Agarose nanofiber and manufacturing method thereof

Abstract

To provide a technique for manufacturing nanofibers using a solution prepared by dissolving only agarose in a solvent.SOLUTION: A manufacturing method of an agarose nanofiber according to the present invention comprises dissolving agarose in a solvent consisting of hexafluoroisopropanol or in a solvent formed by mixing hexafluoroisopropanol or ethanol with water to prepare an agarose solution, and spinning the agarose solution by an electrospinning method to produce the agarose nanofiber.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] The present invention relates to an agarose nanofiber and a method for producing the same. [Background technology]

[0002] Agarose, a natural polysaccharide extracted from seaweed, has a large difference between its melting and gelling temperatures, and after dissolving in water at 80-90°C, it easily gels by cooling to room temperature. Taking advantage of this property, agarose hydrogels, fibrous materials, and sponge-like materials are widely used in fields such as tissue engineering, drug delivery, and biosensors.

[0003] In addition to agarose, there are many other types of natural polysaccharides, including cellulose, chitin, chitosan, alginic acid, and dextrin. Nanofibers made from these natural polysaccharides have high viscosity and water retention properties, and are highly biocompatible, making them attractive for use in food, medical, and cosmetic applications. Furthermore, because natural polysaccharide nanofibers are lightweight and strong, they are also expected to be used in building materials, automotive parts, electrical materials, and more.

[0004] One method for producing nanofibers is an electrospinning technology called electrospinning. In electrospinning, a syringe is filled with a solution containing the raw materials for the nanofibers, and high voltage is applied to the solution inside the syringe, causing it to be ejected from the nozzle, producing fibers with diameters on the order of several tens of nanometers to several micrometers.

[0005] To produce nanofibers using the electrospinning method, it is necessary to prepare a solution by dissolving the raw material in an appropriate solvent. However, because agarose has a tendency to gel, it is difficult to prepare an agarose solution, which is the material for electrospinning. Therefore, in order to obtain agarose nanofibers, a solution prepared by blending agarose with other polymer compounds such as chitosan has been used (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] S. Teng, P. Wang, H. Kim, Mater. Lett. 2009, 63, 2510. [Non-patent document 2] MA Salati et al., Polymers 2020, 12, 1150-1165. [Non-patent document 3] K. Katsuura, Journal of Industrial Science 1965, 3, 205-209. Summary of the Invention [Problem to be solved by the invention]

[0007] The problem to be solved by the present invention is to provide a technique for producing nanofibers using a solution in which agarose is dissolved in a solvent without being blended with other polymer compounds. [Means for solving the problem]

[0008] The method for producing agarose nanofibers according to the present invention, which has been made to solve the above problems, comprises the steps of: Agarose is dissolved in a solvent consisting of hexafluoroisopropanol, a mixture of water and hexafluoroisopropanol, or a mixture of water and ethanol to prepare an agarose solution; The agarose solution is used to spin fibers by electrospinning to produce agarose nanofibers.

[0009] In the above-mentioned method for producing agarose nanofibers, It is preferable to prepare the agarose hydrogel, freeze-dry the agarose hydrogel, and then dissolve the agarose hydrogel in the solvent to prepare the agarose solution.

[0010] In this case, the agarose concentration of the agarose hydrogel is preferably in the range of 1.5 to 5.0% by weight.

[0011] Furthermore, in the above-mentioned method for producing agarose nanofibers, The solvent for the agarose solution is preferably a mixture of water and hexafluoroisopropanol, which has excellent agarose solubility and is easily volatilized during spinning by electrospinning, allowing high-quality nanofibers to be obtained.

[0012] The mixed solvent is preferably a mixture of water and hexofluoroisopropanol in a ratio of 10:90 to 5:95, and more preferably a mixture of water and hexofluoroisopropanol in a ratio of 7.5:92.5.

[0013] The agarose nanofibers according to the present invention are nanofibers made solely from agarose and having a fiber diameter of 10 to 1000 nm. Such agarose nanofibers can be obtained by the production method described above. [Effects of the Invention]

[0014] According to the present invention, nanofibers can be produced using a solution in which agarose is dissolved in a solvent without blending it with other polymer compounds, and therefore nanofibers that take advantage of the properties of agarose can be obtained. [Brief explanation of the drawings]

[0015] [Figure 1] Formula showing the chemical structure of agarose. [Figure 2] FIG. 1 is a schematic diagram illustrating an example of an electrospinning device. [Figure 3] FIG. 1 shows the results of an experiment conducted to search for a solvent to be used in an agarose solution, as Example 1. [Figure 4] Graph showing the relationship between the viscosity of an agarose solution and the percentage of water in the solvent. [Figure 5] This table shows the solvent, agarose concentration, additives, heater output, applied voltage, syringe movement speed, distance between the syringe nozzle and the cathode plate, and appearance of the product for the agarose solutions of entries 1 to 10 used in Example 2. [Figure 6] Laser microscope images (a) to (f) of the products obtained by the electron spinning method using the agarose solutions of entries 1, 4, 6, and 8 to 10. [Figure 7] (a) is a high-resolution FE-SEM image of the product of entry 7, (b) and (c) are high-resolution FE-SEM images of the product of entry 9, and (d) is a bar graph showing the results of statistically processing the fiber diameter measurements from images (b) and (c). [Figure 8] (a) shows the nanofiber sheet formed by stacking agarose nanofibers, which is the product of entry 9. (b) shows the FT-IR spectra of the hydrogen bonding regions at 3500 cm-1 and 3000 cm-1 of agarose powder and agarose nanofibers. DETAILED DESCRIPTION OF THE INVENTION

[0016] Agarose is a naturally occurring polysaccharide with the chemical structure shown in Figure 1. Agarose is extracted from seaweed and is one of the main components of agar. When agarose powder is dispersed in water and heated to 80-90°C, the agarose in the water dissolves (hydrates) to form an aqueous solution. When the aqueous agarose solution is cooled to around room temperature, it becomes a gel-like hydrogel. Agarose hydrogel is difficult to spin using the electrospinning method due to the strong intramolecular hydrogen bonds. To spin using the electrospinning method, the agarose hydrogel must be heated to a hydrated state, but adjusting the heating temperature is difficult, making it impossible to obtain nanofibers with uniform properties.

[0017] In the method for producing agarose nanofibers of the present invention, a solvent was used for the agarose solution that can dissolve agarose without blending it with other polymer compounds and that does not gel even at room temperature (25-28°C). Specifically, a solvent consisting of hexafluoroisopropanol (hereinafter referred to as HFIP), a mixture of water and HFIP, or a mixture of water and ethanol was used. Therefore, the method for producing agarose nanofibers of the present invention allows the production of nanofibers using only agarose as a raw material.

[0018] In the production method of the present invention, the agarose used in preparing the agarose solution can be prepared by preparing a hydrogel with an agarose concentration of 1.5% to 5.0% by weight, freezing it in liquid nitrogen, and then freeze-drying it using a freeze dryer at temperatures of -196°C to -20°C. The freeze-dried agarose hydrogel can be dissolved in the solvent by adding it to the solvent either as is or after pulverization and stirring, making it easy to prepare the agarose solution. In the following explanation, % means % by weight unless otherwise specified.

[0019] In the production method of the present invention, as described above, the solvent for the agarose solution may be a solvent consisting of HFIP or a solvent consisting of a mixture of HFIP or ethanol with water. Among these solvents, a mixed solvent of water and HFIP is particularly preferred, and the mixed solvent is preferably a mixture of water and HFIP in a ratio (weight ratio) of 10:90 to 5:95, more preferably a mixture of water and HFIP in a ratio of 7.5:92.5.

[0020] In the manufacturing method of the present invention, agarose nanofibers are produced by spinning an agarose solution using the electrospinning method. In the electrospinning method, a solution containing raw materials is sprayed under high voltage, evaporating the solvent in the solution to produce nanofibers made from the raw materials. While various types of electrospinning devices are used, such as nozzle-type, wire-type, and cylinder-type, the manufacturing method of the present invention preferably uses a nozzle-type electrospinning device such as the one shown in Figure 2. The electrospinning device in Figure 2 includes a syringe 1, a grounded cathode plate (collector) 2, and a power supply 3 that applies high voltage between the syringe 1 and the cathode plate 2. In this device, the nozzle 11 of the syringe 1 and the cathode plate 2 are arranged opposite each other. A high voltage is applied between the material solution (dope) 4 filled in the syringe 1 and the cathode plate 2, causing the material solution 4 to spray from the nozzle 11 of the syringe 1, depositing nanofibers 41 on the cathode plate 2. 2, the component labeled with the reference numeral 5 is a sheet-like heater for heating the material solution 4 filled in the syringe 1. The heater 5 will be described later.

[0021] 2, the agarose solution is filled into syringe 1, and when a high voltage is applied between syringe 1 (agarose solution in syringe 1) and cathode plate 2, the solvent HFIP easily volatilizes when the agarose solution is ejected from nozzle 11. As a result, the agarose solution becomes highly concentrated as the HFIP evaporates, and is ejected from nozzle 11, allowing fine, long nanofibers to be obtained.

[0022] The production method of the present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0023] [Example 1] [Search for solvents for agarose solutions] In this example, we conducted an experiment to identify solvents for agarose solutions used in the production of agarose nanofibers. The results are shown in Figure 3. First, we conducted an experiment to identify solvents in which agarose can be dissolved without heating and which are easily volatilized during electrospinning. Water, DMSO (dimethyl sulfoxide), DMF (dimethylformamide), glycerin, ethylene glycol, and HFIP (hexafluoroisopropanol) were used as solvents in the experiment. We investigated the necessity of heating when dissolving agarose in these solvents, as well as the agarose concentration (%), agarose solubility, gelation, polarity, boiling point (°C), and volatility.

[0024] In Example 1 and Example 2 described below, a hydrogel with a concentration of 1.5 wt % to 5.0 wt % agarose was prepared as the agarose to be dissolved in the solvent. 10 mL of this hydrogel was frozen in liquid nitrogen and then freeze-dried at temperatures of -196°C to -20°C using a freeze dryer (FDU-1200, manufactured by EYELA).

[0025] Also, in Figure 3, the results for three solvents (formamide, N-methylformamide, and BmimCl (1-butyl-3-methylimidazolium chloride)) marked with the letter [1] on the right superscript, and the results for one solvent (dimethylamine and water) marked with the letter "2" on the right superscript are not the results of experiments conducted by the present inventors, but are listed for reference. Specifically, the results for the three solvents marked with the letter "1" on the right superscript are transcribed from Non-Patent Document 2, and the results for the one solvent marked with the letter "2" on the right superscript are transcribed from Non-Patent Document 3.

[0026] As can be seen from Figure 3, there were three solvents that could dissolve agarose without heating and did not gel after dissolution: DMSO, HFIP, and HFIP+water. Of these three solvents, DMSO has a high boiling point of 189°C and low volatility, while HFIP and HFIP+water both have low boiling points of 58°C and high volatility. Therefore, these two solvents (HFIP and HFIP+water) were selected as candidate solvents for the agarose solution.

[0027] [Relationship between the solvent (HFIP + water) mixing ratio, agarose concentration, and viscosity of the agarose solution] We investigated the relationship between agarose concentration and viscosity when the HFIP / water ratio in a mixed solvent was varied. In this experiment, the viscosity of agarose solutions was measured when the water content in the mixed solvent was varied from 5% to 10% and the agarose concentrations were 1.5%, 2%, 2.5%, and 3%. The results are shown in Figure 4. As can be seen from Figure 4, the viscosity was highest when the HFIP / water ratio was 92.5:7.5, regardless of the agarose concentration. Therefore, when using a HFIP / water mixed solvent as the solvent for agarose solutions, it was found that adjusting the HFIP / water ratio to 92.5:7.5 makes it easy to adjust the viscosity of the agarose solution to an appropriate level.

[0028] [Example 2] Based on the results of Example 1, nanofibers were produced by electrospinning using agarose solutions in TFA (trifluoroacetic acid) / DCM (dichloromethane), water, water / ethanol, HFIP, and water / HFIP. The agarose solutions used in this example are shown in Figure 5 (entries 1 to 10). Of these 10 agarose solutions, citric acid was added as an additive to the agarose solutions in entries 2 and 3, and glucose was added as an additive to the agarose solution in entry 4.

[0029] Furthermore, spinning by electrospinning requires breaking the intramolecular hydrogen bonds of agarose to solvate it. To control the hydrogen bonds, a sheet-like heater 5 was attached to the syringe 1 of the electrospinning apparatus (see Figure 2), and the nozzle length, which is usually 3.7 cm, was shortened to 1.2 cm. Figure 5 shows the electrospinning conditions for each entry: heater output (kV), voltage (kV) applied between the syringe 1 and the cathode plate 2, syringe 1 movement speed (mm / sec), and distance (cm) between the tip of the nozzle 11 and the cathode plate 2. The rightmost column in Figure 5 shows the appearance of the products obtained by electrospinning. Figure 6 shows laser microscope images of the products of entries 1, 4, 6, 8-10.

[0030] In this example, we first attempted to produce nanofibers by electrospinning using the agarose solution of entry 1 under the conditions shown in Figure 5, but only a particulate product was observed, as shown in Figure 6(a). The conditions for entry 1 (solvent, voltage, syringe movement speed, and distance between the nozzle tip and the cathode plate) were set with reference to the conditions for producing nanofibers by electrospinning from a mixed solution of chitosan and agarose described in Non-Patent Document 1.

[0031] Next, based on the experimental results of Entry 1, electrospinning was attempted using the agarose solutions of Entries 2 to 10. The reason for using ethanol instead of HFIP, which was selected as a solvent candidate for the agarose solution in Example 1, is that, like HFIP, it has a low boiling point and dissolves agarose without heating. Furthermore, citric acid and glucose were added as additives to the agarose solutions of Entries 2, 3, and 4 because citric acid and glucose prevent the agarose solution from gelling at room temperature.

[0032] However, in entries 2 and 3, which used water as the solvent, only particulate products were observed even when citric acid was added, and in entry 4, which used a water-ethanol mixed solvent, only lintel-like products were observed even when glucose was added (see Figure 6(b)). These results suggest that if an additive is contained in the agarose solution, the additive remaining in the agarose solution ejected from the nozzle may make it impossible to control the intermolecular hydrogen bonds of the agarose.

[0033] Therefore, in entries 5 and 6, a mixed solvent of water and ethanol was used instead of adding additives, and spinning by electrospinning was attempted while preventing gelation of the agarose solution by heating the syringe with heater 5. As a result, short fiber-like products were obtained in entries 5 and 6. In particular, in entry 6, where the agarose concentration was 2.0 wt%, the fiber diameter was smaller than in entry 5, where the agarose concentration was 1.0 wt%, and fine fibers with an average fiber diameter of 1.30 ± 0.37 μm were obtained (Figure 6(c)).

[0034] In particular, when the syringe was heated with the sheet heater 5, gelation of the agarose solution was observed at the nozzle tip for most samples. This gel clogged the nozzle, which required removal, making it impractical. Furthermore, heating the syringe caused excessive evaporation of the ethanol in the agarose solution, making it difficult to control the intermolecular hydrogen bonding of the agarose. For these reasons, it was concluded that heating the syringe is not desirable for the production of agarose nanofibers.

[0035] Next, for entry 7, which used HFIP as a solvent, and entries 8 to 10, which used a mixed solvent of water and HFIP, spinning was carried out by the electrospinning method.

[0036] As a result, a fibrous product containing a small amount of particles was observed in entry 7, in which 5.0 wt% agarose was dissolved in HFIP. Therefore, in entry 8, in order to change the hydrogen bonding interactions in the agarose, an agarose solution in which 5.0 wt% agarose was dissolved in a mixed solvent of HFIP and water at a ratio of water / HFIP = 1 / 9 (v / v) was used, and the spinning conditions of entry 7 were adjusted and the resulting fiber was spun by electrospinning. The amount of fiber increased compared to entry 7 (Figure 6(d)).

[0037] Furthermore, in entry 9, the agarose concentration was changed to 3.0 wt%, the voltage applied between the syringe and the cathode plate was changed to 15 kV, and the syringe movement speed was changed to 0.06 mm / sec, and spinning was performed using the electrospinning method. As a result, many more fibers were clearly observed compared to entry 8 (Figure 6(e)).

[0038] Furthermore, in entry 10, an agarose solution was prepared by dissolving agarose at a concentration of 4.0 wt% in a water / HFIP mixed solvent with a water / HFIP ratio of 7.5:92.5 (v / v), which is the ratio at which the viscosity of the agarose solution is highest. The spinning conditions were adjusted and the agarose solution was spun by electrospinning, resulting in a large number of clearly visible fibers (Figure 6(f)).

[0039] Figure 7(a) is a high-resolution field emission scanning electron microscope (FE-SEM) image of the product obtained when the agarose solution of entry 7 was used to spin fibers by the electrospinning method, and Figures 7(b) and (c) are high-resolution FE-SEM images of the product obtained when the agarose solution of entry 9 was used to spin fibers by the electrospinning method.

[0040] In the case of entry 7, structures resembling collapsed large particles were observed in several places, and the fibers appeared to be integrated (Figure 7(a)). In contrast, in the case of entry 9, very beautiful fibers were observed (Figures 7(b) and (c)), and when observed at high magnification, it was found that the fiber surface was smooth (Figure 7(c)). The fiber diameter was measured from the images in Figures 7(b) and (c), and the results were statistically processed and shown in a bar graph (Figure 7(c)). From these results, the estimated fiber diameter of the fibrous product of entry 9 was 68±33 nm, indicating that nanofibers with diameters on the order of several tens of nanometers could be obtained.

[0041] Furthermore, the agarose nanofibers obtained using the agarose solution of entry 9 could be stacked to produce a nanofiber sheet (Figure 8(a)).

[0042] Furthermore, to investigate the properties of the agarose nanofibers obtained using the agarose solution of entry 9, a 3500 cm -1 and 3000 cm -1 When the FT-IR spectrum of the hydrogen-bonding region of agarose nanofibers was compared with that of agarose powder, it was observed that the peak top of the hydroxyl stretching peak of agarose nanofibers shifted to a higher wavenumber region than that of agarose powder (Figure 8(b)). This suggests that the hydrogen bonds of agarose nanofibers are weaker than those of agarose powder. Appropriate hydrogen bonds are necessary for the formation of agarose nanofibers, and for this to occur, it is thought that appropriate hydrogen bonds must be formed in the raw material agarose powder (frozen agarose) or agarose solution. [Explanation of symbols]

[0043] 1. Syringe 11...Nozzle 2...Cathode plate 3…Power supply 4...Material solution 5...Heater

Claims

1. Agarose is dissolved in a solvent consisting of hexafluoroisopropanol, a mixture of water and hexafluoroisopropanol, or a mixture of water and ethanol to prepare an agarose solution; A method for producing agarose nanofibers, comprising spinning the agarose solution by electrospinning to produce agarose nanofibers.

2. The method for producing agarose nanofibers according to claim 1, A method for producing agarose nanofibers, comprising preparing a hydrogel of the agarose, freeze-drying the agarose hydrogel, and then dissolving the agarose hydrogel in the solvent to prepare the agarose solution.

3. The method for producing agarose nanofibers according to claim 2, The method for producing agarose nanofibers, wherein the agarose concentration of the agarose hydrogel is 1.5 to 5.0% by weight.

4. The method for producing agarose nanofibers according to any one of claims 1 to 3, A method for producing agarose nanofibers, wherein the solvent is a mixed solvent of water and hexofluoroisopropanol.

5. The method for producing agarose nanofibers according to claim 4, A method for producing agarose nanofibers, wherein the mixed solvent is a mixture of water and hexofluoroisopropanol in a ratio of 10:90 to 5:

95.

6. The method for producing agarose nanofibers according to claim 5, A method for producing agarose nanofibers, wherein the mixed solvent is a mixture of water and hexofluoroisopropanol in a ratio of 7.5:92.

5.

7. Agarose nanofibers made solely from agarose, with fiber diameters of 10 to 1000 nm.

Images