KR101677097B1 - Adhesive sealant for treating of fistula, perforation or anastomosis leak and for connecting of internal organ - Google Patents

Abstract

The present invention relates to a cationic protein comprising a catechol derivative, in particular an adhesive sealant for bonding internal organs using a coacervate based on mussel adhesive protein and for treating fistula, perforation or anastomotic leakage, and a method for producing the same. The sealant according to the present invention can be produced by using an underwater adhesive composition comprising a cationic protein including a catechol derivative and a coacervate crosslinked with an anionic polymer, whereby the sealant is excellent in biocompatibility, It is possible to treat the fusion of the internal organs and the leakage of the fistula, perforation or anastomotic site, and it is possible to treat the fistula of the bladder repeatedly contracting and expanding.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to an adhesive sealant for the treatment of fistulae, fissures, perforations or anastomotic leakage,

The present invention relates to a cationic protein comprising a catechol derivative, in particular an adhesive sealant for bonding internal organs using a coacervate based on mussel adhesive protein and for treating fistula, perforation or anastomotic leakage, and a method for producing the same.

Mussel, a marine creature, produces and secretes adhesive proteins and is firmly attached to various surfaces in water. It is also resistant to external shocks such as waves and buoyancy effects of seawater. The mussel adhesive protein can be bonded to various surfaces such as metals, plastics, glass, Teflon, and biomaterials, as well as strong adhesion that is comparable to that of a synthetic polymer adhesive. In addition, mussel adhesive proteins are known to not cause immune reactions and can be applied in medical fields such as adhesion of internal tissues or broken teeth. However, in the case of adhesive substances naturally extracted from mussels, only 1 gram of natural extract can be obtained from 10 million mussels. Therefore, there are many limitations in using industrially used mussel adhesive proteins.

On the other hand, the coacervate is a kind of colloidal material formed when the anionic polyelectrolyte and the cationic polyelectrolyte are mixed under specific conditions. When the coacervate is formed, the absorbance of the solution increases, and the external solution It exists separately. During formation of the coacervate, the participating electrolyte separates from the solution and condenses and remains liquid. At this time, the surface tension decreases and the viscosity changes. The coacervate can also be formed by mixing a protein with a polymer electrolyte of opposite nature. In addition, a coacervate consisting of a cationic mussel adhesive protein and an anionic polymer is also known, and its adhesive activity is known to be superior to the mussel adhesive protein.

Suturing of the wound is the most basic requirement for surgical operation, but it can also threaten the life of a person if it is not done properly. Sutures using sutures are the most commonly used, but some organs in the body are easily damaged by physical pressure or exposed to body fluids, which can lead to leakage (anastomosis leak) through the gap of the wound . In this case, the use of sutures is extremely limited. The most typical example is a fistula, which is a large hole in the bladder, or a perforation in which fluid leakage occurs at the junction of the small intestine or the large intestine. Such fistula or perforation is caused by a pathologic change or trauma to a part of the tissue, and the pit or hole penetrates the part other than the organ, and the pathology and the lesion are very diverse, so prevention and treatment are very difficult. In particular, fistulas that occur in the bladder, which are constantly exposed to urine and that repeatedly contract and expand, are one of the most difficult and curable diseases. Currently, the most commonly used method for treatment of internal organs, anastomotic leakage, and perforation is a physical suture using a suture. This is a method of using a suture to bond the wound formed in the organ. It simply sutures both ends of the wound and can not prevent leakage of body fluids such as urine. In the case of organs that repeatedly expand and contract like a bladder, Pressure can also cause damage to surrounding tissue. Therefore, studies on biocompatible sealants that can overcome the problems of physical seals and prevent leaking of body fluids by blocking fistula or perforation and facilitating treatment on the affected part have been carried out.

To effectively seal the internal organs and fistulae, perforations, and anastomotic leakage, the currently studied sealants are largely divided into synthetic adhesives and naturally derived glue, . However, in the case of a substance composed of a polymer such as a polymer, its use is limited to the skin due to the heat necrosis caused by the polymerization process and the toxicity of the reactant generated upon decomposition. In addition, the use of biologically derived materials such as fibrin glue, albumin and glutaraldehyde is limited in its use due to the problems of stability such as allergic reactions or infection spreading from other species and very low tissue adhesion .

Accordingly, there is a desperate need to develop a new coarse-to-wear sealant that is effective for bonding internal organs and closing fissures, perforations, or anastomotic leaks, which are biocompatible and stable and strong in vivo tissue.

KR 10-2013-0096424

The inventors of the present invention found that a sealant using an underwater adhesive composition containing a cationic protein including a catechol derivative, particularly a mothball adhesive protein-based coacervate, is excellent in biocompatibility, and an organic substance and an inorganic substance The present inventors have confirmed that the adhesion of the internal organs and the fistula, perforation, or anastomotic leakage can be treated, and particularly the fistula of the bladder repeatedly contracting and expanding can be treated.

Accordingly, the present invention is to provide an underwater adhesive composition comprising a cationic protein comprising a catechol derivative and a coacervate crosslinked with an anionic polymer, and a method for producing the same.

The present invention also provides an adhesive sealant comprising the above-mentioned underwater adhesive composition.

The present invention also provides a kit for sealing, joining or closing an organ, comprising the adhesive sealant.

In order to achieve the above object,

The present invention provides an underwater adhesive composition comprising a cationic protein comprising a catechol derivative and a coacervate crosslinked with an anionic polymer.

Further, the present invention provides an adhesive sealant comprising the above-mentioned underwater adhesive composition.

The present invention also provides a kit for sealing, joining or closing an organ, which comprises the adhesive sealant.

The present invention also relates to a method for producing a catechol derivative, comprising the steps of: (1) mixing a cationic protein and an anionic polymer comprising a catechol derivative; And (2) mixing the mixture with a cross-linking agent.

Hereinafter, the present invention will be described in detail.

The present invention provides an underwater adhesive composition comprising a cationic protein comprising a catechol derivative and a coacervate crosslinked with an anionic polymer.

 The catechol derivative means a compound containing a dihydroxy group, which imparts an adhesive force to the cationic protein through a crosslinking action. Specifically, it may be 3,4-dihydroxyphenylalanine (DOPA), dopa o-quinone, topaz (2,4,5-trihydroxyphenylalanine, TOPA), topaquinone and derivatives thereof , And is preferably waveguide.

The catechol derivative is preferably formed by converting a tyrosine residue in a cationic protein, and preferably 10 to 100% of the total tyrosine residue is converted into a catechol derivative. The specific gravity of tyrosine in the entire amino acid sequence of most cationic proteins may be about 1-50%. Tyrosine in cationic proteins can be converted to DOPA, a catechol derivative, by the addition of OH groups through hydration. However, since the tyrosine residues in the cationic protein produced in Escherichia coli are not transformed, it is preferable to carry out a modification reaction in which tyrosine is converted into a waveguide by a separate enzyme and a chemical treatment method. The method of modifying the tyrosine residue included in the cationic protein by the waveguide may be any method known in the art and is not particularly limited. As a preferred example, a tyrosinase can be used to modify the tyrosine residue to a dopamine residue. In one embodiment of the present invention, an in vitro enzyme reaction using mushroom tyrosinase can produce a cationic protein satisfying the above-described waveguide conversion rate.

The cationic protein means a protein comprising a cationic amino acid residue. The cationic amino acid residue may be arginine (Arg), lysine (Lys) or histidine (His), and one or more of these may be included in the cationic protein.

The cationic protein may be a mussel adhesive protein or a variant thereof.

The mussel adhesive protein is a mussel-derived adhesive protein, preferably Mytilus edulis, Mytilus galloprovincialis or Mytilus coruscus, , ≪ / RTI > or a variant thereof.

(B) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5; (c) a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 from 1 to 10 times And (d) a polypeptide fused with two or more selected from the group consisting of the polypeptides (a), (b), and (c). In (c), the polypeptide may be, but is not limited to, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7. Also, the polypeptide fused in (d) is not limited thereto, but preferably it may be a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

In the present invention, the mutants of the mussel adhesive protein preferably include additional sequences at the carboxyl terminal or amino terminal of the mussel adhesive protein under the condition that the adhesive property of the mussel adhesive protein is maintained, or some amino acids are substituted with other amino acids Lt; / RTI > More preferably, a polypeptide consisting of 3 to 25 amino acids including RGD is linked to the carboxyl terminal or amino terminal of the mussel adhesive protein, or a polypeptide comprising 1 to 100% of the total number of tyrosine residues constituting the mussel adhesive protein, And 5 to 100% thereof may be substituted with 3,4-dihydroxyphenyl-L-alanine (DOPA).

3 to 25 amino acids including RGD include, but are not limited to, RGD (Arg Gly Asp, SEQ ID NO: 8), RGDS (Arg Gly Asp Ser, SEQ ID NO: 9), RGDC (Arg Gly Asp Cys, 10), RGDV (Arg Gly Asp Val, SEQ ID NO: 11), RGDSPASSKP (Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro, SEQ ID NO: 12), GRGDS (Gly Arg Gly Asp Ser, SEQ ID NO: Gly Asp Thr Pro, SEQ ID NO: 14), GRGDSP (Gly Arg Gly Asp Ser Pro, SEQ ID NO: 15), GRGDSPC (Gly Arg Gly Asp Ser Pro Cys, SEQ ID NO: 16) and YRGDS (Tyr Arg Gly Asp Ser, ) May be used.

A mutant of a mussel adhesive protein to which a polypeptide consisting of 3 to 25 amino acids including RGD is linked at the carboxyl terminal or amino terminal of the mussel adhesive protein is not limited thereto but preferably a polypeptide consisting of the amino acid sequence of SEQ ID NO: Lt; / RTI >

The mussel adhesive protein is not limited thereto, but can be mass-produced by a genetic engineering method by inserting the mussel adhesive protein so that the mussel adhesive protein can be expressed in a conventional vector designed for expressing an external gene. The vector may be suitably selected according to the type and characteristics of the host cell for producing the protein, or may be newly produced. A method of transforming the vector into a host cell and a method of producing a recombinant protein from the transformant can be easily carried out by a conventional method. Methods for selecting, producing, transforming and expressing recombinant proteins described above can be easily performed by those skilled in the art, and some modifications are also included in the present invention in ordinary methods.

The anionic polymer can be used without limitation as long as it is a polymer substance capable of forming a coacervate by binding with the cationic protein. Preferably, the anionic polymer is a polymer lower than the pI (isoelectric point) of the cationic protein, more preferably a pI value May be a polymer having 2 to 6, more preferably a polymer having a pI value of 2 to 4. If the pI value is more than or less than the above-mentioned pI value, it is difficult to form coacervate, so that it is preferable to use an anionic polymer within the pI range.

In the present invention, the anionic polymer includes, for example, hyaluronic acid, ferredoxin, polystyrene sulfonic acid, gum arabic, gelatin, albumin, Carbopol, high or low methoxyl pectin, sodium carboxymethyl guar gum, xanthan gum, whey protein, faba bean legumin, carboxymethyl cellulose, alginate, carrageenan, sodium hexametaphosphate, sodium casinate, hemoglobin, heparin, and cells And exopolysaccharide B40, and the average molecular weight of the anionic polymer is not limited thereto, but is preferably in the range of 1 kDa to 300 kDa. It may have a selected molecular weight, and may have more preferably from 10kDa to 100kD, more preferably a molecular weight of 17kDa to about 59kDa, and most preferably 17kDa, 35kDa, or 59kD. If the molecular weight is more than or less than the molecular weight, coacervate may not be formed.

The coacervate refers to a kind of colloid formed by combining the cationic protein and the anionic polymer. That is, in the present invention, the coacervate is formed by mixing the anionic polymer with the cationic protein.

The coacervate may be prepared in a water-soluble solvent, more preferably methanol, ethanol, propanol, acetone, acetic acid aqueous solution, more preferably aqueous acetic acid solution, even more preferably 0.1% to 10% Of an aqueous acetic acid solution, even more preferably 0.5 to 8% of an aqueous acetic acid solution.

When preparing the coacervate in the solvent, the optimum pH is limited to, but is preferably pH 2.0 to pH 10.0, more preferably pH 2.0 to pH 8.0, more preferably pH 2.5 to pH 5.5 . If the pH is more than or less than the above-mentioned pH, coacervate may not be formed or deformation of the polymer may occur. The amount of the cationic protein and the anionic polymer to be added to the solvent is not limited thereto, but may be preferably 0.001 to 100% (w / v), more preferably 0.01 to 30% (w / v).

The coacervate may be formed by mixing the cationic protein and the anionic polymer at a weight ratio of 1: 0.01 to 1:10, more preferably 1: 0.25 to 1: 2.5, More preferably in a weight ratio of 1: 0.25 to 1: 2.33. If the mixing ratio is more than or less than the mixing ratio, coacervate may not be effectively formed.

According to one embodiment of the present invention, the coacervate has an advantage that it can be easily passed through a very thin syringe or pipette and is not diluted or spread in water at all, and can be used in an environment exposed to a large amount of water or in water.

The underwater adhesive composition may further include a cross-linking agent to oxidize the catechol derivative to form cross-linking between the respective molecules.

As the crosslinking agent, glutaraldehyde, glyoxal, genipin, glycerol, sodium periodate, hydrogen peroxide and the like can be used. However, biocompatibility It is most preferred to use sodium periodate with excellent and low cytotoxicity.

It is preferable that the cross-linking agent is varied in the concentration of the cross-linking agent and the treatment time in order to obtain the strongest underwater adhesive force depending on the adhesive substrate. According to one embodiment, a crosslinking agent concentration of 10 to 50 mM for a metal surface and a crosslinking time of 10 to 50 hours is applied, and a crosslinking agent concentration of 6 to 150 mM and a crosslinking time of 10 to 50 hours are applied to a biological surface .

Further, the present invention provides an adhesive sealant comprising the above-mentioned underwater adhesive composition.

The adhesive sealant may be adhered to at least one substrate selected from the group consisting of a biomaterial, plastic, glass, metal, and polymer synthetic resin, and may be used for adhering or fixing the substrate. In addition, it can be used not only for a fine adhesion system such as a cell adhesion activity but also for adhesion of a large-capacity adhesive system, for example, a metal material such as aluminum.

In addition, the adhesive sealant can maintain the adhesive force even in the presence of moisture and can be used for underwater adhesion. For example, the adhesive of the present invention can be used for sealing an environment-friendly adhesive for maintenance and maintenance of an underwater structure, specifically, cracks in swimming pools, bathtubs, ships and the like and is useful as a medical adhesive agent, For example, skin adhesives that replace the staples and staples when closing the lips and / or incisions) and hard (hardened) tissue adhesives (such as bone or dental adhesives).

In addition, the adhesive sealant may comprise (1) adhesion between substrates in water (water or saline water); (2) orthopedic treatments such as bones, ligaments, tendons, meniscus and muscle treatments and artificial material implants; (3) ophthalmic adhesions such as perforation, fissure, incision, corneal transplantation, and artificial corneal incision; (4) dental junctions such as correction devices, prosthetic dentures, crown placement, shaking teeth fixation, broken tooth treatment, and filler fixation; (5) surgical treatment such as vascular occlusion, cell tissue grafting, artificial material grafting, wound closure; (6) junctions in plants such as plant graft joining, wound healing; And (7) drugs, hormones, biological agents, medicinal products, physiological or metabolic monitoring devices, antibiotics and cell transplantation.

Particularly, the adhesive sealant can be effectively used for sealing, joining or closing the internal organs exposed to water at all times. Specifically, it is possible to treat fistula, perforation or anastomotic leakage of internal organs.

In addition, the adhesive sealant can be treated by sealing, joining or closing the fistula of the bladder repeatedly contracting and expanding, and is capable of withstanding the maximum pressure imposed on the bladder during the bladder sealing, Lt; / RTI > According to one embodiment, the adhesive sealant exhibited superior underwater adhesion to bladder tissues compared to commercial biomaterials, Tisseel (R) and 2-octyl cyanoacrylate (Dermabond), and 81 cm H ₂ O Of the bladder, that is, a bladder pressure of 100 to 120 cm H ₂ O. This indicates that the fistula of the bladder can be effectively sealed, bonded or closed.

The present invention also provides a kit for sealing, joining or closing an organ, which comprises the adhesive sealant.

The present invention also relates to a method for producing a catechol derivative, comprising the steps of: (1) mixing a cationic protein and an anionic polymer comprising a catechol derivative; And (2) mixing the mixture with a cross-linking agent.

The step (1) is a step of preparing a coacervate by electrostatic attraction by mixing a cationic protein including a catechol derivative and an anionic polymer. The step (2) is a step of forming a cross-link between each molecule through oxidation of the catechol derivative by mixing a mixture of the step (1) with a cross-linking agent.

The adhesive sealant according to the present invention is biocompatible and has a stable and continuous leakage of internal organs such as fistula, perforation or anastomotic leakage by including an underwater adhesive coacervate crosslinked with a cationic protein containing an catechol derivative and an anionic polymer It is possible to close the opening.

The adhesive sealant according to the present invention can be produced by using an underwater adhesive composition comprising a cationic protein including a catechol derivative and an anionic polymer and a cross-linked coacervate, so that it is excellent in biocompatibility and strong on both organic and inorganic surfaces It shows an underwater adhesive force, and it is possible to treat joints of internal organs, leakage of fistula, perforation or anastomotic leakage, and particularly it is possible to treat fistula of bladder which repeatedly contracts and expands.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing amino acid composition analysis for quantifying the conversion of a tyrosine residue to a dopa (DOPA) residue in the mussel adhesive protein fp-151. FIG.
FIG. 2 is a diagram showing a result of observing that a recombinant mussel adhesive protein meets hyaluronic acid, an anionic polymer, and coacervate in the form of water droplets is well formed by an optical microscope.
FIG. 3 is a graph showing the results of analysis of a recombinant mussel adhesive protein having a positive charge and hyaluronic acid having a negative charge using a zeta potential analyzer in pH 5.5 acetate buffer, which is a condition for forming coacervate.
FIG. 4 is a graph showing the result of UV-vis spectroscopy analysis that the most abundant coacervate is formed when a recombinant mussel adhesive protein having positive and negative charges and hyaluronic acid are mixed at specific volume ratios .
FIG. 5 is a diagram showing that coacervate produced when a mucoadhesive protein having a positive charge and a polymer having a negative charge are mixed is not spread or dispersed in water at all, and can be easily injected through a syringe or a pipette.
FIG. 6 is a schematic view showing a method of measuring the adhesive force in water using a coacervate prepared by mixing a cationic mussel adhesive protein and an anionic polymer.
FIG. 7 is a view showing a result of treating the surface of aluminum oxide with a coacervate comprising mussel adhesive protein and an anionic polymer, and then optimizing the concentration of the cross-linking agent exhibiting the strongest water adhesion through tensile testing. Here, the error bar represents the standard deviation, and the statistical significance is expressed by † p value <0.05, * p value <0.02, ** p value <0.01, and *** p value <0.001.
8 is a view showing a result of treating the surface of aluminum oxide with a coacervate consisting of a mussel adhesive protein and an anionic polymer, and then optimizing the treatment time of the cross-linking agent exhibiting the strongest water adhesion through tensile strength measurement. Here, the error bar represents the standard deviation, and the statistical significance is expressed by † p value <0.05, * p value <0.02, ** p value <0.01, and *** p value <0.001.
FIG. 9 is a graph showing the result of treating the surface of porcine skin with mussel adhesive protein and anchor polymeric coacervate, and then optimizing the concentration of the cross-linking agent showing the strongest water adhesion strength through measurement of tensile strength. Here, the error bar represents the standard deviation, and the statistical significance is expressed by † p value <0.05, * p value <0.02, ** p value <0.01, and *** p value <0.001.
FIG. 10 is a graph showing the result of treating the surface of porcine skin with mussel adhesive protein and anchor polymeric coacervate, and then optimizing the treatment time of the cross-linking agent exhibiting the strongest water adhesion through tensile strength measurement. Here, the error bar represents the standard deviation, and the statistical significance is expressed by † p value <0.05, * p value <0.02, ** p value <0.01, and *** p value <0.001.
Fig. 11 is a graph comparing the water adhesion of a coacervate composed of a mussel adhesive protein and an anionic polymer on a bonding surface such as aluminum oxide, titanium oxide, porcine skin, and rat bladder tissue, using typical tissue adhesives fibrin glue and 2-octyl cyanoacrylate Fig. 5 is a graph showing a result of comparison with an underwater adhesive force. Here, the error bar represents the standard deviation, and the statistical significance is expressed by † p value <0.05, * p value <0.02, ** p value <0.01, and *** p value <0.001.
FIG. 12 is a schematic diagram of a blister fouling closure effect of the adhesive sealant prepared according to Example 1, using a water cystometer. FIG.
FIG. 13 is a graph showing the results obtained by treating the adhesive sealant prepared in Example 1, fibrin glue, and 2-octyl cyanoacrylate with a fistula, and then measuring the maximum pressure applied to the outer wall of the bladder, FIG. 5 is a graph showing the results of measurement using a water cystometry method. FIG. Here, the error bar represents the standard deviation, and the statistical significance is expressed by † p value <0.05, * p value <0.02, ** p value <0.01, and *** p value <0.001.
14 is a diagram showing the stability evaluation results of the adhesive sealant, fibrin glue, and 2-octyl cyanoacrylate prepared according to Example 1 treated for bladder fist closure over time.
Fig. 15 is a view showing the closure effect of the adhesive sealant produced according to Example 1 on repetitive expansion and contraction of bladder. Fig.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example  1. Mussel adhesive protein-based Coaseherbate  Manufacture of Adhesive Sealant

1-1. DOPA - Preparation of Recombinant Recombinant Mussel Adhesion Protein

First, the mussel adhesive protein fp-151 (SEQ ID NO: 1) is fp-1 ( Mytilus ) consisting of a decapeptide (AKPSYPPTYK) which is repeated six times in the amino acid sequence of the natural mussel adhesive protein fp-1 (Genbank No. Q27409) mussel foot protein type 1) mutant was synthesized and E. coli was prepared by inserting the gene of Mgfp-5 (Genbank No. AAS00463) between two fp-1 mutants. The preparation of the mussel adhesive protein fp-151 is the same as that described in International Patent Publication No. WO 2005/092920, which patent application is incorporated herein by reference in its entirety.

The tyrosine residue of the mussel adhesive protein fp-151 was then converted to DOPA (dihydroxyphenylalanine) by performing an in vitro enzyme reaction using mushroom tyrosinase (SIGMA) enzyme. Specifically, 1.50 mg / mL fp-151 solution and 100 μg / mL tyrosinase were reacted in a buffer solution (100 mM sodium phosphate, 20 mM boric acid, 25 mM ascorbic acid, pH 6.8) for 1 hour and dialyzed against 1% acetic acid solution .

In order to analyze the modification efficiency of the mussel adhesive protein fp-151, amino acid composition analysis was performed. As a result, it was confirmed that about 50% of all tyrosinase residues were converted to DOPA, and the results are shown in FIG.

1-2. Preparation of coacervate using DOPA-containing recombinant mussel adhesive protein and anionic polymer

Coacervate was prepared using the DOPA-containing mussel adhesive protein fp-151 (modified fp-151, m fp-151) prepared in Example 1-1 and an anionic polymer.

First, since the formation of the coacervate is due to the intermolecular electrostatic attraction, it is confirmed by using a zeta potential analyzer that m fp-151 has a positive charge and hyaluronic acid has a negative charge (see FIG. 2). Thereafter, m fp-151 and hyaluronic acid, an anionic electrolyte polymer, were completely dissolved in an acetate buffer solution of pH 5.5, respectively, and the two solutions were mixed to form a water droplet-type coacervate (see FIG. 3).

Since the formation of the coacervate is greatly influenced by the intermolecular mixing ratio, the protein concentration is measured by the bradford method (Bio-Rad) in order to find the optimum condition in which the coacervate is most formed, Concentration of anionic polymer were mixed in various ratios. The amount of coacervate formed at this time was proportional to the absorbance, and the ratio of the highest absorbance to the intermolecular mixture was confirmed (see FIG. 4). As a result, it was confirmed that the highest absorbance was obtained when the mussel adhesive protein m fp-151 and hyaluronic acid were mixed at a ratio of 6: 4 (v / v). Thereafter, a coacervate phase in a high concentration was obtained by centrifuging the suspension of coacervate at 4 DEG C at 9000 rpm for 10 minutes.

In addition, the water resistance of the prepared coacervate was measured, and it was confirmed that the coacervate was easily passed through a very thin syringe or pipette and was not diluted or spread at all in the water (see FIG. 5).

1-3. Preparation of coacervate for underwater bonding

The adhesive sealant of the present invention was prepared by oxidizing dopa (DOPA) using a cross-linking agent to improve cross-linking between the molecules to improve the underwater adhesive force of the coacervate prepared in Example 1-2. As a crosslinking agent, sodium periodate, which is less cytotoxic, was used.

Further, in order to optimize the crosslinking agent concentration and crosslinking time, the coacervate prepared in Example 1-2 was treated with aluminum oxide or pig skin, and sodium periodate, which is a crosslinking agent, was treated at various concentrations and for various times, (See Fig. 6). As a result, an optimum value of the crosslinking agent concentration (see FIG. 7) of 10 to 50 mM and the crosslinking time of 20 hours (see FIG. 8) was obtained when the surface of the aluminum oxide was treated, An optimized value of a crosslinking agent concentration of 150 mM (see FIG. 9) and a crosslinking time of 6 to 50 hours (see FIG. 10) was obtained.

Experimental Example  1. Underwater adhesion measurement of adhesive sealant

The underwater adhesive force to the various surfaces of the adhesive sealant (coacervate for adhesion in water) prepared in Example 1 was measured. (a) aluminum oxide, (b) titanium oxide, (c) porcine skin, and (d) rat bladder tissue surfaces with a commercially available fibrin glue (Tisseel) and 2-octyl cyanoacrylate Dermabond®), and the results are shown in FIG.

As shown in FIG. 11, the adhesive sealant prepared in Example 1 exhibited the highest water adhesion on the surface of organic matter of swine skin and rat bladder tissue, and commercial fibrin glue had the lowest water adhesion on both organic and inorganic surfaces Respectively. From the above results, it can be seen that the adhesive sealant of the present invention exhibits a strong adhesive force even in an internal organs, which is always exposed to water, particularly, a tissue such as a bladder.

Experimental Example 2. Measurement of the closure effect of the adhesive sealant for tissue bonding

The adhesion of the adhesive sealant (coacervate for underwater bonding) prepared in Example 1 to the closure of the bladder caused by bladder was measured using water cystometry (see Fig. 12).

2-1. The highest bladder pressure tolerance that an adhesive sealant for tissue bonding can withstand

Each of the fibrin glue (Tisseel (R), 2-octyl cyanoacrylate (Dermabond), and the adhesive sealant of the present invention) is applied to the fistula, followed by the maximum pressure (Maximum bladder pressure) was measured. The results are shown in Fig.

As shown in FIG. 13, 2-octyl cyanoacrylate and the adhesive sealant of the present invention exhibited a bladder pressure of 81 cm H ₂ O or higher, and in particular, the adhesive sealant of the present invention had a bladder pressure of 100 to 120 cm H ₂ O Respectively. In general, anesthetized rats are known to have urination as a result of reflex movements by the cerebellum to prevent bladder injury when the bladder pressure reaches about 81 cm H ₂ O (P. Sadananda et al., 2011 , Frontiers in neuroscience 5). On the other hand, in conscious rats, urination will be more likely to occur in response to a lower bladder pressure. Thus, it can be seen that the adhesive sealant of the present invention, which can withstand bladder pressure of 81 cm H ₂ O or higher, can prevent incontinence through fistulae.

2-2. Confirmation of fistula closure effect over time (stability evaluation)

Each of the fibrin glue (Tisseel (R), 2-octyl cyanoacrylate (Dermabond) and the adhesive sealant of the present invention) was treated with a fulminated bladder, and the stability of each sealant with time was measured. Respectively.

As shown in Fig. 14, the fibrin glue and 2-octyl cyanoacrylate gradually decrease in endurance pressure that can withstand the longer the water is left in the water, but the sealant of the present invention is able to withstand increasing pressure of the bladder . This result suggests that the adhesive sealant according to the present invention, which can maintain the closing effect continuously, is most suitable for closure of the feces, considering the actual treatment situation in which the closure effect must be maintained until the bladder tissue regeneration is continuously performed in the urine. .

2-3. Identify durability against repeated expansion and contraction

The actual bladder constantly expands and contracts repeatedly as the urine flows in and out. Therefore, it was measured whether the adhesive sealant of the present invention retains the fugitive closure effect even with such repeated expansion and contraction, and the results are shown in FIG.

As shown in FIG. 15, it was confirmed that the adhesive sealant of the present invention can withstand the bladder pressure of 0 to 80 cm H ₂ O even when the expansion and contraction exceeded 40 times, and after one or two days, And it was confirmed that it can withstand the same level of bladder pressure.

<110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Adhesive sealant for treating fistula, perforation or          anastomosis leak and for connecting internal organs <130> DPP20102958KR <150> KR2009-0078666 <151> 2009-08-25 <160> 17 <170> Kopatentin 1.71 <210> 1 <211> 196 <212> PRT <213> Artificial Sequence <220> <223> FP-151 <400> 1 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr   1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala              20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro          35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu      50 55 60 Gly Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser  65 70 75 80 Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys                  85 90 95 Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly             100 105 110 Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr         115 120 125 Lys Lys Tyr Tyr Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr     130 135 140 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 145 150 155 160 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys                 165 170 175 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro             180 185 190 Pro Thr Tyr Lys         195 <210> 2 <211> 202 <212> PRT <213> Artificial Sequence <220> <223> FP-151-RGD <400> 2 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr   1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala              20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro          35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ser Ser Glu      50 55 60 Gly Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His Tyr His Ser  65 70 75 80 Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr Lys Gly Lys                  85 90 95 Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys Asn Ser Gly             100 105 110 Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg Lys Gly Tyr         115 120 125 Lys Lys Tyr Tyr Gly Gly Ser Ser Ala Lys Pro Ser Tyr Pro Pro Thr     130 135 140 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser 145 150 155 160 Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys                 165 170 175 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro             180 185 190 Pro Thr Tyr Lys Gly Arg Gly Asp Ser Pro         195 200 <210> 3 <211> 172 <212> PRT <213> Artificial Sequence <220> <223> FP-131 <400> 3 Met Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr   1 5 10 15 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala              20 25 30 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro          35 40 45 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Pro Trp Ala      50 55 60 Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly Gly  65 70 75 80 Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp Asn                  85 90 95 Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr Gly Ser Ala             100 105 110 Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro         115 120 125 Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro     130 135 140 Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr 145 150 155 160 Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Leu                 165 170 <210> 4 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> FP-3 <400> 4 Ala Asp Tyr Tyr Gly Pro Lys Tyr Gly Pro Pro Arg Arg Tyr Gly Gly   1 5 10 15 Gly Asn Tyr Asn Arg Tyr Gly Arg Arg Tyr Gly Gly Tyr Lys Gly Trp              20 25 30 Asn Asn Gly Trp Lys Arg Gly Arg Trp Gly Arg Lys Tyr Tyr          35 40 45 <210> 5 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> FP-5 <400> 5 Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Thr Tyr His   1 5 10 15 Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly Tyr              20 25 30 Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr Lys          35 40 45 Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His Arg      50 55 60 Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser  65 70 75 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> fragment sequence derived from FP-1 <400> 6 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys 1510 1514 1519 <210> 7 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> FP-1 <400> 7 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro   1 5 10 15 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys              20 25 30 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr          35 40 45 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys      50 55 60 <210> 8 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 1 <400> 8 Arg Gly Asp   One <210> 9 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 2 <400> 9 Arg Gly Asp Ser   One <210> 10 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 3 <400> 10 Arg Gly Asp Cys   One <210> 11 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 4 <400> 11 Arg Gly Asp Val   One <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 5 <400> 12 Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro   1 5 10 <210> 13 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 6 <400> 13 Gly Arg Gly Asp Ser   1 5 <210> 14 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 7 <400> 14 Gly Arg Gly Asp Thr Pro   1 5 <210> 15 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 8 <400> 15 Gly Arg Gly Asp Ser Pro   1 5 <210> 16 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 9 <400> 16 Gly Arg Gly Asp Ser Pro Cys   1 5 <210> 17 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> RGD Group 10 <400> 17 Tyr Arg Gly Asp Ser   1 5

Claims (18)

Wherein the catechol derivative-bound cationic protein and the anionic polymer are cross-linked with a coacervate and sodium periodate cross-linking agent. The method according to claim 1,
The catechol derivative may be selected from the group consisting of 3,4-dihydroxyphenylalanine (DOPA), dopa quinone, 2,4,5-trihydroxyphenylalanine (TOPA), topaquinone and derivatives thereof Wherein the bladder is at least one member selected from the group consisting of a bladder, a bladder and a bladder. The method according to claim 1,
Wherein the catechol derivative is formed by converting a tyrosine residue in a cationic protein. The method of claim 3,
And wherein 10 to 100% of the tyrosine residue is converted to a catechol derivative. The method according to claim 1,
Wherein said cationic protein comprises at least one cationic amino acid residue selected from the group consisting of arginine (Arg), lysine (Lys) and histidine (His) / RTI &gt; The method according to claim 1,
Wherein the cationic protein is a mussel adhesive protein or a variant thereof, wherein the cationic protein is a mussel adhesive protein or a variant thereof. The method according to claim 6,
The mussel adhesive protein or a variant thereof may be selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: Characterized in that it comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: Bonded or closed. The method according to claim 1,
The anionic polymer may be selected from the group consisting of hyaluronic acid, ferredoxin, poly styrene sulfonic acid, gum arabic, gelatin, albumin, carbopol, High or low methoxyl pectin, sodium carboxymethyl guar gum, xanthan gum, whey protein, faba bean legumin, carboxymethyl But are not limited to, carboxymethyl cellulose, alginate, carrageenan, sodium hexametaphosphate, sodium casinate, hemoglobin, heparin and extracellular polysaccharide B40 (exopolysaccharide B40 ) Of at least one member selected from the group consisting of polyvinylpyrrolidone and polyvinylpyrrolidone. The method according to claim 1,
Wherein the coacervate is formed by mixing the cationic protein and the anionic polymer at a weight ratio of 1: 0.01 to 1:10 at a pH of from 2.0 to 10: 10, wherein the cationic protein and the anionic polymer are mixed in water for sealing, Adhesive composition. delete An adhesive sealant for sealing, joining or closing a bladder fist, comprising the inventive underwater adhesive composition of any one of claims 1 to 9. delete 12. The method of claim 11,
Wherein the adhesive sealant is for underwater adhesion. The adhesive sealant for sealing, joining or closing a bladder fist. delete delete 12. The method of claim 11,
Wherein said adhesive sealant maintains a closing force to withstand the maximum pressure exerted on the bladder during urination upon sealing, joining or closing of the bladder fist. A kit for sealing, joining or closing a bladder, comprising the adhesive sealant of claim 11. (1) mixing an anionic polymer and a cationic protein to which a catechol derivative is bound; And
(2) mixing the mixture with sodium periodate as a cross-linking agent; and (2) mixing the mixture with a sodium periodate as a cross-linking agent.

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