KR102226724B1 - Preparing Method of Hyaluronic Acid Hydrogel by Physical Treatment - Google Patents

Abstract

The present invention relates to a method for producing a hyaluronic acid hydrogel, and more particularly, to a method for producing a highly functional hyaluronic acid hydrogel by a crosslinking reaction of hyaluronic acid itself by a physical reaction rather than a toxic chemical crosslinking agent.
The method of preparing the hyaluronic acid hydrogel includes the steps of: (a) dissolving hyaluronic acid having a molecular weight of 0.5 to 3.0 MDa in a solvent to prepare a hyaluronic acid solution; (b) inducing physical crosslinking by adjusting the pH to 2-5 while strongly stirring the hyaluronic acid solution; And (c) obtaining a hyaluronic acid hydrogel formed through physical crosslinking.
According to the present invention, a hyaluronic acid hydrogel can be prepared by controlling the pH in a hyaluronic acid solution without using a chemical crosslinking agent with strong cytotoxicity and applying an external shear force. It has the advantage of excellent physical properties. In addition, according to the molecular weight of hyaluronic acid and the type and content of excipients to be added, physical properties can be adjusted by adjusting gelation, so it can be used as various types of cosmetic skin external preparations, joint injections, injections for molding, and the like.

Description

Preparation Method of Hyaluronic Acid Hydrogel by Physical Treatment {Preparing Method of Hyaluronic Acid Hydrogel by Physical Treatment}

The present invention relates to a method for producing a hyaluronic acid hydrogel, and more particularly, to a method for producing a highly functional hyaluronic acid hydrogel by a crosslinking reaction of hyaluronic acid itself by a physical reaction rather than a toxic chemical crosslinking agent.

With increasing interest in hyaluronic acid (Sodium Hyaluronate or Hyaluronic Acid) around the world, various products using this are being developed in the fields of food, cosmetics, and pharmaceuticals.

In particular, due to its viscoelasticity and excellent biocompatibility, hyaluronic acid is a tissue protectant during surgery in fields such as medicine, tissue engineering, and drug delivery systems, as a filler used for skin and tissue spaces (visco augmentation), and tissue during surgery and transplantation. It has been reported that it can be used as a visco supplementation, such as a visco separation, joint fluid, and the like.

In addition, hyaluronic acid is known to be involved in various human physiological activities and has various physiological activities according to molecular weight.

High-molecular hyaluronic acid is used as a space-filler, has functions such as anti-angiogenic and immuno-suppressive, and hyaluronic acid of medium molecule has immuno-stimulatory activity. And low-molecular hyaluronic acid is known to be involved in apoptosis and protein induction (Tammi et al., J. Biol. Chem. 2002, 277, 4581~4584; Kogan et al., Biotechnology Letter. 2006, 29) , 17-25).

On the other hand, hyaluronic acid is a biopolymer having various functions and excellent viscoelastic properties, but due to its large molecular weight and structural properties, there is a problem in that the viscosity is easily reduced due to decomposition by physical forces such as shear stress. This is a characteristic of a typical polymer solution. In general, a polymer network formed at a high concentration has a property of non-newtonian flow, and it has been reported that the change in physical properties of hyaluronic acid corresponds to this phenomenon. In addition, hyaluronic acid has the property that molecular decomposition and aggregation easily occur due to changes in temperature and pH, and changes in the physical properties of the solution occur even by the addition of salts or metal ions (Ladislav et al., Carbohydrate Research. 2007). , 342, 1071-1077).

The viscosity of joint fluid and synovial fluid decreases due to diseases such as arthritis, osteoporosis, rheumatism, etc., which is caused by oxidative stress such as reactive oxygen species or free radicals occurring in the inflammatory mechanism. It is known that hyaluronic acid, which is abundant in synovial fluid, is decomposed by hyaluronidase.

As such, hyaluronic acid is a biocompatible polymer material having excellent properties, but it has low stability and cannot form a hydrogel in an aqueous solution, so there are many restrictions on clinical application.

Therefore, in order to improve the decomposition resistance of hyaluronic acid, it is chemically denatured through a chemical crosslinking reaction, etc., and is used as a medical treatment or adjuvant, and studies have been conducted to reduce the decomposition phenomenon using antioxidants or radical scavengers (Praest. et al., Carbohydrate Research. 1997, 303, 153-157; Soltes et al., Carbohydrate Research. 2006, 341, 2826-2834).

When hyaluronic acid is chemically modified, the carboxyl group (-COOH) and hydroxyl group (-OH) of hyaluronic acid are the main functional groups for modification, and hyaluronic acid substituted or modified by the esterification reaction accordingly is industrially developed. Improvement of decomposition stability and resistance through cross-linking, viscosity increase, etc. are constantly being studied, and the effects of various cross-linkers such as divinyl sulfone, glycidyl ethers, bis (epoxides), glutaraldehyde, formaldehyde, carbodiimides, etc. (Balazs, The Biology of Hyaluronan, Wiley, Chichester, 1989, pp. 265-280; Tomihata et al., Biomaterials. 18, 1997, 189-195).

However, since most of the chemical crosslinking agents have cytotoxicity, it is essential to remove the toxic crosslinking agent from the reaction product, and the modified hyaluronic acid produced by the crosslinking reaction is not pure hyaluronic acid, and is additionally toxic and safe. Since verification must be confirmed, there are restrictions in using it for medical purposes, etc.

Accordingly, the present inventors have tried to solve the above problems, and confirmed that when the pH of the hyaluronic acid solution is strongly stirred and the pH is adjusted to acidity, the hyaluronic acid hydrogel can be prepared without the use of a chemical crosslinking agent having strong cytotoxicity. The present invention has been completed.

An object of the present invention is a method for preparing a pure hyaluronic acid hydrogel having excellent physical properties and stability even in aqueous solution by crosslinking reaction of hyaluronic acid itself without using a chemical crosslinking agent with strong cytotoxicity, hyaluronic acid hydrogel prepared therefrom, And it is to provide an injection containing this.

In order to achieve the above object, the present invention comprises the steps of: (a) preparing a hyaluronic acid solution by dissolving hyaluronic acid having a molecular weight of 0.5 to 3.0MDa in a solvent; (b) inducing physical crosslinking by adjusting the pH to 2-5 while strongly stirring the hyaluronic acid solution; And (c) obtaining a hyaluronic acid hydrogel formed through physical crosslinking.

In the present invention, the concentration of the hyaluronic acid solution is characterized in that 0.1 to 2% by weight.

In the present invention, the stirring is characterized in that it is carried out for 1 hour in 10 seconds at a speed of 10 ~ 200rpm.

In the present invention, the method for preparing the hyaluronic acid hydrogel is characterized in that it further comprises preparing a hyaluronic acid solution and then adding an excipient.

In the present invention, the excipient is characterized by adding 0.5 to 2.5 parts by weight based on 100 parts by weight of hyaluronic acid dissolved in the hyaluronic acid solution.

The present invention also provides a hyaluronic acid hydrogel prepared by the method for preparing the hyaluronic acid hydrogel.

The present invention also provides an injection comprising the hyaluronic acid hydrogel.

In the present invention, the injection is characterized in that it is for cosmetic or pharmaceutical use.

According to the present invention, a hyaluronic acid hydrogel can be prepared by adjusting the pH in a hyaluronic acid solution without using a chemical crosslinking agent with strong cytotoxicity and applying an external shear force. It has the advantage of excellent physical properties. In addition, according to the molecular weight of hyaluronic acid and the type and content of added excipients, it is possible to adjust the physical properties through the adjustment of gelation, so it can be used as various types of cosmetic skin external preparations, joint injections, injections for molding, and the like.

1 is a flow chart of a method of manufacturing a hyaluronic acid hydrogel according to the present invention.

In the present invention, even without the use of a chemical crosslinking agent having strong cytotoxicity, it was attempted to confirm that hyaluronic acid hydrogel having excellent physical properties and stability can be prepared when the pH is adjusted to acid while strongly stirring the hyaluronic acid solution.

In the present invention, a hyaluronic acid solution was prepared by dissolving hyaluronic acid in purified water, and then the pH was adjusted to acidity and strongly stirred to induce physical crosslinking of hyaluronic acid to prepare a hyaluronic acid hydrogel. As a result of confirming the viscosity, gelation degree, and resistance to hyaluronic acid degrading enzyme of the prepared hyaluronic acid hydrogel, it was confirmed that not only the viscosity and gelation degree of hyaluronic acid hydrogel was excellent, but also excellent resistance to hyaluronic acid degrading enzyme. Could.

Accordingly, the present invention, in one aspect, (a) preparing a hyaluronic acid solution by dissolving hyaluronic acid having a molecular weight of 0.5 ~ 3.0MDa in a solvent; (b) inducing physical crosslinking by adjusting the pH to 2-5 while strongly stirring the hyaluronic acid solution; And (c) obtaining a hyaluronic acid hydrogel formed through physical crosslinking.

In order to prepare a hyaluronic acid hydrogel by a physical reaction according to the present invention, hyaluronic acid is first dissolved in a solvent to prepare a hyaluronic acid solution.

The hyaluronic acid may have a molecular weight of 0.5 to 3.0 MDa, but hyaluronic acid of 1.5 to 1.5 MDa or more is expensive, so it is preferable to use hyaluronic acid of 0.5 to 1.5 MDa.

The solvent for dissolving hyaluronic acid may be used without limitation as long as it is harmless to the human body and capable of dissolving hyaluronic acid, and purified water, other cosmetic preservatives, and pharmaceutical preservatives may be exemplified according to the use.

The concentration of the hyaluronic acid solution is characterized in that 0.1 to 2% by weight. When the concentration of the hyaluronic acid solution is less than 0.1% by weight, there is a problem in that the physical properties of the hydrogel are weak, and when the concentration of the hyaluronic acid solution is more than 2% by weight, there is a problem in using it for general purposes due to too high viscosity.

Next, while strongly stirring the hyaluronic acid solution, the pH is adjusted to 2-5 to induce physical crosslinking.

In the present invention, the agitation is for physical crosslinking of hyaluronic acid, and is characterized in that it is carried out at 10 to 200 rpm for 10 seconds to 1 hour. However, the rpm and time can be adjusted depending on the shape of the stirrer bar and the amount of hydrogel to be manufactured.

When the stirring speed is less than 10 rpm, physical crosslinking does not occur, so that the hyaluronic acid hydrogel is not formed, and when it exceeds 200 rpm, there is a problem that the physical properties of some hydrogels are weakened due to a load. In addition, when the stirring time is less than 10 seconds, it is difficult to form a hydrogel, and when it exceeds 1 hour, the physical properties of the hydrogel are also weakened.

In the present invention, when the hyaluronic acid solution is stirred, the pH is adjusted to 2 to 5 to prevent self-decomposition by inducing a ^COO- group through dissociation ^{of -COOH or -COONa H +} and Na ^{+ in hyaluronic acid.} . At this time, all acidic reagents can be used for pH control.

Finally, the hyaluronic acid hydrogel formed through physical crosslinking can be used directly without any additional process.

On the other hand, in the present invention, when an excipient is added before preparing a hyaluronic acid solution and then stirring, the degree of gelation of the hyaluronic acid hydrogel can be controlled, and a hyaluronic acid hydrogel having more excellent physical properties and stability is obtained. It was intended to confirm that it can be manufactured.

In the present invention, a hyaluronic acid solution was prepared by dissolving hyaluronic acid in purified water, and then guar gum was added as an excipient, and then the pH was adjusted to acidity and strongly stirred to induce physical crosslinking of hyaluronic acid to prepare a hyaluronic acid hydrogel. . As a result of confirming the viscosity, degree of gelation, and resistance to hyaluronic acid degrading enzyme of the prepared hyaluronic acid hydrogel, when an excipient was added, the hyaluronic acid hydrogel was more excellent in viscosity and gelation than when an excipient was not added. In addition, it was confirmed that the resistance to hyaluronic acid degrading enzyme was also more excellent.

Therefore, the method for preparing a hyaluronic acid hydrogel of the present invention is characterized in that it further comprises a step of preparing a hyaluronic acid solution and then adding an excipient. The excipient may include a thickener and a gel stabilizer, and the thickener may include guar gum and xanthan gum, and the gel stabilizer may include alginic acid.

Since the excipient may lower the permeability of hyaluronic acid, it is preferable to use one decolorized with activated carbon depending on the application.

The excipient is characterized by adding 0.5 to 2.5 parts by weight based on 100 parts by weight of hyaluronic acid dissolved in the hyaluronic acid solution. When the excipient is added less than 0.5 parts by weight based on 100 parts by weight of hyaluronic acid, the effect of increasing the viscosity, gelation degree, or resistance to hyaluronic acid degrading enzyme is insignificant, and when added in excess of 2.5 parts by weight, the permeability of the hydrogel decreases. There is a concern.

In the method for preparing the hyaluronic acid hydrogel of the present invention, when an excipient is added, conventional auxiliary agents and carriers such as vitamins, antioxidants, solubilizers, pigments, fragrances, etc. may be further added according to the intended use.

That is, the method of manufacturing the hyaluronic acid hydrogel of the present invention is the physical properties of the hyaluronic acid hydrogel by adjusting the degree of gelation of the hydrogel by adjusting the molecular weight of hyaluronic acid and the type and amount of excipients added according to the application. Can be adjusted. For example, when high molecular weight hyaluronic acid is used or excipients are added, joint injections and hyaluronic acid hydrogels having high elasticity injectable physical properties can be prepared, and low molecular weight hyaluronic acid or excipients are not used. In this case, a hyaluronic acid hydrogel having physical properties that can be used as a mask pack for external skin preparation or other cosmetics or anti-adhesion agent can be prepared.

In another aspect, the present invention relates to a hyaluronic acid hydrogel prepared by the method for preparing the hyaluronic acid hydrogel.

The hyaluronic acid hydrogel prepared by a physical reaction according to the present invention has no cytotoxicity, has excellent viscosity, and has excellent resistance to hyaluronic acid degrading enzymes, so it can be usefully used as a cosmetic or pharmaceutical injection. Examples of the pharmaceutical injections include injections for joints and molded injections for fillers.

[Example]

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples. In addition, in the present invention, examples were presented using sodium hyaluronate of Jinwoo Bio.

Example 1: hyaluronic acid Hydrogel Produce

Hyaluronic acid (molecular weight: 1.0MDa) is dissolved in purified water to prepare a hyaluronic acid solution having a concentration of 1% by weight, and then the pH is adjusted to 4.5 using 0.1N-HCl, while a general stirrer for imparting shear stress A hyaluronic acid hydrogel in which hyaluronic acid is physically crosslinked was prepared by stirring for 3 minutes at a speed of 50 to 100 rpm.

Comparative Example 1: Preparation of hyaluronic acid 1% solution

Hyaluronic acid (molecular weight: 1.0MDa) was dissolved in purified water to prepare a hyaluronic acid solution having a concentration of 1% by weight.

Example 2: Preparation of hyaluronic acid hydrogel added with guar gum

Hyaluronic acid (molecular weight: 1.0MDa) was dissolved in purified water to prepare a hyaluronic acid solution having a concentration of 1% by weight, and then guar gum was added to a concentration of 0.01% by weight, and external shear force while adjusting the pH to 4.5 using 1N-HCl In order to impart (Shear stress), hyaluronic acid hydrogel in which hyaluronic acid is physically crosslinked was prepared by stirring for 3 minutes at a speed of 50 to 100 rpm using a general stirrer.

Comparative Example 2: Preparation of hyaluronic acid/guar gum solution to which guar gum is added

Hyaluronic acid (molecular weight: 1.0MDa) was dissolved in purified water to prepare a hyaluronic acid solution having a concentration of 1% by weight, and then guar gum was added to a concentration of 0.01% by weight.

Example 3: Preparation of hyaluronic acid hydrogel

A hyaluronic acid hydrogel was prepared in the same manner as in Example 1, except that hyaluronic acid (molecular weight: 0.5MDa) was used instead of hyaluronic acid (molecular weight: 1.0MDa).

Example 4: Preparation of hyaluronic acid hydrogel

A hyaluronic acid hydrogel was prepared in the same manner as in Example 2, except that hyaluronic acid (molecular weight: 0.5MDa) was used instead of hyaluronic acid (molecular weight: 1.0MDa).

Experimental Example 1: Measurement of viscosity and gelation degree of hyaluronic acid hydrogel

The viscosity of the hyaluronic acid hydrogel prepared in Examples 1 to 4 and the hyaluronic acid solution prepared in Comparative Examples 1 to 2 was measured using a viscometer (Brookfield RV-II) under conditions of 25° C., Spindle 62, 100 rpm, The results are shown in Table 1.

Example 1 Example 2 Example 3 Example 4 Comparative Example 1 Comparative Example 2 cps Not measurable Not measurable Not measurable Not measurable 6,500 15,000

From Table 1, in the case of a 1% solution of hyaluronic acid having a molecular weight of 1.0 MDa (Comparative Example 1), the viscosity was about 6,500 cps, showing a high viscosity, and the viscosity was improved by about 15,000 cps by adding guar gum (Comparative Example 2) by 3 times. I was able to confirm it. However, when the hydrogel was formed by a physical reaction (Examples 1 to 4), the viscosity could not be measured with the viscometer used in this patent due to its excellent viscoelasticity.

In addition, in order to confirm the gel-forming ability of the hyaluronic acid hydrogel, the degree of gelation of the hydrogel was measured as the amount of water per 1 g of HA, and is shown in Table 2.

Gelation degree = water content (g) / amount of hyaluronic acid (g)

Example 1 Example 2 Example 3 Example 4 Comparative Example 1 Comparative Example 2 Gelation degree (g) 70 100 30 70 0 0

As shown in Table 2, in the case of pure hyaluronic acid to which no crosslinking agent is added, as in Comparative Example 1 and Comparative Example 2, it is dissolved in water and exhibits high viscosity, but there is no ability to maintain a certain shape by containing water. The degree of gelation was measured as zero. That is, the hydrogel was not formed.

On the other hand, the hyaluronic acid hydrogel prepared by physically crosslinking the hyaluronic acid solution was found to contain about 30 to 70 g of water depending on the molecular weight (Examples 1 and 3), and guar gum was added before stirring. In the case, it was found that the water content was increased to about 70 to 100 g (Examples 2 and 4).

From this, it was seen that the moisture content of the hyaluronic acid in the hydrogel changes depending on the molecular weight of the hyaluronic acid and the use of the excipient, so it was found that the physical properties of the hyaluronic acid hydrogel can be adjusted depending on the molecular weight of the hyaluronic acid used in the formation of the hydrogel and the use of the excipient. .

Experimental Example 2: Measurement of resistance to degradation against hyaluronic acid degrading enzyme

Hyaluronidase (Activity: 400unit/mg), a hyaluronic acid degrading enzyme, was added to each solution and hydrogel in Sodium Phosphate Buffer (pH 6.4), and the viscosity of each sample within 1 minute after enzyme treatment at 37℃ in a shaking incubator for 10 minutes. And the gelation degree change measurement to confirm the degradation resistance to the enzyme, and the results are shown in Tables 3 and 4.

Example 1 Example 2 Example 3 Example 4 Comparative Example 1 Comparative Example 2 Initial cps Not measurable Not measurable Not measurable Not measurable 6,500 15,000 Final cps Not measurable Not measurable Not measurable Not measurable 50 12,000

From Table 3, in the case of the hyaluronic acid solution, it was confirmed that the initial viscosity rapidly decreased from 6,500 cps to 50 cps, so that the decomposition of hyaluronic acid was very rapidly progressed by the enzyme (Comparative Example 1). In addition, in the case of the hyaluronic acid solution to which guar gum is added, the initial viscosity decreases from 15,000 cps to 12,000 cps, but it is very small compared to the reduction rate of Comparative Example 1, confirming that the enzymatic degradation is delayed compared to the hyaluronic acid alone. It was possible (Comparative Example 2)

On the other hand, in the case of Examples 1 to 4, even if the hyaluronic acid degrading enzyme was treated, the hydrogel retained its physical properties and the viscosity was still very high, so the viscosity could not be measured with a viscometer.

Example 1 Example 2 Example 3 Example 4 Comparative Example 1 Comparative Example 2 Initial gelation degree 70 100 30 70 0 0 Final gelation degree 50 90 25 65 0 0

From Table 4, in the case of the hyaluronic acid hydrogel to which guar gum was not added, the initial gelation degree slightly decreased from 30 and 70 to 25 and 50, respectively, indicating that the hyaluronic acid hydrogel was partially decomposed by the enzyme. It could be confirmed (Example 3 and Example 1).

On the other hand, in the case of hyaluronic acid hydrogel to which guar gum is added, the initial gelation degree is slightly reduced from 70 and 100 to 65 and 90, respectively, but the reduction rate is small compared to hyaluronic acid hydrogel without guar gum, so enzymatic degradation It was confirmed that the progress was delayed compared to the hyaluronic acid alone (Examples 4 and 2).

That is, it was found that when guar gum was added as an excipient, physical properties such as viscosity and gelation degree and resistance to enzyme degradation were improved.

In general, in the case of pharmaceutical injections, high molecular weight hyaluronic acid is used to have excellent mechanical properties and decomposition resistance in the body. That is, in order to obtain high molecular weight hyaluronic acid, a process of removing low molecular weight hyaluronic acid is performed in the purification process. Therefore, the yield of the high-molecular hyaluronic acid is lowered and the refining cost is increased, and the price of high molecular hyaluronic acid is also increasing.

However, in the present invention, not only can the hyaluronic acid hydrogel be prepared through a physical reaction without the use of a cytotoxic chemical crosslinking agent, but also hyaluronic acid of a commonly used molecular weight, not of an expensive high molecular weight hyaluronic acid, is used. It can be used to prepare a hydrogel of excellent physical properties.

In particular, the hyaluronic acid hydrogel produced by the manufacturing method of the present invention has improved resistance to decomposition against hyaluronic acid degrading enzymes compared to conventional hyaluronic acid. It is expected that the patient's discomfort due to frequent administration can be greatly reduced as the duration of the patient can be extended.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments for those of ordinary skill in the art, and the scope of the present invention is not limited thereby. something to do. Accordingly, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

Claims (8)

(a) preparing a hyaluronic acid solution by dissolving hyaluronic acid having a molecular weight of 0.5 to 3.0 MDa in a solvent;
(b) inducing physical crosslinking by adjusting the pH to 2 to 5 while stirring the hyaluronic acid solution at 10 to 200 rpm for 10 seconds to 1 hour; And
(c) A method for producing a hyaluronic acid hydrogel comprising the step of obtaining a hyaluronic acid hydrogel formed through physical crosslinking.
The method of claim 1, wherein the concentration of the hyaluronic acid solution is 0.1 to 2% by weight.
delete The method of claim 1, further comprising preparing a hyaluronic acid solution and then adding an excipient.
The method of claim 4, wherein the excipient is 0.5 to 2.5 parts by weight based on 100 parts by weight of hyaluronic acid dissolved in the hyaluronic acid solution.
It is prepared by the method of any one of claims 1, 2, 4 and 5, and is composed of hyaluronic acid having a molecular weight of 0.5 to 3.0 MDa, and the gelation degree measured by the following method is 30 to 70. Hyaluronic acid hydrogel.

Gelation degree = water content (g) / amount of hyaluronic acid (g)
An injection comprising the hyaluronic acid hydrogel of claim 6.
The injection according to claim 7, wherein the injection is for cosmetic or pharmaceutical use.

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