KR20110076838A - Hydrgel, preparation method and use thereof - Google Patents

Abstract

PURPOSE: Hydrogel is provided to enable the gelation within a short time under a physiology condition, to exhibit fast gelation rate, and to have high adhesive property and high fracture strength. CONSTITUTION: Hydrogel comprises a crosslinking material. The crosslinking material is such that γ-polyglutamic acids and a polylysine or polyethylene glycol-based polymer having a plurality of nucleophilic functional groups are crosslinked, wherein at least one part of carboxyl groups is activated. The activated γ-polyglutamic acids are activated by bonding a compound of chemical formula 1 and succineimide ester with at least a part of carboxy group. In chemical formula 1, R represents hydrogen or -SO3Na.

Description

Hydrogel, preparation method and use thereof {HYDRGEL, PREPARATION METHOD AND USE THEREOF}

The present invention relates to hydrogels, methods for their preparation and uses. More specifically, the present invention relates to a hydrogel, which is short in gelation time, has biocompatibility and biodegradability, exhibits excellent adhesion and burst strength, and is applicable to biotissue adhesion, a method and a preparation thereof.

Hydrogels have been applied to various tissue adhesives applied to living bodies. Such hydrogels can be prepared from synthetic polymers, natural polymers or mixtures thereof. In addition, a tissue adhesive such as fibringlu is used, but such fibringlu has a low adhesive strength and easily falls from the surgical site, and since it is a blood product, there is a risk of disease transmission such as a viral infection.

Thus, there has been known a hydrogel prepared using various synthetic polymers or natural polymers and tissue adhesives using the same. For example, although hydrogels using albumin, gelatin, and aldehyde crosslinking agents are known, some of them are used, but all of them use animal-derived proteins, which may cause infections, and toxic problems of aldehyde crosslinking agents may cause problems of poor biocompatibility. have.

On the other hand, γ-polyglutamic acid is a biosynthetic polymer obtained by culturing microorganisms, and since the repeating unit contains a carboxyl group, crosslinking reaction with other substances is possible, and thus water solubility, biodegradability and biocompatibility are shown. Hydrogels using a crosslinking reaction of polyglutamic acid have also been known.

For example, there has been known a hydrogel using a crosslinking reaction of γ-polyglutamic acid and an epoxy compound, a complex prepared by combining γ-polyglutamic acid and a quaternary amine salt such as chitosan. However, these hydrogels have a disadvantage in that the time required for the crosslinking reaction (gelling time) is too long or the crosslinking reaction at room temperature is difficult, and thus, it is difficult to form a hydrogel directly at the medical field and apply it to tissue adhesion.

In addition, in order for the hydrogel to be applied to a tissue adhesive or the like, in addition to biodegradability, absorbency, biocompatibility, and the like, excellent adhesion and burst strength are required. Most of the previously known hydrogels do not satisfy these required properties or the gelation time described above. This was too long.

Accordingly, there is a continuing need for a hydrogel that has a short gelation time, exhibits biocompatibility and biodegradability, is excellent in adhesive strength and rupture strength, and can be preferably applied to tissue adhesion or the like.

One embodiment of the present invention is to provide a hydrogel that is short in gelation time, has biocompatibility and biodegradability, exhibits excellent adhesion and burst strength, and is applicable to adhesion of biological tissues.

Another embodiment of the present invention is to provide a method for preparing the hydrogel.

Another embodiment of the present invention is to provide a kit for preparing the hydrogel.

Another embodiment of the present invention is to provide a tissue adhesive composition comprising a component that enables the formation of the hydrogel.

The present invention provides a hydrogel comprising at least part of a carboxyl group-activated γ-polyglutamic acid and a crosslinked crosslinked ε-polylysine or polyethyleneglycol polymer having a plurality of nucleophilic functional groups.

The present invention also provides a method for producing a hydrogel comprising crosslinking a γ-polyglutamic acid having at least a portion of a carboxyl group and an ε-polylysine or polyethylene glycol polymer having a plurality of nucleophilic functional groups.

The present invention also provides a kit for producing a hydrogel comprising at least a portion of a carboxyl group-activated γ-polyglutamic acid and an ε-polylysine or polyethyleneglycol polymer having a plurality of nucleophilic functional groups.

The present invention also provides a tissue adhesive composition comprising γ-polyglutamic acid with at least part of a carboxyl group activated and an ε-polylysine or polyethyleneglycol polymer having a plurality of nucleophilic functional groups. Such tissue adhesive compositions can be applied and cured to living tissue to form hydrogels to adhere the living tissue.

Hereinafter, a hydrogel according to an embodiment of the present invention, a manufacturing method thereof, and a tissue adhesive will be described in detail.

According to one embodiment of the invention, there is provided a hydrogel comprising a crosslinked cross-linked γ-polyglutamic acid having at least a portion of carboxyl groups and an ε-polylysine or polyethyleneglycol polymer having a plurality of nucleophilic functional groups. .

Such hydrogels can exhibit biocompatibility and biodegradability suitable for application to biological tissues, as they include crosslinks of polyamino acids, such as γ-polyglutamic acid, and ε-polylysine or polyethyleneglycol polymers that exhibit biocompatibility. . In addition, the experimental results of the present inventors, it was found that the hydrogel containing the cross-linked product of the activated γ-polyglutamic acid and ε-polylysine or polyethylene glycol-based polymer can exhibit excellent adhesion and burst strength.

Furthermore, as will be described in more detail below, when at least a part of γ-polyglutamic acid is reacted with a specific compound to be converted into a succinimide ester group or the like, the activated γ-polyglutamic acid is converted into an amine group, thiol group, or hydroxy group. It was found that hydrogel can be formed by causing rapid gelation (crosslinking reaction) at room temperature with ε-polylysine or polyethyleneglycol polymer having a nucleophilic functional group. Therefore, the hydrogel is very suitable to be formed as a gel state in the medical field and applied to tissue adhesion and the like.

In addition, the activated succinimide ester group of γ-polyglutamic acid may be attached to the tissue with excellent adhesion while reacting with an amino group (-NH2), a thiol group (-SH), or a hydroxyl group (-OH) of a biological tissue. have. Therefore, the hydrogel can be very preferably applied to the adhesion of biological tissue.

Referring to the hydrogel of this embodiment in more detail as follows.

The "hydrogel" may be defined as meaning a polymer matrix capable of swelling, and may have a crosslinked structure including a covalent bond or a non-covalent bond. In addition, the hydrogel may include a three-dimensional network structure composed of the crosslinked structure, and may absorb water to form an elastic gel.

According to one embodiment of the invention, the hydrogel comprises a cross-linked cross-linked γ-polyglutamic acid with at least a portion of the carboxy group and ε-polylysine or polyethylene glycol-based polymer having a plurality of nucleophilic functional groups, The carboxyl group may be activated by combining with a compound of formula 1 (eg, N-hydroxysuccinimide or N-hydroxysulfosuccinimide) to form a succinimide ester group:

[Formula 1]

In Formula 1, R represents hydrogen or -SO ₃ Na.

Such succinimide ester groups may crosslink with a nucleophilic functional group contained in an ε-polylysine or polyethylene glycol polymer such as an amine group, a thiol group or a hydroxyl group at a very high rate to form an amide bond or a thioamide bond. The result is a very fast gelation of the hydrogel. In particular, such a crosslinking reaction may occur in an aqueous solution of pH 7.2 ~ 11.0, 37 ℃ corresponding to physiological conditions, and even under these conditions gelling occurs within 10 minutes, specifically within 2 minutes, more specifically within 5 to 30 seconds Hydrogel can be formed. Therefore, the hydrogel is formed in a gel state at the medical site and applied to a living tissue, and thus can be preferably used for tissue adhesion.

The molar ratio of the carboxyl group activated by the succinimide ester group or the like in the carboxyl group of γ-polyglutamic acid may be 0.1 to 0.7, specifically 0.1 to 0.5, and more specifically 0.1 to 0.3. By activating the carboxyl group of γ-polyglutamic acid in such a molar ratio, it is possible to shorten the gelation time of the hydrogel and optimize the adhesion to biological tissues. In addition, there is almost no additional effect of increasing the molar ratio, so the above range is appropriate.

The γ-polyglutamic acid preferably has at least a portion of a carboxyl group which is not activated by the succinimide ester group or the like in the form of an alkali metal salt or an alkaline earth metal salt, for example, a sodium salt. As at least a part of the carboxyl groups of the γ-polyglutamic acid have a form of a metal salt such as sodium salt, the activated γ-polyglutamic acid may exhibit water solubility. In view of such water solubility, the molar ratio of the metal salt such as sodium salt in the carboxy group of γ-polyglutamic acid is preferably 0.2 or less, specifically 0.1 to 0.2.

As such, the category of γ-polyglutamic acid that can be activated with succinimide ester groups and the like can encompass not only γ-polyglutamic acid in the free state, but also metal salts thereof, and also mixtures of free and metal salt states. have. Specifically, γ-polyglutamic acid before activation may have a structure represented by the following general formula (1):

[Formula 1]

In Chemical Formula 1, M is H, an alkali metal or an alkaline earth metal (eg, Na, K, Ca or Mg), or two or more selected from them (eg, γ-polyglutamic acid and a metal salt state in a free state). When a mixture of γ-polyglutamic acid is used); n is 390 to 15,500, specifically 3,900 to 7,800.

The γ-polyglutamic acid before the activation can be obtained by natural or synthetic, specifically by fermentation of a microorganism, Bacillus subtilis , it is reacted with the compound of Formula 1 to obtain the activated γ-polyglutamic acid Can be. .

The weight average molecular weight of the γ-polyglutamic acid may be 500,000 to 2,000,000 Daltons, specifically 1,000,000 to 2,000,000 Daltons. If the weight average molecular weight is too small, the gelation time for forming the crosslinked body and the hydrogel may be long, or it may be difficult to provide a hydrogel having a sufficient level of adhesive strength or burst strength. On the contrary, when the molecular weight of gamma -polyglutamic acid becomes too large, there exists a possibility that a nonuniform or turbid hydrogel may be provided.

In addition, the intrinsic viscosity of the activated γ-polyglutamic acid may be 1 to 5 dL / g, specifically 1.5 to 4.5 dL / g. The intrinsic viscosity reflects the molecular weight of the activated γ-polyglutamic acid, and as the intrinsic viscosity range is optimized, a hydrogel having excellent physical properties such as uniformity and strength may be provided.

On the other hand, since the ε-polylysine forming a crosslinked product with the γ-polyglutamic acid has a nucleophilic functional group such as an amine group in the repeating unit, it is possible to form an amide bond with the activated γ-polyglutamic acid, thereby crosslinking The structure can be formed to form a crosslinked body and a hydrogel having a three-dimensional network structure. In particular, the ε-polylysine causes a rapid crosslinking reaction with the activated γ-polyglutamic acid to enable the provision of a hydrogel having a short gelling time.

Such ε-polylysine may encompass not only ε-polylysine in the free state represented by the general formula (2), but also acid addition salts thereof, particularly acid addition salts applicable to a living body such as bromate, hydrochloride or fluorate, and in the free state And mixtures of acid addition salts can also be encompassed:

[Formula 2]

Wherein m is 8 to 54, specifically 23 to 38.

The ε-polylysine may be produced using microorganisms ( Streptomyces ) or enzymes, and excellent in solubility in water and stability in the human body. Accordingly, the hydrogel of one embodiment may exhibit biocompatibility suitable for application to tissue adhesion and the like.

In addition, the weight average molecular weight of the ε-polylysine may be 1,000 to 20,000 Daltons, specifically 1,000 to 6,000 Daltons. If the weight average molecular weight is too small, less than 1,000 Daltons, the gelation time for the formation of the crosslinked body and the hydrogel may be long, or the gelation reaction (such as the formation of the crosslinked body) may not occur well, on the contrary, ε-polylysine If the molecular weight of is too large, the biodegradability of the crosslinked product and the hydrogel may be lowered.

Meanwhile, the polyethyleneglycol polymer, which is an example of another polymer forming a crosslinked product with γ-polyglutamic acid, may be modified to have a nucleophilic functional group such as, for example, an amine group, a thiol group, or a hydroxy group. Specifically, the polyethyleneglycol polymer may be one in which an amine group or a thiol group is bonded to the terminal. For example, such a polyethyleneglycol polymer has a structure in which a polyethyleneglycol repeating unit is bonded to each hydroxy group of a divalent or higher, preferably 2 to 12, polyhydric alcohol, and an amine group, thiol group, or hydroxy group is bonded to the terminal thereof. Can have More specifically, such polyethylene glycol-based polymer may be represented by Formula 2:

[Formula 2]

I-[(CH ₂ CH ₂ O) n-CH ₂ CH ₂ X] m

In the above formula, I is a radical derived from a 2 to 12 valent polyhydric alcohol, wherein hydrogen is substituted with-(CH ₂ CH ₂ O) n-CH ₂ CH ₂ X in each of the hydroxyl groups of the polyhydric alcohol to form an ether bond thereto. X represents an amine group, a thiol group or a hydroxy group, n is 19 to 170, m is an integer of 2 to 12, which is the same as the number of hydroxy groups of the polyhydric alcohol derived from I.

In Formula 2, specific examples of I include diols such as ethylene glycol, propanediol, butandiol, butandiol, pentandiol, hexanediol, and the like; Or glycerol, erythritol, thritol, pentaerythritol, pentaerythritol, xylitol, adonitol, sorbitol, mannitol palatinose radicals derived from trisaccharides such as disaccharides such as palatinose, maltose monohydrate or maltitol, or trisaccharides such as D-raffinose pentahydrate. Can be mentioned. More specifically, I may be a radical derived from 4 to 12 valent polyhydric alcohol.

Examples of the polyethylene glycol polymer belonging to the scope of Formula 2 include a polymer in which a polyethylene glycol repeating unit having a terminal nucleophilic functional group is bonded to a radical derived from pentaerythritol or sorbitol, for example, the following Formula 3 Or polymers of four:

(3)

[Formula 4]

In Formulas 3 and 4, X and n are as defined above.

The polyethyleneglycol polymer may form an amide bond, a thioamide bond or an ester bond with the activated γ-polyglutamic acid by including a plurality of nucleophilic functional groups such as an amine group, a thiol group or a hydroxy group. The crosslinked structure can be formed to form a crosslinked body and a hydrogel having a three-dimensional network structure. In particular, such polyethyleneglycol-based polymers may cause a rapid crosslinking reaction with the activated γ-polyglutamic acid, thereby providing a hydrogel having a short gelling time.

In addition, such a polyethylene glycol-based polymer is a representative biocompatible polymer among synthetic polymers, and the hydrogel including its crosslinked body may exhibit biocompatibility suitable for tissue adhesion or the like. The polyethylene glycol polymer may be prepared according to a conventional method such as addition polymerization of ethylene oxide to a polyhydric alcohol and introduction of nucleophilic functional groups.

In addition, the weight average molecular weight of the polyethylene glycol polymer may be 5,000 to 30,000 Daltons, specifically 10,000 to 20,000 Daltons. If the weight average molecular weight is too small, the gelation time for the formation of the crosslinked body and the hydrogel may be long, or the gelation reaction may not occur well. On the contrary, if the molecular weight of the polyethylene glycol polymer is too large, the biodegradation of the crosslinked body and the hydrogel is excessive. The castle may be degraded.

In addition, in the hydrogel of the embodiment, with respect to the activated carboxyl group (eg, succinimide ester group) of γ-polyglutamic acid, the nucleophilic functional group of the ε-polylysine or polyethylene glycol polymer is 0.2 to 2.0, Specifically, it may have a molar ratio of 0.2 to 1.0, more specifically 0.4 to 0.6. By satisfying this molar ratio, the adhesion and burst strength of the hydrogel to the biological tissue can be excellent, and the crosslinking of the hydrogel can also be optimized. However, when the molar ratio is too low, the crosslinking point is less, the strength of the hydrogel may be weakened.

On the other hand, according to another embodiment of the invention, there is provided a method for producing the hydrogel described above. The method for preparing a hydrogel may include a step of crosslinking a γ-polyglutamic acid having at least a portion of a carboxyl group and an ε-polylysine or polyethyleneglycol polymer having a plurality of nucleophilic functional groups. According to such a manufacturing method, a hydrogel having a crosslinked structure formed within a fast gelation time can be obtained even under physiological conditions near room temperature, and in particular, the hydrogel can be readily applied to tissue adhesion and the like even in a medical field.

This preparation method may further comprise forming the activated γ-polyglutamic acid before the crosslinking reaction. In the step of forming the activated γ-polyglutamic acid, γ-polyglutamic acid in a free state, a metal salt state, or a mixture thereof may be reacted with the compound of Formula 1. This reaction step is described in detail as follows.

Since the activated γ-polyglutamic acid constitutes a hydrogel applied to a living body, it must be water-soluble, and at the same time, it is required to be dissolved in an appropriate aprotic solvent. However, the activated γ-polyglutamic acid may lose activity by directly reacting with the solvent in the presence of water or a protonic solvent, so that the reaction of the γ-polyglutamic acid with the compound of Formula 1 proceeds in an aprotic solvent. desirable.

In addition, in order to make the γ-polyglutamic acid water-soluble, at least part of the carboxy groups preferably have the form of an alkali metal salt or an earth metal salt such as sodium salt, as described above. To this end, in the reaction with the compound of Formula 1, at least a part of the carboxyl group is reacted with sodium bicarbonate (NaHCO ₃ ) and the like in a free state of γ-polyglutamic acid under an aprotic solvent to form a sodium salt or the like. React with at least one compound (eg, N-hydroxysuccinimide; NHS) to activate at least a portion of the carboxyl group (see Scheme 1 below). In this case, the reaction with the compound of Formula 1 may be carried out in the presence of a carbodiimide-based compound such as, for example, dicyclohexylcarbodiimide (DCC), and thus the reaction step may be performed more efficiently. have.

Scheme 1

As a result of the experiments of the present inventors, Ion coupled 20% by mol of the carboxyl group was converted into the sodium salt form when the free γ-polyglutamic acid dissolved in an aprotic solvent such as DMSO was reacted with an excess of sodium bicarbonate for 16 hours at room temperature. Plasmon spectroscopy (ICP) confirmed. Increasing the reaction temperature or reaction time did not significantly increase the molar ratio of carboxyl groups converted to sodium salt form, and it was confirmed that γ-polyglutamic acid in which the above molar ratio of carboxyl groups were converted to sodium salts exhibited desirable water solubility. Thus, the activated γ-polyglutamic acid may be prepared by reacting γ-polyglutamic acid in which some carboxyl groups are converted to sodium salt form with a compound of Formula 1 in the presence of a carbodiimide-based compound such as DCC.

The molecular weight change of γ-polyglutamic acid with reaction time can be measured by measuring the inherent viscosity (I.V.). As reaction time elapses, decomposition occurs and molecular weight decreases. The higher the substituted compound of Formula 1, the more the crosslinking points forming the three-dimensional network structure of the hydrogel produced by the crosslinking reaction, the higher the physical strength such as compressive strength and tensile strength of the hydrogel, and the higher the tissue adhesion. do. However, if the molecular weight of the polymer is too low, the strength of the produced hydrogel is low, if too high it is difficult to mix with the aqueous solution of the polymer having a nucleophilic functional group may be low strength of the hydrogel.

In consideration of this point, the reaction time with the compound of Formula 1 is suitably 1 to 16 hours, specifically 3 to 4 hours, and the degree to which the compound of Formula 1 is substituted is 0.1 to 0.7 as the molar ratio of the carboxyl group. 0.3 to 0.6 are preferred. In addition, the carbodiimide-based compound is preferably added in a molar ratio of 0.1 to 3, specifically 1 to 2, based on the number of moles of carboxyl groups contained in γ-polyglutamic acid before activation, the compound of Formula 1 is It is appropriate to add a molar ratio of 0.1 to 3, specifically 1 to 2, based on the number of moles. The reaction temperature is preferably 0 to 150 ° C, specifically 20 to 70 ° C.

On the other hand, after preparing the γ-polyglutamic acid activated by the above-described method, it can be cross-linked with an ε-polylysine or polyethylene glycol polymer having a plurality of nucleophilic functional groups to prepare a crosslinked body and a hydrogel. This crosslinking reaction can be carried out in an aqueous solution in which the activated γ-polyglutamic acid and ε-polylysine or a polyethylene glycol polymer are mixed. For example, the first liquid including the activated aqueous solution of γ-polyglutamic acid and the second liquid including the aqueous solution of ε-polylysine or polyethylene glycol polymer may be mixed.

At this time, in order to obtain a uniform hydrogel in mix | blending a 1st liquid and a 2nd liquid, it is preferable to mix the solution of both appropriate concentrations. Crosslinking density may be controlled by the concentration of the polymer in addition to the functional group of the polymer. The higher the concentration of the polymer, the higher the degree of crosslinking, but it may be difficult to produce a uniform hydrogel due to precipitation above a certain concentration. If the concentration of the polymer is too low, the strength of the hydrogel may be weak or not properly formed. . In view of this point, the concentration of γ-polyglutamic acid and the total polymer of the ε-polylysine or polyethylene glycol polymer in the aqueous solution is 1 to 20% by weight, specifically 2 to 10% by weight, more specifically 5 to 7% by weight. % Is appropriate.

In addition to the distilled water, as the aqueous solvent for preparing the first and second liquids, non-toxic agents such as physiological saline, buffers such as sodium hydrogencarbonate (NaHCO ₃ ), boric acid, or phosphoric acid may be used.

Solid and aqueous components of the first and second liquids can be easily sterilized by radiation sterilization, preferably by gamma irradiation of 10 to 50 kGy, more preferably gamma irradiation of 20 to 30 kGy. Can be. Such sterilization can be carried out by setting the conditions so as not to adversely affect the gelation time or the physical properties of the other hydrogels.

And, the crosslinking reaction can be carried out at 0 to 50 ℃, specifically 25 to 40 ℃, from 1 second to 200 seconds, preferably from 2 seconds to 100 seconds, more preferably from 3 seconds to 50 seconds from the mixing The crosslinking reaction (gelling) can be initiated. In addition, the time for the crosslinking reaction may vary depending on the reaction temperature and the like, but may be performed for 5 seconds to 20 minutes, specifically 10 seconds to 10 minutes. And, when mixing the aqueous solution for the reaction can be mixed in a conventional manner using a magnetic stirrer or the like. In addition, when the crosslinking reaction (gelation) proceeds in the medical field to form and apply the hydrogel immediately, the aqueous solution for the crosslinking reaction can be mixed using a device such as a double barrel syringe.

As described above, according to the above-described manufacturing method, a hydrogel having excellent properties can be obtained through rapid gelation even under physiological conditions, and thus, the hydrogel can be very preferably applied for tissue adhesion in a medical field.

The hydrogel prepared by the above-described method may be freeze-dried to be provided in the form of a sponge or a sheet, or may be powdered, and may be used as an anti-adhesion agent, an absorbent, or a drug delivery agent in such a form. In addition, as will be described in more detail below, a kit or composition containing each component (eg, activated γ-polyglutamic acid and an ε-polylysine or polyethyleneglycol polymer) for formation of a hydrogel is used in the medical field. By carrying out the crosslinking reaction (gelation) or the like to form the hydrogel described above, it can be applied to adhesion of living tissue. For application to such tissue adhesion, after forming a mixture containing activated γ-polyglutamic acid and ε-polylysine or polyethylene glycol polymer, for example, a mixed aqueous solution, on the biological tissue, the aqueous solution is crosslinked. Reaction (gelling) can form a hydrogel and a coating on living tissue.

According to another embodiment of the present invention, there is provided a kit for the preparation of the hydrogel described above. Such a hydrogel manufacturing kit may include γ-polyglutamic acid having at least a portion of carboxyl groups activated and an ε-polylysine or polyethyleneglycol polymer having a plurality of nucleophilic functional groups.

As described above, the hydrogel of one embodiment can be obtained at a high gelation rate even under physiological conditions. Therefore, such hydrogel can be used for tissue adhesion or the like in the form of a kit or composition before gelation including each component for its formation. For example, after the kit or composition including each of the above components is applied to the living tissue, gelation may be performed on the living tissue to form a hydrogel, and may exhibit the function of tissue adhesion. For this purpose and the like, the kit of another embodiment can be used.

According to a specific embodiment, such a hydrogel manufacturing kit may include a first liquid containing an aqueous solution of activated γ-polyglutamic acid and a second liquid including an aqueous solution of a polymer having nucleophilic functional groups. Such a kit can be used by mixing an aqueous solution of the first liquid and the second liquid.

The mixing and use of the first liquid and the second liquid can be carried out by various methods. For example, mixing is performed by applying one of the stock solutions of the first and second liquids to the surface of an adherend such as a living tissue, and then applying the other stock solution of the first and second solutions to the surface of the other adherend. can do. In addition, the first liquid and the second liquid may be mixed in an applicator such as a double barrel syringe to apply the coating. The mixing ratio (volume ratio) of the first liquid and the second liquid is usually set to 0.5 to 2.0.

When the first liquid and the second liquid are mixed, an amide, thioamide, or ester bond is formed between the activated carboxyl group of γ-polyglutamic acid (such as succinimide ester group) and the nucleophilic functional group, which becomes a crosslinking point and becomes three-dimensional. A hydrogel having a network structure can be formed. As a result, gelation can occur from 1 second to 200 seconds, preferably from 2 seconds to 100 seconds, more preferably from 3 seconds to 50 seconds from mixing. However, although the preferred time from mixing to gelation may vary somewhat depending on the application, when the gelling time is 2 seconds or less, smooth application may be difficult, and if it is too long, it may flow into the solution before the gel is formed. This is not easy.

On the other hand, according to another embodiment of the invention, there is provided a tissue adhesive composition using the above-mentioned hydrogel. As described above, the hydrogel is preferably in the form of a composition before gelation including each component for its formation, and may be preferably used for adhesion to a living tissue. Accordingly, one embodiment of the tissue adhesive composition is an ε-polylysine or polyethylene glycol having each component for preparing a hydrogel, for example, γ-polyglutamic acid having at least some carboxyl groups activated thereon and a plurality of nucleophilic functional groups. It may include a polymer.

A composition of one embodiment comprising at least a portion of a carboxyl group-activated γ-polyglutamic acid and an ε-polylysine or polyethyleneglycol polymer having a plurality of nucleophilic functional groups, has a temperature range of 25-40 ° C. It can be applied in the body in the vicinity of the conditions, the gelation may occur within 10 minutes, specifically 2 minutes, more specifically 5 to 30 seconds when mixed to form a hydrogel. Therefore, since such a composition can be gelled within a short time after application to living tissue, the tissue can be adhered to, and thus, preferable application in the medical field is possible.

Since the method of applying the above-described tissue adhesive composition has already been described with respect to the kit for preparing hydrogel, further description thereof will be omitted.

The tissue adhesive composition may be applied for a variety of uses such as surgery such as topical wound closure, gastrointestinal anastomosis, vascular anastomosis, ophthalmic surgery.

The hydrogel according to the present invention is capable of gelation within a short time under physiological conditions, showing a high gelation rate. In addition, the hydrogel has a high adhesive strength and high burst strength enough to be used as an adhesive in biological tissues.

Therefore, the hydrogel can be very preferably used for various purposes such as adhesion to biological tissues.

1 is a diagram showing the degradation behavior (in vitro degradation) of the hydrogel prepared by the crosslinking reaction of the activated γ-polyglutamic acid and ε-polylysine

Hereinafter, the invention will be described in more detail with reference to Examples. However, these examples are only intended to illustrate the present invention, but the scope of the invention is not limited by these examples.

Preparation Example 1 Preparation of Activated γ-polyglutamic Acid

Into a dried 500 ml round two-necked glass flask was added γ-polyglutamic acid (γ-PGA, weight average molecular weight 50, 500, 1,000 kDa) by 40 mmol (5.16 g) based on the carboxyl unit, and 250 ml of dimethyl sulfoxide (DMSO). The mixture was stirred for 5 hours at 50 ° C. to be uniformly dissolved, and 80 mmol (6.7 g) of sodium bicarbonate was added and reacted at 50 ° C. for 16 hours. As a result, it was confirmed by Ion coupled Plasmon spectroscopy (ICP) that 20mol% of the carboxyl group is converted to the sodium salt form. After the temperature of the reaction solution was lowered to room temperature (25 ° C.), N-hydroxysuccinimide (NHS) was quantitatively added according to the molar ratio shown in Table 1, and water was removed under reduced pressure. Dicyclohexylcarbodiimide (DCC) was quantified according to the molar ratio shown in Table 1 in a dried 100 ml round two-necked glass flask, and dimethyl sulfoxide (15w / v%) was added to dissolve and water was removed under reduced pressure. The dicyclohexylcarbodiimide solution was added to the γ-polyglutamic acid solution under nitrogen through a cannula and then reacted under nitrogen for 1 to 5 hours while stirring to ensure uniform mixing.

After the completion of the reaction, the reaction solution was filtered and precipitated in 1200 ml of a mixed solvent of tetrahydrofuran (THF) and chloroform (Chloroform) to remove urea. After washing three times with THF to completely remove the unreacted NHS and DCC and dried in a vacuum oven maintained at 90 ℃ for more than 16 hours to remove the remaining solvent. Finally activated γ-polyglutamic acid was obtained.

Substituted NHS content was determined by the following method through UV-VIS spectroscopy measurement using Hydroxamate method, and NHS substitution degree for DCC and NHS dosages is shown in Table 1.

<quantification of NHS introduced into γ-polyglutamic acid>

The content of NHS introduced into the activated γ-polyglutamic acid was measured using the hydroxamate method (H. Iwata et al., Biomaterials, 19, 1869, 1998). 0.2 ml of 0.2N NaOH solution was added to 1 ml of 0.1-0.2umol NHS solution, and the mixture was left at 60 ° C for 10 minutes. After 10 minutes, 1.5 ml of 0.85N HCl solution was added and acidified. In order to form complexes with NHS to induce color development, ferric chloride (FeCl ₃ ) dissolved in 5% in 0.1N HCl solution was added, and absorbance was measured at 500 nm. A standard curve was prepared from the absorbance obtained according to the concentration of NHS. The absorbance of the activated γ-polyglutamic acid was measured in the same manner as above, and the content of NHS introduced into γ-polyglutamic acid was calculated from the standard curve.

Introduction ratio of NHS to γ-PGA according to DCC and NHS input (reaction time, 2 hours) number γ-PGA
Weight average molecular weight (kDa) [NHS] / [COOH] [DCC] / [COOH] Introduction ratio of NHS to γ-PGA:
[COO-NHS] /
([COOH] + [COO-NHS]) T-1 500 0.2 0.2 0.05 T-2 500 0.4 0.4 0.13 T-3 500 0.6 0.6 0.15 T-4 500 0.8 0.8 0.24 T-5 500 1.0 1.0 0.25 T-6 500 1.2 1.2 0.25 T-7 500 1.2 0.7 0.17 T-8 500 1.2 1.5 0.29 T-9 500 1.2 2.0 0.28

 Referring to Table 1, when reacted for 2 hours at room temperature, the NHS and the degree of DCC increased until the same amount of carboxyl groups was added, the degree of substitution of NHS increased. However, even when excess NHS and DCC were reacted with the carboxyl group, only 30 mol% of the carboxyl group was substituted.

To determine the change in reaction time, the content of NHS and DCC was fixed at [NHS] / [COOH] = 1.2, [DCC] / [COOH] = 1.5, and the reaction time was changed to 1-5 hours, Table 2 shows the measurement results of the molecular weight NHS substitution degree of γ-polyglutamic acid. NHS substitution degree for γ-PGA increased up to 3 hours at room temperature, but thereafter, the substitution degree did not increase significantly to 45-49 mol%.

Inherent viscosity (Inherent Viscosity, IV) was also measured to investigate the trend of decreasing molecular weight of γ-polyglutamic acid during NHS substitution reaction. After dissolving NHS-substituted γ-polyglutamic acid at 0.1 g / dl in pH 7.0 disodium phosphate buffer aqueous solution, the solution was filtered through a 0.45 μm Nylon syringe filter and the viscosity was measured at 25 ° C. (AUTOVISC ^R I, Cannon, Japan) The results are shown in Table 2. It can be seen that the molecular weight of γ-polyglutamic acid is continuously decreased until the reaction time of 5 hours.

NHS introduction ratio and intrinsic viscosity change of γ-PGA with reaction time ([NHS] / [COOH] = 1.2, [DCC] / [COOH] = 1.5) exam [COO-NHS] /
([COOH] + [COO-NHS]) Intrinsic Viscosity (dL / g) γ-PGA MW
Reaction time 50 kDa 500 kDa 1,000kDa 50 kDa 500 kDa T-10 1h 0.18 0.22 0.21 3.22 4.52 T-11 2h 0.29 0.35 0.34 2.16 4.04 T-12 3h 0.38 0.45 0.49 1.65 3.11 T-13 4h 0.35 0.49 0.48 1.17 2.56 T-14 5h 0.37 0.49 0.45 1.05 1.23

Examples 1-5: Crosslinking Reaction of Activated γ-polyglutamic Acid with ε-polylysine

Activated γ-polyglutamic acid (weight average molecular weight 50, 500, 1,000 kDa) and ε-polylysine synthesized according to T-12 conditions of Table 2 were crosslinked. Activated γ-polyglutamic acid was dissolved in 1 ml of Na ₂ HPO ₄ 7% aqueous solution in the amount shown in Table 3 to prepare an aqueous solution (first liquid). In the same manner, ε-polylysine bromate (ε-PL.HBr) was dissolved in 1 ml of a 7% aqueous solution of Na ₂ HPO _{4 in the} amounts shown in Table 3 to prepare an aqueous solution of ε-polylysine (second solution). 0.5 ml of the 1st liquid and the 2nd liquid were extract | collected into the syringe of 1 ml volume, respectively. Two syringes were mounted in a dual barrel syringe and applied to the primary mixture in the needle.

Crosslinking reaction condition of activated γ-polyglutamic acid (buffer: 7% aqueous solution of Na ₂ HPO ₄ ) and ε-polylysine (buffer: 7% aqueous solution of Na ₂ HPO ₄ ) Example γ-polyglutamic acid weight average molecular weight (kDa) ε-polylysine weight average molecular weight (Da) Polymer concentration of the first liquid (%) Polymer concentration of the second liquid (%) Polymer
Concentration (g / ml) Example 1 50 4,000 10 2 6 Example 2 500 4,000 8 2 5 Example 3 500 4,000 10 2 6 Example 4 500 4,000 12 2 7 Example 5 1,000 4,000 10 2 6

Examples 6-7: Crosslinking Reaction of Activated γ-polyglutamic Acid with ε-polylysine

Activated γ-polyglutamic acid and ε-polylysine synthesized according to T-12 conditions of Table 2 were crosslinked. Activated γ-polyglutamic acid was dissolved in 1 ml of Na ₂ HPO ₄ 7% aqueous solution in the amount shown in Table 4 to prepare an aqueous solution (first liquid). In the same manner, ε-polylysine bromate (ε-PL.HBr) was dissolved in 1 ml of a mixed solution of 7% Na ₂ HPO ₄ and 0.3 M PBS in an amount of Table 4 to prepare an aqueous solution of ε-polylysine (second solution). . 0.5 ml of the 1st liquid and the 2nd liquid were extract | collected into the syringe of 1 ml volume, respectively. Two syringes were mounted in a dual barrel syringe and applied to the primary mix in the needle.

Crosslinking reaction conditions of activated γ-polyglutamic acid (buffer: Na ₂ HPO ₄ 7% aqueous solution) and ε-polylysine (buffer: Na ₂ HPO ₄ 7% + 0.3 M PBS mixed solution) Example γ-polyglutamic acid weight average molecular weight (kDa) ε-polylysine weight average molecular weight (Da) Polymer concentration of the first liquid (%) Polymer concentration of the second liquid (%) Polymer
Concentration (g / ml) Example 6 1,000 4.000 10 2 6 Example 7 1,000 4,000 12 2 7

Examples 8-9: Crosslinking Reaction of Activated γ-polyglutamic Acid with Polyethylene Glycol-Based Polymer of Formula 3 (X is an Amine Group)

Activated γ-polyglutamic acid (weight average molecular weight: 1,000 kDa) synthesized according to T-12 conditions of Table 2 was crosslinked with a polyethylene glycol polymer (X is an amine group). Activated γ-polyglutamic acid was dissolved in 1 ml of Na ₂ HPO ₄ 7% aqueous solution in the amount shown in Table 5 to prepare an aqueous solution (first liquid). Similarly, polyethylene glycol polymer (weight average molecular weight: 20 kDa) was dissolved in 1 ml of Na ₂ HPO ₄ 7% aqueous solution in the amount shown in Table 5 to prepare an aqueous solution of polyethylene glycol polymer (second solution). 0.5 ml of the 1st liquid and the 2nd liquid were extract | collected into the syringe of 1 ml volume, respectively. Two syringes were mounted in a dual barrel syringe and applied to the primary mix in the needle.

Crosslinking reaction conditions of the activated γ-polyglutamic acid (buffer: Na ₂ HPO ₄ 7% aqueous solution) and the polyethylene glycol polymer of formula 3 (X is an amine group) (buffer: Na ₂ HPO ₄ 7% aqueous solution) Example γ-polyglutamic acid weight average molecular weight (kDa) Polyethyleneglycol polymer weight average molecular weight (kDa) Polymer concentration of the first liquid (%) Polymer concentration of the second liquid (%) Polymer
Concentration (g / ml) Example 8 1,000 20 10 10 10 Example 9 1,000 20 10 20 15

Test Example 1 Measurement of Gelation Time

0.5 ml of the first liquid and the second liquid prepared according to Examples 1 to 9 were each collected in 1 ml syringes, and then the two liquids were agitated in a 24-well cell culture plate made of transparent polystyrene. Using a magnetic stirrer with a diameter of 4 mm and a length of 12 mm, the mixture was stirred at a speed of 500 rpm at room temperature. The time from the first liquid and the second liquid to the stop of the magnetic stirrer was measured with a stopwatch. 6 is shown.

In the crosslinking reaction between the activated γ-polyglutamic acid and ε-polylysine, as the molecular weight of γ-polyglutamic acid increases, the degree of substitution of NHS increases, and gelation time decreases. For γ- polyglutamic acid having a molecular weight of 1,000 kDa Na ₂ HPO ₄ 7 so that the gel time of the solution in% short but, in the case of using the mixed aqueous solution in PBS 0.3M Na ₂ HPO ₄ 7% aqueous solution has either a gelation time delay It was confirmed.

In addition, the crosslinking reaction between the activated γ-polyglutamic acid and the polyethylene glycol-based polymer increased the amine group as the polymer concentration increased, but the gelation time was decreased, but the gelation time was longer than the reaction with polylysine.

Test Example 2 Measurement of Adhesive Strength

The first liquid and the second liquid prepared in Example 9 were each collected 0.15 ml in a 1 ml syringe. Frozen pig skin was thawed at room temperature and then degreased and cut into 1 × 5 cm. The first liquid and the second liquid collected in the 1ml syringe were mixed by 0.1ml each using a dual barrel syringe so that the volume of the reaction solution was 0.2ml, and was applied to the surface of the pig skin 1 × 1 cm ² . After splicing pig skin of the same size, 50 g of the load was applied and left for 10 minutes to allow the gel to cure. After 10 minutes, remove the load and use a tensile tester (H5K-T, Hounsfield) to continuously apply shear force at a rate of 100mm / min until the bonded pig skin is peeled from each other and adhere the load load when peeled. It was as a robbery.

The results are shown in Table 6. When ε-polylysine was used as the second liquid, the adhesive strength increased with increasing molecular weight. In the case of Example 9 it was confirmed that the gelation time takes longer, but its adhesive strength is high.

Test Example 3 Measurement of Burst Strength

Burst strength was measured by the method specified in ASTM2392. The first liquid and the second liquid prepared in Examples 1 to 9 were each collected 0.35 ml in a 1 ml syringe. The collagen casing was washed twice with water and ethanol, respectively, to remove glycerin from the collagen casing, and then cut to 3 × 3 cm and punched a 3 mm hole using a skin biopsy punch to use as a tissue substitute. Thereafter, a collagen casing having a hole of 3 mm was fixed with Teflon as a support. Thereafter, the first liquid and the second liquid were mixed with 0.3 ml each using a dual barrel syringe to have a volume of 0.6 ml, and then applied to the holes of the collagen casing, and left to cure for 5 minutes. The adhesive-coated collagen casing was then separated from the Teflon support and fixed to a burst strength meter prepared by the method specified in ASTM2392, and then the water pressure was measured. The water pressure measured as the cured gel was broken was taken as the bursting strength. The results are shown in Table 6. The bursting strength was similar to that of the adhesive strength. When ε-polylysine was used as the second liquid, the burst strength also increased as the molecular weight of γ-polyglutamic acid increased. In the case of Example 9, the gelation time takes longer, but it can be seen that it has a high burst strength.

Properties of Hydrogel Example γPGA-NHS
NHS substitution degree Gel time
(second) Adhesive strength
(gf / cm ² ) Bursting strength
(mmHg) Example 1 0.38 60 84 45 Example 2 0.45 15 159 105 Example 3 0.45 28 171 119 Example 4 0.45 40 168 110 Example 5 0.50 0.5 74 25 Example 6 0.50 5 180 150 Example 7 0.50 8 170 121 Example 8 0.50 25 155 130 Example 9 0.50 15 181 145

Test Example 4: Swelling Test

In order to determine the degree of in situ swelling of the hydrogel of the example, 2 g of hydrogel prepared in the same manner as in Examples 2 and 3 were placed in 50 ml of distilled water and 0.01 M PBS buffer (pH 7.4) at 37 ° C. and 50 rpm in a thermostat. Immerse for hours. The degree of swelling was measured through the weight change of the hydrogel. The experiment was repeated three times, and the swelling degree was calculated by the following equation.

[Equation 1]

Swelling degree = Wg1 (weight of water swelling gel after immersion) / Wg2 (weight of hydrogel)

The degree of swelling of the hydrogel is shown in Table 7. The swelling results did not show any difference according to the molecular weight. The weight increase was 1.5 ~ 1.6 times in PBS buffer and 3.5 ~ 4.0 times in distilled water.

Swelling degree of hydrogel γ-polyglutamic acid weight average molecular weight (kDa) Immersion solvent Distilled water PBS buffer 50 3.59 ± 0.23 1.62 ± 0.02 500 4.05 ± 0.57 1.57 ± 0.13

Test Example 5: Degradation Test

In order to determine the in situ decomposition behavior of the hydrogel, 2 g of hydrogel prepared in the same manner as in Example 6 was placed in 50 ml of distilled water and soaked in a constant temperature bath at 37 ° C. and 50 rpm for a period of time (1, 2, 4, 6, 8 weeks). Observed after making. The weight change by hydrolysis of the hydrogel was measured by the weight ratio before and after the decomposition through lyophilization. The weight loss result of hydrolysis of the hydrogel is shown in FIG. 1. Almost no degradation was observed during the first two weeks, then gradually decreased from two weeks to six weeks (20% degradation) and then degraded at eight weeks.

Test Example 6 In vivo Implantation Test

In order to confirm the In situ tissue response of the hydrogel, a hydrogel prepared in the same manner as in Example 6 was implanted into the rat.

SD rats (male, 6 weeks old, n = 2) were used for the experiment. After the first liquid and the second liquid were prepared in the same manner as in Example 6, 1 ml of the aqueous solution was collected in a 1 ml syringe. The rats were implanted under the general anesthesia using a needle to inject the gel subcutaneously into the back of the rat. After 2, 4, 6, and 8 weeks, the degree of decomposition of hydrogel and the tissue response of the tissues around the hydrogel were observed. The implanted hydrogels disintegrated within 8 weeks.

As described above, the hydrogel of the example shows biocompatibility, biodegradability and absorbency (swellability) with fast gelation time, and shows excellent adhesion and burst strength, so that it can be preferably used for adhesion of biological tissues, etc. It became.

Claims (20)

Γ-polyglutamic acid with at least part of a carboxyl group activated,
Hydrogel comprising a crosslinked cross-linked ε-polylysine or polyethylene glycol polymer having a plurality of nucleophilic functional groups.
The hydrogel of claim 1 wherein the activated γ-polyglutamic acid is activated by at least a portion of the carboxyl groups linked to a succinimide ester with a compound of formula (I):
[Formula 1]

In Formula 1, R represents hydrogen or -SO ₃ Na.
The hydrogel of claim 1, wherein the γ-polyglutamic acid has at least a portion of a carboxy group in the form of an alkali metal salt or an alkaline earth metal salt. The hydrogel of claim 1 wherein the weight average molecular weight of γ-polyglutamic acid is from 500,000 to 2,000,000 daltons.
The hydrogel according to claim 1, wherein the molar ratio of activated carboxyl groups in the carboxyl groups of? -Polyglutamic acid is 0.1 to 0.7.
The hydrogel of claim 1 wherein the nucleophilic functional group is an amine group, thiol group or hydroxy group.
The hydrogel of claim 1, wherein the polyethylene glycol polymer is represented by Chemical Formula 2.
(2)
I-[(CH ₂ CH ₂ O) n-CH ₂ CH ₂ X] m
Wherein I is a radical derived from a 2 to 12 valent polyhydric alcohol, X represents an amine group, thiol group or hydroxy group, n is 19 to 170, m is an integer from 2 to 12, wherein I is derived from It is the same as the hydroxyl group number of a polyhydric alcohol.
The hydrogel according to claim 7, wherein the polyethylene glycol polymer is represented by the following Chemical Formula 3 or 4:
(3)

[Chemical Formula 4]

In Formulas 3 and 4, X represents an amine group, a thiol group or a hydroxyl group, n is from 19 to 170.
The hydrogel of claim 1 wherein the weight average molecular weight of ε-polylysine is 1,000 to 20,000 Daltons.
The hydrogel of claim 1, wherein the polyethylene glycol polymer has a weight average molecular weight of 5,000 to 30,000 Daltons.
The hydrogel of claim 1 wherein the molar ratio of nucleophilic functional groups relative to the activated carboxyl group is from 0.2 to 2.0.
A method for producing a hydrogel comprising crosslinking at least a portion of a carboxyl group-activated γ-polyglutamic acid with an ε-polylysine or polyethylene glycol polymer having a plurality of nucleophilic functional groups.
The method of claim 12, further comprising the step of reacting γ-polyglutamic acid with a compound of Formula 1 to form an activated γ-polyglutamic acid before the crosslinking reaction.
[Formula 1]

In Formula 1, R represents hydrogen or -SO ₃ Na.
The method for producing a hydrogel according to claim 13, wherein γ-polyglutamic acid having at least a portion of the carboxyl group in the form of an alkali metal salt or an alkaline earth metal salt is reacted with a compound of formula (1).
The method according to claim 12, wherein the crosslinking reaction proceeds in an aqueous solution in which the activated γ-polyglutamic acid and the ε-polylysine or polyethylene glycol polymer are mixed.
The method for producing a hydrogel according to claim 15, wherein the concentration of γ-polyglutamic acid in the aqueous solution and the total polymer of the ε-polylysine or polyethylene glycol polymer is 1 to 20% by weight.
A hydrogel manufacturing kit comprising at least part of a carboxyl-activated γ-polyglutamic acid and an ε-polylysine or polyethylene glycol polymer having a plurality of nucleophilic functional groups.
A tissue adhesive composition comprising γ-polyglutamic acid with at least a portion of carboxyl groups activated and an ε-polylysine or polyethyleneglycol polymer having a plurality of nucleophilic functional groups.
19. The crosslinked product of γ-polyglutamic acid and ε-polylysine or a polyethylene glycol polymer is prepared by crosslinking the activated γ-polyglutamic acid with an ε-polylysine or polyethylene glycol polymer. Tissue adhesive composition to form a hydrogel containing.
19. The tissue adhesive composition of claim 18 used for topical wound closure, gastrointestinal anastomosis, vascular anastomosis or surgery.

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