forming a structure represented by the following formula (D). [ka] In formula (D) above, "Drug" represents the drug portion excluding the primary amino group. In the above equation (D), R 1 The definitions and preferred embodiments of rings P and Y are as follows: R in formula (A) above 1 And it is the same as the definition and preferred embodiment of ring P.

[0135] (Drugs containing a primary amino group) The drug is not particularly limited as long as it has one or more primary amino groups in its molecule. The drug may be a synthetic product or a natural product. Examples include at least one selected from low molecular weight compounds, medium molecular weight compounds, peptides, nucleic acids, nucleic acid derivatives, aptamers, vitamins, monoamines, amino acids, polyamines, antibodies, fluorescent dyes, and contrast agents. The primary amino group may also be a hydrazide group (-C(=O)-NH-NH2). The hydrazide group may be an aldehyde group or ketone group (-CR) of a polysaccharide derivative. 1 It can react with (=O) to form a hydrazone bond (-C(=O)-NH-N=CH-). Hydrazone bonds are more stable than those of ordinary Schiff bases, and delayed sustained release can be expected.

[0136] Examples of low molecular weight compounds (e.g., molecular weight 500 or less) and medium molecular weight compounds (e.g., molecular weight 500 to 2000) having a primary amino group include doxorubicin, gemcitabine, aminosalicylic acid, pemetrexed, methotrexate, andronate, eribulin, memantine, remdesivir, ambicillin, amoxicillin, aztreonam, tigemonam, vancomycin, cephalosporin C, gentamicin, trimethoprim, sulfamethoxazole, oseltamivir, mexiletine, and mi Examples include dodrine, levodopa, tenofovir, darunavir, procaine, ethyl aminobenzoate, procainamide, fluvoxamine, milnacipran, baclofen, benserazide, carbidopa, droxidopa, gusperimus, ubenimex, fingolimod, amfenac, sulfamine, triamterene, methiciletine, amlodibin, azelnidipine, methyldopa, hydralazine, mosapride, sitagliptin phosphate hydrate, valacyclovir, acyclovir, and celecoxib. Peptides are not particularly limited as long as they have a primary amino group at the N-terminus or elsewhere. Examples of peptides include methionine enkephalin, leucine enkephalin, dynorphin A, β-endorphin, bacitracin, daptomycin, colistin, erkatonin, and oxytocin. The nucleic acid bases adenine, thymine, guanine, and cytosine have a primary amino group, and nucleic acids and nucleic acid derivatives composed of these nucleic acid bases also have a primary amino group and can be used in the present invention. Examples of nucleic acid derivatives include cytarabine, cladribine, and fludarabine. Nucleic acid (DNA / RNA) aptamers and peptide aptamers can be used as aptamers. Examples of vitamins containing a primary amino group include folic acid, vitamin B1 (thiamine), vitamin B6 (pyridoxamine), vitamin B12 (cyanocobalamin), and nicotinamide. Examples of monoamines containing a primary amino group include dopamine, norepinephrine, serotonin, histamine, thiamine, and octobamin. Examples of amino acids include various amino acids that have a primary amino group in their molecule. The amino acids may be natural amino acids or artificial amino acids such as tranexamic acid. Examples of polyamines include molecules that exist in living organisms and contain multiple primary amines, such as spermine, spermidine, and putrescine. Examples of fluorescent dyes include fluorescein-5-thiosemicarbazide (FTSC).

[0137] Drugs can be used individually or in combination of two or more types. The drug may be water-soluble or water-insoluble. The drug may be in salt form, hydrate form, or solvate form, as long as it contains a primary amino group.

[0138] (Drug adoption rate) The drug delivery rate of a modifying group (A) in a polysaccharide derivative-drug conjugate is not particularly limited, but is, for example, the percentage of modifying groups (A) introduced into the polysaccharide derivative that are bonded to a drug (Schiff base). The drug delivery rate is not particularly limited, but can be, for example, 1 to 100%.

[0139] (Slow-release characteristics) Aldehyde or ketone group (-CR) of polysaccharide derivatives 1 The Schiff base formed between (=O) and the primary amino group of the drug remains stable under neutral to basic pH conditions, but dissociates under low pH conditions, releasing the drug. In this specification, “low pH conditions” means a pH below 7, but typically refers to a pH between 3.5 and 7.0. In addition, some drug release will occur in neutral to alkaline (high pH) environments (e.g., pH 7.4 or higher), but this release will be slow. On the other hand, under low pH conditions, the rate of drug release will be rapid, resulting in the release of a large amount of drug. While physiological pH, such as that of blood, is around pH 7.4, inflammatory tissues and tumor tissues (pH 6.5-7.2), and the interiors of lysosomes and endosomes (pH 4.5-5.5) are known to have low pH environments. Since this form of polysaccharide derivative-drug conjugate can release drugs in response to low pH, for example, at pH 7.4, which corresponds to blood pH, it is possible to suppress drug release and stably retain the drug, while efficiently releasing the drug at target sites in low pH environments. [ka] In the above formula, "Drug" represents the drug portion excluding the primary amino group. In the above formula, R 1 The definitions and preferred embodiments of rings P and Y are as follows: R in formula (A) above 1 And it is the same as the definition and preferred embodiment of ring P.

[0140] (Manufacturing method) A further embodiment of the present invention relates to a method for producing a polysaccharide derivative-drug conjugate. The method of production in this embodiment involves mixing the above-mentioned polysaccharide derivative with a drug containing a primary amino group in a solvent. By mixing, the aldehyde group or ketone group (-CR) of the polysaccharide derivative is converted. 1 (=O) reacts with the amino group of the drug to form a Schiff base, yielding a polysaccharide derivative-drug conjugate. This method of production allows for the simple, one-pot, and stable production of polysaccharide derivative-drug conjugates under mild and safe reaction conditions in a single step. Since this method eliminates the need for drug modification before conjugate formation and does not require complex experimental equipment or catalysts for conjugate formation, it also enables the in-situ preparation of polysaccharide derivative-drug conjugates from polysaccharide derivatives and drugs.

[0141] The solvent is not particularly limited, and any solvent capable of dissolving the polysaccharide used should be appropriately selected. For example, due to their excellent solubility of polysaccharides, it is preferable to use an aqueous solvent or a polar solvent such as dimethyl sulfoxide (DMSO). Examples of aqueous solvents include water, ether-based solvents such as tetrahydrofuran (THF) and 1,4-dioxane (to the extent that water and polysaccharides do not precipitate), alcohol-based solvents such as methanol, ethanol, and 2-propanol, and polar solvents such as N,N-dimethylformamide and dimethyl sulfoxide (DMSO). The temperature during mixing is not particularly restricted, but it can be carried out in the range of 0°C to 50°C, for example. The preferred temperature range is 15°C to 40°C. By carrying out the reaction at a temperature close to room temperature, it is possible to achieve both an appropriate reaction rate and prevention of volume reduction.

[0142] The pH of the reaction between the polysaccharide derivative and the drug should be such that the drug dissolves without denaturation, but from the viewpoint of reaction rate, conditions of pH 4.0 to 11.0 (more preferably pH 6.0 to 9.0, and even more preferably pH 7.0 to 8.0) are preferred. By proceeding the reaction while adjusting the pH within this range, the rate of drug introduction to the modifying group (A) can be further improved. Hydrochloric acid or sodium hydroxide is preferably used to adjust the pH range. Furthermore, when the reaction between a polysaccharide derivative and a drug is carried out under light-shielding conditions, it is preferable to do so if the drug has poor photostability. The reaction time is not particularly limited, but is approximately 1 minute to 24 hours, preferably 10 minutes to 2 hours. The mixing ratio of the polysaccharide derivative to the drug is appropriately set according to the number of modifying groups (A) introduced into the polysaccharide derivative, the desired drug introduction rate, and the solubility of the resulting polysaccharide derivative-drug conjugate. For example, 0.001 to 10 moles, preferably 0.05 to 3 moles, and more preferably 0.1 to 1 mole are mixed with 1 mole of the modified groups (A) introduced into the polysaccharide derivative. The method of mixing the polysaccharide derivative and the drug is not particularly limited, as long as both components can be mixed uniformly.

[0143] The polysaccharide derivative-drug conjugate (reaction solution) obtained by mixing may be used as is without purification. After conjugate formation, purification treatments such as dialysis, salting out, gel filtration, ion exchange chromatography, and electrophoresis, or freeze-drying may be performed.

[0144] 4. Composition Further embodiments of the present invention relate to compositions comprising the polysaccharide derivative of the above embodiment or the polysaccharide derivative-drug conjugate of the above embodiment. In some embodiments, compositions are provided comprising a polysaccharide derivative of the above form or a polysaccharide derivative-drug conjugate of the above form and an aqueous solvent. In the composition, the weight ratio of the polysaccharide derivative or conjugate to the aqueous solvent is, for example, 0.0001:1 to 0.3:1, preferably 0.001:1 to 0.1:1. In some embodiments, compositions are provided comprising a polysaccharide derivative of the above form and a drug having a primary amino group. In the composition, the molar ratio of the polysaccharide derivative to the drug is, for example, a ratio of A to drug of 1:0.001 to 1:10, preferably 1:0.05 to 1:3, and more preferably 1:0.1 to 1:1. In some embodiments, compositions are provided comprising a polysaccharide derivative of the above form, a drug having a primary amino group, and an aqueous solvent. In the composition, the ratio of the total weight of the polysaccharide derivative and the drug to the weight of the aqueous solvent is, for example, 0.0001:1 to 0.3:1, preferably 0.001:1 to 0.1:1. In the composition, the molar ratio of the polysaccharide derivative to the drug is, for example, the ratio of A to the drug is in the range of 1:0.001 to 1:10, preferably 1:0.05 to 1:3, more preferably 1:0.1 to 1:1. In some embodiments, a composition is provided comprising a crosslinked structure of a polysaccharide derivative, as described later, and an aqueous solvent. In this composition, the weight ratio of the crosslinked structure to the aqueous solvent is, for example, 0.0001:1 to 0.3:1, preferably 0.001:1 to 0.1:1. In some embodiments, a composition is provided comprising a crosslinked structure of a polysaccharide derivative-drug conjugate (crosslinked structure-drug conjugate) described later and an aqueous solvent. In this composition, the weight ratio of the crosslinked structure to the aqueous solvent is, for example, 0.0001:1 to 0.3:1, preferably 0.001:1 to 0.1:1. As aqueous solvents, those listed in the sections on methods for producing polysaccharide derivatives and methods for producing conjugates can be used in the same way.

[0145] The polysaccharide derivative or polysaccharide derivative-drug conjugate of the present invention can be incorporated into a pharmaceutical composition with additives such as commonly known pharmaceutical carriers and diluents, and can be administered parenterally or orally to mammals, including humans, or non-mammals. Examples of mammals, though not limited to them, include humans, chimpanzees, apes, monkeys, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, and guinea pigs. Examples of non-mammals, though not limited to them, include birds, fish, and reptiles. One embodiment provides a pharmaceutical composition comprising a polysaccharide derivative of the above form, a drug, and a pharmaceutically acceptable additive. One embodiment provides a pharmaceutical composition comprising the polysaccharide derivative-drug conjugate of the above form and a pharmaceutically acceptable additive. Examples of pharmaceutically acceptable additives include excipients, fillers, bulking agents, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, preservatives, solubilizers, antiseptics, flavoring and odor-correcting agents, analgesics, stabilizers, and isotonic agents, which are commonly used in drug manufacturing. Pharmaceutical compositions can be prepared by conventional methods using these additives selected as appropriate.

[0146] The polysaccharide derivative or polysaccharide derivative-drug conjugate of the present invention can be combined with additives commonly used in the food industry (for example, at least one component such as various nutritional supplements (amino acids, vitamins, minerals, etc.), sugars, dairy products, sweeteners, flavorings, flavorings, antioxidants, preservatives, coloring agents, emulsifiers, pH adjusters, organic acids, buffers, fruit juices, etc.) to form a food composition.

[0147] The composition in the above form can be used as a tissue adhesive material or for manufacturing a tissue adhesive material.

[0148] 5.Crosslinked structure The polysaccharide derivative may form a crosslinked structure. One embodiment of the present invention relates to a crosslinked structure containing a polysaccharide derivative.

[0149] (1) Crosslinked structures of polysaccharide derivatives via crosslinking agents In some forms, a crosslinked structure is a crosslinked structure of a polysaccharide derivative or a crosslinked structure of a polysaccharide derivative-drug conjugate (also called a "crosslinked structure-drug conjugate"). A crosslinked structure of a polysaccharide derivative is formed when a polysaccharide derivative is crosslinked via crosslinking groups to form a three-dimensional network structure. A crosslinked structure of a polysaccharide derivative-drug conjugate (crosslinked structure-drug conjugate) is formed when a polysaccharide derivative-drug conjugate is crosslinked via crosslinking groups to form a three-dimensional network structure. A crosslinked structure of a polysaccharide derivative can be obtained by subjecting a polysaccharide derivative having crosslinking groups to a crosslinking reaction using a crosslinking agent. A crosslinked structure of a polysaccharide derivative-drug conjugate (crosslinked structure-drug conjugate) can typically be obtained by subjecting a polysaccharide derivative-drug conjugate having crosslinking groups to a crosslinking reaction using a crosslinking agent, or by first producing a crosslinked structure of a polysaccharide derivative and then linking the crosslinked structure and the drug via a Schiff base. Examples of crosslinking agents include metal ions; amino group-containing polymers and amino group-containing low molecular weight compounds containing two or more primary amino groups, hydrazide groups, or aminooxy groups; and compounds containing two or more aldehyde groups, such as glutaraldehyde.

[0150] For example, unmodified carboxyl groups can function as crosslinking groups and form crosslinked structures via crosslinking agents such as divalent metal ions. A crosslinked structure can be formed by reacting a composition containing a polysaccharide derivative or a polysaccharide derivative-drug conjugate containing unmodified carboxyl groups in a solution containing a crosslinking agent. The shape of these cross-linked structures is not particularly limited, but examples include tubular structures, fibrous structures, fibers, beads, gels, roughly spherical gels, capsules, sponges, sheets, films, and the like. In certain embodiments, gels, capsules, sponges, beads, fibers, tubes, sheets, or films are provided that include polysaccharide derivatives, polysaccharide derivative-drug conjugates, crosslinked structures, or crosslinked structure-drug conjugates in the forms described above.

[0151] Specifically, a crosslinked structure can be obtained by partially crosslinking a solution containing a polysaccharide derivative with unmodified carboxyl groups by dropping it into a solution containing divalent metal ions as a crosslinking agent. Alternatively, a crosslinked structure can be obtained by partially crosslinking a solution containing a polysaccharide derivative-drug conjugate with unmodified carboxyl groups by dropping it into a solution containing divalent metal ions. The cross-linked structures produced in this manner can take the form of, for example, capsules, beads, fibers, tubes, or films.

[0152] Alternatively, a crosslinked structure can be obtained by partially crosslinking a polysaccharide derivative containing unmodified carboxyl groups and a solution containing divalent metal ions as a crosslinking agent onto a substrate. The shape of a crosslinked structure produced by this method may be a gel (hydrogel).

[0153] By freeze-drying the gel, a sponge-like cross-linked structure can be obtained.

[0154] As a method for attaching a drug to a crosslinked structure via a Schiff base after the crosslinked structure has been formed, a method similar to that used for producing polysaccharide derivative-drug conjugates can be used. For example, a method can be used in which the crosslinked structure and a drug containing a primary amino group are mixed in a solvent after the crosslinked structure has been formed. By mixing, the aldehyde or ketone group (-CR) of the crosslinked structure is attached. 1 (=O) reacts with the amino group of the drug to form a Schiff base, yielding a cross-linked structure-drug conjugate.

[0155] A cross-linked polysaccharide derivative structure on which a drug is supported can also be obtained by chemically binding or physically attaching the drug to the cross-linked polysaccharide derivative structure. For example, a cross-linked polysaccharide derivative structure on which a drug is supported can be obtained by coating a solution containing a drug onto the cross-linked polysaccharide derivative structure, immersing the cross-linked polysaccharide derivative structure in the solution, or printing the drug onto the cross-linked polysaccharide derivative structure.

[0156] Examples of divalent metal ions used as crosslinking agents in the above crosslinking reaction include, for example, calcium ions, magnesium ions, barium ions, strontium ions, and zinc ions, with calcium ions being preferred. More specifically, divalent metal ion compounds such as CaCl2, MgCl2, CaSO4, ZnCl2, BaCl2, and SrCl2 (preferably CaCl2, CaSO4, ZnCl2, SrCl2, and BaCl2) can be used.

[0157] In addition to the above-mentioned divalent metal ions (divalent metal ion compounds), crosslinking agents include Fe 3+ Trivalent metal ion compounds such as FeCl3, or crosslinking reagents having 2 to 4 amino groups in the molecule may be used. Crosslinking reagents having 2 to 4 amino groups in the molecule include diaminoalkanes that may have a lysyl group (-COCH(NH2)-(CH2)4-NH2) on the nitrogen atom, i.e., diaminoalkanes and their derivatives in which the amino group is replaced by a lysyl group to form a lysylamino group. Specifically, examples include diaminoethane, diaminopropane, and N-(lysyl)-diaminoethane.

[0158] In addition to the above, if a drug has multiple primary amino groups, the drug itself, when introduced into a polysaccharide, may function as a crosslinking group. For example, a polysaccharide derivative can be crosslinked via a medium-sized compound (peptide) (e.g., bacitracin) having multiple primary amino groups to form a three-dimensional network structure. Furthermore, as will be described later, amino group-containing polymers and amino group-containing low molecular weight compounds containing two or more primary amino groups, hydrazide groups, or aminooxy groups can function as crosslinking agents and can be crosslinked by covalent bonding with the modifying group (A) of the polysaccharide derivative via a Schiff base. Furthermore, compounds containing two or more aldehyde groups, such as glutaraldehyde, can function as crosslinking agents and form crosslinked structures (crosslinked structures) via the hydroxyl groups of polysaccharide derivatives.

[0159] The amount of crosslinking agent used should be appropriately adjusted depending on the amount and molecular weight of the polysaccharide derivative used, and the type of polysaccharide constituting the polysaccharide derivative. For example, when calcium ions are used as the crosslinking agent, the calcium ion concentration of the solution containing calcium ions is not particularly limited, but for example, it can be 1 mM to 1 M, preferably 5 mM to 500 mM, and more preferably 10 mM to 300 mM. The degree of crosslinking can be adjusted by controlling the amount of crosslinking agent used.

[0160] (2) Crosslinked structures of polysaccharide derivatives and amino group-containing polymers / amino group-containing low molecular weight compounds In some forms, the crosslinked structure comprises a polysaccharide derivative and at least one amino group-containing polymer and an amino group-containing low molecular weight compound containing two or more primary amino groups, hydrazide groups, or aminooxy groups, and the crosslinking is achieved by covalent bonding via a Schiff base between the primary amino groups, hydrazide groups, or aminooxy groups contained in the amino group-containing polymer and amino group-containing low molecular weight compound and the group represented by formula (A) (modifying group (A)) contained in the polysaccharide derivative. That is, the amino group-containing polymer and amino group-containing low molecular weight compound function as crosslinking agents and form a crosslinked structure (crosslinked structure) via the crosslinking group of the polysaccharide derivative (e.g., the aldehyde group or carbonyl group of the modifying group (A)). Specifically, the aldehyde group or ketone group (-CR) of the modifying group (A) of the polysaccharide derivative 1 (=O) reacts with the primary amino group, hydrazide group, or aminooxy group of an amino group-containing polymer and an amino group-containing low molecular weight compound to form a Schiff base (-CR1 The hydrazide group is represented as -C(=O)-NH-NH2 and reacts with the aldehyde or ketone group of the modifying group (A) to form a hydrazone bond. The aminooxy group is represented as -O-NH2 and the terminal -NH2 reacts with the aldehyde or ketone group of the modifying group (A) to form an oxime bond. An amino group-containing polymer is a substance containing two or more primary amino groups, hydrazide groups, or aminooxy groups, with a molecular weight of 1,000 or more. In this specification, "amino group-containing polymer" also includes molecules with molecular weights of approximately 1,000 to 10,000, such as those commonly called oligomers. A low molecular weight compound containing amino groups refers to a substance with a molecular weight of less than 1,000 that contains two or more primary amino groups, hydrazide groups, or aminooxy groups. Amino group-containing polymers and amino group-containing low molecular weight compounds may be produced by synthesis or may be naturally occurring.

[0161] An amino group-containing polymer (also simply called an "amino group-containing polymer") containing a primary amino group, a hydrazide group, or an aminooxy group is not particularly limited as long as it contains a total of two or more of at least one of these groups. The primary amino group, hydrazide group, or aminooxy group may be present at the ends of the polymer, in the side chains of the polymer, or in pendant groups within the polymer.

[0162] Examples of amino group-containing polymers include polyamines; polyalkylene glycols substituted with amino groups, hydrazide groups, or aminooxy groups; polyallylamines; polyvinylamines; polyacrylamines; amino group-containing polysaccharides; amino group-containing proteins; and polyamino acids, with at least one selected from these. These may be commercially available products or synthesized using conventionally known methods.

[0163] The polyamine may be linear, branched, or dendritic. "Dendritic" means that the polyamine has a dendritic, hyperbranched form, and is a polymer having multiple arms of equal or unequal length. Polyamines are not particularly limited, but examples include linear, branched, or dendritic polyalkyleneimines and branched or dendritic polyetheramines.

[0164] Polyalkyleneimines refer to polymers that contain alkyleneimine structures as repeating units and have primary amino groups at their terminals. The alkylene and imine portions in the alkyleneimine structure may be substituted. Linear polyalkyleneimines contain alkyleneimine structures having secondary amino groups. Branched polyalkyleneimines and dendritic polyalkyleneimines contain alkyleneimine structures containing primary, secondary, or tertiary amino groups. Polyalkyleneimines are preferably those having lower (e.g., C1-C6, C1-C3) alkyleneimine structures, and specifically include, but are not limited to, polyethyleneimine and polypropyleneimine.

[0165] Branched or dendritic polyetheramines refer to polymers that contain alkylene oxide structures as repeating units and have primary amino groups at their ends. Examples include, but are not limited to, amino-terminated star-shaped polyethylene oxide, amino-terminated dendritic polyethylene oxide, amino-terminated comb-shaped polyethylene oxide, amino-terminated star-shaped polypropylene oxide, amino-terminated dendritic polypropylene oxide, amino-terminated comb-shaped polypropylene oxide, amino-terminated star-shaped polyethylene oxide-polypropylene oxide copolymer, amino-terminated dendritic polyethylene oxide-polypropylene oxide copolymer, and amino-terminated comb-shaped polyethylene oxide-polypropylene oxide copolymer. Examples of amino-terminated star-shaped polymers include polymers having 3, 4, 6, or 8 arms terminated with a primary amine. For example, amino-terminated star-shaped polyethylene glycols include star-shaped polyethylene glycols having 3, 4, 6, or 8 arms terminated with a primary amine (star-shaped PEGamines with 3, 4, 6, or 8 arms). However, they are not limited to these. As branched or dendritic polyetheramines, commercially available products such as polyoxyalkylene triamines sold by Huntsman LLC. (Houston, TX) under the trade name Jeffamine® triamine can be used.

[0166] Examples of polyalkylene glycols substituted with amino groups, hydrazide groups, or aminooxy groups include polyethylene glycol (PEG), polypropylene glycol, or polyethylene oxide-polypropylene oxide copolymer substituted with multiple (two or more) amino groups, hydrazide groups, or aminooxy groups.

[0167] Polyallylamine refers to a polymer containing an allylamine structure as a repeating unit. The allyl portion in the allylamine structure may be substituted. In addition to the repeating units having the allylamine structure, polyallylamine may also contain other repeating units as copolymer components. Polyvinylamine refers to a polymer containing a vinylamine structure as a repeating unit, where the vinyl portion of the vinylamine structure may be substituted. In addition to the repeating units having a vinylamine structure, polyvinylamine may also contain other repeating units as copolymer components. Polyacrylamine refers to a polymer containing an acrylic structure with a side chain containing a primary amino group, and the vinyl portion of the acrylic structure may be substituted. For example, an acrylic polymer containing an amine structure with a polyalkylene imine grafted onto the side chain can be used. In addition to repeating units having an acrylic structure with a side chain containing a primary amino group, polyacrylamine may also contain other repeating units as copolymer components. Commercially available products such as aminoethylated acrylic polymers sold under the trade name Polyment (registered trademark) (Nippon Shokubai Co., Ltd.) can be used.

[0168] Examples of amino group-containing polysaccharides include polysaccharides containing amino groups, such as chitosan. Alternatively, amino group-containing polysaccharides may be obtained by modifying polysaccharides that do not contain amino groups to introduce amino groups (amination polysaccharides). Examples of amination polysaccharides include those obtained by introducing amino groups into aminodextran. Examples of amino group-containing proteins include fibrinogen, albumin, gelatins, and collagens. Examples of polyamino acids (polypeptides) include polylysine, polyarginine, polyglutamic acid, and polyaspartic acid.

[0169] The lower limit of the weight-average molecular weight of an amino group-containing polymer is not particularly limited, but is generally 1.00 or higher. The upper limit of the weight-average molecular weight of an amino group-containing polymer is not particularly limited, but is generally 1,000,000 or lower.

[0170] The amino group-containing low molecular weight compound is not particularly limited as long as it contains a total of two or more of at least one of the following: a primary amino group, a hydrazide group, or an aminooxy group. Examples include hydrazide-based crosslinking agents such as adipic acid dihydrazide, sebacate acid dihydrazide, dodecanediohydrazide, and isophthalic acid dihydrazide.

[0171] The amino groups in an amino group-containing polymer or amino group-containing low molecular weight compound may form salts. Examples of salts of an amino group-containing polymer or amino group-containing low molecular weight compound include halide salts (e.g., hydrochloride), phosphates, phosphites, carbonates, bicarbonates, sulfates, bisulfites, hydroxides, nitrates, persulfates, sulfites, acetates, ascorbic acid, citrates, oxalates, succinates, tartrates, taurocholates, or cholates.

[0172] Figure 34 shows a schematic diagram of a crosslinked structure formed from a polysaccharide derivative (AL-ABA) and an amino group-containing polymer (DPI; polyethyleneimine) prepared in Example I-17 described later. As shown in Figure 34, the crosslinked structure has a crosslinked structure in which the primary amino group contained in polyethyleneimine, an amino group-containing polymer, and the benzaldehyde group contained in the polysaccharide derivative are covalently bonded via a Schiff base (-C=N-).

[0173] Crosslinked structures of polysaccharide derivatives and amino group-containing polymers or amino group-containing low molecular weight compounds are typically produced by mixing the polysaccharide derivative and the amino group-containing polymer or amino group-containing low molecular weight compound in a solvent. From a biocompatibility standpoint, aqueous solvents (e.g., water, physiological saline) are preferred as the solvent.

[0174] After obtaining a crosslinked structure of a polysaccharide derivative and an amino group-containing polymer or amino group-containing low molecular weight compound, the structure may be further crosslinked via the crosslinking groups (e.g., unmodified carboxyl groups) contained in the crosslinked structure using a crosslinking agent (e.g., divalent metal ions) different from the amino group-containing low molecular weight compound. Specifically, the polysaccharide derivative and the amino group-containing polymer or amino group-containing low molecular weight compound are mixed in a solvent, and then a solution containing divalent metal ions as a crosslinking agent is added dropwise to a solution containing the crosslinked structure; or, the polysaccharide derivative and the amino group-containing polymer or amino group-containing low molecular weight compound are mixed in a solvent, and then a solution containing the crosslinked structure is added dropwise to a solution containing divalent metal ions as a crosslinking agent to partially crosslink the structure and obtain the crosslinked structure. The type and amount of crosslinking agent used are not particularly limited, but the same type and amount of crosslinking agent can be used in the crosslinking reaction of the polysaccharide derivative described above.

[0175] The shape of the crosslinked structure between the polysaccharide derivative and the amino group-containing polymer or amino group-containing low molecular weight compound is not particularly limited, but examples include tubular structures, fibrous structures, fibers, beads, gels, approximately spherical gels, capsules, sponges, sheets, films, and the like. In one embodiment, the crosslinked structure of a polysaccharide derivative and an amino group-containing polymer or amino group-containing low molecular weight compound may be a gel (hydrogel). By freeze-drying the gel, a sponge-like crosslinked structure can be obtained.

[0176] 6.Tissue adhesive material In some embodiments, the tissue adhesive material comprises the polysaccharide derivatives, polysaccharide derivative-drug conjugates, crosslinked structures, and crosslinked structure-drug conjugates. In certain embodiments, the tissue adhesive material is a biotissue adhesive or sealant used to bond biological tissues.

[0177] In some embodiments, tissue-adhesive materials are provided that include the above-mentioned polysaccharide derivative or crosslinked structure. The above-mentioned polysaccharide derivative or crosslinked structure includes an aldehyde group or ketone group (-C(=O)R) of the modifying group (A) introduced thereto. 1 ) can adhere to biological tissues by interacting with the functional groups of those tissues. In certain embodiments, the polysaccharide derivative or crosslinked structure is a modified group (A) introduced therein, which is an aldehyde group or a ketone group (-C(=O)R 1 ) can adhere to biological tissue by reacting with primary amino groups in biological tissue to form Schiff bases.

[0178] In some embodiments, the tissue-adhesive material comprises a polysaccharide derivative-drug conjugate or a crosslinked structure-drug conjugate. Preferably, the polysaccharide derivative-drug conjugate or crosslinked structure-drug conjugate has a drug having a primary amino group attached to a portion of the modifying group (A), and has an unreacted modifying group (A) to which the drug is not attached. According to these embodiments, the drug is delivered to the adhesion site while the aldehyde group or ketone group (-C(=O)R of the unreacted modifying group (A) is 1 ) can adhere to biological tissue by reacting with primary amino groups in biological tissue to form Schiff bases.

[0179] The shape of the tissue adhesive material is not particularly limited, but examples include tubular, fibrous, fiber, beads, capsules, gel, approximately spherical gel, sponge, sheet, film, etc. Beads, capsules, gel or approximately spherical gel or sponge are preferred, capsules or hydrogel or sponge are more preferred, and hydrogel is even more preferred. Hydrogel can be useful as a tissue adhesive or sealant in medical applications where more rapid degradation is required, such as preventing undesirable tissue-to-tissue adhesions resulting from trauma or surgery.

[0180] There are no particular restrictions on the method of using tissue adhesive materials. In some embodiments, the tissue adhesive material is a tissue adhesive used to bond two biological tissues together. In some embodiments, two biological tissues are bonded together by applying the tissue adhesive containing the polysaccharide derivative or crosslinked structure to at least one adhesion site of biological tissue and bringing two or more sites into contact. In some embodiments, a tissue adhesive containing the polysaccharide derivative or crosslinking structure is applied to at least one adhesion site of biological tissue, then a crosslinking agent (e.g., a solution containing divalent metal ions) is applied to the adhesion site, and two or more sites are brought into contact to bond the two biological tissues (layering method). In some embodiments, a crosslinking agent (e.g., a solution containing divalent metal ions) is applied to at least one adhesion site of biological tissue, then a tissue adhesive containing the polysaccharide derivative or crosslinking structure is applied to the adhesion site, and two or more sites are brought into contact to bond the two biological tissues (layering method). In some forms, the tissue adhesive containing the polysaccharide derivative or crosslinking structure and the crosslinking agent (e.g., a solution containing divalent metal ions) are mixed, the mixture is applied to the biological tissue, and two or more sites are brought into contact to bond the two biological tissues (mixing method). In this mixing method, it is preferable to apply the mixture to the biological tissue immediately after mixing in order to obtain sufficient adhesive strength. By applying the crosslinking agent, a hydrogel can be formed or the degree of crosslinking of the hydrogel can be increased.

[0181] In some embodiments, the tissue adhesive material is a sealant used to seal air / fluid leaks in living tissue or to seal or fill small cavities or defects in living tissue. In some embodiments, a sealant containing the polysaccharide derivative or crosslinked structure is applied to biological tissue and allowed to stand to seal air / fluid leaks in the biological tissue, or to seal or fill small cavities or defects in the biological tissue. In some embodiments, a sealant containing the polysaccharide derivative or crosslinking structure is applied to biological tissue, and then a crosslinking agent (e.g., a solution containing divalent metal ions) is applied to the applied area and allowed to stand to seal air / fluid leaks in the biological tissue, or to seal or fill small cavities or defects in the biological tissue (layering method). In some embodiments, a crosslinking agent (e.g., a solution containing divalent metal ions) is applied to biological tissue, and then a sealant containing the polysaccharide derivative or crosslinking structure is applied to the applied area and allowed to stand to seal air / fluid leaks in the biological tissue, or to seal or fill small cavities or defects in the biological tissue (layering method). In some forms, a sealant containing the polysaccharide derivative or crosslinking structure and a crosslinking agent (e.g., a solution containing divalent metal ions) are mixed, the mixture is applied to biological tissue, and allowed to stand to seal air / fluid leaks in the biological tissue, or to seal or fill small cavities or defects in the biological tissue (mixing method). In this mixing method, it is preferable to apply the mixture to the biological tissue immediately after mixing in order to obtain sufficient adhesion and sealing ability. By applying the crosslinking agent, it is possible to form a hydrogel or increase the degree of crosslinking of the hydrogel. In some embodiments, this mounting agent may be combined with tissue fragments derived from biological tissue, such as skin flaps and periosteum. These tissue fragments combined with the mounting agent can be used by attaching them to the tissue.

[0182] In some embodiments, this encapsulant may be combined with biodegradable and bioindegradable nonwoven fabrics, sheets, and films, and can also be used to fix and adhere these nonwoven fabrics, sheets, and films. Examples of biodegradable nonwoven fabrics, sheets, and films include those made from polyglycolic acid, L-lactide-ε-caprolactone polymer, polylactic acid, glycolic acid / lactic acid polyester, and sodium alginate, while examples of bioindegradable nonwoven fabrics, sheets, and films include those made from polytetrafluoroethylene (PTFE). These nonwoven fabrics, sheets, and films combined with the encapsulant can be used by attaching them to tissue. By using polysaccharide derivatives in combination with nonwoven fabrics, sheets, films, etc., the mechanical strength of the polysaccharide derivative structure (such as hydrogels) is improved, making it possible to prevent rupture even at higher pressure levels. In some embodiments, the tissue-adhesive material further comprises a nonwoven fabric, sheet, or film. In certain embodiments, the tissue-adhesive material is a encapsulant, and the encapsulant comprises the polysaccharide derivative and a nonwoven fabric, sheet, or film. In one embodiment, the tissue-adhesive material is a encapsulant used for repairing dura mater damage, and the encapsulant comprises the polysaccharide derivative and a nonwoven fabric, sheet, or film. Commercially available nonwoven fabrics, sheets, or films may be used, such as Neovil® from Gunze Medical Japan Ltd (GMJ) or Vicryl® Mesh from Ethicon.

[0183] The method of use when used in combination with nonwoven fabrics, sheets, or films is not particularly limited, but for example, one can first place or attach the nonwoven fabric, sheet, or film onto the tissue (e.g., the site of tissue damage), then apply the tissue adhesive or sealant containing the polysaccharide derivative or crosslinked structure onto the nonwoven fabric, sheet, or film, or between the nonwoven fabric, sheet, or film and the tissue, and then apply a crosslinking agent (e.g., a solution containing divalent metal ions) to the applied area and leave it to stand. According to this method, the site of tissue damage can be easily and reliably repaired. Alternatively, the following methods may be used: applying a tissue adhesive or sealant containing the polysaccharide derivative or crosslinked structure onto the tissue (e.g., the site of tissue damage), then placing or attaching the nonwoven fabric, sheet, or film to the applied area, then applying a crosslinking agent (e.g., a solution containing divalent metal ions) and allowing it to stand; or applying a tissue adhesive or sealant containing the polysaccharide derivative or crosslinked structure onto the tissue (e.g., the site of tissue damage), then applying a crosslinking agent (e.g., a solution containing divalent metal ions), and then placing or attaching the nonwoven fabric, sheet, or film to the applied area before gelation occurs.

[0184] In certain embodiments, tissue adhesive materials (tissue adhesives, sealants) are applied to biological tissue in the form of an aqueous solution or dispersion. In certain embodiments, tissue adhesive materials (tissue adhesives, sealants) are applied to biological tissue in the form of gels, sponges, sheets, or films.

[0185] The tissue-adhesive material may further contain various additives depending on the intended application. These additives may include, for example, at least one selected from pH adjusters, antimicrobial agents, colorants, and surfactants.

[0186] As described above, in the first embodiment, the tissue adhesive material is preferably used for rigid, thick tissues and / or connective tissue, but it can also be used for a wide range of biological tissues other than rigid, thick tissues and connective tissue. The biological tissues to which the tissue adhesive material can be applied are not particularly limited and include, for example, the dura mater, skin, oral cavity, periodontal ligament, gums, alveolar bone, esophagus, stomach, small intestine, large intestine (rectum), bone, nerves, exons, cartilage, perichondrium, elastic cartilage (e.g., auricular cartilage, nasal cartilage, epiglottic cartilage), blood vessels, cornea, muscle, fascia, brain, prostate, breast, endometrium, lungs, spleen, liver, testes, ovaries, cervix, lymph nodes, bone marrow, and kidneys. Among these, the dura mater, perichondrium, periodontal ligament, elastic cartilage (e.g., auricular cartilage, nasal cartilage, epiglottic cartilage), skin, mucosa of the digestive tract (oral cavity, esophagus, stomach, small intestine, large intestine), and the submucosa of the digestive tract (sites of mucosal injury). In some embodiments, the biological tissue to which the tissue adhesive material is applied is a biological tissue in which amino groups are exposed on the surface. In such cases, the aldehyde group or ketone group (-C(=O)R of the modifying group (A) of the tissue adhesive material is used. 1 A covalent bond is formed between the amino group and the Schiff base, which can improve adhesion strength. Examples of biological tissues where the amino group is exposed on the surface include skin (especially areas where collagen is exposed after surgery), healthy mucosa, the submucosa of the digestive tract (areas of mucosal damage), dura mater, perichondrium, and periodontal ligament. Application to biological tissues such as the lungs, heart, pancreas, kidneys, liver, and bladder is also preferable.

[0187] There are no particular restrictions on the specific areas of biological tissue to which the material can be applied; areas where tissue adhesive materials have conventionally been used can be suitably adopted. Examples of such application areas include surgical suture sites, bleeding sites, fracture fragment fixation sites, peripheral nerve and microvessel anastomosis sites, tendon adhesion and tendon suturing sites, and organ wound adhesion sites. Tissue adhesive materials can be used for applications such as dural adhesion, nerve anastomosis, microvascular anastomosis, preservation of vascular sutures, closure of traumatic tympanic membrane defects, sealing of prosthetic vessels and preservation of suture sites, liver destruction, adhesion of hepatic and cholecystolectomy sites, wound treatment during partial nephrectomy, nephrode destruction and prostatectomy, preservation of intestinal anastomosis, adhesion of fissured skin flaps, closure of bone and cartilage wound cavities (especially in patients at risk of bleeding), cartilage and bone adhesion, reinforcement of tendon sutures, pleural adhesion in pneumothorax, alveolar closure after tooth extraction when bleeding is a concern, tonsillar closure using collagen fiber bundles (especially in patients at risk of bleeding), and free skin grafting in burn patients. As mentioned above, its use as a tissue adhesive is not particularly limited, but for example, in neurosurgery, it is used in surgery for brain injury, brain tumors, and intracerebral hemorrhage to repair the dura mater, the outermost layer of the meninges, that is, to repair and restore the dura mater after an incision, or as an adhesive when filling defects with artificial dura mater (dura mater repair and protection). In orthopedics, it is used in spinal and spinal cord surgery to repair and protect the dura mater in particular. In otolaryngology, it is also used as an adhesive when transplanting or repairing elastic cartilages such as auricular cartilage, nasal cartilage, and epiglottic cartilage. In addition, in gastrointestinal surgery, it is used for tissue adhesion at gastrointestinal anastomoses, tissue repair during anal fistula and anal formation, and to prevent pancreatic fistula and bile leakage during pancreatic and hepatectomy, as well as for protecting and repairing damaged areas of the gastrointestinal wall during endoscopic surgery. In respiratory surgery, it is used as a lung sealant (for lung tissue protection) to prevent air leakage during lung resection, for tissue adhesion such as tracheal cartilage, as well as for tissue protection during surgery in urological surgery, to prevent urine leakage and repair anastomoses by adhering to ureteral anastomose sites, and for tissue protection and repair during surgery in obstetrics and gynecology.

[0188] According to some embodiments, a kit is also provided which includes a tissue adhesive material used to be applied to and bond the biological tissue, and instructions for using the tissue adhesive material. According to some embodiments, a kit is also provided which includes a precursor of a tissue adhesive material used to be applied to and bond the biological tissue, and instructions for use of the tissue adhesive material. In one embodiment, the precursor of the tissue adhesive material includes an uncrosslinked polysaccharide derivative or a partially crosslinked crosslinked structure, or a conjugate thereof with a drug, and a crosslinking agent (e.g., a solution containing a divalent metal ion).

[0189] II. Second Form A second embodiment of the present invention is a crosslinked structure comprising (a) a polysaccharide derivative in which a group represented by the following formula (A) is introduced into an acidic, basic, or amphoteric polysaccharide, and (b) at least one of an amino group-containing polymer and an amino group-containing low molecular weight compound (hereinafter also referred to as "amino group-containing substance (b)") containing two or more primary amino groups, hydrazide groups, or aminooxy groups and a group represented by the following formula (x1) (hereinafter also referred to as "modifying group (x1)"), wherein the primary amino groups, hydrazide groups, or aminooxy groups contained in the amino group-containing polymer and the amino group-containing low molecular weight compound are crosslinked by covalent bonding via a Schiff base between the group represented by formula (A) contained in the polysaccharide derivative. [ka] (In formula (A), R 1 is a hydrogen atom or C 1-4 Represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or pyridine ring may be substituted with one or more substituents independently selected from a halogen atom, -CF3, -NO2, a carboxyl group, and -SO3H. Y is -NH-, -C(=O)-, -S-, -O-, C 1-6 Alkylene, -(CH2CH2O) n- represents a divalent base selected from the group consisting of , and any combination thereof, where n is an integer from 1 to 9. * indicates a linkage with a polysaccharide. [ka] In equation (x1), k is an integer between 2 and 5, preferably between 2 and 3. In formula (x1), L 5 L is a linker group. 5 It is either a single bond or -C(=O)-, -S-, -O-, alkylene, -(CH2CH2O) n - and a divalent base selected from the group consisting of any combination thereof, where n is an integer from 1 to 9. In some embodiments, L 5 is -C(=O)- or an alkylene having 1 to 5 carbon atoms. In one embodiment, L 5 This is -C(=O)-. In formula (x1), * represents a linkage with an amino group-containing polymer or an amino group-containing low molecular weight compound.

[0190] In the crosslinked structure, the aldehyde group or ketone group (-CR) of the modifying group (A) of the polysaccharide derivative 1 (=O)) reacts with the primary amino group, hydrazide group, or aminooxy group of the amino group-containing substance (b) to form a covalent bond (Schiff base (-CR) 1 It forms a hydrazone bond or oxime bond (=N-). In the crosslinked structure, modifying groups (x1) such as catechol (1,2-dihydroxybenzene) groups and pyrogallol (1,2,3-trihydroxybenzene) groups can form hydrogen bonds with hydrogen atoms on the tissue surface and promote adhesion. The modifying group (x1) in the crosslinked structure and the modifying group (A) in the unreacted polysaccharide derivative can function as functional groups for tissue adhesion and other purposes.

[0191] In the second embodiment, (a) as the polysaccharide derivative, those described in section "1. Polysaccharide Derivatives" of the first embodiment above can be used in the same manner.

[0192] In the second embodiment, the amino group-containing substance (b) can be a modified version of the "amino group-containing polymer" and "amino group-containing low molecular weight compound" from the first embodiment, to which a modifying group (x1) has been introduced.

[0193] There are no particular restrictions on the method of introducing the modifying group (x1) into the amino group-containing substance (b). For example, the modifying group (x1) can be introduced by reacting a precursor compound having a modifying group (x1) such as gallic acid and a carboxyl group with the amino group-containing substance (b) in a carbodiimide reaction, thereby bonding the carboxyl group to the amino group of the amino group-containing substance (b).

[0194] In some embodiments, the amino group-containing substance (b) is a polyalkylene imine modified with a modifying group (x1).

[0195] In the second embodiment, the ratio of the polysaccharide derivative to the amino group-containing substance (b) is not particularly limited. In some embodiments, the ratio of the polysaccharide derivative to the amino group-containing substance (b) is in the range of 1:1000 to 1000:1 (equivalent ratio) between the modifying group (A) of the polysaccharide derivative and the amino group of the amino group-containing substance (b). If the amino group of the amino group-containing substance (b) is highly reactive, it tends to react rapidly with the modifying group (A) (aldehyde group or ketone group) of the polysaccharide derivative to form a Schiff base. In this case, if the amount of the modifying group (A) of the polysaccharide derivative is less than the equivalent amount of the amino group of the amino group-containing substance (b), almost all of the modifying group (A) of the polysaccharide derivative will react with the amino group of the amino group-containing substance (b) to form a Schiff base, resulting in a small or no unreacted modifying group (A). From this perspective, when using the crosslinked structure of this form for applications such as tissue adhesion materials described later, it is preferable to combine the polysaccharide derivative and the amino group-containing substance (b) such that a sufficient amount of unreacted modifying group (A) remains to react with the amino groups of the tissue. For example, the ratio of the polysaccharide derivative to the amino group-containing substance (b) is in the range of 1:100 to 100:1 (equivalent ratio) between the modifying group (A) of the polysaccharide derivative and the amino group of the amino group-containing substance (b).

[0196] The method for producing the crosslinked structure and its form are the same as those described in "4. Crosslinked Structures" "(2) Crosslinked structure of polysaccharide derivative and amino group-containing polymer / amino group-containing low molecular weight compound" of the first form, except that the amino group-containing substance (b) has a modifying group (x1).

[0197] The second form of the crosslinked structure may be a crosslinked structure formed from a polysaccharide derivative-drug conjugate, which is obtained by pre-conjugating a drug onto a polysaccharide derivative, and an amino group-containing substance (b), similar to the first form. As the "polysaccharide derivative-drug conjugate," those described in section "3. Polysaccharide derivative-drug conjugate" of the first form above can be used in the same way. Several embodiments provide compositions comprising a second form of crosslinked structure. The form of the "composition" is the same as in section "4. Composition" of the first embodiment described above.

[0198] (Application) The applications of the second form of bridged structure are not particularly limited.

[0199] Crosslinked structures and crosslinked structure-drug conjugates and compositions can be preferably used as tissue adhesive materials for use in biological tissues. Embodiments of the "tissue adhesive material" (such as shape, method of use, and specific usage) are the same as those described in section "6. Tissue Adhesive Material" of the first embodiment above. The tissue adhesive material of the second embodiment can be applied to a wide range of biological tissues, similar to the first embodiment. That is, the tissue adhesive material of the second embodiment can be used for a wide range of biological tissues, including soft tissues in the body, as well as hard, thick tissues and / or connective tissue, as described in section "6. Tissue Adhesive Material" of the first embodiment above.

[0200] Furthermore, the above-mentioned crosslinked structures, crosslinked structure-drug conjugates, or compositions can be used in a wide range of fields, including food, medicine, cosmetics, daily necessities, textiles, and papermaking. Their shapes are not particularly limited and are selected according to the application. Examples include tubular structures, fibrous structures, fibers, beads, gels, approximately spherical gels, capsules, sponges, sheets, films, etc. In one embodiment, the shape of the crosslinked structure, crosslinked structure-drug conjugate, or composition may be a gel, sponge, film, or capsule.

[0201] Cross-linked structures can be used in a wide range of fields, including food, medicine, cosmetics, daily necessities, textiles, and papermaking, as an alternative to conventional polysaccharide materials.

[0202] The above-mentioned cross-linked structures, cross-linked structure-drug conjugates, or compositions are suitable for use as medical materials due to their excellent biodegradability and biocompatibility. When used as medical materials, examples include drug release devices, sutures, materials for hemostasis, adhesion, and adhesion prevention in the body, tissue adhesion materials, wound dressings, materials for preventing leakage of tissue fluid, lung sealants to prevent air leakage, cell culture substrates, cell transplantation substrates, regenerative engineering materials, antithrombotic materials, diagnostic agents, and dialysis carriers. Medical materials can be used on mammals or non-mammals. In particular, polysaccharide derivatives can bind to drugs having a primary amino group under neutral and high pH conditions and release them under low pH conditions (pH-responsive drug release), making them suitable for use as drug delivery carriers. One embodiment of the present invention provides a drug delivery device comprising the above-described crosslinked structure, crosslinked structure-drug conjugate, or composition containing these. The drug delivery device can be used as a means of selectively and efficiently introducing a drug supported on a polysaccharide derivative into a target tissue. Another embodiment provides a method for releasing a desired encapsulated drug in a target tissue, or a method for controlling the release.

[0203] Examples of cross-linked structures for use as medical materials include tubular, fibrous, fiber, beads, capsules, gels, approximately spherical gels, sponges, sheets, films, etc. Beads, capsules, gels, or approximately spherical gels and sponges are preferred, and capsules, hydrogels, and sponges are more preferred.

[0204] According to some embodiments, an anti-adhesion material is provided which includes the above-mentioned crosslinked structure and crosslinked structure-drug conjugate.

[0205] Cross-linked structures and drug conjugates exhibit excellent biocompatibility and can therefore be used as bioabsorbable materials and medical devices.

[0206] Since the cross-linked structure binds to drugs containing primary amino groups, it can be used as a separation material. Examples of separation materials include chromatographic carriers, nonwoven fabrics, and membrane materials.

[0207] Cross-linked structures and cross-linked structure-drug conjugates can be used as foods, supplements, and food additives. An example of this cross-linked structure is described, for example, in Example I-30, which will be discussed later.

[0208] III. Third Form A third embodiment of the present invention relates to a tissue-adhesive material comprising a polysaccharide derivative containing an amino group. Several embodiments of the present invention are tissue-adhesive materials comprising a polysaccharide derivative in which a group represented by the following formula (A-2) (hereinafter also referred to as "modifying group (A-2)") is introduced into a polysaccharide containing an amino group, wherein the group represented by formula (A-2) is introduced into the polysaccharide by substituting a hydrogen atom of the amino group of the polysaccharide to form an amide bond. In some embodiments, the third form of tissue-adhesive material is in gel form. [ka] (In equation (A-2), R1 is a hydrogen atom or C 1-4 Represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or pyridine ring may be substituted with one or more substituents independently selected from a halogen atom, -CF3, -NO2, a carboxyl group, and -SO3H. L 2 It is either a single bond or -NH-, -S-, -O-, C 1―6 Alkylene, -(CH2CH2O) n - Represents a divalent group selected from the group consisting of any combination thereof. n is an integer from 1 to 9. * indicates a linkage with a polysaccharide.

[0209] In the third embodiment, the "polysaccharide derivative containing an amino group" in the first embodiment can be used in the same manner as the "polysaccharide derivative containing an amino group" in the first embodiment. The "polysaccharide containing an amino group" and the "group represented by formula (A-2)" are also in the same form as in the first embodiment.

[0210] The third form of tissue-adhesive material exhibits good adhesion due to the modification group (A-2) and / or amino group of the polysaccharide derivative. Specifically, the aldehyde group or ketone group (-C(=O)R of the modification group (A-2) of the polysaccharide derivative. 1 The polysaccharide derivative exhibits good adhesion to tissue by forming a Schiff base between the amino group of the biological tissue and the aldehyde of the polysaccharide derivative. Furthermore, interactions such as electrostatic interactions or hydrogen bonding occur between the amino group of the polysaccharide derivative and the carboxyl group of the biological tissue, which can improve adhesion to tissue. In some embodiments, the amino group of the polysaccharide derivative is reacted with ammonium ions (NH₃) at low pH (e.g., pH 5 or less, or pH less than 5, or pH 1 to 5, pH 4 or less, or pH 1 to 4). 3+) results in electrostatic interactions with the carboxyl groups of biological tissue, which can improve adhesion to the tissue. In some embodiments, the amino groups of the polysaccharide derivatives interact with the hydroxyl and carboxyl groups of biological tissue, such as hydrogen bonding, at high pH levels (e.g., pH 5 or higher, or pH greater than 5, or pH 5-10, or pH 5-8, or pH 6-10, or pH 6-8), which can improve adhesion to the tissue. Thus, the third form of tissue adhesive material, by containing a polysaccharide derivative with amino groups, can exhibit excellent adhesion at a wide range of pH levels and can be applied to various parts of biological tissue.

[0211] The shape of the third form of tissue-adhesive material is not particularly limited and includes tubular, fibrous, fiber, bead, capsule, gel, approximately spherical gel, sponge, sheet, film, etc. In some embodiments, the tissue-adhering material is in gel form. (Gel form) The shape of the tissue-adhesive material in gel form is not particularly limited, but examples include tubes, fibers, discs, beads, sheets, and films. The fact that the tissue-adhesive material is in gel form can be confirmed, for example, by the vial inversion test described in Example V-2 later.

[0212] In some embodiments, the tissue-adhering material is self-crosslinking. In some embodiments, the amino group contained in the polysaccharide derivative and the group represented by formula (A-2) are crosslinked by forming a covalent bond via a Schiff base, thereby forming a crosslinked structure and exhibiting a gel morphology. In some embodiments, the tissue-adhering material has pH-responsive self-crosslinking (pH-responsive self-crosslinking). As shown in Figure 88(A), a polysaccharide derivative containing an amino group crosslinks with the aldehyde group or ketone group (-C(=O)R) of the group represented by formula (A-2) under high pH conditions. 1A covalent bond is formed between the two via a Schiff base, resulting in a gel state. On the other hand, in polysaccharide derivatives containing amino groups, the amino groups in the polysaccharide derivative become ammonium ions (cations) at low pH, resulting in a solution state. In some embodiments, the tissue-adhering material exhibits a gel morphology when the polysaccharide derivative self-crosslinks under a pH of 5 or higher (e.g., pH 5-10 or pH 5-8). The pH of tissue-adhesive materials can be adjusted using an acid (e.g., hydrochloric acid) or an alkali (e.g., sodium hydroxide). By adjusting the pH of a solution containing a polysaccharide derivative to, for example, a pH of 5 or higher (e.g., pH 5-10, or pH 5-8), a hydrogel of the polysaccharide derivative can be obtained, and a tissue-adhesive material containing a hydrogel of the polysaccharide derivative can be obtained. In some embodiments, the third form of tissue-adhesive material can be produced by a method similar to the "Method for producing a polysaccharide derivative represented by formula (C2)" in "2. Method for producing a polysaccharide derivative" above. Specifically, the third form of tissue-adhesive material can be produced by reacting a polysaccharide containing an amino group with compound (a2) represented by formula (a2) to obtain a polysaccharide derivative represented by the following formula (C2) in which a group represented by formula (A-2) is introduced into the polysaccharide, and then gelling the mixture by adjusting the pH to, for example, 5 or higher (for example, pH 5 to 10, or pH 5 to 8).

[0213] In some embodiments, the tissue-adhering material has a structure (crosslinked structure) formed by crosslinking polysaccharide derivatives via crosslinking groups (e.g., carboxyl groups, aldehyde groups or carbonyl groups, amino groups, hydroxyl groups, etc.) using a crosslinking agent (e.g., divalent metal ions such as calcium ions, magnesium ions, barium ions, strontium ions, zinc ions; amino group-containing polymers and amino group-containing low molecular weight compounds containing two or more primary amino groups, hydrazide groups, or aminooxy groups; compounds containing two or more aldehyde groups such as glutaraldehyde, etc.), and exhibits a gel form.

[0214] In some embodiments, the tissue-adhering material is measured at a shear rate of 0.001 s at 25°C. -1 The viscosity of the composition in (η 0.001 ) preferably 10 9 ~1 mPa·s, fua10 9 ~10 3 mPa·s, more preferably 10 9 ~10 4 This is within the range of mPa·s. In some embodiments, the tissue-adhering materials were measured at 25°C with a shear rate of 1000 s. -1 The viscosity of the composition in (η 1000 ) is preferably 0.1 to 10 4 mPa·s, comfort level 1-10 3 The range is mPa·s, more preferably 1 to 100 mPa·s. The tissue-adhering materials of several embodiments were measured at 25°C with a shear rate of 0.001 s². -1 viscosity (η) 0.001 ) and shear rate 1000s -1 viscosity (η) 1000 ) ratio (η 0.001 / η 1000 ) is preferably 10 or more, more preferably 100 or more, and even more preferably 10 4 The above, particularly preferably 10 5 That's all. 0.001 / η 1000 The higher the value, the better, and there is no particular upper limit, but for example, 10 9 The following applies: η 0.001 / η 1000 For example, preferably 10 to 10 9 comfort level 100-10 9 , more preferably 10 4 ~10 9 , particularly preferably 10 5 ~10 9 It is within the range. Shear rate 0.001s -1 viscosity (η) 0.001 ) and shear rate 1000s -1 viscosity (η) 1000 ) ratio (η 0.001 / η 1000 Tissue adhesive materials whose properties fall within the above range have high shear-thinning properties, which has the advantage of reducing injection resistance, for example, with catheters and injection needles.

[0215] In the third form of tissue-adhesive material, as in the first form, the polysaccharide derivative may be a polysaccharide derivative-drug conjugate formed by conjugating a drug with the polysaccharide derivative. As the "polysaccharide derivative-drug conjugate," those described in section "3. Polysaccharide derivative-drug conjugate" of the first form above can be used in the same way.

[0216] (tissue adhesive material) A third form of tissue adhesive material is applied to the biological tissue The embodiments of the "tissue adhesive material" (such as shape, method of use, and specific usage) are the same as those described in section "6. Tissue Adhesive Material" of the first embodiment above. The tissue adhesive material of the third embodiment can be applied to a wide range of biological tissues. That is, the tissue adhesive material of the third embodiment can be used on a wide range of biological tissues, including soft tissues in living organisms, as well as hard, thick tissues and / or connective tissue, as described in section "6. Tissue Adhesive Material" of the first embodiment above.

[0217] A hydrogel (a composition in gel form) containing the above-mentioned polysaccharide derivative containing an amino group can also be used for applications other than tissue adhesion materials (for example, the wide range of applications described in the second form).

[0218] Furthermore, all publications cited herein, such as prior art documents, and published gazettes, patent gazettes, and other patent documents, are incorporated herein by reference.

[0219] Furthermore, the object, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description herein, and those skilled in the art will be able to easily implement the present invention from the description herein. The best mode for carrying out the invention and specific examples are provided for illustrative or explanatory purposes only and do not limit the present invention to them. It will be apparent to those skilled in the art that the intent and scope of the present invention disclosed herein can be modified in various ways based on the description herein. [Examples]

[0220] The present invention will be further described below with reference to examples and comparative examples, but the present invention is not limited to these examples. In this specification, "room temperature" generally refers to a temperature range of approximately 10°C to 35°C. Unless otherwise specified, "%" indicates a weight percentage. In this specification, the term "approximately" may mean ±10%. Unless otherwise specified, nuclear magnetic resonance spectra ( 1 For the 1H NMR (H NMR) measurements, an ALPHA FT-NMR spectrometer (500 MHz) JEOL JNM-A500 (JEOL) was used, along with the deuterated solvent D2O.

[0221] The abbreviations used in the examples are common abbreviations well known to those skilled in the art. Some of these abbreviations are listed below. AL: Alginate (sodium) Alg: Alginate (sodium) ABA: 4-aminobenzaldehyde AL-ABA: Benzaldehyde-modified alginate HOBt: 1-hydroxybenzotriazole WSCD / HCl,EDC·HCl:1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride THF: Tetrahydrofuran DMSO: Dimethyl sulfoxide NaCl: Sodium Chloride eq: equivalent Van: Vancomycin PBS: Phosphate Buffered Saline FTSC: Fluorescein-5-thiosemicarbazide FBS: Fetal Bovine Serum DMEM: Dulbecco's Modified Eagle Medium Bac: Bacitracin HA: Hyaluronic Acid PMX: Pemetrexed CMC: Carboxymethyl Cellulose DCC: N,N'-Dicyclohexylcarbodiimide CMD, CMDX: Carboxymethyl Dextran Mw: Weight Average Molecular Weight In the synthetic schemes and reaction formulas shown in the following examples, the polysaccharides (AL, HA, CMC, CMDX) are shown in the free form (the form with carboxyl groups), but the carboxyl groups (-COOH) in the polysaccharides (AL, HA, CMC, CMDX) shown in the synthetic schemes and reaction formulas can be in the ionized state (-COO - ) or in the salt form (-COOX). For example, in Example 1 below, sodium alginate, which is the sodium salt, is used as AL, and the carboxyl group (-COOH) in AL in Synthetic Scheme 1 can be in the ionized state (-COO - ) or in the sodium salt form (-COONa).

[0222] I. Alginate Derivatives 1. AL-ABA [Example I-1] Benzaldehyde-Modified Alginate (AL-ABA) <Synthesis of AL-ABA(1)> [Chemical Formula] In Scheme 1 above, for convenience, a reaction in which a modifying group derived from 4-aminobenzaldehyde (ABA) is introduced to the carboxyl group of the guluronic acid unit (the monosaccharide unit on the left) is shown. However, the ABA-derived modifying group may also be introduced to the carboxyl group of the mannuronic acid unit (the monosaccharide unit on the right). That is, in Scheme 1, AL-ABA has the ABA-derived modifying group introduced to both the carboxyl group of the guluronic acid unit (the monosaccharide unit on the left) and the carboxyl group of the mannuronic acid unit (the monosaccharide unit on the right).

[0223] According to the above synthesis scheme, AL-ABA(1) was synthesized by amidation reaction via carbodiimide following the steps (1)-(6) below. (Synthesis procedure) (1) 200 mg (1 mmol) of sodium alginate (AL) (IL-6G, manufactured by Kimika Co., Ltd., viscosity 50-80 mPa·s (1%), weight-average molecular weight: Mw = 750,000-850,000) was dissolved in 50 mL of pure water and stirred overnight to prepare an alginate solution. (2) Dissolve 242.9 mg (2 mmol, 1 eq) of ABA in 20 mL of THF and stir overnight to prepare an ABA solution. (3) 270.3 mg (2 mmol, 2 eq) of 1-hydroxybenzotriazole (HOBt) was dissolved in 10 mL of DMSO and added dropwise to the alginic acid solution prepared in (1) above. Then, 383.7 mg (2 mmol, 2 eq) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCD / HCl) was dissolved in 5 mL of distilled water and added dropwise to the alginic acid solution. (4) The ABA solution prepared in (2) above was added dropwise to the alginic acid solution obtained in (3) above. (5) The solution obtained in (4) above was adjusted to pH 5.5, stirred for 3-4 hours, and then diluted with 100 mL of distilled water. (6) The solution obtained in (5) above was filtered under reduced pressure and then dialyzed with NaCl for 2 days and with pure water for 2 days. After that, it was frozen with liquid nitrogen and freeze-dried for 3 days to obtain AL-ABA(1).

[0224] <1 1H NMR spectroscopy, UV-visible absorption spectroscopy (UV-vis) measurement, and FT-IR spectroscopy measurement. Regarding AL-ABA(1), 1 1H NMR spectroscopy, ultraviolet-visible absorption spectroscopy (UV-vis) spectroscopy, and Fourier transform infrared (FT-IR) spectroscopy were performed. The results are shown in Figures 1 to 3. 1 In the 1H NMR spectrum (Figure 1), peaks originating from ABA (peaks b and c originating from the benzene ring and peak d originating from the aldehyde) were observed in the modified AL-ABA. Furthermore, in UV-vis (Figure 2), characteristic peaks originating from ABA (peaks at approximately 235 nm and 330 nm) were observed in the modified AL-ABA, and in the FT-IR spectrum (Figure 3), absorption (approximately 1740 cm⁻¹) originating from the C=O amide bond site of ABA was observed in the modified AL-ABA. -1 A peak was observed. These results confirmed the synthesis of benzaldehyde-modified alginate (AL-ABA), in which the amino group of 4-aminobenzaldehyde and the carboxyl group of alginate are bonded. 1 From the 1H NMR spectrum, the ABA modification rate for the carboxyl group (-COOH) of alginic acid was calculated to be 0.069.

[0225] <Cytotoxicity evaluation of benzaldehyde-modified alginate (AL-ABA) (WST assay)> (Experimental procedure) MeT-5A (human mesothelial cell line), NIH / 3T3 (mouse embryonic fibroblasts), HUVEC (human umbilical vein endothelial cells), and RAW264.7 cells (mouse macrophage-like cell line) were seeded in 24-well plates and cultured for 24 hours. The culture medium was then replaced with medium containing AL-ABA and AL at different concentrations (0.01 mg / mL, 0.1 mg / mL, 1 mg / mL). Cell viability (%) was measured 48 hours after sample addition using the WST-8 assay (Cell Counting Kit-8, Dojindo). The AL-ABA used was AL-ABA(1) synthesized in Example I-1. (Result) The results are shown in Figure 4. AL-ABA showed a cell viability equivalent to that of AL. It was confirmed that AL-ABA has low cytotoxicity and high biocompatibility.

[0226] [Example I-2] Benzaldehyde-modified alginic acid (AL-ABA) AL-ABA(2) to AL-ABA(3) were synthesized by the alternative method described below.

[0227] <Synthesis of AL-ABA(2)> 1 g (0.0046 mol) of AL-500 (manufactured by Mochida Pharmaceutical Co., Ltd., sodium alginate, viscosity: 400 - 600 mPa·s) was dissolved in 300 ml of distilled water and stirred for 6 - 8 hours. 2.81 g (0.020 mol) of HOBt was dissolved in 10 ml of DMSO and added dropwise to the solution of AL-500. Subsequently, 3.97 g (0.020 mol) of WSCD / HCl was dissolved in 10 ml of distilled water and added dropwise to the solution of AL-500. The solution of AL-500 was further stirred for 10 minutes to obtain an alginic acid solution. 0.84 g (0.0039 mol) of 4-aminobenzaldehyde (ABA) in 10 ml of DMSO was added dropwise to the alginic acid solution. The pH of the reaction mixture was maintained at pH 7.5 with 1 N NaOH and stirred at room temperature for 16 - 20 hours. The reaction mixture was transferred to a 50 ml centrifuge tube and centrifuged at 3000 rpm for 10 minutes at room temperature, and the supernatant was collected. Then, using a dialysis tube (MWCO: 6 - 8 kDa) (Spectra / Pro (registered trademark)), the solution was dialyzed against deionized water for 72 hours and then lyophilized to obtain AL-ABA(2).

[0228] <Synthesis of AL-ABA(3)> AL-ABA(3) was obtained in the same manner as AL-ABA(2), except that the amount of HOBt used was changed to 1.04 g (0.007 mol), the amount of WSCD / HCl used was changed to 1.98 g (0.010 mol), and the pH of the reaction mixture was changed to 5.5.

[0229] < 11H NMR spectroscopy and FT-IR spectroscopy > Regarding AL-ABA(2) to AL-ABA(3), 1 1H NMR and FT-IR spectroscopy measurements were performed (not shown). These results confirmed the synthesis of ABA-modified alginate (AL-ABA), in which the amino group of ABA and the carboxyl group of alginate are bonded. Also, 1 The ABA modification rate of the carboxyl group (-COOH) of alginic acid in AL-ABA(2) to AL-ABA(3) was calculated from the 1H NMR spectra. The results, along with the reaction conditions for AL-ABA(2) to AL-ABA(3), are shown in Table 1 below.

[0230] <Quantification of aldehydes by colorimetric aldehyde assay> Using the colorimetric aldehyde assay kit, Blue (MAK140), the aldehydes in AL-ABA(2) and AL-ABA(3) were quantified, and the ABA modification rate of the carboxyl group (-COOH) of alginic acid was calculated. (Assay procedure) (1) Dilute 10 μl of 10 mM standard solution with 990 μl of assay buffer to prepare a 100 μM standard solution. Further dilute the 100 μM standard solution twofold with assay buffer. Add 50 μL of the diluted standard solution to a 96-well plate to generate 0 (blank), 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 μM standards. (2) Add 50 μL of Master Reaction Mix to each well. Mix thoroughly using a horizontal shaker or by pipetting, and incubate the reaction mixture at room temperature for 20-30 minutes. Protect the plate from light during incubation. (3) Add 50 μL of Blue Enhancer to each well. (4) Incubate the reaction mixture at room temperature for a further 20 minutes, protecting it from light. (5) After the incubation period, measure the absorbance at 620-660 nm. (result) The ABA modification rates for the carboxyl group (-COOH) of alginic acid, calculated from the aldehyde content, were 0.56 or 0.64 for AL-ABA(2) and 0.33 for AL-ABA(3). The results are shown in Table 1 below. [Table 1] In Table 1, DS represents the degree of substitution, which corresponds to the ABA modification rate converted to a percentage (ABA modification rate × 100). DS NMR is 1 The values ​​are calculated from the 1H NMR spectrum, while DS Colorimetric is calculated from the colorimetric aldehyde assay. *1 and *2 of AL-ABA(2) are AL-ABA prepared in different batches. In subsequent examples, *1 may be referred to as AL-ABA(2)(Low DS) and *2 as AL-ABA(2)(High DS).

[0231] 2.AL-AAP, AL-ADFBA, AL-APCA, AL-ANA Alginic acid modified with 4-aminoacetophenone (AAP), 4-amino-2,6-difluorobenzaldehyde (ADFBA), 2-amino-3-pyridinecarboxaldehyde (APCA), or 6-aminonicotinaldehyde (ANA) was obtained in the same manner as in Example I-1. In the following scheme, for convenience, the reaction in which the modifying group derived from AAP, ADFBA, APCA, or ANA is introduced to the carboxyl group of the guluronic acid unit (the monosaccharide unit on the left) is shown, but these modifying groups can also be introduced to the carboxyl group of the mannuronic acid unit (the monosaccharide unit on the right). That is, in AL-AAP, AL-ADFBA, AL-APCA, and AL-ANA, the modifying group derived from AAP, ADFBA, APCA, or ANA is introduced to both the carboxyl group of the guluronic acid unit (the monosaccharide unit on the left) and the carboxyl group of the mannuronic acid unit (the monosaccharide unit on the right).

[0232] [Example I-3] AAP-modified alginate (AL-AAP) [ka]

[0233] 0.5 g (0.0023 mol) of AL-500 (sodium alginate, viscosity: 400-600 mPa·s, manufactured by Mochida Pharmaceutical Co., Ltd.) was dissolved in 100 ml of distilled water and stirred for 6-8 hours. 1.4 g (0.010 mol) of HOBt was dissolved in 10 ml of DMSO and added dropwise to the AL-500 solution. Subsequently, 1.97 g (0.010 mol) of WSCD / HCl was dissolved in 10 ml of distilled water and added dropwise to the AL-500 solution. The AL-500 solution was stirred for a further 10 minutes to obtain an alginate solution. 0.310 g (0.0023 mol) of 4'-aminoacetophenone (AAP) in 10 ml of DMSO was added dropwise to the alginate solution. The pH of the reaction mixture was maintained at pH 5.5 with 1N NaOH and stirred at room temperature for 16-20 hours. The reaction mixture was transferred to a 50 ml centrifuge tube and centrifuged at 3000 rpm for 10 minutes at room temperature, and the supernatant was collected. The solution was then dialyzed against deionized water for 72 hours using a dialysis tube (MWCO: 6-8 kDa) (Spectra / Pro®), followed by lyophilization to obtain AL-AAP.

[0234] < 1 1H NMR spectroscopy measurement, FT-IR spectroscopy measurement > Regarding AL-AAP, 1 1H NMR and FT-IR spectroscopy measurements were performed. The results are shown in Figures 5 and 6. These results confirmed the synthesis of AAP-modified alginate (AL-AAP), in which the amino group of AAP is bonded to the carboxyl group of alginate. Also, 1 From the 1H NMR spectrum, the AAP modification rate for the carboxyl group (-COOH) of alginic acid was calculated to be 0.31. <Quantification of aldehydes by colorimetric aldehyde assay> The AAP modification rate of the carboxyl group (-COOH) of alginic acid, calculated from the amount of aldehyde measured using the colorimetric aldehyde assay kit, blue (MAK140), was 0.34.

[0235] [Example I-4] ADFBA-modified alginate (AL-ADFBA) [ka]

[0236] An alginate solution was obtained in the same manner as in Example I-3. 0.361 g (0.0023 mol) of 4'-4-amino-2,6-difluorobenzaldehyde (ADFBA) in 10 ml of DMSO was added dropwise to the alginate solution. The pH of the reaction mixture was maintained at pH 5.5 with 1N NaOH and stirred at room temperature for 16-20 hours. The reaction mixture was transferred to a 50 ml centrifuge tube and centrifuged at 3000 rpm for 10 minutes at room temperature, and the supernatant was collected. The solution was then dialyzed against deionized water for 72 hours using a dialysis tube (MWCO: 6-8 kDa) (Spectra / Pro®), and then freeze-dried to obtain AL-ADFBA.

[0237] < 1 1H NMR spectroscopy measurement, FT-IR spectroscopy measurement > Regarding AL-ADFBA, 1 1H NMR and FT-IR spectroscopy were performed. The results are shown in Figures 7 and 8. These results confirm the synthesis of ADFBA-modified alginate (AL-ADFBA), in which the amino group of ADFBA is bonded to the carboxyl group of alginate. Also, 1 From the 1H NMR spectrum, the ADFBA modification rate for the carboxyl group (-COOH) of alginic acid was calculated to be 0.12.

[0238] <Quantification of aldehydes by colorimetric aldehyde assay> The ADFBA modification rate for the carboxyl group (-COOH) of alginic acid, calculated from the amount of aldehyde measured using the colorimetric aldehyde assay kit, blue (MAK140), was 0.18.

[0239] [Example I-5] APCA-modified alginate (AL-APCA) [ka]

[0240] An alginate solution was obtained in the same manner as in Example I-3. 0.28 g (0.0023 mol) of 2-amino-3-pyridinecarboxaldehyde (APCA) in 10 ml of DMSO was added dropwise to the alginate solution. The pH of the reaction mixture was maintained at pH 5.5 with 1N NaOH and stirred at room temperature for 16-20 hours. The reaction mixture was transferred to a 50 ml centrifuge tube and centrifuged at 3000 rpm for 10 minutes at room temperature, and the supernatant was collected. The solution was then dialyzed against deionized water for 72 hours using a dialysis tube (MWCO: 6-8 kDa) (Spectra / Pro®), and then lyophilized to obtain AL-APCA.

[0241] < 1 1H NMR spectroscopy measurement, FT-IR spectroscopy measurement > Regarding AL-APCA, 1 1H NMR and FT-IR spectroscopy measurements were performed. The results are shown in Figures 9 and 10. These results confirmed the synthesis of APCA-modified alginate (AL-APCA), in which the amino group of APCA is bonded to the carboxyl group of alginate. Also, 1 From the 1H NMR spectrum, the APCA modification rate of the carboxyl group (-COOH) of alginic acid was calculated to be 0.41.

[0242] <Quantification of aldehydes by colorimetric aldehyde assay> The APCA modification rate of the carboxyl group (-COOH) of alginic acid, calculated from the amount of aldehyde measured using the colorimetric aldehyde assay kit, blue (MAK140), was 0.33.

[0243] [Example I-6] ANA-modified alginate (AL-ANA) [ka]

[0244] An alginate solution was obtained in the same manner as in Example I-3. 0.280 g (0.0023 mol) of 6-aminonicotinaldehyde (ANA) in 10 ml of DMSO was added dropwise to the alginate solution. The pH of the final reaction mixture was maintained at pH 5.5 with 1N NaOH and stirred at room temperature for 16-20 hours. The reaction mixture was transferred to a 50 ml centrifuge tube and centrifuged at 3000 rpm for 10 minutes at room temperature, and the supernatant was collected. The solution was then dialyzed against deionized water for 72 hours using a dialysis tube (MWCO: 6-8 kDa) (Spectra / Pro®), and then freeze-dried to obtain AL-ANA.

[0245] < 1 1H NMR spectroscopy measurement, FT-IR spectroscopy measurement > Regarding AL-ANA, 1 1H NMR and FT-IR spectroscopy were performed. The results are shown in Figures 11 and 12. These results confirm the synthesis of ANA-modified alginate (AL-ANA), in which the amino group of ANA is bonded to the carboxyl group of alginate. Also, 1 From the 1H NMR spectrum, the ANA modification rate of the carboxyl group (-COOH) of alginic acid was calculated to be 0.17.

[0246] <Quantification of aldehydes by colorimetric aldehyde assay> The ANA modification rate of the carboxyl group (-COOH) of alginic acid, calculated from the amount of aldehyde measured using the colorimetric aldehyde assay kit, blue (MAK140), was 0.26.

[0247] In the table below, 1 The modification rates of the modifying group (A), determined from 1H NMR spectra (NMR) and colorimetric aldehyde assays (Colorimetric), are summarized. [Table 2]

[0248] 3. Drug conjugate of AL-ABA [[ID=⑤]][Example I-7] Benzaldehyde-modified alginic acid-vancomycin conjugate (AL-ABA-Van) <Synthesis of AL-ABA-Van>

Chemical formula

[0249] Vancomycin (Van) is an antibiotic having a primary amino group. According to the above synthesis scheme, a conjugate (AL-ABA-Van) in which Van is bound to AL-ABA was synthesized by the following procedure through a Schiff base reaction between the aldehyde group of AL-ABA and the primary amino group of Van. Specifically, 50 mg of AL-ABA (1) obtained in Example I-1 was dissolved in 70 mL of pure water and stirred for 1 hour or more. 60 mg of vancomycin hydrochloride was dissolved in 60 mL of pure water and added to the AL-ABA solution. 0.1 M NaOH was added dropwise to adjust the pH to around 7.0, and the reaction was carried out with stirring overnight at room temperature under light-shielded conditions. After dilution with 150 mL of pure water, dialysis was performed with pure water for 2 days (apparatus: Spectra / Pro (registered trademark) 1 Dialysis Membrane Standard RC Tubing MWCO: 6-8 kD), and freeze-drying was carried out for 3 days to recover 86.8 mg of white AL-ABA-Van. (Yield: 78.9%)

[0250] < 1 1H NMR spectrum measurement, ultraviolet-visible light absorption spectrum (UV-vis) measurement, and FT-IR spectrum measurement> Regarding the obtained AL-ABA-Van 1 1H NMR spectrum measurement, ultraviolet-visible light absorption spectrum (UV-vis) measurement, and FT-IR spectrum measurement were carried out. The results are shown in FIGS. 13 to 15. 1In the 1H NMR spectrum (Figure 13), peaks originating from ABA and vancomycin (Van) were observed in AL-ABA-Van. Furthermore, in UV-vis (Figure 14), peaks originating from ABA and vancomycin (Van) (indicated by arrows in the figure; a shift to lower wavelengths was observed) were observed in AL-ABA-Van. In the FT-IR spectrum (Figure 15), absorption from the C=O amide bond site between AL and ABA was observed in AL-ABA-Van (approximately 1740 cm⁻¹). -1 A peak was observed. These results confirmed the formation of a conjugate between AL-ABA and Van. 1 From the 1H NMR spectrum, the conjugation rate of Van as a drug to the carboxyl group (-COOH) of alginate was 5.9%. This indicates that 82.4% of the ABA in AL-ABA reacted with Van. The results above demonstrate that conjugation is completed instantaneously simply by mixing AL-ABA and Van in an aqueous solvent. Furthermore, the reaction product is only water, eliminating the need for subsequent purification. Since this reaction can be applied to various primary amine-containing drugs, AL-ABA is expected to become a convenient and versatile platform for developing alginate-drug conjugates.

[0251] <Experiment on the release of Van from benzaldehyde-modified alginate-vancomycin conjugate (AL-ABA-Van)> (Experimental procedure) The release behavior of van from AL-ABA-Van solutions at different pH levels was investigated using a dialysis membrane (Pore size: 50kDa). Specifically, 1 mL of AL-ABA-Van solution, prepared by dissolving the AL-ABA-Van obtained in Example I-7 in pure water at a concentration of 3 mg / mL, was injected into a dialysis membrane cut to 9 cm, and both ends of the membrane were tied with string. This was placed in an 80 mL security container, 79 mL of PBS solution was added, and the mixture was stirred at maximum rotation speed using a 1 cm diameter stirrer tip in a 37°C constant temperature bath. 1.0 mL samples were taken from the solution in the security container at predetermined time points (1, 2, 3, 4, 6, 8, 12, 24, and 48 hours). The UV-vis spectrum of the sampled solution was measured, and the drug concentration in the solution was quantified from the absorbance, and the release rate of the drug van (Vancomycin Release %) was calculated. The above measurements were performed on PBS solutions at pH 5.0, 6.0, and 7.4 (n=4 for each pH sample).

[0252] For comparison, as a control experiment, instead of the AL-ABA-Van solution, the above release experiment was performed using a mixed solution of sodium alginate (AL) and vancomycin (Van) prepared by dissolving 3 mg / mL of sodium alginate (AL) (IL-6G, manufactured by Kimika Co., Ltd., viscosity 50-80 mPa·s (1%)) and 8.6 mg / mL of vancomycin (Van) in pure water (AL+Van), and a solution of 8.6 mg / mL of vancomycin (Van) dissolved in pure water (Van only).

[0253] (result) The results are shown in Figure 16. Figure 16A shows the release behavior of Van from the AL-ABA-Van solution (cumulative release rate of Van, %), Figure 16B shows the release behavior of Van from the AL+Van mixed solution (cumulative release rate of Van, %), and Figure 16C shows the release behavior of Van from the Van solution (cumulative release rate of Van, %). After 10 hours, in the AL-ABA-Van solution, over 80% of Van was released at pH 5.0, while slightly less than 50% remained at pH 7.4. This result indicates that Van is released more rapidly as the pH decreases. This is thought to be due to the fact that the imine bond between AL-ABA and Van is more easily dissociated at low pH. On the other hand, in the AL+Van solution and Van solution (control experiment), it was found that most of the Van was released within the first 10 hours. Furthermore, no difference in release behavior was observed depending on pH. From the above, it was shown that the AL-ABA-Van conjugate enables sustained release of Van compared to the control group (AL+Van, Van only), and furthermore, the drug is selectively released at low pH, and the Van release rate can be altered in a pH-dependent manner. [ka]

[0254] [Example I-8] FTSC-supported AL-ABA microcapsules (AL-ABA-FTSC capsules) (Capsule fabrication) [ka] Fluorescein-5-thiosemicarbazide (FTSC) is a fluorescent dye containing a primary amino group. AL-ABA capsules supported with FTSC were prepared. Specifically, AL-ABA(1) prepared in Example I-1 was dissolved in pure water to prepare a 2% by weight AL-ABA solution. This was added dropwise to an aqueous solution of 50 mM CaCl2 to obtain capsules (AL-ABA capsules). FTSC was dissolved in DMEM (Dulbecc's modified Eagle medium) containing physiological saline and 10% FBS (fetal bovine serum), and the obtained AL-ABA capsules were immersed in this solution to support the FTSC in the capsules by reaction diffusion. For comparison, a control experiment was conducted in the same manner as above, except that AL-ABA was replaced with sodium alginate (AL) (IL-6G, manufactured by Kimika Co., Ltd., viscosity 50-80 mPa·s (1%)). Capsules (AL capsules) were obtained. The obtained AL capsules were similarly immersed in physiological saline containing FTSC to confirm whether FTSC was supported.

[0255] (result) AL and AL-ABA capsules immersed in FTSC solution were observed using a confocal microscope. The obtained transmission image, fluorescence image (Amine-Fluorescein), and merged image are shown in Figure 17. Figure 17 shows that, similar to AL, microscale capsules were fabricated when using AL-ABA. Furthermore, while almost no fluorescence from FTSC was detected in the AL capsules, fluorescence from FTSC was clearly detected in the AL-ABA capsules, indicating that FTSC was supported in the capsule in situ. This suggests that benzaldehyde-modified alginate (AL-ABA), like alginate (AL), can support Ca 2+ It was confirmed that capsules could be easily fabricated while maintaining crosslinking ability, and that amine compounds could be supported simply by immersion.

[0256] [Example I-9] Van-supported AL-ABA microcapsules (AL-ABA-Van capsules) (Capsule fabrication) AL-ABA(1) prepared in Example I-1 was dissolved in pure water to prepare a 2% by weight AL-ABA solution. 15 mg of vancomycin (Van) was added to this to obtain a mixed solution of AL-ABA and Van. The mixed solution of AL-ABA and Van was added dropwise to an aqueous solution of 50 mM CaCl2 to obtain capsules (AL-ABA-Van capsules). For comparison, a control experiment was conducted in the same manner as above, except that AL-ABA was replaced with sodium alginate (AL) (IL-6G, manufactured by Kimika Co., Ltd., viscosity 50-80 mPa·s (1%)) to obtain capsules (AL-Van capsules).

[0257] (Result) Even when using a mixed solution of AL-ABA and Van, benzaldehyde-modified alginic acid (AL-ABA) retains the ability to crosslink with Ca 2+ and it was confirmed that capsules can be easily prepared, similar to alginic acid (AL).

[0258] ><Van Release Experiment from AL-ABA-Van Capsules> (Experimental Procedure) The AL-ABA-Van capsules prepared above were collected and added to a 10 mM CaCl2 solution in 1 mL of physiological saline. The external solution (sustained release solution) was collected at predetermined time points (3, 24, and 48 hours), and the total amount of the external solution was replaced with a 10 mM CaCl2 solution in fresh 1 mL of physiological saline. UV-vis spectrum measurement was performed on the external solution (sustained release solution) collected at predetermined time points, the drug concentration in the solution was quantified from the absorbance, and the release rate of the drug Van (Vancomycin Release %) was calculated.

[0259] For comparison, as a control experiment, the above release experiment was performed using AL-Van capsules instead of AL-ABA-Van capsules.

[0260] (Result) The results are shown in Fig. 18. Fig. 18A shows the cumulative release rate (%) of Van from each capsule, and Fig. 18B shows the transition of the release rate (%) of Van at each time point from each capsule. From Fig. 18A and Fig. 18B, it was confirmed that both AL-Van capsules and AL-ABA-Van capsules gradually released vancomycin, and about 60% of Van was released at the 48-hour time point. There was no significant difference between the two in the cumulative sustained release amount (Fig. 18A), which is probably because the rate of Van release from the microcapsules by diffusion was slower than the rate of Van liberation due to the dissociation of the Schiff base with ABA, making the effect of Schiff base-mediated loading less visible.

[0261] <Bacterial Growth Inhibition Experiment> To verify the antibacterial effect of vancomycin-loaded AL-ABA microcapsules, the sustained-release solutions collected at each time point in the Van release experiment from AL-ABA-Van capsules conducted above were used, and the growth inhibitory effect of each sustained-release solution was evaluated using a halo test with Staphylococcus aureus. The specific experimental procedure is as follows. (Experimental procedure) (1) Inoculation of Staphylococcus aureus onto agar plates 1.1 Agar plates were prepared by adding 15 g of agar to 1 L of Mueller-Hinton medium (BD), dissolving and sterilizing it by autoclaving, and then adding 15 mL to each 100 mm dish and cooling at room temperature. 1.2 700 μl of Mueller-Hinton medium was added to a 1.5 mL microtube (Azunol sterile tube), and a stock of Staphylococcus aureus was inoculated to it using a toothpick. The cultures were then incubated overnight in a 37°C incubator. 1.3 Bacterial solution 1.0 × 10 7 The solution was diluted to cells / ml, and 100 μl of it was added to an agar plate and spread evenly with a convex rod. (2) Impregnation of the sustained-release solution into the filter paper and administration onto the agar plate 2.1 One mL of each sustained-release solution collected in the release experiment was sterilized by filter using a syringe filter (0.22 μm). 2.2 A piece of filter paper (AS ONE: MFWG4780) was cut into a circle with a diameter of 16 mm. 2.3 Place the cut-out filter paper in the center of the agar plate inoculated with Staphylococcus aureus. 2.4 40 μl of sustained-release solution was uniformly dropped onto the filter paper and allowed to impregnate it. 2.5 The dish was transferred to an incubator and incubated at 37°C for 24 hours. 2.6 The dish was removed from the incubator, and the growth of the bacteria was photographed with a camera. Image analysis using Image J (provided by NIH) was performed to measure the area where growth was inhibited.

[0262] (result) The results of observing the growth of Staphylococcus aureus on agar medium under various conditions are shown in Fig. 19. For both Van-loaded AL capsules (AL-Van) and Van-loaded AL-ABA capsules (AL-ABA-Van), circular regions where no bacteria grew were observed around the filter paper, confirming the growth inhibitory effect. Furthermore, the area where growth was inhibited was calculated by image analysis, and the results of comparison for each condition are shown in Fig. 20. In the case of AL capsules (AL-Van), the growth inhibition area decreased as time passed at 3 hours, 24 hours, and 48 hours after the start of sustained release. In contrast, for AL-ABA capsules (AL-ABA-Van), this decrease was suppressed, showing a tendency that the difference in area between 3 hours and 48 hours was small. From these results, it was suggested that in AL-Van capsules, vancomycin was released in large amounts initially, and the release amount decreased over time, resulting in a decrease in the antibacterial effect. In contrast, in AL-ABA-Van capsules, the initial release of vancomycin was suppressed, and it was possible that the effective concentration was maintained over a longer period.

[0263] [Example I-10] Benzaldehyde-modified alginic acid-bacitracin conjugate (AL-ABA-Bac) <Synthesis of AL-ABA-Bac> [Chemical formula] Bacitracin (Bac) is a polypeptide antibacterial agent in the medium molecular weight range having two primary amino groups. Benzaldehyde-modified alginic acid-bantracin conjugate (AL-ABA-Bac) was synthesized in the same manner as in Example I-7. Specifically, 50 mg of AL-ABA(1) obtained in Example I-1 was dissolved in 70 mL of pure water and stirred for more than 1 hour. 15 mg of bacitracin (Bac) was dissolved in 15 mL of pure water and added to the AL-ABA solution. 0.1 M NaOH was added dropwise to adjust the pH to around 7.0, and the reaction was carried out overnight at room temperature under light-shielding conditions with stirring. After dilution with 150 mL of pure water, the solution was dialyzed with pure water for 2 days (apparatus: Spectra / Pro(registered trademark) 1 Dialysis Membrane Standard RC Tubing MWCO: 6-8kD), and freeze-dried for 3 days to recover 47.6 mg of gel-like AL-ABA-Bac. (Yield: 73.2%)

[0264] <Ultraviolet-Visible Light Absorption Spectrum (UV-vis) Measurement and FT-IR Spectrum Measurement> The obtained AL-ABA-Bac was subjected to ultraviolet-visible light absorption spectroscopy (UV-vis) and FT-IR spectroscopy measurements. The results are shown in Figures 21 and 22. In UV-vis (Figure 21), peaks originating from bacitracin (Bac) and ABA (indicated by arrows in the figure; a shift to lower wavelengths was observed) were also observed in AL-ABA-Bac. In the FT-IR spectrum (Figure 16), absorption originating from the C=O amide bond site between AL and ABA was observed in AL-ABA-Bac (approximately 1740 cm⁻¹). -1 A peak was observed. These results confirmed the formation of a conjugate between AL-ABA and Bac.

[0265] The recovered AL-ABA-Bac had formed a hydrogel. It is thought that the two amino groups in Bac formed bonds with the aldehyde group of ABA, causing Bac itself to function as a crosslinking agent (gelling agent) and gel. This gel structure is expected to enable sustained release over a long period of time. In addition to bacitracin (Bac), there are many polypeptide antibacterial drugs that have two or more amino groups (for example, daptomycin, colistin, ercatonin, etc.). In these drugs, the drug itself is expected to function as a crosslinking agent and form a gel structure.

[0266] [Example I-11] Benzaldehyde-modified alginic acid-dopamine conjugate (AL-ABA-DOPA) <Synthesis of AL-ABA-DOPA> [Chemical formula] Dopamine (DOPA) is a neurotransmitter that plays an important role in the brain and the body, and is an organic chemical substance of the catecholamine and phenethylamine families. DOPA has a primary amino group. The benzaldehyde-modified alginic acid-dopamine conjugate (AL-ABA-DOPA) was synthesized according to the following procedure. Dopamine (DOPA) is a neurotransmitter that plays an important role in the brain and the body and has a primary amino group. The benzaldehyde-modified alginic acid-dopamine conjugate (AL-ABA-DOPA) was synthesized according to the following procedure.

[0267] 0.25 g (0.00115 mol) of AL-ABA(2) (Low DS; modification rate (Colorimetric): 0.56) prepared in Example I-​​2 was dissolved in 100 ml of PBS (pH 7.4) and stirred for 6 to 8 hours. 0.43 g (0.0023 mol) of dopamine hydrochloride (Sigma Aldrich) was dissolved in 10 ml of PBS (pH: 7.4) and added dropwise to the AL-ABA solution. The solution was dialyzed against deionized water for 72 hours using a dialysis tube (MWCO: 6-8 kDa) (Spectra / Pro (registered trademark)) to obtain an AL-ABA-DOPA solution, which was then lyophilized.

[0268] < 1 1H NMR spectrum measurement and FT-IR spectrum measurement> For AL-ABA-DOPA, 11H NMR and FT-IR spectroscopy were performed to confirm the formation of a conjugate between AL-ABA and dopamine. The results are shown in Figures 23-24. 1 From the 1H NMR spectrum, the conjugate rate of DOPA as a drug to ABA in AL-ABA was 47% (DOPA modification rate to ABA: 0.47). The ABA modification rate in AL-ABA was: 1 Since the modification rate (Colorimetric) calculated by the colorimetric aldehyde assay is more accurate than the modification rate (NMR) calculated by the 1H NMR spectrum, the conjugate rate of DOPA was calculated from the modification rate (Colorimetric) value obtained by the colorimetric aldehyde assay. In the following examples, if the modification rate (Colorimetric) is measured, the conjugate rate of the drug to ABA is calculated based on the modification rate (Colorimetric) value.

[0269] [Example I-12] Sponge supported with AL-ABA-DOPA (AL-ABA-DOPA sponge) (Manufacturing procedure) AL-ABA (modification rate (NMR): 0.60) was synthesized under the same conditions as AL-ABA(2) in Example I-2. AL-ABA was dissolved in PBS (pH 7.4) to prepare a 0.5% AL-ABA solution. To this, two equivalent molar amounts of dopamine (DOPA) were added to ABA, and the solution was stirred at room temperature for 16-20 minutes. The solution was then dialyzed against pure water for 72 hours, followed by freeze-drying to obtain AL-ABA-DOPA. AL-ABA-DOPA was dissolved in PBS to obtain a 1.0 wt% AL-ABA-DOPA solution. The AL-ABA-DOPA solution was mixed with 1.0 ml of 10 mM CaCl2 aqueous solution in a 35 mm Petri dish. After hydrogel formation, it was frozen overnight at -20°C and then freeze-dried. Freeze-drying yielded an AL-ABA-DOPA sponge. Crosslinking with calcium ions enabled the formation of a hydrogel and a sponge of AL-ABA-DOPA, a polysaccharide derivative-drug conjugate.

[0270] [Example I-13] Benzaldehyde-modified alginic acid-serotonin conjugate (AL-ABA-Serotonin) <Synthesis of AL-ABA-Serotonin> [Chemical formula] Serotonin (5-hydroxytryptamine) is a monoamine neurotransmitter and has a primary amino group. Benzaldehyde-modified alginic acid-serotonin conjugate (AL-ABA-Serotonin) was synthesized according to the following procedure. 0.25 g (0.00115 mol) of AL-ABA(2) (Low DS, modification rate (Colorimetric): 0.56) prepared in Example I-2 was dissolved in 100 ml of PBS (pH 7.4) and stirred for 6 to 8 hours. 0.489 g (0.0023 mol) of serotonin hydrochloride (Sigma Aldrich) was dissolved in 10 ml of PBS (pH: 7.4) and added dropwise to the AL-ABA solution. The solution was dialyzed against deionized water for 72 hours using a dialysis tube (MWCO: 6-8 kDa) (Spectra / Pro (registered trademark)) and then lyophilized.

[0271] <000261​​​​​​​​​​​​ [Example I-14] Benzaldehyde-modified alginic acid-celecoxib conjugate (AL-ABA-Celecoxib) <Synthesis of AL-ABA-Celecoxib>[[]] [Chemical formula][[]] Celecoxib is a non-steroidal anti-inflammatory and analgesic drug having a primary amino group. Benzaldehyde-modified alginic acid-celecoxib conjugate (AL-ABA-Celecoxib) was synthesized according to the following procedure.[[]] 0.25 g (0.00115 mol) of AL-ABA(2) (Low DS; modification rate (Colorimetric): 0.56) prepared in Example I-2 was dissolved in 100 ml of PBS (pH: 7.4) and stirred for 6 to 8 hours. 0.877 g (0.0023 mol) of celecoxib hydrochloride (TCI) was dissolved in 10 ml of DMSO and added dropwise to the AL-ABA solution. The solution was dialyzed against deionized water for 72 hours using a dialysis tube (MWCO: 6-8 kDa) (Spectra / Pro (registered trademark)), and then lyophilized.[[]]

[0273] [[]] 1 <1H NMR spectrum measurement and FT-IR spectrum measurement>[[]] For AL-ABA-Celecoxib,[[]] 1 1H NMR spectrum measurement and FT-IR spectrum measurement (not shown) were carried out, and it was confirmed that a conjugate of AL-ABA and celecoxib was formed.[[]] 1 The results of 1H NMR spectrum measurement are shown in Fig. 27.[[]] 1 From the 1H NMR spectrum, the conjugation rate of celecoxib as a drug to ABA of AL-ABA was 11% (celecoxib modification rate to ABA: 0.11).[[]]

[0274] [Example I-15] Benzaldehyde-modified alginic acid-HGF aptamer conjugate (AL-ABA-Apt)[[]] For the purpose of developing a novel anti-adhesion hydrogel having a sustained release function of HGF aptamer (Lot: 1649399-3, manufactured by Eurofins Genomics K.K.) with a promoting effect on mesothelial cell proliferation, synthesis of benzaldehyde-modified alginic acid-HGF aptamer conjugate (AL-ABA-Apt) and a sustained release experiment of HGF aptamer from AL-ABA-Apt were conducted. The experimental procedure is shown in Fig. 28.

[0275] (Experiment) <Synthesis of AL-ABA-Apt> Under the same conditions as AL-ABA (1) in Example I-1, AL-ABA (modification rate (NMR) 4.0%) was synthesized. AL-ABA was dissolved in phosphate buffered saline (PBS, pH = 7.4) to obtain a 0.4 wt% AL-ABA solution. An HGF aptamer containing 1.0 equivalent amino group with respect to the aldehyde group of AL-ABA was added to the AL-ABA solution and stirred for 1 hour. Then, in order to remove the unreacted HGF aptamer, dialysis was performed with pure water for 3 days and freeze-dried. Ultraviolet-visible absorption spectrum (UV-vis) measurements were performed for each of the AL-ABA-HGF aptamer conjugate (AL-ABA-Apt) and the HGF aptamer, and the presence or absence of the loading of the HGF aptamer (Apt) by AL-ABA was evaluated from the peak value at 256 nm of the UV-vis spectrum.

[0276] <Release experiment of Apt from AL-ABA-Apt> 1.0 wt% of AL-ABA was dissolved in PBS (pH = 7.4). Subsequently, an HGF aptamer containing 1.0 equivalent of amino groups with respect to the aldehyde groups of AL-ABA was added into the AL-ABA aqueous solution. By stirring this for 1 hour, an AL-ABA-HGF aptamer conjugate (AL-ABA-Apt) was synthesized. Then, the AL-ABA-HGF aptamer was placed into a dialysis membrane (MWCO = 50 kDa) and stirred in PBS (pH = 7.4). At each time point from the start of stirring, 1 mL of the external solution was collected each time, and the cumulative release rate (%) of the HGF aptamer was calculated by UV-vis measurement. As a control experiment, a mixture of normal alginic acid and the HGF aptamer (ALG-Apt) and the HGF aptamer alone (Apt) were each dissolved in PBS, placed into a dialysis membrane, and the same sustained release experiment was conducted to compare the sustained release rates.

[0277] <AL-ABA-HGF aptamer / Ca 2+ Preparation of gel> The AL-ABA-HGF aptamer (AL-ABA-APt) synthesized above was dissolved in pure water at a concentration of 1.0 wt% to obtain an AL-ABA-HGF aptamer aqueous solution. The presence or absence of gelation was examined by mixing 300 μL each of the AL-ABA-HGF aptamer aqueous solution and a 100 mM CaCl2 aqueous solution in a microtube.

[0278] (Results) <Synthesis of AL-ABA-HGF aptamer> In the UV-vis spectrum of the synthesized AL-ABA-HGF aptamer (not shown), a peak derived from the HGF aptamer was confirmed near 256 nm, indicating that the synthesis of the AL-ABA-HGF aptamer (AL-ABA-Apt) was successful.

[0279] <Sustained release of HGF aptamer from AL-ABA-HGF aptamer> Figure 29 shows the results of sustained-release experiments of HGF aptamers (Apt) from AL-ABA-HGF aptamers (AL-ABA-Apt), a mixture of alginic acid and HGF aptamers (ALG-Apt), and HGF aptamers alone (Apt). The release of HGF aptamers (Apt) was delayed in the AL-ABA-Apt group compared to the ALG-Apt and Apt groups. This indicates that sustained-release of HGF aptamers can be achieved under physiological pH conditions by conjugating HGF aptamers with AL-ABA.

[0280] <AL-ABA-HGFアプタマー / Ca 2+ Gel preparation > Ca 2+ Crosslinking instantly gelled the AL-ABA-HGF aptamer, successfully producing a gel. As described in Examples I-16 and I-17 below, AL-ABA is Ca 2+ It has been confirmed that crosslinking causes instantaneous gelation. Therefore, it was suggested that conjugation of HGF aptamers to AL-ABA has little effect on the gelation performance of AL-ABA. We successfully synthesized an AL-ABA-HGF aptamer carrying an HGF aptamer that promotes mesothelial cell proliferation, and demonstrated that the HGF aptamer can be released sustainedly from the AL-ABA-HGF aptamer. Furthermore, Ca 2+ We successfully fabricated an AL-ABA-HGF aptamer (AL-ABA-Apt) gel by crosslinking. This suggests its potential use as an anti-adhesion material.

[0281] 4. AL-ABA sponge, hydrogel [Example I-16] Calcium-crosslinked benzaldehyde-modified alginate sponge (AL-ABA sponge) (Manufacturing procedure) A total of 20 mg of sodium alginate (AL) (IL-6G, manufactured by Kimika Co., Ltd., viscosity 50-80 mPa·s (1%)) and AL-ABA (1) obtained in Example I-1 were dissolved in 2 ml of pure water in weight ratios of 75:25, 50:50, and 25:75, respectively, to prepare an AL-ABA aqueous solution. This was mixed with 2 ml of a 10 mM CaCl2 aqueous solution on a dish to obtain an AL-ABA hydrogel. After freezing overnight in a -20°C freezer, the mixture was freeze-dried in a freeze-dryer for 3 days to obtain an AL-ABA sponge. For comparison, a control experiment was conducted in the same manner as above, except that AL-ABA was replaced with AL only (AL 100% by weight), to obtain a sponge (AL sponge).

[0282] (result) Optical images of the AL-ABA sponge and the AL sponge used as a control obtained in the above example are shown in Figure 30, and SEM images are shown in Figure 31. It was confirmed that a benzaldehyde-modified alginate sponge (AL-ABA sponge) can be fabricated using a simple method similar to that of AL. Figure 31 confirms that the AL-ABA sponge also has a porous structure similar to that of the AL sponge. It is thought that the unmodified carboxyl groups of AL and AL-ABA are crosslinked via calcium (Ca) ions, forming a crosslinked structure. By mixing AL and AL-ABA, it is possible to control the content of the benzaldehyde (ABA) modifying group and the crosslinking density in the sponge obtained.

[0283] [Example I-17] Hydrogel of calcium-crosslinked benzaldehyde-modified alginate (Ca-crosslinked AL-ABA hydrogel) A hydrogel (calcium-crosslinked AL-ABA hydrogel) was prepared by crosslinking AL-ABA(2) (High DS, Modification rate (Colorimetric): 0.64) prepared in Example I-2 with calcium. Specifically, 20 mg of AL-ABA(2) (High DS, Modification rate (Colorimetric): 0.64) prepared in Example I-2 was dissolved in 1 ml of pure water to prepare an AL-ABA aqueous solution. This was mixed with 1 ml of a 100 mM CaCl2 aqueous solution on a dish to obtain a calcium-crosslinked AL-ABA hydrogel.

[0284] <In vitro swelling and decomposition test> The Ca-crosslinked AL-ABA hydrogels obtained above were swollen in phosphate-buffered saline (PBS) at pH 7.4. Specifically, four freshly prepared hydrogel samples were prepared, the weight of each sample was measured, and each was placed in 25 ml of PBS solution at 37°C and swollen for 72 hours. The degradation of each sample after 72 hours of swelling was monitored. The weight of the hydrogel samples was measured every 24 hours for two weeks. The samples were replaced weekly with fresh PBS that had been pre-equilibriumized at 37°C. The fully swollen Ca-crosslinked AL-ABA hydrogels were freeze-dried, and their morphology was observed using a scanning electron microscope (SEM).

[0285] (result) Figure 32 shows the swelling and decomposition profile (change in hydrogel weight) of Ca-crosslinked AL-ABA hydrogel. The weight change values ​​are the average values ​​of four samples. Figure 33 shows an SEM image (magnification X500; 24 hours after the start of swelling) of a cross-section of a sponge obtained by freeze-drying Ca-crosslinked AL-ABA hydrogel, cut with a scalpel. The porous structure of the dried hydrogel is confirmed.

[0286] 5. Hydrogel containing AL-ABA and amino group-containing polymer [Example I-18] Hydrogel of AL-ABA and DPI (AL-ABA-DPI) [ka] AL-ABA(2) (Low DS, Colorimetric modification rate: 0.56) prepared in Example I-2 was dissolved in physiological saline as a solvent to prepare a 2% w / v AL-ABA solution. Dendritic poly(ethyleneimine) (DPI) (manufactured by BASF, product name: PS, weight average molecular weight M: 750,000) was dissolved in water to prepare a 10% w / v DPI solution. Two solutions were mixed so that the aldehyde groups of the AL-ABA solution and the primary amino groups (-NH2) of the DPI solution were equimolar (1:1). Specifically, 250 μL of a 10% w / v DPI solution (amino groups: 2.59×10 -4 mol) was mechanically mixed with 5 mL of a 2% w / v AL-ABA solution (aldehyde groups: 2.59×10 -4 mol) with a pipette. A hydrogel was formed within 20 to 30 seconds (Figure 35). The hydrogel was stable in water for two weeks.

[0287] [Example I-19] Hydrogel of AL-ABA and PEG(4k)-dihydrazide (AL-ABA-PEGDH) <Synthesis of PEGDH> [Chemical formula] For the preparation of PEGDH, a two-step reaction was carried out. First, 10 g of PEG (Mw 4000) (0.0025 mol) 162 - 09115, Wako) was dissolved at room temperature in a 300 ml eggplant-shaped flask containing 100 ml of dichloromethane (DCM) solvent. After 2.0 g (0.02 mol) of succinic anhydride, 2.57 g (0.0125 mol) of N,N'-dicyclohexylcarbodiimide and 1.83 g (0.015 mol) of 4-dimethylaminopyridine were added to the PEG solution, the flask was closed with a rubber septum and stirred at room temperature for 24 hours. Then, the reaction mixture was filtered through filter paper, and the filtrate was evaporated at 50 °C using a rotary evaporator. After completely evaporating DCM, a viscous reaction mixture was left in the flask, then 100 ml of distilled water was added and stirred for another 1 hour, and then the solution was dialyzed against pure water for 48 hours using a dialysis tube (MWCO 1 kDa) (Spectra / Pro (registered trademark)). After dialysis, the solution was lyophilized, 1 characterized by 1H NMR (using D2O solvent). The yield was about 73%, and the conversion rate from PEG to PEG-COOH was about 100%. The second step is a carbodiimide reaction. 4 g (0.00097 mol) of newly prepared PEG-COOH was taken in a 200 ml Erlenmeyer flask and dissolved with 50 ml of distilled water for 2 - 4 hours. 1.05 g (0.0070 mol) of HOBt was dissolved in 10 ml of DMSO (Wako), then 1.49 g (0.0078 mol) of WSCD.HCl (Peptide Institute) was dissolved in 10 ml of distilled water and added dropwise to the PEG-COOH solution. The solution was stirred for another 10 minutes. 3.399 g (0.019 mol) of adipic dihydrazide was dissolved in 15 ml of pure water and added to the PEG-COOH solution. The pH of the final reaction mixture was maintained between 7.5 pH with 1N NaOH and stirred at room temperature for 16 - 20 hours. Then, using a dialysis tube (MWCO 1 kDa) (Spectra / Pro (registered trademark)), the solution was thoroughly dialyzed against deionized water for 48 hours and subsequently lyophilized. 1 The binding of PEGDH was confirmed using 1H NMR. The degree of substitution was about 91%.

[0288] <Synthesis of AL-ABA-PEGDH> The AL-ABA(2) (High DS, Modification Rate (Colorimetric): 0.64) prepared in Example I-2 was dissolved in physiological saline as a solvent to prepare a 1.5% w / v AL-ABA solution (2). The PEGDH prepared above was dissolved in water to prepare a 10% w / v PEGDH solution. The two solutions were mixed so that the aldehyde groups of the AL-ABA(2) solution and the hydrazide groups of the PEGDH solution were equimolar (1:1). Specifically, 1 mL of 1.5% w / v AL-ABA solution (aldehyde groups: 6.9 × 10) was mixed. -5 (mol) Add 0.5 mL of 10% w / v PEGDH solution (hydrazide group: 2.27 × 10 -5 The mixture (in moles) was mechanically mixed using a pipette. A hydrogel formed within 30 seconds (Figure 36).

[0289] <Dynamic Viscoelasticity Measurement> The storage modulus G' and loss modulus G'' were measured using a rheometer. The results are shown in Figure 37. Within the measurement frequency range, AL-ABA-PEGDH showed G'>G'', confirming the formation of a hydrogel.

[0290] [Example I-20] Benzaldehyde-modified alginate film (uncrosslinked and crosslinked AL-ABA film) <Film preparation> (1) Uncrosslinked AL-ABA film Uncrosslinked AL-ABA films were prepared by solution casting and vacuum heating drying processes. Specifically, AL-ABA(2) (High DS, modification rate (Colorimetric): 0.64) prepared in Example I-2 was dissolved in distilled water to prepare a 1.5% w / v AL-ABA solution. After complete dissolution, bubbles were removed by sonication for 15 minutes. The solution was then poured into 60 mm Petri dishes, and the Petri dishes were held in a vacuum dryer at 50°C for 40-48 hours. After that, the film was peeled off and stored at 4°C. The film was a non-porous, transparent film with a thickness of approximately 63 μm.

[0291] (2) Cross-linked AL-ABA film The uncrosslinked AL-ABA film obtained in (1) above was crosslinked with PEGDH to obtain a crosslinked AL-ABA film. Specifically, the PEGDH synthesized in Example I-19 above was dissolved in an aqueous butanol solution (butanol:water = 9:1), and the film obtained in (1) above was immersed in the solution and maintained at room temperature for 6 to 8 hours. The film was removed from the PEGDH solution, dried in the air, and stored at 4°C. The obtained crosslinked AL-ABA film was a non-porous and transparent film.

[0292] <Film swelling and dissolution test> (Experimental procedure) The uncrosslinked AL-ABA film obtained in (1) above, the crosslinked AL-ABA film obtained in (2) above, and the AL film were immersed in PBS (pH=7.4) for 2 to 4 weeks, and the swelling and dissolution of the films were observed. Here, the AL film was prepared by the method of (1) above, except that AL-500 (sodium alginate, viscosity: 400-600 mPa·s, manufactured by Mochida Pharmaceutical Co., Ltd.) was used instead of AL-ABA (2) prepared in Example I-2. (result) The results are shown in the table below. [Table 3]

[0293] [Example I-21] Hydrogel of AL-ABA and polyallylamine (AL-ABA-PAA1, AL-ABA-PAA2, AL-ABA-PAA3, AL-ABA-PAA4) [ka] (i) PAA1: Product name: Allylamine maleic acid copolymer (Product number: PAA-1151, Nitto Boseki Medical Co., Ltd., viscosity 20 wt%, 20 cp at 20°C) (ii) PAA2: Diallyldimethylammonium chloride acrylamide copolymer (product number: PAS-J-81, Nitto Boseki Medical Co., Ltd., viscosity 25 wt% 900 cp at 20°C), weight-average molecular weight Mw: 180000 (iii) PAA3: Allylamine hydrochloride dimethylallylamine copolymer (product number: PAA-1112CL, Nitto Medical Co., Ltd., viscosity 15 wt%, 5 cp at 20°C) (iv) PAA4: Allylamine hydrochloride polymer (product number: PAA-HCl-10L, Nitto Boseki Medical Co., Ltd., viscosity 40 wt% 1500 cp (20℃)), weight-average molecular weight Mw: 150000 The polyallylamines (PAA1 to PAA4) described in (i) to (iv) above were each dissolved in water to prepare 10% w / v PAA solutions (PAA1 solution, PAA2 solution, PAA3 solution, and PAA4 solution). AL-ABA(2) (Low DS, Modification rate (Colorimetric): 0.56) prepared in Example I-2 was dissolved in physiological saline as a solvent to prepare a 2% w / v AL-ABA solution. The two solutions were mixed so that the aldehyde groups of the AL-ABA solution and the primary amino groups (-NH2) of the PAA solution were equimolar (1:1). Specifically, 5 mL of 2% w / v AL-ABA solution (aldehyde groups: 2.59 × 10⁻¹⁶) was mixed. -4 (mol) 10% w / v each PAA solution (PAA1 solution, PAA2 solution, PAA3 solution, PAA4 solution) (amino groups in each solution: 2.59 × 10 -4 The mixtures (in moles) were mechanically mixed using a pipette. In all cases, hydrogels formed within 20-30 seconds (Figure 38).

[0294] 6. Tissue adhesion materials using AL-ABA [Example I-22] Benzaldehyde-modified alginate (AL-ABA) as a tissue adhesion material (adhesion to the submucosa) <Evaluation of adhesion behavior to mucous membranes> The adhesion of AL-ABA to the submucosa was investigated. The submucosa is rich in collagen and therefore contains a large amount of amino groups. The following experiments confirmed that cross-linking structures are formed between the amino groups in the submucosa and the aldehyde groups of AL-ABA using Schiff bases, potentially improving adhesion. The specific experimental procedure is as follows.

[0295] (Experimental procedure) 1. The pig esophagus was cut lengthwise and divided into 2cm x 2cm sections. The inner mucosal layer of the esophagus was removed, exposing the submucosa. 25mL of physiological saline solution with 0.1% sodium benzoate was placed in each of 80mL security containers. One esophageal section with the submucosa exposed was immersed in each container, and the containers were placed in a shaker in a 37°C incubator and shaken overnight. The weight of the esophageal sections was measured after shaking. (Esophageal sections: n=4) 2. AL-ABA and unmodified AL(IL-6G) were dissolved in pure water at a concentration of 2 w / v% for comparison. The AL-ABA used was AL-ABA(1) prepared in Example I-1. 3. Place the alginate solution (AL-ABA aqueous solution or AL aqueous solution) prepared in step 2 into one end of a double syringe, and a 50 mM CaCl2 aqueous solution into the other end. 4. Remove the sections prepared in step 1 from the security container and place them in a petri dish. 0.5 mL of the alginate solution prepared in step 3 and 0.5 mL of the 50 mM CaCl2 aqueous solution were simultaneously sprayed onto the sections using a double syringe and nitrogen gas. The nitrogen gas flow rate at this time was 2 L / min. For gelation, the sections were left to stand at room temperature (25°C) for 10 minutes. This treatment converted benzaldehyde-modified alginate (AL-ABA) or alginate (AL) into calcium (Ca 2+ The resulting material was cross-linked to form a hydrogel (AL-ABA gel, AL gel). 5. The gel on the petri dish was rinsed with pure water, and the moisture from the esophageal section was wiped off. The weight of the esophageal section was then measured. The weight of the gel on the esophageal section was calculated by subtracting the weight of the esophageal section measured in step 1 from this weight. This was defined as the gel weight at 0 hours. 6. The saline solution containing 0.1% sodium benzoate in the security container was replaced with fresh saline solution. The esophageal section was then immersed in this solution with the gel side facing downwards. This was then placed on a shaker and shaken in a 37°C incubator. 7. The weight of the esophageal section was measured at predetermined time intervals (1, 2, 3, 4, 6, 8, 12, 24, and 48 hours), and the weight of the remaining gel was calculated by subtracting the weight of the esophageal section measured in step 1 above. From the gel weight at 0 hours and the remaining gel weight, the percentage of gel remaining at each time point [(remaining gel weight) / (gel weight at 0 hours) × 100] was determined. The appearance of the gel at each time point was also observed. The above procedure was performed on four samples each of AL and AL-ABA, and the average percentage of remaining gel at each time point was defined as the gel adhesion rate (%).

[0296] (result) The results are shown in Figures 39 and 40. Figure 39 shows the appearance of the AL gel and AL-ABA gel at each time point. Figure 40 shows the adhesion rate (%) of the AL gel and AL-ABA gel at each time point. As shown in Figure 39, in the group sprayed with AL (ALG(IL-6G), AL gel), the gel peeled off in 3 out of 4 samples after 1 hour. The remaining sample also peeled off after 2 hours. In the group sprayed with AL-ABA (ALG-ABA, AL-ABA gel), the gel gradually peeled off starting after 2 hours, and a small amount of gel remained visible even after 12 hours. Furthermore, Figure 40 shows that the adhesion rate (%) of the gel in the AL-sprayed group (ALG(IL-6G), AL gel) became zero after 3 hours, while the AL-ABA-sprayed group (ALG-ABA, AL-ABA gel) was able to maintain an adhesion rate of approximately 40%. These results suggest that modifying AL with ABA improves its adhesion.

[0297] [Example I-23] AL-ABA as a tissue adhesive material (evaluation of adhesion to the esophageal mucosa and submucosa and Ca 2+ (Evaluation of the effect of concentration) (37℃)

[0298] <Adhesion experiment on the mucosal layer and submucosal layer> The adhesion of AL-ABA and AL to the esophageal mucosa and submucosa was investigated under a 37°C environment. Samples with the mucosa intact were used as controls, and samples with the mucosa removed and the submucosa exposed were used as ESD samples. In addition, Ca 2+ We investigated two concentrations, 50M and 100mM, and Ca 2+ We investigated the change in adhesion due to changes in concentration. The experimental procedure is shown in Figure 42. The specific procedure is as follows:

[0299] (material) AL-ABA: AL-ABA (modification rate (NMR): 0.052) prepared under the same conditions as AL-ABA(1) in Example I-1 was dissolved in pure water at a concentration of 2 w / v%. AL:IL-6G (IL-6G manufactured by Kimika Co., Ltd., viscosity 50-80 mPa·s (1%)) was dissolved in pure water at a concentration of 2 w / v%. CaCl2 aqueous solution (for crosslinking): 50 mM or 100 mM Esophageal section (Control: mucosal layer present; ESD: mucosal layer dissected, submucosa exposed)

[0300] (Experimental procedure) 1. The pig esophagus was cut lengthwise and divided into 2cm x 2cm sections. In the submucosal group (ESD), the mucosal layer was peeled off with scissors. 2. The mass of the septal pig esophageal sections (Control) and the sections with exposed submucosa (ESD) was measured. 3. AL or AL-ABA was dissolved in pure water at a concentration of 2 w / v%. 4. Place the solution prepared in step 3 and the CaCl2 solution into a 2.5 mL syringe and set it in a double syringe. (0.5 mL of alginate solution + 0.5 mL of CaCl2 solution per sample) 5. A spray nozzle was attached to a double syringe, and esophageal sections were sprayed with nitrogen gas at a rate of 4 L / min. (n=4 per sample) 6. It was left to stand for 10 minutes until it gelled. This process converted benzaldehyde-modified alginic acid (AL-ABA) or alginic acid (AL) into calcium (Ca 2+ The resulting hydrogel was formed by cross-linking (AL-ABA gel, AL gel). The mass of the esophageal section was then measured. 7. Place 25 mL of physiological saline solution containing 0.1% methyl benzoate and 1.25 mM CaCl2 into a 6 cm diameter glass petri dish, immerse the esophageal section in it, and shake with a shaker. 1.25 mM Ca 2+ The calcium contained in saliva 2+ It is an imitation of [another work]. 8. The mass of esophageal sections was measured at predetermined time intervals (1, 2, 3, 4, 6, 8, 12, 24, 48, and 72 hours), and the mass of the remaining gel was determined. From the gel weight at 0 hours and the remaining gel weight, the percentage of gel remaining at each time point [(remaining gel weight) / (gel weight at 0 hours) × 100] was calculated. The appearance of the gel at each time point was also observed. The above procedure was applied to esophageal sections (Control) and submucosal exposed sections (ESD), Ca 2+ The test was performed on four samples each of AL and AL-ABA at concentrations of 50 mM or 100 mM, and the average percentage of gel remaining at each time point was defined as the gel adhesion rate (%).

[0301] (result) Figure 43 shows the appearance of the gel at time points (2, 4, 12, 24, 48, and 72 hours after death). Figure 44 shows the percentage of gel remaining (adhesion rate, %) at each time point. The study showed that AL-ABA sprayed onto the submucosa (AL-ABA ESD) and AL-ABA sprayed onto the mucosa (AL-ABA Control; AL-ABA Con) maintained gel adhesion for a longer period compared to the group sprayed with AL (AL ESD and AL Control (Con)). It was confirmed that modifying AL with ABA improves adhesion. In particular, it was shown that spraying AL-ABA onto the submucosa (AL-ABA ESD) resulted in even greater adhesion compared to spraying AL-ABA onto the mucosa (AL-ABA Control; AL-ABA Con). It is presumed that the strong adhesion was achieved by ABA binding to the amino groups in the submucosa. Furthermore, it was shown that the gel's adhesion could be maintained for a longer period when the CaCl2 concentration was high (100 mM). It is thought that the increased Ca ion content improved the crosslinking point density, resulting in increased mechanical strength and longer decomposition time of the gel. Since cohesive failure occurred at the maximum breaking strength measured in the tensile test, it is thought that the increased mechanical strength of the gel led to increased adhesive strength.

[0302] <Adhesion evaluation by tensile testing> In the above adhesion experiment, both gel delamination and gel dissolution occurred simultaneously. A tensile test was performed to verify only the adhesion of AL-ABA. The procedure for the tensile test is shown in Figure 45. (material) AL-ABA: AL-ABA (modification rate (NMR): 0.052) prepared in the same manner as in Example I-1 was dissolved in pure water at a concentration of 2 w / v%. AL:IL-6G (IL-6G manufactured by Kimika Co., Ltd., viscosity 50-80 mPa·s (1%)) was dissolved in pure water at a concentration of 2 w / v%. CaCl2 aqueous solution (for crosslinking): 50 mM or 100 mM Esophageal section (Control: mucosal layer present; ESD: mucosal layer dissected, submucosa exposed) (Device) Physical property measurement rheometer (Shiro Sangyo Co., Ltd., M993R-3000S) (Experimental procedure) 1. The pig esophagus was cut lengthwise and divided into 1cm x 3cm sections. For the mucosal group (Control), the cut sections were used as is. For the submucosal group (ESD), the mucosal layer of the sections was removed (n=3). 2. Alginate solution (AL-ABA or AL) was added to a 1cm x 1cm area at one end of each section. 3. CaCl2 solution was added to the alginate solution to induce gelation. 4. A weight (an 80mL container filled with 30mL of water) was placed on top to remove the air. The sample was left undisturbed in a constant temperature bath at 5.37°C for 1 hour. 6. The fracture strength (N) was measured using a rheometer. 7. The tensile strength (N) and the area (m²) of the gel (region to which alginic acid solution was added) as described in 6. above. 2 The fracture pressure (Pressure, Pa) was determined from the given data.

[0303] (result) The results are shown in Figure 46. It was confirmed that the AL-ABA hydrogel exhibited significantly greater adhesion strength to the mucosa and submucosa compared to the AL hydrogel. Furthermore, the crosslinking agent (Ca 2+ It was confirmed that the adhesive strength changes depending on the concentration of ions.

[0304] [Example I-24] AL-ABA as a tissue adhesive material (evaluation of adhesion to submucosa and skin tissue by overlapping shear method) The adhesion strength of AL-ABA, AL, and conventional tissue adhesives to biological tissues was evaluated using the lap shear test. Specifically, the following Pregel solutions were used, and porcine submucosa and porcine skin tissue were used as biological tissues. The adhesion strength of each material to biological tissues was evaluated according to the procedure described below. The experimental procedure for the lap shear test is shown in Figure 47. (Pregel solution) • Aqueous solution of AL-ABA(2) (High DS; modification rate (Colorimetric): 0.64) prepared in Example I-2 with AL-ABA: 4 w / v% • AL500: 4 w / v% AL-500 (manufactured by Mochida Pharmaceutical Co., Ltd., sodium alginate, viscosity: 400-600 mPa·s) • Fibrin Glue: Fibrin glue (Beriplast P, manufactured by CSL Behring Co., Ltd.) • Hydrofit® (Registered Trademark) (Urethane-based hemostatic agent; Terumo Corporation) • Dermabond® (Cyanoacrylate-based skin adhesive; Johnson & Johnson) The gelation of the Pregel solution was carried out as follows. For AL-ABA and AL500, after applying the Pregel solution (AL-ABA or AL500) and overlapping the biological tissue, 200 μL of 50 mM or 100 mM CaCl2 aqueous solution was applied to each side of the biological tissue (200 μL x 2). The Fibrin Glue was gelled using a double syringe. Hydrofit® is a one-component solution and was applied directly. Dermabond® is a one-component solution and was applied directly. (Biological tissue) Submucosa: The pig esophagus was cut into sections measuring 40 mm in length and 10 mm in width. The mucosal layer on the inner side of the cut esophageal tissue was then removed to expose the submucosa. Skin tissue: Pig skin tissue cut into pieces measuring 40 mm in length and 10 mm in width was prepared. (Device) Dynamic Mechanical Analyzer (DMA), Sun Scientific Co., Ltd., CR3000-EX

[0305] (Experimental procedure for the lap shear method) 0.5 mL of Pregel solution was applied to the overlapping area (10 mm x 10 mm) at the ends of two pieces of biological tissue (40 mm long x 10 mm wide), overlapped, and then gelled. Twenty minutes after gelling, an overlapping shear test was performed using a dynamic mechanical analyzer (DMA) at a speed of 5 mm / min. The adhesive energy was measured by a 180° peel test. The adhesive strength was calculated using the following formula. Adhesive strength = force (N / m 2 ) / Overlap area (m 2 )

[0306] (result) The results are shown in Figure 48. AL-ABA hydrogels have been shown to be usable as tissue adhesives for biological tissues such as the submucosa and skin tissue.

[0307] [Example I-25] AL-ABA as a tissue adhesive material (tissue sealant) (evaluation of its effectiveness as a sealant for submucosal and skin tissues by bursting test) To investigate its effectiveness as a tissue sealant, a burst test was performed using a device designed with slight modifications to the apparatus reported by Lei Zhou et al., ADVANCED FUNCTIONAL MATERIALS, Volume 31, Issue 14, 2021, 2007457, “Injectable Self-Healing Natural Biopolymer-Based Hydrogel Adhesive with Thermoresponsive Reversible Adhesion for Minimally Invasive Surgery”. Specifically, the following Pregel solution was used, and porcine submucosa and porcine skin tissue were used as biological tissues. The burst test was performed according to the burst test procedure described below. The apparatus and burst test procedure used are shown in Figure 49. (Pregel solution) • Aqueous solution of AL-ABA(2) (High DS; Modification rate (Colorimetric): 0.64) prepared in Example I-2 with AL-ABA: 4 w / v% • AL500: 4 w / v% AL-500 (manufactured by Mochida Pharmaceutical Co., Ltd., sodium alginate, viscosity: 400-600 mPa·s) • Fibrin Glue: Fibrin glue (Beriplast P, manufactured by CSL Behring Co., Ltd.) • Hydrofit® (Registered Trademark) (Urethane-based hemostatic agent; Terumo Corporation) • Dermabond® (Cyanoacrylate-based skin adhesive; Johnson & Johnson) The gelation of the Pregel solution was carried out as follows. For AL-ABA and AL500, the procedure involved injecting Pregel solution (AL-ABA or AL500) into the puncture site, then dropping 500 μL of 100 mM CaCl2 aqueous solution from above and allowing it to crosslink for 5-10 minutes. The Fibrin Glue was gelled using a double syringe. Hydrofit® is a one-component solution and was applied directly. Dermabond® is a one-component solution and was applied directly. (Biological tissue) Submucosa: The pig esophagus was cut into 50 mm long x 50 mm wide sections, and the mucosal layer on the inner side of the cut esophageal tissue was excised to expose the submucosa. Skin tissue: Pig skin tissue cut into pieces measuring 50 mm in length and 50 mm in width was prepared. (Burst test procedure) A biological tissue sample was fixed to the upper surface of a burst pressure device, and a 2 mm diameter hole was created in the center of the tissue by puncture. 2.5-3 mL of Pregel solution was injected into the 2 mm diameter hole (puncture site) and allowed to gel. The measuring device was connected to a syringe pump filled with PBS solution. Finally, the PBS solution was injected into the device, and the maximum burst pressure (mmHg) was recorded using a digital pressure gauge. The experiment was repeated five times.

[0308] (result) The results are shown in the table and Figure 50 below. [Table 4] As shown in Figure 50, the AL-ABA hydrogel showed no significant difference compared to Fibrin Glue, demonstrating its potential use as a tissue sealant.

[0309] 7. Tissue adhesion materials using AL-ABA, AL-AFA, AL-AAP, AL-APA, and AL-ANA [Example I-26] Modified alginate as a tissue adhesion material to dura mater (evaluation of adhesion to dura mater by overlapping shear method) To evaluate the adhesive properties and bonding capacity of alginate derivatives (AL-ABA, AL-AFA, AL-AAP, AL-APA, AL-ANA), a lap shear test was performed according to the ASTM D1002 test procedure. The bonding capacity of the alginate derivatives was compared with several commercially available adhesives (Fibrin Glue (Beriplast® P), Duraseal®, Dermabond®, Hydrofit®)) and unmodified sodium alginate (AL-500).

[0310] <Sample> Alginic acid derivatives (AL-ABA, AL-AFA, AL-AAP, AL-APA, AL-ANA) were prepared in the same manner as AL-ABA(2) in Example I-2 and in Examples I-3 to I-6. The modification rates below represent the modification rate of the carboxyl group (-COOH) of alginic acid, calculated from the amount of aldehyde measured using the colorimetric aldehyde assay kit, blue (MAK140). • AL-ABA: Alginic acid modified with benzaldehyde (ABA) (modification rate: 0.56 (56%)) • AL-AFA (AL-ADFBA): Alginic acid modified with 4'-4-amino-2,6-difluorobenzaldehyde (ADFBA) (modification rate: 0.12 (12%)) • AL-AAP: Alginic acid modified with 4'-aminoacetophenone (AAP) (modification rate: 0.25 (25%)) • AL-APA (AL-APCA): Alginic acid modified with 2-amino-3-pyridinecarboxaldehyde (APA, APCA) (modification rate: 0.31 (31%)) • AL-ANA: Alginic acid modified with 6-aminonicotinaldehyde (ANA) (modification rate: 0.17 (17%)) • AL-500: Sodium alginate (manufactured by Mochida Pharmaceutical Co., Ltd., viscosity: 400-600 mPa·s) • Fibrin Glue: Fibrin glue adhesive (Beriplast® P, manufactured by CSL Behring Co., Ltd.) • Duraseal (registered trademark) (Duration sealant: manufactured by Integra Japan) • Dermabond® (Cyanoacrylate-based skin adhesive; Johnson & Johnson) • Hydrofit® (Registered Trademark) (Urethane-based hemostatic agent; Terumo Corporation) <Biological tissue> The porcine dura mater was purchased from Tokyo Shibaura Zouki Co. Ltd.

[0311] The experiment was carried out as follows, following the procedure shown in Figure 74(A). (experiment) First, each alginate derivative and unmodified sodium alginate (AL-500) were dissolved in PBS (pH 7.4) for 48 hours to prepare 3% w / v solutions (Pregel solutions) of each. Then, porcine dura mater was thawed and washed with distilled water, and cut into several rectangular pieces of similar size measuring 3 cm in length and 1 cm in width to prepare dura mater tissue. Approximately 0.1 mL of Pregel solution (3% alginate derivative or alginate, w / w) was applied to 1 × 1 cm 2 The solution was applied to the edge of the dura mater tissue in the region, and two pieces of dura mater tissue were placed on top of it to cover the applied area. The tissue sample was immediately immersed in a 100 mM CaCl2 solution for 2 minutes, followed by incubation at 37°C for 1 hour to gel the Pregel solution (RCom MARU Delux Max Digital incubator). During incubation, the tissue sample was held with several damp tissues to prevent moisture loss. The obtained samples were subjected to overlapping shear adhesion tests using a tensile testing apparatus (CR-3000EX-S; Sun Science Co., Ltd.). The samples were fixed between both crambs of the tensile testing machine, and the test was performed at a speed of 5 mm / min until the two overlapping tissue samples separated from each other. This test was performed three times for each sample. The adhesion strength was calculated from the maximum force at which the samples were peeled off using the following formula. Adhesive strength = maximum force (N / m) 2 ) / Overlap area (m 2 )

[0312] For commercially available adhesives, the experiment was conducted using the same procedure as above, except that instead of immersion in CaCl2 solution, gelation was performed using the following method. The Fibrin Glue was gelled using a double syringe. Duraseal (registered trademark) was gelled using the included applicator. Dermabond® is a one-component solution and was applied directly. Hydrofit® is a one-component solution and was applied directly.

[0313] (result) The results are shown in the table and Figure 74(B) below. [Table 5]

[0314] Adhesion data measured by overlapping shear tests using dura mater as a substrate showed that various alginate derivatives exhibited significantly higher adhesive strength to dura mater. Among the alginate derivatives, AL-APA showed the highest adhesive strength (88.5±9.3 kPa), while AL-AAP showed the lowest adhesive strength (54.8±5.3 kPa). All alginate derivatives showed significantly higher adhesive strength than unmodified alginate (AL-500). Furthermore, all alginate derivatives showed significantly higher adhesive strength than the commercially available dura mater sealant Duraseal® (28.7±5.8 kPa). In addition, all alginate derivatives showed significantly higher adhesive strength than Fibrin Glue (46.8±5.1 kPa). On the other hand, all alginate derivatives showed significantly lower adhesive strength than Hydrofit® (176.1 ± 14.2 kPa) and Dermabond® (189.9 ± 3.6 kPa). The reason why alginate derivatives exhibit higher adhesion is the involvement of Schiff base reactions between the amino groups of the dura mater and the aldehydes of the alginate derivatives. Furthermore, hydrophobic interactions and hydrogen bonding between the alginate derivatives and dura mater proteins are also thought to contribute to improved adhesion strength.

[0315] [Example I-27] Alginate derivative as a tissue adhesion material (tissue sealant) for dura mater tissue (evaluation of sealing effect and adhesive ability to dura mater tissue by burst test) To compare the sealing effect and adhesive properties of alginate derivatives (AL-ABA, AL-AFA, AL-AAP, AL-APA, AL-ANA) with several commercially available adhesives (Fibrin Glue (Beriplast® P, sometimes referred to as "FG"), Duraseal®, Dermabond®, Hydrofit®), Bio Glue®, and unmodified sodium alginate (AL-500), burst tests were conducted using custom-designed equipment.

[0316] The alginate derivatives, commercially available adhesives other than Bio Glue®, and unmodified sodium alginate were the same as those used in Example I-26. • Bio Glue®: Glutaraldehyde-based adhesive (manufactured by Cyiolife) The porcine dura mater was purchased from Tokyo Shibaura Zouki Co. Ltd. A 3% w / v solution of alginate derivative (Pregel solution) was prepared by dissolving the alginate derivative in PBS (pH 7.4) for 48 hours.

[0317] The experiment was conducted using the apparatus shown in Figure 49(A) as follows. The experimental procedure is shown in Figure 75(A). (Experiment 1: Experiment without using nonwoven fabric) Fresh porcine dura mater was gently washed with distilled water, cut into approximately 50 mm diameter circles, and a hole was created in the center using a 2 mm biopsy punch (Kai Japan). Then, 2-3 ml of a pre-prepared 3% w / v solution of alginate derivative (Pregel solution) was spread over the central hole of the dura mater sample. The sample was then transferred to a 100 mM CaCl2 (Wako CAS RN(registered trademark): 10043-52-4, molecular formula: CaCl2, molecular weight: 110.98) solution, and hydrogel was formed (gelled) over 5-10 minutes to allow proper crosslinking of the alginate derivative and diffusion of the CaCl2 solution to deeper surfaces. Next, the sample was kept in the incubator for 5-10 minutes after incubation, and for the burst test, the sample was firmly fixed between support rings, and the measuring device was connected to a syringe pump filled with PBS solution. Finally, PBS (pH 7.4) solution was injected into the device at a flow rate of 10 ml / min. The maximum burst pressure was recorded using a digital pressure gauge. The pump injected PBS solution into the burst pressure device and continued injecting PBS until it began to leak from the applied hydrogel. The pressure change was recorded after the pump was stopped. The experiment was repeated 4-5 times for each sample.

[0318] (Experiment 2: Experiment using nonwoven fabric) To improve the burst pressure performance of alginate derivatives, samples containing both the nonwoven fabric (Gunze Medical Japan Ltd (GMJ) Neovil®) or Ethicon Vicryl® Mesh) as a support membrane and the alginate derivative (nonwoven fabric-containing samples) were prepared. Gunze Medical Japan Ltd (GMJ) Neovil (registered trademark): Material: Glycolic acid ester, Thickness: 0.15~0.5mm Ethicon Vicryl® Mesh: Material: Glycolic acid / lactic acid polyester (90 / 10), Thickness: 0.1~0.5mm The nonwoven fabric is 3 x 3 cm. 2The nonwoven fabric was cut to the specified size for use. Experiments using nonwoven fabric-containing samples were conducted using the following procedure. Specifically, the nonwoven fabric, trimmed to the predetermined size, was attached to the tissue defect (central pore of the dura mater sample), tissue adhesive was applied on top of it, and then it was immersed in a CaCl2 solution to gel, as in Experiment 1.

[0319] For unmodified sodium alginate (AL-500), the experiment was conducted using the same procedure as above (Experiment 1, Experiment 2), except that the alginate derivative was changed to unmodified sodium alginate. For commercially available adhesive materials, the experiment was conducted using the same procedure as above (Expe...

Claims

1. A tissue adhesive material for use in rigid, thick tissues and / or connective tissue, comprising a polysaccharide derivative, The aforementioned polysaccharide derivative is (i) A polysaccharide derivative in which a group represented by the following formula (A-1) is introduced into an acidic, basic, or amphoteric polysaccharide containing a carboxyl group, wherein the group represented by formula (A-1) is introduced into the polysaccharide by substituting the -OH of the carboxyl group of the polysaccharide to form an amide bond, or (ii) A polysaccharide derivative in which a group represented by the following formula (A-2) is introduced into an acidic, basic, or amphoteric polysaccharide containing an amino group, wherein the group represented by formula (A-2) is introduced into the polysaccharide by substituting a hydrogen atom of the amino group of the polysaccharide and forming an amide bond. The polysaccharide is selected from alginic acid, its derivatives or salts thereof, hyaluronic acid, its derivatives or salts thereof, carboxymethylcellulose, its derivatives or salts thereof, carboxymethyl dextran, its derivatives or salts thereof, carboxymethyl starch, its derivatives or salts thereof, heparin, its derivatives or salts thereof, heparan sulfate, its derivatives or salts thereof, chondroitin sulfate, its derivatives or salts thereof, dermantan sulfate, its derivatives or salts thereof, chitosan, its derivatives or salts thereof, regenerated oxidized cellulose, its derivatives or salts thereof, and pectinic acid, its derivatives or salts thereof, as a tissue adhesive material. 【Transformation 73】 (In equations (A-1) and (A-2), R １ is a hydrogen atom or C １-４ Represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or pyridine ring is a halogen atom, -CF ３ , -NO ２ carboxyl group and -SO ３ It may be substituted with one or more substituents independently selected from H, L1 is selected from a single bond, C1-6 alkylene, and -(CH2CH2O)n-, L2 represents a divalent group selected from the group consisting of a single bond, -NH-, -S-, -O-, C1-6 alkylene, and -(CH2CH2O)n-. n is an integer from 1 to 9. * indicates the linkage with polysaccharides.

2. The aforementioned polysaccharide is alginic acid, its derivatives, or salts thereof. The tissue-adhering material according to claim 1, wherein the polysaccharide derivative comprises a structural unit represented by the following formula (c11) and / or (c12). 【Chemistry 74】 (wherein, R １１ , R １２ , R １３ , and R １４ are each independently selected from a hydrogen atom, C １－６ alkyl, and -C(=O)-C １－６ alkyl, R １ is a hydrogen atom or C １－４ Represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or pyridine ring is a halogen atom, -CF ３ , -NO ２ carboxyl group and -SO ３ It may be substituted with one or more substituents independently selected from H, Y is -L １ -NH- represents, and in this case L １ It is bonded to ring P, L １ C is a single bond. １－６ Alkylene and -(CH ２ CH ２ O) n - Selected from, n is an integer between 1 and 9.

3. The group represented by the above formula (A-1) is 【Chemistry 75】 (In the formula, * represents the linkage with the polysaccharide.) A tissue-adhesive material according to claim 1 or 2, selected from the group consisting of the following.

4. A tissue adhesive material for use in rigid, thick tissues and / or connective tissue, comprising a crosslinked structure in which at least a portion of the polysaccharide derivative is crosslinked, The aforementioned polysaccharide derivative is (i) A polysaccharide derivative in which a group represented by the following formula (A-1) is introduced into an acidic, basic, or amphoteric polysaccharide containing a carboxyl group, wherein the group represented by formula (A-1) is introduced into the polysaccharide by substituting the -OH of the carboxyl group of the polysaccharide to form an amide bond, or (ii) A polysaccharide derivative in which a group represented by the following formula (A-2) is introduced into an acidic, basic, or amphoteric polysaccharide containing an amino group, wherein the group represented by formula (A-2) is introduced into the polysaccharide by substituting a hydrogen atom of the amino group of the polysaccharide and forming an amide bond. The polysaccharide is selected from alginic acid, its derivatives or salts thereof, hyaluronic acid, its derivatives or salts thereof, carboxymethylcellulose, its derivatives or salts thereof, carboxymethyl dextran, its derivatives or salts thereof, carboxymethyl starch, its derivatives or salts thereof, heparin, its derivatives or salts thereof, heparan sulfate, its derivatives or salts thereof, chondroitin sulfate, its derivatives or salts thereof, dermantan sulfate, its derivatives or salts thereof, chitosan, its derivatives or salts thereof, regenerated oxidized cellulose, its derivatives or salts thereof, and pectinic acid, its derivatives or salts thereof, as a tissue adhesive material. 【Transformation 73】 (In equations (A-1) and (A-2), R1 represents a hydrogen atom or a C1-4 alkyl group. Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or pyridine ring may be substituted with one or more substituents independently selected from a halogen atom, -CF3, -NO2, a carboxyl group, and -SO3H. L1 is selected from a single bond, C1-6 alkylene, and -(CH2CH2O)n-, L2 represents a divalent group selected from the group consisting of a single bond, -NH-, -S-, -O-, C1-6 alkylene, and -(CH2CH2O)n-. n is an integer from 1 to 9. * indicates the linkage with polysaccharides.

5. A tissue-adhering material according to any one of claims 1 to 4, which is in the form of a gel, sponge, sheet, or film.

6. The tissue-adhering material according to any one of claims 1 to 4, further comprising a nonwoven fabric, a sheet, or a film.

7. (a) Polysaccharide derivatives, (b) an amino group-containing polymer and an amino group-containing low molecular weight compound comprising two or more primary amino groups, hydrazide groups, or aminooxy groups and a group represented by the following formula (x1), The aforementioned polysaccharide derivative is (i) A polysaccharide derivative in which a group represented by the following formula (A-1) is introduced into an acidic, basic, or amphoteric polysaccharide containing a carboxyl group, wherein the group represented by formula (A-1) is introduced into the polysaccharide by substituting the -OH of the carboxyl group of the polysaccharide to form an amide bond, or (ii) A polysaccharide derivative in which a group represented by the following formula (A-2) is introduced into an acidic, basic, or amphoteric polysaccharide containing an amino group, wherein the group represented by formula (A-2) is introduced into the polysaccharide by substituting a hydrogen atom of the amino group of the polysaccharide and forming an amide bond. z The polysaccharide is selected from alginic acid, its derivatives or salts thereof, hyaluronic acid, its derivatives or salts thereof, carboxymethylcellulose, its derivatives or salts thereof, carboxymethyl dextran, its derivatives or salts thereof, carboxymethyl starch, its derivatives or salts thereof, heparin, its derivatives or salts thereof, heparan sulfate, its derivatives or salts thereof, chondroitin sulfate, its derivatives or salts thereof, dermantane sulfate, its derivatives or salts thereof, chitosan, its derivatives or salts thereof, regenerated oxidized cellulose, its derivatives or salts thereof, and pectinic acid, its derivatives or salts thereof. A crosslinked structure in which a primary amino group, hydrazide group, or aminooxy group contained in the amino group-containing polymer and amino group-containing low molecular weight compound is crosslinked via a Schiff base between the group represented by formula (A-1) or formula (A-2) contained in the polysaccharide derivative. 【Transformation 73】 (In equations (A-1) and (A-2), R1 represents a hydrogen atom or a C1-4 alkyl group. Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or pyridine ring may be substituted with one or more substituents independently selected from a halogen atom, -CF3, -NO2, a carboxyl group, and -SO3H. L1 is selected from a single bond, C1-6 alkylene, and -(CH2CH2O)n-, L2 represents a divalent group selected from the group consisting of a single bond, -NH-, -S-, -O-, C1-6 alkylene, and -(CH2CH2O)n-. n is an integer from 1 to 9. * indicates the linkage with polysaccharides. 【Transformation 78】 (In equation (x1), k is an integer between 2 and 5, L ５ These are single bonds, or -C(=O)-, -S-, -O-, alkylene, and -(CH ２ CH ２ O) ｎ - A divalent group selected from the group consisting of, n is an integer from 1 to 9, * indicates a linkage with an amino group-containing polymer or amino group-containing low-molecular-weight compound.

8. The crosslinked structure according to claim 7, wherein the amino group-containing polymer is at least one selected from linear, branched, or dendritic polyamines; polyalkylene glycols substituted with amino groups, hydrazide groups, or aminooxy groups; polyallylamines; polyvinylamines; polyacrylamines; amino group-containing polysaccharides; amino group-containing proteins; and polyamino acids.

9. The aforementioned polysaccharide is alginic acid, its derivatives, or salts thereof. The crosslinked structure according to claim 7 or 8, wherein the polysaccharide derivative comprises a constituent unit represented by the following formula (c11) and / or (c12). 【Chemistry 79】 (In the formula, R １１ , R １２ , R １３ , and R １４ These are, independently, hydrogen atoms and C １－６ Alkyl and -C(=O)-C １－６ Selected from alkyl groups, R １ is a hydrogen atom or C １－４ Represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or pyridine ring is a halogen atom, -CF ３ , -NO ２ carboxyl group and -SO ３ It may be substituted with one or more substituents independently selected from H, Y is -L １ -NH- represents, and in this case L １ It is bonded to ring P, L １ C is a single bond. １－６ Alkylene and -(CH ２ CH ２ O) n - Selected from, n is an integer between 1 and 9.

10. A tissue-adhesive material comprising a crosslinked structure according to any one of claims 7 to 9.

11. A tissue-adhesive material, The polysaccharide derivative includes a polysaccharide containing an amino group into which a group represented by the following formula (A-2) has been introduced. The group represented by formula (A-2) is introduced into the polysaccharide by substituting a hydrogen atom of the amino group of the polysaccharide, thereby forming an amide bond. The aforementioned polysaccharide is chitosan, its derivatives, or salts thereof. A tissue-adhering material in gel form. 【Chemistry 80】 (In equation (A-2), R １ is a hydrogen atom or C １-４ Represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or pyridine ring is a halogen atom, -CF ３ , -NO ２ carboxyl group and -SO ３ It may be substituted with one or more substituents independently selected from H, L ２ It is either a single bond or -NH-, -S-, -O-, C １―６ Alkylene and -(CH ２ CH ２ O) n Represents a divalent group selected from the group consisting of - n is an integer from 1 to 9, * indicates the linkage with polysaccharides.

12. Shear rate of 0.001 s, measured at 25°C. －１ viscosity (η) ０．００１ ) and shear rate 1000 s －１ viscosity (η) １０００ ) ratio (η ０．００１ / η １０００ ) is 10-10 ９ The composition according to claim 11.

13. The tissue-adhering material according to claim 11 or 12, wherein the polysaccharide derivative is crosslinked by forming a covalent bond between the amino group contained in the polysaccharide derivative and the group represented by formula (A-2) via a Schiff base, thereby forming a crosslinked structure.

14. The tissue-adhering material according to any one of claims 11 to 13, wherein the polysaccharide derivative includes a structural unit represented by the following formula (c16). 【Chemistry 81】 (In the formula, R ８１ and R ８２ These are, independently, hydrogen atoms and C １－６ Alkyl and -C(=O)-C １－６ Selected from alkyl groups, R １ is a hydrogen atom or C １－４ Represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or pyridine ring is a halogen atom, -CF ３ , -NO ２ carboxyl group and -SO ３ It may be substituted with one or more substituents independently selected from H, Y is -L ２ -C (=O)- represents, and in this case L ２ It is bonded to ring P, L ２ is selected from single bond, C １―６ alkylene, -(CH ２ CH ２ O) n -, -(CH ２ ) ｍ１ -(CH ２ CH ２ O) n -(CH ２ ) ｍ２ -, and -(CH ２ ) ｍ１ -O-(CH ２ CH ２ O) n -(CH ２ ) ｍ２ - and is selected from n is an integer from 1 to 9, m1 and m2 are each independent integers between 1 and 9.

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