KR100973779B1 - Methods of preparing Fibroin Microsphere, Emulsion including the same and Dispersion Liquid including the same - Google Patents

Abstract

The present invention provides fibroin microspheres, emulsions containing fibroin microspheres and methods for preparing dispersions comprising fibroin microspheres. A mixed solution is prepared by adding a fibroin aqueous solution containing fibroin and water to a hydrophobic solution containing an organic solvent and a surfactant. The mixed solution is stirred to prepare an emulsion containing fibroin microspheres. The emulsion containing fibroin microspheres can stably maintain fibroin having a property of easily coagulating in water or an organic solvent without aggregation for a long time.

Description

Fibroin microspheres, emulsions comprising the same and methods for preparing dispersions comprising the same {Methods of preparing Fibroin Microsphere, Emulsion including the same and Dispersion Liquid including the same}

The present invention relates to a process for preparing fibroin microspheres, emulsions containing fibroin microspheres and dispersions comprising fibroin microspheres. More particularly, the present invention relates to a method for preparing fibroin microspheres, emulsions containing fibroin microspheres and dispersions containing fibroin microspheres, which can be stably preserved for a long period of time.

Proteins or peptides can show excellent drug efficacy in small amounts, have fewer side effects, and have a unique physiological activity. In addition, in the case of proteins or peptides having a specific function, it is included in cosmetics, as well as drugs, may be prepared and used in an emulsion state. However, because such proteins or peptides are unstable, it is difficult to maintain proper concentrations in the manufacture of pharmaceuticals, and can be easily degraded by external stimuli during storage.

On the other hand, fibroin is a kind of silk protein, and is known as a material having excellent biocompatibility and which does not adversely affect any surrounding tissue. Therefore, beverages, cosmetics and dressings or films including the fibroin have been developed. In addition, the fibroin can be crystallized in a β-sheet structure, so that the physical strength is strong and excellent in stability compared to other proteins.

As described above, the fibroin is developed as a drug delivery particle for delivering the protein or peptide to protect other relatively unstable proteins or peptides that can function as drugs because the fibroin is not only biocompatible but also structurally stable. Efforts are being made a lot.

In order for the fibroin to function as a drug delivery particle, it must be able to maintain a stable state for a long time without decomposition in solution or air, and it is required to have an appropriate size and shape as necessary. In particular, in order to be used as a substance capable of delivering the drug, a technique for preparing nano or micro sized fibroin particles is particularly necessary.

However, dissolving the fibroin in water or oil or fat easily forms a gel. Therefore, when preparing a solution including the fibroin, fibroin is not uniformly dispersed in the solution and the like, it is easy to form agglomerates, and it is not easy to prepare to have a desired shape. In addition, since the gel is easily formed as described above in water or fat or oil, it is not easy to prepare fibroin particles having a desired size.

Therefore, there is a need for a method for stably dispersing the fibroin in a solution, maintaining a stable state in the air, and easily controlling the size and decomposition rate or decomposition time of the fibroin.

It is therefore an object of the present invention to provide a method for preparing an emulsion comprising fibroin microspheres in which fibroin is stably dispersed without aggregation.

It is another object of the present invention to provide a method for preparing fibroin microspheres having excellent biocompatibility.

Still another object of the present invention is to provide a method for preparing a dispersion including fibroin microspheres stably dispersed in an aqueous solution.

The method for preparing an emulsion including fibroin microspheres according to embodiments for achieving the above object of the present invention prepares a hydrophobic solution comprising an organic solvent and a surfactant. Fibroin aqueous solution containing fibroin and water is added to the hydrophobic solution to prepare a mixed solution, and the mixed solution is stirred to prepare an emulsion containing fibroin microspheres.

In embodiments of the present invention, the hydrophobic solution may include the organic solvent and the surfactant in a volume ratio of about 1: 0.07 to 0.4.

In embodiments of the present invention, the fibroin aqueous solution may include about 0.5% to about 3.5% by weight of the fibroin.

In embodiments of the present invention, the fibroin aqueous solution may further include a metal salt or an ammonium salt.

In embodiments of the present invention, the fibroin aqueous solution may further NaCl, NaHCO ₃ , KCl, MgCl ₂ LiCl, (NH ₄ ) ₂ SO ₄ , NH ₄ Cl, KH ₂ PO ₄ or Ca ₃ (PO ₄ ) 2 It may include.

In embodiments of the present invention, the fibroin aqueous solution is about NaCl, NaHCO ₃ , KCl, MgCl ₂ LiCl, (NH ₄ ) ₂ SO ₄ , NH ₄ Cl, KH ₂ PO ₄ or Ca ₃ (PO ₄ ) 2 About It may comprise 1.5 wt% or less.

In embodiments of the present invention, the fibroin aqueous solution may be added to the hydrophobic solution at a rate of about 0.5 mL / min to about 1.5 mL / min.

In embodiments of the present invention, the mixed solution may include the hydrophobic solution and the fibroin aqueous solution in a volume ratio of about 1: 0.05 to 0.15.

The method for preparing fibroin microspheres according to the embodiments for achieving the above-described other object of the present invention prepares a hydrophobic solution containing an organic solvent and a surfactant. Fibroin aqueous solution containing fibroin and water is added to the hydrophobic solution to prepare a mixed solution, and the mixed solution is stirred to prepare an emulsion containing fibroin microspheres. The emulsion is dried to produce fibroin microspheres.

In embodiments of the present invention, the method may further include filtering the emulsion before drying the emulsion.

In embodiments of the present invention, the emulsion may be naturally dried at room temperature or forcedly dried using an evaporator.

In embodiments of the present invention, the fibroin microspheres may further comprise the step of washing with alcohol.

In embodiments of the present invention, the fibroin microspheres may have a diameter of about 4 ㎛ to about 40 ㎛.

According to another aspect of the present invention, a method of preparing a dispersion including fibroin microspheres includes preparing a hydrophobic solution including an organic solvent and a surfactant. Fibroin aqueous solution containing fibroin and water is added to the hydrophobic solution to prepare a mixed solution, and the mixed solution is stirred to prepare an emulsion containing fibroin microspheres. The emulsion is dried to prepare fibroin microspheres, and the fibroin microspheres are dispersed in water or an acidic aqueous solution to prepare a dispersion including the fibroin microspheres.

The emulsion containing the fibroin microspheres according to the present invention described above can maintain the fibroin in a hydrophobic solution for a long time without aggregation. In addition, the fibroin microsphere can easily adjust the diameter and the like through the stirring speed of the emulsion and the drying method of the emulsion. In addition, the dispersion containing the fibroin microspheres can be maintained for a long time without agglomerated the fibroin in the aqueous solution stably. In addition, when the fibroin microspheres are prepared, when appropriate additives are added, a large number of pores may be generated on the surface of the fibroin microspheres in the water or an acidic aqueous solution, or the fibroin microspheres may be broken and controlled to have a hemispherical shape. can do.

As the inventive concept allows for various changes and numerous modifications, the embodiments will be described in detail in the text. However, this is not intended to limit the present invention to a specific disclosed form, it should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. Like reference numerals are used for like elements in describing each drawing. The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting of the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting of the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this application, the terms "comprise" or "consist of" are intended to indicate that there is a feature, number, step, operation, component, part, or combination thereof described on the specification, but one or more other features. It should be understood that they do not exclude in advance the possibility of the presence or addition of numbers, numbers, steps, components, parts or combinations thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art. Terms such as those defined in the commonly used dictionaries should be construed as having meanings consistent with the meanings in the context of the related art, and, unless expressly defined in this application, are construed in ideal or excessively formal meanings. It doesn't work.

Hereinafter, a method of preparing an emulsion including fibroin microspheres and a dispersion including fibroin microspheres according to embodiments of the present invention will be described in detail.

fibroin Microspheres  Containing Emulsion  Manufacturing method

1 is a flowchart illustrating a method of preparing an emulsion containing fibroin microspheres according to embodiments of the present invention.

According to the method for preparing an emulsion containing the fibroin microsphere of the present invention, a hydrophobic solution is prepared by mixing an organic solvent and a surfactant (S10). The mixed solution is prepared by adding an aqueous solution of fibroin including fibroin and water to the hydrophobic solution (S20). The mixture is stirred to prepare an emulsion containing fibroin microspheres (S30).

Referring to FIG. 1, a hydrophobic solution including an organic solvent and a surfactant is prepared (S10).

The organic solvent serves to impart hydrophobicity to the hydrophobic solution. Therefore, the organic solvent is not particularly limited as long as it is a material that is not mixed with a hydrophilic material.

According to embodiments of the present invention, the organic solvent may be a hydrocarbon compound including an oil, an aliphatic saturated hydrocarbon having 5 or more carbon atoms, an aromatic hydrocarbon having 6 or more carbon atoms, or a hydrocarbon-based polymer.

The oil may be a vegetable oil, an animal oil or a synthetic oil. For example, the oil may be a polyalcohol ester oil, almond oil, apricot seed oil, avocado oil, theobroma oil, carrot seed oil, castor oil, citrus seed oil, coconut oil, corn oil, cottonseed oil, cucumber oil, egg oil, Jojoba oil, lanolin oil, linseed oil, mineral oil, mink oil, olive oil, palm oil, peanut oil, rape seed oil, safflower oil, sesame oil, shark liver oil, soybean oil, sunflower seed oil, almond oil, whale oil, squalene or wheat germ oil And so on.

The hydrocarbon compound is not particularly limited as long as it is an aliphatic saturated hydrocarbon having a carbon number of 5 or more, an aromatic hydrocarbon having a carbon number of 6 or more, or a hydrocarbon polymer, and a compound having hydrophobicity such that it is not mixed with water. Do not. For example, the hydrocarbon compound may be a polymer such as polyisobutene, silicone polymer, alkylsilicone polymer, acrylic polymer, acrylic silicone polymer, urethane polymer, and the like, octane, nonane, decane, undecane, dodecane, tri It may be an aliphatic hydrocarbon of alkanes, alkenes or alkynes having 5 or more carbon atoms such as decane, tetradecane, pentadecane, hexadecane, heptadecane or octadecane, and aromatic hydrocarbons such as benzene, toluene, aniline or xylene, and the like. Can be.

According to embodiments of the present invention, the surfactant functions as an emulsifier such that the prepared emulsion does not separate the hydrophilic layer and the hydrophobic layer from each other. Therefore, the surfactant is not particularly limited as long as it can perform the function of the emulsifier, it may be used by mixing two or more types of surfactant. For example, the surfactant may be polyoxyalkylene alkyl ether, polyoxyalkylene alkyl phenol ether, polyoxyalkylene aryl phenol ether, polyoxyalkylene fatty acid ester, polyoxyalkylene sorbitan fatty acid ester, polyoxyalkyl Nonionic surfactants such as ethylene alkyl amines, sorbitan fatty acid esters, alkyl alcohol amines or aryl alcohol amines.

According to embodiments of the present invention, the hydrophobic solution may be prepared by adding the surfactant to the organic solvent in a volume ratio of about 1: 0.07 to 0.4. If the volume ratio of the surfactant to the organic solvent is less than about 0.07, the fibroin microspheres may not be produced even when an emulsion is prepared by adding an aqueous solution of fibroin. On the other hand, when the volume ratio of the surfactant to the organic solvent exceeds about 0.4, aggregation may occur between the generated fibroin microspheres. When the volume ratio of the organic solvent and the surfactant is about 1: 0.2 to 0.3, the emulsion is dried to obtain the fibroin microspheres, and then the microspheres can be maintained even when redispersed in water or an aqueous solution. Excellent in terms of Therefore, the hydrophobic solution may be prepared by mixing the organic solvent and the surfactant in a ratio of about 1: 0.07 to 0.4, preferably, by mixing in a ratio of about 1: 0.2 to 0.3.

The mixed solution is prepared by adding a fibroin aqueous solution containing the fibroin and water to the hydrophobic solution (S20). The fibroin aqueous solution exhibits hydrophilicity unlike the hydrophobic solution. When the hydrophilic aqueous solution of the fibroin is slowly added to the hydrophobic solution, the hydrophobic solution may contain a surfactant, and thus a mixed solution in which the fibroin aqueous solution component is dispersed in the hydrophobic solution may be formed without layer separation.

According to embodiments of the present invention, the fibroin aqueous solution may include about 0.5% to about 3.5% by weight of the fibroin. When the fibroin aqueous solution contains less than about 0.5 wt% of the fibroin, the fibroin microspheres may not be formed. On the other hand, if the aqueous solution of the fibroin includes more than about 3.5% by weight of the fibroin, while the fibroin agglomeration occurs, the production yield of the fibroin microspheres may be reduced.

When the aqueous solution of fibroin contains about 1.5 to about 2.5% by weight of the fibroin, the emulsion is dried to obtain a fibroin microsphere, and then, even when redispersed again in water or an acidic aqueous solution, the fibroin microsphere does not decompose for a long time. You can keep the shape.

Thus, the aqueous solution of fibroin may include about 0.5 wt% to about 3.5 wt% of the fibroin, and preferably about 1.5 wt% to about 2.5 wt% of the fibroin.

According to embodiments of the present invention, the mixed solution may be prepared by mixing the hydrophobic solution and the fibroin aqueous solution in a volume ratio of about 1: 0.05 to about 0.15. If the volume ratio of the aqueous solution of fibroin to the hydrophobic solution is less than about 0.05, the content of the fibroin is too low may not form the fibroin microspheres. On the other hand, when the volume ratio of the fibroin aqueous solution to the hydrophobic solution exceeds about 0.15, the formed fibroin microspheres may aggregate with each other.

According to embodiments of the present invention, the fibroin aqueous solution may further include a metal salt or an ammonium salt. If the fibroin aqueous solution further comprises the metal salt or ammonium salt, it is possible to prepare a dispersion containing a microspheres of porous or hemispherical fibroin. Therefore, when the fibroin aqueous solution includes the metal salt or the ammonium salt, the fibroin microsphere is destroyed without applying external pressure, or a hole is formed in the fibroin microsphere surface to discharge the material inside the fibroin microsphere. You can.

The metal salt or ammonium salt is not particularly limited in kind. For example, the metal salt may include NaCl, NaHCO ₃ , KCl, MgCl _{2 ,} KH ₂ PO ₄ , Ca ₃ (PO ₄ ) _2, or LiCl. Examples of the ammonium salt include (NH ₄ ) ₂ SO ₄ or NH ₄ Cl.

According to embodiments of the present invention, the fibroin aqueous solution may include about 1.5 wt% or less of the metal salt or ammonium salt. If the fibroin aqueous solution contains more than about 1.5% by weight of the metal salt or ammonium salt, the fibroin microspheres can be easily broken during the drying of the emulsion.

According to embodiments of the present invention, the fibroin aqueous solution may be added to the hydrophobic solution at a rate of 0.5 mL / to about 1.5 mL / minute or less. Fibroin microspheres may not be formed when the addition rate of the fibroin aqueous solution is slower than about 0.5 mL / min. On the other hand, if the addition rate of the fibroin aqueous solution is faster than about 1.5mL / min, the resulting fibroin microspheres can be aggregated with each other without being dispersed in the hydrophobic solution.

The mixture is stirred to prepare an emulsion containing fibroin microspheres (S30). When the mixed solution is stirred, an emulsion containing fibroin microspheres may be prepared while the hydrophilic fibroin aqueous solution is uniformly dispersed throughout the hydrophobic solution.

According to embodiments of the present invention, the diameter of the fibroin microspheres may be adjusted according to the stirring speed of the mixed solution. When the emulsion is stirred at about 600 rpm, the fibroin microspheres may have a diameter of about 15 μm to about 35 μm, and when the mixed solution is stirred at about 700 rpm, the fibroin microspheres may have a diameter of about 12 μm to about 29 μm When the mixed solution is stirred at about 800rpm, the fibroin microspheres may have a diameter of about 9 μm to about 24 μm. When the mixed solution is stirred at a speed exceeding about 800 rpm, fibroin microspheres having a flat shape or a pressed shape may be formed, and thus it may be difficult to control the shape or diameter to an appropriate degree. Therefore, the stirring speed of the emulsion can be adjusted appropriately, but preferably can be stirred at a speed of about 800rpm or less.

The emulsion comprising the fibroin microspheres prepared by the above method is uniformly dispersed in the hydrophobic solution without fibrinating with each other for a long time, so that the fibroin can be stably maintained. In addition, it is possible to adjust the diameter of the fibroin microspheres and the like by the stirring speed of the emulsion, it is possible to easily form a fibroin microsphere having a desired diameter.

fibroin Microspheres  Manufacturing method

According to the method for preparing the fibroin microsphere of the present invention, a hydrophobic solution is prepared by mixing an organic solvent and a surfactant. Fibroin aqueous solution containing fibroin and water is added to the hydrophobic solution to prepare a mixed solution. The mixture is stirred to prepare an emulsion containing fibroin microspheres. The emulsion is dried to give fibroin microspheres.

An aqueous solution containing fibroin microspheres is added to the hydrophobic solution containing the organic solvent and the surfactant, followed by stirring, followed by stirring with a fibroin solution containing fibroin and water. When the hydrophilic fibroin aqueous solution is added to the hydrophobic solution as described above at an appropriate content and at an appropriate addition rate, an emulsion in which micro-sized fibroin spheres are uniformly distributed can be prepared. Since the emulsion containing the fibroin microsphere of the present invention has been described in detail above, further detailed description will be omitted in order to avoid duplication.

According to embodiments of the present invention, the emulsion containing the fibroin microspheres may be filtered before the emulsion is dried. By the filtration it is possible to obtain the fibroin microspheres containing a large amount of the organic solvent and the surfactant. The filtration method is not limited as long as the fibroin microspheres are not destroyed. For example, the emulsion may be a filter or the like. However, the filtration process is to shorten the drying time, and may be omitted for simplicity of the process.

The emulsion is dried to give fibroin microspheres. The drying allows the organic solvent and the surfactant component contained in the emulsion to evaporate to obtain the fibroin microspheres.

According to one embodiment of the invention, the emulsion may be naturally dried at room temperature. When the emulsion is naturally dried at room temperature, fibroin microspheres having an average diameter of about 10 μm to about 40 μm can be obtained. According to another embodiment of the invention, the emulsion may be forced to dry using an evaporator. Forced drying of the emulsion using an evaporator yields fibroin microspheres with an average diameter of about 4 μm to about 20 μm. Therefore, the size of the fibroin microspheres can be adjusted according to the drying method.

According to embodiments of the present invention, the fibroin microspheres obtained by drying the emulsion may be washed with alcohol. The washing may further remove the organic solvent component and the surfactant component remaining in the fibroin microspheres. For example, the fibroin microspheres can be washed using ethanol, methanol or isopropanol and the like. However, the process of washing the obtained microspheres may be omitted when the organic solvent and the surfactant are completely removed by drying in the fibroin microspheres.

According to an embodiment of the present invention, the fibroin microspheres may have a structure in which the inside of the mycross sphere is filled with fibroin. According to another embodiment of the present invention, when the fibroin aqueous solution further includes a metal salt or an ammonium salt, the fibroin microspheres are located in the internal space of the fibroin microspheres with the metal salts. Therefore, when the fibroin microspheres are dispersed in water or an aqueous solution or the like, the fibroin microspheres may be formed while the materials are released from the fibroin microspheres.

According to embodiments of the present invention, the fibroin microspheres are not well degraded by proteolytic enzymes. When the fibroin microspheres obtained as described above are brought into contact with proteolytic enzymes, a change in the roughness of the surface of the fibroin microspheres occurs, but the fibroin microspheres can be stably maintained in prolonged contact.

The fibroin microspheres produced by the method of the present invention can be preserved in a dried state without breaking for a long time. In addition, the diameter of the fibroin microspheres can be appropriately adjusted by adjusting the drying method of the emulsion. In addition, by changing the addition of other substances to the aqueous solution of fibroin and the type of the added substance, the internal structure of the fibroin microspheres can be appropriately controlled, and not greatly degraded against various proteolytic enzymes capable of degrading fibroin. Can maintain a stable state.

Method for producing a dispersion containing fibroin microspheres

According to the method for preparing the dispersion containing the fibroin microsphere of the present invention, a hydrophobic solution is prepared by mixing an organic solvent and a surfactant. Fibroin aqueous solution containing fibroin and water is added to the hydrophobic solution to prepare a mixed solution. The mixture is stirred to prepare an emulsion containing fibroin microspheres. The emulsion is dried to give the fibroin microspheres. The obtained fibroin microspheres are dispersed in water or an acidic aqueous solution to prepare a dispersion containing the fibroin microspheres.

Fibroin aqueous solution containing fibroin and water is added to the hydrophobic solution containing the organic solvent and the surfactant, followed by stirring to prepare an emulsion containing the fibroin microsphere and drying the emulsion to obtain a fibroin microsphere. Since the fibroin microsphere of the present invention has been described in detail above, further detailed description will be omitted in order to avoid duplication.

The obtained fibroin microspheres are dispersed in water or an acidic aqueous solution to prepare a dispersion containing the fibroin microspheres.

According to an embodiment of the present invention, when the fibroin aqueous solution does not include a metal salt or an ammonium salt, the fibroin microspheres as described above may be uniformly dispersed in water and maintain a stable shape without decomposing for a considerable period of time. Can be.

According to another embodiment of the present invention, when the fibroin aqueous solution includes a metal salt or an ammonium salt, the fibroin microspheres may have porosity or large holes in the fibroin microspheres or may have a hemispherical shape.

For example, when the fibroin aqueous solution includes NaCl, pores may be formed on a surface of the fibroin microsphere dispersed in water. When the fibroin aqueous solution containing the NaCl is used, precipitation of NaCl occurs inside the fibroin microspheres while the emulsion is dried. As the NaCl precipitate adheres to the surface of the microsphere, pressure is applied to the surface of the fibroin microsphere. As a result, when the fibroin microspheres are dispersed in water, the NaCl may be dissolved to generate pores on the surface of the fibroin microspheres.

When the fibroin aqueous solution includes NaHCO ₃ , the fibroin microspheres prepared may be dispersed in an acidic aqueous solution. When the fibroin microspheres prepared using the aqueous solution of fibroin including NaHCO ₃ are dispersed in an acidic aqueous solution, large holes are generated on the surface of the fibroin microsphere, or the fibroin microsphere has a hemispherical shape. NaHCO ₃ in the fibroin microsphere Carbon dioxide gas is generated when it comes into contact with an acidic substance in an acidic aqueous solution. As the carbon dioxide gas is discharged from the fibroin microsphere, a large hole may be formed on the surface of the fibroin microsphere, or a fibroin microsphere having a hemispherical shape may be formed.

The dispersion containing the fibroin microspheres prepared according to the method of the present invention can maintain the fibroin microspheres in a stable state for a long time in water or an aqueous solution. In addition, when the aqueous solution of fibroin further comprises a material such as a metal salt or an ammonium salt, it is possible to prepare a microsphere having a pore or hemispherical fibroin. By manufacturing the fibroin microspheres in addition to other materials in addition to the fibroin, the fibroin microspheres can be easily decomposed under an aqueous solution while maintaining a stable state. Therefore, the dispersion containing the fibroin microspheres can be usefully used to deliver the desired material where necessary.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples. However, the following examples are provided to illustrate the present invention, and the present invention is not limited to the following examples and may be variously modified and changed.

fibroin Microspheres  Containing emulsion

Example 1

A hydrophobic solution was prepared by adding about 5 mL of a surfactant Span 80 (Sigma Aldrich, USA) and about 5 mL of Tween 80 (Sigma Aldrich, USA) to about 40 mL of n-decane (n-decane, Sigma Aldrich, USA). About 5 mL of an aqueous solution of fibroin containing about 2% by weight of fibroin (Fineco Co. Ltd., Korea) was added dropwise to the hydrophobic solution at a rate of about 1 mL / min to prepare a mixed solution. The mixture was stirred at a speed of about 600 rpm to prepare an emulsion containing fibroin microspheres.

Example 2

An emulsion containing fibroin microspheres was prepared in substantially the same manner as in Example 1 except that Span 80 and Tween 80, each of surfactants, were used.

Example 3

An emulsion including fibroin microspheres was prepared in substantially the same manner as in Example 1 except that 4 mL of surfactants Span 80 and Tween 80 were used.

Example 4

An emulsion including fibroin microspheres was prepared in substantially the same manner as in Example 1 except that Span 80 and Tween 80, each of surfactants, were used.

Example 5

An emulsion comprising fibroin microspheres was prepared in substantially the same manner as in Example 1 except that an aqueous solution of fibroin containing about 1 wt% fibroin was used.

Example 6

An emulsion comprising fibroin microspheres was prepared in substantially the same manner as in Example 1 except that an aqueous solution of fibroin containing about 3 wt% fibroin was used.

Example 7

An emulsion containing fibroin microspheres was prepared in substantially the same manner as in Example 1 except that the mixed solution in which the fibroin aqueous solution was added to the hydrophobic solution was stirred at about 700 rpm.

Example 8

An emulsion containing fibroin microspheres was prepared in substantially the same manner as in Example 1 except that the mixed solution in which the fibroin aqueous solution was added to the hydrophobic solution was stirred at about 800 rpm.

Example 9

An emulsion comprising fibroin microspheres was prepared in substantially the same manner as in Example 1 except that the aqueous solution of fibroin contained about 2 wt% fibroin and about 1 wt% NaCl.

Example 10

An emulsion comprising fibroin microspheres was prepared in substantially the same manner as in Example 1 except that the aqueous solution of fibroin included about 2 wt% fibroin and about 1 wt% NaHCO ₃ .

Comparative Example 1

An emulsion including fibroin microspheres was prepared in substantially the same manner as in Example 1 except that the surfactants Span 80 and Tween 80 were not used.

Comparative Example 2

An emulsion including fibroin microspheres was prepared in substantially the same manner as in Example 1 except that Span 80 and Tween 80, each of surfactants, were used.

Comparative Example 3

An emulsion containing fibroin microspheres was prepared in substantially the same manner as in Example 1 except that an aqueous solution of fibroin containing about 4 wt% fibroin was used.

The content of each component used in the preparation of the emulsion of Examples 1 to 10 and Comparative Examples 1 to 3 is shown in Table 1 below.

Experimental Example  One

The emulsion of Example 1, Example 2, Example 3, Comparative Example 1 and Comparative Example 2 was observed using an optical microscope (CX40, Olympus, Japan), the results are shown in Figures 2a to 2c. FIG. 2A shows the emulsion of Example 2, FIG. 2B shows Example 3, and FIG. 2C shows the emulsion of Example 4. FIG.

In the case of the emulsions of Comparative Examples 1 and 2, fibroin microspheres were not observed. Thus, it was found that fibroin microspheres are not formed when the content of Span 80 and Tween 80 is about 0.05 of the volume of n-decane or does not include the surfactant of Span 80 and Tween 80.

On the other hand, as can be seen in Figures 2a to 2c, the content of the Span 80 and Tween 80 is about 0.1 of the volume of the n- decane. In the case of 0.2 and 0.3, fibroin microspheres were produced, and it can be seen that the fibroin microspheres are dispersed in the solution stably without aggregation with each other.

Experimental Example 2

The emulsion of Example 1, Example 5, Example 6 and Comparative Example 3 was observed using an optical microscope (manufacturer), the results are shown in Figures 3a to 3d. 3A corresponds to Example 5, FIG. 3B corresponds to Example 1, FIG. 3C corresponds to Example 6, and FIG. 3D corresponds to Comparative Example 3.

As can be seen in Figures 3a to 3d, when the concentration of the fibroin in the aqueous solution of fibroin is about 1% by weight, 2% by weight, 3% by weight and 4% by weight, all of the fibroin microspheres were formed. However, as shown in FIG. 3D, when the concentration of the fibroin is about 4% by weight, the resulting fibroin microspheres are not dispersed in the solution but combine with each other to form agglomerates.

Method for preparing fibroin microspheres

Example 11

The emulsion prepared in Example 1 was evaporated at about 60 ° C. for about 30 minutes using a rotary evaporator (JP / N 1000S-W, EYELA, Japan) to obtain a precipitate. The precipitate was washed three times with ethanol to remove excess n-decane, Span 80 and Tween 80 from the precipitate, and dried again at room temperature for 1 hour to recover the fibroin microspheres in powder form.

Example 12

The emulsion prepared in Example 1 was dried at room temperature of about 25 ℃ for one day to obtain a precipitate. The precipitate was washed three times with ethanol to remove excess n-decane, Span 80 and Tween 80 from the precipitate, and dried again at room temperature for 1 hour to recover the fibroin microspheres in powder form.

Example 13

Fibroin microspheres were prepared in substantially the same manner as in Example 12, except that the emulsion prepared in Example 3 was used.

Example 14

Fibroin microspheres were prepared in substantially the same manner as in Example 12, except that the emulsion prepared in Example 4 was used.

Example 15

Fibroin microspheres were prepared in substantially the same manner as in Example 12, except that the emulsion prepared in Example 7 was used.

Example 16

Fibroin microspheres were prepared in substantially the same manner as in Example 12, except that the emulsion prepared in Example 8 was used.

Example 17

Fibroin microspheres were prepared in substantially the same manner as in Example 12, except that the emulsion prepared in Example 9 was used.

Example 18

Fibroin microspheres were prepared in substantially the same manner as in Example 12, except that the emulsion prepared in Example 10 was used.

The content of each component used in the preparation of the fibroin microspheres of Examples 11 to 18 is shown in Table 2 below.

Experimental Example  3

After dissolving 2 mg of the fibroin microspheres of Example 11 in about 2 mL of sodium carbonate buffer solution of pH 9 and 0.1 M at room temperature, the solution was prepared, and then the solution was dimethyl sulfoxide containing about 50 μL of about 1 mg / mL FITC. Mixtures were prepared by mixing with the side (DMSO) solution. After leaving the mixture in the dark for about 4 hours, the mixture was filtered using a filter paper of about 0.45 mu m. The solid material obtained by filtration was washed with phosphate buffered saline (PBS) and then dried. The dried solid material was observed using a confocal microscope (FLUROVIEW-FV300, Olympus, Japan), and the results are shown in FIG.

As can be seen in Figure 4, the cross-section cut by (A) to (I) of the fibroin microspheres of Example 11 are all green fluorescence, from which the fibroin microspheres of Example 11 It can be seen that the interior has a structure filled with fibroin.

Experimental Example 4

The surface and shape of the fibroin microspheres of Example 11 were observed using a scanning electron microscope (S-4300, HITACHI, Japan), the results are shown in Figures 5a and 5b. 5A is a photograph observed at a magnification of 5000 times and FIG. 5B is a photograph observed at a magnification of 2000 times.

As can be seen in Figures 5a and 5b, it can be seen that the fibroin microsphere of Example 11 has a spherical shape.

Experimental Example 5

Fibroin microspheres of Examples 11, 15 and 16 were observed using a scanning electron microscope (S-4300, HITACHI, Japan) and the size of 100 particles on the runner electron micrograph was measured. The measurement results are shown in the graph of FIG. 6, and FIGS. 7A to 7C are scanning electron micrographs of Examples 11, 15 and 16, respectively.

As can be seen in FIGS. 6 and 7A to 7C, the slower the stirring speed, the more the fibroin microspheres having the larger diameter are produced.

Experimental Example 6

The fibroin microspheres of Example 11 and Example 12 were observed using an optical microscope (CX40, Olympus, Japan) and a scanning electron microscope, the results are shown in Figures 8a to 8d below. 8A and 8B are optical micrographs and scanning micrographs of the fibroin microspheres of Example 11, respectively, and FIGS. 8C and 8D are optical micrographs and scanning micrographs of the fibroin microspheres of Example 12, respectively. The optical microscope limbs were observed at a magnification of 200 times, and the scanning micrographs were each photographed at a magnification of 1000 times.

As can be seen in Figures 8a to 8d, fibroin microspheres obtained by forcibly drying the emulsion using an evaporator are fibroin microspheres having a relatively smaller diameter than the fibroin microspheres obtained by forcibly drying the emulsion. It can be seen that is obtained.

Experimental Example 7

The diameters of the particles of the fibroin microspheres of Examples 11 and 12 were measured using a scanning electron microscope (S-4300, HITACHI, Japan) photograph. Similarly, the magnification was 1000 times, and the number of particles contained in the same photograph area was measured, and the results are shown graphically in FIG. 9.

As can be seen in FIG. 9, the forcedly dried fibroin microspheres, as in FIGS. 8A to 8D, have a relatively smaller average diameter.

Experimental Example 8

After mixing 1 mg of the fibroin microspheres of Example 11 with about 0.1 mg of trypsin, the mixture was left at room temperature for about 11 days, and then the surface and shape of the fibroin microspheres were scanned using a scanning electron microscope (S-4300, HITACHI, Japan). The results are shown in FIGS. 10A and 10B. 10A is a photograph observed at a magnification of 5000 times and FIG. 10B is a photograph observed at a magnification of 200 times.

As can be seen in FIGS. 10A and 10B, the fibroin microspheres have changed part of the surface by trypsin, but the fibroin microspheres have maintained a constant shape for 11 days and have not been decomposed. It can be seen. In other words, it can be seen that the fibroin microspheres maintain a stable state against proteolytic enzymes.

fibroin Microspheres  Dispersion preparation method containing

Example 19

About 1 g of the fibroin microspheres prepared in Example 13 was added to about 100 g of distilled water to prepare a dispersion including the fibroin microspheres.

Example 20

A dispersion including fibroin microspheres was prepared in substantially the same manner as in Example 19 except that the fibroin microspheres prepared in Example 14 were used.

Example 21

A dispersion comprising fibroin microspheres was prepared in substantially the same manner as in Example 19 except that the fibroin microspheres prepared in Example 17 were used.

Example 22

About 1 g of the fibroin microspheres prepared in Example 18 was added to about 100 g of an aqueous hydrochloric acid solution of about 0.1 N to prepare a dispersion containing the fibroin microspheres.

Experimental Example 9

Dispersions containing the fibroin microspheres prepared in Examples 19 and 20 were observed using an optical microscope, and the results are shown in FIGS. 11A and 11B. 11A and 11B are photographs observed at a magnification of 200 times.

As can be seen in 11a and 11b, it can be seen that the fibroin microspheres are uniformly dispersed in the water without aggregation with each other.

Experimental Example 10

The dispersions containing the fibroin microspheres of Examples 21 and 22 were observed using a scanning electron microscope (S-4300, HITACHI, Japan), and the results are shown in FIGS. 12A and 12B and FIGS. 13A to 13D. . 12A and 12B are scanning micrographs of the dispersion containing the fibroin microspheres of Example 21, and FIGS. 13A to 13D are scanning micrographs of the dispersion containing the fibroin microspheres of Example 22.

As can be seen in FIGS. 12a and 12b, the fibroin microspheres prepared by adding NaCl may have cracks formed on the surface of the microspheres in the water. Therefore, in the dispersion containing fibroin microspheres prepared by adding NaCl, the fibroin microspheres exhibit porosity as a plurality of pores are formed on a surface thereof.

Meanwhile, the fibroin microspheres prepared by adding NaHCO ₃ have a hemispherical shape while the fibroin microspheres are broken in the dispersion as shown in FIGS. 13A to 13C or as shown in FIG. 13D, one part of the fibroin microspheres is completely turned off. Indicates.

In the emulsion containing the fibroin microspheres prepared according to the method of the present invention described above, the fibroin microspheres can be uniformly distributed in the hydrophobic solution without aggregation. In addition, the fibroin microspheres prepared according to the above-described method of the present invention may have various diameters according to the stirring speed of the emulsion and the drying method of the emulsion, and may be decomposed by proteolytic enzymes while having a micro-scale size. , The diameter and decomposition rate of the fibroin microspheres can be easily adjusted. In addition, the dispersion containing the fibroin microspheres according to the above-described method can be stably preserved for a long time uniformly the fibroin microspheres in water or aqueous solution. In addition, when the fibroin microspheres are formed using NaCl or NaHCO ₃ and the like, when dispersed in water or an acidic aqueous solution, pores may be formed on the surface of the fibroin microspheres or the fibroin microspheres may be broken, so the inside of the fibroin microspheres May release the substance out.

Therefore, emulsions and dispersions containing the fibroin microspheres prepared by the above method can stably preserve fibroin for a long time and the fibroin microspheres are prepared by adding other substances, such as drugs such as proteins or peptides therein. It can also be used as a drug delivery particle that can be stored for a long time and released under appropriate circumstances.

As described above with reference to the preferred embodiment of the present invention, those skilled in the art without departing from the spirit and scope of the present invention described in the claims below various modifications and It will be appreciated that it can be changed.

1 is a flow chart illustrating a process for preparing an emulsion comprising fibroin microspheres in accordance with embodiments of the present invention.

2A to 2C and 3A to 3D are photographs of optical emulsions of emulsions containing fibroin microspheres according to embodiments of the present invention.

4 is a photograph of a fibroin microsphere according to embodiments of the present invention under a confocal microscope.

5A and 5B are photographs of a fibroin microsphere according to embodiments of the present invention under a scanning electron microscope.

Figure 6 is a graph showing the change in diameter of the fibroin microspheres according to the emulsion stirring speed of the fibroin microspheres according to embodiments of the present invention.

7A to 7C are photographs of a fibroin microsphere according to embodiments of the present invention under a scanning electron microscope.

8A to 8D are photographs of a fibroin microsphere according to embodiments of the present invention observed with an optical microscope and a scanning electron microscope.

9 is a graph showing the diameter change of the fibroin microspheres according to the emulsion drying method of the fibroin microspheres according to embodiments of the present invention.

10A and 10B are photographs obtained by scanning electron microscopy of a fibroin microsphere according to embodiments of the present invention with trypsin.

11A and 11B are photographs of an optical microscope of a dispersion including fibroin microspheres according to embodiments of the present invention.

12A and 12B and 13A to 13D are photographs of a dispersion including fibroin microspheres according to embodiments of the present invention under a scanning electron microscope.

Claims (15)

Preparing a hydrophobic solution comprising an organic solvent and a surfactant; Preparing a mixed solution by adding an aqueous solution of fibroin including fibroin and water to the hydrophobic solution; Emulsion method comprising a fibroin microspheres comprising the step of stirring the mixture. The method of claim 1, wherein the volume ratio of the organic solvent and the surfactant in the hydrophobic solution is 1: 0.07 to 0.4. The method of claim 1, wherein the fibroin aqueous solution comprises fibroin microspheres, characterized in that it comprises 0.5% to 3.5% by weight of the fibroin. The method of claim 1, wherein the fibroin aqueous solution further comprises a metal salt or an ammonium salt. The method of claim 1, wherein the fibroin aqueous solution is selected from the group consisting of NaCl, NaHCO ₃ , KCl, MgCl ₂ LiCl, (NH ₄ ) ₂ SO ₄ , NH ₄ Cl, KH ₂ PO ₄ and Ca ₃ (PO ₄ ) ₂ Emulsion production method comprising a fibroin microsphere, characterized in that it further comprises at least one. The method of claim 5, wherein the fibroin aqueous solution is selected from the group consisting of NaCl, NaHCO ₃ , KCl, MgCl ₂ LiCl, (NH ₄ ) ₂ SO ₄ , NH ₄ Cl, KH ₂ PO ₄ and Ca ₃ (PO ₄ ) ₂ Emulsion production method comprising a fibroin microsphere, characterized in that it comprises at least one 1.5% by weight or less. The method of claim 1, wherein the fibroin aqueous solution is added to the hydrophobic solution at a rate of 0.5 mL / min to 1.5 mL / min. The method of claim 1, wherein the volume ratio of the hydrophobic solution and the fibroin aqueous solution in the mixed solution is 1: 0.05 to 0.15, the emulsion production method comprising a fibroin microspheres. Preparing a hydrophobic solution comprising an organic solvent and a surfactant; Preparing a mixed solution by adding an aqueous solution of fibroin including fibroin and water to the hydrophobic solution; Preparing an emulsion comprising fibroin microspheres by stirring the mixed solution; And Fibroin microsphere manufacturing method comprising the step of drying the emulsion to obtain a fibroin microspheres. 10. The method of claim 9, prior to drying the emulsion, Fibroin microspheres manufacturing method further comprising the step of filtering the emulsion. The method of claim 9, wherein the drying of the emulsion, Fibroin microspheres manufacturing method characterized in that the emulsion is naturally dried at room temperature or forcibly dried using an evaporator. 10. The method of claim 9, further comprising the step of washing the obtained fibroin microspheres with alcohol. The method of claim 9, wherein the fibroin microspheres have a diameter of 4 μm to 40 μm. Preparing a hydrophobic solution comprising an organic solvent and a surfactant; Preparing a mixed solution by adding an aqueous solution of fibroin including fibroin and water to the hydrophobic solution; Preparing an emulsion comprising fibroin microspheres by stirring the mixed solution; Drying the emulsion to obtain the fibroin microspheres; And Dispersing method comprising a fibroin microspheres comprising the step of dispersing the fibroin microspheres in water or an acidic aqueous solution. The method of claim 14, wherein the fibroin aqueous solution is selected from the group consisting of NaCl, NaHCO ₃ , KCl, MgCl ₂ LiCl, (NH ₄ ) ₂ SO ₄ , NH ₄ Cl, KH ₂ PO ₄ and Ca ₃ (PO ₄ ) ₂ . Method for producing a dispersion comprising a fibroin microspheres, characterized in that it further comprises at least one.

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