KR20210007631A - Development of injectable and sprayable tissue adhesive hydrogel based on tyrosinase from Streptomyces avermitilis and its application - Google Patents

Abstract

The present invention relates to a method for manufacturing an injecting and spraying type hydrogel composition having adhesion to biological tissue using Streptomyces avermitilis-derived tyrosinase as a catalyst. To this end, according to the present invention, after a phenol derivative capable of being oxidized by tyrosinase is introduced into a biopolymer, cross-linking is induced with Streptomyces avermitilis-derived tyrosinase. The Streptomyces-derived tyrosinase used as the catalyst for crosslinking can rapidly oxidize the phenol derivative while exhibiting broad substrate specificity, thereby being suitable for manufacturing an injecting and spraying type adhesive hydrogel composition. Accordingly, the adhesive hydrogel composition can be usefully applied to tissue engineering.

Description

BACKGROUND OF THE INVENTION {Development of injectable and sprayable tissue adhesive hydrogel based on tyrosinase from Streptomyces avermitilis and its application}

The present invention relates to an injection-type and spray-type hydrogel having tissue adhesion prepared using actinomycetes-derived tyrosinase, a preparation method thereof, and an application thereof.

Hydrogel is one of the materials widely used in tissue engineering and regenerative medicine because it has high moisture content, high biocompatibility, and can easily modify physical properties. Recently, the development of hydrogels having tissue adhesion and shear reduction properties has been actively made, because it has the advantage of being applicable to minimally invasive surgery and drug delivery systems.

Proteins and polysaccharides derived from an extracellular matrix (ECM) are mainly used in the manufacturing process of a hydrogel for intracorporeal injection, and these materials generally exhibit negative charges. In addition, since biological tissues also have negative charges, when the materials derived from the extracellular matrix and the biological tissues meet, they repel each other due to electrostatic repulsion, and thus there is a problem in that the adhesion of the hydrogel is inferior. Therefore, in order to increase the adhesion between the hydrogel and the living tissue, it is required to perform various covalent or non-covalent bonds even in a wet environment.

In this regard, in the field of tissue adhesion, crosslinking and tissue adhesion applications using phenolic derivatives are being studied most actively. This is a method of simulating mussel protein adhesion, and has the advantage of having strong adhesion even in a wet environment. In particular, a reaction in which a phenolic derivative is introduced into an extracellular matrix-derived protein or polysaccharide and then cross-linked using an enzyme as a catalyst has been studied.

As such enzyme crosslinking agents, the use of peroxidase and tyrosinase has been recently reported. In particular, many studies have been conducted on the use of horseradish peroxidase (HRP) as a crosslinking agent among peroxidase. Specifically, Patent Document 1 shows the results of obtaining an intra-body injection hydrogel by bonding gelatin and tyramine, a phenol derivative, using a polyethylene glycol (PEG) linker and then cross-linking it with HRP, and Patent Document 2 shows the result of obtaining a tyramine-modified hydrogel. The results of crosslinking the carboxymethylcellulose derivative and pullulan with HRP to obtain an intracorporeal hydrogel are shown.

However, in the case of HRP, the presence of hydrogen peroxide is required as a proton donor, and this may lead to cell death, so there is a limitation that an appropriate amount of hydrogen peroxide must be used within a range that does not cause cytotoxicity.

Meanwhile, tyrosinase is an enzyme that directly catalyzes the oxidation reaction of phenols, and is an enzyme that can be easily observed around us, such as determining the skin color of animals and causing browning of fruits. Tyrosinase catalyzes the hydroxylation and oxidation reactions of monophenol-type structural materials to produce catechol-type and quinone-type structural materials, and finally polymerizes to produce melanin. Using the reactivity of such a tyrosinase, an example in which a polymer compound containing a tyrosine residue was crosslinked with a tyrosinase has been reported. There was a low problem.

Korean Patent Publication No. 10-2011-0002741 Korean Patent Publication No. 10-2016-0063154

An object of the present invention is to provide an adhesive hydrogel composition capable of being delivered in an injection or spray form, a method of manufacturing the same, and an application invention.

Accordingly, the present inventors in order to solve the above problems, Streptomyces ( Streptomyces ) As a result of crosslinking a polymer containing a phenol derivative using the derived tyrosinase, an adhesive hydrogel composition capable of being delivered in an injection or spray form could be prepared.

Streptomyces-derived tyrosinase of the present invention has a structure having a relatively wide entrance to the active site and a shallow depth when the structure is compared with other known microbial-derived tyrosinase. This structural advantage facilitates the access of the phenolic derivative to the tyrosinase, and as a result, the tyrosinase of the present invention has a broader substrate specificity than the tyrosinase derived from other known microorganisms.

Specifically, the present invention provides the following.

(1) Streptomyces polymer comprising a phenol derivative (Streptomyces) A method for producing an adhesive hydrogel composition comprising the step of crosslinking using the derived tyrosinase.

(2) The polymer according to (1), wherein the polymer containing the phenol derivative,

Hyaluronic acid, 　 alginate, 　 chondroitin sulfate, 　 chitosan or a compound obtained by introducing a phenol derivative into polyethylene glycol; or

Hyaluronic acid, 　 alginate, 　 chondroitin sulfate, 　 a mixture of a compound obtained by introducing a phenol derivative into chitosan or polyethylene glycol, and gelatin,

Method for producing an adhesive hydrogel composition.

(3) The polymer according to (2), wherein the polymer containing the phenol derivative,

Compounds obtained by introducing a phenol derivative into hyaluronic acid; or

A mixture of gelatin and a compound obtained by introducing a phenol derivative into hyaluronic acid,

Method for producing an adhesive hydrogel composition.

(4) The method for producing an adhesive hydrogel composition according to (1), wherein the tyrosinase is derived from Streptomyces avermitilis .

(5) The method for producing an adhesive hydrogel composition according to (1), wherein the phenol derivative is tyrosine, tyramine, 4-hydroxyphenylacetic acid or 3-(4-hydroxyphenyl)propionic acid.

(6) The method for producing an adhesive hydrogel composition according to (5), wherein the phenol derivative is tyrosine or tyramine.

(7) The method for producing an adhesive hydrogel composition according to (1), wherein the step of crosslinking is performed under neutral conditions.

(8) The method for producing an adhesive hydrogel composition according to (7), wherein the neutral condition is a pH of 7 to 8.

(9) An adhesive hydrogel composition prepared by the method of any one of (1) to (8).

(10) The adhesive hydrogel composition according to (9), which can be injected through injection or injection.

(11) The adhesive hydrogel composition according to (10), which is decomposed after being injected into a living tissue through injection or injection.

(12) The adhesive hydrogel composition according to (9), which is for delivering cells or drugs into a living body.

(13) The adhesive hydrogel composition according to (9), which is for coating tissues or organs.

In the present invention, a phenolic derivative capable of maximizing the efficacy of tyrosinase is introduced into a hydrogel, and gelatin, which has in vivo activity and contains a small amount of phenolic derivatives, is applied to the tissue adhesion field to maximize tissue adhesion. can do.

The hydrogel described above is prepared through a reaction under physiological conditions, and in particular, since it is a biocompatible reaction that does not generate heat or by-products during the reaction, it has the advantage of increasing the success rate of hydrogel implantation.

The hydrogel described above can be delivered in an injection type through a syringe through a rapid crosslinking reaction, and thus can be injected into the body by a minimally invasive method. In addition, since it can be delivered in a spray type, it is possible to coat tissues and organs within a short time by being mounted on a catheter and injected into the body by a minimally invasive method.

1 shows a schematic diagram of a reaction for generating an injection or sprayable hydrogel obtained by crosslinking a tyramine-hyaluronic acid conjugate (HA_t) and gelatin with a Streptomyces avermitilis-derived tyrosinase (SA_Ty).
2 is a solution (HG_gel) obtained by mixing a gelatin 3% solution, a HA_t 1% solution, and a gelatin 6% solution and a HA_t 2% solution in a 1:1 volume ratio on an experimental vessel, respectively, as a Tyro derived from Streptomyces avermitilis. After cross-linking reaction for 1 hour using synase or tyrosinase derived from another previously reported microorganism, the reaction result and the flowability when the container is erected at 90 degrees are compared. The control group did not add tyrosinase. (Ctrl: control, AB: Agaricus bisporus derived tyrosinase, BM: Bacillus megaterium derived tyrosinase, SA: Streptomyces avermitilis derived tyrosinase).
FIG. 3 is a comparison of the reactivity of Agaricus bisporus-derived tyrosinase (AB_Ty), Bacillus megaterium-derived tyrosinase (BM_Ty), and Streptomyces avermitilis-derived tyrosinase (SA_Ty) according to substrates. . Figure 3A is a comparison of the enzyme activities of AB_Ty, BM_Ty and SA_Ty against 1 mM L-tyrosine, 3% (w/v) gelatin and 0.2% (w/v) HA_t, and Figure 3B is in the reaction shown in Figure 3A. The initial reaction rate (V ₀ ) was compared.
FIG. 4 is a schematic diagram showing a rheological mutation experiment process of a hydrogel performed to test the adhesion of the hydrogel to mouse skin.
5 shows an image of a series of rheological mutation experiments for testing the adhesion of the hydrogel to mouse skin. 5A is a process for testing the adhesion of a hydrogel obtained by crosslinking an HG_gel solution with SA_Ty, and FIG. 5B shows a process for testing the adhesion of a hydrogel obtained by crosslinking a 3% gelatin solution and a 1% HA_t solution with SA_Ty. will be.
6 shows the results of testing the adhesion of the hydrogel to the skin of a mouse. FIG. 6A is a stress-variation graph of an adhesion experiment, and FIG. 6B is a bar graph showing the degree of adhesion by changing the amount.
7 shows the possibility of injection and in vivo injection of HG_gel hydrogel. Fig. 7A shows that HG_gel is mixed with SA_Ty and then injected into saline through a syringe. In order to facilitate observation, red paint was mixed with the mixed solution. 7B is a diagram showing the injection of the hydrogel into the subcutaneous fat of the mouse and a photograph of the hydrogel after the actual injection. 7C shows the results of histological analysis to confirm the biocompatibility and resolution of the hydrogel injected into the mouse. The upper part of the dotted line represents the hydrogel.
Figure 8 shows that the HG_gel hydrogel of the present invention can be sprayed. 8A is an image showing an image of spraying HG_gel on a slide glass through an airbrush after mixing HG_gel with SA_Ty, and FIG. 8B is a series of checking the elasticity of the HG_gel hydrogel after removing the sprayed HG_gel hydrogel from the slide glass. It shows the process of.
9 shows the result of spray coating HG_gel hydrogel on mouse heart tissue. FIG. 9A is a schematic diagram showing that HG_gel is mixed with SA_Ty and then spray-coated ex vivo on mouse heart tissue, and FIG. 9B is after spraying a HG_gel solution carrying a fluorescent substance and SA_Ty onto the mouse heart tissue using a nebulizer , It is the result of confirming that HG_gel hydrogel is coated on mouse heart tissue through fluorescence observation. 9C shows the results of histological analysis after spray coating an HG_gel solution on mouse heart tissue ex vivo.

Hereinafter, the present invention will be described in detail. However, the present invention may be changed and implemented in various forms, and is not limited to the embodiments described herein.

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as understood by an expert skilled in the art to which the present invention belongs. In general, the nomenclature used in this specification and the experimental methods described below are well known and commonly used in the art.

The present invention is an invention of a hydrogel composition having tissue adhesion and a method for producing the same prepared using tyrosinase, wherein the tyrosinase is a Streptomyces-derived tyrosinase (SA_Ty).

That is, the hydrogel composition of the present invention comprises a hydrogel crosslinked by using a polymer compound into which a phenol derivative is introduced and a tyrosinase derived from Streptomyces as a catalyst.

The tyrosinase of the present invention is a polyphenol oxidase, which converts phenol into catechol by introducing a hydroxyl group into the aromatic ring in the presence of oxygen, and generates quinone from the catechol through an additional oxidation reaction. Since the highly reactive quinone is rapidly bonded to an amine group or thiol group by a nucleophile reaction, not only carbon-carbon and carbon-oxygen bonds, but also carbon-nitrogen and carbon-sulfur bonds can be formed. In addition, since the reaction proceeds in the presence of oxygen without other cofactors, there is an advantage of having milder reaction conditions.

The tyrosinase used as a catalyst for crosslinking in the present invention is derived from the genus Streptomyces, and preferably is derived from Streptomyces avermitilis .

On the other hand, as the polymer compound used for the production of the hydrogel of the present invention, a compound in which a phenol derivative is introduced into hyaluronic acid, 　 alginate, 　 chondroitin sulfate, 　 chitosan or polyethylene glycol (PEG) may be used, and also Gelatin can also be used. Preferably, a compound or gelatin into which a phenol derivative is introduced into hyaluronic acid may be used.

In addition, as the polymer compound used for the production of the hydrogel of the present invention, a mixture of hyaluronic acid, 　 alginate, 　 chondroitin sulfate, 　 chitosan or a mixture of a compound obtained by introducing a phenol derivative into polyethylene glycol, and gelatin may be used. Preferably, a mixture of gelatin and a compound obtained by introducing a phenol derivative into hyaluronic acid can be used.

As the phenol derivative, tyramine, tyrosine, 4-hydroxyphenylacetic acid or 3-(4-hydroxyphenyl)propionic acid may be used, and tyrosine or tyramine may be used preferably.

In one embodiment of the present invention, the method of preparing the crosslinking material of the present invention comprises the following steps:

(a) introducing a phenol derivative to hyaluronic acid, 　 alginate, 　 chondroitin sulfate, 　 chitosan or polyethylene glycol (PEG) to prepare a polymer compound into which the phenol derivative is introduced, and

(b) crosslinking by adding tyrosinase derived from Streptomyces as a catalyst to the polymer compound into which the obtained phenol derivative has been introduced.

In this case, the polymer compound into which the phenol derivative is introduced in the step (a) can be obtained through a method known to those skilled in the art.

The step (b) is characterized in that it is performed under a neutral condition, and the neutral condition is preferably a pH of 7 to 8.

In addition, the step (b) is characterized in that it is carried out in a temperature range of 4 ~ 37 ℃, is preferably carried out at a temperature range of 25 ~ 37 ℃, more preferably carried out at 30 ~ 37 ℃. The reaction time may be performed for 5 minutes to 24 hours, but it is preferable to react for 5 minutes to 1 hour.

In the present invention, the Streptomyces-derived tyrosinase includes not only purified tyrosinase, but also a cell culture medium or cell extract containing a microorganism expressing the tyrosinase. In addition, the tyrosinase-expressing microorganism may be used as a whole cell catalyst.

With regard to the amount of tyrosinase, the catalyst of the present invention, since tyrosinase can also act as an impurity, the crosslinking efficiency can be maximized only when the minimum amount possible is used. From this point of view, the preferred amount of tyrosinase used as a catalyst is 100 nM to 10 μM.

In another embodiment of the present invention, an adhesive hydrogel composition that can be injected through injection or injection is prepared. In particular, the polymer compound into which the phenol derivative is introduced may be mixed with tyrosinase, and then injected or sprayed into a living tissue to be delivered in vivo. The adhesive hydrogel composition is characterized in that it is decomposed after being injected into a living tissue through injection or injection.

The adhesive hydrogel composition of the present invention may be used for delivery of cells or drugs in vivo, and may also be used for coating tissues or organs. Cells or drugs that can be used with the hydrogel composition of the present invention may include all known cells or drugs that can be injected into a living body.

The present invention will be described in more detail through the following examples. However, the following examples are only for embodiing the contents of the present invention, and the present invention is not limited by the examples.

[Example]

Preparation Example 1: Preparation of Streptomyces avermitilis-derived tyrosinase (SA_Ty)

Streptomyces avermitilis-derived tyrosinase (SA_Ty) gene inserted pET28a vector was introduced into E. coli BL21 (DE3) strain, transformed, and then applied to LB solid medium to obtain a colony of 50 µg/mL kanamycin. (kanamycin) was inoculated into 3 mL of LB liquid medium and cultured in a shaking incubator at 37°C for 8 hours. For subculture, 50 µg/mL kanamycin and 500 µl of seed culture were added to 50 mL of LB liquid medium, and cultured in a shaking incubator at 37°C.

When the OD ₆₀₀ (optical density at 600 nm) reached about 0.6, 0.2 mM IPTG and 1 mM CuSO ₄ were added, and then protein overexpression was induced at 37°C for 20 hours. To obtain a protein, the cells were harvested by centrifugation at 4,000 rpm for 10 minutes, and 5 mL of 50 mM Tris-HCl buffer (pH 8.0) was added to wash the cells. After adding 5 mL of 50 mM Tris-HCl buffer (pH 8.0) again, cell disruption was performed using an ultrasonic disruptor (Vibra & cell, USA). Thereafter, the cell lysate was dispensed into a 1.7 mL tube and centrifuged at 16,000 rpm for 30 minutes to obtain a cell extract containing protein.

The cell extract was purified by His-tag using a Ni-NTA column. First, using 1 column volume of pre-binding buffer (5 mM imidazole, 300 mM NaCl, 50 mM Tris-HCl buffer), the activity of Ni-NTA was induced, and the cell extract was transferred to a Ni-NTA column. Impurities that are not strongly bound to the Ni-NTA column using a wash buffer (25 mM imidazole, 300 mM NaCl, 50 mM Tris-HCl buffer) of twice the column volume after passing through the column to bind tyrosinase to the column. Were removed. Finally, tyrosinase was separated from the Ni-NTA column using an elution buffer (250 mM imidazole, 50 mM Tris-HCl buffer), and then 50 mM using a 10k filter to dilute the imidazole to a level of 1/2500. Purified tyrosinase was obtained by replacing the buffer with Tris-HCl. Agaricus bisporus-derived tyrosinase (AB_Ty) and Bacillus megaterium-derived tyrosinase (BM_Ty) were also prepared in the same manner as above, except that the AB_Ty gene or the BM_Ty gene was used instead of the SA_Ty gene. In the crosslinking reaction shown in the following Preparation Examples and Examples, the purified tyrosinase was used.

Preparation Example 2: Preparation of a hydrogel crosslinked by SA_Ty

After dissolving hyaluronic acid (Lifecore Biomedical, LLC, 40-64 kDa) in primary distilled water, EDC (ethyl(dimethylaminopropyl) carbodiimide, ThermoFisher), Sulfo-NHS (sulfo-N-hydroxysulfosuccinimide, ThermoFisher) and tyramine hydrochloride (tyramine hydrochloride, Sigma-Aldrich) was added so that the molar ratio was 1:1:1, and reacted at room temperature for 24 hours. After the reaction was completed, after dialysis for two days using a dialysis membrane (Snakeskin, MWCO: 3 kDa, ThermoFisher), the remaining solution was freeze-dried to obtain a hyaluronic acid-tyramine conjugate (HA_t).

Thereafter, the synthesized HA_t and gelatin (Porcine skin, Type B, Sigma-Aldrich) were dissolved in distilled water to be 2% and 6% (w/v), respectively, and then the two solutions were mixed in a volume ratio of 1:1. , 200 nM SA_Ty was added to the mixed solution (HG_gel) to induce a crosslinking reaction. Then, it was reacted at 37° C. for 1 hour to obtain a hydrogel.

Preparation Example 3: Preparation of injectable hydrogel

The HG_gel prepared according to Preparation Example 2 was mixed with SA_Ty, loaded on a syringe, and then injected through a syringe. When the viscosity of HG_gel needs to be adjusted, NaCl was added to a concentration of 0.3-1 M.

Preparation Example 4: Preparation of spray-type hydrogel

The HG_gel prepared according to Preparation Example 2 was mixed with SA_Ty and loaded on an airbrush (nozzle size 0.3 mm, capacity 7 ml, air pressure 15-35 psi, Monster), and then sprayed through oxygen injection. When the viscosity of HG_gel needs to be adjusted, NaCl was added to a concentration of 0.3-1 M.

Example 1: Comparison of the reactivity of gelatin, hyaluronic acid-tyramine conjugate (HA_t) and hyaluronic acid-tyramine conjugate and gelatin mixture (HG_gel) to SA_Ty

After preparing HA_t and HG Gel in the same manner as in Preparation Example 2, for each of gelatin 3% solution, HA_t 1% solution or HG_gel, SA_Ty (SA) as a catalyst for crosslinking, tyrosinase derived from Agaricus bisporus ( AB_Ty: AB) or Bacillus megaterium-derived tyrosinase (BM_Ty: BM) was added to perform the reaction, and then the reaction result was observed.

When the oxidation reaction by tyrosinase proceeds, the degree of progress of the reaction can be known through color change. Control (Ctrl) indicates that no tyrosinase was added.

As a result, after 1 hour of reaction, in the case of a crosslinking reaction by SA, it was confirmed that the color became dark in the order of gelatin, HA_t, and HG_gel (FIG. 2). This indicates that, while gelatin or HA_t alone did not sufficiently crosslink, HG_gel exhibited more crosslinking due to the synergistic effect of gelatin and HA_t.

On the other hand, in the case of the hydrogel cross-linked by BM_Ty, it was found that the cross-linking reaction did not proceed sufficiently, and in the case of AB_Ty, the tendency was similar to that of SA_Ty.

In addition, the crosslinked hydrogel was tilted at 90 degrees to compare flowability. As a result, in the case of the hydrogel crosslinked by SA_Ty, it was confirmed that the gelatin hydrogel flowed down, while the HA_t and HG_gel hydrogels were adhered to the container as they were (FIG. 2 ). On the other hand, it was confirmed that the hydrogel cross-linked by BM_Ty flowed down all of the gelatin, HA_t and HG_gel hydrogels.

Example 2: Comparison of reactivity of SA_Ty, AB_Ty and BM_Ty according to the type of substrate

In order to compare the difference in the reactivity of SA_Ty, AB_Ty and BM_Ty according to the type of substrate, the oxidation reaction rates of SA_Ty, AB_Ty or BM_Ty to L-tyrosine, gelatin or HA_t were measured, respectively.

For this, the reaction was initiated by adding 100 nM of purified tyrosinase to 200 μl buffer containing 10 μM CuSO ₄ and substrate (200 μM L-tyrosine, 0.3 w/v% gelatin or 0.2 w/v% HA_t). After that, the change in the substrate concentration over time was measured. As the buffer, for SA_Ty and BM_Ty, 50 mM Tris-HCl buffer (pH 8.0) was used, and for AB_Ty, 50 mM sodium phosphate buffer (pH 7.0) was used. In addition, the change in the substrate concentration is the absorbance at 505 nm (ε = 29000 M ^-1 cm ^-1 ) of the conjugate of quinone and MBTH (3-methyl-2-benzothiazolinone hydrazone) produced in the oxidation reaction. It was observed by measuring.

As a result, it was found that AB_Ty had the highest reactivity with respect to tyrosine, while SA_Ty had the highest reactivity with gelatin and HA_t (Fig. 3A). In addition, as a result of calculating the initial reaction rate (V ₀ ) from the results shown in FIG. 3A, it was found that the initial reaction rate of SA_Ty was the fastest for gelatin and HA_t similarly (FIG. 3B ).

This is because the structure of the substrate-binding portion of AB_Ty is advantageous for binding to small molecules such as tyrosine, but not suitable for binding to high molecular compounds such as gelatin or HA_t, whereas SA_Ty binds to phenol derivatives bound to polymers. It indicates that it has an advantageous structure. That is, from the above results, it can be seen that SA_Ty is the most suitable catalyst for the crosslinking reaction of polymers such as gelatin or HA_t.

Example 3: Comparison of tissue adhesion of gelatin 3%, HA_t 1%, and HG_gel hydrogel

As shown in Preparation Example 2, gelatin 3% solution, HA_t 1% solution, and HG_gel were crosslinked with SA_Ty, respectively, to obtain a hydrogel, and the tissue adhesion of these hydrogels was compared.

Tissue adhesion comparison experiment was carried out as follows: mouse skin tissue was cut to a diameter of 8 mm, and the cut skin tissue was attached to the upper and lower probes of a rheometer (302 MCR, Anton-Paar), The hydrogel was injected between the cut skin tissues. Thereafter, after pressing the gap 1 mm for 5 minutes, the probe was lifted at a rate of 10 mm/min, and the measurement was performed (FIGS. 4 and 5).

As a result, it was found that the HG_gel is the most excellent for the adhesion strength and adhesion work of the hydrogel (FIGS. 6A and 6B). This indicates that numerous cross-links were formed between the phenol group of HA_t and the primary amine and thiol groups of gelatin, and also many covalent bonds were formed between the hydrogel and the skin tissue.

Example 4: Preparation and biocompatibility experiment of injectable HG_gel

As a result of loading the mixed solution of HG_gel and SA_Ty in a syringe and injecting it into distilled water, it was confirmed that the mixture was present in distilled water as it maintained the shape of the hydrogel by the rapid crosslinking ability of SA_Ty (FIG. 7A).

In addition, the hydrogel was injected into the mouse skin tissue as shown in FIG. 7B, and histological analysis was performed to observe changes in the tissue over time. The histological analysis was carried out as follows: 1, 2 and 4 weeks after the hydrogel was injected into the mouse skin tissue, and then the mouse skin tissue containing the injected hydrogel was recovered and 4% (w/v) para. Fixed with formaldehyde (Sigma-Aldrich, St Louis, MO) for 24 hours. The fixed samples were washed with PBS buffer for 5 minutes and then dehydrated with 50, 75, 90, 95, and 100% ethanol solutions, respectively. Thereafter, the dehydrated sample was embedded in paraffin and then cut to a thickness of 10 μm with a multipurpose flaker, and the tissue was observed through hematoxylin and eosin (Sigma) staining.

As a result, it was confirmed that the injected hydrogel showed biocompatibility and gradually decomposed over time (FIG. 7C). This indicates that the produced hydrogel can be used for drug delivery or cell delivery in the body.

Example 5: Preparation of spray-type HG_gel and mouse heart tissue coating experiment

The mixed solution of HG_gel and SA_Ty was loaded on an airbrush and sprayed onto the slide glass (FIG. 8A). As a result, it was confirmed that the hydrogel in the form of a thin film was generated by the fast crosslinking ability of SA_Ty (FIG. 8B).

On the other hand, as shown in FIG. 9A, after spray-coating a mixed solution of HG_gel and SA_Ty containing a fluorescent substance on the heart tissue for 10 seconds using an airbrush, the degree of fluorescence and the histological analysis result were observed. HG_gel containing a fluorescent substance is a 3% solution of FITC-labeled gelatin obtained by reacting 2.6 mmol of FITC (fluorescein isothiocyanate) solution with 8% (w/v) gelatin solution for 24 hours, HA_t 1% solution and SA_Ty Prepared by mixing with.

As a result, it was confirmed that a thin and constant-thick hydrogel was formed on the heart surface (FIGS. 9B and 9C). This indicates that even after the hydrogel is sprayed, a large amount of crosslinking occurs at a high speed enough to maintain its shape well, and at the same time, it shows excellent adhesion to living tissue.

The adhesive hydrogel composition of the present invention can be used for cell or drug delivery, and can also be used for coating tissues or organs, and thus can be applied to various fields including pharmaceutical fields such as use in stem cell therapeutics.

Claims (13)

Streptomyces polymer comprising a phenol derivative (Streptomyces) A method for producing an adhesive hydrogel composition comprising the step of crosslinking using the derived tyrosinase. The method of claim 1,
The polymer containing the phenol derivative,
Compounds obtained by introducing a phenol derivative into hyaluronic acid, alginate, chondroitin sulfate, chitosan or polyethylene glycol; or
Hyaluronic acid, alginate, chondroitin sulfate, chitosan, or a mixture of gelatin and a compound obtained by introducing a phenol derivative into polyethylene glycol,
Method for producing an adhesive hydrogel composition. The method of claim 2,
The polymer containing the phenol derivative,
Compounds obtained by introducing a phenol derivative into hyaluronic acid; or
A mixture of gelatin and a compound obtained by introducing a phenol derivative into hyaluronic acid,
Method for producing an adhesive hydrogel composition. The method of claim 1,
The tyrosinase is Streptomyces avermitilis ( Streptomyces avermitilis ) that is derived from, the method for producing an adhesive hydrogel composition. The method of claim 1,
The phenol derivative is tyrosine, tyramine, 4-hydroxyphenylacetic acid or 3-(4-hydroxyphenyl)propionic acid, a method for producing an adhesive hydrogel composition. The method of claim 5,
The phenol derivative is tyrosine or tyramine, a method for producing an adhesive hydrogel composition. The method of claim 1,
The crosslinking step is performed in a neutral condition, a method of producing an adhesive hydrogel composition. The method of claim 7,
The neutral condition is a pH of 7 to 8 conditions, the method for producing an adhesive hydrogel composition. An adhesive hydrogel composition prepared by the method of any one of claims 1 to 8. The method of claim 9,
The adhesive hydrogel composition that can be injected through injection or injection. The method of claim 10,
An adhesive hydrogel composition, characterized in that it is decomposed after being injected into a living tissue through injection or injection. The method of claim 9,
To deliver cells or drugs in vivo, adhesive hydrogel composition. The method of claim 9,
To coat tissues or organs, adhesive hydrogel composition.

Images