KR20220067417A - The manufacturing Method of cross-linked poly-γ-glutamic acid hydrogel - Google Patents

Abstract

The present invention relates to a preparation method of a cross-linked poly-gamma-glutamic acid hydrogel. According to the preparation method of the present invention, a cross-linked poly-gamma-glutamic acid hydrogel with excellent viscoelasticity can be obtained in a short time by optimizing the conditions during a cross-linking reaction. The cross-linked poly-gamma-glutamic acid hydrogel prepared by the method can be widely used in the field of medical devices comprising biomaterials for tissue regeneration such as wound dressings such as tissue adhesion prevention agents after surgery, artificial implants, and facial fillers.

Description

The manufacturing method of cross-linked poly-γ-glutamic acid hydrogel

The present invention relates to a cross-linked poly-gamma-glutamic acid hydrogel and a method for producing the same, and more particularly, to a cross-linked poly-gamma-glutamic acid hydrogel prepared by dissolving, cross-linking, refining/desalting, and sterilizing poly-gamma-glutamic acid and a method for producing the same it's about

Poly-gamma-glutamic acid (Poly-γAcid, γ is an anionic hydrophilic amino acid polymer (γ-polypeptide) in which the γ-carboxyl group of glutamic acid and the α-amino group of glutamic acid are amide bonded. It is produced from Bacillus subtilis isolated from Cheonggukjang, natto in Japan, Kinema in Nepal, etc. In addition, polygamma-glutamic acid is a natural high molecular substance with a sticky consistency obtained through edible microbial fermentation. It is a biocompatible material with very good safety, so polygamma-glutamic acid can be ingested as food, and as the immune activation effect is recently known, the field of application to cosmetics and pharmaceuticals is expanding.

Although polygamma-glutamic acid has the advantage of having a high hydration capacity, the hydrated hydrogel has low viscoelasticity, so its application has been limited in the field of medical devices that need to maintain its shape in vivo. There have been attempts, the representative examples of which are as follows.

Japanese Patent Laid-Open No. 1999-276572 discloses an example of using a polygamma-glutamate complex in which a quaternary amine salt such as chitosan is hydrogen-bonded to the anion carboxyl group of polygammaglutamate as a surgical suture, wound covering material, anti-adhesion agent, hemostatic agent, etc. is starting

Japanese Patent Laid-Open No. 1999-343339 discloses a crosslinking reaction using a polyepoxy compound such as diethylenegylcol diglycidyl ether in polygammaglutamic acid as a crosslinking agent, and applying this as an anti-adhesion agent is an international patent. Publication No. 2007/132785 is disclosed. However, it is impossible to apply it to the field because it is a principle that must be used after in situ gelation in the medical field.

In addition, Japanese Patent Laid-Open No. 2002-128899 discloses an example in which a water-soluble carbodiimide is used to promote the reaction between the carboxyl group and the nucleophile of polygamma-glutamic acid. Also disclosed is an example in which polygammaglutamic acid, fructose, lysine, chitosan, etc. were prepared as a double crosslinked compound using a water-soluble carbodiimide 3-(3-dimethyl aminopropyl)-1-ethylcarbodiimide (EDC).

Korean Patent Application Laid-Open No. 10-2010-0000040 proposes a method of dissolving polygamma-glutamic acid by adding a salt and then performing crosslinking through irradiation with gamma rays, but radiation such as gamma rays is prohibited in general laboratories, so accessibility Not only is this low, but mass production is required for actual commercialization, but gamma irradiation is not suitable for this. In addition, there is a possibility that an unexpected by-product may be generated due to a secondary reaction of the main material through gamma rays.

Japanese Patent Laid-Open No. 1999-276572 Japanese Patent Laid-Open No. 1999-343339 International Patent Publication No. 2007/132785 Japanese Patent Laid-Open No. 2002-128899 Republic of Korea Patent Publication No. 10-2010-0000040

Accordingly, the present inventors studied the main factors affecting the formation of a cross-linked product during a cross-linking reaction using a cross-linking agent, and as a result, the viscosity/elasticity of the cross-linked product and the formation of the cross-linked product depend on the pH of the solvent before the cross-linking reaction, the temperature during the cross-linking reaction, etc. The present invention was completed by confirming that time was changed.

Therefore, the present invention solves a cross-linked poly-gamma-glutamic acid hydrogel production method in which poly-gamma-glutamic acid is dissolved in an acidic solvent before cross-linking reaction and then cross-linked at a high temperature. Providing a gel is a specific challenge.

In order to solve the above problems, the present invention discloses the following means.

In one aspect, a process for dissolving polygamma-glutamic acid in a solvent, a crosslinking process at a high temperature, and a method for preparing through a purification process are disclosed.

Specifically, (Step 1) dissolving polygamma-glutamic acid in an acidic solvent;

(Step 2) adding a crosslinking agent to the dissolved mixture at a molar concentration of 0.1 to 0.25 compared to polygamma-glutamic acid, and allowing the crosslinking reaction to proceed by standing at high temperature for 6 hours or more (comparative stirring);

(Step 3) purifying the cross-linked polygamma-glutamic acid under buffer conditions for 300 to 350 hours; and

(Step 4) Filling a glass syringe with the purified liquid, sterilizing at 121 to 125° C. for 10 to 20 minutes, and allowing to stand at room temperature for 24 hours or more;

Discloses a method for producing a cross-linked polygamma-glutamic acid hydrogel, characterized in that it comprises a.

The polygamma-glutamic acid may have a weight average molecular weight of 100 to 1000 kDa, more preferably 900 kDa, but is not limited thereto. In addition, the concentration of the polygamma-glutamic acid may be dissolved in 75 to 150 mg/ml, preferably 100 to 130 mg/ml.

The pH of the acidic solvent in step 1 is 4 or less, and 0.1 to 3% hydrochloric acid solution may be used. Poly-gamma-glutamic acid is an anionic polymer to which a highly reactive α-carboxyl group is exposed. In the aqueous solution of poly-gamma-glutamic acid, the structure shows different shapes depending on the degree of ionization. In addition, since the ionized polygamma-glutamic acid has a random coil structure and may have a different structure depending on the pH of the aqueous solution, the crosslinking pattern may be different depending on the pH when dissolving before crosslinking. Therefore, in step 1, the pH may act as a factor that has a decisive influence on the viscoelasticity of the final cross-linked polygamma-glutamic acid. In addition, the acidic condition of the solvent induces a catalytic reaction so that the α-carboxyl group of polygammaglutamic acid can form a covalent bond more quickly.

The high temperature of the said 2 process is 40-60 degreeC, Preferably it is 50 degreeC. In the case of a high temperature of 60° C. or higher, a phenomenon in which the viscosity decreases may occur.

The crosslinking agent in step 2 is a material that can crosslink the polymer chains of the polygamma-glutamic acid by chemical bonding, and a polyfunctional compound having a functional group capable of forming a covalent bond by reacting with a carboxyl group is used. can The crosslinking agent used in the present invention is 4-butanediol diglycidyl ether (1,4-butanediol diglycidyl ether, BDDE), polyethylene glycol diglycidyl ether (Poly (ethylene glycol) diglycidyl ether, PEGDE), ethylene glycol di Ethylene glycol diglycidyl ether (EGDGE), divinyl sulfone (DVS), polystyrene sulfonate, poly(3,4-ethylenedioxythiophene) (Poly(3,4-ethylenedioxythiophene) , PEDOT), and can be used in combination of two or more.The specific substituent (epoxide group or sulfone group) of these crosslinking agents strongly reacts with the α-carboxyl group of polygammaglutamic acid to form crosslinked polygammaglutamic acid covalently linked. can be formed.

Step 3 may be performed with or without a dialysis membrane, and purification is performed under 1X Dulbecco's phosphate buffered saline (DPBS) (pH 7.4) buffer conditions. If the dialysis membrane is not used, it is suitable within 3 days, and it is preferable to terminate the purification on the first day (when the hydration rate reaches 20-24%).

The sterilized solution in step 4 is left at room temperature for at least 24 hours to prepare a final cross-linked polygamma-glutamic acid hydrogel.

In another aspect, it is possible to provide a cross-linked polygamma-glutamic acid hydrogel according to the hydrogel manufacturing method of the present invention.

The cross-linked polygamma-glutamic acid hydrogel can be used as a raw material for medical devices such as artificial implants, anti-adhesion agents, and biomaterials for tissue repair.

More specifically, the method for preparing the cross-linked polygamma-glutamic acid hydrogel is as follows.

(first step) dissolution step

-PGA를 75~125mg/ml로 용해시킨다. 이때 용해 공정은 상온에서 약 2시간 동안 교반하여 진행한다.In the present invention, in order to prepare the cross-linked poly-gamma-glutamic acid hydrogel, a dissolution process of poly-gamma-glutamic acid is required. First, a 0.1~3% dilute hydrochloric acid solution was used as a solvent.

-Dissolve PGA at 75-125 mg/ml. At this time, the dissolution process is carried out by stirring at room temperature for about 2 hours.

(Second process) Cross-linking reaction step

In the first step, a crosslinking agent is added at a molar concentration of 0.1 to 0.25 compared to polygamma-glutamic acid to the mixed solution that has been dissolved in the first step, and the crosslinking reaction is performed by standing (comparative stirring) at 40 to 60°C for 6 hours or more.

(Step 3) Purification (dialysis)

In the second step, after the crosslinking reaction is completed, purification (dialysis) is carried out for 300 to 350 hours under 1XDPBS (pH 7.4) buffer conditions with or without the hardened crosslinked polygamma-glutamic acid placed in the dialysis membrane. The buffer solution is changed once every 8 hours or more.

If a dialysis membrane is not used, dialyze within 3 days under 1XDPBS (pH 7.4) buffer conditions. Preferably, dialysis is terminated on the 1st day (when the hydration rate reaches 20-24%).

(Step 4) Sterilization

Fill a glass syringe with the purified liquid in the third step, sterilize it at 121~125℃ for 10~20 minutes, and cool it quickly. The sterilized solution is left at room temperature for at least 24 hours to complete the preparation of the final cross-linked polygamma-glutamic acid hydrogel.

The cross-linked polygamma-glutamic acid hydrogel of the present invention has much higher viscoelasticity than existing polygamma-glutamic acid, so it can be used as a medical device for maintaining its shape in vivo for a certain period of time, and has durability as a biomaterial for tissue restoration, so it can be used as an anti-adhesion agent, artificial implant, It can be used in various medical fields such as cosmetic fillers.

1 is a result of measuring the viscoelasticity of a hydrogel prepared by the method of the present invention and a conventional hydrogel.
2 is a measurement result of the viscoelasticity of a hydrogel using various crosslinking agents.
3 is a measurement result of storage stability of the hydrogel prepared by the method of the present invention.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

Example 1. Preparation of cross-linked polygamma-glutamic acid structure under acidic conditions

-glutamic acid, Lot No. : LB190202, 제조사 : lubon industry co., ltd.)을 125mg/ml의 농도로 1% HCl 용액에 2시간 교반하여 용해한 후, 1,4-부탄디올 디글리시딜에테르(BDDE)를 1/6 몰농도로 넣어주고, 50℃에서 6시간 이상 정치하여 가교하였다. Polygamma glutamic acid (Molecular weight: about 900 kDa, product name:

-glutamic acid, Lot No. : LB190202, manufacturer: lubon industry co., ltd.) was dissolved in 1% HCl solution at a concentration of 125 mg/ml by stirring for 2 hours, 1/6 mol of 1,4-butanediol diglycidyl ether (BDDE) was added The concentration was added, and the mixture was crosslinked by standing at 50° C. for 6 hours or more.

Comparative Example 1. Preparation of cross-linked polygamma-glutamic acid structure under neutral conditions

Proceeded in the same manner as in Example 1, but distilled water was used instead of 1% HCl solution in Example 1.

Comparative Example 2. Preparation of cross-linked polygamma-glutamic acid structure under basic conditions

Proceeded in the same manner as in Example 1, except that in Example 1, a 1% NaOH solution was used instead of a 1% HCl solution.

Experimental Example 1. Crosslinking reaction rate according to dissolution conditions

The crosslinking reaction rates were measured in Example 1, Comparative Example 1 and Comparative Example 2, and the results are shown in Table 1 below. In the acidic solvent, the crosslinking reaction was completed quickly enough to be completed within about 5 hours, whereas in the neutral condition, the crosslinking reaction was not completely completed even after 12 hours had elapsed, and the reaction itself did not proceed in the basic condition.

That the crosslinking reaction can proceed quickly means a reduction in process time. This means that the manufacturing time required can be minimized through the manufacturing method of the present invention, and ultimately the manufacturing cost can be reduced. In particular, the present invention is a It makes sense.

Example 1
(acid solvent) Comparative Example 1
(neutral solvent) Comparative Example 2
(basic solvent) solvent composition 1% HCl Distilled water 1% NaOH pH of solution with 20% γ-PGA added 3-4 7-8 9-10 Cross-linking reaction result Fast (about 5 hours) Slow (about 12 hours) No reaction proceeding

Example 2. Preparation of cross-linked polygamma-glutamic acid hydrogel

-glutamic acid, Lot No. : LB190202, 제조사 : lubon industry co., ltd.)을 125mg/ml의 농도로 1% HCl 용액에 2시간 교반하여 용해한 후, 1,4-부탄디올 디글리시딜에테르(BDDE)를 1/6 몰농도로 넣어주고, 50℃에서 6시간 이상 정치하여 가교하였다. 이후 1XDPBS 조건 하에서 312시간 동안 정제를 진행하였다. 정제가 완료된 액을 유리주사기에 충진하여, 121℃에서 15분간 멸균하고 빠르게 식힌 후 24시간 이상 실온에 정치하여 최종 가교 폴리감마글루탐산 하이드로겔을 제조하였다. Polygamma glutamic acid (Molecular weight: about 900 kDa, product name:

-glutamic acid, Lot No. : LB190202, manufacturer: lubon industry co., ltd.) was dissolved in 1% HCl solution at a concentration of 125 mg/ml by stirring for 2 hours, 1/6 mol of 1,4-butanediol diglycidyl ether (BDDE) was added The concentration was added, and the mixture was crosslinked by standing at 50° C. for 6 hours or more. Thereafter, purification was carried out for 312 hours under 1XDPBS conditions. The purified liquid was filled in a glass syringe, sterilized at 121° C. for 15 minutes, cooled quickly, and left at room temperature for at least 24 hours to prepare a final cross-linked polygamma-glutamic acid hydrogel.

Comparative Example 3. Preparation of cross-linked polygamma-glutamic acid hydrogel

1 mol of polygamma-glutamic acid and 50 ml of phosphate solvent (pH 5.5) were stirred for 3 hours to completely dissolve. 1 mol of BDDE was added to this solution, stirred for 1 hour, and stored at 25° C. for 4 hours. Agitated for 30 minutes using a stirrer, and stored in an incubator at 25° C. for 20 hours to prepare a final cross-linked polygamma-glutamic acid hydrogel.

Experimental Example 2. Measurement of viscoelasticity of hydrogels

The viscoelasticity of the cross-linked polygamma-glutamic acid hydrogels prepared in Example 2 and Comparative Example 3 was measured, and the results are shown in FIG. 1 . Specifically, the prepared hydrogel was left at room temperature for 24 hours or more. And after putting 500ul of hydrogel on the upper parallel plate of Anton Parr Rheometer, viscosity and elasticity were measured under the conditions of Measuring system: pp25, Gap size: 1.0mm, Temperature: 25℃, Shear rate: 0.1/s.

From the results of Figure 1, the viscosity and elasticity of the hydrogel prepared by the manufacturing method of the present invention shows superior viscoelasticity compared to the hydrogel of Comparative Example 3, in which polygamma-glutamic acid was dissolved in weakly acidic conditions and then cross-linked at room temperature conditions was able to confirm that

Example 3. Preparation of cross-linked polygamma-glutamic acid hydrogel using DVS (Divinylsulfone)

Proceeded in the same manner as in Example 2, but using DVS (Divinylsulfone) instead of BDDE as a crosslinking agent to prepare a hydrogel of Example 3.

Example 4. Preparation of cross-linked polygamma-glutamic acid hydrogel using EGDGE (Ethyleneglycol diglycidyl ether)

Proceeded in the same manner as in Example 2, but using EGDGE (Ethyleneglycol diglycidyl ether) instead of BDDE as a crosslinking agent, a hydrogel of Example 4 was prepared.

Example 5. Preparation of cross-linked polygamma-glutamic acid hydrogel using PEDGE (Polyethyleneglycol diglycidyl ether)

Proceeds in the same manner as in Example 2, but using PEDGE (Polyethyleneglycol diglycidyl ether) instead of BDDE as a crosslinking agent to prepare a hydrogel of Example 5.

Experimental Example 3. Measurement of viscoelasticity of hydrogels using various crosslinking agents

The viscoelasticity of the hydrogels prepared in Examples 3 to 5 were measured in the same manner as in Experimental Example 2, and the results are shown in Table 2 and FIG. 2 .

From the results of Table 2 and FIG. 2, it was confirmed that the preparation method of the present invention can provide a hydrogel having excellent viscoelasticity regardless of the type of crosslinking agent.

Viscosity [Pa s] Elasticity [Pa] Example 2 (BDDE) 435.98 379.164 Example 3 (DVS) 457.32 315.73 Example 4 (EGDGE) 402.85 317.371 Example 5 (PEDGE) 415.74 321.087

Experimental Example 4. Storage stability evaluation

Immediately after preparing the hydrogel of Example 2, the viscosity was measured in the same manner as in Experimental Example 2 at the time points of 1 hour, 2 hours, 5 hours, 7 hours, 10 hours, 13 hours, 17 hours, and 20 hours, The results are shown in Table 3 and FIG. 3 .

From the results of FIG. 3, it was confirmed that the hydrogel of the present invention maintained stability even after long-term storage.

Time after manufacture (days) Viscosity (Pa s) 0 435.98 One 438.45 2 439.54 5 435.46 7 448.64 10 430.48 13 426.87 17 430.74 20 438.65

The cross-linked polygamma-glutamic acid hydrogel prepared by the production method of the present invention can be used in the field of cell regeneration medicine and cosmetics by showing effects such as moisturizing and regenerating effects or acting as a nutrient component.

Claims (6)

(Step 1) dissolving polygamma-glutamic acid in an acidic solvent;
(Step 2) adding a crosslinking agent to the dissolved mixture at a molar concentration of 0.1 to 0.25 compared to polygamma-glutamic acid, and allowing the crosslinking reaction to proceed by standing at high temperature for 6 hours or more (comparative stirring);
(Step 3) purifying the cross-linked polygamma-glutamic acid under buffer conditions for 300 to 350 hours; and
(Step 4) Filling a glass syringe with the purified liquid, sterilizing at 121 to 125° C. for 10 to 20 minutes, and allowing to stand at room temperature for 24 hours or more;
A method for producing a cross-linked polygamma-glutamic acid hydrogel comprising a. The method according to claim 1, wherein the pH of the acidic solvent in step 1 is 4 or less. The method according to claim 1, wherein the high temperature in step 2 is 40 to 60°C. The method according to claim 1, wherein the crosslinking agent in step 2 is 4-butanediol diglycidyl ether (BDDE), polyethylene glycol diglycidyl ether (PEGDE), ethylene glycol diglycidyl ether (EGDGE), divinyl sulfone ( DVS), polystyrene sulfonate, and poly(3,4-ethylene dioxythiophene) (PEDOT) at least one selected from the group consisting of a cross-linked polygamma-glutamic acid hydrogel manufacturing method. The method for producing a cross-linked polygamma-glutamic acid hydrogel according to claim 1, wherein in step 3, purification is performed using a dialysis membrane. The method according to claim 1, wherein the buffer solution in step 3 is 1X Dulbecco's phosphate buffered saline (DPBS) (pH 7.4).

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