KR102273628B1 - Development of Adhesive Mussel Protein Hydrogel Formulation by Introducing Histidine-based Protein Domain from Mussel Byssus - Google Patents

Abstract

The present invention relates to a hydrogel comprising a histidine-introduced mussel adhesion protein and a metal ion, wherein the metal ion is bound to histidine on the surface of the mussel adhesion protein, and the hydrogel according to the present invention has excellent biocompatibility and excellent extrudability. It is possible to apply freely in the form of application and has strong adhesiveness.

Description

Development of Adhesive Mussel Protein Hydrogel Formulation by Introducing Histidine-based Protein Domain from Mussel Byssus}

It relates to an adhesive mussel protein hydrogel in which a histidine-based protein derived from mussels is introduced.

Recently, mussel adhesion proteins have become available for mass production from genetic transformants. A conventional hydrogel based on mussel adhesive protein was formed through cross-linking of 3,4-dihydroxyphenylalanine (DOPA), an amino acid present in mussel adhesive protein. At this time, there was a method of completely oxidative crosslinking of waveguide and a method of crosslinking through iron ions. However, in the case of complete oxidative crosslinking, dopa, a key amino acid that can show adhesiveness in mussel adhesive protein, is oxidized and completely loses adhesiveness. Also, in the crosslinking method through iron ions, the waveguide, which should have contributed to the adhesion, contributed to the crosslinking, and the adhesion was significantly reduced. In addition, in the conventional hydrogel system, both adhesion and crosslinking of the formulation were formed due to waveguide, so it was difficult to balance the two, and there were many limitations in the field of application.

Accordingly, the present inventors have developed a new hydrogel based on mussel adhesive protein using the interaction between a specific amino acid and a metal ion pair to overcome the limitations of the prior art. The mussel adhesive protein used in the present invention can be mass-produced from a genetic transformant, and it was confirmed that the mussel adhesive protein produced through this forms a hydrogel through interaction with various metal ions.

The fusion protein of the present invention can form a hydrogel by cross-linking through a coordination bond between an amino acid and a metal ion present in the mussel adhesive protein. The hydrogel thus formed was cross-linked by histidine by applying the mechanism of the mussel family, and it was confirmed that the existing waveguide formed a pure adhesive force, and thus had superior adhesion compared to the existing material. In addition, as crosslinks are formed by coordination bonds, when compared to hydrogels through conventional oxidative crosslinking, the hydrogel of the present invention has the ability to self-repair damage even when damage is applied to the formulation due to interaction with metal. , has excellent tissue engineering utility in that it has excellent cell viability due to the cell-friendly crosslinking method.

Republic of Korea Patent Publication No. 10-2014-0027031 Republic of Korea Patent Publication No. 10-2016-0026441

In the present invention, a new recombinant protein was developed by fusing the protein present in the mussel family and the adhesive protein of the mussel. The protein present in the mussel's foot is a protein rich in histidine, and it is free of coordination with metal ions.

In addition, the hydrogel formed by the interaction of the newly developed recombinant protein with metal ions can self-restore the damage and strength of the formulation, so it can be applied to tissues in various environments.

In addition, it was confirmed that the hydrogel formed using the cross-linking method in which the waveguide was preserved has excellent adhesion compared to the hydrogel made of the conventional mussel adhesive protein.

In addition, since it is possible to self-recover the damage of the formulation, it is possible to apply through extrusion, so the form of application is free, and through this, the formation of a three-dimensional structure is also possible.

The present invention includes a mussel adhesion protein to which histidine has been introduced and a metal ion, wherein the mussel adhesion protein has a catechol derivative on its surface, and the metal ion is bound to histidine on the surface of the mussel adhesion protein, a hydrogel provides

Also, the present invention

(1) introducing histidine into the mussel adhesion protein; and

(2) adding metal ions to the histidine-introduced mussel adhesion protein aqueous solution;

It provides a method for producing a hydrogel comprising a.

In the present invention, by developing a histidine-based mussel adhesion protein, an adhesive hydrogel with increased adhesion using interaction with metal ions was developed. This hydrogel not only has biocompatibility, but also has the ability to recover the strength of the formulation even from damage caused by external force, so that it can be applied through extrusion. In addition, the hydrogel of the present invention is expected to be able to induce rapid regeneration of biological tissues because it is easy to apply without losing adhesion.

1 shows the basic DNA information and the amino acid sequence of the protein and mussel adhesion protein sequence derived from the foot family used for the development of the recombinant protein in the present invention.
FIG. 2 shows the results of production and purification of a histidine-based mussel adhesive protein capable of interacting with various types of metal ions as SDS Pages.
3 shows staining data showing that dopa can be generated by reacting histidine-based mussel adhesive protein made using recombinant technology with mushroom-derived tyrosinase. This confirmed that not only histidine but also dopa can be reliably included in the histidine-based mussel adhesion protein.
4 is a comparison of the adhesive strength of hydrogels formed using pig skin, and hydrogels crosslinked with copper using histidine-based mussel adhesive protein rather than hydrogels crosslinked with dopa and iron as in the conventional method. It was confirmed that the adhesive strength was better.
5 shows the formation of a hydrogel capable of maintaining a fixed structure by mixing the produced protein with various types of metal ions.
6 shows the results of rheological experiments to quantify the self-healing properties of the formed hydrogel.
7 is a graph showing changes in rheological properties according to the concentration of a hydrogel formed by the binding of a histidine-based mussel adhesive protein and copper.
8 shows an example of applying the self-healing ability of the formulation measured in FIGS. 6 and 7 to actual cases.
Figure 9 shows the results of the cytotoxicity test when extruded by supporting myoblasts, fibroblasts and stem cells in a hydrogel.

The present invention provides a hydrogel comprising a mussel adhesion protein to which histidine is introduced and a metal ion, the mussel adhesion protein has a catechol derivative on its surface, and the metal ion is bound to histidine on the surface of the mussel adhesion protein. it's about Here, the catechol derivative on the surface of the mussel adhesive protein may exist while maintaining adhesive force.

In the present invention, the mussel adhesion protein is a mussel selected from the group consisting of Mytilus edulis , Mytilus galloprovincialis , and Mytilus coruscus. As a natural protein derived from the family shellfish, a mutant thereof, or a recombinant protein, histidine may be introduced.

The mussel adhesive protein may include at least one amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 4, and combinations thereof. Here, at least one may mean 2, 3, 4, 10, 20, 30, 40, 50, 100 or more.

As a preferred example, it may be an amino acid sequence (SEQ ID NO: 2) when the DNA sequence (SEQ ID NO: 1) as shown in FIG. 1 is expressed. In addition, as another preferred example, it may be an amino acid sequence (SEQ ID NO: 4) when the DNA sequence (SEQ ID NO: 3) as shown in FIG. 1 is expressed. The amino acid sequence of SEQ ID NO: 2 and the amino acid sequence of SEQ ID NO: 4 may be cross-linked alone or together to form the hydrogel of the present invention. In the present invention, the amino acid sequence of SEQ ID NO: 2 and/or 4 may be included in at least two or more hydrogels.

According to the present invention, the mussel adhesive protein may be modified within the range including a conservative amino acid sequence capable of maintaining the properties of the mussel adhesive protein mentioned above. That is, 70% or more, preferably 80% or more, even more preferably 90% or more, that is, 95%, 96%, 97%, 98%, 99 of the amino acid sequence of the SEQ ID NOs showing substantially equivalent effects. Amino acid sequences having % or greater sequence identity are also included within the scope of the present invention.

The protein used in the present invention has an advantage in that it can be attached to and applied to various existing mussel adhesion proteins, and thus has high utility.

In the hydrogel according to the present invention, the metal ion may be one or more selected from the group consisting ^{of Zn 2+} , Ni ²⁺ , Co ²⁺ , Cu ²⁺ and Fe ^{3+ , and is an amino acid of the mussel adhesive protein.} It can coordinate with histidine.

In the hydrogel according to the present invention, the mussel adhesive protein may be a catechol derivative introduced therein. Here, the catechol derivative may be one in which the tyrosine residue of the mussel adhesion protein is converted. In most mussel adhesion proteins, tyrosine accounts for about 20-30% of the total amino acid sequence. Tyrosine in the natural mussel adhesive protein can be converted into a catechol derivative by adding a hydroxyl group through the hydration process. The mussel adhesive protein produced in E. coli can convert a tyrosine residue into a catechol derivative by a separate enzymatic and chemical treatment method. As a method for converting a tyrosine residue contained in the mussel adhesive protein into a catechol derivative, as known in the art, a method using tyrosinase may be used, but is not limited thereto. Preferably, tyrosine can be converted into dopa (3,4-dihydroxyphenylalanine, DOPA) through an in vitro enzymatic reaction using mushroom tyrosinase.

The catechol derivative is a compound containing a dihydroxy group, and may be one capable of coordinating with a metal. Specifically, dopa, dopaquinone (Dopa o-quinone), dopamine (dopamine), norepinephrine (norepinephrine), epinephrine (epinephrin), epigallocatechin gallate (epigallocatechin gallate) and derivatives thereof, etc. may be used, preferably It can be daunting.

When the catechol derivative is introduced by changing the tyrosine residue in the mussel adhesive protein into which the catechol derivative is introduced, 10 to 100% of the total tyrosine residue present in the mussel adhesive protein can be converted into a catechol derivative.

In addition, the present invention relates to a stem cell delivery system comprising the hydrogel and stem cells according to the present invention.

The present invention also relates to a method for preparing a hydrogel, comprising the steps of (1) introducing histidine into a mussel adhesive protein, and (2) adding metal ions to an aqueous solution of mussel adhesive protein to which histidine has been introduced.

In the method for producing a hydrogel according to the present invention, the step of (3) tyrosine crosslinking may be further included. By crosslinking the tyrosine contained in the mussel adhesive protein with each other, the strength of the prepared hydrogel can be improved. In the preparation method, the mussel adhesive protein may include at least one or more amino acid sequences selected from the group consisting of the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 4, and combinations thereof.

The present invention provides a composition for biocompatible agents comprising the hydrogel.

The present invention provides a composition for cell delivery comprising the hydrogel and cells. The composition according to the present invention delivers cells to the affected area or an appropriate site for delivery through high bioadhesive capacity without the toxicity of the hydrogel, and through this, promotes adhesion of cells to the affected area or an appropriate site for delivery. It greatly enhances its action. The cells are not limited thereto, but may be myoblasts, fibroblasts, stem cells, and the like.

Examples and Experimental Examples

Hereinafter, the present invention will be described in more detail through examples. However, the examples are merely illustrative of the present invention, and the scope of the present invention is not limited by these examples.

Example 1. Preparation of dopa-introduced mussel adhesive protein

Tyrosine of the mussel adhesive protein of SEQ ID NO: 2 was converted into dopa through an in vitro enzymatic reaction using mushroom tyrosinase on the mussel adhesive protein. The SDS page results of the prepared mussel adhesive protein are shown in FIG. 2 . The part marked with a red square is a band showing the produced mussel adhesive protein, and no other bands were observed in the same line, which means that the protein to be prepared was generated and purified.

Example 2. Preparation of Hydrogel Containing Mussel Adhesive Protein and Metal Ions

A hydrogel was prepared by preparing an aqueous solution (concentration of 5% by weight) of mussel adhesive protein prepared according to Example 1, ^{adding Zn 2+} , and stirring.

In the same manner, ^{a hydrogel was prepared in which Ni 2+} , Co ²⁺ , Cu ²⁺ and Fe ³⁺ were added, respectively, the appearance of which is shown in FIG. 5 . Each hydrogel exhibited a different color depending on the added metal ion.

Experimental Example 1. Confirmation of NBT staining results

The hydrogel prepared in Example 2 was immersed in a sodium borate solution to the extent that it was moistened, and rotationally cultured for 10 minutes. Thereafter, the sodium borate solution was discarded and rotation incubated with deionized water for 10 minutes. Discard deionized water, add 30 ml of sodium glycinate and 500 μl of NBT solution, and dye until color is developed. After staining was completed, the solution was discarded, and the remaining hydrogel was immersed in sodium borate solution and incubated for 10 minutes by rotation. Thereafter, the sodium borate solution was discarded, and the result was shown in FIG. 3 by rotation in deionized water for 10 minutes. As a result, it was confirmed that only the histidine-based mussel adhesive protein after the reaction was accurately stained, and it was confirmed that dopa as well as histidine could be reliably included in the histidine-based mussel adhesive protein.

Experimental Example 2. Confirmation of adhesive ability of hydrogel

The hydrogel prepared in Example 2 was added to a pH 7.4 PBS solution after adding 0.05 ml of fibrin glue, a hydrogel using iron, and a hydrogel using copper between 1 cm x 1 cm pig skin specimens. The result of measuring the adhesive force after bonding for 6 hours is shown in FIG. 4 . As a result, it was confirmed that the hydrogel of Example 2 cross-linked with copper using a histidine-based mussel adhesive protein was superior to the hydrogel cross-linked using dopa and iron as in the conventional method.

Experimental Example 3. Confirmation of self-healing properties of hydrogels

In Examples, ^{hydrogels formed using Zn 2+} , Ni ²⁺ , Co ²⁺ were used as test subjects. The experiment was carried out while increasing the shear stress up to the critical strain level, and the same experiment was repeated again after giving a recovery time of 60 seconds. At this time, it was shown that the same level of strength was restored again (FIG. 6).

Experimental Example 4. Confirmation of structure formation and shape maintenance through extrusion of hydrogel

Fig. 7 shows the results of measuring the change in rheological properties according to the concentration of the hydrogel formed by the binding of copper and the histidine-based mussel adhesive protein prepared by the preparation method of Example 2 above. Self-healing properties were clearly confirmed at all concentrations, and it was confirmed that mechanical properties also increased in proportion to the concentration.

In addition, it was confirmed that the tyrosine cross-linked formulation after extrusion preserves the shape for more than 7 days when stored in PBS solution after cross-linking using tyrosine. (Fig. 8)

Experimental Example 5. Confirmation of cytotoxicity

Chemical additives necessary for tyrosine crosslinking were added to the hydrogel formed using Zn ²⁺ , and myoblasts, fibroblasts, and stem cells were each supported on the hydrogel and extruded, followed by a cytotoxicity test.

As a result of analysis through Live/Dead imaging, it was confirmed that the hydrogel according to the present invention exhibited a toxicity suitable for cell delivery by showing a high cell viability (FIG. 9).

As described above, the present invention has been described through examples. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. Therefore, the above-described embodiments are to be understood in all respects as illustrative and not restrictive. The scope of the present invention is indicated by the following claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be construed as being included in the scope of the present invention.

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Claims (9)

and a histidine-incorporated mussel adhesion protein and a metal ion,
The mussel adhesive protein has a catechol derivative on the surface,
The metal ion is bound to histidine on the surface of the mussel adhesive protein, hydrogel. According to claim 1,
The bond is a coordination bond, hydrogel. According to claim 1,
The metal ion is one or more selected from the group consisting ^{of Zn 2+} , Ni ²⁺ , Co ²⁺ , Cu ²⁺ and Fe ^{3+ , the hydrogel.} The hydrogel according to claim 1, wherein the catechol derivative is a tyrosine residue of the mussel adhesion protein converted. The hydrogel of claim 1, wherein the catechol derivative is at least one selected from the group consisting of dopa, dopaquinone, dopamine, norepinephrine, epinephrine and epigallocatechin gallate. The hydrogel of claim 1, wherein the mussel adhesive protein comprises at least one amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 4, and combinations thereof. (1) introducing histidine into the mussel adhesion protein; and
(2) adding metal ions to the histidine-introduced mussel adhesion protein aqueous solution;
A method for producing a hydrogel comprising a. 8. The method of claim 7,
(3) crosslinking tyrosine;
A method for producing a hydrogel further comprising a. The method according to claim 7, wherein the mussel adhesive protein comprises at least one or more amino acid sequences selected from the group consisting of the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 4, and combinations thereof. .

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