JP5907489B2 - Hydrogels derived from chitosan derivatives - Google Patents

Description

  The present invention relates to a novel hydrogel, and particularly to a hydrogel derived from a chitosan derivative useful as a wound dressing or the like.

  Wound dressings are required to promote wound treatment. Hydrogel wound dressings that can provide a moist environment to the wound site have been reported to promote granulation tissue formation and re-epidermalization at the wound site and promote healing.

  Chitosan is a natural polysaccharide having antibacterial and wound healing effects, and due to its properties, chitosan has been widely used as a wound dressing material. However, since chitosan is poorly water-soluble at physiological pH, it is difficult to produce a hydrogel-like chitosan wound dressing. On the other hand, a chitosan derivative that is water-soluble at physiological pH has been reported, but in order to prepare a hydrogel from this chitosan derivative, a cross-linking agent such as glutaraldehyde must be used, and it remained in the gel. The biotoxicity of the crosslinking agent is a problem.

  JP-T-2009-520705 (Patent Document 1) describes a chitosan composition that gels with heat by mixing chitosan and a disaccharide such as trehalose or a polyol such as glycerol. It is mentioned that this thermal gelation property is reconstituted after lyophilization. Japanese Patent Application Publication No. 2001-513367 (Patent Document 2) discloses a mixture of chitosan (derivative) and a polyol such as glycerol or a salt of sugar (for example, monophosphate dibasic salt). Compositions that gel at 0C are described. JP-T-2003-503367 (Patent Document 3) describes a hydrogel composed of chitosan and a polyalkylene oxide such as polyethylene glycol. Japanese Patent Application Laid-Open No. 2008-174671 (Patent Document 4) discloses a chitosan derivative in which a phenolic hydroxyl group is introduced into chitosan and describes that it is gelled by subjecting it to an environment in which the phenolic hydroxyl group is oxidized. ing. JP 2010-533154 (Patent Document 5) describes a hydrogel composed of a carboxyalkylamide of chitosan and intended for cosmetic and dermatological uses.

  The above references relate to hydrogels using chitosan or chitosan derivatives, but all merely show that hydrogels are formed and prepare chitosan-derived hydrogels in a shape adapted to the particular application. No such technique is disclosed in any document.

Special table 2009-520705 gazette JP-T-2001-513367 Japanese translation of PCT publication No. 2003-503367 JP 2008-174671 A Special table 2010-533154 gazette

  An object of the present invention is to provide a new technology for obtaining a hydrogel derived from chitosan that can be easily formed into an arbitrary shape without using an additive harmful to a living body and is useful as a wound dressing or the like. It is in.

  As a result of repeated studies, the present inventors have focused on chitosan derivatives that are soluble at physiological pH, and found that an aqueous solution containing the chitosan derivatives forms a hydrogel only by freezing and thawing. Derived.

  Thus, the present invention provides a method for preparing a hydrogel derived from a chitosan derivative in which aldonic acid is bonded to the amino group of the glucosamine unit of chitosan by dehydration condensation, and the aqueous solution containing the chitosan derivative is formed into a mold having a predetermined shape. And a step of freezing the chitosan derivative-containing aqueous solution in the mold, and a step of melting the frozen chitosan derivative-containing aqueous solution to obtain a gel having a shape corresponding to a predetermined shape. A method for preparing a hydrogel derived from chitosan is provided.

  The present invention further provides a hydrogel (hydrogel structure) obtained by the above method. The present invention further provides the above hydrogel mixed with polyvinyl alcohol (PVA).

  It is possible to prepare hydrogels of various shapes suitable for medical use such as wound dressings by providing a moist environment to the wound area from chitosan derivatives having a specific structure without using harmful additives such as cross-linking agents. it can.

The chemical structural formula of aldonic acid used for the chitosan derivative used in this invention is illustrated. A reaction scheme for synthesizing a chitosan derivative used in the present invention is illustrated. When measuring the introduction rate of gluconic acid in the chitosan derivative used in the present invention, the relationship between the amount of sodium hydroxide aqueous solution added to the chitosan derivative aqueous solution and the conductivity is shown. The relationship between the pH of a chitosan derivative aqueous solution measured in order to evaluate the water solubility of the chitosan derivative used in this invention and turbidity is shown. A molded gel obtained from a chitosan derivative according to the present invention is illustrated along with its template. As strength evaluation regarding the molded gel obtained from the chitosan derivative according to the present invention, (a) the result of compressive strength obtained by measuring strain (%) and stress (mN / mm ² ), and (b) the moisture content (%) of the gel Results are shown. (A) Results of water separation rate (%) relating to water retention and (b) to (d) photographs immersed in an aqueous lysozyme solution relating to enzyme degradation resistance, for a molded gel obtained from a chitosan derivative according to the present invention. As a wound healing promotion evaluation of a molded gel obtained from the chitosan derivative according to the present invention, (a) a subcutaneous image of the back of a mouse in which the hydrogel according to the present invention and 5 (weight / volume) polyvinyl alcohol gel (control) were implanted was photographed. And (b) a photograph of the change over time of the wound part, and (c) a result of the change over time of the wound area. The result of the evaluation experiment of the cytotoxicity of the chitosan derivative used in this invention is shown. (A) Gluconic acid-modified chitosan, (b) Threonic acid-modified chitosan. The relationship between pH and turbidity of a chitosan derivative aqueous solution measured in order to evaluate the water solubility of another chitosan derivative used in the present invention is shown.

The chitosan used for synthesizing the chitosan derivative used in the present invention is a deacetylated product of chitin. As is well known, the conversion of chitin to chitosan (deacetylation reaction) does not proceed completely, and part of the sugar chain contains N-acetylglucosamine, and the degree of deacetylation of commercially available chitosan is usually It is in the range of 50 to 100%, especially about 70 to 90%.
The chitosan used in the context of the present invention means those having a degree of deacetylation of 50 to 100% unless otherwise specified, and therefore also includes those having a degree of deacetylation that is not 100%.

  The aldonic acid used for synthesizing the chitosan derivative used in the present invention is a generic name of carboxylic acids in which the formyl group at the 1-position of aldose is changed to a carboxyl group among the derivatives obtained by oxidizing a monosaccharide. For example, it can be represented by the following general formula (I).

In the above formula, n is an integer of 2 to 4.

  A preferred aldonic acid used for the synthesis of the chitosan derivative used in the present invention includes gluconic acid, and threonic acid and xylonic acid can also be suitably used. In addition, any carboxylic acid known as aldonic acid such as galactonic acid, mannonic acid, lyxonic acid, erythronic acid, ribbon acid, arabinonic acid, aronic acid, altronic acid, gulonic acid, idonic acid, and taronic acid can be used. . FIG. 1 shows some chemical structural formulas of these aldonic acids according to the Fischer projection formula.

  As understood from the above description, the chitosan derivative used in the present invention generally has a site where an aldonic acid is bonded to an amino group of a glucosamine unit and an acetylglucosamine unit as shown in the following formula (II). It can be expressed as having a repeating unit (repeating unit) consisting of the remaining site.

In the above formula, n is an integer of 2 to 4.

  Therefore, when the aldonic acid is gluconic acid, the chitosan derivative used in the present invention can be generally expressed as having a repeating unit represented by the following formula (III).

  The chitosan derivative used in the present invention can be synthesized by condensing and dehydrating aldonic acid to chitosan and binding (introducing) aldonic acid to the amino group at the 2-position of the glucosamine unit of chitosan. The aldonic acid is generally reacted as a suitable salt (eg, Na salt). FIG. 2 shows a reaction scheme for the case where gluconic acid is used as aldonic acid.

  This condensation dehydration reaction can be carried out at room temperature under acidic conditions using a condensing agent (dehydration condensing agent). 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) which functions as a condensing agent in the condensation dehydration reaction between chitosan and aldonic acid and which functions to activate the carboxyl group of aldonic acid And N-hydroxysuccinimide (NHS) that functions to suppress side reactions, but is not limited thereto.

  The rate of introduction of aldonic acid such as gluconic acid into chitosan can be adjusted by changing the relative amount of aldonic acid and the amount of condensing agent used relative to chitosan. The introduction rate can be increased by increasing the amount of use. Although the aldonic acid introduction rate can be up to about 50%, in the present invention using the obtained chitosan derivative (aldonic acid-modified chitosan) as a hydrogel, the aldonic acid introduction rate into chitosan is deacetylated as described later. Using chitosan having a degree of conversion of 75 to 85%, it is usually preferably 10 to 30%.

Here, the introduction rate (to chitosan) of aldonic acids such as gluconic acid is represented by the following formula (A).
Aldonic acid introduction rate (%) = [Y / (X + Y)] × 100 (A)
X = substance amount (number of moles) of glucosamine units contained in the chitosan-introduced body, Y = substance amount (number of moles) of aldonic acid-introduced glucosamine units contained in the chitosan derivative.

  This rate of introduction measures the change in conductivity when a chitosan derivative (aldonic acid-modified chitosan) is dissolved in an aqueous hydrochloric acid solution of an appropriate concentration and the hydrochloric acid and an equimolar aqueous sodium hydroxide solution are added. This can be calculated.

This measurement method will be described in detail with reference to FIG. 3 with respect to gluconic acid-modified chitosan obtained by using gluconic acid as aldonic acid (hereinafter sometimes abbreviated as GC): Gluconic acid-modified chitosan When 0.1 M sodium hydroxide is added to a 0.1 M hydrochloric acid aqueous solution in which is dissolved, H ⁺ in the solution decreases due to the neutralization reaction, and the conductivity of the aqueous solution decreases accordingly (FIGS. 3A-B). ). When the neutralization reaction is completed, deprotonation of the protonated amino group in the glucosamine unit begins, and the conductivity does not change until the deprotonation is completed (FIGS. 3B-C). When the deprotonation is completed, OH ⁻ in the aqueous solution increases, so that the conductivity increases (FIGS. 1C-D). Here, the amount of sodium hydroxide added to the aqueous solution in BC in FIG. 3 corresponds to the amount of protonated amino group in the glucosamine unit. In addition, in consideration of the fact that amino groups in most glucosamine units are protonated in this aqueous hydrochloric acid solution, the amount of sodium hydroxide added to the aqueous solution with BC in FIG. 1 is not modified with gluconic acid. Corresponds to the amount of glucosamine units. Therefore, the amount of glucosamine units in gluconic acid-modified and unmodified chitosan is calculated by this measurement method, and the introduction rate of gluconic acid can be determined by comparing them.

  The chitosan derivative (aldonic acid-modified chitosan) used in the present invention is water-soluble in the acidic region and has a property that the region exhibiting water-solubility extends to a region having a high pH as the introduction rate of aldonic acid increases. . For example, when chitosan having a deacetylation degree of 75 to 85% is used and the introduction rate of aldonic acid is 10 to 30%, the chitosan derivative of the present invention is in the vicinity of neutral pH 7 (physiological pH range). Have also been found to exhibit good water solubility. Surprisingly, it has been found that a hydrogel is formed only by freezing and then thawing an aqueous chitosan derivative solution having such a physiological pH value.

  Thus, according to the present invention, an aqueous solution containing a chitosan derivative (aldonic acid-modified chitosan) is poured into a mold (template) having a predetermined shape, frozen, and then melted to obtain a shape corresponding to the predetermined shape. A gel can be obtained. Freezing is, for example, −20 ° C., and thawing is generally performed at room temperature, but the temperature and time for freezing and thawing are not particularly limited, and may be adjusted as necessary.

  Although the detailed mechanism for forming hydrogels by freezing and thawing chitosan derivatives according to the present invention has not yet been fully elucidated, chitosan derivative (aldonic acid-modified chitosan) molecules are entangled with each other through hydrophobic bonds and hydrogen bonds. After the structure is fixed by freezing, it is presumed that the structure is gelled by being loosened somewhat by melting.

  Furthermore, as a result of intensive studies, the present inventor has also found that a hydrogel having improved various properties is formed by mixing polyvinyl alcohol (PVA) with the chitosan derivative and performing the same operation. That is, by mixing polyvinyl alcohol with an aqueous solution containing the chitosan derivative (an aqueous solution poured into a mold), and freezing and thawing, a hydrogel in which polyvinyl alcohol is mixed with the chitosan derivative is obtained.

When obtaining a hydrogel in which polyvinyl alcohol is mixed with a chitosan derivative, the amount of polyvinyl alcohol in the hydrogel is such that the chitosan derivative / polyvinyl alcohol [(weight / volume)% ratio] is 1/10 or more and less than 2/5. It is preferably 1/5. For example, it can be formed at a blending ratio of chitosan derivative [1 (weight / volume)%] and polyvinyl alcohol [5 (weight / volume)%]. The molecular weight of the polyvinyl alcohol applied to the present invention should be 6.6 × 10 ⁴ Da (Dalton) or more and 2.5 × 10 ⁵ Da (Dalton) or less because of the ease of gel formation. For example, a material having a density of 1.5 × 10 ⁵ Da can be used. When the molecular weight is smaller than 6.6 × 10 ⁴ Da, high strength cannot be obtained even when gelled, and when it is larger than 2.5 × 10 ⁵ Da, the viscosity of the aqueous solution is high and the mixture of polyvinyl alcohol and chitosan derivative is mixed. This is because it is difficult.

  The hydrogel thus obtained has extremely excellent properties such as strength, moisture retention, and enzyme degradation resistance while maintaining high biocompatibility due to the contribution of polyvinyl alcohol. See example). For this reason, the hydrogel according to the present invention is preferably mixed with polyvinyl alcohol when used in applications requiring strength, moisture retention, and enzyme degradation resistance, for example, used as a wound dressing. In this case, while maintaining biocompatibility, it is possible to prevent the gel strength from decreasing and to maintain the wound in a moist state for a longer period of time.

The gel formed from the chitosan derivative of the present invention is generally an elastic hard hydrogel. According to the shape of the mold (mold) to be used, it can be formed into various shapes such as a sheet shape or a spherical shape depending on the application. Further, as understood from the above description, no cross-linking agent is used at the time of gelation, and the use of additives harmful to the living body is avoided.
Hereinafter, in order to describe the characteristics of the present invention more specifically, examples in the case of using several aldonic acids including gluconic acid will be shown, but the present invention is not limited by these examples. Absent.

Synthesis of gluconic acid-modified chitosan (GC) 3.0 g of chitosan (trade name “chitosan LL”, deacetylation degree) in 300 ml of distilled water (pH 4.0) in which 2-morpholinoethanesulfonic acid was dissolved at a concentration of 23.5 mM 80%, manufactured by Yaizu Suisan Chemical Co., Ltd.) was dissolved, and the pH was adjusted to 4.0 by adding a 1M aqueous hydrochloric acid solution. As shown in Table 1, sodium gluconate (hereinafter abbreviated as GA), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (hereinafter abbreviated as EDC) and N-hydroxysuccinimide were added to this aqueous solution. (Hereafter, abbreviated as NHS) was dissolved and stirred at room temperature for 24 hours to introduce gluconic acid into the amino group at the 2-position of the glucosamine unit of chitosan (see FIG. 2).

  Subsequently, the pH of the reaction solution was adjusted to 8.0 by adding 1 M aqueous sodium hydroxide solution, and then 99.5% ethanol was added to precipitate gluconate-modified chitosan and unreacted sodium gluconate, followed by centrifugation. The precipitate was recovered by operation. Thereafter, the precipitate was sealed in a dialysis membrane, and sodium gluconate was removed by immersing in distilled water for 1 week. At this time, distilled water was changed twice a day. Subsequently, the precipitate and the aqueous solution in the dialysis membrane were recovered, 99.5% ethanol was added to precipitate gluconic acid-modified chitosan, and the precipitate was recovered. Thereafter, it was frozen and dried and stored.

  The reaction results are shown in Table 1 below. As described above, the gluconic acid introduction rate is obtained by dissolving 0.2 g of gluconic acid-modified chitosan dry powder in 40 ml of 0.1 M hydrochloric acid aqueous solution and adding 0.1 M sodium hydroxide aqueous solution to the aqueous solution. Calculated by measuring the change.

Was subjected to chitosan FT-IR measured on the products and unmodified For gluconate introduction rate 18.7%, both, the peak was observed at 3400 cm ^-1 and 1590 cm ^-1, the peak height The difference was greater for the product in which gluconic acid was introduced. In FT-IR measurement, the peak height of 3400 cm ^-1 is dependent on the hydroxyl groups in the molecule, the peak height of 1590 cm ^-1 is dependent on the number of amino groups of glucosamine units. Difference between the peak height of the peak height and 1590 cm ^-1 in 3400 cm ^-1 are theoretically towards gluconate modified chitosan is greater than unmodified chitosan. Indeed, the difference between the peak height of the peak height and 1590 cm ^-1 in 3400 cm ^-1, the direction of gluconic acid modified chitosan is gluconic acid modified chitosan of chitosan since larger than unmodified chitosan was introduced gluconate Generation was confirmed.

Evaluation of water solubility of gluconate-modified chitosan 0.5 g of dry gluconate-modified chitosan powder is dissolved in 50 ml of distilled water (pH 4.0) in which 2-morpholinoethanesulfonic acid is dissolved at a concentration of 50 mM, and 1M hydrochloric acid aqueous solution is added. The pH was adjusted to 4.0. Subsequently, the pH was raised by adding a 0.1 M or 1.0 M sodium hydroxide aqueous solution, and the turbidity (wavelength 600 nm) of the solution at that time was measured. As a result, as the gluconic acid introduction rate increases, the pH at which turbidity begins to rise shifts to the alkali side, and aqueous solutions in which chitosan derivatives with introduction rates of 10.5% and 18.7% are dissolved are in the vicinity of pH 7. Even in the sex region, it was hardly clouded, and it was shown that it was soluble even at physiological pH (see FIG. 4). Further measurement revealed that no white turbidity was observed in the physiological pH range at an introduction rate of 10 to 30%, and that the chitosan derivative (gluconic acid-modified chitosan) was water-soluble.

Cytotoxicity evaluation of gluconic acid-modified chitosan L929 fibroblasts were seeded in a 96-well plate at a density of 1.0 × 10 ⁻⁴ cells / well and cultured for 24 hours. A minimum essential medium (pH 7.0) in which the gluconic acid-modified chitosan derivative synthesized in Example 1 was dissolved at different concentrations was added to the wells, and further cultured for 24 hours. Subsequently, the cell viability was measured using cell counting kit 8 (Dojin Chemical). The survival rate of cells cultured in a medium not containing a chitosan derivative was taken as 100%.
The result is shown in FIG. It shows almost 100% within the measurement error range, and it is understood that the gluconic acid-modified chitosan derivative has extremely low cytotoxicity like chitosan.

Preparation of hydrogel by freezing and thawing 0.1 g of dry powder of chitosan derivative synthesized in Example 1 having a gluconic acid introduction rate of 18.7% was added to 10 ml of distilled water, and 0.1 M hydrochloric acid aqueous solution was added to completely remove the powder. Dissolved. Subsequently, the pH is adjusted to 7.0 by adding a 0.1 M or 1.0 M aqueous sodium hydroxide solution, and the obtained aqueous solution is placed in a star-shaped metal mold and frozen at −20 ° C. for 12 hours. did. Then, when it was made to melt | dissolve by standing at room temperature, the production | generation of the star-shaped gel according to the shape of a metal casting_mold | template was confirmed (refer FIG. 5). As a control, gelation of chitosan into which gluconic acid was not introduced was attempted in the same manner, but gelation did not occur.

Evaluation of cell adhesion of hydrogel 1% (w / v) aqueous solution (pH 7.0) of gluconic acid-modified chitosan synthesized in Example 1 having a gluconic acid introduction rate of 18.7% was poured into a 48-well plate, and half of the bottom surface was poured. It was covered with a polymer solution comprising the chitosan derivative. Subsequently, the polymer solution was gelled by freezing at -20 ° C. (24 hours) and then thawing at room temperature (2 hours). When the L929 fibroblasts were seeded in the 48-well plate and the cell morphology was observed microscopically after 24 hours of culture, it was found that the cell adhesion by the gel was very low.

Preparation of Hydrogel Mixed with Polyvinyl Alcohol Chitosan derivative dry powder having a gluconic acid introduction rate of 19.3% was dissolved in physiological saline at pH 4.0 to obtain a 2 (weight / volume)% chitosan derivative solution. Also, polyvinyl alcohol powder (molecular weight 1.5 × 10 ⁵ Da, manufactured by Sigma-Aldrich) was added to another physiological saline and heated to dissolve to obtain a 10% (weight / volume) polyvinyl alcohol solution. . The chitosan derivative solution and the polyvinyl alcohol solution were mixed at 1: 1 (volume ratio), and 0.1M or 1.0M aqueous sodium hydroxide solution was added therein, thereby adjusting the pH of the aqueous solution to 7.0, 1 ml of the obtained aqueous solution was put into a cylindrical container having an inner diameter of 11 mm and frozen at −30 ° C. for 24 hours. Then, the production | generation of the hydrogel which mixed polyvinyl alcohol was confirmed by leaving still and melting at room temperature (20 degreeC) for 2 hours.

Evaluation of properties of hydrogel Several properties of hydrogel derived from gluconic acid-modified chitosan (GC) prepared according to the above-mentioned examples and obtained by mixing GC with polyvinyl alcohol (PVA) were evaluated as follows. did.
(1) Mechanical strength (compressive strength)
The disc-shaped gel was taken out from the cylindrical container, and its compressive strength was measured. FIG. 6A shows the results of measuring strain (%) and stress (mN / mm ² ) when this disk-shaped gel is compressed. As shown in FIG. 5A, the hydrogel according to the present invention alone has sufficient compressive strength, but by further mixing polyvinyl alcohol, 1 (weight / volume)% GC (gluconic acid modified chitosan) It was found that the compressive strength was dramatically improved up to 5 times and 2 (weight / volume)% GC. Furthermore, the result of having calculated the moisture content (%) of the gel as a ratio of (gel wet weight / gel dry weight) is shown in FIG. 6 (b). In general, the higher the mechanical strength of the gel, the lower the moisture content of the gel. Therefore, the hydrogel according to the present invention alone has a sufficiently low moisture content of the gel, as shown in FIG. The water content of the gel decreased to 1/4 with respect to 1 (weight / volume)% GC and 1/3 with respect to 2 (weight / volume)% GC. It was found that the strength improved dramatically.

(2) Install a cell strainer (manufactured by Japan BD) consisting of a mesh with a pore size of 40 μm in the mouth of a 50 ml centrifuge tube with a water retention capacity, and put the disk-shaped gel (volume: 2 ml) into this cell strainer. Centrifugation was performed at 1200 rpm for 15 minutes. FIG. 7 shows the results of calculating the water separation rate (%) as a ratio of (water weight accumulated in the centrifuge tube after centrifugation / water weight in the gel before centrifugation) from the weight of the water accumulated in the centrifuge tube after centrifugation. Shown in a). As shown in FIG. 5A, the hydrogel according to the present invention has a sufficiently high water retention ability by itself, but by further mixing polyvinyl alcohol, 1 / (weight / volume)% GC Since the water separation rate was suppressed to 1/15 with respect to 2 (weight / volume)% GC up to 10, it was found that the moisture retention was dramatically improved.

(3) Enzymatic degradation resistance In order to confirm the enzymatic degradation resistance of the hydrogel according to the present invention, the hydrogel according to the present invention is immersed in an aqueous lysozyme solution containing lysozyme (a chitosan degrading enzyme also present in the human body). The change in gel shape over time was observed. FIGS. 7 (b) and 7 (c) show a photograph immediately after immersion in a lysozyme aqueous solution and a photograph after 24 hours. In particular, as shown in FIG. 4D, the hydrogel mixed with polyvinyl alcohol maintained a clear gel shape similar to that immediately after the immersion in the lysozyme aqueous solution even after 24 hours from the immersion in the lysozyme aqueous solution. As described above, the hydrogel according to the present invention alone has sufficient enzyme degradation resistance, but it has been found that the enzyme degradation resistance is dramatically improved by further mixing polyvinyl alcohol.

Evaluation of promotion of wound healing by hydrogel In order to confirm the immunostimulatory ability (wound healing promoting effect) of the hydrogel according to the present invention in which polyvinyl alcohol (PVA) is mixed with gluconic acid-modified chitosan (GC), the gel is subcutaneously applied to the back of the mouse. Implanted weekly and followed up. The photograph which image | photographed the mouse | mouth back part of the mouse | mouth which implanted the hydrogel based on this invention and 5 (weight / volume)% polyvinyl alcohol gel (control) is shown to Fig.8 (a). As shown in FIG. 5A, after 1 week, excessive white blood cells (immune cells) were accumulated in the vicinity of the hydrogel according to the present invention, but 5 (weight / volume)% polyvinyl as a control. In the vicinity of the alcohol gel, accumulation of leukocytes was not observed. It has been found that the hydrogel according to the present invention maintains the immunostimulatory ability inherent in chitosan even after mixing with polyvinyl alcohol.

  Furthermore, the hydrogel according to the present invention was applied to a skin full-thickness model of rats (blood glucose concentration:> 300 mg / dl) rendered diabetic by administration of streptozotocin. Male Wistar rats (diabetic, 7 weeks old) have four skin full-thickness wounds (1 cm in diameter) on the back, hydrogel according to the present invention, and basic fibroblast growth factor (bFGF) in the hydrogel according to the present invention. The changes over time in a total of 4 cases of gauze and Buegel (registered trademark) were verified. The photograph which image | photographed the time-dependent change of a wound part is shown in FIG.8 (b). Moreover, the time-dependent change of a wound area is shown in FIG.8 (c).

  From the results of FIGS. 8 (b) and (c), it was found that the hydrogel according to the present invention significantly promotes wound healing than commercially available gauze and view gel. Furthermore, it was found that the addition of basic fibroblast growth factor (bFGF) to the hydrogel according to the present invention significantly promotes wound healing, particularly in the early stage of wound healing.

Synthesis of Threonic Acid-Modified Chitosan and Xylonic Acid-Modified Chitosan, Evaluation of Water Solubility and Preparation of Hydrogel Using the same method as described in Example 1, using threonic acid and xylonic acid as aldonic acid, threonic acid-modified chitosan and xylon Acid-modified chitosan was synthesized. The reaction results are shown in Table 3 below. In the same manner as described in Example 1, the introduction rate of each aldonic acid was determined by adding the hydrochloric acid and an equimolar aqueous sodium hydroxide solution to an aqueous solution in which a chitosan derivative (aldonic acid-modified chitosan) was dissolved in the aqueous hydrochloric acid solution. It was determined by measuring the change in conductivity when added.

Next, the water solubility of the threonic acid-modified chitosan and the xylonic acid-modified chitosan synthesized as described above was evaluated by measuring the change in turbidity with respect to pH by the same method as in Example 2. The result is shown in FIG. As shown in the figure, both the threonic acid-modified chitosan and the xylonic acid-modified chitosan are similar to the gluconic acid-modified chitosan in the neutral region (physiological pH region) around pH 7 as compared with the untreated chitosan. It is recognized that the water solubility is high.
Furthermore, it was confirmed that the synthesized threonic acid-modified chitosan and xylonic acid-modified chitosan were frozen at −20 ° C. for 12 hours by the same method as in Example 4 and then melted at room temperature.

Cytotoxicity evaluation of threonate-modified chitosan and xylonic acid-modified chitosan L929 fibroblasts were seeded in a 96-well plate at a density of 1.0 × 10 ⁴ cells / well and cultured for 24 hours. A minimum essential medium (pH 7.0) in which each of threonic acid-modified chitosan and xylonic acid-modified chitosan synthesized in Example 9 was dissolved was added to the wells, and further cultured for 24 hours. Subsequently, the cell viability was measured using cell counting kit 8 (Dojin Chemical). The survival rate of cells cultured in a medium containing no chitosan derivative was taken as 100%. The measurement results in the case of a threonic acid-modified chitosan derivative are shown in FIG. Similar to glucone-modified chitosan, threonic acid-modified chitosan and xylonic acid-modified chitosan were found to have low cytotoxicity.

Evaluation of cell adhesion of hydrogel A 1% (w / v) aqueous solution (pH 7.0) of threonic acid-modified chitosan and xylonic acid-modified chitosan synthesized in Example 9 was poured into a 48-well plate, and half of the bottom surface thereof was their aldonic acid. Covered with a polymer solution consisting of modified chitosan. Subsequently, the polymer solution was gelled by freezing at -20 ° C. (24 hours) and then thawing at room temperature (2 hours). The 48-well plate was seeded with L929 fibroblasts, and the cell morphology was observed under a microscope after 24 hours of culture. In both cases of the gel derived from threonic acid-modified chitosan and the gel derived from xylonic acid, the cell adhesion by the gel was low.

Claims (6)

  A method for preparing a hydrogel derived from a chitosan derivative formed by binding aldonic acid to an amino group of a glucosamine unit of chitosan by dehydration condensation, the step of pouring an aqueous solution containing the chitosan derivative into a mold of a predetermined shape, A hydrogel preparation comprising: freezing the chitosan derivative-containing aqueous solution therein; and thawing the frozen chitosan derivative-containing aqueous solution to obtain a gel having a shape corresponding to the predetermined shape. Method.   The method for preparing a hydrogel according to claim 1, wherein the chitosan derivative is chitosan having a deacetylation degree of 75 to 85% and the introduction rate of aldonic acid into chitosan is 10 to 30%.   The method for preparing a hydrogel according to claim 2, wherein the aldonic acid is gluconic acid.   The method for preparing a hydrogel according to claim 2, wherein the aldonic acid is threonic acid.   The method for preparing a hydrogel according to claim 2, wherein the aldonic acid is xylonic acid. The method for preparing a hydrogel according to claim 1, wherein polyvinyl alcohol is mixed in the aqueous solution containing the chitosan derivative.