JP3893468B2 - Polysaccharide hydrogel and method for producing the same - Google Patents

Description

  The present invention relates to a polysaccharide hydrogel and a method for producing the same, and particularly to a hyaluronic acid hydrogel that can be used for biomedical materials such as cell scaffolds and drug carriers, and matrix materials for cosmetics or foods, and a method for producing the same.

  Hyaluronic acid is one of typical mucopolysaccharides and is widely present in the vitreous body of the eye, umbilical cord, connective tissue, blood, joint fluid, arteriovenous wall, and the like. As a synthesis method, a fermentation synthesis method has been established industrially so far and is supplied as a raw material for cosmetics and a raw material for pharmaceuticals. Main applications of hyaluronic acid include high-grade cosmetic moisturizing ingredients, ophthalmic surgical drugs, arthropathy therapeutics, and postoperative adhesion prevention agents. Biomedical as a matrix material that enables various gels of biopolymers such as hyaluronic acid to express their functions at desired times by including functionally expressed substances such as drugs and cells. It is used in a wide range of fields such as cosmetics and food.

Hyaluronic acid gel is known to be obtained as a physical gel by cooling an acidic aqueous solution, for example, but has the problem of lacking stability and durability due to the reversible nature of the aqueous solution. (Patent Documents 1, 2, and 5).
In order to improve such properties of hyaluronic acid, a crosslinked hydrogel obtained by chemical modification of hyaluronic acid is known. Representative crosslinking agents include bisepoxides, divinylsulfone, dihydrazine, dihydrazide, and polyisocyanate (Patent Documents 3 and 6). Moreover, the synthesis | combination of bridge | crosslinking hydrogel is reported by introduce | transducing vinyl groups, such as methacrylic ester, into hyaluronic acid, and the subsequent radical polymerization of a vinyl group (patent document 4).

Japanese Patent Laid-Open No. 05-58881 Japanese Patent Laid-Open No. 05-140201 JP 09-59303 A JP-A-10-67687 JP 2000-230003 A JP 2001-348401 A

  However, since the conventional cross-linking method using a cross-linking agent requires the use of an acid or an alkali, the function-expressing substances (drugs, cells, etc.) to be included in the gel are gelled for use as a matrix material of the generated hydrogel. There was a problem that it was denatured or deactivated during the production process. In addition, since most of the crosslinking agents are bifunctional, there is a possibility that the function-expressing substance may be modified or deactivated by the reaction between the crosslinking agent and the function-expressing substance during gelation. Not suitable for sustained release of expressed substance. Even in the case of gelation by vinyl polymerization, the initiator may cause modification or deactivation of the function-expressing substance. Furthermore, the hyaluronic acid gel obtained in Patent Documents 3 and 6 is not biodegradable and is a crosslinking method that requires the addition of a highly toxic metal catalyst such as dibutyltin dilaurate (Patent Document 6). Further, there is a possibility that a metal catalyst may remain in the obtained hydrogel, which is not suitable as a biomedical material.

  Therefore, the first object of the present invention is to provide a polysaccharide hydrogel such as a hyaluronic acid hydrogel that is biodegradable and harmless to the living body and the environment. A second problem is to provide a method for producing a polysaccharide hydrogel such as a hyaluronic acid hydrogel that can stably contain a function-expressing substance.

In order to solve the problem, the method for producing a polysaccharide hydrogel of the present invention is characterized in that a polymerizable compound is bound to a polysaccharide using a condensing agent and then polymerized and cured by treatment with an enzyme. .
According to the method of the present invention, since the polysaccharide derivative obtained by binding the polymerizable compound to the polysaccharide is polymerized by treating with the enzyme, the function-expressing substance prepared together with the polysaccharide is denatured during the gel production process. It remains in the gel while maintaining its function. Further, according to this method, a polysaccharide derivative and an enzyme can be prepared and gelled only when necessary.

In the method of the present invention, since the enzyme functions as a catalyst and does not bind to the product, the produced polysaccharide hydrogel is a polymer of a condensate of a polysaccharide and a polymerizable compound. To do.
The polysaccharide is an acidic polysaccharide, and the polymerizable compound needs to have a structure represented by the following general formula (1).

（式中、Ｒは、アルコール性水酸基、１級アミノ基、２級アミノ基またはカルボキシル基；ａは、１〜１０の整数；Ａ¹〜Ａ⁵のうち１つまたは２つはフェノール性水酸基で、残りは水素原子または炭素数１〜６のアルコキシ基である。Ａ¹〜Ａ⁵のうち２つがフェノール性水酸基である場合、２つのフェノール性水酸基はオルトまたはパラ位の位置関係にあるものとする。）

Wherein R is an alcoholic hydroxyl group, a primary amino group, a secondary amino group or a carboxyl group; a is an integer of 1 to 10; one or two of A ^{1 to} A ⁵ are phenolic hydroxyl groups The remainder is a hydrogen atom or an alkoxy group having 1 to 6 carbon atoms, and when two of A ^{1 to} A ⁵ are phenolic hydroxyl groups, the two phenolic hydroxyl groups are in the ortho or para position. To do.)

By making the said polysaccharide into polysaccharide containing acid groups, such as carboxylic acid, R of the polymeric compound couple | bonds with the acid group, and polysaccharide becomes a polysaccharide derivative which has a phenolic hydroxyl group. And since a polysaccharide derivative has a phenolic hydroxyl group, it can superpose | polymerize easily with an enzyme.
R is particularly preferably an alcoholic hydroxyl group, a primary amino group or a secondary amino group. This is because these functional groups specifically condense with the acid groups of the acidic polysaccharide, making it easy to control the reaction.

The polysaccharide is preferably hyaluronic acid, but may be other polysaccharides such as alginic acid. This is because alginic acid is easily condensed with the polymerizable compound via a carboxylic acid group, polymerized by the action of an enzyme, and other polysaccharides are the same.
Examples of the condensing agent include one or more selected from 1,1-carbonyldiimidazole, dicyclohexylcarbodiimide, and 3- (3-dimethylaminopropyl) -1-ethylcarbodiimide / hydrochloride.
Examples of the enzyme include one or more selected from laccase, 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 when used in combination with an oxidizing agent such as hydrogen peroxide, and is suitable as an instantaneous adhesive or hemostatic agent at medical sites.

  According to the present invention, since an enzyme catalyst is used, a polysaccharide hydrogel such as a crosslinked hyaluronic acid hydrogel can be easily obtained at normal temperature and near neutrality. Therefore, if a function-expressing substance is prepared together with the starting polysaccharide, the function-expressing substance can be included in the resulting gel. Since this polysaccharide hydrogel such as hyaluronic acid hydrogel has transparency and biodegradability, it can be used in a wide range of fields such as pharmaceuticals, cosmetics and foods.

  The polymerizable compound used for the production of the polysaccharide derivative is a compound having a phenolic hydroxyl group as a polymerizable functional group and an alcoholic hydroxyl group, a primary, secondary amino group or a carboxyl group as a functional group for binding to the polysaccharide. Examples of compounds having one phenolic hydroxyl group include tyramine and homovanillic acid derivatives, and examples of compounds having two phenolic hydroxyl groups include catecholamine derivatives such as dopamine, noradrenaline or adrenaline, particularly tyramine derivatives. Are preferred.

Examples of the solvent used for producing the polysaccharide derivative include dimethylformamide and dimethylsulfoxide, and dimethylformamide is particularly preferable. This is because hyaluronic acid is well dispersed.
The origin of the enzyme used in the polymerization curing process is not particularly limited, but includes the following. First, examples of the origin of copper enzymes such as laccase and tyrosinase (phenol oxidase) include urushi, mushrooms (Tuchikaburi, mushrooms), and molds (Polyporus vericolor). The origins of hydroperoxidases such as catalase and peroxidase are, for example, bovine liver, horse blood cells, human blood cells, M. lysodeikticus, horseradish, soybean, radish, turnip, thyroid, milk, intestine, leukocytes, erythrocytes, yeast, Caldariomyces Examples include fumago and Steptococcus faecalis. Of these, tyrosinase is preferably derived from mushrooms, and peroxidase is preferably derived from horseradish.

The present invention will be described in detail with reference to examples. In addition, this invention is not limited to an Example.
[Production of hyaluronic acid derivatives]

Production Example 1
Under an argon atmosphere, 2.30 g of hyaluronic acid (manufactured by Chisso, neutralized with hydrochloric acid) was added to 230 ml of dry dimethylformamide (solvent) and dispersed by stirring. 1.15 g of 1,1-carbonyldiimidazole (condensing agent) was added, and the mixture was further stirred for 6 hours. Next, 0.18 g of tyramine (polymerizable compound) was added and stirred for 48 hours. Thereafter, the solvent was distilled off under reduced pressure, and the residue was washed with acetone and subsequently with methanol. The residue was dispersed in 50 ml of water and dissolved by adding sodium hydroxide. The obtained solution was put into a dialysis tube having an excluded molecular weight of 2,000, and the polymer in the solution was purified by dialysis. After dialysis, the hyaluronic acid derivative was isolated by lyophilizing the aqueous solution in the tube.

Yield 1.40 g, number average molecular weight 4,500, molecular weight distribution 1.8, phenolic hydroxyl group introduction rate 17% determined from ¹ H-NMR.
¹ H-NMR (D ₂ O, ppm) 1.9 (s, CH ₃ C = O), 2.7 (br, CH ₂ Ar), 3.1-3.9 (m, CH ₂ N, CHN, CHO, CH ₂ OH), 4.5 (br, CHO at 1 and 1` positions of hyaluronic acid), 6.7, 7.0 (br, Ar)

Production Example 2
A reaction was carried out in the same manner as in Production Example 1 except that the amount of tyramine was changed to 0.44 g. As a result, 2.30 g of a hyaluronic acid derivative was obtained. Number average molecular weight 16,000, molecular weight distribution 3.5, 36% introduction of phenolic hydroxyl group determined from ¹ H-NMR.

Production Example 3
A reaction was carried out in the same manner as in Production Example 1 except that the amount of tyramine was changed to 0.087 g. As a result, 2.20 g of a hyaluronic acid derivative was obtained. Number average molecular weight 220,000, molecular weight distribution 2.5, phenolic hydroxyl group introduction rate 13% determined from ¹ H-NMR.

[Hardening of hyaluronic acid derivatives]
Curing example 1
When 50 mg of the hyaluronic acid derivative of Production Example 1 was dissolved in 1 ml of water and 50 units of laccase derived from Mycelopthora was added, a transparent hydrogel was formed in 2 hours.

Curing example 2
When 50 mg of the hyaluronic acid derivative of Production Example 1 was dissolved in 1 ml of water and 500 units of mushroom-derived tyrosinase was added, a transparent hydrogel was formed in 4 hours.

Curing example 3
When 50 mg of the hyaluronic acid derivative of Production Example 1 and 1 mg of horseradish peroxidase were dissolved in 1 ml of water and 50 μl of 30% hydrogen peroxide solution was added, a transparent hydrogel was instantaneously formed.

Curing example 4
When 50 mg of the hyaluronic acid derivative of Production Example 2 was dissolved in 1 ml of water and 50 units of laccase derived from the genus Mycelopthora were added, a transparent hydrogel was formed in 1 hour.

Curing example 5
When 50 mg of the hyaluronic acid derivative of Production Example 2 and 1 mg of horseradish peroxidase were dissolved in 1 ml of water and 50 μl of 30% hydrogen peroxide solution was added, a transparent hydrogel was instantaneously formed.

Curing example 6
When 100 mg of the hyaluronic acid derivative of Production Example 3 and 2 mg of horseradish peroxidase were dissolved in 2 ml of 0.1 M phosphate buffer (PBS, pH 7) and 50 μl of 30% hydrogen peroxide solution was added, a transparent hydrogel was instantaneously formed. Generated.

Curing example 7
When 100 mg of the hyaluronic acid derivative of Production Example 3 was dissolved in 1.9 ml of PBS same as that used in Curing Example 6 and 0.1 ml of PBS solution of 600 units of tyrosinase derived from mushrooms was added, a transparent hydrogel was obtained in 6 hours. Generated. When the degree of swelling of this gel was determined by the gravimetric method, it was 50 times after 2 days in PBS.

Comparative Example 1
Although 50 mg of unmodified hyaluronic acid was dissolved in 5 ml of water and 50 μl of an aqueous laccase solution was added and left for 3 days, no gel was obtained.
Comparative Example 2
Although 50 mg of unmodified hyaluronic acid and 1 mg of horseradish peroxidase were dissolved in 5 ml of water and 50 μl of 30% hydrogen peroxide solution was added and left for 3 days, no gel was obtained.

[Enzymatic degradation of hyaluronic acid hydrogel]
The gel prepared in Curing Example 7 was cut into a flat plate and immersed in a 50 unit / ml hyaluronidase PBS solution at 37 ° C., and the weight loss of the gel was measured over time. For comparison, the cut gel was immersed in a PBS solution not containing hyaluronidase in the same manner, and the weight loss was measured. The measurement results are shown in FIG. In the figure, white circles are measurement results in a solution containing hyaluronidase, and white squares are measurement results in a solution not containing white squares.

  As seen in FIG. 1, no gel weight change was observed in PBS without hyaluronidase, whereas the gel weight decreased linearly over time in PBS solution with hyaluronidase. . From this result, it was found that hyaluronic acid hydrogel has good biodegradability, and the gel is degraded from the surface by the enzyme.

It is the graph which showed the decomposition rate by the presence or absence of the hyaluronidase enzyme of the hyaluronic acid hydrogel synthesize | combined in the hardening example 7.

Claims (8)

A polysaccharide hydrogel, which is a polymer of a condensate of an acidic polysaccharide and a polymerizable compound represented by the following general formula (1) .

Wherein R is an alcoholic hydroxyl group, a primary amino group, a secondary amino group or a carboxyl group; a is an integer of 1 to 10; one or two of A ^{1 to} A ⁵ are phenolic hydroxyl groups The remainder is a hydrogen atom or an alkoxy group having 1 to 6 carbon atoms, and when two of A ^{1 to} A ⁵ are phenolic hydroxyl groups, the two phenolic hydroxyl groups are in the ortho or para position. To do.) The polysaccharide hydrogel according to claim 1 , wherein R is an alcoholic hydroxyl group, a primary amino group, or a secondary amino group. Wherein the acidic polysaccharide is polysaccharide hydrogel according to any one of claims 1-2 is hyaluronic acid. A method for producing a polysaccharide hydrogel, characterized in that a polymerizable compound represented by the following general formula (1) is bound to an acidic polysaccharide by using a condensing agent and then polymerized and cured by treatment with an enzyme.

Wherein R is an alcoholic hydroxyl group, a primary amino group, a secondary amino group or a carboxyl group; a is an integer of 1 to 10; one or two of A ^{1 to} A ⁵ are phenolic hydroxyl groups The remainder is a hydrogen atom or an alkoxy group having 1 to 6 carbon atoms, and when two of A ^{1 to} A ⁵ are phenolic hydroxyl groups, the two phenolic hydroxyl groups are in the ortho or para position. To do.) The production method according to claim 4 , wherein R is an alcoholic hydroxyl group, a primary amino group, or a secondary amino group. The production method according to claim 4 , wherein the acidic polysaccharide is hyaluronic acid. The condensing agent is 1,1-carbonyldiimidazole, dicyclohexylcarbodiimide and 3- (3-dimethylaminopropyl) -1- claim 4 is at least one selected from among ethyl carbodiimide hydrochloride The manufacturing method as described in. The production method according to claim 4 , wherein the enzyme is one or more selected from laccase, tyrosinase, catalase, and peroxidase.

Images