KR102071029B1 - Biodegradable polymer hydrogel complex improved in biostability and mechanical properties and method for producing the same - Google Patents

Abstract

The present invention relates to a biodegradable polymer hydrogel complex and a method for preparing the same, wherein the biodegradable hydrogel complex having improved resistance to hyaluronidase, antibacterial activity, or antioxidant activity can be prepared.

Description

Biodegradable polymer hydrogel complex improved in biostability and mechanical properties and method for producing the same}

The present invention relates to a biodegradable polymer hydrogel complex and a method for preparing the same.

As the bioengineering research for growing cells into implantable tissues by culturing cells in vitro using a support material based on a biomaterial such as hydrogel having excellent biocompatibility, Research is also active. Hydrogels are largely composed of natural hydrogels such as hyaluronic acid, alginate, collagen, alginic acid, heparin and heparan, and polyethylene glycol (poly (ethylene glycol). PEG) or polylactic acid-glycolic acid copolymer (poly (latic-co-glycolic acid); PLGA) based on the synthetic hydrogel can be classified, the natural hydrogel is a substance present in the body It is a biodegradable polymer that is highly compatible and can be naturally absorbed by specific enzymes in the body (ACS NANO, 6 (4), p. 2960-2968 (2012.03.12)).

Among them, hyaluronic acid is the most widely used natural polymer and is present in connective tissues of skin, cartilage, bone, synovial fluid, and the like. Hyaluronic acid has excellent properties as a biomaterial because of its viscoelasticity, non-immunogenic response, biodegradability, and swelling properties. Thus, it can be used in a variety of applications such as viscoseplepleation, drug delivery, and scaffolds for wound healing.

However, since hyaluronic acid is rapidly degraded by specific enzymes (hyaluronidase) and radicals generated in tissues, it is necessary to lower the degradation rate to increase bio stability in order to use it as a support for tissue engineering. In addition, the hyaluronic acid hydrogel is made of 95% or more water, the mechanical properties are very weak, a method of mixing a crosslinking system or additives to increase the mechanical strength has been studied. Therefore, the role of additives to control the mechanical properties, biodegradation behavior, biological properties, etc. of the hyaluronic acid hydrogel is important and a technology for this is required (Korean Patent Publication No. 10-1605528 (2016.03.16)).

In general, an enzyme inhibitor may be added to increase the decomposition resistance of hyaluronic acid, but the cytotoxicity and simple mixing of the additive may cause the body to be washed out by the body fluid, thereby preventing its proper function as an additive. Therefore, attempts have been made to increase the decomposition resistance of hyaluronic acid by adding a chemical crosslinking agent. However, even if the mechanical properties of the hyaluronic acid gel is improved by increasing the degree of crosslinking, the gel produced is not only toxic by the crosslinking agent but also loses biological activity, and the chemical properties of the material change the unique properties of the material.

Recently, studies have been conducted to add polyphenols to biomaterials that show antibacterial, anti-inflammatory, anticancer and antiviral functions as well as antioxidants. Tannic acid extracted from plants in polyphenols has an inhibitory effect on hyaluronidase enzymes, but research has been limited because it does not show a unique interaction with hyaluronic acid.

Accordingly, the present inventors have made diligent efforts to prepare biodegradable polymer hydrogel composites having improved biostability and mechanical properties. As a result, when polyphenol is added to a biodegradable polymer hydrogel crosslinked with a crosslinking agent having an ether bond to the biodegradable polymer, The present invention was completed by confirming that polyphenols form a hydrogen bond with a crosslinking agent having an ether bond, thereby improving resistance to hyaluronidase, antibacterial activity or antioxidant activity.

One object of the present invention is to prepare a biodegradable polymer hydrogel by (1) cross-linking the biodegradable polymer with a crosslinking agent having an ether bond; And (2) adding a polyphenol to the biodegradable polymer hydrogel, wherein the polyphenol is cross-linkage having an ether bond of the biodegradable polymer hydrogel. It is to provide a method of producing a biodegradable polymer hydrogel composite to bind to).

It is another object of the present invention to (a) a biodegradable polymer hydrogel crosslinked with a crosslinking agent having an ether bond, and (b) a poly-bonded cross-linkage having an ether bond of the biodegradable polymer hydrogel. It is to provide a biodegradable polymer hydrogel complex containing phenol.

Another object of the present invention to provide a support for tissue engineering comprising the biodegradable polymer hydrogel complex.

Hereinafter, the present invention will be described in more detail.

Meanwhile, each of the descriptions and the embodiments disclosed herein may be applied to each of the other descriptions and the embodiments. That is, all combinations of the various elements disclosed herein are within the scope of the present invention. In addition, the scope of the present invention is not limited by the specific description described below.

In addition, one of ordinary skill in the art can recognize or identify many equivalents to certain aspects of the invention described in this application using conventional experiments only. In addition, such equivalents are intended to be included in the present invention.

In accordance with one aspect of the present invention, there is provided a biodegradable polymer hydrogel by crosslinking a biodegradable polymer with a crosslinking agent having an ether bond; And (2) adding a polyphenol to the biodegradable polymer hydrogel, wherein the polyphenol is cross-linkage having an ether bond of the biodegradable polymer hydrogel. To bind to, provides a method for producing a biodegradable polymer hydrogel complex.

Step (1) is a step of preparing a biodegradable polymer hydrogel containing a crosslink having an ether bond using a biodegradable polymer and a crosslinking agent having an ether bond.

In the present invention, "crosslinking agent having an ether bond" refers to a material having an ether bond, and a polymer is bonded to each other by a chemical bond to make a polymer of a three-dimensional network structure. Specifically, the crosslinking agent having an ether bond may be a crosslinking agent that hydrogen bonds with the polyphenol through an oxygen atom of the ether bond. The crosslinking agent having an ether bond may be used without limitation to crosslink the polymer and bond with the polyphenol through one or more ether bonds, specifically, polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxy At least one selected from the group consisting of ethylenated polyol, polyvinyl alcohol and polyvinyl ethyl ether.

Specifically, the crosslinking agent having the ether bond is hydroxyl group (OH), amine group (NH 2), succinimidyl succinate, succinic acid, thiol group (SH), acrylate (Acrylate), epoxy group ( Epoxide, maleimide, nitrophenyl carbonate, pyridyl disulfide, tosylate, azide, phosphoric acid group, oligoamine group ([-CH2-CH2- NH-] n), diglycidyl ether, catechol and catechol amine may include any one or more functional groups selected from the group consisting of, but not limited to.

The molecular weight of the crosslinking agent having an ether bond is not limited as long as it crosslinks the polymer and can be combined with one or more polyphenols, but it is specifically 10 to 500,000 Da, 100 to 50,000 Da, 200 to 25,000 Da, 500 to 10,000 Da or It may be 1,000 to 5,000 Da, but is not limited thereto.

As used herein, the term "biodegradable polymer" refers to a generic term for a polymer that decomposes itself in vivo or in a natural environment. Specifically, the biodegradable polymer may be a biodegradable polymer decomposed in vivo for medical purposes, but is not limited thereto. The biodegradable polymer is not limited so long as it can be cross-linked to form a hydrogel. Biodegradable polymers can also be prepared from homologous or heterologous monomers.

Specifically, the biodegradable polymer is not limited as long as it can prepare a hydrogel, but selected from the group consisting of hyaluronic acid, collagen, alginate, starch, chitosan, gelatin, dextrin, cellulose, alginic acid, chondroitin sulfate, heparin and heparan It may be any one or more.

In addition, the molecular weight of the biodegradable polymer may be 10,000 Da to 100,000,000 Da, specifically 50,000 Da to 100,000,000 Da, 50,000 Da to 50,000,000 Da, or 100,000 Da to 10,000,000 Da, but is not limited thereto.

In the present invention, "hydrogel" refers to a gel containing water as a dispersion medium, wherein the hydrosol loses fluidity due to cooling, or a hydrophilic polymer having a three-dimensional network structure and a microcrystalline structure contains water and expands. Can be formed. Hydrogels of electrolyte polymers often exhibit high absorbency and are widely used as absorbent polymers. Some hydrogels undergo a phase transition at a temperature, pH, or the like and discontinuously change the expansion ratio.

In the present invention, the hydrogel may be prepared using a biodegradable polymer, and specifically, the biodegradable polymer may be prepared by crosslinking with a crosslinking agent having an ether bond. Therefore, the hydrogel in the present invention has a crosslink containing an ether bond, it can be bonded to the polyphenol through the ether bond of the crosslink. In the present invention, the hydrogel is mixed with a biodegradable polymer hydrogel.

The biodegradable polymer hydrogel has 0.0001 to 90% (w / w), 0.0005 to 80% (w / w), 0.001 to 70% (w / w), and 0.01 to 60% (w /) crosslinking having an ether bond. w), 0.1 to 50% (w / w), 1 to 45% (w / w), and may include 5 to 40% (w / w), but is not limited thereto.

Step (1) is a cross-linking agent having an ether bond with a biodegradable polymer in a weight ratio of 1: 0.05 to 1: 5, 1: 0.05 to 1: 2, 1: 0.1 to 1: 1, 1: 0.2 to 1: 0.8 The reaction may be, but is not limited thereto.

When the crosslinking agent is used in a weight ratio of less than 0.05 compared to the biodegradable polymer, it may be difficult to achieve the desired elasticity of the biodegradable polymer hydrogel due to insufficient crosslinking, and using the crosslinking agent in a weight ratio of more than 5 compared to the biodegradable polymer. In this case, due to excessive crosslinking, the biodegradable polymer may lose its unique properties or may not be suitable as a biodegradable polymer hydrogel composite due to the deterioration of rheological properties.

Step (2) is a step of combining the biodegradable polymer hydrogel and polyphenol prepared in step (1).

In the present invention, "polyphenol" means a substance having two or more hydroxyl groups (-OH) in the benzene ring. Specifically, the polyphenol can be used without limitation when having a polyvalent hydroxyl group capable of binding to cross-linkage having an ether bond of the biodegradable polymer hydrogel. Specifically, the polyphenol is chlorogenic acid (Chlorogenic acid), catechol (catechol), flavones (flavones), anthocyan (anthocyan), catechin (catechin), tannic acid (tannic acid), resorcinol, gallic acid (gallic acid) ) And combinations thereof, but is not limited thereto. In addition, the polyphenol has a resistance to hyaluronidase, antimicrobial activity or antioxidant capacity, the biodegradable polymer hydrogel complex comprising the same may have a resistance to hyaluronidase, antibacterial activity or antioxidant capacity.

The polyphenol is 0.005 to 1.5% (w / v), 0.005 to 1.0% (w / v), 0.01 to 0.5% (w / v), 0.01 to 0.3% (w / v), 0.02 to 0.2% (w / v), 0.05 to 0.15% (w / v) polyphenol solution, but is not limited thereto.

In the present invention, "solution" may be a liquid type generally used in the art.

The biodegradable polymer hydrogel complex of the present invention may be formed by combining a polyphenol with a crosslink having an ether bond of a biodegradable polymer hydrogel, and through such a bond, resistance to hyaluronidase, antibacterial activity or antioxidant activity is improved. Can be.

The biodegradable polymer hydrogel complex may include 0.0001 to 50% (w / w) of polyphenol, specifically 0,0001 to 40% (w / w), and 0.0001 to 30% (w / w). This is not restrictive. When the polyphenol is used in less than 0.0001% (w / w) in the biodegradable polymer hydrogel composite, the biodegradable polymer hydrogel and polyphenols are not sufficiently combined, so as to resist the desired hyaluronidase, Antioxidant activity may be difficult to achieve, and when used in excess of 50% (w / w), the resistance to hyaluronidase may be excessively increased to lose biodegradability and may not be suitable as a polymer hydrogel composite. In addition, too much weight of polyphenols may cause undesirable toxic reactions in the body and may be undesirable.

The preparation method of the present invention may further comprise the step of washing the hydrogel or complex after step (1) and / or after step (2). The washing may be a general method used in the art.

Another embodiment of the present invention comprises a) a biodegradable polymer hydrogel crosslinked with a crosslinking agent having an ether bond, and (b) a polyphenol bonded to a crosslink having an ether bond of the biodegradable polymer hydrogel. It provides a biodegradable polymer hydrogel complex.

The biodegradable polymer hydrogel complex of the present invention may be formed by combining a polyphenol with a crosslink having an ether bond of a biodegradable polymer hydrogel, and through such a bond, resistance to hyaluronidase, antibacterial activity or antioxidant activity is improved. Can be.

Biodegradable polymers, hydrogels, crosslinkers and ethers with ether linkages are as described above.

Since the mechanical properties of the biodegradable polymer hydrogel composite are determined according to the degree of bonding of the crosslinking agent and the polyphenol of the biodegradable polymer hydrogel, the G '(viscoelastic modulus) value of the biodegradable polymer hydrogel composite indicates the degree of the two reactions. It can be a known standard. Therefore, the measurement of the G 'according to the frequency can be seen that the mechanical properties tend to change. The increase in G 'value with polyphenol content at a fixed frequency (rad / s) may be attributed to the physical crosslinking of the polyphenols added to the biodegradable polymer hydrogel.

The G 'value of the biodegradable polymer hydrogel composite is when the frequency is 0.1 to 10 rad / s, 2500 Pa or more, specifically 2500 Pa to 100000 Pa, 2500 Pa to 80000 Pa, 2500 Pa to 50000 Pa, 2500 Pa to 10000 Pa, or 2500 Pa to 7500 Pa, but is not limited thereto.

The biodegradable polymer hydrogel composite may have improved resistance to hyaluronidase, antimicrobial activity or antioxidant activity, compared to biodegradable polymer hydrogel.

In the present invention, "hyaluronidase" is an enzyme that lowers the molecular weight of hyaluronic acid and acts on carbohydrates or glycosides to hydrolyze glycoside bonds. In the present invention, the biodegradable polymer hydrogel complex includes polyphenols bound to cross-linkage having an ether bond of the biodegradable polymer hydrogel, thereby improving resistance to hyaluronidase, antibacterial activity, and antioxidant activity.

Another aspect of the invention provides a support for tissue engineering comprising the biodegradable polymer hydrogel complex.

The biodegradable polymer hydrogel composite is as described above.

As used herein, the term "tissue engineering support" refers to a substance that replaces a part of an organ or tissue damaged in vivo and that can supplement or replace their function. Specifically, the polymer support may include a biodegradable polymer material, which may be maintained until the support sufficiently performs its function and role and then completely decomposed in vivo.

In order for the tissue engineering scaffold of the present invention to perform a function of replacing a living body for a period of time, it is preferable that it is physically stable. The support for tissue engineering of the present invention includes a polyphenol bound to a cross-linkage having an ether bond of a biodegradable polymer hydrogel, thereby improving resistance to hyaluronidase, antimicrobial activity, and antioxidant activity, thereby improving stability. Can be used as

Biodegradable polymer hydrogel complex of the present invention is improved resistance to hyaluronidase, antimicrobial activity or antioxidant bar, can be utilized as a support for various tissue engineering.

1 is a schematic diagram of a crosslinking reaction of hyaluronic acid with a polyethylene glycol derivative and a method of adding tannic acid.
Figure 2 relates to the viscoelastic coefficient (G ', Storage modulus) value for the frequency (rad / s) according to the tannic acid content of the hyaluronic acid / tannic acid hydrogel complex made by the present invention. Where "HA" is a hydrogel complex without tannic acid, "HA-0.05% TA" is a hyaluronic acid / tannic acid hydrogel complex containing 0.05 (w / v)% tannic acid, and "HA-0.10% TA" is 0.10 (w / v)% hyaluronic acid / tannic acid hydrogel complex and “HA-0.15% TA” means hyaluronic acid / tannic acid hydrogel complex comprising 0.15 (w / v)% tannic acid. As the content of tannic acid increases, the viscoelastic coefficient of the hyaluronic acid / tannic acid hydrogel complex increases, indicating that the mechanical properties are improved.
Figure 3 relates to the degradation behavior of hyaluronidase over time of the hyaluronic acid / tannic acid hydrogel complex made with the present invention. Higher tannic acid content indicates that the hyaluronic acid / tannic acid hydrogel complex has increased resistance to hyaluronidase.
Figure 4 relates to the antioxidant capacity according to the tannic acid content of the hyaluronic acid / tannic acid hydrogel complex made by the present invention. The higher the content of tannic acid, the higher the degree of radical inhibition of the hyaluronic acid / tannic acid hydrogel complex.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention and the scope of the present invention is not limited thereto.

Example  One. Polyethylene glycol  Hyaluronic Acid Using Derivatives Hydrogel  Manufacturing steps

After dissolving 1 g of hyaluronic acid in 5 ml of 0.3 M NaOH, 0.55 g of PEGDE (polyethylene glycol diglycidyl ether, molecular weight 2000 Da) was added, mixed in a homogeneous container, sealed, and reacted at 25 ° C. for 24 hours. After completion of the reaction, the mixture in the form of gel is washed with physiological saline (0.9% NaCl) at least five times.

Example  2. Hyaluronic acid Tannic acid  Step of adding

Hyaluronic acid hydrogel prepared in Example 1 was added to 0 (control), 0.05 (w / v)%, 0.1 (w / v)%, 0.15 (w / v)% (tannic acid / saline) tannic acid solution 2 days React liver. According to the above concentrations, the experimental group was treated with HA (without tannic acid), HA-0.05% TA (contains 0.05 (w / v)% tannic acid), HA-0.10% TA (contains 0.10 (w / v)%), HA-0.15 It was named% TA (including 0.15 (w / v)% of tannic acid). After completion of the reaction, the gel was washed three times or more with saline and soaked in PBS to prepare a sample.

Experimental Example  Hyaluronic Acid / Tannic acid Hydrogel  Mechanical property analysis of the composite

The hyaluronic acid / tannic acid hydrogel complex prepared according to the above example was measured by a rheological method to measure viscoelastic modulus, and the results are shown in FIG. 2. As shown in FIG. 2, the hyaluronic acid hydrogel complex treated with tannic acid increased the viscoelastic coefficient from 2000 Pa to 7000 Pa than when tannic acid was not treated. As the tannic acid content increased, the viscoelastic coefficient increased with the measurement frequency (rad / s). Since tannic acid crosslinks and contributes to the physical crosslinking role of the biodegradable polymer hydrogel complex, the higher the content of tannic acid, the higher the viscoelastic coefficient is, resulting in the hyaluronic acid / tannic acid hydrogel composite. Mechanical properties are improved.

Experimental Example  2. Hyaluronic Acid / Tannic acid Hydrogel  Determination of Biodegradation Behavior of Complexes

The biodegradation behavior of the hyaluronic acid / tannic acid hydrogel complex prepared according to the above example was immersed in an enzyme solution (hyaluronidase, 50 U / ml), and the mass change of the gel was measured. The results are shown in FIG. 3. The hydrogel complex that was not treated with tannic acid was almost decomposed and disappeared within two days, but all of the hydrogel complexes treated with tannic acid remained in shape for more than seven days. As a result, the higher the content of tannic acid increases the resistance to hyaluronidase, it can be seen that the hyaluronic acid / tannic acid hydrogel complex is improved in the body.

Experimental Example  3. Hyaluronic Acid / Tannic acid Hydrogel  Antioxidant Capacity of Complex

The antioxidant capacity of the hyaluronic acid / tannic acid hydrogel complex prepared according to the above example was measured by ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) assay method. It is based on the action of inhibiting the ABTS (blue-green color) of the results, and the results are shown in Figure 4. The tannic acid-treated hyaluronic acid hydrogel complex was confirmed to have little antioxidant capacity, tannin acid treated hyaluronic acid The lononic acid / tannic acid hydrogel complex showed antioxidative activity and its ability to increase with tannic acid content, meaning that it can inhibit the decomposition of hyaluronic acid by highly reactive radicals present in the body by antioxidant action. The hyaluronic acid / tannic acid hydrogel complex can be confirmed that the element that contributes to the improvement of the persistence in the body.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (15)

(1) preparing a biodegradable polymer hydrogel by crosslinking the biodegradable polymer with a crosslinking agent having an ether bond; And
(2) a method for producing a biodegradable polymer hydrogel composite comprising adding polyphenol to the biodegradable polymer hydrogel,
The polyphenol is to bind to the cross-linkage having an ether bond of the biodegradable polymer hydrogel, method of producing a biodegradable polymer hydrogel composite.
According to claim 1, wherein the step (1) is a method of producing a biodegradable polymer hydrogel complex, wherein the biodegradable polymer and a crosslinking agent having an ether bond in a weight ratio of 1: 0.05 to 1: 5.
The method of claim 1, wherein the polyphenol is 0.005 to 1.5% (w / v) of a polyphenol solution.
The method of claim 1, wherein the biodegradable polymer is any one or more selected from the group consisting of hyaluronic acid, collagen, alginate, starch, chitosan, gelatin, dextrin, cellulose, alginic acid, chondroitin sulfate, heparin and heparan. Method for preparing gel complex.
The method of claim 1, wherein the biodegradable polymer has a molecular weight of 100,000 Da to 10,000,000 Da.
The method of claim 1, wherein the polyphenol is selected from chlorogenic acid (Chlorogenic acid), catechol (catechol), flavones (flavones), anthocyan (anthocyan), catechin (tanchin), tannic acid (resorcinol), Gallic acid (gallic acid) and a combination thereof, the method of producing a biodegradable polymer hydrogel composite.
The biodegradable according to claim 1, wherein the crosslinking agent having an ether bond is at least one selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, and polyvinyl ethyl ether. Method for producing a polymer hydrogel composite.
The crosslinking agent having an ether bond according to claim 1, wherein the crosslinking agent having an ether bond is hydroxyl group (OH), amine group (NH 2), succinimidyl succinate, succinic acid, thiol group (SH), and acrylate (Acrylate). ), Epoxy, maleimide, nitrophenyl carbonate, orthopyridyl disulfide, tosylate, azide, phosphoric acid group, oligoamine group ([- CH2-CH2-NH-] n), diglycidyl ether (diglycidyl ether), catechol and catechol amine containing any one or more functional groups selected from the group consisting of amine, Manufacturing method.
According to claim 1, The molecular weight of the crosslinking agent having an ether bond is 100 to 50,000 Da, the method of producing a biodegradable polymer hydrogel composite.
The method of claim 1, wherein the biodegradable polymer hydrogel comprises 0.001 to 70% (w / w) of a crosslink having an ether bond.
The method of claim 1, wherein the biodegradable polymer hydrogel complex comprises 0.0001 to 30% (w / w) polyphenol.
A biodegradable polymer hydrogel composite comprising (a) a biodegradable polymer hydrogel crosslinked with a crosslinking agent having an ether bond, and (b) a polyphenol bonded to a crosslink having an ether bond of the biodegradable polymer hydrogel.
The biodegradable polymer hydrogel composite of claim 12, wherein the G '(storage modulus) value of the biodegradable polymer hydrogel composite is 2500 Pa or more when the frequency is 0.1 to 10 rad / s.
The biodegradable polymer hydrogel composite of claim 12, wherein the biodegradable polymer hydrogel complex has improved resistance to hyaluronidase, antimicrobial activity, or antioxidant activity, compared to the biodegradable polymer hydrogel.
13. A tissue engineering support comprising the biodegradable polymer hydrogel complex of claim 12.

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