JP2018188561A - Denatured polysaccharide and use therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a denatured polysaccharide in which a hydroxy aryl group is introduced into a polysaccharide, which facilitates the oxidation coupling efficiency of the hydroxy aryl group and allows the gelation.SOLUTION: The present invention provides a denatured polysaccharide in which (a1) a group having a carbon-carbon unsaturated double bond and (a2) a group having a hydroxy aryl group are introduced into a polysaccharide.SELECTED DRAWING: None

Description

  The present invention relates to modified polysaccharides and uses thereof.

  When a part of living tissue such as cartilage and bone is damaged, the damaged part is replaced with an artificial medical instrument using hydrogel. As such hydrogels, the following polymer networks (gels) are known. That is, a polymer network formed by oxidative coupling of a modified polysaccharide in which a hydroxyphenyl group is introduced into a polysaccharide using an enzyme such as horseradish peroxidase and hydrogen peroxide as a catalyst system at the hydroxyphenyl group ( Gel) (see Patent Documents 1 to 4).

U.S. Pat. No. 8,287,906 US Pat. No. 6,982,298 US Published Patent No. 2012/301441 US Pat. No. 9,132,201

  According to the study of the present inventors, in a modified polysaccharide in which a hydroxyaryl group is introduced into a polysaccharide, the efficiency of oxidative coupling of the hydroxyaryl group is poor depending on the introduction site of the hydroxyaryl group into the polysaccharide and the type of the polysaccharide. It became clear that gelation was impossible.

  An object of the present invention is to provide a modified polysaccharide in which a hydroxyaryl group is introduced into a polysaccharide, whereby the efficiency of oxidative coupling of the hydroxyaryl group is increased and gelled, and the modified polysaccharide contains the modified polysaccharide. It is providing the composition obtained, the gel obtained from the said modified polysaccharide, the molded object formed from the said gel, and the medical device which has the said molded object.

The present inventor has intensively studied to solve the above problems. As a result, it has been found that a modified polysaccharide having the following constitution can solve the above-mentioned problems, and the present invention has been completed.
The present invention includes, for example, the following [1] to [10].

[1] A modified polysaccharide in which a group (a1) having a carbon-carbon unsaturated double bond and a group (a2) having a hydroxyaryl group are introduced into the polysaccharide.
[2] The modified polysaccharide according to [1], wherein the polysaccharide is hyaluronic acid or a derivative thereof.
[3] The modified polysaccharide according to [1] or [2], wherein the group (a2) having the hydroxyaryl group is a group represented by the following formula (2).

［式（２）中、＊は、多糖中の水酸基残基との結合手であり、Ｒ¹は、炭素数１〜２０のアルカンジイル基または−（ＣＨ₂）_n−Ｏ−（ＣＨ₂）_m−で示される２価の基であり、ｎおよびｍは、それぞれ独立に１〜１０の整数を示し；Ｒ²〜Ｒ⁶は、それぞれ独立に水素原子、水酸基、アルコキシ基、または置換基を有してもよいアルキル基であり、ただしＲ²〜Ｒ⁶のうち少なくとも１つは水酸基である。］

[In formula (2), * is a bond with a hydroxyl group residue in the polysaccharide, and R ¹ is an alkanediyl group having 1 to 20 carbon atoms or — (CH ₂ ) _n —O— (CH ₂ ). _{m- represents a} divalent group, and n and m each independently represent an integer of 1 to 10; R ^{2 to} R ⁶ each independently represents a hydrogen atom, a hydroxyl group, an alkoxy group, or a substituent. It is an alkyl group which may have, provided that at least one of R ^{2 to} R ⁶ is a hydroxyl group. ]

[4] The modified polysaccharide according to any one of [1] to [3], wherein the group (a1) having a carbon-carbon unsaturated double bond is a (meth) acryloyl group.
[5] A composition comprising the modified polysaccharide according to any one of [1] to [4], an enzyme, and water.
[6] The composition according to [5], further containing a peroxide.
[7] A gel having a crosslinked structure by oxidative coupling of a hydroxyaryl group contained in the modified polysaccharide according to any one of [1] to [4].
[8] The gel according to [7], which is a hydrogel containing 50 to 99.9% by mass of moisture.
[9] A molded article containing 50% by mass or more of the gel according to [7] or [8].
[10] A medical instrument having the molded article according to [9].

  ADVANTAGE OF THE INVENTION According to this invention, in the modified polysaccharide by which the hydroxyaryl group was introduce | transduced into the polysaccharide, the efficiency of the oxidative coupling of a hydroxyaryl group can be improved, and the modified polysaccharide which can be gelatinized can be provided, and also contains the said modified polysaccharide. The composition obtained, the gel obtained from the modified polysaccharide, the molded product formed from the gel, and the medical device having the molded product can be provided.

FIG. 1 is a ¹ H NMR chart of the modified polysaccharide obtained in Synthesis Example 1. FIG. 2 is a ¹ H NMR chart of the modified polysaccharide obtained in Example 1. FIG. 3 is a ¹ H NMR chart of the modified polysaccharide obtained in Example 2.

DESCRIPTION OF EMBODIMENTS Hereinafter, embodiments for carrying out the present invention will be described including preferred embodiments.
[Modified polysaccharide]
The modified polysaccharide of the present invention is obtained by introducing a group (a1) having a carbon-carbon unsaturated double bond and a group (a2) having a hydroxyaryl group into the polysaccharide. Since the modified polysaccharide is more efficiently oxidatively coupled to the hydroxyaryl group than the conventional modified polysaccharide into which only the hydroxyaryl group is introduced, a gel of the modified polysaccharide can be obtained.

  As will be described later, a compound having a functional group such as an alcoholic hydroxyl group, a carboxy group, and an amino group and a group capable of reacting with the functional group and a carbon-carbon unsaturated double bond or a hydroxyaryl group, as described later. By reacting, the groups (a1) and (a2) can be introduced into the polysaccharide.

<Polysaccharide>
Examples of polysaccharides before modification, that is, before introduction of the groups (a1) and (a2) include, for example, hyaluronic acid, alginic acid, xanthan gum, cellulose, guar gum, pullulan, dextran, fructan, mannan, carrageenan, chitin, chitosan, pectin, starch Natural polysaccharides such as glycogen, dextrin and gellan gum; and derivatives of said natural polysaccharides.

  Examples of the natural polysaccharide derivatives include groups other than the group (a1) having a carbon-carbon unsaturated double bond and the group (a2) having a hydroxyaryl group, such as a methyl group and an ethyl group. , Derivatives having a substituent such as hydroxyethyl group, hydroxypropyl group, hydroxyethylmethyl group, hydroxypropylmethyl group, carboxymethyl group, acetyl group, stearoxy group, glycerol, propylene glycol and the like. The derivative may have the substituent alone or in combination.

  Specific examples of the polysaccharide having a substituent include acetylated hyaluronic acid, propylene glycol hyaluronate, propylene glycol alginate, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, stearate. Examples include roxyhydroxypropyl methylcellulose, methyl guar gum, ethyl guar gum, hydroxyethyl guar gum, hydroxypropyl guar gum, hydroxyethyl methyl guar gum, and hydroxypropyl methyl guar gum.

  The polysaccharide may be a salt, and examples thereof include acid addition salts, metal salts, and ammonium salts. Examples of the acid addition salt include inorganic acid salts such as hydrochloride, sulfate, and phosphate, and organic acid salts such as acetate, maleate, fumarate, tartrate, and citrate. Examples of the metal salt include alkali metal salts such as sodium salt and potassium salt, Group 2 metal salts of the periodic table such as calcium salt and magnesium salt, aluminum salt and zinc salt. Specifically, the metal salt of the polysaccharide which has a carboxy group is mentioned.

  Among the polysaccharides, hyaluronic acid, dextran and pullulan, and derivatives thereof are preferable, and hyaluronic acid and derivatives thereof are more preferable because the group (a2) having a hydroxyaryl group can be easily introduced into the polysaccharide. In the modified hyaluronic acid and derivatives thereof in which only the group (a2) having a hydroxyaryl group was introduced, for example, the horseradish peroxidase / hydrogen peroxide catalyst system was thickened but could not be gelled. Any modified hyaluronic acid and derivative thereof into which a group (a1) having a carbon unsaturated double bond has been introduced can be gelled by the catalyst system.

<Group (a1) having carbon-carbon unsaturated double bond>
The modified polysaccharide of the present invention contains a group (a1) having a carbon-carbon unsaturated double bond.
The carbon-carbon unsaturated double bond is preferably a radically polymerizable double bond, for example, as long as it is contained in the modified polysaccharide as a (meth) acryloyl group, a vinyl group or the like, and the (meth) acryloyl group is Preferably, an acryloyl group is more preferable. The reason why the gel formation proceeds well in the present invention is not clear, but if the polysaccharide is partially modified with a (meth) acryloyl group, a vinyl group or the like that can be radically polymerized and has a small steric hindrance, It is presumed that the unsaturated double bond is first cross-linked to bring the hydroxyaryl groups closer to each other, and therefore the oxidative coupling of the hydroxyaryl group can proceed more efficiently.

  The group (a1) is preferably a group ((meth) acryloyl group) represented by the following formula (1). For example, the modified polysaccharide of the present invention includes a group represented by the formula (1) bonded to a hydroxyl group residue in the polysaccharide.

式（１）中、Ｒ^Aは水素原子またはメチル基を示す。
＊は、多糖中の水酸基残基（−Ｏ−）との結合手である。例えば、ヒアルロン酸を構成するＮ−アセチルグルコサミン単位中のヒドロキシメチル基における水酸基残基と、式（１）に示す基とが結合していることが好ましい。

In the formula (1), R ^A represents a hydrogen atom or a methyl group.
* Is a bond with a hydroxyl group residue (—O—) in the polysaccharide. For example, it is preferable that the hydroxyl group residue in the hydroxymethyl group in the N-acetylglucosamine unit constituting hyaluronic acid is bonded to the group represented by the formula (1).

  As a method for introducing a group (a1) having a carbon-carbon unsaturated double bond into a polysaccharide, for example, a polysaccharide having a functional group such as an amino group, an alcoholic hydroxyl group and a carboxy group can react with the functional group. A compound having a functional group (eg, carboxy group, acid halide thereof, amino group, alcoholic hydroxyl group, epoxy group, oxetanyl group) and a carbon-carbon unsaturated double bond (hereinafter referred to as “double bond-containing compound (11)”) (Also called).

  The double bond-containing compound (11) has a carboxy group or an acid halide thereof (group represented by —COX (X is a halogen atom such as a chlorine atom)) capable of reacting with an alcoholic hydroxyl group possessed by many polysaccharides. Compounds are preferred, for example, unsaturated monocarboxylic acids such as (meth) acrylic acid and crotonic acid; unsaturated dicarboxylic acids such as maleic acid, fumaric acid, citraconic acid, mesaconic acid and itaconic acid; and anhydrides of the unsaturated dicarboxylic acids A mono [(meth) acryloyloxyalkyl] ester of a polyvalent carboxylic acid such as mono [2- (meth) acryloyloxyethyl] succinate, mono [2- (meth) acryloyloxyethyl] phthalate, and the like; Acid halides such as (meth) acrylic acid chloride are mentioned.

  Other examples of the double bond-containing compound (11) include glycidyl (meth) acrylate, 2-methylglycidyl (meth) acrylate, 3,4-epoxybutyl (meth) acrylate, and (meth) acrylic acid 6,7. Epoxy group-containing (meth) acrylates such as epoxyheptyl, 3,4-epoxycyclohexylmethyl (meth) acrylate; o, m or p-vinylphenylglycidyl ether, o, m or p-isopropenylphenylglycidyl ether, Epoxy group-containing vinyl compounds such as o, m or p-vinylbenzylglycidyl ether, o, m or p-isopropenylbenzylglycidyl ether; 3-((meth) acryloyloxymethyl) oxetane, 3-((meth) acryloyloxy Methyl) -2-methyloxetane, 3-((meth) ac Royloxymethyl) -3-ethyloxetane, 3-((meth) acryloyloxymethyl) -2-phenyloxetane and other oxetanyl group-containing (meth) acrylates; 3- (p-vinylphenyloxymethyl) -3-methyloxetane , Oxetanyl group-containing vinyl compounds such as 3- (p-isopropenylphenyloxymethyl) -3-methyloxetane; 2-hydroxyethyl (meth) acrylate, 3-hydroxypropyl (meth) acrylate, (meth) acrylic acid Examples include hydroxyalkyl (meth) acrylates such as 4-hydroxybutyl.

  For example, a polysaccharide and a double bond-containing compound (11) containing a polysaccharide and a double bond-containing compound (11) or a hydroxyaryl group-containing group (a2) in the presence or absence of a basic compound. And react. The reaction temperature of the reaction is usually −10 to 50 ° C., preferably −5 to 30 ° C., and the reaction time is usually 1 to 48 hours.

Examples of the basic compound include trimethylamine, triethylamine, tributylamine, pyridine, N, N-dimethyl-4-aminopyridine, and piperidine.
In the reaction, the double bond-containing compound (11) is usually 0.1 to 50 parts by mass with respect to 100 parts by mass of the polysaccharide or 100 parts by mass of the modified polysaccharide containing the group (a2) having a hydroxyaryl group. Preferably it is 0.3-20 mass parts, More preferably, it can be used in the amount of 0.5-10 mass parts.

  The reaction is preferably performed in a solvent. Examples of the solvent include water, methanol, ethanol, isopropyl alcohol, N, N′-dimethylformamide, dimethyl sulfoxide, and dimethylacetamide. The reaction may be performed in an inert gas atmosphere such as nitrogen or argon.

<Group (a2) having hydroxyaryl group>
The modified polysaccharide of the present invention includes a group (a2) having a hydroxyaryl group.
As a hydroxyaryl group, C6-C14 hydroxyaryl groups, such as a hydroxyphenyl group and a hydroxy naphthyl group, are mentioned, for example. The number of hydroxyl groups in the hydroxyaryl group is usually 1 to 3, preferably 1. Further, the hydroxyaryl group may have an alkoxy group, an alkyl group which may have a substituent, or the like in addition to the hydroxyl group.

  The group (a2) is preferably a group represented by the following formula (2). For example, the modified polysaccharide of the present invention includes a group represented by the formula (2) bonded to a hydroxyl group residue in the polysaccharide.

式（２）中の各記号について以下に説明する。
＊は、多糖中の水酸基残基（−Ｏ−）との結合手である。例えば、ヒアルロン酸を構成するＮ−アセチルグルコサミン単位中のヒドロキシメチル基における水酸基残基と、式（２）に示す基とが結合していることが好ましい。

Each symbol in the formula (2) will be described below.
* Is a bond with a hydroxyl group residue (—O—) in the polysaccharide. For example, it is preferable that the hydroxyl group residue in the hydroxymethyl group in the N-acetylglucosamine unit constituting hyaluronic acid is bonded to the group represented by the formula (2).

R ¹ is an alkanediyl group having 1 to 20 carbon atoms or a divalent group represented by — (CH ₂ ) _n —O— (CH ₂ ) _m —, and n and m are each independently 1 to 10 Indicates an integer.
Examples of the alkanediyl group include methane-1,1-diyl group, ethane-1,1-diyl group, ethane-1,2-diyl group, propane-1,1-diyl group, propane-1,2- Diyl group, propane-1,3-diyl group, propane-2,2-diyl group, butane-1,2-diyl group, butane-1,3-diyl group, butane-1,4-diyl group, pentane- 1,4-diyl group, pentane-1,5-diyl group, hexane-1,5-diyl group, hexane-1,6-diyl group, heptane-1,7-diyl group, octane-1,8-diyl Groups. Carbon number of alkanediyl group becomes like this. Preferably it is 1-10, More preferably, it is 2-5.

R ^{2 to} R ⁶ are each independently a hydrogen atom, a hydroxyl group, an alkoxy group, or an alkyl group that may have a substituent, provided that at least one of R ^{2 to} R ⁶ is a hydroxyl group. It is preferable that only R ⁴ is a hydroxyl group among R ^{2 to} R ⁶ because oxidative coupling proceeds preferentially in the gelation described later and a gel superior in mechanical strength is obtained. It is preferable that a hydrogen atom is bonded to a carbon atom in at least one ortho position with respect to the hydroxyl group.

As an alkoxy group, C1-C6 alkoxy groups, such as a methoxy group, an ethoxy group, a propoxy group, a butoxy group, a pentyloxy group, a hexyloxy group, are mentioned, for example.
As an alkyl group, C1-C6 alkyl groups, such as a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, are mentioned, for example. One or more hydrogen atoms in the alkyl group may be substituted. Examples of the substituent include halogen atoms such as fluorine atom, chlorine atom and bromine atom, phenyl group, hydroxyl group, acetoxy group, alkoxy group, carboxy group, A cyano group and a nitro group are mentioned.

Of the R ^{2 to} R ⁶ , the remaining group other than the hydroxyl group is preferably a hydrogen atom or an alkoxy group, and more preferably a hydrogen atom.
Examples of a method for introducing a group (a2) having a hydroxyaryl group into a polysaccharide include a functional group capable of reacting with the functional group (for example, a polysaccharide having a functional group such as an amino group, an alcoholic hydroxyl group and a carboxy group). And a method of reacting a compound having a carboxy group, an acid halide thereof, an amino group, an alcoholic hydroxyl group, an epoxy group, an oxetanyl group) and a hydroxyaryl group. For example, by reacting a hydroxyl group in a polysaccharide with —COX in a compound represented by the following formula (21) (hereinafter also referred to as “compound (21)”), a hydroxyphenyl group derived from the compound (21) is converted into an ester. It can be introduced into a polysaccharide via a bond.

式（２１）中、Ｒ¹〜Ｒ⁶は式（２）中の同一記号と同義である。Ｘは、−ＯＨまたはハロゲン原子である。ハロゲン原子としては、例えば、フッ素原子、塩素原子、臭素原子が挙げられ、塩素原子が好ましい。

In formula (21), R < ¹ > -R < ⁶ > is synonymous with the same symbol in Formula (2). X is —OH or a halogen atom. As a halogen atom, a fluorine atom, a chlorine atom, and a bromine atom are mentioned, for example, A chlorine atom is preferable.

  Examples of the compound (21) include 4-hydroxyphenylacetic acid, 3- (4-hydroxyphenyl) propionic acid, 4-hydroxyphenylbutanoic acid, 2- (2,5-dihydroxyphenyl) acetic acid, and acid halides thereof. A compound.

  For example, the polysaccharide and the compound (21) or the modified polysaccharide containing the group (a1) having a carbon-carbon unsaturated double bond and the compound (21) are reacted. For example, the solvent is distilled off from the solution containing the polysaccharide or the modified polysaccharide containing the group (a1) and the compound (21), and then the residue is subjected to heat dehydration condensation. The solvent is distilled off, for example, under reduced pressure, usually at 0 to 150 ° C., preferably at 20 to 80 ° C. Heat dehydration condensation is performed by heating the residue after evaporation of the solvent under reduced pressure, and the heating temperature here is usually 50 to 150 ° C., preferably 80 to 130 ° C., and the reaction time is usually 1 -92 hours, preferably 2-48 hours. The decompression condition is, for example, less than 760 mmHg, preferably 1 to 500 mmHg. The product after heat dehydration condensation may be dissolved in water and purified by reprecipitation with a poor solvent such as ethanol, ethyl acetate, or n-hexane.

  In the reaction, the compound (21) is usually 1 to 100 parts by mass, preferably 100 parts by mass of the polysaccharide or 100 parts by mass of the modified polysaccharide containing a group (a1) having a carbon-carbon unsaturated double bond. Can be used in an amount of 5 to 80 parts by mass, more preferably 10 to 60 parts by mass.

  The reaction is preferably performed in a solvent. Examples of the solvent include water, methanol, ethanol, isopropyl alcohol, N, N′-dimethylformamide, dimethyl sulfoxide, and dimethylacetamide. The reaction may be performed in an inert gas atmosphere such as nitrogen or argon.

<Preferred embodiment>
The modified polysaccharide of the present invention is preferably a modified polysaccharide in which a (meth) acryloyl group and a hydroxyphenyl group are introduced into the polysaccharide, and more preferably a modified polysaccharide in which the above group is introduced into hyaluronic acid, dextran and pullulan, and derivatives thereof. Particularly preferred are modified polysaccharides in which the above groups are introduced into hyaluronic acid and its derivatives.

In the modified polysaccharide of the present invention, a group (a1) having a carbon-carbon unsaturated double bond is introduced into the polysaccharide. The degree of substitution (DS _D ) by the group (a1) in the modified polysaccharide, preferably the degree of substitution (DS _A ) by the group ((meth) acryloyl group) shown in the formula (1) is usually 0.01-30. Yes, preferably 0.1-15, more preferably 0.5-10.

In the modified polysaccharide of the present invention, a group (a2) having a hydroxyaryl group is introduced into the polysaccharide. The degree of substitution (DS _Ar ) by the group (a2) in the modified polysaccharide, preferably the degree of substitution (DS _P ) by the group shown in the formula (2) is usually 0.1 to 40, preferably 0.5 -20, more preferably 1-15.

The ratio of the degree of substitution (DS _Ar) and degree of substitution _{(DS D) (DS Ar /} DS D) is preferably 0.1 to 20, more preferably from 0.5 to 10.
The degree of substitution can be determined from ¹ H NMR. The degree of substitution of the modified polysaccharide indicates the average number of groups (for example, the group (a1) or (a2)) introduced per 100 units of the constituent monosaccharide of the modified polysaccharide. Since the modified polysaccharide has such a substitution degree, the gelation rate of the modified polysaccharide is improved.

  The degree of substitution of the modified polysaccharide can be controlled, for example, by changing the reaction time, reaction temperature, and concentration of each reaction component described above. For example, the substitution degree can be lowered by shortening the reaction time, lowering the reaction temperature, or lowering the concentration of each reaction component.

  The number average molecular weight by the absolute molecular weight measurement of the modified polysaccharide of the present invention is usually 1,000 to 10,000,000, preferably 3,000 to 1,000,000, more preferably 5,000 to 500,000. is there.

[Composition]
The composition of the present invention contains the above-described modified polysaccharide, an enzyme, and water, and preferably further contains a peroxide. One embodiment of the composition comprises a first solution comprising a modified polysaccharide, comprising either a peroxide and an enzyme, but not both, and a first solution of the aforementioned peroxide and enzyme. And a second solution including the one not included.

  The composition of the present invention contains the above-described modified polysaccharide. In the composition, the content of the modified polysaccharide is usually 0.05 to 40% by mass, preferably 0.2 to 30% by mass. The modified polysaccharides can be used alone or in combination of two or more.

  The composition of the present invention preferably contains a peroxide. An example of the peroxide is hydrogen peroxide. In the composition, the amount of peroxide is usually 0.01 to 1000 mol, preferably 0.1 to 500 mol, more preferably 0.5 to 1 mol of phenolic hydroxyl group in the modified polysaccharide. ~ 200 moles. The phenolic hydroxyl group is, for example, a phenolic hydroxyl group contained in a hydroxyaryl group in the group (a2).

  The composition of the present invention contains an enzyme. Examples of the enzyme include phenol oxidases such as laccase and tyrosinase, catalase, and peroxidase. Laccase and tyrosinase are excellent in that there is no possibility that the function-expressing substance is denatured during gelation because there is no need to add any other chemicals that do not constitute gel components. Peroxidase can be instantly gelled by using it together with a peroxide such as hydrogen peroxide. Examples of the origins of copper enzymes such as laccase and tyrosinase include urushi, mushrooms (Tuchikaburi, mushrooms), and molds (Polyporus vericolor). The origin of catalase and peroxidase is, for example, bovine liver, horse blood cells, human blood cells, M. lysodeikticus, horseradish (horseradish), soybean, radish, turnip, thyroid, milk, intestine, leukocytes, erythrocytes, yeast, Caldariomyces fumago, Steptococcus faecalis. Among these, tyrosinase is preferably derived from mushrooms and peroxidase is preferably derived from horseradish.

In the present invention, from the viewpoint of proceeding with the crosslinking reaction of the carbon-carbon unsaturated double bond contained in the modified polysaccharide, it is preferable to use a peroxide and at least one or more selected from peroxidase and catalase as an enzyme. It is more preferable to use at least.
Enzymes can be used alone or in combination of two or more.

  In the composition of the present invention, the amount of the enzyme is preferably 0.01 U / mL or more, more preferably 1 U / mL or more; 1,000 U / mL or less, more preferably 500 U / mL or less. U represents a unit of enzyme activity, and is the amount of enzyme that can change 1 micromole of substrate per minute at a temperature of 30 ° C. under optimum conditions.

  The composition of the present invention contains water. The composition of the present invention may further contain sodium chloride. Thus, the composition of the present invention may contain an aqueous sodium chloride solution such as physiological saline.

  The water content of the composition of the present invention is usually 50 to 99.9% by mass, preferably 60 to 99% by mass, and more preferably 70 to 98% by mass. With such an embodiment, the mechanical strength of the resulting gel tends to be excellent.

  The composition of the present invention may further contain a polysaccharide other than the modified polysaccharide. As this polysaccharide, the polysaccharide described in the column of [modified polysaccharide] is mentioned, for example. Polysaccharides can be used alone or in combination of two or more.

  The composition of this invention may contain the function expression substance according to the use of the gel. Thereby, a function expression substance can be included in the gel obtained. Examples of the function expressing substance include drugs and cells. Gels containing a function-expressing substance can be used in a wide range of fields such as medical instruments.

  The composition of this invention can be used suitably as a raw material composition of the gel demonstrated below. In addition, the composition of the present invention can be used, for example, as a lubricant in a joint cavity used for the treatment of osteoarthritis or a damage covering material. Osteoarthritis can occur in each joint of the body such as knees, shoulders, hips, hips, ankles, wrists, and fingers, but the composition of the present invention can be applied to any joint.

[gel]
The gel of the present invention has a crosslinked structure by oxidative coupling of hydroxyaryl groups contained in the above-described modified polysaccharide, that is, contains a crosslinked product of the modified polysaccharide. Details of the oxidative coupling will be described later. Moreover, it is preferable that the gel of this invention also has the crosslinked structure by addition polymerization of the carbon-carbon unsaturated double bond contained in the modified polysaccharide mentioned above in one embodiment.

  In one embodiment, the gel is a hydrogel containing water in an amount of usually 50 to 99.9% by mass, preferably 60 to 99% by mass, more preferably 70 to 98% by mass. The hydrogel usually contains 50 to 0.1% by mass, preferably 40 to 1% by mass, and more preferably 30 to 2% by mass of the crosslinked polysaccharide. The hydrogel is excellent in mechanical strength and flexibility.

  The hydrogel of the present invention can be obtained by mixing a modified polysaccharide and an enzyme in water and crosslinking the modified polysaccharide as described later. Moreover, a xerogel can be obtained by removing the water | moisture content by drying the said hydrogel suitably. Alternatively, the gel of the present invention is gelled by light irradiation using a photosensitizer such as eosin Y, methylene blue, or rose bengal together with a modified polysaccharide as described in Macromolecules (2015) 2624-2630, for example. It can also be obtained.

  A gel can be formed by using the composition of the present invention. For example, in one embodiment, the modified polysaccharide, a peroxide such as hydrogen peroxide, and an enzyme are mixed in water to crosslink the modified polysaccharide to form a hydrogel. Here, it is presumed that addition polymerization of carbon-carbon unsaturated double bonds in the modified polysaccharide proceeds, further, the hydroxyaryl group is oxidized, oxidative coupling (crosslinking) between the groups occurs, and gelation proceeds. Is done. In more detail about oxidative coupling, for example, when a hydroxyaryl group of a modified polysaccharide is reacted with a peroxide in the presence of an enzyme, a hydrogen atom is extracted from the phenolic hydroxyl group to form a free radical species, which is isomerized. It is considered that a free-radical species in which unpaired electrons exist on the carbon at the ortho position is formed, and then the two free-radical species are combined and then enolized to form a crosslinked structure.

  In one embodiment of the method for producing a hydrogel of the present invention, for example, a modified polysaccharide, an enzyme, and, if necessary, a peroxide are mixed in water to crosslink the modified polysaccharide to produce a hydrogel. The modified polysaccharide, enzyme, and peroxide may be used as respective aqueous solutions.

  Another embodiment of the method for producing a hydrogel of the present invention is, for example, (1) preparing a first solution containing a modified polysaccharide and containing either a peroxide or an enzyme but not both, (2 ) Prepare a second solution containing one of the above-mentioned peroxides and enzymes not included in the first solution, and (3) mixing the first solution and the second solution to obtain the modified polysaccharide. Crosslinking to produce a hydrogel. Specifically, a first solution containing a modified polysaccharide, an enzyme and water and a second solution containing a peroxide and water are mixed to crosslink the modified polysaccharide. Here, for example, a mold having a cavity having a desired shape is prepared, and the first solution and the second solution are mixed and reacted in the cavity to thereby obtain a hydrogel having the shape. Can be formed.

The aqueous solution temperature (polymerization / crosslinking temperature) during the formation of the hydrogel is usually 4 to 50 ° C., preferably 10 to 45 ° C., more preferably 20 to 40 ° C., and the reaction time is usually 1 minute to 20 hours. , Preferably 5 minutes to 10 hours. Hydrogel formation may be performed under pressure, normal pressure (atmospheric pressure), or reduced pressure, but normal pressure is preferred. By using the composition of the present invention, a hydrogel can be formed under mild conditions of about room temperature.
The gel and hydrogel of the present invention can be suitably used as, for example, artificial cartilage and artificial bone when a part of living tissue such as cartilage and bone is damaged.

[Molded body and medical device]
The molded object of this invention contains the gel mentioned above. For example, the gel of the present invention is a molded body formed into a desired shape. In the molded body, the amount of gel is usually 50% by mass or more, preferably 60% by mass or more, and more preferably 70% by mass or more.

  The medical instrument of this invention has the said molded object. Since the said medical instrument has the said molded object which is excellent in mechanical strength and a softness | flexibility, it can be used suitably for an implant material especially among medical instruments. Examples of the implant material include an implant material used when a part of biological tissue such as cartilage and bone is damaged.

  EXAMPLES Hereinafter, although this invention is demonstrated further more concretely based on an Example, this invention is not limited to these Examples. In the following description of Examples and the like, “part” means “part by mass” unless otherwise specified.

[Synthesis Example 1] Synthesis of hydroxyphenyl group-modified hyaluronic acid (A1) In a 100 ml eggplant-shaped flask, 7.58 g of hyaluronic acid (trade name “HA-LF5-A” manufactured by Kewpie Co., Ltd.) was added, and 80 g of distilled water was added. It was made to melt | dissolve, 3- (4-hydroxyphenyl) propionic acid 3.32g was added and stirred here, and it was set as the uniform liquid. This liquid was distilled off under reduced pressure while heating at 60 ° C. to obtain a residue. Next, the eggplant-shaped flask containing the residue was heated at 120 ° C. for 24 hours while reducing the pressure at 400 mmHg. After completion of the reaction, 80 g of distilled water was added and dissolved in the reaction solution, and this aqueous solution was reprecipitated in a mixed solution of 200 ml of ethanol, 100 ml of ethyl acetate, and 50 ml of n-hexane. The precipitate was suction filtered, and the resulting precipitate was decompressed while heating at 60 ° C. to distill off the solvent, and the hydroxyphenyl group-modified hyaluronic acid (A1) (hereinafter “modified hyaluronic acid (A1)”) Got.

When the hydroxyphenyl group substitution degree (DS _P ) of the modified hyaluronic acid (A1) was calculated by ¹ H NMR, it was 10.8. ^{A 1} H NMR chart is shown in FIG. ¹ H-NMR spectrum was measured using an apparatus name “JNM-ECX400P” (manufactured by JEOL Ltd., heavy solvent is heavy water).

[Example 1] Synthesis of hydroxyphenyl group and acryloyl group-modified hyaluronic acid (A11) In a 50 ml eggplant-shaped flask, 515 mg of modified hyaluronic acid (A1) was added, 25 g of N, N-dimethylformamide was added, and 100% was added under a nitrogen atmosphere. The mixture was heated at 0 ° C. to obtain an N, N-dimethylformamide solution. Next, at room temperature, 189 mg of triethylamine and 1.5 mg of N, N-dimethyl-4-aminopyridine were added to the solution and stirred. Next, a solution of 45 mg of acrylic acid chloride in 10% by mass of N, N-dimethylformamide was added dropwise with stirring under ice cooling. After completion of dropping, the mixture was stirred at room temperature for 24 hours. Next, 1 mg of 2,6-di-tert-butyl-4-methylphenol was added, and N, N-dimethylformamide was distilled off at 65 ° C. under reduced pressure. The residue obtained after distillation was dissolved in 2 g of distilled water, this solution was reprecipitated with isopropyl alcohol, the precipitate was filtered off with suction, dried in vacuo at 45 ° C. for 12 hours, and modified with hydroxyphenyl group and acryloyl group-modified hyaluronic acid. (A11) (hereinafter “modified hyaluronic acid (A11)”) was obtained.

DS _P and acryloyl group substitution degree of the modified hyaluronic acid (A11) and (DS _A) was calculated by ¹ H NMR, it was 10 and 5, respectively. A chart of ¹ H NMR is shown in FIG.

[Example 2] Synthesis of hydroxyphenyl group and acryloyl group-modified hyaluronic acid (A12) In the synthesis of modified hyaluronic acid (A11) of Example 1, the same operation was performed except that 22.5 mg of acrylic acid chloride was used. Hydroxyphenyl group and acryloyl group-modified hyaluronic acid (A12) (hereinafter, “modified hyaluronic acid (A12)”) was obtained. The DS _P and DS _A modified hyaluronic acid (A12) was calculated by ¹ H NMR, it was respectively 9.5 and 1.9. A chart of ¹ H NMR is shown in FIG.

[Synthesis Example 2] Synthesis of Hydroxyphenyl Group-Modified Hyaluronic Acid (A2) In the synthesis of the modified hyaluronic acid (A1) of Synthesis Example 1, 1.66 g of 3- (4-hydroxyphenyl) propionic acid was used and reprecipitation with ethanol. A hydroxyphenyl group-modified hyaluronic acid (A2) (hereinafter “modified hyaluronic acid (A2)”) was obtained in the same manner as described above. The DS _P of modified hyaluronic acid (A2) was calculated by ¹ H NMR, it was 7.

[Example 3] Synthesis of hydroxyphenyl group and acryloyl group-modified hyaluronic acid (A21) In the synthesis of modified hyaluronic acid (A11) of Example 1, modified hyaluronic acid (A2) was used instead of modified hyaluronic acid (A1). The same operation was performed except that 498 mg was used, to obtain a hydroxyphenyl group and acryloyl group-modified hyaluronic acid (A21) (hereinafter, “modified hyaluronic acid (A21)”). The DS _P and DS _A modified hyaluronic acid (A21) was calculated by ¹ H NMR, it was respectively 3.9 and 4.

[Example 4] Synthesis of hydroxyphenyl group and acryloyl group-modified hyaluronic acid (A22) In the synthesis of modified hyaluronic acid (A11) of Example 1, modified hyaluronic acid (A2) was used instead of modified hyaluronic acid (A1). The same operation was carried out except that 498 mg was used, 22.5 mg of acrylic acid chloride was used, and reprecipitation was performed with ethanol to obtain a hydroxyphenyl group and acryloyl group-modified hyaluronic acid (A22) (hereinafter, “modified hyaluronic acid (A22)”). It was. When a DS _P and DS _A modified hyaluronic acid (A22) was calculated by ¹ H NMR, were respectively 6 and 1.

[Synthesis Example 3] Synthesis of Hydroxyphenyl Group-Modified Hyaluronic Acid (A3) In the synthesis of the modified hyaluronic acid (A1) of Synthesis Example 1, 0.82 g of 3- (4-hydroxyphenyl) propionic acid was used and re-treated with isopropyl alcohol. Except for precipitation, the same operation was performed to obtain hydroxyphenyl group-modified hyaluronic acid (A3) (hereinafter, “modified hyaluronic acid (A3)”). The DS _P of modified hyaluronic acid (A3) was calculated by ¹ H NMR, it was 3.5.

[Synthesis Example 4] Acryloyl group-modified hyaluronic acid (A4)
In a 50 ml eggplant-shaped flask, 2.06 g of hyaluronic acid (trade name “HA-LF5-A” manufactured by Kewpie Corporation) was added, 250 g of N, N-dimethylformamide was added, and heated at 100 ° C. in a nitrogen atmosphere. An N, N-dimethylformamide solution was obtained. Next, at room temperature, 823 mg of triethylamine and 7 mg of N, N-dimethyl-4-aminopyridine were added to this solution and stirred. Next, a solution of 3.95 g of N, N-dimethylformamide in 10% by mass of N, N-dimethylformamide was added dropwise with stirring under ice cooling. After completion of dropping, the mixture was stirred at room temperature for 24 hours. Next, 3 mg of 2,6-di-tert-butyl-4-methylphenol was added, and N, N-dimethylformamide was distilled off at 65 ° C. under reduced pressure. The residue obtained after the distillation was dissolved in 20 g of distilled water, this solution was reprecipitated in a mixture of 200 ml of isopropyl alcohol and 100 ml of ethyl acetate, the precipitate was filtered off with suction, and dried in vacuo at 45 ° C. for 12 hours, Acrylyl group-modified hyaluronic acid (A4) (hereinafter, “modified hyaluronic acid (A4)”) was obtained.
The degree of acryloyl group substitution (DS _A ) of the modified hyaluronic acid (A4) was calculated by ¹ H NMR and found to be 9.

[Synthesis Example 5] Acrylyl group-modified hyaluronic acid (A5)
In the synthesis of the modified hyaluronic acid (A4) of Synthesis Example 4, the same operation was performed except that 2.47 g of acrylic acid chloride was used, and acryloyl group-modified hyaluronic acid (A5) (hereinafter referred to as “modified hyaluronic acid (A5)”). Got. The DS _A modified hyaluronic acid (A5) was calculated by ¹ H NMR, it was 7.

[Synthesis Example 6] Acrylyl group-modified hyaluronic acid (A6)
In the synthesis of the modified hyaluronic acid (A4) of Synthesis Example 4, the same operation was carried out except that 0.99 g of acrylic acid chloride was used and reprecipitated with isopropyl alcohol to obtain an acryloyl group-modified hyaluronic acid (A6) (hereinafter “modified hyaluronic acid”). Acid (A6) ") was obtained. The DS _A of the modified hyaluronic acid (A6) was calculated by ¹ H NMR and found to be 1.9.
Synthesis Example 1-6, the DS _P and DS _A modified hyaluronic acid synthesized in Examples 1 to 4 shown in Table 1 below.

[Examples 5-8, Comparative Examples 1-6] Production of Composition and Gel, and Evaluation of the Compositions A 10% by mass aqueous solution of the modified hyaluronic acid of Examples 1-4 and Synthesis Examples 1-6, horseradish peroxidase (sum Product types “169-10791”, abbreviated as HRP, aqueous solution (concentration: 150 U / ml) and hydrogen peroxide solution (0.3 mass%) in this order, the types shown in Table 2 below And the amount was mixed to produce a composition.
After mixing, the presence or absence of gelation was evaluated after 10 minutes at 23 ° C. The presence or absence of gelation was evaluated according to the following criteria. The results are shown in Table 2 below.
X: The composition has lost fluidity. Y: The composition has fluidity and exhibits behavior as a liquid.

  In the examples, the composition could be gelled. On the other hand, in the comparative example, although the amounts of HRP and hydrogen peroxide were increased relative to the examples, gelation could not be performed.

Claims (10)

To polysaccharides,
A group (a1) having a carbon-carbon unsaturated double bond, and a group (a2) having a hydroxyaryl group
Is a modified polysaccharide.   The modified polysaccharide according to claim 1, wherein the polysaccharide is hyaluronic acid or a derivative thereof. The modified polysaccharide according to claim 1 or 2, wherein the group (a2) having a hydroxyaryl group is a group represented by the following formula (2).

[In formula (2), * is a bond with a hydroxyl group residue in the polysaccharide, and R ¹ is an alkanediyl group having 1 to 20 carbon atoms or — (CH ₂ ) _n —O— (CH ₂ ). _{m- represents a} divalent group, and n and m each independently represent an integer of 1 to 10; R ^{2 to} R ⁶ each independently represents a hydrogen atom, a hydroxyl group, an alkoxy group, or a substituent. It is an alkyl group which may have, provided that at least one of R ^{2 to} R ⁶ is a hydroxyl group. ]   The modified polysaccharide according to any one of claims 1 to 3, wherein the group (a1) having a carbon-carbon unsaturated double bond is a (meth) acryloyl group.   A composition comprising the modified polysaccharide according to any one of claims 1 to 4, an enzyme, and water.   The composition according to claim 5, further comprising a peroxide.   The gel which has a crosslinked structure by the oxidative coupling of the hydroxyaryl group contained in the modified polysaccharide of any one of Claims 1-4.   The gel according to claim 7, which is a hydrogel containing 50 to 99.9% by mass of water.   The molded object containing 50 mass% or more of gels of Claim 7 or 8.   A medical instrument having the molded body according to claim 9.

Images