WO2013012119A1

WO2013012119A1 - Method for manufacturing nanofiber coated with carbon nanotube

Info

Publication number: WO2013012119A1
Application number: PCT/KR2011/006155
Authority: WO
Inventors: 김해원; 신원상; 김광진
Original assignee: 단국대학교 산학협력단
Priority date: 2011-07-15
Filing date: 2011-08-19
Publication date: 2013-01-24
Also published as: KR101323427B1; KR20130009476A

Abstract

The present invention relates to a method for manufacturing nanofiber coated with a carbon nanotube for introducing a hydrophilic property to the surface of the nanofiber by effectively coating the carbon nanotube on the surface of the nanofiber, and for increasing the degree of cell adhesion and cultivation by increasing the surface area.

Description

Manufacturing method of nanofiber coated with carbon nanotube

The present invention relates to a method for producing carbon nanotube-coated nanofibers that can be used for culturing cells.

In order to treat the damage of the nerve tissue neural tissue regeneration, the important consideration is the regeneration of the neurite process. For this purpose, a method of culturing neurites outside the living body and implanting the same may be used, wherein a support for cell culture is used for external culture.

A polymer scaffold may be used as the support, and it is known that the polymer scaffold may be suitably used for cell attachment and growth of neurites. In particular, it has been suggested that electrospun nanofibrous scaffolds can be used for neural tissue regeneration (Chew et al .; Adv Funct Mater 2007; 17: 1288-96), which supports the attachment of cells. It has been reported that neurites can be grown along the axis of the fibers (1) Lee et al .; Biomaterials 2005; 26: 1261-70, (2) Schnell et al .; Biomaterials 2007; 28: 3012-25). In addition, it has been reported that neurites grow more effectively when the diameter of the nanofibers is from several hundred nanometers to several micrometers ((1) Corey et al .; Neurosci Res 1991; 30: 300-7; (2) Corey et al .; IEEE Trans Biomed Eng 1996; 43: 944-55; (3) Rajnicek et al .; J Cell Sci 289 1997; 110: 2905-13; (4) Wen et al .; J Biomed Mater Res A 2006; 76: 626-37).

Meanwhile, PLCL (Polylactic Acid-co-caprolactone) may be used for the polymer support, and PLCL may be used as a support for tissue regeneration as a synthetic, biocompatible, and biodegradable copolymer. Nanotubes are hollow cylinder-like materials in which graphene is wound (Baughman et al .; Science 2002; 297: 787-92), which have characteristic structural, electrical, and mechanical properties. For example, the structure of carbon nanotubes has been reported to enable cell attachment and growth upon contact with cells (1) Garibaldi et al .; Nanotechnology 2006; 17: 391. (7) (2) Meng et al .; J Biomed Mater Res A 2006; 79: 298-306) In addition, the growth of neurites in carbon nanotubes has also been reported ((1) Hu et al .; Nano Lett 2004; 4: 507-11; (2) Mattson et al .; J Mol Neurosci 2000; 14: 175-82). Depending on the number of layers wound, it can be divided into single-walled carbon nanotubes, double-walled carbon nanotubes or multi-walled carbon nanotubes.

When the carbon nanotubes are included in the polymer support, in particular, when the carbon nanotubes are coated on the surface of the polymer support, it is expected to be more suitable for tissue regeneration. For example, Republic of Korea Patent No. 10-0895631 and Republic of Korea Patent No. 10-0851431 in the process of producing nanofibers by electrospinning the polymer, before mixing the carbon nanotubes to the polymer before the electrospinning, Disclosed is a method of preparing a polymer support comprising electrospun carbon nanotubes. However, this is not a coating on the surface of the polymer support, in particular, there is a problem that various conditions are required for uniform dispersion in the process of mixing carbon nanotubes in the polymer. In addition, according to the above method, since the carbon nanotubes are not coated on the surface but buried inside the polymer, excellent electrical properties of the carbon nanotubes may be reduced.

In order to coat the carbon nanotubes on the surface of the polymer support, an interface interaction is required between the polymer support and the carbon nanotubes, but there is a problem in that the coating is not performed well because the interface interaction between the polymer and the carbon nanotubes is very small. Therefore, even when the polymer support and the carbon nanotubes are mixed, the carbon nanotubes are not located on the surface of the polymer and the coating is hardly performed.

In addition, carbon nanotubes have a strong agglomeration phenomenon. In order to coat the carbon nanotubes, the carbon nanotubes should be dispersed and used in a solvent. However, due to the coagulation phenomenon, the carbon nanotubes are not dispersed well in the solvent. There is a tendency for the coating to concentrate only on the location.

In addition, carbon nanotubes have a problem in that hydrophobicity is strong. Since the polymer support used for cell culture has a biocompatibility, it is often hydrophilic. However, the hydrophilic polymer support and the hydrophobic carbon nanotube have the problem that the coating is not performed well.

Therefore, the inventors of the present invention, while studying a method of coating the carbon nanotubes on the surface of the nanofibers, when the carbon nanotubes using the ion reforming method, it was confirmed that the method of coating carbon nanotubes on the surface of the nanofibers, The nanofibers prepared according to the present invention have been found to be useful for the proliferation of cells and the growth of nerve cells.

The present invention effectively coated the carbon nanotubes on the surface of the nanofibers, thereby introducing hydrophilicity on the surface of the nanofibers, and also increasing the surface area, thereby coating the carbon nanotubes that can increase the degree of cell adhesion and culture. It is to provide a method for producing a nanofiber.

In order to solve the above problems, the present invention is the step of reacting carbon nanotubes with 1-butyl-3-methyl-imidazolium undeca fluoroantimonate and tetrahydrofuran to prepare carbon nanotubes-SbF ₆ (Step 1); Preparing carbon nanotube-tartrate by reacting the carbon nanotube-SbF ₆ with potassium sodium tartrate (step 2); Dissolving the carbon nanotube-tartrate in a solvent to prepare a carbon nanotube-tartrate solution (step 3); And immersing the nanofibers in the carbon nanotube-tartrate solution to coat the carbon nanotubes on the surface of the nanofibers (step 4).

Step 1 is a step for bonding SbF ₆ to the carbon nanotubes, and reacting the carbon nanotubes with 1-butyl-3-methyl-imidazolium undeca fluoroantimonate to form carbon nanotubes-SbF ₆ To prepare.

As used herein, the term 'carbon nanotube' refers to a material in which one carbon atom forms an sp2 bond with three other carbon atoms, has a hexagonal honeycomb structure, and has a diameter of several nanometers to several tens of micrometers. . Carbon nanotubes have excellent mechanical and electrical properties, and the present invention is for coating such carbon nanotubes on the surface of nanofibers. Carbon nanotubes that can be used in the present invention may be used single-walled carbon nanotubes, double-walled carbon nanotubes or multi-walled carbon nanotubes, preferably multi-walled carbon nanotubes.

The term "carbon nanotube-SbF ₆ " used in the present invention means a material in which the carbon nanotubes and SbF ₆ are bonded. In order to bind SbF ₆ to carbon nanotubes, 1-butyl-3-methyl-imidazolium undeca fluoroantimonate ([bmim] [Sb ₂ F ₁₁ ]) and tetrahydrofuran (THF) were added. It acts as a Lewis acid, and carbon nanotubes act as Lewis bases, so SbF ₆ reacts with carbon nanotubes and 1-butyl-3-methyl-imidazolium undeca fluoroantimonate. Can be combined with carbon nanotubes.

In the reaction of step 1, 1-butyl-3-methyl-imidazolium undeca fluoroantimonate and THF is used in a molar ratio of 0.5-1.5: 1 relative to the mole of C ₆ of the carbon nanotubes, respectively. It is preferable and it can use more preferably in molar ratio of 1: 1. In addition, the reaction of Step 1 is preferably carried out in a dichloromethane solvent.

Step 2 is a reaction of binding tartic acid to carbon nanotubes, wherein the carbon nanotube-SbF ₆ prepared in step 1 is reacted with potassium sodium tartrate to prepare carbon nanotube-tartrate.

As used herein, the term “carbon nanotube-tartrate” refers to a material in which carbon nanotubes and tartic acid are combined. Potassium sodium tartrate used in the reaction is used to provide tart acid, and ion exchange occurs with SbF ₆ bonded to carbon nanotubes, thereby binding tartic acid to carbon nanotubes. At this time, potassium sodium tartrate is SbF ₆ carbon nanotube -SbF ₆ ^- is preferably reacted in an equivalent ratio of 5 times to 15 times.

After preparing the carbon nanotube tartrate in step 2, it is preferable to additionally filter, wash with water and ethanol, and dry, thus, can be produced in high purity carbon nanotube-tartrate.

Step 3 is to prepare a carbon nanotube-tartrate solution for immersing the nanofibers, dissolving the carbon nanotube-tartrate prepared in step 1 in a solvent to prepare a carbon nanotube-tartrate solution Step.

The solvent is not limited as long as it dissolves carbon nanotube-tartrate. Preferably, ethanol may be used. At this time, it is preferable to further sonicate to promote dissolution.

Step 4 is a step for coating the carbon nanotubes on the surface of the nanofibers, the step of coating the carbon nanotubes on the surface of the nanofibers by immersing the nanofibers in the carbon nanotube-tartrate solution.

As used herein, the term 'nanofiber' refers to an ultrafine fiber composed of tens to hundreds of nanometers in diameter, and generally refers to a fiber produced by electrospinning a polymer. The polymer that can be used at this time may be appropriately selected according to the use of the nanofibers, for example, PLCL and the like can be used, but is not limited thereto.

Generally, even if the nanofibers are immersed in a solution containing carbon nanotubes, the carbon nanotubes cannot be effectively coated on the nanofibers because the interfacial interaction between the nanofibers and the carbon nanotubes is weak. However, in the present invention, since the tartrate of carbon nanotubes is used, the interfacial interaction between the nanofibers and the carbon nanotubes can be enhanced, and thus the carbon nanotubes can be effectively coated on the nanofibers.

The immersion time of the nanofibers is preferably 1 second to 60 seconds. In addition, the prepared carbon nanotube coated nanofibers are preferably washed and dried with ethanol.

According to the manufacturing method, in order to coat carbon nanotubes on the nanofibers, the carbon nanotubes may be coated on the nanofibers by combining tartic acid with the carbon nanotubes and contacting the nanofibers. By the manufacturing method, the interface interaction between the conventional carbon nanotubes and the nanofibers is low, which can solve the problem of poor coating. In addition, since the coating time is only a few seconds, the coating time can be significantly shortened. Furthermore, since an effective coating is possible on the surface of the nanofibers, the function of the carbon nanotubes can be introduced into the nanofibers, thereby extending the application range of the nanofibers.

In addition, the prepared nanofibers are characterized by easy attachment and growth of cells. Nanofibers have a network similar to the tissue structure of a living body, and are often used as a support for cell growth. The surface of the prepared nanofibers is coated with carbon nanotubes, and the hydrophilicity of the nanofibers is increased. By introducing and increasing the surface area, the adhesion and growth of cells can be increased. According to one embodiment of the present invention, comparing the carbon nanotubes coated nanofibers and the degree of adhesion and culture of the cells to the other case, it was confirmed that the carbon nanotubes coated nanofibers are more effective. Therefore, the nanofibers coated with carbon nanotubes prepared according to the present invention can be applied to culture of cells.

According to the production method of the present invention, the carbon nanotubes can be effectively coated on the surface of the nanofibers, thereby introducing hydrophilicity to the surface of the nanofibers, and also increasing the surface area, thereby increasing the degree of cell adhesion and culture. Can be.

1 shows an SEM image of nanofibers. 1A and 1B show SEM images of nanofibers of the present invention without carbon nanotubes coated, and FIGS. 1C and 1D show SEM images of nanofibers prepared in one embodiment of the present invention.

2 is a graph showing the cell proliferation in the nanofibers according to the incubation time.

3 shows an image of neurites in nanofibers. In Figure 3, D3, D6, D9 means 3 days, 6 days, 9 days after the seeding of the cells, respectively. In addition, the scale bar of A-E means 20 μm, and the scale bar of F means 50 μm.

Figure 4 is a graph showing the average value of the nanofibers of the production example is not coated with carbon nanotubes and neurites on the nanofibers prepared in one embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be presented to assist in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

제조예: 나노섬유의 제조Preparation Example: Preparation of Nanofibers

First, PLCL solution (12.5% w / v) was prepared by dissolving 1.87 g of PLCL in solvent (3 ml 20% ethanol and 15 ml 80% dichloromethane). Electrospinning was performed using an electrospinner (manufactured by NanoNC), and the solution was added to a 10 ml syringe attached to a 21G blunted stainless steel needle by using a syringe pump. Inject at a rate of ml / h and add 13 kV. Nanofibers were prepared using a cylinder rotating at 4000 rpm, and the nanofibers were collected in aluminum foil 15 cm from the needle tip. The prepared nanofibers were dried at room temperature under vacuum.

실시예: 탄소나노튜브가 코팅된 나노섬유의 제조Example: Preparation of Carbon Fiber Coated Nanofibers

Step 1

200 mg of multi-walled carbon nanotubes (MWCNT, Iljin Nanotech, purity> 95%) were added to the reactor, and dried under reduced pressure, and then argon (Ar) gas was charged into the reactor. Dichloromethane was added as a solvent, 1-butyl-3-methyl-imidazolium undecafluoroantimonate was added, and then evenly dispersed using an ultrasonic wave. Tetrahydrofuran was added slowly for 30 minutes under sonicator operation. At this time, 1-butyl-3-methyl-imidazolium undecafluoroantimonate and tetrahydrofuran were used in the amount of 1.0 equivalent each based on the C ₆ unit which is the basic unit of carbon nanotubes. Reaction temperature was 25 degreeC and reaction time was 15 minutes. After the reaction, a small amount of water was added to the reaction mixture to terminate the reaction. The modified carbon nanotubes in the reaction mixture were separated on the filter paper. The modified carbon nanotubes collected on the filter paper were first washed several times with acetone, and then repeatedly washed several times with dichloromethane. The washed product was dried completely under reduced pressure. The obtained modified carbon nanotube (carbon nanotube-SbF ₆ ) was 371.2 mg.

Step 2

The modified carbon nanotubes (carbon nanotubes-SbF ₆ ) prepared in step 1 were prepared as a dispersion, and potassium sodium tartrate was added thereto, followed by stirring for 3 hours. Next, the filtrate was washed with water and ethanol to prepare carbon nanotube-tartrate.

Step 3

Carbon nanotube-tartrate prepared in step 2 was added to ethanol and sonicated for 3 minutes to prepare a carbon nanotube-tartrate solution (0.1 mg / ml).

Step 4

In the carbon nanotube tartrate solution prepared in step 3, the nanofibers prepared in the preparation example were immersed for about 1 second to coat the carbon nanotubes, and the nanofibers were washed several times with ethanol and dried at room temperature. .

실험예 1: 탄소나노튜브가 코팅된 나노섬유의 SEM 확인Experimental Example 1: SEM confirmation of nanofibers coated with carbon nanotubes

The following experiment was performed to confirm the nanofibers coated with the carbon nanotubes prepared in the above example by scanning electron microscopy (Hitachi 3000, Japan).

First, the nanofibers of the preparation example without the coating of carbon nanotubes and the nanofibers prepared in the example were fixed in PBS solution (glutaraldehyde 2.5%) for 10 minutes at room temperature, followed by ethanol (75%, 95% and 100%). The concentration was sequentially increased to%) and dried for 5 minutes. After coating a thin gold thin film on the carbon nanotubes, the SEM was confirmed at 15 kV (the diameter of the nanofibers was calculated from the SEM image), and the results are shown in FIG. 1.

1A and 1B are SEM images of nanofibers of the preparation example without the carbon nanotubes coated, and FIGS. 1C and 1D are SEM images of the nanofibers prepared in the examples. The diameter of the nanofibers of the preparation example without the carbon nanotubes coated was 1.45 ± 0.58 μm, and the diameters of the nanofibers prepared in the examples were observed to be 1.30 ± 0.46 μm.

In addition, as can be seen in Figures 1C and 1D, it can be seen that the carbon nanotubes are uniformly coated on the surface of the nanofibers. The coating amount of carbon nanotubes from the TGA analysis was determined to be 8% by weight in the total nanofibers.

실험예 2: 세포의 배양 및 신경돌기의 성장 측정Experimental Example 2: Measurement of Cell Culture and Neurites

PC-12 cells were cultured in DMEM medium (Dulbecco's Modified Eagle Medium; Gibco, USA) fed with 10% FBS (fetal bovine serum). When the culture was about 80%, the cells were detached with 0.05% trypsin, and then the number of cells visually confirmed by trypsin blue assay was confirmed. In order to evaluate the growth of neurites, the nanofibers of the production example without carbon nanotubes and each of the nanofibers prepared in Example were placed on a 4-well plate, and the cells were placed in DMEM medium containing 0.5% FBS. Under conditions, seeding was performed at a density of 5 × 10 ³ per nanofiber. The cells were treated with 50 ng / ml NGF on the 3rd, 6th and 9th days from the seeding day.

Cell proliferation was measured by kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Japan). During each incubation period, 10 μl of CCK-8 solution was added to each well and incubated at 37 ° C. for 3 hours. Absorption at 450 nm of each well was measured by a microplate reader (Molecular Devices, Sunnyvale, CA), and the results are shown in FIG. 2.

As shown in Figure 2, compared with the nanofibers of the production example is not coated with carbon nanotubes, it was confirmed that the cell proliferation is more active in the nanofibers prepared in the example.

In addition, the growth of neurites was confirmed as follows. On the 3rd, 6th and 9th day from the seeding, the cells were fixed with 4% paraformaldehyde, treated with 0.2% Triton X-100, and then treated with 1% (w / v) was treated for 30 minutes in PBS containing bovine serum albumin (BSA). Next, incubated with 20 nM Alexa Fluor 546-conjugated phalloidin diluted in PBS for 30 minutes. Images were obtained with an inverted microscope (IX-71; Olympus, Tokyo, Japan) equipped with a DP-72 digital camera. Ten images were obtained at randomly selected areas on each of at least three nanofibers under each condition. At least 250 cells were evaluated in each experiment. In order to quantify the growth of neurites, the longest neurites of each cell were measured by image analysis software (DP2-BSW, Olympus Co.) and their mean values were calculated. The results are shown in FIGS. 3 and 4, respectively.

Figure 3 shows an image of each neurite, and it can be seen that the length of the neurite in the nanofibers prepared in the example is longer enough to be visually confirmed. In addition, the average value of the longest neurites was calculated and shown in FIG. The results were expressed as mean ± standard error (SE). The difference between the experimental group and the control group was evaluated by one-way analysis of variance (ANOVA) and Student's t-tests using SPSS software (SPSS, version 13.0). 0.05 was evaluated as an important difference. As shown in Figure 4, it was confirmed that the length of the neurites longer in the nanofibers prepared in the example was quantitative. In addition, the longer the culture period was confirmed that the difference increases.

Claims

Reacting the carbon nanotubes with 1-butyl-3-methyl-imidazolium undeca fluoroantimonate and tetrahydrofuran to prepare carbon nanotubes-SbF 6 (step 1);

Preparing carbon nanotube-tartrate by reacting the carbon nanotube-SbF 6 with potassium sodium tartrate (step 2);

Dissolving the carbon nanotube-tartrate in a solvent to prepare a carbon nanotube-tartrate solution (step 3); And

Coating carbon nanotubes on the surface of nanofibers by dipping nanofibers in the carbon nanotube-tartrate solution (step 4)

Carbon nanotubes coated with a method for producing a nanofiber comprising a.
The method of claim 1, wherein the carbon nanotubes are single-walled carbon nanotubes, double-walled carbon nanotubes, or multi-walled carbon nanotubes.
The method of claim 1, wherein the 1-butyl-3-methyl-imidazolium undeca fluoroantimonate and tetrahydrofuran of step 1 are each 0.5-1.5: 1 based on the mole of C 6 of the carbon nanotubes. Method for producing a reaction by the molar ratio of.
The method of claim 1 wherein the potassium sodium tartrate of step 2) is SbF 6 of the carbon nanotube -SbF 6 - production method, comprising a step of reacting an equivalent ratio of 5 times to 15 times of.
The method according to claim 1, wherein the prepared carbon nanotube-tartrate of step 2 is additionally filtered, washed with water and ethanol, and dried.
The method of claim 1, wherein the solvent of step 3 is ethanol.
The method of claim 6, wherein the solvent of step 3 is further sonicated.
The method of claim 1, wherein the immersion time of step 4 is 1 second to 60 seconds.
The method according to claim 1, wherein the carbon nanotubes coated with the nanofibers prepared in step 4 are additionally washed and dried with ethanol.
The method of claim 1, wherein the nanofibers of step 4 are manufactured by electrospinning.
The method of claim 10, wherein the nanofibers are produced by electrospinning PLCL.