JP3094182B2 - Microcapsule using keratin S-sulfo salt as a raw material for wall and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microcapsule containing keratin as a wall material and suitable for inclusion of dyes, fragrances, pharmaceuticals, agricultural chemicals, enzymes and other drugs, or for immobilization of enzymes and the like, and a method for producing the same. About.

[0002]

2. Description of the Related Art Conventional microcapsule production methods include chemical techniques (interfacial polymerization method and in situ polymerization method), physical and chemical techniques (phase separation method from aqueous solution and organic solvent, spray coagulation granulation method, etc.). ), Physical and mechanical techniques. In the interfacial polymerization method, a monomer in an aqueous phase and a monomer in an oil phase are polymerized at an interface to form an insoluble polymer film. But,
Only non-natural monomers are available for this reaction, and the resulting polymer coating is limited or non-biocompatible and poorly biodegradable. When the core substance reacts with the reactant (for example,
However, there is also a drawback that the core substance can be changed by the reaction in the case of having a reactive amino group or carboxyl group like a protein or enzyme).

[0003] The phase separation method most well known as a physical / chemical technique is a method of precipitating a concentrated phase of a polymer from an aqueous solution or an oil solution of a polymer on the surface of a core material by some method and microencapsulating the polymer. , The acidity of the system,
Since these conditions are strongly influenced by the concentration of the polymer and the like, these conditions must be well understood. In physical / mechanical techniques, spray drying, in which a stock solution for encapsulation is sprayed and brought into contact with hot air to evaporate and dry volatile components, is typical, but the equipment is relatively inflexible. In addition, the physical properties of the microcapsules cannot be largely changed by the same apparatus, and the liquid to be dried must be transported into the drying chamber. In addition, a surfactant such as sodium dodecyl sulfate is often contained as a stabilizing agent in the material constituting the wall of the capsule, even if in a trace amount, or when a capsule is encapsulated using collagen or the like, it is used as a cross-linking agent in the living body. Often, toxic substances must be used.

[0004]

It is an object of the present invention to use keratin S-sulphosalts which are easily prepared from natural keratin-containing substances as a constituent material for the wall of the capsule. Another object of the present invention is to provide a microcapsule suitable for inclusion of a drug by using a simple apparatus and method without using a surfactant such as sodium dodecyl sulfate or a highly toxic cross-linking agent when applied to a living body. To provide. In addition, in this specification, "keratin S-
“Sulfo salt” means that the sulfhydryl group (—SH) of the cysteine residue among the amino acid residues constituting keratin is —
_{^{S-SO 3 - X + (}} X + is Na ⁺ or K ^+, etc.) refers to that has been converted to.

[0005]

Wool Means for Solving the Problems], animal hair, an aqueous solution of a keratin-containing substances such as feathers _{M 2 S0 3 -M 2 S 4} O 6 (M represents a sodium or potassium.) (PH7-
9.5), an aqueous solution of keratin S-sulfo salt can be easily obtained [Encyclopedia
of Polymer Science and Technology, Edited by NM Bikales, Volume 8, Interscience Publishers, New York (196
4), page 1. Hereinafter, it is referred to as “Document 1”. ]. Although the keratin S-sulfo salt varies depending on the keratin-containing substance used as the raw material, the sulfhydryl group has -S-SO ₃
^- X ⁺ (X is sodium or potassium) and the converted cysteine residues in the form of a 100 residue per normal 4 to 10 amino acids.

The present inventors have studied the possibility of producing microcapsules based on keratin S-sulfo salt, and obtained the following results, thereby completing the present invention. (1) An aqueous solution of keratin S-sulfo salt is mixed with an organic solvent insoluble or hardly soluble in water, such as toluene or hexane, and the mixture is irradiated with ultrasonic waves at 0 to 50 ° C. for 10 seconds to 10 minutes to obtain the organic compound. It has been found that a microcapsule in which a solvent is efficiently contained as a core substance can be obtained. The mixing ratio varies depending on the concentration of the aqueous solution of keratin S-sulfo salt and the purpose of use of the microcapsules.
It has been found that it is preferable that (organic solvent volume) = 0.3 to 3.

[0007] (2) The above items (1) to the mixture -S-SO ₃ keratin S- sulfo salt ^- a small amount of hydrogen peroxide corresponding to the number of X ⁺ group, an oxidizing agent such as sodium periodate After addition, it was found that similar microcapsules could be obtained by irradiating ultrasonic waves or vigorously stirring with a vortex mixer or a stirring motor.

(3) The mixture of the above item (1) is vigorously stirred with an ultrasonic device, a vortex mixer, a stirring motor, or the like in an atmosphere of nitrogen gas or other gas having no oxidizing ability to form an emulsion. It has been found that microcapsules can be obtained even when the oxidizing agent described in the above item (2) is added and mixed.

(4) An aqueous solution containing keratin S-sulfo salt and another protein or peptide having a sulfhydryl group or a disulfide bond, or keratin S-sulfo salt
It has been found that microcapsules can be produced even when an aqueous solution containing a sulfo salt and a non-protein compound having a sulfhydryl group or a disulfide bond is used as a raw material for forming a wall and treated by the method described in items (1) to (3). Was.

(5) By dissolving in advance the substances such as dyes, fragrances and pharmaceuticals in the organic solvent to be used, these can be efficiently formed into microcapsules together with the solvent by the methods of the above items (1) to (4). Found to be included.

That is, the present invention provides a method for producing microcapsules, which comprises mixing an aqueous solution containing keratin S-sulfo salt with an organic solvent insoluble or hardly soluble in water and subjecting the mixed solution to ultrasonic treatment and / or vigorous stirring. is there. Further, the present invention provides a microcapsule, wherein an oxidizing agent is added to the aqueous solution before ultrasonic treatment or the like, or the oxidizing agent is added and stirred after ultrasonic irradiation or the like in a gas atmosphere lacking oxidizing ability. It is a manufacturing method of. The present invention also provides keratin S-
A method for producing microcapsules, comprising treating an aqueous solution containing a sulfo salt and a protein or peptide having a sulfhydryl group or a disulfide bond or another compound having a sulfhydryl group or a disulfide group in the same manner as described above. is there.

Hereinafter, known components and a production method used for producing the microcapsules of the present invention will be described. (I) Keratin S-sulfo salt-containing aqueous solution: The following keratin S-sulfo salt aqueous solution (item ia) alone, and keratin S-sulfo salt (item ia) described below in item (ib) or (i) -C) or a mixture of keratin S-sulfo salt (ia) to which both items (ib) and (ic) are added.

(Ia) Keratin S-sulfo salt aqueous solution: It was prepared from a substance containing keratin such as wool, human hair, chicken wings, and dog hair by a known method (see the above-mentioned document 1).

(Ib) Other proteins or peptides mixed with keratin S-sulfo salt: proteins having a mercapto group or disulfide bond, such as keratin, collagen, gelatin, fibrinogen, silk, egg white lysozyme, and insulin; glycyl-glycyl -Peptides such as cysteine (Gly-Gly-Cys) and (glycyl-glycyl-cystine) ₂ ((Gly-Gly-Cyt) ₂ ).

(Ic) Non-protein having a mercapto group or a disulfide group: a high molecular weight compound such as polyvinyl alcohol carrying a sulfhydryl group (for example, one having 1 to 5 sulfhydryl groups based on an average molecular weight of 2,000) An aqueous solution of a molecule or an aqueous solution of an organic disulfide compound such as ₂ -mercaptoethyl ether (HSCH ₂ CH ₂ OCH ₂ CH ₂ SH).

(Ii) Organic solvents: hydrocarbon solvents which are hardly soluble in water, such as toluene, xylene, hexane, decane,
Cyclohexane and the like are most preferable, but ether solvents such as diethyl ether and halogen-containing hydrocarbons such as fluorocyclohexane and Freon 113 can also be used. But,
The solvent is not limited to these as long as the solvent has low solubility in water.

(Iii) Oxidizing agents: Air, oxygen, hydrogen peroxide, sodium periodate, ammonium persulfate, potassium iodate and the like are preferably used. Further, together with these oxidizing agents, for example, iron ions can be used in combination as oxidation promoters or catalysts.

(Iv) Manufacturing method of microcapsules: (iv-1) Ultrasonic method: Any ultrasonic device may be used as long as it can irradiate a sample with ultrasonic waves. A probe-type device that generates ultrasonic waves from the tip of a metal probe such as titanium is preferable for increasing the height. The ultrasonic irradiation conditions are appropriately adjusted depending on the components and the volume of the sample, but generally, irradiation may be performed at 30 to 50 W for 10 seconds to 5 minutes at a total volume of 10 mL of the keratin S-sulfo salt aqueous solution and the organic solvent. Depending on the organic solvent used, the microcapsule generation efficiency may be low, but in this case, if a small amount of an oxidizing agent such as hydrogen peroxide is added before the ultrasonic treatment,
The production efficiency can be increased. Alternatively, an oxidizing agent may be added to the resulting emulsion mixture by performing ultrasonic treatment in a gas atmosphere lacking oxidizing power such as nitrogen gas. This technique is
When the core substance is easily oxidized, it has an effect of forming microcapsules while preventing oxidation of the core substance, and is particularly useful. The amount of the oxidizing agent used is generally an amount corresponding to 1 to 6 times the number of molecules of the oxidizing agent with respect to the S-sulfo group (-S-SO ₃ ^- group) in the raw material. Keratin S-sulfo salt and other wall constituent materials [items (ib) and (i)
-C)] can be used in an amount of 1 to 500% by weight based on the keratin S-sulfo salt. The amount of the organic solvent varies depending on the solubility of the core substance, but is 0.1% based on the total amount of the aqueous solution of the keratin S-sulfo salt and the raw material for forming the wall.
Use 1 to 5 times volume, usually 0.5 to 2 times volume.

(Iv-2) Stirring method: The wall constituting material is the same as in the above item (iv-1), but instead of ultrasonic treatment, it is vigorously vibrated by a vortex mixer and vigorously stirred, or vigorously by a stirring motor. Stir. In addition,
Before the treatment, a small amount of the oxidizing agent (1 to 6 times the number of the oxidizing agent molecules per one S-sulfo group) is added, or the oxidizing agent is added to the emulsified mixture produced by stirring. Thereafter, the mixture may be stirred gently so that the oxide is well mixed.

(V) The microcapsules can be isolated as follows. (V-1) The treatment liquid is directly concentrated or dried. Drying can be performed, for example, by freeze-drying.

(V-2) The treatment liquid is centrifuged to separate and fractionate microcapsules. As it is, as it is, there may be impurities such as wall constituent materials and oxidizing agents that did not contribute to the generation of the microcapsules outside the microcapsules, so water and a buffer solution were added to the microcapsule fraction, followed by centrifugation, followed by centrifugation. The microcapsules are separated and fractionated again. After repeating this operation several times, the microcapsule dispersion is used as it is, or concentrated or dried (by freeze-drying or the like).

(V-3) Using a semipermeable membrane such as a cellophane membrane, the treatment liquid is dialyzed against water, a buffer solution, or an aqueous solution in which a fragrance, a dye, a biologically active substance, or the like is dissolved. Either use the dialysate as it is, or concentrate or dry (eg, by freeze-drying).

The mechanism by which the aqueous solution containing keratin S-sulfo salt is insolubilized to form capsule walls is not clear in detail. However, it is well known that ultrasonic waves in water generate H ₂ O ₂ and HO ₂ with strong oxidizing power from water molecules [for example, B. Lippitt, JM McCord, I. Fridovic
.. h, J. Biol Chem, 247, 4688 (1972) ], whereas, the present inventors ^{_{have, R-S-SO 3 -}} ( in R methyl, a hydrocarbon group of hexyl) Na ⁺ solution oxygen presence of By sonication under the corresponding disulfide compound (R-
(SSR) occurs (K. Yamauch
i, Bull. Chem. Soc. Jpn.). Estimated from this finding, among the amino acid residues of keratin S-sulfo salt, -S-SO of S-sulfonated cysteine residue
₃ ^- group is considered to be due to the result of cross-linking between the polymer chains by converting to a disulfide bond. It is also considered that the addition of an oxidizing agent promotes the conversion to a disulfide bond.

Although the diameter of the capsule varies depending on the type of the aqueous solution containing keratin S-sulfo salt, the volume ratio of the organic solvent to the aqueous solution containing keratin S-sulfo salt, the mode of ultrasonic treatment or stirring, the time, etc., it cannot be specified unconditionally. , 1.5
1% by weight aqueous solution of keratin S-sulfo salt and toluene
When a 1-volume mixture (6 mL) was subjected to ultrasonic treatment at room temperature for 3 minutes at 30 W, it was determined by light scattering that it was a microsphere mainly composed of 1 to 3 μm. Observed at, the wall thickness was about 0.02μ
m and was very thin, like a paper balloon.

[0025]

[Example 1]

Preparation of keratin S-sulfo salt aqueous solution (X ⁺ = Na ⁺ ): wool (washed with water and defatted with dichloromethane);
_{g), Na 2 SO 3 (} 1.3g), Na 2 S 4 O 6 · 2
H ₂ O (1.5 g), urea (24 g) and 0.1 M tr
The mixture of is-buffer (pH 9; 50 mL) was stirred at 25 ° C. for 24 hours. After removing insoluble matter by filtration, the filtrate was added to a cellophane tube (Spectra / Pore 4).
Keratin S-sulfo salt aqueous solution (110 mL; 1.3 to 1.8% by weight) which is dialyzed against deionized water and is colorless and transparent
I got

Example 2 A keratin S-sulfo salt aqueous solution (sodium salt; concentration: 1.5% by weight) was placed in a wide-mouth test tube.
(5 mL) and toluene (3 mL) were added, and the mixture was irradiated with ultrasonic waves at 25 ° C. for 3 minutes at an output of 30 W while the mixture was indirectly stirred with a magnet bar. The resulting suspension is 200
The mixture was centrifuged at 0 rpm for 15 minutes to separate a cloudy solid, water (10 mL) was added, and the mixture was stirred and centrifuged in the same manner. After the same washing operation was further repeated twice, it was freeze-dried. According to transmission electron microscopy, the obtained white powdery substance (about 0.06 g) was a relatively uniform microcapsule, the wall thickness was about 0.02 μm, and the diameter was 1 to 3 μm.

Example 3 A keratin S-sulfo salt aqueous solution (sodium salt; concentration: 1.5% by weight) was placed in a wide-mouth test tube.
(5 mL) and toluene (3 mL) were added, and the mixture was irradiated with ultrasonic waves at 25 ° C. for 5 minutes in a nitrogen gas atmosphere. A 30% aqueous hydrogen peroxide solution (0.07 mL) was added to the resulting white suspension, and the mixture was shaken gently and allowed to stand for 15 minutes. The mixture was then centrifuged at 2000 rpm for 15 minutes to separate a white solid,
mL), and the mixture was stirred and centrifuged in the same manner. After repeating the same washing operation two more times, it was immediately freeze-dried. According to transmission electron microscopic observation of the obtained white powdery substance (about 0.05 g), the diameter of the resulting microcapsules was 2 to 5 μm, although there was some variation.

Example 4 A 1.3% aqueous solution (5 mL) of keratin prepared by the following method was added to a wide-mouth test tube, and then an aqueous solution of keratin S-sulfo salt (sodium salt; the concentration was 1.5% by weight) (5 mL), and the mixture was vibrated and stirred sufficiently. Then, toluene (5 mL) containing dissolved Sudan IV (7 mg) was added, and the mixture was sonicated at 25 ° C. for 3 minutes at a power of 30 W while indirectly stirring the mixture with a magnet bar. The resulting suspension is cooled at 2000 rpm for 15 minutes.
After centrifugation for minutes, the upper layer solid was separated and dispersed in pure water (5 mL). The dispersion is freeze-dried to obtain about 0.1.
12 g of powder were obtained. Electron microscopic observation of the dispersion showed relatively uniform particles (1 to 3 μm in diameter). The fact that the microcapsules contained most of the added red dye in the inside was confirmed by the result of ultraviolet spectroscopic analysis of the lyophilized product dispersed in benzene to leak the dye into the external solution.

The aqueous keratin solution used in this example was produced as follows. 10 g of defatted wool (Merino)
A mixed solution of 6.0 g of sodium dodecyl sulfate, 16 g of sodium bisulfite and 300 mL of 8 molar urea was treated with a bath-type ultrasonic apparatus at 50 to 55 ° C. for 1 hour after being sealed. The insoluble matter was removed by filtration, the filtrate was put into a cellophane tube, dialyzed with a 0.2% by weight aqueous sodium bisulfite solution (3 L) as an external solution, and a small amount of the insoluble matter was removed from the dialysate by centrifugation to remove keratin. 1.3
About 330% by weight (Lowry method)
mL was obtained. According to amino acid analysis, this keratin has 7.6 cysteines and 0.8 cystine per 100 amino acid residues. In polyacrylamide gel electrophoresis, proteins having a molecular weight of about 40,000 and 60,000 (30 to 40%, respectively) (50 to 60%) as the main component.

Continuation of front page (58) Field surveyed (Int.Cl. ⁷ , DB name) B01J 13/02 A61K 9 / 50,47 / 42

Claims (5)

(57) [Claims] 1. A method for producing microcapsules, comprising mixing an aqueous solution containing keratin S-sulfo salt with an organic solvent insoluble or hardly soluble in water, and subjecting the mixed solution to ultrasonic treatment and / or vigorous stirring. 2. The method according to claim 1, wherein the oxidizing agent is added before sonication and / or vigorous stirring. 3. An aqueous solution containing keratin S-sulfo salt is mixed with an organic solvent insoluble or hardly soluble in water in a gas atmosphere lacking oxidizing ability, and the mixture is subjected to ultrasonic treatment and / or vigorous stirring to form an emulsion. A method for producing microcapsules, comprising adding an oxidizing agent and stirring after preparation. 4. The aqueous solution containing a keratin S-sulfo salt, wherein the aqueous solution further contains a protein or peptide having a sulfhydryl group or a disulfide bond, or polyvinyl alcohol having a sulfhydryl group or a disulfide bond. 3. The production method according to any one of 3. 5. A microcapsule using keratin S-sulfo salt as a raw material for forming a wall.