KR102366564B1 - Chitosan nanofiber-based bioadhesive composition and preparation method thereof - Google Patents

Abstract

The present invention relates to a bioadhesive composition comprising chitin or chitosan nanofibers modified with anionic functional groups, having low viscosity sprayable liquid properties, and excellent adhesion even when exposed to UV, heating and oxygen It can be provided as a hydrogel having hemostatic properties or an adhesive for use in living tissues.

Description

Chitosan nanofiber-based bioadhesive composition and method thereof {Chitosan nanofiber-based bioadhesive composition and preparation method thereof}

The present invention relates to a bioadhesive composition based on chitin or chitosan nanofibers, and more particularly, to a composition having the properties of a biocompatible adhesive by chemically modifying the chitin or chitosan.

A bio-adhesive refers to a material having adhesion properties to various biological samples such as cell membranes, cell walls, lipids, proteins, DNA, growth factors, cells, and tissues of living things. It is used in various biomedical fields such as tissue adhesive, hemostatic agent, tissue engineering scaffold, drug delivery carrier, tissue filling agent, wound treatment, or intestinal adhesion prevention.

The bio-adhesive can be used not only for biological samples but also for adhesion of hydrogels using bio-derived polymers. As the use of skin-attachable devices using hydrogels increases, an adhesive for bonding hydrogels containing a large amount of moisture is required.

Since bioadhesives are in direct contact with the skin, biocompatibility is required. In addition, since it is usually used in vivo, it should not have toxicity and risk, and it should be a biodegradable material. In addition, it is important to select a material that can be sterilized without disturbing the self-healing properties of the living body while having the ease so that the adhesion can be terminated instantaneously under mild conditions and strongly bonding the living tissue.

Hydrogels or bioadhesives exhibit strong adhesion even in moisture and must maintain their function in the living body for a long time. Materials for bioadhesives that are currently commercialized include cyanoacrylate, fibrin, gelatin, and polyurethane.

However, the bioadhesive using a synthetic polymer has a problem of showing very weak adhesion in an environment with a lot of moisture in the body. In addition, in the case of a cyanoacrylate-based adhesive, it is pointed out that it is a big limitation to cause an immune reaction side effect in the human body. In the case of the fibrin-based adhesive currently used in actual patients, the side effect is small, but its use is limited because its adhesive ability is at a very low level. In gelatin adhesives, formalin or glutaraldehyde, which are used as crosslinking agents, also cause a crosslinking reaction with proteins in the living body, causing tissue toxicity.

A characteristic of traditional bio-adhesives, including water-based adhesives, is that they are sticky and highly viscous. It must be accompanied by a chemical reaction that can cause crosslinking, and its stickiness and viscosity make it difficult to spray or apply. And the crosslinking chemical reaction must occur during adhesion, but can also occur during storage and transportation. Existing bio-adhesives work by chemical reaction, so they lose their function when heated or irradiated with UV, and in many cases their function is weakened only by exposure to the air. In this case, the adhesive strength gradually decreases, so the traditional bio-adhesive must be stored in an air-tight container or stored at a low temperature.

Therefore, in order to overcome these problems, it is not sticky, there is no chemical reaction, so it is easy to store, and it has strong adhesion and crosslinking ability on wet biological tissues and hydrogel surfaces. I need this.

A research team in France has developed an underwater adhesive using a solution of silica nanoparticles. However, even if the adhesive strength is excellent, silica particles are not biodegradable in vivo, so they are not suitable for use as a bioadhesive.

Chitin and chitosan are natural polymers with N-acetylglucosamine and glucosamine as repeating units, respectively. In general, it is difficult to accurately distinguish between chitin and chitosan because N-acetylglucosamine and glucosamine exist in a mixed state in nature. In general, the case where N-acetylglucosamine is higher than that of glucosamine is called chitin, and the opposite is called chitosan. The deacetylation process can convert chitin to chitosan. Chitin and chitosan are present in crustacean shells and are the second most abundant natural polymers on Earth after cellulose. They are biodegradable in the human body and have a hemostatic function, so they are spotlighted as medical polymer materials.

Severine et al., Nature, Dec 11, 2013, Vol.505, p.382-385,'Nanoparticle solutions as adhesives for gels and biological tissues'

In order to solve the above problem, the present inventors prepared a low-viscosity aqueous solution in which chemically modified chitin or chitosan nanofibers are dispersed using chitin and chitosan, which are natural polymers harmless to the human body and biodegradable, and thus prepared aqueous solution The present invention was completed by confirming that this adhesive for living body exhibits an excellent effect.

An object of the present invention is to provide a composition comprising functionalized chitin or chitosan nanofibers, which can be used as an adhesive for living tissues and hydrogels.

Another object of the present invention is to provide a method for preparing a biodegradable, low-viscosity and chemically stable bio-adhesive.

In order to achieve the above object, the present invention provides a bioadhesive composition comprising chitin or chitosan nanofibers modified with an anionic functional group, and a solvent.

In the bioadhesive composition according to an embodiment of the present invention, the anionic functional group may be any one or two or more selected from the group consisting of carboxylic acid, phosphoric acid, sulfuric acid and salts thereof.

In one embodiment of the present invention, the diameter of the nanofiber may be 2 to 500 nm.

In one embodiment of the present invention, the anionic functional group may be included in 0.1 to 2 mmol/g based on the dry weight of chitin or chitosan.

In the bioadhesive composition according to an embodiment of the present invention, heat resistance may satisfy the following [Formula 1].

[Equation 1]

In the bioadhesive composition according to an embodiment of the present invention, light resistance may satisfy the following [Formula 2].

[Equation 2]

In the bioadhesive composition according to an embodiment of the present invention, water resistance may satisfy the following [Formula 3].

[Equation 3]

In one embodiment of the present invention, the nanofibers may be present in a dispersed form in a solvent.

In one embodiment of the present invention, the modified chitin or chitosan nanofibers may be included in an amount of 0.05 to 30% by weight based on the total composition.

In one embodiment of the present invention, the solvent is any one or two or more selected from water, C1 to C4 alcohol, and C1 to C10 organic acid, and the pH of the composition may be less than 7.

In the bioadhesive composition according to an embodiment of the present invention, the loss tangent is measured for rotational rheological properties under the conditions of 10 rad/s frequency and 0.5% strain after heating at 90 ° C. for 3 hours or more, and 5 at 25 ° C. may be more than

In addition, the present invention comprises the steps of dispersing chitin or chitosan in a solvent to prepare a dispersion; oxidizing the hydroxyl group of chitin or chitosan using an N-oxyl-based compound as a catalyst; generating nanofibers by ultrasonically treating the oxidized chitin or chitosan; and dispersing the nanofibers in an acid solvent to anionize them; provides a method for preparing a bioadhesive composition comprising a.

In one embodiment of the present invention, the N-oxyl compound may be a compound represented by any one of Chemical Formulas 1 to 3 below.

[Formula 1]

[Formula 2]

[Formula 3]

In the above formula, R ¹¹ to R ¹⁵ , R ²¹ to R ²⁵ , and R ³¹ to R ³⁵ may each independently be hydrogen or a C1-C4 alkyl group.

The bioadhesive composition according to the present invention bonds wet hydrogels or biological tissues by physical adsorption without chemical crosslinking. Because it provides adhesion by physical adsorption without chemical reaction, it is very stable to air and heat and is easy to store.

In addition, the bioadhesive composition according to the present invention has a low viscosity, so it is possible to use it in a spray form, and it is safe because it is biodegradable and disappears after use while exhibiting hemostatic properties, so that it is possible to prevent a foreign body reaction.

The composition for bioadhesive according to the present invention provides excellent adhesion without adding a chemical crosslinking agent in a hydrogel or living tissue having a high water content.

1 shows a photograph of bonding a gelatin hydrogel using the bioadhesive composition according to Example 1 of the present invention.
Figure 2 shows a photograph of bonding the experimental pig skin using the bioadhesive composition according to Example 1 of the present invention.
Figure 3 shows a photograph immersed in water after bonding the gelatin hydrogel using the bioadhesive composition according to Example 1 of the present invention.
Figure 4 shows the SEM image of the surface oxidation-treated chitosan nanofibers prepared according to Example 1 of the present invention.
5 shows a catalyst and a carboxyl group introduction process for surface oxidation treatment of chitin nanofibers.
6 shows a representative conductivity graph during titration of surface-modified anionic functional groups of chitin or chitosan nanofibers.

Hereinafter, the bioadhesive composition according to the present invention will be described in detail.

Unless otherwise defined, terms used in this specification should be interpreted as content commonly understood by those of ordinary skill in the art. The drawings and embodiments of the present specification are for those of ordinary skill in the art to easily understand and practice the present invention, and content that may obscure the gist of the present invention may be omitted from the drawings and embodiments, and the present invention is not limited to the drawings and embodiments is not limited to

Also, unless otherwise defined, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the present invention is for the purpose of effectively describing particular embodiments only and is not intended to limit the present invention.

In the following, when it is said that any one component "includes" another component, it is not construed as being limited to only the component, unless otherwise stated, and it is understood that other components may be further included. should be

The present invention provides a bioadhesive composition comprising chitin or chitosan nanofibers modified with an anionic functional group, and a solvent.

The anionic functional group may be any one or two or more selected from the group consisting of carboxylic acid, phosphoric acid, sulfuric acid, and salts thereof. Specifically, for example, a carboxylate salt, lithium carboxylate, sodium carboxylate, potassium carboxylate, rubitium carboxylate, cesium carboxylate, francium carboxylate, zinc carboxylate or calcium carboxylateyl However, it is not particularly limited thereto. In the form of sulphate, it may be lithium sulphate, sodium sulphate, potassium sulphate, leubitium sulphate, cesium sulphate, franc sulphate, zinc sulphate or calcium sulphate; in the case of phosphate, lithium phosphate, sodium phosphate, potassium phosphate, leubitium phosphate, phosphate It may be cesium, francium phosphate, zinc phosphate, or calcium phosphate, but is not particularly limited thereto.

The anionic functional group may impart physical adhesion to a living tissue or hydrogel through an ionic bond with a cation on the hydrogel surface or a biological tissue in the form of an aqueous solution. The anionic functional group may preferably be a carboxylic acid or a salt thereof. A carboxyl group is introduced to the surface of chitin or chitosan nanofibers to form an ionic bond with a cation of a living tissue or hydrogel in the form of COO ^- ions in an aqueous solution, and by forming a hydrogen bond with a hydroxy group or an amine group included in the living tissue or hydrogel It can provide excellent physical adhesion.

In this case, when the anionic functional group is included in an amount of 0.1 to 2 mmol/g based on the dry weight of chitin or chitosan nanofibers, excellent adhesion can be exhibited without interfering with the repulsive force between the anions. Preferably, the content of 0.5 to 1.0 mmol/g has the effect of exhibiting excellent adhesive properties.

In the present invention, chitin or chitosan nanofibers may have a concentration gradient of anionic functional groups formed between the inside and the surface. This may mean that the surface of the chitin or chitosan nanofiber is modified and an anionic functional group is introduced into the surface of the nanofiber.

At this time, the diameter of the nanofiber may be 2 to 500 nm. Preferably, the diameter is 3 to 50 nm, more preferably 5 to 20 nm. The length may be 100 to 800 nm, preferably 200 to 500 nm. Their average aspect ratio (aspect ratio) is 10 to 100, preferably 20 to 50, while sufficiently securing a surface area, it exhibits a property of being easily dispersed, which is good for improving adhesion.

In the present invention, chitin or chitosan nanofibers may exist in a dispersed form in a solvent. In this case, the solvent may be any one or two or more selected from water, C1 to C4 alcohol, and C1 to C10 organic acid. Specifically, as an aqueous solution containing water, for example, it may further include any one or more from the group consisting of methanol, ethanol, ethylene glycol, acetone, acetic acid, succinic acid, and citric acid, but is not limited thereto.

In one embodiment of the present invention, chitin or chitosan nanofibers are included in an amount of 0.1 to 20% by weight, preferably 0.1 to 10% by weight, more preferably 1 to 10% by weight based on the weight of the aqueous solution containing water. and, most preferably, it may exhibit excellent adhesion to be included in an amount of 2 to 10% by weight.

If the pH of the dispersion in which chitin or chitosan nanofibers are dispersed in a solvent is less than 7, excellent adhesion may be exhibited, and more preferably, when the pH is 3 to 5, the adhesion is excellent. In this sense, the solvent may be a mixture of water and an organic acid of C1 to C10. When mixing water and organic acid, the amount of water is preferably 10 to 50% by weight based on the total composition, but the weight is not limited in consideration of the acidity of the organic acid.

In one embodiment of the present invention, the heat resistance of the bioadhesive composition may satisfy the following [Formula 1].

[Equation 1]

The present invention is a composition that exhibits adhesion using physical adsorption, and there is almost no chemical change even in high-temperature heat treatment, and there is no change in adhesive force. The heat resistance result according to [Equation 1] may be 0.85 or more, preferably 0.9, and more preferably 0.95 or more. Due to its high heat resistance, it does not deteriorate when used for bioadhesive purposes as well as during storage, so it can maintain excellent adhesion.

In one embodiment of the present invention, the light resistance of the bioadhesive composition is

2] may be satisfied.

[Equation 2]

In the bioadhesive composition according to the present invention, when UV light is irradiated according to the conditions of [Equation 2], there is hardly any change in adhesive strength. Light resistance according to the conditions of [Equation 2] may be 0.8 or more, preferably 0.85 or more, and more preferably 0.9 or more. The present invention is a composition to which a chemical crosslinking agent is not added. Even when exposed to UV, a chemical reaction does not easily occur, so there is little change in adhesive strength, and excellent adhesive strength can be maintained.

In one embodiment of the present invention, the water resistance of the bioadhesive composition may satisfy the following [Formula 3].

[Equation 3]

Since biological tissue contains a large amount of moisture, if the adhesive strength after immersion is significantly reduced compared to the adhesive strength before immersion, it may not be suitable for use as a biological adhesive. In this regard, the water resistance measured according to Equation 3 is preferably 0.5 or more, preferably 0.65 or more, and more preferably 0.7 or more.

In one embodiment of the present invention, when the rotational rheological properties of the bioadhesive composition are measured, the loss tangent may be 5 or more. After heating at 90 °C for 3 hours or more, the rheological properties were measured under the conditions of 10 rad/s frequency and 0.5% strain, which means the loss tangent at 25 °C. In this case, the loss tangent may be calculated as storage modulus (G′)/loss modulus (G″).

Theoretically, when the loss tangent value is less than 1, it hardens like a solid, and when it is greater than 1, it has liquid-like properties. The loss tangent value of the bioadhesive composition according to the present invention is 5 or more, and has liquid properties, and the loss tangent value hardly changes even after heating. Due to this, it is not gelled even after heating, so that it can maintain a liquid state, and can be used in a spray form as a low viscosity.

The present invention comprises the steps of dispersing chitin or chitosan in a solvent to prepare a dispersion; oxidizing the hydroxyl group of chitin or chitosan using an N-oxyl-based compound as a catalyst; generating nanofibers by ultrasonically treating the oxidized chitin or chitosan; and dispersing the nanofibers in an acid solvent to anionize them; provides a method for producing a bioadhesive composition comprising a.

Chitin or chitosan is a natural polymer and has an agglomerated nanofiber form, so chitin or chitosan nanofibers can be manufactured by a top-down method so that the agglomerated surface has a uniformly dispersed nanofiber form through ultrasonic treatment there is. However, the present invention is not necessarily limited thereto, and a bottom-up method of manufacturing known in the art through electrospinning and ultrasonication is also available.

In one embodiment of the present invention, the N-oxyl compound may be a compound represented by any one of Chemical Formulas 1 to 3 below.

[Formula 1]

[Formula 2]

[Formula 3]

In the above formula, R ¹¹ to R ¹⁵ , R ²¹ to R ²⁵ , and R ³¹ to R ³⁵ may each independently be hydrogen or a C1-C4 alkyl group. Preferably, R ¹¹ to R ¹⁴ , R ²¹ to R ²⁴ and R ³¹ to R ³⁴ may each independently be any one selected from the group consisting of a methyl group, an ethyl group, a propyl group and a butyl group, and R ¹⁵ , R ²⁵ and R ³⁵ may be hydrogen.

More preferably, the N-oxyl compound is a 2,2,6,6-tetramethyl-1-piperidin-oxy radical (hereinafter, TEMPO), (2,2,6,6-tetramethylpiperidin-1-yl) oxidanyl or (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl) and 4-hydroxy-2,2,6,6-tetramethyl-1-piperidine-oxy radicals (hereinafter referred to as 4-hydroxyl hydroxy TEMPO) may be a compound that generates any one or two or more selected from the group consisting of.

In addition, when using a 4-hydroxy TEMPO derivative in which the hydroxyl group of 4-hydroxy TEMPO is substituted with an alkoxy, acetamido group, or sulfonic acid ester group as a catalyst for surface modification, uniformly surface-modified chitin or chitosan nanofibers It is preferable to obtain

Specifically, for example, the 4-hydroxy TEMPO derivative is any one or two selected from the group consisting of TEMPO, 4-hydroxy-TEMPO, 4-amino-TEMPO, 4-carboxy-TEMPO, and 4-acetamido-TEMPO. may be more than

In one embodiment of the present invention, the N-oxyl compound may be used in an amount of 0.1 to 10% by weight based on the dry weight of the chitin or chitosan nanofibers. Preferably it is contained in an amount of 0.2 to 5% by weight, more preferably 0.8 to 1.2% by weight, so that catalytic activity can be excellent.

Hereinafter, the bioadhesive 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 bioadhesive composition including chitin nanofiber (T-chW) introduced with carboxylic acid by surface oxidation treatment

1-1. Manufacture of chitin nanofibers introduced with carboxylic acid by surface oxidation treatment

0.08 g of 2,2,6,6-tetramethyl-1-piperidine-oxy radical (TEMPO) and 0.5 g of NaBr were dissolved in 500 mL of deionized water (DIW). 5 g of α-chitin powder was dispersed in 500 ml of TEMPO aqueous solution, and 37 g of NaClO solution was slowly added to the chitin-containing dispersion. The pH of the chitin-containing dispersion was slowly decreased, and a 0.5 M aqueous NaOH solution was slowly added to maintain the pH at 10. When the pH of the dispersion did not decrease, 10 ml of ethanol (EtOH) was added to terminate the reaction. The dispersion was centrifuged at 10,000 rpm for 10 minutes, the supernatant was removed, and the precipitate was redispersed in 500 ml of DIW. This dilution process was repeated 3 times. The dispersion was then dialyzed with DIW until the pH reached 7. The concentration of the dispersion was adjusted to 1% by weight by adding DIW. The purified dispersion was sonicated with a probe ultrasonic processor for 10 minutes (750W, amplitude 50%, 10 sec pulse and 5 sec pulse off) to prepare carboxylic acid-introduced chitin nanofibers.

1-2. Preparation of bioadhesive composition including chitin nanofibers introduced with carboxylic acid

The aqueous dispersion of the prepared chitin nanofiber itself or its freeze-dried form was maintained at 4 °C. This was dispersed so as to be 1,2,3 wt% in a dilute aqueous acetic acid solution of pH 4 to prepare a bioadhesive composition.

Comparative Example 1. Preparation of a bioadhesive composition comprising chitosan nanofibers (chW)

5 g of α-chitin powder was immersed in 150 mL of 3 M HCl aqueous solution, and the suspension was heated at 120° C. for 3 hours in a nitrogen atmosphere. The suspension was centrifuged at 10,000 rpm for 10 min. And the supernatant was removed and the precipitate was redispersed in 150 mL of distilled water. This acid dilution process was repeated 3 times. The suspension was then dialyzed with distilled water until a pH of 7 was reached. The purified suspension was sonicated with a 750W probe ultrasonic processor for 10 minutes (amplitude 50%, 10 sec pulse and 5 sec pulse off) to prepare chitosan nanofibers.

A bioadhesive composition was prepared by maintaining the aqueous chitosan nanofiber suspension itself or its freeze-dried form at 4 °C, and dispersing it in a pH 4 dilute aqueous acetic acid solution to 1, 2, 3 wt%.

Comparative Example 2. Preparation of bioadhesive composition containing silica nanoparticles

A bioadhesive composition was prepared by dispersing silica nanoparticles (CAS Number 7631-86-9, Sigma-Aldrich) in a pH 4 dilute aqueous acetic acid solution to 1, 2, 3 wt%. Comparative Example 3. Preparation of a bioadhesive composition comprising cellulose nanocrystals (CNC)

Cellulose nanocrystals (CNC) (The University of Maine) were dispersed in a pH 4 dilute aqueous acetic acid solution to 1,2,3 wt% to prepare a bioadhesive composition. The diameter of the CNC is 20 nm and the length is 200 nm.

Comparative Example 4. Preparation of fibrin glue

Fibrin glue (Green Cross) was dispersed in a pH 4 dilute aqueous acetic acid solution to 1,2,3 wt% to prepare a bioadhesive composition.

Comparative Example 5. Chitosan Dopamine Adhesive Preparation

The material made by binding dopamine to chitosan using EDC/NHS reagent (Thermoscientific) was freeze-dried and then dissolved again in water to obtain an aqueous solution. In the case of chitosan-dopamine adhesive, in general, by adding a curing agent such as NaIO ₄ (sodium periodate), a chemical reaction is induced to show adhesive ability. .

Experimental Example 1. Adhesion evaluation

1-1. Gelatin hydrogel synthesis

12 g of dry gelatin powder (Sigma Aldrich) extracted from pig skin was added to 20 ml of distilled water in a glass vial. Then, an additional 20 ml of water was added to immerse the gelatin powder, and the vial was sealed and placed in an oven at 50° C. for complete dissolution of the gelatin and maintained for 24 hours. The resulting solution was degassed at 50% amplitude in a sonication bath for 2 hours to remove trapped air bubbles. The gelatin solution was poured into a 150 mm diameter Petri dish and covered with a lid to prevent water evaporation. To shorten the gelation process time, it was placed on a hot plate at 50° C. for 30 minutes. Thereafter, it was cooled to 25 °C to proceed with gelation. The resulting hydrogel was made uniformly with a thickness of 2 mm. Thereafter, it was tightly sealed with paraffin film and kept at 4° C. in a refrigerator before use. For the adhesion test, the gelatin hydrogel was cut into rectangular pieces with dimensions of 50 mm Х 10 mm Х 2 mm.

1-2. Underwater adhesion evaluation test

An aqueous adhesive solution according to Example 1 and Comparative Examples 1 to 5 was prepared. For the adhesive substrate, 23 wt% of gelatin hydrogel and experimental pig skin were cut to be 50 mm in length (l), 10 mm in width (w), and 2 mm in thickness (h), and the two cut adhesive substrates were overlapped. 30 μl of adhesive was applied in between. After 10 minutes, the two bonded specimens were pulled using a tensile strength tester (ASTM F2255-05) so that the overlapping area was 10 mm Х 10 mm = 100 mm ² , and the two adhesive substrates at a speed of 10 mm/min. pulled The bonded hydrogel and experimental pig skin were soaked in distilled water for 3 days and stored, and after 3 days had elapsed, they were taken out and the two bonded specimens were pulled using a tensile strength tester to measure the adhesive force again.

The concentrations of the aqueous adhesive solution used for the gelatin hydrogel and experimental pig skin are as shown in Tables 1 and 2 below.

1-3. Adhesion evaluation after UV irradiation

After irradiating for 1 hour or more with UV of 385 nm wavelength and 1,000 (mW/cm 2 ) intensity using a UV curing machine, adhesive strength was evaluated in the same manner as in 1-2, and is shown in Table 3 below.

In the case of Comparative Example 1, the degree of loss of adhesive ability due to UV curing is not large, but compared to Example 1, the adhesive strength is low, and in Comparative Examples 4 and 5, which exhibit adhesive ability by chemical reaction, adhesion after UV curing It was confirmed that the performance was completely lost or reduced to less than half.

1-4. Adhesion evaluation after 1 week storage in the air

After storage for one week in the air, adhesion was evaluated in the same manner as 1-2, and is shown in Table 4 below.

In the case of Example 1 according to the present invention, even after exposure to the air for one week, the adhesive force did not decrease at all, in the case of Comparative Example 1, the adhesive force was slightly decreased, and in Comparative Examples 4 and 5, the adhesive force was sharply decreased. In the case of Comparative Examples 4 and 5, since they are crosslinked by a chemical reaction to exhibit adhesive ability, they are expected to deteriorate when exposed to the atmosphere for a long period of time, thereby significantly reducing adhesiveness.

1-5. Adhesion evaluation after heating at 90 °C for 3 hours

After heating at 90 °C in the air for 3 hours, the adhesive strength was evaluated in the same manner as in 1-2 and shown in Table 5 below.

In Example 1 according to the present invention, the change in adhesive strength was not large even after heating, but Comparative Example 1 showed a greater decrease in adhesive strength than this, and in Comparative Examples 4 and 5, which exhibit adhesive ability by chemical reaction, the adhesive strength was It was confirmed that it was completely lost.

1-6. Adhesion evaluation according to pH

In order to confirm the change in adhesive strength according to pH, the adhesive strength at pH 4.0, pH 6.0, pH 7.0 and pH 8.0 was evaluated as shown in Table 6 below.

It was confirmed that excellent adhesion was exhibited in acidic conditions of pH 7.0 or less, through that both Example 1 and Comparative Example 1 maintained similar adhesion at pH 7.0 or less.

Experimental Example 2. Hemostasis evaluation

Thromboelastography (TEG) Hemostatic performance was evaluated by measuring thromboelastography using a hemostatic analyzer system 5000 (Haemonetics, Braintree, MA, USA). Cannie blood was supplied from the Korean Animal Blood Bank by venous blood collection in CPDA solution, and the compositions according to Examples 1 and Comparative Examples 1 to 5 were dissolved in PBS buffer at a concentration of 5 μm to evaluate the hemostatic ability. . After putting the TEG cup (Haemonetics) into the TEG device, 320 μl of dog blood, 20 μl of 0.2 M CaCl ₂ solution (Haemonetics), and 20 μl of the adhesive composition according to Examples 1 and Comparative Examples 1 to 5 were added to the TEG exclusive cup. put All parameters of interest were measured by rotating the torsion wire and pin on the sample until the endpoint was reached. The variables measured are the reaction time (R), the clotting time (K), the kinetic angle (α) and the maximum amplitude (MA) of the coagulation phenomenon, the shorter R, the shorter the K, the larger the angle α, the larger the MA. It can be analyzed that the hemostatic ability is excellent. Table 7 below shows the value of Comparative Example 3 (cellulose nanocrystal) as a relative numerical value.

Example 1 showed that the reaction time and solidification time were 15% and 25% shorter than that of CNC under the same conditions, and the dynamic angle and maximum amplitude were 15% and 22% larger.

Experimental Example 3. Rheological properties evaluation

Rheological properties were evaluated with a rotary rheometer (MCR 501, Physica). A 1 ml aliquot of the dispersion was placed between two 25 mm parallel plates and the rheological properties were measured with a gap of 1 mm. Loss tangent values were measured at 10 rad/s preconception, 0.5% strain, and 25 °C. In addition, in order to evaluate safety by heating, the loss tangent was measured after heating at 90° C. for 3 hours.

Theoretically, the loss tangent value may mean that when it is less than 1, it hardens like a solid, and when it is greater than 1, it has liquid-like properties.

Since the bioadhesive composition including the surface oxidation-treated chitosan nanofibers according to Example 1 exerts adhesive force by physical adsorption, it exhibited a loss tangent value greater than 1 regardless of whether heated or not. This is a low-viscosity liquid that does not gel even when heated, and it can be confirmed that it can be used in a spray form.

Experimental Example 4. Measurement of anionic functional group concentration

To titrate anionic functional groups on the surface of chitin or chitosan nanofibers, 0.05 g of a dry sample was dispersed in 50 ml of distilled water, and a few drops of 0.5 M HCl solution were added to the mixture to set the pH to 2.0. The anionic functional group used for titration in this Experimental Example is a carboxy group. Conductivity was measured while raising the anionic functional group to pH 12 while dropping 0.1 N NaOH, a basic solution, into an acidic suspension at a flow rate of 0.1 ml/min using a pH-stat titration system equipment. The mol amount of the anionic functional group could be obtained by monitoring the conductivity change through the equipment. As shown in the graph shown in FIG. 6 , the conductivity decreased at the initial stage of adding aqueous NaOH solution, and then stopped for a certain period of time. After that, the conductivity increased by reacting again. The number of moles of NaOH consumed during the stationary phase corresponds to the number of moles of modified anionic functional groups in the chitin or chitosan nanofibers contained in the solution.

The concentration of anionic functional groups increased in proportion to the oxidation time required for surface modification. Using a TEMPO catalyst, the oxidation time was further increased or decreased to prepare a chitin or chitosan nanofiber adhesive having a carboxyl group at the concentration shown in Table 9 above, Comparative Examples 6, 7 and Examples 1 to 5 at such concentrations Adhesion evaluation results are shown. Adhesion evaluation was performed using the same method as described in Experimental Example 1.

In the case of 0.1 to 2 mmol based on 1 g of dry weight of chitin or chitosan nanofibers, excellent adhesion was exhibited compared to that of non-surface-modified chitin or chitosan nanofibers, and when the anionic functional group concentration was 0.5 to 1 mmol, much It showed better adhesion properties. The higher the degree of surface modification of the anionic functional group, the greater the adhesive strength, but when the anionic functional group exceeded 2 mmol/g, the adhesive strength decreased sharply. This appears to be the action by the repulsive force between the anions.

Claims (13)

As a bioadhesive composition comprising chitin or chitosan nanofibers modified with anionic functional groups, and a solvent,
The nanofiber has a diameter of 2 to 500 nm,
The anionic functional group is introduced to the surface of the nanofiber, a concentration gradient of the anionic functional group is formed between the inside and the surface of the nanofiber,
The anionic functional group forms an ionic bond with a cation contained in a living tissue, and forms a hydrogen bond with a hydroxy group or an amine group contained in the living tissue, characterized in that it provides physical adhesion, a bioadhesive composition. The method of claim 1,
The anionic functional group is any one or two or more selected from the group consisting of carboxylic acid, phosphoric acid, sulfuric acid and salts thereof. delete The method of claim 1,
The anionic functional group will be contained in an amount of 0.5 to 1 mmol/g based on the dry weight of chitin or chitosan, the bioadhesive composition. The method of claim 1,
The heat resistance of the bioadhesive composition satisfies the following [Formula 1].
[Equation 1]

The method of claim 1,
The light resistance of the bioadhesive composition satisfies the following [Formula 2].
[Equation 2]

The method of claim 1,
The water resistance of the bioadhesive composition satisfies the following [Formula 3].
[Equation 3]

The method of claim 1,
The nanofiber is a bioadhesive composition that is present in a dispersed form in a solvent. The method of claim 1,
The modified chitin or chitosan nanofibers are contained in an amount of 0.05 to 30% by weight based on the total composition for bioadhesive composition. The method of claim 1,
The solvent is any one or two or more selected from water, C1 to C4 alcohol and C1 to C10 organic acid, and the pH of the composition is less than 7 for a bioadhesive composition. The method of claim 1,
The loss tangent of the bioadhesive composition is 5 or more at 25 °C as measured for rotational rheological properties under conditions of 10 rad/s frequency and 0.5% strain after heating at 90 ° C. for 3 hours or more. Dispersing chitin or chitosan in a solvent to prepare a dispersion;
oxidizing the hydroxyl group of chitin or chitosan using an N-oxyl-based compound as a catalyst;
generating nanofibers by ultrasonically treating the oxidized chitin or chitosan; and
Dispersing the nanofibers in an acid solvent to anionize them;
The nanofiber has a diameter of 2 to 500 nm,
An anion concentration gradient is formed between the inside and the surface of the nanofiber,
The anion forms an ionic bond with a cation contained in a living tissue, and forms a hydrogen bond with a hydroxy group or an amine group contained in the living tissue, thereby providing physical adhesion. 13. The method of claim 12,
The N-oxyl compound is a compound represented by any one of Chemical Formulas 1 to 3 below.
[Formula 1]

[Formula 2]

[Formula 3]

(In the formula, R ¹¹ to R ¹⁵ , R ²¹ to R ²⁵ , and R ³¹ to R ³⁵ are each independently hydrogen or a C1-C4 alkyl group).

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