KR20220043917A - Aqueous adhesive composition comprising silk fibroin protein modified with kosmotropic salt and method for preparing the same - Google Patents

Abstract

The present invention relates to an aqueous adhesive composition comprising silk fibroin protein modified with a kosmotropic salt, a method for preparing the same, and a medical aqueous adhesive. Provided are advantages of implementing semi-permanent adhesive force and exhibiting adhesive force in a short time without addition of chemical materials by adjusting internal structure transition of silk fibroin protein.

Description

Aqueous adhesive composition comprising silk fibroin protein modified with kosmotropic salt and method for preparing the same

The present invention relates to an aqueous adhesive composition comprising silk fibroin protein modified with a cosmotropic salt and a method for preparing the same.

Medical adhesives are used for suturing or applying tissue during surgery or as hemostasis, body fluids and blood blocking agents. A wide range of materials are available. Medical adhesives are used in biomedical fields such as tissue adhesives for biological samples, hemostatic agents, tissue engineering scaffolds, drug delivery carriers, tissue fillers, wound healing, or intestinal adhesion prevention, fibrin glue for soft tissue, gelatin glue, bone cement for bone tissue. It is being used as a commercialized product.

In general, since medical adhesives are used in the body, there is always a problem of lowering of adhesion by body fluids or blood and detachment thereof. An ideal adhesive as a medical adhesive should have the characteristics of instantaneous termination with minimal external energy under mild conditions.

Accordingly, studies related to water adsorption to realize adhesion using water are gradually being reported. Most studies involve polymerization, so adhesives contain chemicals such as initiators and monomers. When a chemical substance is diffused by the water present on the surface of the area to be used, strong external energy such as UV is applied to initiate polymerization of the polymer to form an adhesive force.

However, this technique depends on the rate of diffusion of chemical substances and the rate of polymerization of polymers and requires a short time in minutes to form an adhesive force. Considering that it is for medical use, however, the time in minutes is not appropriate.

On the other hand, as a medical adhesive, cyanoacrylate provides strong tissue adhesion, but flexibility is not secured due to the problem of slow adhesive fixation time and curing speed for bonding one substrate to another, and the cured product becomes excessively hard. Compared to methyl and ethyl cyanoacrylate, n-butyl cyanoacrylate and octyl cyanoacrylate, which are less toxic than methyl and ethyl cyanoacrylate, exhibit a rapid reaction pattern when in contact with water because their reaction rate to water is very fast, and is still toxic to tissues. There is a limit in that it shows

Therefore, it is necessary to develop a material that is suitable for medical use without toxicity and risk, and exhibits underwater adhesion in seconds even in mild conditions and strongly binds living tissue.

Republic of Korea Patent Publication No. 10-2013-0034382 (2013.04.05.) Republic of Korea Patent Publication No. 10-2012-0046596 (2012.05.10.)

In order to overcome the limitations of the medical adhesives described above, the inventor of the present invention has repeatedly studied to modify silk fibroin protein with excellent biocompatibility. could come up with

The present invention realizes excellent adhesion in an aqueous environment by regulating the transition of the internal atypical structure of silk fibroin protein. An object of the present invention is to provide a water-based adhesive composition.

In addition, an object of the present invention is to provide a semi-permanent water-based adhesive composition that is not based on a chemical reaction, but provides adhesion by controlling the internal structure of the protein.

In addition, an object of the present invention is to provide a method for preparing a water-based adhesive composition that exhibits significantly improved adhesion in seconds, as opposed to conventional commercialized products exhibiting adhesion in minutes.

In addition, an object of the present invention is to provide a medical water-based adhesive that uses a natural polymer harmless to the human body, has high biocompatibility, exhibits high body water absorption and excellent adhesion.

In order to solve the above technical problem, the present invention provides a composition for water-based adhesion comprising a silk fibroin protein modified with a kosmotropic salt.

In one aspect of the present invention, the cosmotropic salt is an alkali metal ion or alkaline earth metal ion and sulfate (SO ₄ ^2- ), phosphate (HPO ₄ ^2- ), acetate (CH ₃ COO ^- ), hydroxide (OH) ^- ), chloride (Cl ^- ), bromide (Br ^- ) and formate (HCOO ^- ) may be composed of one or a combination of two or more selected from the group consisting of.

In one aspect of the present invention, the cosmotropic salt is one or two or more selected from calcium chloride (CaCl ₂ ), lithium bromide (LiBr), potassium chloride (KCl), sodium chloride (NaCl) and sodium acetate (CH ₃ COONa). there is.

In one aspect of the present invention, the cosmotropic salt may be included in an amount of 1 to 20 parts by weight based on 100 parts by weight of silk fibroin protein.

In one aspect of the present invention, it may be a structure in which a hydrophilic amino acid is exposed on the surface of the atypical structure of the silk fibroin protein.

In one aspect of the present invention, the hydrophilic amino acid may be one or more selected from the group consisting of serine, threonine, cysteine, aspartic acid, glutamine, and tyrosine.

In one aspect of the present invention, the hydroxyl group in the hydrophilic amino acid and the amino group of the adherend may form a hydrogen bond.

In one aspect of the present invention, the content of serine exposed to the surface of the amorphous structure may be 5 to 20% as measured by solid ¹³ C-NMR.

In one aspect of the present invention, the shear strength according to ASTM F2255-05 standard at 36 ° C. of the water-based adhesive composition may be 5 to 50 kPa.

The present invention also comprises the steps of obtaining a first solution by adding a cosmotropic salt to an acidic solvent; obtaining a second solution by adding silk fibroin protein to the first solution and dissolving it by heating to 50 to 70° C. for 30 minutes to 3 hours; and drying the second solution at room temperature; provides a method for producing a water-based adhesive composition comprising a.

At this time, in one aspect of the present invention, the step of stabilizing by heating the dried second solution to 50 to 70 ℃; may further include.

In one aspect of the present invention, the cosmotropic salt may be included in an amount of 1 to 20 parts by weight based on 100 parts by weight of silk fibroin protein.

In one aspect of the present invention, the second solution may contain silk fibroin whose content of serine exposed on the surface of the atypical structure is 5 to 20% as measured by solid ¹³ C-NMR.

In one aspect of the present invention, the acidic solvent may be a C1-C7 organic acid.

In addition, the present invention provides a medical aqueous adhesive comprising a water-based adhesive composition having the same technical idea as the aforementioned aqueous adhesive composition.

The modified silk fibroin protein of the water-based adhesive composition according to the present invention is rich in non-binding functional groups of an amorphous domain, and hydration-adaptive crystallization is suppressed even in an aqueous environment, resulting in high levels within seconds. It is possible to provide a water-based adhesive composition capable of implementing adhesive strength.

The aqueous adhesive composition according to the present invention can exhibit excellent adhesion by exposing hydrophilic amino acids to the surface of the atypical structure of silk fibroin protein by controlling the concentration of cosmotropic salts, thereby participating in hydrogen bonding. In addition, it can implement reversible adhesion by using a hydrogen bond rather than a covalent bond.

The water-based adhesive composition according to the present invention does not add potentially toxic chemicals, and as it realizes adhesion through modification of silk fibroin protein, it stably maintains adhesion even over a long period of time, and easily exhibits excellent water-based adhesion. can make it

In addition, since the medical water-based adhesive according to the present invention exhibits excellent adhesion to various substrates, it can be utilized as a double-sided tape for bonding various medical devices to tissues.

1 shows a conceptual diagram analyzing the presence or absence of adhesion in a dry and wet state of a water-based adhesive composition according to the present invention.
2 is an image showing various types of designs of silk wet gauze (SWG), which is an embodiment, as a composition for water-based adhesion including silk fibroin protein modified from a cocoon. Specifically, the right (i) is a miniaturized structure of 4 to 0.25 cm2 for local disease management (eg, for periodontitis treatment), (ii) is a structure for antiseptic masking of a 3D tubular organ, and (iii) shows a functional mesh structure for breathable treatment applicable to large-area burns.
Figure 3 (a) shows an embodiment in which the water-based adhesive composition according to the present invention is used as a disinfection mask for peritoneal dialysis management. The red fluorescence image shows the secondary infected kidney, and after 72 hours according to SWG (silk wet gauze) treatment, the organ is completely healed (purple and blue fluorescence images mean the fully healed organ). (b) is a schematic diagram showing the crystallization tendency of proteins in an aqueous environment according to the concentration of active serine. When the concentration of active serine is insufficient, crystallization proceeds as a protein modification according to hydration-adaptation, but when the concentration of active serine is high enough, a stable hydration shell is formed, crystallization is inhibited, and as a result, adhesion with an adherend is improved. can be
Figure 4 (a) is a schematic diagram showing the internal structure of the silk fibroin protein in which the crystal structure is dispersed in an atypical structure matrix. (b) is a schematic diagram showing that the protein is modified by hydration from the interface in contact with water from the dry parallel state toward the center of the material.
5 is a schematic diagram showing that the adhesive strength and hydration degree of the water-based adhesive composition according to the present invention are in a concave function relationship. Serine, a hydrophilic amino acid in the α-helix structure, is exposed to the surface to obtain an appropriate degree of hydration. The concept that high adhesion can be formed when shown is illustrated.
6 is a graph showing the shear strength according to the concentration of active serine. Orange column corresponds to co-SWG, and blue column means ad-SWG.
Figure 7 shows a graph of the relationship between the profile and the extension vs force on the biotic-biotic interaction for SWG. (a) strain vs. The stress curve shows two types of cohesive force (co-SWG) as the interaction between A and B and adhesion force (ad-SWG) as the interaction between A* and B in pigskin skin. (b) is a photograph showing the cohesive force (co-SWG) and adhesion (ad-SWG) in medical plastics (PETG), when co-SWG and ad-SWG show the best adhesion. Specific conditions are as follows, and co-SWG (orange column) is calculated as shear strength (kPa), while ad-SWG (blue column), which is a form that slowly stretches and falls off, is adhesive energy density (adhesive energy density, kJ/m ³ ) can be calculated.
- co-SWG: 7.90% active serine + 5 seconds of hydration
- ad-SWG: active serine 9.23% + hydration 30 seconds
Figure 8a (a) shows the experimental procedure using a mouse model of peritonitis for peritoneal dialysis (PD) performed according to Experimental Example 2-1 of the present invention, (b) is the introduction of ad-SWG into peritonitis mice and 1 After one day, it was stably attached to the affected part, and it is a photograph showing that it can be detached without trauma.
Figure 8b is a time when applying, after CLP, surgical tape, ad-SWG, and ad-SWG (ad-SWG + Vanco) with antibiotics added according to Experimental Example 2-1 of the present invention It is a graph showing the survival rate according to
8c is an image of intraperitoneal infection by monitoring for 0, 6 and 12 hours using a fluorescently-labeled bacterial detection probe after CLP surgery of a peritonitis mouse (SKH1), performed according to Experimental Example 2-1 of the present invention. . Peritoneal dialysis (PD) was performed 6 times (once every 12 h) after CLP surgery, and diseased sites were treated after each PD, surgical tape, ad-SWG, and ad-SWG (ad-SWG + vancomycin) with abdominal infusion. Vanco). White arrows indicate the peritonitis area.
Figure 8d shows the bacterial infection in (i) lung, (ii) spleen, (iii) liver and (iv) kidney, performed according to Experimental Example 2-1 of the present invention, and the red fluorescence image shows the infected organ. and purple and blue mean healed organs.
Figure 8e shows the skin condition after disease management of mice undergoing repeated PD, performed according to Experimental Example 2-1 of the present invention. The yellow arrow indicates severe necrosis, and the blue arrow indicates the incision site free of infection.
9a shows an image of H&E staining performed according to Experimental Example 2-1 of the present invention. Black arrows indicate structural damage to organs (scale bar: 250 μm).
Figure 9b is a graph confirming whether organ damage through a marker after 72 hours of PD in peritonitis mice, performed according to Experimental Example 2-1 of the present invention. Specifically, (a) shows the results of ALT and AST, which are liver damage markers, (b) shows the results of creatinine and BUN, which are kidney damage markers, and (c) shows the results of LDH and CRP, which are organ damage markers.
10 is a graph showing the measurement result of the number of neutrophils, an inflammatory marker, according to Experimental Example 2-2 of the present invention.
11 is a graph showing the concentrations of cytokines (IL-1β, IL-4, IL-6 and IFN-γ) in plasma according to Experimental Example 2-3 of the present invention.

Hereinafter, the aqueous adhesive composition comprising the modified silk fibroin protein according to the present invention, a manufacturing method thereof, and a medical aqueous adhesive using the same will be described in detail. At this time, unless otherwise defined, all technical and scientific terms have the meanings commonly understood by those of ordinary skill in the art to which this invention belongs, and the terms used in the description of the present invention are only specific examples is intended to effectively describe, and is not intended to limit the present invention.

In addition, in the following description, descriptions of well-known effects and configurations that may unnecessarily obscure the gist of the present invention will be omitted.

In the following specification, the units used without special mention are based on weight, and for example, the unit of % or ratio may mean weight % or weight ratio.

In addition, in describing the components of the present invention, terms such as first, second, A, B (a), (b), etc. may be used. These terms are only for distinguishing the elements from other elements, and the essence, order, or order of the elements are not limited by the terms.

Also, the singular form used in the specification of the present invention may be intended to include the plural form as well, unless the context specifically dictates otherwise.

In the present specification, 'wet adhesion' is defined as meaning a phenomenon in which adhesion is generated by a liquid such as water, broadly, body fluid or blood.

In the present specification, refined silk fibroin (degummed silk fibroin) is defined as meaning silk fibroin remaining after removing silk sericin from Bombyx Mori silk cocoons.

Hereinafter, the water-based adhesive composition according to the present invention will be described in detail.

The present invention provides a composition for water-based adhesion comprising a silk fibroin protein modified with a kosmotropic salt.

Cosmotropic salts contribute to stabilization by hydration with water molecules in an aqueous environment, and are known to contribute to changes in the strength of intramolecular interactions in macromolecules such as proteins. The cosmotropic salt is an alkali metal ion or alkaline earth metal ion and sulfate (SO ₄ ^2- ), phosphate (HPO ₄ ^2- ), acetate (CH ₃ COO ^- ), hydroxide (OH ^- ), chloride (Cl ^- ) , bromide (Br ^- ) and formate (HCOO ^- ) may be any one or a combination of two or more selected from the group consisting of. Specifically, it may be any one or two or more selected from calcium chloride, lithium bromide, potassium chloride, sodium chloride and sodium acetate, but is not particularly limited thereto.

In general, protein polymers are crystallized by water, resulting in significant loss of adhesion in an aqueous environment. This is called hydration-adaptive crystallization. An object of the present invention is to provide an adhesive composition that provides excellent adhesion even in an aqueous environment by inhibiting hydration-adapted crystallization by controlling the sequence structure of specific amino acids of the silk fibroin protein.

Accordingly, in one embodiment of the present invention, the silk fibroin protein may be modified with calcium chloride. Specifically, the silk fibroin protein modified with an excess of calcium chloride has a ratio of an atypical structure such as α-helix or a random coil, and is relatively rich in unbound hydrophilic functional groups to the surrounding environment. and new combinations are possible. In addition, by forming a hydrogen bond with water molecules when placed in an aqueous environment, it may be possible to inhibit hydration-adapted crystallization by improving the stability of water molecules.

Specifically, the hydrophilic functional group may be a hydroxyl group (-OH), a thiol group (-SH), a carboxyl group (-COOH), or an amide group (-CONH ₂ ). More specifically, in a non-limiting example of the present invention, the hydrophilic amino acid may be serine.

Serine is a polar amino acid containing a hydroxyl group, and in particular, active serine ( Φ _{Active Ser} ) in an α-helix form interacts with a surface functional group of an adherend through hydrogen bonding in an aqueous environment. Active serine may exhibit aggregation and adhesion behavior depending on the degree of hydration. In the case of a short hydration time, for example, less than 30 seconds, it is difficult to induce rotational mobility of the active serine, and the cohesive force may be weakened. However, in the case of having a hydration time of 30 seconds or more, a significant improvement in adhesion may be exhibited due to the formation of a sufficient hydration shell in specifically 120 seconds or less.

Referring specifically to FIG. 6 , a horizontal dash line corresponding to a shear strength of 3 kPa and an adhesion energy density of 30 kJ/m ³ indicates fibrin glue. Fibrin glue has the advantage of almost no rejection or tissue toxicity in vivo, but as described above, it has a weak adhesive strength in an aqueous environment and a low adhesion energy density of a liquid adhesive, making it difficult to apply to an accurate site. The aqueous adhesive composition comprising silk fibroin protein fortified with active serine according to the present invention has significantly higher shear strength and adhesion energy density compared to currently commercialized and used fibrin glue, so it can realize much better adhesion than fibrin glue adhesive. It can be predicted that there is

As shown in FIG. 4, silk fibroin protein is a form in which a crystal structure is dispersed in a matrix of an atypical structure, and the crystal structure is formed by stacking β-sheets in a layered form by hydrogen bonding. . In the silk fibroin protein modified with cosmotropic salt, the hydrophilic amino acid of the atypical structure is exposed to the surface by the cosmotropic salt, and the ratio of the atypical structure and the non-bonding functional group is significantly increased. In this case, the unbound functional group is a functional group exposed on the surface of the atypical structure, and in particular, may be a hydroxyl group in a hydrophilic amino acid. The hydroxyl group may provide adhesion in an aqueous environment by forming a hydrogen bond with water or an amino group of an adherend. In addition, sufficient hydrogen bonding between the unbonded functional group exposed on the surface of the amorphous structure and the water molecule can stabilize the water molecule, thereby inhibiting hydration-adapted crystallization.

In one embodiment of the present invention, the cosmotropic salt may be included in an amount of 1 to 20 parts by weight based on 100 parts by weight of silk fibroin protein. Preferably, it is included in an amount of 2 to 18 parts by weight, and more preferably 3 to 15 parts by weight, thereby increasing the proportion of the atypical structure of the silk fibroin protein and remarkably improving adhesion.

As for the representative nucleotide sequence of silk fibroin extracted from the cocoon (Bombyx mori), GAGAGS accounts for about 53% and GAGAGY accounts for about 18%. As such, hydrophobic amino acids such as glycine (G) and alanine (A) occupy a large portion, and hydrophilic amino acids such as serine (S) and tyrosine (Y) are irregularly contained in the crystal structure do.

In one embodiment of the present invention, the modified silk fibroin protein may be in a form in which serine is more exposed on the surface of the atypical structure. Specifically, in the modified silk fibroin protein, the cosmotropic salt strongly binds to water in an aqueous environment to achieve stable solvation. For example, the binding enthalpy of calcium chloride and water is -163 kJ/mol, which is stabilized while releasing heat when calcium chloride is combined with water. At this time, the formed water film interferes with the formation of the crystal structure of the β-sheet, and the serine present in the atypical structure is exposed to the surface of the atypical structure. be able to

Specifically, looking at the involvement of serine in hydrogen bonding according to the surrounding environment, when serine is distributed in an amorphous matrix such as an α-helix or a random coil, its involvement in hydrogen bonding is quite high.

The content of serine exposed to the surface of the amorphous structure may be 5 to 20%, 7 to 15%, 7.5 to 11, or 8 to 10% as measured by solid ¹³ C-NMR. At this time, solid ¹³ C-NMR measurement conditions are as follows.

- Radio frequency: 400 MHz

- Spinning rate: 10 kHz

- Calibration: TMS (0 ppm)

In one embodiment of the present invention, the shear strength at 36 ℃ of the water-based adhesive composition measured according to ASTM F2255-05 may be 5 to 50 kPa. Preferably it may be 10-40 kPa, More preferably, it may be 20-30 kPa. The adhesive energy density per volume (kJ/m ³ ) may be 40 to 160 kJ/m ³ .

In one embodiment of the present invention, the modified silk fibroin protein may have a thickness of 30 to 500 μm, preferably 50 to 400 μm, more preferably 100 to 350 μm. When the thickness is maintained in the above range, the transparency of silk fibroin is maintained high, so that the utilization can be excellent. The transparency may be measured as a transmittance of visible light, and may be specifically 50% or more, more specifically 70% or more, and most preferably 80% or more in a wavelength region of 400 to 700 nm. However, the transparency can be easily adjusted according to the use of the water-based adhesive composition.

In addition, the present invention provides a method for preparing an aqueous adhesive composition comprising a silk fibroin protein modified with a cosmotropic salt. Specifically, it may be characterized by including the following steps.

obtaining a first solution by adding a cosmotropic salt to an acidic solvent; obtaining a second solution by adding silk fibroin protein to the first solution and dissolving it by heating to 50 to 70° C. for 30 minutes to 3 hours; and drying the second solution at room temperature; and may further include stabilizing the dried second solution by heating it to 50 to 70°C.

In one embodiment of the present invention, the acidic solvent may be an organic acid containing 1 to 7 carbon atoms. Specifically, for example, formic acid, acetic acid, propionic acid, butyric acid, lactic acid, fumaric acid, malic acid, It may be citric acid or a mixed solvent thereof. However, it is not particularly limited thereto.

First, a cosmotropic salt is added and dispersed in an acidic solvent to prepare a first solution. When the cosmotropic salt is dispersed, a homogeneous solution can be obtained by ultrasonication for 30 minutes to 2 hours. Thereafter, a step of adding refined silk fibroin protein to the first solution is performed. Refined silk fibroin protein refers to silk fibroin remaining after removing silk sericin from a cocoon. In general, it can be obtained by supporting and refining a cocoon in an alkaline soap solution, washing it 2-3 times with distilled water, and then drying it at room temperature. It is necessary to add the refined silk fibroin protein to the first solution and dissolve it at a high temperature. Specifically, it is heated to 50 to 70° C. and dissolved for 1 to 5 hours to obtain a second solution. Thereafter, the second solution may be cast-dried on the substrate and additionally subjected to a drying step at a high temperature. In this case, it is easily detached from the substrate. In this case, the substrate may be a polyethylene terephthalene, polystyrene, polyethylene or nitrile polymer substrate, but is not limited thereto, and may be easily adhered to various polymer surfaces.

The water-based adhesive composition according to the present invention exhibits fast and strong adhesion in an aqueous environment, that is, an environment with water, body fluid, blood, etc., and is not accompanied by a violent chemical reaction, so tissue damage can be minimized, and as a bioadhesive composition It can be widely used.

According to the present invention, it is possible to provide a medical adhesive, an anti-adhesion material, a wound covering material, or a hemostatic material obtained by applying the above-described aqueous adhesive composition to a dressing material processing process. Specifically, the dressing material processing process may apply a processing process known in the art according to the type of dressing material.

According to an embodiment of the present invention, the water-based adhesive composition may be gauze wet with modified silk fibroin protein (silk wet gauze, SWG), and the SWG provides a disinfection masking for topical disinfection of the affected area. In this case, it plays the role of a gauze dressing that locally elutes the disinfectant and the role of a surgical tape so that the affected tissue can be stably arranged after surgery. Through this, the present invention can prevent secondary diseases including infection at the surgical site, and furthermore, it is possible to provide a platform for pre- and post-surgery management with dramatically improved efficacy.

In addition, the present invention can provide a method of adhesion, adhesion prevention, wound coating or hemostasis of living tissue comprising the step of applying the aqueous adhesive composition to a living tissue in need of adhesion, adhesion prevention, wound coating or hemostasis.

Hereinafter, the water-based adhesive composition according to the present invention and a manufacturing method thereof will be described in more detail through Examples. However, the following examples are only a reference for describing the present invention in detail, and the present invention is not limited thereto, and may be implemented in various forms.

[Example 1] Preparation of silk wet gauze (SWG) with modified silk fibroin protein

The cocoon obtained from Bombyx mori was cut thinly to the size of a coin (diameter 18 mm), and immersed in 0.02 M sodium carbonate solution (~90 ℃), and the refining process was performed for 1 hour. The refined silk fibroin was then washed with deionized water and dried overnight at ambient conditions. 3 wt%, 6 wt%, or 10 wt% of formic acid containing calcium chloride (CaCl ₂ ) is sonicated for 30 minutes to obtain a transparent homogeneous solution, and refined silk fibroin is added to the solution, so that the aqueous silk fibroin turns yellow. until dissolved at 60 °C for 1 hour. The viscous silk fibroin was cast overnight in polystyrene molds of three sizes (diameter 3.5 cm, diameter 5.0 cm, diameter 10.5 cm) at 25° C. and 65% relative humidity, and dried. After drying again by heat treatment at 60 ° C. for 2 hours, SWG was obtained.

[Experimental Example 1] Water-based adhesion evaluation

Lap shear evaluation was performed on the modified silk fibroin according to Example 1 based on ASTM F2255-05 and ASTM D3163-01. The adhesive substrate was 1 mm polyethylene terephthalate (PETG) with excellent flatness, and an underwater adhesive specimen was prepared to be 1.27 cm × 2.50 cm, attached to the PETG lower plate, and immersed in distilled water for 30 seconds to impart adhesion. The PETG upper plate and the PETG lower plate to which the specimen was attached were overlapped by 1.25 cm, and pressed with a force of 2 kPa for 30 seconds. Thereafter, using a universal testing machine (UTM; Model 3366, Instron, USA), while maintaining 25 °C and 44% relative humidity, the bonded specimen was pulled at a speed of 10 mm/min to evaluate the adhesion. The results are shown in FIG. 6 . As shown in Fig. 6, the shear strength achieved the best performance when the active serine concentration was 9.23% ( Φ _{Active Ser} =9.23). Therefore, the conditions with the best performance, active serine concentration of 9.23% and hydration for 30 seconds, were referred to as ad-SWG and used in preclinical experiments.

[Experimental Example 2] Evaluation of treatment effect through in vivo

2-1. Verification of disease management effect before and after surgery through preclinical experiments

CLP (cecal ligation and puncture) surgery was performed on Hairless SKH1, a mouse model of peritonitis, (i) CLP surgical group (CLP), (ii) surgical tape treated group (surgical tape), (iii) ad -SWG treated group (ad-SWG), (iv) vancomycin (10 mg/kg) and ad-SWG treated group (ad-SWG + Vanco), divided into 4 groups (n = 5), 12 A total of 6 peritoneal dialysis (PD) sessions were performed once every hour. After 72 hours of CLP, mice were euthanized, and lung, spleen, liver, and kidney tissues were removed for histological examination. Phenotypic change analysis and H&E (Hematoxylin & Eosin) staining were performed using standard protocols. The results are shown in FIGS. 8 and 9 .

Figure 8c is an image taken through FOBI imaging equipment (CELLGENTEK, Cheongju, Korea) after intravenous administration of a NIR fluorescently labeled bacterial detection probe (Perkinelmer, 133398) after CLP surgery and disease management through disinfection masking, intraperitoneal infection As a result of the degree of analysis, more than half reduction was observed in the ad-SWG and ad-SWG + Vanco groups compared to the group treated only with surgical tape.

Figure 8d shows the fluorescence images of the lung, spleen, liver and kidney, and in particular, when an antibiotic (vancomycin) was administered together, it was observed that the degree of infection was significantly lowered. In addition, as shown in FIG. 8E , it was observed that the damage to the skin tissue was significantly reduced without tissue necrosis due to bacterial infection.

9A, it was confirmed that vascular leakage and structural damage to organs were significantly reduced through histological examination of lung, kidney, and liver tissues.

Through the above experiment, it was confirmed that, when the water-based adhesive composition according to the present invention is used, effective disease management is possible and the effect of remarkably inhibiting bacterial infection is exhibited. Through this, the water-based adhesive composition of the present invention is expected to have high utility value as a clinical platform that can control the risk before and after surgery of surgical patients requiring repetitive disease management.

2-2. Neutrophil count analysis

In a mouse model of peritonitis, a 2 cm incision was made in the middle of the abdomen to expose the cecum and the adjacent intestine, and the cecum was tightly ligated using a 3.0 silk suture at a site 5 mm away from the tip of the cecum and punctured with a 22-gauge needle. Thereafter, excreta was pushed out of the perforation site by gently squeezing, and the cecum was returned to the abdominal cavity. The laparotomy site was sutured using 4.0 silk sutures. In the Sham operation, the animal's cecum was surgically exposed, but without ligation or perforation, it was repositioned into the abdominal cavity.

After the mice were euthanized, the abdominal cavity was washed with 5 ml of physiological saline, and the number of infiltrated neutrophils in the abdominal cavity was investigated. Neutrophils were counted using an automatic hematology analyzer (Mindray, bc-5000 Vet), and as shown in FIG. decreased results.

2-3. Cytokine trend analysis

AST, ALT, creatinine, BUN, LDH and CRP levels were analyzed using a biochemical kit (MyBioSource) and an ELISA plate reader (Tecan, Austria GmbH, Austria) to investigate the plasma cytokine levels of septic mice. Student's t-test was performed to evaluate statistical significance at P-values <0.05(*), <0.01(**), <0.001(***).

As shown in FIG. 11 , the level of inflammatory cytokines including IL-1β, IL-4, IL-6 and IFN-γ decreased by approximately half after PD using the SWG platform (ad-SWG) according to the present invention. it can be checked that This is expected to significantly improve bacterial infections and severe inflammatory responses that occur during recurrent PD.

Although the preferred embodiment of the present invention has been described above, the present invention is not limited thereto, and various modifications can be made within the scope of the claims, the description of the invention, and the accompanying drawings, and this is also It goes without saying that it falls within the scope of the invention.

Claims (15)

A water-based adhesive composition comprising a silk fibroin protein modified with a kosmotropic salt. The method of claim 1,
The cosmotropic salt is an alkali metal ion or alkaline earth metal ion and sulfate (SO ₄ ^2- ), phosphate (HPO ₄ ^2- ), acetate (CH ₃ COO ^- ), hydroxide (OH ^- ), chloride (Cl ^- ) , bromide (Br ^- ) and formate (HCOO ^- ) will consist of one or a combination of two or more selected from the group consisting of, the water-based adhesive composition. The method of claim 1,
The cosmotropic salt is one or more selected from calcium chloride (CaCl ₂ ), lithium bromide (LiBr), potassium chloride (KCl), sodium chloride (NaCl) and sodium acetate (CH ₃ COONa), aqueous adhesive composition. The method of claim 1,
The cosmotropic salt is included in an amount of 1 to 20 parts by weight based on 100 parts by weight of silk fibroin protein, an aqueous adhesive composition. The method of claim 1,
A composition for water-based adhesion, wherein a hydrophilic amino acid is exposed on the surface of the atypical structure of the silk fibroin protein. 6. The method of claim 5,
The hydrophilic amino acid is at least one selected from the group consisting of serine, threonine, cysteine, aspartic acid, glutamine, and tyrosine, an aqueous adhesive composition. 6. The method of claim 5,
A hydroxy group in the hydrophilic amino acid and the amino group of the adherend form a hydrogen bond. 7. The method of claim 6,
The content of serine exposed to the surface of the atypical structure is 5 to 20% as measured by solid ¹³ C-NMR, the composition for water-based adhesion. The method of claim 1,
A water-based adhesive composition having a shear strength of 5 to 50 kPa according to ASTM F2255-05 standard at 36° C. of the water-based adhesive composition. obtaining a first solution by adding a cosmotropic salt to an acidic solvent;
obtaining a second solution by adding silk fibroin protein to the first solution and dissolving it by heating to 50 to 70° C. for 30 minutes to 3 hours; and
Drying the second solution at room temperature; A method for producing a water-based adhesive composition comprising a. 11. The method of claim 10,
Heating the dried second solution to 50 to 70 ℃ stabilizing; further comprising a method for producing a water-based adhesive composition. 11. The method of claim 10,
The cosmotropic salt is included in an amount of 1 to 20 parts by weight based on 100 parts by weight of silk fibroin protein, a method for producing a water-based adhesive composition. 11. The method of claim 10,
The second solution contains silk fibroin, wherein the content of serine exposed on the surface of the atypical structure is 5 to 20% as measured by solid ¹³ C-NMR. 11. The method of claim 10,
The acidic solvent is a C1-C7 organic acid, a method for producing a water-based adhesive composition. A medical water-based adhesive comprising the composition for water-based adhesive according to any one of claims 1 to 9.

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