KR102115167B1 - Mechanically improved collagen/chitin nanofibers membrane and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a method for manufacturing a nanofiber membrane manufactured by an electrospinning process from an electrospinning solution containing collagen and chitin, and a nanofiber membrane manufactured therefrom. The present invention provides the nanofiber membrane obtained by an electrospinning process from an electrospinning solution in which collagen and chitin are dissolved together. The nanofiber membrane of the present invention not only increases mechanical properties such as toughness, tensile strength and Young′s modulus by new combination of collagen and chitin, but also has excellent biocompatibility like collagen.

Description

A collagen / chitin nanofiber membrane with increased strength and a method for manufacturing the same {MECHANICALLY IMPROVED COLLAGEN / CHITIN NANOFIBERS MEMBRANE AND MANUFACTURING METHOD THEREOF}

The present invention relates to a method for manufacturing a nanofiber membrane comprising collagen and chitin and a nanofiber membrane produced therefrom.

Collagen is a type of light protein that is a major component of tissues that protect and support human and animal organisms. It is distributed in animal bones, skin, organs, cartilage, hair, etc. It is composed of collagen. Collagen is mainly composed of glycine, proline, and hydroxyproline, and has a triple spiral structure in which three polypeptide chains composed of them are twisted.

In particular, collagen is used as a biodegradable shielding membrane with the ability to block certain types of cell inflow or limit it to a specific location in periodontal tissue regeneration. Unlike other non-absorbent masks, the shields made of collagen have excellent absorption and decomposition ability in vivo, and occupy most of the membrane market in recent years thanks to the simple procedure that does not require secondary surgery. However, collagen has poor mechanical properties, so it lacks the role of a support and has excellent absorption ability, so it has a disadvantage in its function as a shielding film due to its faster decomposition rate than expected.

Glucosamine saccharides, represented by chitin and chitosan, are the components that make up the exoskeleton of arthropods such as shrimp and crabs, and are richer after cellulose among saccharides present on the earth. Chitin is a polysaccharide substance composed of countless N-acetyl-D-glucosamine units, and chitosan is a polysaccharide substance composed of countless deacetylation-produced units in chitin.

Chitin and chitosan are natural ingredients with excellent biocompatibility, and have high utility value in various fields such as burn, medicine, suture, artificial membrane, medicine delivery, as well as industrial fields such as cosmetics, plastics, and textiles. In addition, chitin and chitosan, unlike synthetic polymers, have the property of being slowly absorbed in vivo and have excellent mechanical properties. Therefore, when used in combination with various functional materials having weaker mechanical properties, it can exhibit the effect of compensating each other's shortcomings.

The types of chitin are classified into alpha (α) -chitin, beta (β) -chitin, and gamma (γ) -chitin according to the intermolecular bond structure. α- Chitin is mainly distributed in the shells of crustaceans and accounts for about 2/3 of all chitins present on the earth. α-chitin has an extremely hard physical property because the intermolecular chain structures are arranged in an inverse equilibrium structure. β-chitin is mainly present in marine mollusks and has a structure in which the intermolecular chain structure is balanced, so it is looser than α-chitin, so it is easy to form derivatives and has good reactivity, so it is highly available. The gamma-structure is a structure in which the arrangement of alpha-chitin and beta-chitin is mixed, and mainly constitutes the carapace of insects.

Unlike bulk materials, nano-structured materials have unique structures that are physically and chemically different, and recently, academic research has been actively conducted. Electrospinning technology is widely used as an efficient and economical method for manufacturing such nanomaterials. Since various complexes such as polymer nanofibers mixed with functional materials can be easily manufactured, various studies are being conducted in applications such as chemistry, medicine, and electronic materials.

Chitin is difficult to electrospin because of the structure with strong attraction between each other, so the techniques have lowered the molecular weight of chitin or used an expensive ionic liquid to facilitate dissolution. Therefore, electrospinning that utilizes chitin in an economical and convenient way can have a high priority competitiveness in the nanofiber market.

In the existing technologies and inventions, ionic liquids (ILs) are used as a solvent for dissolving high molecular weight chitin, or methods of dissolving in polar solvents after lowering the molecular weight by applying energy such as radiation are used. . However, since the ionic liquid is very expensive and has a limitation in expecting good mechanical properties of chitin when lowering the molecular weight, it is a feature of the present invention to electrospin high molecular weight chitin using an appropriate polar solvent.

 Jamil A. Matthews et al, Biomacromolecules, Vol. 3, 232-238, 2002

Accordingly, the inventors of the present invention completed the present invention by confirming that excellent mechanical properties appear due to a new interaction between collagen and chitin when collagen and chitin are added while developing a biodegradable shield based on collagen. .

The present invention aims to provide a method for producing a nanofiber membrane based on collagen and chitin and a nanofiber membrane based on collagen and chitin.

As one aspect of the present invention for achieving the above object

(1) preparing a electrospinning solution by adding collagen and chitin to a mixed solvent of HFIP (Hexafluoro-2-propanol) and TFA (Trifluoroacetic acid);

(2) preparing a nanofiber membrane from the electrospinning solution through an electrospinning process; And

(3) removing residual solvent from the prepared nanofiber membrane;

It provides a method for producing a nanofiber membrane comprising a.

The electrospinning process used in the present invention is a process in which a polymer having a diameter of hundreds to hundreds of nanometers is obtained by instantaneously spinning a solution or a solid polymer in the form of a fiber using high-voltage electrostatic attraction. Since the nanofiber of the present invention has a very small diameter compared to conventional spinning methods, it has an advantage of maximizing the effect occurring on the surface because it has a larger surface area than the existing fiber.

The collagen and chitin contained in the nanofibers prepared in the present invention are made of an electrospinning solution to form hydrogen bonds and hydrophobic interactions during electrospinning to form a new structure.

Collagen can be extracted from animal or human cartilage or organs and includes extraction using chemicals such as acids, alkalis or salts, or extraction using physical methods such as ultrasound.

In one embodiment of the present invention, in the step of preparing the electrospinning solution of step (1), α-chitin or β-chitin is added to the mixed solvent to dissolve for 10 to 30 hours, and collagen is added to dissolve for 5 to 20 hours. According to a specific embodiment of the present invention, α-chitin or β-chitin is added to dissolve for about 24 hours, and collagen is added to dissolve for about 12 hours. However, the present invention is not limited thereto, and an appropriate order and dissolution time required for dissolution can be freely selected.

In one embodiment of the present invention, the mixed solvent of step (1) has an HFIP and TFA weight ratio of 7: 3 to 9: 1, and according to a specific embodiment of the present invention, is about 8.5: 1.5. If outside this ratio, α-chitin may not be soluble in the solvent. Specifically, in the case of α-chitin, since it is more difficult to dissolve in a solvent because it has a stronger hydrogen bond than each other, β-chitin can be dissolved in a solvent even if it exceeds the corresponding ratio.

In one embodiment of the present invention, the electrospinning solution of step (1) comprises collagen and chitin in a weight ratio of 10: 1 to 15: 1, according to a specific embodiment of the present invention, collagen and chitin are about 13: It is included in a weight ratio of 1. However, it is not limited thereto, and an appropriate weight ratio required to control the properties of the nanofiber membrane may be selected.

In one embodiment of the present invention, in the electrospinning process of step (2), a voltage of 30 to 35 kV is applied to the needle injecting the electrospinning solution. When the voltage is less than 30kV, the surface tension of the solution may be stronger than the applied voltage, resulting in a problem that radiation cannot be made, and when the voltage is more than 35kV, a solution may be spun in the form of a spray and fibers may not be formed.

In one embodiment of the present invention, in the electrospinning process of step (2), the feed rate of the electrospinning solution is 0.15 to 0.25 ml / h, and according to a specific embodiment of the present invention, the feed rate is about 0.25 ml / h. . As the supply speed of the prepared electrospinning solution increases, the productivity of the nanofiber membrane increases. If it exceeds 0.25ml / h, it is not suitable because the solution may be agglomerated before the fibers are formed, which may fall into droplets and damage the nanofiber membrane being manufactured.

In one embodiment of the present invention, the electrospinning process of step (2) may be performed for 60 to 240 minutes, preferably 60 to 120 minutes, and according to a specific embodiment of the present invention, the electrospinning process May be performed for about 90 minutes, but is not limited thereto, and an appropriate time may be selected for the capacity or concentration of the solvent or solution used in the electrospinning process.

In one embodiment of the present invention, the step of removing the residual solvent in step (3) may be used by heating the membrane to evaporate the solvent to dry, but is not limited thereto, and may be used to evaporate the solvent. Various methods can be used.

Another aspect of the present invention for achieving the above object is a nanofiber membrane manufactured by any one of the above manufacturing methods.

The nanofiber membrane has a toughness of 100 to 400 KJ / m ³ , and according to a specific embodiment of the present invention, is about 100 to 350 KJ / m ^3, but is not necessarily limited thereto.

Toughness of the present invention refers to the ability of a sample to flexibly change with stretching, and the ability of the sample to absorb energy until the sample is completely cut off, and has a unit of kJ / m ³ .

The tensile strength of the nanofiber membrane is 0.5 to 1.5 MPa, according to a specific embodiment of the present invention, about 0.5 to 1.1 MPa, but is not necessarily limited thereto.

Tensile strength (Pa) of the present invention is a value obtained by dividing the pulling force by a cross-section, and has a unit of N / m ² , and the elongation (Strain,%) is divided by the first length divided by the first length and multiplied by 100 to%. It indicates the increased ratio.

The Young's modulus of the nanofiber membrane is 10 to 30 MPa, and according to a specific embodiment of the present invention, about 15 to 25 MPa, but is not necessarily limited thereto.

The Young's modulus of the present invention is a modulus of elasticity representing the relationship between strain and pressure that appears in a sample. It is a value obtained by dividing the change value of the force (tensile strength) required for deformation by the change value of the strain, and the unit is tensile strength same.

Another aspect of the present invention for achieving the above object is a nanofiber membrane that is formed from an electrospinning solution comprising a mixed solvent of Hexafluoro-2-propanol (HFIP) and Trifluoroacetic acid (TFA), collagen and chitin.

The overlapping contents are omitted in consideration of the complexity of the present specification, and terms that are not otherwise defined herein have meanings commonly used in the technical field to which the present invention pertains.

In the present invention, by manufacturing a nanofiber membrane based on collagen and chitin by an electrospinning process, it is confirmed that mechanical properties such as toughness, tensile strength and Young's modulus are improved due to new interaction between collagen and chitin, and thus the disadvantages of the existing shielding film Improved.

The collagen / chitin nanofiber membrane prepared by the manufacturing method of the present invention maintains excellent biocompatibility as compared to the collagen-only membrane prepared by electrospinning, and has excellent mechanical properties and is used for periodontal tissue regeneration when used as a biomaterial. It can be usefully used as a guided bone regeneration (GBR) membrane.

FIG. 1 shows the results of experiments in which high-purity α-chitin (Sigma Aldrich, C9752-1G) having a molecular weight of 100,000 or more is dissolved in a mixed solvent of Hexafluoro-2-propanol (HFIP) and Trifluoroacetic acid (TFA) in various ratios. .
Figure 2 (a) is an electron scanning microscope image of collagen nanofibers obtained by electrospinning collagen alone by the method of Example 2 of the present invention, (b) is collagen and α by the method of Example 2 of the present invention -Electron scanning microscopic image of collagen / α-chitin nanofibers obtained by electrospinning an electrospinning solution of chitin, and (c) is an electrospinning mixture of collagen and β-chitin by the method of Example 2 of the present invention This is an electron scanning microscope image of collagen / β-chitin nanofibers obtained by electrospinning a solution.
3 (a), (e), and (f) are collagen raw materials, α- chitin raw materials, and β- chitin raw materials, respectively. Differential Scanning Calorimetry measures the change in calories by temperature (b), (c), and (d) is a collagen, collagen / α-chitin, and collagen / β-chitin nanofiber membranes prepared by the manufacturing method of the present invention, respectively, by measuring the change in calories by temperature by differential scanning calorimetry.
The mechanical strength of the nanofiber membrane of the present invention of FIG. 4 is measured (a) is a nominal stress (force) -elongation representative diagram, (b) is toughness, (c) is tensile strength, and (d) is Young's modulus. It is shown.

Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

Example 1. Preparation of collagen / chitin electrospinning solution

As shown in FIG. 1, when a commercially available general α- chitin having a molecular weight of 100,000 or more is dissolved in a mixed solution of various ratios of Hexafluoro-2-propanol (HFIP) and Trifluoroacetic acid (TFA), a specific ratio (HFIP: TFA = 7: 3, 8: 2, 9: 1) it can be seen that α- chitin was completely dissolved. In the case of α-chitin, since it is more difficult to dissolve in a solvent because it has a stronger hydrogen bond than that of β-chitin, the ratio of polar solvents was confirmed based on α-chitin. β-chitin has superior solubility compared to α-chitin, and thus dissolution proceeds at a specific ratio under the same conditions.

For the production of collagen chitin nanofiber-based membrane, 1% (w / v) solution and β- chitin of 0.02 g of α-chitin added to a mixed solvent having a weight ratio of 2 ml of HFIP: TFA (8.5: 1.5) are added. A solution of 1% (w / v) added with g was prepared and dissolved for 24 hours. Then, 0.26 g of collagen was added to both solutions and dissolved for 12 hours to prepare collagen to have a weight ratio of 13% (w / v) so that the weight ratio of collagen: chitin was 13: 1. As a collagen control to which chitin was not added, 0.28 g of collagen was added to a mixed solvent of 2 ml of HFIP: TFA (8.5: 1.5) to prepare collagen to have a weight ratio of 14% (w / v).

Example 2. Preparation of collagen / chitin based nanofiber membrane

For electrospinning, the electrospinning solution was injected into a syringe with a 26-gauge stainless steel needle with an inner diameter of 4.24 mm. At this time, a voltage of 30-35 kV is applied to the needle. The feed rate of the solution was adjusted to 0.25 ml / h. The electrospinning process took 90 minutes to prepare the membrane.

The electrospun nanofiber membrane was uniformly formed on the top surface of a specially manufactured metal block. The membrane composed of nanofibers was carefully separated from the metal block, and the remaining solvent was removed by evaporation in an oven at 40 degrees.

Example 3. Electron scanning microscopy observation of collagen / chitin nanofibers

In order to find out the shape of the collagen chitin nanofibers prepared in Example 2, they were observed with an electron scanning microscope (SEM) and the results are shown in FIG. 2.

As shown in FIG. 2, nanofibers having a uniform and homogeneous diameter (collagen: 210 nm, collagen / α-chitin: 271 nm, collagen / β-chitin: 223 nm) without formation of beads in the wrong form of spinning It was confirmed that it was prepared.

Example 4. Analysis of structural differences using Differential Scanning Calorimetry (DSC)

In order to confirm different molecular interactions with the control group of the collagen / β-chitin nanofiber membrane prepared in Example 2, it was confirmed that the heat change required when phase transformation was performed. Collagen / α-chitin nanofiber membranes prepared under the same conditions and collagen nanofiber membranes and raw material collagen not dissolved in a solvent were compared with α-chitin and β-chitin. According to Figure 3, when compared to the nanofiber membrane containing β- chitin compared to the collagen nanofiber membrane, it was confirmed that the thermal properties of collagen are maintained, and the structure of collagen is not deteriorated.

Example 5. Confirmation of mechanical strength

The collagen, collagen / α-chitin and collagen / β-chitin nanofiber membranes prepared in Example 2 were evaluated for mechanical strength. The sample was cut to a size of 5 mm X 20 mm so that the length of the portion to be stretched so that the portion caught by the jig was 5 mm above and below each was 10 mm. As a device for analyzing mechanical tensile strength, a universal testing machine (UTM, INSTRON) was used. The tensile speed was adjusted to 10 mm / min.

In FIG. 4, (a) is a nominal stress (force)-elongation representative diagram, (b) is toughness, (c) is tensile strength, (d) is Young's modulus.

The strengths of the collagen membrane, collagen / α-chitin nanofiber membrane and collagen / β-chitin nanofiber membrane prepared according to the conditions of Example 2 of the present invention were 24.9, 123.8, and 319.4 KJ / m ³ , respectively. Through this, it was found that the toughness of the collagen / β-chitin nanofiber membrane is 12.83 times higher than that of the collagen nanofiber membrane and 2.58 times higher than that of the collagen / α-chitin nanofiber membrane.

In addition, the tensile strength of the collagen membrane, collagen / α-chitin nanofiber membrane and collagen / β-chitin nanofiber membrane prepared according to the conditions of Example 2 of the present invention were 0.097, 0.687, and 1.055 MPa, respectively. Through this, it was found that the tensile strength of the collagen / β-chitin nanofiber membrane is 10.88 times higher than that of the collagen nanofiber membrane and 1.53 times higher than the tensile strength of the collagen / α-chitin nanofiber membrane.

In addition, Young's modulus of the collagen membrane, collagen / α-chitin nanofiber membrane, and collagen / β-chitin nanofiber membrane prepared according to the conditions of Example 2 of the present invention were 2.69, 17.86, and 24.63 MPa, respectively. Through this, it was found that the Young's modulus of the collagen / β-chitin nanofiber membrane is 9.16 times higher than the Young's modulus of the collagen nanofiber membrane, and 1.38 times higher than the Young's modulus of the collagen / α-chitin nanofiber membrane.

From the above results, it can be seen that when β-chitin was used, a combination of collagen and β-chitin formed a hydrogen bond and a hydrophobic interaction that did not appear in the mixture of collagen and α-chitin, resulting in a significant increase in mechanical strength. there was.

The present invention has been described through the examples as described above. Those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in other specific forms without changing its technical spirit or essential features. Therefore, the above-described experimental examples are to be understood as illustrative and non-limiting in all respects. The scope of the present invention is indicated by the following claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted to be included in the scope of the present invention.

Claims (13)

(1) preparing a electrospinning solution by adding collagen and chitin to a mixed solvent of HFIP (Hexafluoro-2-propanol) and TFA (Trifluoroacetic acid),
Step (1) is to dissolve for 10 to 30 hours by adding α-chitin or β-chitin to the mixed solvent, and for 5 to 20 hours by adding collagen,
The mixed solvent has an HFIP and TFA weight ratio of 7: 3 to 9: 1;
(2) preparing a nanofiber membrane from the electrospinning solution through an electrospinning process; And
(3) removing the residual solvent from the prepared nanofiber membrane; nanofiber membrane manufacturing method comprising a. delete delete According to claim 1,
The electrospinning solution of step (1) is characterized in that it comprises collagen and chitin in a weight ratio of 10: 1 to 15: 1, wherein the nanofiber membrane manufacturing method. According to claim 1,
The electrospinning process of step (2) is a method of manufacturing a nanofiber membrane, in which a voltage of 30 to 35 kV is applied to a needle injecting an electrospinning solution. According to claim 1,
The electrospinning process of step (2) is a nanofiber membrane manufacturing method wherein the supply speed of the electrospinning solution is 0.15 to 0.25 ml / h. According to claim 1,
The electrospinning process of step (2) is performed for 60 to 240 minutes. According to claim 1,
The step of removing the residual solvent of step (3) is to heat the membrane and evaporate the solvent to dry the nanofiber membrane. Nanofiber membrane produced by the production method according to any one of claims 1 and 4 to 8. The nanofiber membrane according to claim 9, wherein the toughness is 100 to 400 KJ / m ³ . The nanofiber membrane of claim 9, wherein the tensile strength is 0.5 to 1.5 MPa. The nanofiber membrane according to claim 9, wherein the Young's modulus is 10 to 30 MPa. A nanofiber membrane formed from an electrospinning solution comprising a mixed solvent of Hexafluoro-2-propanol (HFIP) and Trifluoroacetic acid (TFA), collagen and chitin,
The chitin is α-chitin or β-chitin,
The mixed solvent is HFIP and TFA nanofiber membrane having a weight ratio of 7: 3 to 9: 1.

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