JP2000248002A - Self-crosslinking hyaluronic acid, its production method and its use - Google Patents

Abstract

(57) [Summary] [PROBLEMS] To maximize the biocompatibility characteristic inherent in hyaluronic acid itself, without using any chemical crosslinking agent or chemical modifier, and without using cationic agents. To provide a self-crosslinked hyaluronic acid which is excellent in safety and biocompatibility without being complexed with a polymer. SOLUTION: The self-crosslinked hyaluronic acid is characterized by partially containing a molecular weight fraction having a degree of branching of 0.5 or more.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel self-crosslinked hyaluronic acid and a method for producing the same, and further to those medical materials.

[0002]

2. Description of the Related Art Hyaluronic acid is a linear high molecular polysaccharide in which β-DN-acetylglucosamine and β-D-glucuronic acid are alternately bonded. Hyaluronic acid is known to be distributed in connective tissues of mammals and also present in chicken crusts, capsular streptococci, and the like. In addition to chicken crest, umbilical cord and the like being used as an extraction material, purified products have also been prepared from streptococcal cultures.

[0003] Hyaluronic acid is polydisperse in molecular weight, but has no species or organ specificity and is known to exhibit excellent biocompatibility even when implanted or injected into a living body. . Further, in order to obtain a hyaluronic acid derivative having new physical properties, various chemical modifications and chemical cross-links have been proposed. Many techniques for chemically modifying hyaluronic acid and making water hardly soluble by chemical crosslinking have been reported, and formation of a hyaluronic acid gel by three-dimensional crosslinking is also known.

[0004] A typical example thereof is a highly swellable crosslinked hyaluronic acid gel obtained by using a bifunctional reagent such as divinyl sulfone, bisepoxides or formaldehyde as a crosslinking agent. (U.S. Pat. No. 4,582,865, JP-B-6-37575, JP-A-7-97401, JP-A-60-13)
No. 0601).

Further, a method for chemically modifying hyaluronic acid utilizing the feature that the tetrabutylammonium salt of hyaluronic acid is dissolved in an organic solvent such as dimethyl sulfoxide has been disclosed (JP-A-3-105003). Also, a method in which a tetrabutylammonium salt of hyaluronic acid is added to triethylamine and 2-chloro-1-methylpyridinium iodide in dimethyl sulfoxide and reacted to form an ester bond between a carboxyl group and a hydroxyl group of hyaluronic acid. It has been disclosed (European Patent No. 0341745).
A1).

As a method not using a chemical reagent for forming a covalent bond, hyaluronic acid and a high molecular compound having an amino group or an imino group are used by combining a carboxyl group of the hyaluronic acid with an amino group or an imino group of the high molecular compound. For preparing a hyaluronic acid polymer complex by binding to an ionic complex (Japanese Patent Laid-Open No. 6-7 / 1990).
No. 3103).

[0007] Further, an aqueous solution of hyaluronic acid is adjusted to pH 2.0.
A method for producing a hyaluronic acid gel characterized by being placed in the presence of a water-soluble organic solvent of from 20 to 80% by weight (see JP-A-5-58881).

In general, a method of producing a polymer gel by repeating the operation of freezing and thawing from an aqueous polymer solution has also been proposed. Typical examples thereof include polyvinyl alcohol and glucomannan (JP-A-57-190072,
See JP-A-5-161449.

However, when purifying or preserving hyaluronic acid and a biological sample containing hyaluronic acid, freezing and thawing operations and freeze-drying are frequently used as general methods, but are usually controlled under neutral conditions. Therefore, there is no report that a self-crosslinked hyaluronic acid is formed by such a method.

[0010]

SUMMARY OF THE INVENTION In order to make the most of the excellent biocompatibility inherent in hyaluronic acid itself, there is no need to use any chemical cross-linking agent or chemical modifying agent, and it is not necessary to use a cationic cross-linking agent. Self-crosslinking hyaluronic acid that can be used as a biocompatible medical material without forming a complex with the above polymer has not yet been developed.

The present inventors have intensively studied the physicochemical properties of hyaluronic acid itself to achieve the above object. As a result, by (1) adjusting the aqueous solution of hyaluronic acid to a specific pH, freezing and then thawing the aqueous solution at least once, (2) when the hyaluronic acid concentration is 5% by weight or more, It has been found that a self-crosslinked hyaluronic acid can be obtained by forming from a hyaluronic acid acidic aqueous solution containing a carboxyl group and an equimolar or more acid component.

The modified hyaluronic acid according to the conventional method is
Despite many efforts, the use of chemically reactive substances has had the problem of essentially entailing the dangers of toxicity and biological incompatibility due to new modifications.

For example, a hyaluronic acid derivative obtained by chemical modification / crosslinking and an ionic method using a metal salt or the like can improve the storage property in a living body, but the modified hyaluronic acid cannot crosslink a substance such as a crosslinked substance or the like. Since the metal is encapsulated in the molecule of hyaluronic acid by covalent or ionic bonds, the structure is no longer different from that of natural hyaluronic acid, and its physiological actions, biocompatibility, and safety, including toxicity, are essentially the same as hyaluronic acid. It has been difficult to completely avoid the problems of residual toxicity of these cross-linking agents and the like and the safety of cross-linking agents contained in degradation products in vivo.

The present inventors have found that the self-crosslinked hyaluronic acid obtained in the present invention has ideal biocompatibility as a medical material, and have completed the present invention.

[0015]

That is, the present invention provides (1)
Self-crosslinking hyaluronic acid characterized by partially containing a molecular weight fraction having a degree of branching of 0.5 or more, (2) forming an aqueous solution of hyaluronic acid having a pH of 3.5 or less by freezing and then thawing. The self-crosslinking hyaluronic acid according to (1), and (3) the aqueous solution of hyaluronic acid,
(2) The method for producing self-crosslinked hyaluronic acid according to (2), wherein the method is adjusted to 3.5 or less, and the aqueous solution is frozen and then thawed at least once. (4) Hyaluronic acid concentration of 5% by weight (1) The method for producing self-crosslinked hyaluronic acid according to (1), wherein the aqueous solution is formed from an acidic aqueous solution of hyaluronic acid containing at least an equimolar amount of an acid component with the carboxyl group of hyaluronic acid. A medical material characterized by containing self-crosslinked hyaluronic acid partially containing the above molecular weight fraction.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The hyaluronic acid used in the present invention can be used regardless of its origin, whether it is extracted from animal tissues or produced by fermentation. The strain used in the fermentation method is a microorganism having hyaluronic acid-producing ability, such as Streptococcus sp. Isolated from nature, or JP-A-63-1233.
No. 92, Streptococcus equi FM
-100 (Microtechnical Laboratory No. 9027), JP-A-2-2346
No. 89, Streptococcus equii FM
A mutant strain that stably produces hyaluronic acid at a high yield, such as -300 (Microtechnological Laboratory No. 2319) is desirable. Those cultured and purified using the above mutant strains are used.

The self-crosslinked hyaluronic acid according to the present invention is a hyaluronic acid in which hyaluronic acid retains a crosslinked structure and has a branch point, and can be distinguished from linear hyaluronic acid in terms of polymer solution. It is known that hyaluronic acid itself is a linear polymer and does not have a branched structure (polysaccharide biochemistry 1 Chemistry, Kyoritsu Shuppan, 1969).

A method for measuring the molecular weight and the degree of branching of the self-cross-linked hyaluronic acid includes a GPC using a differential refractometer and a multi-angle laser light scattering detector (MALLS) as detectors on a gel permeation chromatogram (GPC). -MALL
The S method, the GPC-LARLS method using a differential refractometer and a low-angle laser light scattering detector as detectors in GPC, and the GPC-viscosity method using a differential refractometer and a viscometer as detectors in GPC are available.

In the present invention, the GPC-MALLS method is used,
The molecular weight and the degree of branching of the molecular weight fraction separated by GPC were continuously measured online. GPC-MALL
In the S method, the molecular weight and radius of gyration of each fraction separated by GPC can be continuously measured. GPC-
In the MALLS method, the relationship between the molecular weight and the radius of gyration of each fraction of the self-cross-linked hyaluronic acid is compared with the relationship between the molecular weight and the radius of gyration of each fraction of the linear hyaluronic acid as a control to calculate the degree of branching. The elution volume method calculates the degree of branching by comparing the molecular weight of hyaluronic acid of the fraction with the same elution volume with the molecular weight of the linear hyaluronic acid as a control.

In the present invention, the degree of branching was measured using the elution volume method. The degree of branching is the number of branch points present per one polymer chain of self-crosslinked hyaluronic acid, and is plotted against the molecular weight of self-crosslinked hyaluronic acid. The molecular weight and radius of gyration of each fraction were calculated using the Jim plot (1) equation at finite concentration, the molecular weight was extrapolated to a scattering angle of 0 °, and the radius of gyration was calculated from the angle-dependent initial gradient according to the following equations. did.

[0021]

(Equation 1)

Here, M is the molecular weight, <S ² > is the mean square radius of inertia, K is the optical constant, R (θ) is the excess reduction scattering intensity at the scattering angle θ, c is the polymer concentration, and P (θ) Is the particle scattering function, λ is the wavelength of the laser beam in the solution, A ₂ is the second virial coefficient, and 0.002 ml for hyaluronic acid.
- it is a mol / g ^2. c is the output of the differential refractometer,
The concentration gradient of the refractive index of the hyaluronic acid solution (dn / dc:
0.153 ml / g).

In the GPC-MALLS method, the molecular weight and the mean square radius of inertia are calculated from the excess reduction scattering intensity, and the measurement accuracy depends on the magnitude of the excess reduction scattering intensity. (1)
From the equation, the excess reduction scattering intensity is a function of both concentration and molecular weight. Depending on the molecular weight of the sample, the sample concentration and injection volume need to be determined. GP fractionated into molecular weight fraction
Along with the selection of the C column, the maximum sample concentration and the maximum injection amount are selected within a range in which the concentration load on the GPC column does not appear.

The degree of branching of each fraction by the elution volume method was calculated according to the following equation (2). Assuming that the molecular weight of the branched polymer is M _b and the molecular weight of the linear polymer is M _{1 in} the fractions having the same elution volume, the shrinkage factor g is obtained.

[0025]

(Equation 2)

Here, a is a Mark-Houwink constant, which is 0.78 for hyaluronic acid. e is a clear factor and is set to 1.0. Assuming tetrafunctional long-chain disordered branching, the number B (degree of branching) of branch points on one polymer chain is calculated from the following equation (3).

[0027]

(Equation 3)

The calculation method of the degree of branching by the elution volume method is GP
This is the same as the measurement of the degree of branching by the C-LALLS method, and the details are described in Size Exclusion Chromatography (Kyoritsu Shuppan, 1991).

The concentration of the self-crosslinked hyaluronic acid was adjusted by diluting it with a GPC solvent, followed by filtering with a 0.2 μm membrane filter and then subjecting it to measurement. In the self-crosslinked hyaluronic acid referred to in the present invention, when there is a crosslinked structure that is stably present,
The branched structure is confirmed by polymer solution theory.

The term "self-crosslinking" as used in the present invention means that no chemical crosslinker or chemical modifier other than hyaluronic acid is used, and that it is not complexed with a cationic polymer. In addition, since the self-crosslinking proceeds to form a gel when the three-dimensional crosslink is formed, it goes without saying that the gel is also included in the self-crosslinked hyaluronic acid according to the present invention.

The chemical crosslinking agent for hyaluronic acid is a polyvalent compound which forms a covalent bond by reacting with a carboxyl group, a hydroxyl group or an acetamido group of hyaluronic acid, and is a polyvalent epoxy compound such as polyglycidyl ether, divinyl sulfone, Examples include formaldehyde, phosphorus oxychloride, a combination use of a carbodiimide compound and an amino acid ester, and a combination use of a carbodiimide compound and a dihydrazide compound.

The chemical modifier of hyaluronic acid is a compound which forms a covalent bond by reacting with a carboxyl group, a hydroxyl group or an acetamido group of hyaluronic acid, and is used in combination with acetic anhydride and concentrated sulfuric acid, trifluoroacetic anhydride and organic acid. And alkyl iodide compounds.

The cationic polymer that forms a complex with hyaluronic acid is a polymer that forms an ionic complex between the carboxyl group of hyaluronic acid and the amino group or imino group of the polymer compound, and includes chitosan, polylysine, Examples include polyvinyl pyridine, polyethylene imine, and polydimethylaminoethyl methacrylate.

The hyaluronic acid used in the present invention may be preferably used, whether it is a purified polymer obtained by animal tissue extraction or fermentation, or a low-molecular-weight product obtained by further hydrolysis. It should be noted that the hyaluronic acid according to the present invention is used in a concept that also includes alkali metal salts thereof, for example, sodium, potassium and lithium salts.

The aqueous solution of hyaluronic acid used in the present invention is obtained by mixing hyaluronic acid powder and water and stirring.

First, self-crosslinked hyaluronic acid obtained by freezing and thawing an acidic solution prepared with hyaluronic acid will be described. As the acid used for adjusting the pH of the aqueous solution of hyaluronic acid, any acid can be used as long as it can adjust the pH to 3.5 or less. In order to reduce the amount of acid used, it is desirable to use a strong acid, for example, hydrochloric acid, nitric acid, sulfuric acid and the like.

The pH of the aqueous solution of hyaluronic acid is adjusted so that the carboxyl group of hyaluronic acid is protonated at a sufficient ratio.
Adjust to H. The equilibrium constant for the dissociation of the acid form of hyaluronic acid is log K ₀ = infinite dilution of hyaluronic acid concentration.
4.25 (Acta Chimica Hungarica-Models in
Chemistry 129 (5) 671-683 1992). The pH to be adjusted is appropriately determined depending on the type of the counter ion of the hyaluronic acid salt, the molecular weight of the hyaluronic acid, the concentration of the aqueous solution, the conditions of freezing and thawing, and various properties such as the generated cross-linked hyaluronic acid,
In the present invention, it is necessary to adjust the pH to 3.5 or less. Preferably, the pH is 2.5 or less.

For freezing and thawing, an operation of placing an acidic aqueous solution of hyaluronic acid in an arbitrary container, freezing at a predetermined temperature, and thawing at a predetermined temperature after freezing is performed at least once. . The temperature and time for freezing and thawing are appropriately determined within the range of the temperature and time for freezing and thawing the acidic aqueous solution of hyaluronic acid depending on the size of the container and the amount of the aqueous solution. Is preferred.

Since the freezing and thawing times can be shortened, more preferably, a freezing temperature of -5 ° C or less and a thawing temperature of 5 ° C or more are selected. The time is not particularly limited as long as it is equal to or longer than the time at which freezing and thawing is completed at that temperature.

The number of repetitions of the operation of freezing and then thawing the adjusted acidic aqueous solution of hyaluronic acid depends on the molecular weight of the hyaluronic acid used, the concentration of the aqueous solution, the pH of the aqueous solution, the temperature and time of freezing and thawing, and the crosslinking generated. It is appropriately determined according to various properties such as hyaluronic acid. Normally, it is preferable to repeat it one or more times. Further, each time the operation of freezing and thawing is repeated, the temperature and time of freezing and thawing may be changed.

The acidic aqueous solution of hyaluronic acid having a hyaluronic acid concentration of 5% by weight or more used in the present invention means an aqueous solution prepared so that the carboxyl group of hyaluronic acid is protonated at a sufficient ratio. The amount of the acid component used for preparing the acidic solution is appropriately determined according to various kinds of properties such as the type of the counter ion of the hyaluronic acid salt, the molecular weight of the hyaluronic acid, the hyaluronic acid concentration, and the strength of the generated gel. The amount of the acid component more than equimolar to the carboxyl group of hyaluronic acid is preferred. As the acid component, any acid can be used as long as it is stronger than hyaluronic acid. In order to reduce the amount of acid used, it is desirable to use a strong acid, for example, hydrochloric acid, nitric acid, sulfuric acid and the like.

Next, self-crosslinked hyaluronic acid obtained from an aqueous solution of hyaluronic acid having a hyaluronic acid concentration of 5% by weight or more will be described. Hyaluronic acid concentration 5 used in the present invention
Before the neutralization of the resulting hyaluronic acid gel, the hyaluronic acid acidic aqueous solution of not less than% by weight requires aging for promoting gelation. This aging temperature and time
The hyaluronic acid acidic aqueous solution is appropriately determined according to the type of the counter ion of the hyaluronic acid salt, the molecular weight of the hyaluronic acid, the hyaluronic acid concentration, and the strength of the generated gel. In order to prevent the decomposition and to suppress the decomposition of hyaluronic acid by an acid, it is preferable to carry out at a temperature of -10 ° C or more and 30 ° C or less.

The method of preparing the aqueous hyaluronic acid solution having a hyaluronic acid concentration of 5% by weight or more used in the present invention is not limited.
Examples thereof include a method of concentrating a low-concentration aqueous solution of hyaluronic acid to a predetermined concentration, and a method of adding an acid component to a high-concentration aqueous solution of hyaluronic acid.

In the method of mixing the solid hyaluronic acid and the acidic aqueous solution, the form of the solid hyaluronic acid to be mixed is not limited, but the powder, the block-shaped molded product obtained by pressing the hyaluronic acid powder, and the hyaluronic acid are mixed. A form such as a cast film obtained by dissolving in distilled water to form an aqueous solution and then ventilation drying or a sponge-like hyaluronic acid obtained after freeze-drying is considered. Examples of the mixing method include a method in which solid hyaluronic acid is impregnated with an acidic aqueous solution so as to have a predetermined concentration, and a method in which an acidic aqueous solution is added to solid hyaluronic acid and kneaded.

In the method of concentrating a low-concentration acidic aqueous solution of hyaluronic acid to a predetermined concentration, first dissolve solid hyaluronic acid in distilled water and then add an acid component, or dissolve solid hyaluronic acid directly in the acidic aqueous solution. By doing so, a low-concentration aqueous solution of hyaluronic acid is prepared. Here, the form of the dissolved solid hyaluronic acid does not matter. The term “low concentration” as used herein means lower than the hyaluronic acid concentration of the target hyaluronic acid gel. However, the hyaluronic acid concentration is preferably 5% by weight or less from the viewpoint of easy handling. Examples of the concentration method include ultracentrifugation, ventilation drying, drying under reduced pressure, and freeze drying.

In the method of adding an acid component to a high-concentration aqueous solution of hyaluronic acid, a high-concentration aqueous solution of hyaluronic acid is first mixed by mixing solid hyaluronic acid and distilled water, or by concentrating a low-concentration aqueous solution of hyaluronic acid. Prepare an aqueous solution. The form of the solid hyaluronic acid used at this time does not matter. Examples of a method for adding an acid component to the obtained high-concentration aqueous solution of hyaluronic acid include a method of exposing an acid in a gaseous state, for example, an atmosphere of hydrogen chloride, and an acid solution of a poor solvent for hyaluronic acid, for example, ethanol-hydrochloric acid. A method of dipping in a solution may be used.

The self-crosslinked hyaluronic acid obtained by freezing and thawing the adjusted acidic solution of hyaluronic acid, and the self-crosslinked hyaluronic acid obtained from the aqueous solution of hyaluronic acid having a hyaluronic acid concentration of 5% by weight or more are obtained by converting the acid of hyaluronic acid into In order to avoid hydrolysis, it is necessary to remove components such as acids used for adjusting the acidity. In order to remove components such as acid, washing or dialysis is usually performed with an aqueous solvent. There is no particular limitation as long as the function of the self-crosslinked hyaluronic acid is not impaired.For example, water, saline, phosphate buffer and the like are used, and preferably, saline, phosphate buffer and the like are used. Used.

The washed self-crosslinked hyaluronic acid is
Depending on the purpose of use, it is provided as a medical material in the form of a solution, a state immersed in a solvent, a wet state containing a solvent, air-dried, dried under reduced pressure, or freeze-dried.

For use as a medical material, high biocompatibility is essential, and it is generally necessary to prove that there is no cytotoxicity. The self-cross-linked hyaluronic acid of the present invention sufficiently satisfies these conditions, and can be used as a biodegradable medical material without particular limitation as long as the hyaluronic acid is used. For example,
Examples include use in anti-adhesion materials, joint injections, soft tissue injections, vitreous substitutes, cosmetics, biomedical products or medical compositions such as medical instruments and medical tools used for diagnosis and treatment.

The self-crosslinked hyaluronic acid and the molded product thereof may be used in a single form, or may be mixed or used in combination with different self-crosslinked hyaluronic acid forms, and further mixed or combined with a hyaluronic acid solution. The effect can be expected to be enhanced.

[0051]

The present invention will be described in more detail with reference to the following examples. Note that the present invention is not limited to this.

Example 1 Sodium hyaluronate having a molecular weight of 3.5 × 10 ⁵ daltons was dissolved in distilled water to prepare a 1.5% by weight aqueous solution of hyaluronic acid. The pH of the adjusted aqueous solution of hyaluronic acid was 6.0. The pH of this aqueous solution was adjusted to pH 1.5 with 1N hydrochloric acid. 15 ml of an acidic aqueous solution of hyaluronic acid was placed in a 30 ml container and placed in a freezer set at -20 ° C. After freezing for 24 hours, 48 hours, and 72 hours, it was thawed at 25 ° C. As a result, crosslinked hyaluronic acid was obtained.

Example 2 In Example 1, an aqueous solution of hyaluronic acid was prepared by dissolving sodium hyaluronate having a molecular weight of 2.2 × 10 ⁵ daltons. Adjustment was performed in the same manner as in Example 1, and the mixture was placed in a freezer set at −20 ° C. After freezing for 24 hours, 48 hours, and 72 hours, it was thawed at 25 ° C. As a result, crosslinked hyaluronic acid was obtained.

Example 3 In Example 1, an aqueous solution of hyaluronic acid was prepared by dissolving sodium hyaluronate having a molecular weight of 6.0 × 10 ⁴ daltons. Adjustment was performed in the same manner as in Example 1, and the mixture was placed in a freezer set at −20 ° C. After freezing for 40 days, it was thawed at 25 ° C. As a result, crosslinked hyaluronic acid was obtained.

Example 4 In Example 1, an aqueous solution of hyaluronic acid was prepared by dissolving sodium hyaluronate having a molecular weight of 4.0 × 10 ⁴ daltons. Adjustment was performed in the same manner as in Example 1, and the mixture was placed in a freezer set at −20 ° C. After freezing for 40 days, 62 days, and 81 days, it was thawed at 25 ° C. As a result, crosslinked hyaluronic acid was obtained.

Example 5 In Example 1, an aqueous solution of hyaluronic acid was prepared by dissolving sodium hyaluronate having a molecular weight of 2.0 × 10 ⁴ daltons. Adjustment was performed in the same manner as in Example 1, and the mixture was placed in a freezer set at −20 ° C. After freezing for 81 days, it was thawed at 25 ° C. As a result, crosslinked hyaluronic acid was obtained.

Example 6 In Example 4, the solution was frozen for 62 days, thawed at 25 ° C., and an aqueous solution of hyaluronic acid having a pH of 1.5 was added to a solution of 0.2N N 2.
Neutralized to pH 7.0 with aqueous aOH. The neutralized aqueous solution of hyaluronic acid was lyophilized to recover cross-linked hyaluronic acid.

Example 7 Sodium hyaluronate having a molecular weight of 2.0 × 10 ⁴ daltons was dissolved in heavy water to prepare a 1.5% by weight aqueous solution of hyaluronic acid. P of adjusted aqueous solution of hyaluronic acid
D was 6.0. The pD of this aqueous solution was adjusted to pD1.1 with a heavy aqueous solution of 1N dehydrochloric acid. 15 ml of an acidic aqueous solution of hyaluronic acid is placed in a 30 ml container,
Put in the freezer set. After freezing for 81 days, 25
Thawed at ℃. As a result, crosslinked hyaluronic acid was obtained.

Example 8 Sodium hyaluronate having a molecular weight of 2.2 × 10 ⁵ daltons was dissolved in a 1N aqueous hydrochloric acid solution to prepare a 20% by weight aqueous solution of hyaluronic acid. Acidic aqueous solution of hyaluronic acid 1
5 ml was placed in a 30 ml container and placed in a freezer set at 5 ° C. After refrigeration for 33 days, crosslinked hyaluronic acid was obtained.

Comparative Example 1 In Example 1, the aqueous solution of hyaluronic acid was frozen without adjusting the pH, frozen for 24 hours, 48 hours, and 72 hours, and then thawed at 25 ° C.

Comparative Example 2 In Example 8, the aqueous solution of hyaluronic acid was refrigerated for 33 days without adjusting the pH.

Example 9 Molecular weight of cross-linked hyaluronic acid The cross-linked hyaluronic acid obtained in Examples 1 to 8 and Comparative Example 1,
The molecular weight of the hyaluronic acid recovered in Step 2 was determined by GPC-MA
It was measured using LLS. The polymer concentration was adjusted to 0.2% by weight, 0.1% by weight, and 0.05% by weight with a GPC solvent, and after filtration through a 0.2 μm membrane filter,
GPC-MALLS was measured by injecting 0.1 ml. One SB806HQ manufactured by Showa Denko KK as a GPC column or Ultrahydrog manufactured by Waters
el500, and 830-RI manufactured by JASCO Corporation and MALLS manufactured by Wyatt D
Using AWNDSP-F, a 0.2 M aqueous solution of a solvent sodium nitrate, a measurement temperature of 40 ° C., and a flow rate of 0.3 ml /
And data acquisition interval once / second. The scattering intensity was measured using 8 detectors having a scattering angle of 28.7 ° to 90 °. Data processing software is ASTR manufactured by Wyatt
A Version 4.10 was used. Table 1 shows the measurement results.

[0063]

[Table 1]

Example 10 Molecular weight distribution of crosslinked hyaluronic acid The molecular weight of the crosslinked hyaluronic acid obtained in Example 4 was 4.0.
The molecular weight distribution of × 10 ⁴ dalton hyaluronic acid is GP
It measured using C. Reduce the polymer concentration to 0 with GPC solvent.
After adjusting to 2% by weight and filtering through a 0.2 μm membrane filter, 0.1 ml was injected and GPC was measured. Waters Ultrah as GPC column
ydrogel 500, 830-RI manufactured by JASCO Corporation as a differential refractive index detector, 0.2 M aqueous solution of sodium nitrate solvent, measurement temperature of 40 ° C., flow rate of 0.3 ml
/ Min, at a data acquisition interval of once / 2 seconds. FIG. 1 shows the measurement results.

Example 11 Measurement of Degree of Branching of Cross-Linked Hyaluronic Acid In Example 4, the cross-linked hyaluronic acid obtained with a reaction time of 81 days and the hyaluronic acid having a molecular weight of 4.0 × 10 ⁴ daltons had a polymer concentration of 0 with a GPC solvent. 0.2% by weight and filtered through a 0.2 μm membrane filter.
GPC-MALLS was measured by injecting 0.1 ml. Waters Ultrah as GPC column
ydrogel 500, 830-RI manufactured by JASCO Corporation as a differential refractive index detector, 0.2 M aqueous solution of sodium nitrate solvent, measurement temperature of 40 ° C., flow rate of 0.3 ml
/ Min, at a data acquisition interval of once / 2 seconds. FIG. 2 shows the measurement results.

From the results shown in Table 1, for example, in Example 1, as the reaction time becomes longer, the weight average molecular weight becomes 3.5 ×
From 10 ⁵ , 4.3 × 10 ⁵ , 4.7 × 10 ⁵ , 6.1
It has increased to × 10 ⁵ . Even when the molecular weight of the hyaluronic acid used as the raw material in Examples 2, 3, 4, and 5 decreases, the molecular weight increases as the reaction time increases.

From the results shown in Table 1, in Example 6, the recovered crosslinked hyaluronic acid was neutralized and freeze-dried, but the molecular weight was increased as in Example 4. In Example 7, heavy water was used as the reaction solvent. The increase in molecular weight is greater than in Example 5 using light water. In Example 8, although the reaction time was 5 ° C, the molecular weight was increased as in Examples 1 to 7 where the reaction temperature was -20 ° C.

From the results shown in FIG. 1, it is understood that the increase in the molecular weight is caused by a change in the molecular weight distribution, that is, the generation of a high molecular weight component. The linear hyaluronic acid (molecular weight 4.0 ×) of the crosslinked hyaluronic acid obtained in Example 4 for a reaction time of 81 days.
FIG. 2 shows the calculation results of the branching degree for 10 ⁴ Dalton).

The degree of branching was calculated from the molecular weights of both fractions having the same elution volume, using Equations 2 and 3. FIG. 2 shows that the degree of branching of the crosslinked hyaluronic acid rapidly increases from a degree of branching of 0.5 or more in a region having a molecular weight of about 30,000 or more. That is, it can be seen that the crosslinked hyaluronic acid obtained in the present invention becomes a branched hyaluronic acid due to the presence of a stable crosslinked structure, and has an increased molecular weight.

Example 12 Cytotoxicity Test of Cross-Linked Hyaluronic Acid In normal human skin-derived fibroblast cultures, the cross-linked hyaluronic acid obtained in the present invention was coexistent, and its cytotoxicity was evaluated by observing the cell growth behavior. Dialyzing the crosslinked hyaluronic acid of Examples 1-5 against phosphate buffered saline,
Cell culture plate with seeded cells (Falcon)
20 mg each. Culture under the following culture conditions,
The cell proliferation ability was evaluated. Culture in the absence of cross-linked hyaluronic acid was used as a control.

Culture conditions Plate: 12-well plate for cell culture Medium: DMEM medium + 10% fetal bovine serum, 2 ml / well Temperature: 37 ° C. (under 5% CO ₂ ) Number of seeded cells: 1 × 10 ⁴ cells / well

After 2 days, 5 days, and 8 days after the start of the culture, the cell density was observed using an inverted microscope.
No matter which cross-linked hyaluronic acid was present, good growth was exhibited as in the control. Therefore, it was suggested that the crosslinked hyaluronic acid obtained in the present invention has no cytotoxic effect and can be used as a medical material.

[0073]

As described above, according to the present invention, self-crosslinked hyaluronic acid can be obtained without using any chemical crosslinking agent or chemical modifying agent. Adverse effects on biocompatibility due to the use of a chemical cross-linking agent or a chemical modifying agent are avoided, which is useful in the field of biocompatible materials.

[Brief description of the drawings]

FIG. 1 is a GPC chromatogram of cross-linked hyaluronic acid and linear hyaluronic acid (molecular weight 4.0 × 10 ⁴ daltons) obtained in Example 4 for a reaction time of 62 days and 81 days.

FIG. 2 shows the linear hyaluronic acid (molecular weight: 4.0 × 10) of the cross-linked hyaluronic acid obtained in Example 4 for a reaction time of 81 days.
⁴ is a graph showing the relationship between the degree of branching and the molecular weight calculated for ( ⁴ Daltons).

[Explanation of symbols]

1 GPC chromatogram of linear hyaluronic acid (molecular weight 4.0 × 10 ⁴ dalton) 2 GPC chromatogram of cross-linked hyaluronic obtained in Example 4 with a reaction time of 62 days 3 Obtained with a reaction time of 81 days in Example 4 Chromatogram of extracted cross-linked hyaluronic acid

Claims (5)

[Claims] 1. Self-crosslinking hyaluronic acid, which partially contains a molecular weight fraction having a degree of branching of 0.5 or more. 2. The self-crosslinked hyaluronic acid according to claim 1, which is formed by freezing and then thawing an aqueous solution of hyaluronic acid having a pH of 3.5 or less. 3. The production of self-crosslinked hyaluronic acid according to claim 2, wherein the pH of the aqueous solution of hyaluronic acid is adjusted to 3.5 or less, and the aqueous solution is frozen and then thawed at least once. Method. 4. The production of self-crosslinked hyaluronic acid according to claim 1, wherein the hyaluronic acid is formed from an aqueous acidic solution of hyaluronic acid having a hyaluronic acid concentration of 5% by weight or more and containing an acid component in an equimolar amount or more with the carboxyl group of hyaluronic acid. Method. 5. A medical material comprising self-crosslinked hyaluronic acid partially containing a molecular weight fraction having a degree of branching of 0.5 or more.