KR100956276B1 - Method for preparing silk fibroin nanoparticles by microemulsion - Google Patents

Abstract

The present invention relates to a method for producing silk fibroin nanoparticles and fluorescent silk fibroin nanoparticles using a water-soluble silk fibroin solution, and specifically, silk fibroin using a microemulsion method divided into an oil phase and an aqueous solution phase by a surfactant. It relates to a method for producing nanoparticles. Method for producing a silk fibroin nanoparticles, (A) dissolving a nonionic surfactant in cyclohexane to prepare an oil phase solution; (B) adding an aqueous solution containing silk fibroin to the oily solution to prepare a microemulsion; (C) removing the nonionic surfactant and crystallizing the silk fibroin to produce silk fibroin nanoparticles; (D) washing the silk fibroin nanoparticles with cyclohexane; And (E) redispersing the silk fibroin nanoparticles.

Silk fibroin, microemulsion, nanoparticle, nonionic surfactant, fluorescent dye

Description

Method for preparing silk fibroin nanoparticles using microemulsion {Method for preparing silk fibroin nanoparticles by microemulsion}

The present invention relates to a method for producing silk fibroin nanoparticles using a water-soluble silk fibroin solution and a method for producing fluorescent silk fibroin nanoparticles including a fluorescent dye therein.

The present invention relates to a method for producing silk fibroin nanoparticles using a water-soluble silk fibroin solution, and to a method for producing fluorescent silk fibroin nanoparticles including a fluorescent dye therein, and specifically, to an organic phase using phase separation of a microemulsion. Fluorescent silk nanoparticles are prepared by adding fibroin aqueous solution.

Nanoparticles have many advantages in terms of electrical, optical, mechanical, and chemical aspects, so they are applied to many technical fields such as computer and information communication, food engineering, biotechnology, and environmental industry. It is expected to lose.

Known methods for producing fine nanoparticles are known as mechanical grinding, co-precipitation, spraying, sol-gel, electrolysis, etc. The disadvantage is that it is difficult to control and costs a lot. For example, the coprecipitation method makes particles in an aqueous solution, and thus, it is impossible to control the size, shape, and size distribution of the particles. Electrolysis and sol-gel methods have disadvantages of high production cost and difficult mass production. Therefore, in recent years, many researches have been conducted on the preparation of nanoparticles using a surfactant molecular self-assembly system having a supermolecular microstructure.

Surfactant, the most representative amphiphilic material, has a constant order by interaction between hydrophilic-hydrophobic groups in a molecule in an aqueous solution or a solvent to form thermodynamically stable colloidal supramolecular aggregates. For example, monolayers, double layers, micelles, reverse micelles, microemulsions, liquid crystals, and liposomes, depending on the type of surfactant and the manufacturing technology. Various kinds of supramolecular microstructures such as) can be obtained. In particular, a nanomaterial manufacturing method using a microemulsion is known as a method for easily producing fine particles having a relatively uniform distribution.

The present invention relates to a method for producing silk fibroin nanoparticles by an inverse microemulsion method. Microemulsions are characterized by thermodynamic stability, which means that the Gibbs free energy has a negative value (ΔG <0) as compared to the separated state when the microemulsion is formed. As a result, the microemulsion is spontaneously formed so that phase separation does not occur under conditions such as temperature change. The particle size of the dispersed phase is also very small, from 5 to 140 nm, making it appear transparent or translucent in appearance. When the particle size is small, the surface area of the interface is very large and the interface tension is small, so that the microemulsion spontaneously forms unlike the macroemulsion. As a result, the energy requirements for making the emulsion can be reduced, thereby reducing the actual production cost. When preparing macroemulsions, many factors must be taken into account, such as equipment, components, mixing strength and time, emulsification time, temperature, mixing order of additives, heating / cooling rate and heat loss, but microemulsions are formed even with gentle stirring. Production costs will be greatly reduced.

Biofibrous silk fibroin proteins are expected to be used in various biomedical materials when manufacturing nanoparticles. Fibroin and sericin, which make up silk proteins, are made up of many different amino acids. Silk proteins, which consist of several amino acids, have a hydrophilic block and a hydrophobic block in the form of random or block copolymers. This unique property of silk has been reported to produce nanoparticles using silk sericin.

Particles prepared using silk sericin have been reported to produce silk polymer nanoparticles having increased stability to biofunctional peptides such as enzymes by combining PEG with a highly reactive hydrophilic portion in the block of silk polymer. The silk protein nanoparticles thus prepared form spherical particles and have also suggested the possibility of drug carriers for the delivery of hydrophobic useful substances such as retinol. In addition, International Patent Publication No. 2007/112679 discloses a method for preparing enzyme-immobilized silk fibroin nanoparticles using a water-friendly organic solvent. In addition, it has been reported that silk particles having nano size were prepared by special treatment in silk fibers (Y. Li, CWM Yuen, JY Hu, and YF Cheng, Journal of Applied Polymer Science, 2006, 100, 268-274). . However, this method has a disadvantage in that it is difficult to control the size and shape of the particles.

The present invention provides a method for producing silk fibroin nanoparticles having a certain size using a microemulsion system and a method for producing fluorescent silk nanoparticles.

It is an object of the present invention to provide a method for producing silk fibroin nanoparticles of uniform size using a microemulsion system in which phase separation occurs by a nonionic surfactant.

Another object of the present invention is to provide a method for producing fluorescent silk fibroin nanoparticles that can be used for biomedical analysis and the like by using an aqueous silk solution into which a fluorescent dye is introduced.

According to the present invention, spherical silk fibroin nanoparticles of uniform size can be prepared by phase separation from a microemulsion formed by mixing a cyclohexane oil phase including a nonionic surfactant and an aqueous phase including silk fibroin.

Silk fibroin nanoparticles can be prepared by a simple method using a silk fibroin aqueous solution using a microemulsion method divided into an oil phase and an aqueous solution phase by a nonionic surfactant, and a fluorescent silk in which fluorescent dyes are introduced through a similar manufacturing method Fibroin nanoparticles can be prepared.

According to a preferred embodiment of the present invention, the silk fibroin nanoparticles are prepared by (A) dissolving a nonionic surfactant in cyclohexane to prepare an oily solution; (B) adding an aqueous solution containing silk fibroin to the oil phase solution to prepare a microemulsion; (C) removing the nonionic surfactant and crystallizing the silk fibroin to produce silk fibroin nanoparticles; (D) washing the silk fibroin nanoparticles with cyclohexane; And (E) redispersing the silk fibroin nanoparticles.

According to another suitable embodiment of the present invention, the aqueous solution further comprises a fluorescent dye.

According to another suitable embodiment of the present invention, the nonionic surfactant is polyoxyethylene octylphenylether.

According to another suitable embodiment of the invention, the oil phase is characterized in that it contains 0.1 to 10 mmol of nonionic surfactant.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

1 is a schematic diagram showing a method for producing spherical silk fibroin nanoparticles by a microemulsion method. The present invention provides a microemulsion method through phase separation of an aqueous phase and an oil phase through mixing with a nonionic surfactant to prepare silk fibroin nanoparticles.

(A) dissolving a nonionic surfactant in cyclohexane to prepare an oily solution; (B) adding an aqueous solution in which silk fibroin is dissolved in an oil phase solution to prepare a microemulsion; (C) removing the nonionic surfactant and crystallizing the silk fibroin to produce silk fibroin nanoparticles; (D) washing the silk fibroin nanoparticles with cyclohexane; (E) redispersing the silk fibroin nanoparticles.

In the present invention, it is preferable to use cyclohexane as the oil phase.

Nonionic surfactants include nonionic ethers such as polyoxyethylene ether surfactants, propoxylate alcohols, ethoxylate block copolymers, propoxylate block copolymers, ethylene glycol esters, propylene glycol esters, sorbitan esters, water Nonionic esters, such as cross ester, polyethylene glycol, and polyoxy ethylene, nonionic amide, etc. can be used.

It is preferable to add 0.004-0.4 mmol with respect to 1 mmol of cyclohexane which are nonionic surfactants in an oil phase. When the amount of the nonionic surfactant added is 0.004 mmol or less, the spherical microemulsion may not be formed and a net structure is formed. In addition, when the amount of the nonionic surfactant added is 0.4 mmol or more, a spherical particle is formed instead of a spherical particle. Or it is also preferable to add 0.1-10 mmol with respect to 7.5 ml of cyclohexane.

Silk fibroin is a kind of silk protein that is dissolved in an aqueous solution to prepare an aqueous phase. It is preferable that an aqueous phase contains 7 to 9 weight% of silk fibroin.

The microemulsion is prepared by adding the aqueous phase while stirring the oil phase. It is preferable to mix an aqueous phase and an oil phase in the ratio of 90:10-99: 1 volume ratio. The prepared microemulsion forms nano sized droplets by phase separation and is stabilized by a nonionic surfactant dissolved in an oil phase.

Since the prepared microemulsion droplets contain nonionic surfactants, the nonionic surfactants must be washed. Since nonionic surfactants act as stabilizers, removing them requires crystallization of silk fibroin. The solvent mixed with methanol and ethanol at 5: 5 can be used to prepare the nonionic surfactant and crystallize the silk fibroin. Silk fibroin has a property of crystallizing with methanol, and since nonionic surfactants are miscible with ethanol, it is preferable to use a mixed solvent of methanol and ethanol.

It is also washed once more sufficiently with cyclohexane to completely remove the nonionic surfactants contained in the oil phase.

The washed micro emulsion was dried at room temperature for 24 hours and then vacuumed at room temperature for 24 hours to obtain silk fibroin nanoparticles. The prepared silk fibroin nanoparticles were put in deionized distilled water and dispersed for 30 seconds in an ultrasonic generator (Ultrasonicator, 25 ° C, 28 kHz, 600 W) to prepare spherical silk fibroin nanoparticles dispersed in an aqueous solution.

Method for producing a fluorescent silk nanoparticles, (A) dissolving a nonionic surfactant in cyclohexane to prepare an oil phase solution; (B) preparing a silk fibroin / fluorescent dye aqueous solution by mixing a fluorescent dye aqueous solution with an aqueous solution of silk fibroin; (C) adding a microfibroin / fluorescent dye aqueous solution to the oily solution to prepare a microemulsion; (D) removing the nonionic surfactant and crystallizing the fluorescent silk fibroin to produce fluorescent silk fibroin nanoparticles; (E) washing the fluorescent silk fibroin nanoparticles with cyclohexane; And (G) redispersing the fluorescent silk fibroin nanoparticles.

It is the same as the method for producing silk fibroin nanoparticles except that a silk fibroin / fluorescent dye aqueous solution is prepared by mixing a fluorescent dye aqueous solution with an aqueous silk fibroin solution. Silk fibroin aqueous solution and fluorescent dye aqueous solution is preferably prepared by mixing in a weight ratio of 1: 1 to 1: 1000.

The fluorescent dyes include lanthanum chelates containing europium, terbium, samarium and dysprosium, general fluorescent organic dyes containing cyanines and rhodamines, colloidal metals, phosphor particles or inorganic semiconductor nanocrystals. Can be. The fluorescent dye aqueous solution is preferably prepared at a concentration of 0.01 ~ 10mg / ml.

Characterization of Silk Spherical Particles and Fluorescent Silk Spherical Particles

The surface characteristics of the silk fibroin nanoparticles and the silk fibroin nanoparticles into which fluorescent dyes were introduced were observed using a field emission scanning electron microscope (S-4300, Hitachi, Japan). Scanning electron micrographs were taken after the sample was placed on an aluminum SEM disc to be fixed and coated with platinum (Pt) particles by sputtering. Measurements were made at an acceleration voltage of 15 kV and an operating distance of 3.2 mm.

Measurement was performed using a Confocal Laser Scanning Microscope to confirm that the fluorescent dye was introduced into the silk fibroin nanoparticles. Confocal laser scanning microscopy was used to measure fluorescent dyes present in fluorescent silk fibroin nanoparticles using an Argon / Krypton laser using an MRC-1024 instrument (Bio-Rad Laboratories, Inc., UK).

Example

Example 1 Preparation of Silk Nanoparticles

First, an oil phase is prepared by dissolving 1.77 g (2.73 mmol) of triton X-100 (manufactured by Union carbide, polyoxyethylene octyl phenyl ether), which is a nonionic surfactant, in 7.5 mL (69.4 mmol, 5.84 g) of cyclohexane. . The aqueous phase uses about 8 wt% silk aqueous solution. A microemulsion is prepared by adding 100 μL of the aqueous silk solution while stirring the prepared oil phase at 1000 rpm. The prepared microemulsion forms nano-sized droplets by phase separation, which is stabilized by Triton X-100, a nonionic surfactant dissolved in an oil phase.

Next, the microemulsion is washed with 10 ml of a solvent in which methanol and ethanol are 5: 5 (volume ratio) and mixed. Silk fibroin nanoparticles are also prepared by thoroughly washing and then redispersing with cyclohexane for complete removal of the nonionic surfactant, a stabilizer contained in the oil phase.

Example 2-Preparation of Fluorescent Silk Nanoparticles

Rhodamine B isothiocyanate-dextran, a fluorescent dye, was dissolved in distilled water to prepare a concentration of 0.01 mg / ml. The prepared fluorescent dye aqueous solution and the 8wt% silk fibroin aqueous solution was prepared in a 1: 1 (volume ratio) mixing ratio to prepare a silk fibroin aqueous solution into which fluorescent dyes were introduced. A fluorescent silk fibroin spherical particle in which a fluorescent dye was introduced into the silk fibroin nanoparticles was prepared in the same manner as in Example 1.

Surface Properties of Silk Fibroin Nanoparticles and Fluorescent Silk Fibroin Nanoparticles

Surface properties of the silk fibroin nanoparticles and the fluorescent silk fibroin nanoparticles prepared according to the above example were measured and shown in FIGS. 2 and 3.

2 is a photograph of spherical silk fibroin nanoparticles observed with a field emission scanning electron microscope. Silk fibroin nanoparticles are spherical, have a uniform size of 1-1000 nm and a smooth surface.

3 is a photograph of spherical fluorescent silk fibroin nanoparticles observed with a field emission scanning electron microscope. Fluorescent silk fibroin nanoparticles are spherical and have a uniform size of 1-1000 nm. However, when the fluorescent dye is introduced, it can be seen that the surface is relatively uneven compared to the silk fibroin nanoparticles, and it can be seen that the pores are formed on the surface of the particles. In addition, when compared with the silk fibroin nanoparticles it can be seen that relatively close to the oval.

Spherical silk fibroin  Nanoparticles and Fluorescence silk fibroin  Structural Properties of Nanoparticles, Stability of Fluorescent Dyes, and Location of Fluorescent Dyes

Silk fibroin nanoparticles and fluorescent silk fibroin nanoparticles were prepared using a microemulsion method as presented in the present invention. Fourier transform infrared spectra were used to confirm whether the prepared silk fibroin nanoparticles and the fluorescent silk fibroin nanoparticles had the characteristics of silk fibroin and were introduced into the fluorescent silk fibroin nanoparticles through the characteristic structure represented by the fluorescent dye. .

FIG. 4 shows Fourier transform infrared spectral curves of silk fibroin nanoparticles according to the present invention (Example 1), fluorescent silk fibroin nanoparticles (Example 2), and fluorescent dye materials observed in the silk fibroin characteristic structure region. Silk fibroin nanoparticles and fluorescent silk fibroin nanoparticles can be seen to show the characteristic structure of silk fibroin, respectively, and it can be seen that there is no change in the silk fibroin characteristic structure by the fluorescent dye introduced.

FIG. 5 shows Fourier transform infrared spectral curves of the silk fibroin nanoparticles (Example 1), the fluorescent silk fibroin nanoparticles (Example 2), and the fluorescent dye material according to the present invention observed in the characteristic structure region of the fluorescent dye. . When comparing the silk fibroin nanoparticles with the fluorescent silk fibroin nanoparticles, the characteristic structure of the fluorescent dye can be confirmed only in the spectral curve of the fluorescent silk fibroin nanoparticles. You can check it.

6 is a photograph of spherical fluorescent silk fibroin nanoparticles observed by confocal laser scanning microscope. Fluorescence images represented by fluorescent dyes in fluorescent silk fibroin nanoparticles were measured. As a result of measurement by confocal laser scanning microscope, it can be confirmed that fluorescent dye is introduced into spherical fluorescent silk fibroin nanoparticles.

Figure 7 shows the amount of accumulation of fluorescent particles emitted through dialysis in a curve to confirm the stability of the fluorescent silk nanoparticles according to the present invention can be used in the fluorescent probe. What can be confirmed through the dialysis process is that since the fluorescent particles are stably introduced into the silk fibroin nanoparticles, it can be confirmed that there is little change over time.

According to the present invention, silk fibroin nanoparticles can be prepared by a simple method using an aqueous solution of silk fibroin by using a microemulsion phase separation method divided into an oil phase and an aqueous solution phase by a nonionic surfactant, and fluorescence through a similar manufacturing method. Fluorescent silk fibroin nanoparticles into which dyes have been introduced can be prepared.

The embodiments presented above are exemplary and one of ordinary skill in the art may make various modifications and modifications to the embodiments presented without departing from the spirit of the present invention. Such modifications and variations are not intended to limit the scope of the invention.

According to the manufacturing method of the present invention, it is possible to manufacture silk fibroin, which has conventionally been difficult to produce spherical nanoparticles of uniform size, through a simple and efficient method. In addition, the spherical fluorescent silk fibroin nanoparticles prepared by introducing a fluorescent dye on the aqueous solution of silk fibroin may be widely applied to various applications depending on the application. For example, it may be used for biomedical analysis and the like through fluorescent silk fibroin nanoparticles in which fluorescent dyes are introduced using silk fibroin having excellent biocompatibility. In addition, by using the kind of fluorescent dyes to be introduced into the silk fibroin can be used in a wide range of applications.

1 is a schematic diagram showing the manufacturing process of the spherical silk fibroin nanoparticles according to the present invention.

2 is a field emission scanning electron micrograph of silk fibroin nanoparticles prepared according to the present invention.

3 is a field emission scanning electron micrograph of fluorescent silk fibroin nanoparticles prepared using an aqueous fluorescent material / silk fibroin solution according to the present invention.

Figure 4 shows a Fourier transform infrared spectral curve observed in the silk fibroin characteristic structure region of the silk fibroin nanoparticles, fluorescent silk fibroin nanoparticles and fluorescent dye material according to the present invention.

FIG. 5 shows Fourier transform infrared spectral curves of the silk fibroin nanoparticles, the fluorescent silk fibroin nanoparticles, and the fluorescent dye material according to the present invention observed in the characteristic structure region of the fluorescent dye.

6 is a photograph measured using a confocal laser scanning microscope to confirm that the fluorescent dye material in the fluorescent silk nanoparticles according to the present invention is included.

Figure 7 shows the amount of accumulation of fluorescent particles emitted through dialysis in a curve to confirm the stability of the fluorescent silk nanoparticles according to the present invention can be used in the fluorescent probe.

Claims (4)

In the method for producing silk fibroin nanoparticles, (A) dissolving a nonionic surfactant in cyclohexane to prepare an oily solution; (B) adding an aqueous solution containing silk fibroin and a fluorescent dye to the oil phase solution to prepare a microemulsion; (C) removing the nonionic surfactant and crystallizing the silk fibroin to produce silk fibroin nanoparticles; (D) washing the silk fibroin nanoparticles with cyclohexane; And (E) a method for producing silk fibroin nanoparticles comprising redispersing silk fibroin nanoparticles. delete The method of claim 1, wherein the nonionic surfactant is polyoxyethylene octylphenylether. The method of claim 1, wherein the oil phase contains 0.004 to 0.4 mmol of a nonionic surfactant with respect to 1 mmol of cyclohexane.

Images