JP2016163851A - Composite type solubilized nanoliposome and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a liposome with the average particle diameter of 18 to 25 nm high in safety and safety, and further, to provide an easily and simple method for producing the liposome.SOLUTION: Provided is a production method comprising a process where a phosphatide solubilized nano-vesicle emulsion (A) is prepared in the first step, a solubilized nano-emulsion (B) as an oil soluble component is prepared in the second step, and the (A) and (B) are mixed and homogenized in the third step. The method for producing a composite type solubilized nano-liposome being a general-purpose production technical method requiring no use of organic solvents, special emulsification devices and particle size regulation devices and economically and industrially excellent. Particularly, from the point that organic solvents are not used, it is a liposome excellent in safety.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a composite-type solubilized nanoliposome having high heat stability and high long-term storage stability and a method for producing the same.

  Liposomes are closed vesicles of lipid bilayers that can enclose hydrophilic substances in the inner aqueous phase or hydrophobic components inside the bilayer. Therefore, in the pharmaceutical field, attention has been focused on therapeutic drugs, diagnostic drugs and the like as drug carriers for DDS. In other cosmetics, nanocapsules are used, and in foods, enzymes, antioxidants, fragrances and the like are enclosed.

  Conventional liposome production methods include (1) Bangham method, (2) sonication method, (3) organic extraction method, (4) surfactant removal method, (5) freeze-thaw method, and (6) A reverse phase evaporation method or the like is known. (1) In the Bangham method, phospholipids are dissolved in chloroform in a container, then chloroform is evaporated to form a lipid film on the inner surface of the container, water is added to the container to hydrate, and the container is shaken. MLV (multilamellar vesicle) shaped liposomes. (2) The sonication method is a method of obtaining SUV (small unilamellar vesicle) shaped liposomes by sonicating MLV shaped liposomes prepared by the Bangham method. (3) The organic solvent extraction method is a method of injecting phospholipids dissolved in an organic solvent into an aqueous solution to obtain SUV or LUV (large unilamellar vesicle) shaped liposomes. (4) In the surfactant removal method, the phospholipid is dissolved in chloroform in the container, and then the chloroform is evaporated to form a lipid thin film on the container surface. Furthermore, after the container is vigorously shaken to form micelles, the surfactant is removed by dialysis. (5) In the freeze-thaw method, a phospholipid thin film dissolved in an organic solvent is dissolved and dispersed in water and then shaken vigorously, and then the phospholipid thin film is frozen in liquid nitrogen and allowed to thaw at room temperature to obtain LUV-shaped liposomes. Is the method. (6) The reverse phase evaporation method is a method in which a phospholipid membrane is dissolved in an organic solvent, then the phospholipid thin film is dissolved in water and shaken vigorously, and then the organic solvent is removed under reduced pressure to obtain LUV-shaped liposomes. is there. In addition, these liposomes after the production process require a special emulsifying device (eg microfluidizer, thin-film swirl high-speed homomixer) and a sizing device (eg extruder) to make the particle size small and uniform. However, a general-purpose manufacturing technique method that is excellent in terms of cost and industry has not been proposed.

  In addition, in the production methods (1) to (6) described above, since the phospholipid is dissolved in a water-insoluble organic solvent such as chloroform or hexane in the production process, the water-insoluble solvent is removed after preparing the liposome. A process is required. Further, it is difficult to completely remove the water-insoluble organic solvent. The heating / depressurization or pressurization process required to remove these water-insoluble organic solvents and to adjust the particle size is a factor that degrades or destroys the liposome membrane, which also adversely affects the stability over time, In addition to causing aggregation / precipitation, the production process is complicated and the price of the emulsion and dispersion process is increased.

  A production method using a hydrocarbon organic solvent is disclosed in Patent Document 1 (Japanese Patent Laid-Open No. 2010-248171) and 2 (Japanese Patent Laid-Open No. 2013-22482). The primary phase of preparing a W / O emulsion by mixing and emulsifying an oil phase solution in which a phospholipid component is dissolved or dispersed in a volatile hydrocarbon organic solvent and an aqueous solution obtained by dissolving the drug to be encapsulated in a water-soluble solvent. An emulsification step, and a secondary emulsification step of preparing a W / O / W emulsion by mixing and emulsifying the W / O emulsion obtained in the above step and an aqueous phase liquid to which an amorphous lipid component has been added. In addition, a solvent removal step of preparing a liposome dispersion by removing the hydrocarbon organic solvent from this W / O / W emulsion, and further removing the aqueous phase liquid to which an amorphous lipid component is added from the liposome dispersion, This is a method for producing single cell liposomes having an average particle size of 50 nm or more and 200 nm or less, comprising an aqueous phase replacement step of preparing a liposome preparation by adding an aqueous phase solution (claims).

  On the other hand, a method for producing liposomes without using an organic solvent is disclosed in Patent Documents 3 (JP 60-7934 A) and 4 (JP 2000-290170 A). In the method disclosed in Patent Document 3, the liposome membrane component substance is kneaded and hydrated at a temperature higher than the phase transition temperature (Tc) between the aqueous solution and the membrane component substance, and then the required amount of aqueous solution is added and stirred at Tc or higher. (Claims). Patent Document 4 discloses a hybrid liposome preparation for cancer treatment characterized in that it is a liposome containing a phospholipid, a non-sugar micelle surfactant, and a sugar micelle surfactant (Claim 1). The Furthermore, the method disclosed in Patent Document 5 (Japanese Patent Application Laid-Open No. 2006-298839) uses PEG-phospholipid having a melting point at 40 to 70 ° C. as a liposome membrane constituent, and the liposome is subjected to specific first to fourth steps. A method for producing a containing preparation (claim 10). However, the production methods disclosed in Patent Documents 3 to 5 have problems with stability over time, and it is difficult to obtain solubilized liposomes having an average particle size of 25 nm or less. Further, in the methods disclosed in Patent Documents 1 and 2, a residual solvent by removing the water-insoluble hydrocarbon-based organic solvent used as a lipid solubilizer under reduced pressure can be considered, and the average particle size having a high stability and safety is 25 nm or less. There is a problem that a solubilized liposome cannot be obtained.

JP 2010-248171 JP 2013-22482 Japanese Unexamined Patent Publication No. 60-7934 JP 2000-290170 A JP 2006-298839 A

  It is an object of the present invention to provide a liposome having an average particle size of 18 to 25 nm, which is highly safe and highly stable, and to provide an easy and simple method for producing the liposome.

  As a result of intensive studies, the inventor prepared a phospholipid-solubilized nanovesicle emulsion (A) in the first step, a solubilized nanoemulsion (B) of an oil-soluble component in the second step, and the third step. Production comprising mixing and homogenizing (A) and (B) in the process, and heating and emulsifying the mixed solubilized nanoemulsion obtained in the third in the fourth process above the cloud point According to the method, the present inventors have found that the above-mentioned problems can be solved and completed the present invention.

That is, this invention consists of the following.
1. A method for producing a composite-type solubilized nanoliposome comprising the following steps:
(1) A step of preparing an O / W-type solubilized nanovesicle emulsion (A) containing a phospholipid and a nonionic surfactant;
(2) a step of preparing an O / W-type solubilized nanoemulsion (B) containing an oil-soluble component and a nonionic surfactant;
(3) A step of preparing the mixed solubilized nanoemulsion by mixing and homogenizing the O / W type solubilized nanovesicle emulsion (A) and the O / W type solubilized nanoemulsion (B);
(4) A step of heating and emulsifying the mixed solubilized nanoemulsion obtained in step (3) above the cloud point.
2. 2. The step 1 described above, wherein the step (1) is a step of dissolving and uniformly mixing a phospholipid having a phase transition temperature of 0 to 75 ° C. in a solution containing a nonionic surfactant and emulsifying and dispersing by a phase inversion temperature emulsification method. Manufacturing method.
3. 3. The method according to item 1 or 2, wherein the phospholipid in the step (1) is a phospholipid that is phosphatidylcholine having an acyl group having 12 to 22 carbon atoms.
4). 4. The production method according to any one of items 1 to 3, wherein the O / W-type solubilized nanovesicle emulsion (A) described in the step (1) contains 0.5 to 5% by mass of phospholipid.
5. 5. The production method according to any one of items 1 to 4, further comprising a tonicity agent in the step (1).
6). 6. The production method according to any one of items 1 to 5, further comprising ethanol in the step (1).
7). 7. The production method according to any one of the preceding items 1 to 6, wherein the step (2) comprises a step of dissolving and mixing an oil-soluble component in a nonionic surfactant and then emulsifying and dispersing by a phase inversion temperature emulsification method.
8). 8. The production method according to any one of items 1 to 7, wherein the oil-soluble component in the step (2) contains at least cholesterol.
9. The nonionic surfactant used in the steps (1) and (2) is polyoxyethylene sterol ether, polyoxyethylene stearyl ether or its isoform, and polyoxyethylene polyoxypropylene alkyl ether, and the affinity thereof Any one of 1 to 8 above, each of which includes one or a plurality of nonionic surfactants selected from surfactants having a property (HLB value) of 10.7 to 18.0, each independently or in common The manufacturing method as described in.
10. 10. Composite-type solubilized nanoliposomes produced by the production method according to any one of 1 to 9 above.
11. 11. The composite-type solubilized nanoliposome according to item 10 above, wherein the liposome has a particle size distribution of 10 to 80 nm.
12 A liposome having an average particle size of 18 to 25 nm, comprising a hydrophilic substance in the inner aqueous phase, and an oil-soluble component enclosed in the bilayer membrane.
13. When the composite-type solubilized nanoliposome contains at least a phospholipid, an oil-soluble component and a nonionic surfactant, and the phospholipid is 1 by weight, the oil-soluble component is contained in an amount of 0.01 to 0.6%. 10. The composite type solubilized nanoliposome according to any one of 10 to 12 above.
14 When the composite-type solubilized nanoliposome contains at least a phospholipid, an oil-soluble component, and a nonionic surfactant, and the weight ratio of each component is phospholipid: oil-soluble component: nonionic surfactant, 2 The composite solubilized nanoliposome according to 13 above, wherein 0.02 to 1.2 is 0.78 to 4.0.
15. 15. The composite-type solubilized nanoliposome according to item 13 or 14, wherein the oil-soluble component contained in the liposome contains at least cholesterol and further contains a function-imparting oil-soluble component.
16. 16. A solubilized liposome emulsion comprising the composite type solubilized nanoliposome according to any one of 10 to 15 above.
17. The solubilized liposome emulsion according to item 16, wherein the particle size distribution of the liposome contained in the solubilized liposome emulsion is 10 to 80 nm.
18. 18. The solubilized liposome emulsion according to item 16 or 17 above, wherein the particle size peak distribution of the liposome contained in the solubilized liposome emulsion consists of one peak.

  The composite-type solubilized nanoliposome of the present invention having a structure containing a nonionic surfactant, a phospholipid, and a hydrophobic substance is an SUV liposome having an average particle size of 18 to 25 nm and is excellent in stability. According to the method for producing a composite-type solubilized nanoliposome of the present invention, it is not necessary to use an organic solvent, and no special emulsifying device or granulating device is required. Since this is a technical method and does not use an organic solvent, a liposome having excellent safety can be obtained.

  The liposome of the present invention is economically superior in terms of equipment and materials in the production process. Furthermore, the liposome of the present invention and the emulsion containing the liposome can be used in many fields such as pharmaceuticals, foods, and cosmetics, and the effect is great from this point.

It is a figure which shows the structure of the composite type solubilized nanoliposome of this invention. It is a figure which shows the concept of the manufacturing process of the composite type | mold solubilized nanoliposome of this invention. It is a figure which shows the particle distribution of each O / W type solubilization nano vesicle emulsion (A) prepared at the process (1). (Example) It is a figure which shows the particle distribution of each O / W type | mold solubilization nanoemulsion (B) prepared at the process (2). (Example) It is a figure which shows the result of having confirmed the effect on drug sustained release property when the composite type | mold solubilized nanoliposome of this invention is used. (Experimental example 1) It is a figure which shows the result of having confirmed the passage stability of the composite type | mold solubilized nanoliposome of this invention. (Experimental example 2)

  The present invention relates to a composite-type solubilized nanoliposome having high heat stability and high long-term storage stability and a method for producing the same. The composite solubilized nanoliposome of the present invention has the structure shown in FIG. 1 (I), an average particle diameter of 18 to 25 nm, and at least phospholipid, nonionic surfactant, oil-soluble. It consists of a structure containing a component (hydrophobic substance). An oil-soluble component is contained inside the phospholipid bilayer. Furthermore, the composite-type solubilized nanoliposomes of the present invention can contain a hydrophilic substance as shown in FIG. 1 (II) in the inner inner water layer formed inside the bilayer membrane of phospholipid.

  First, a method for producing the composite-type solubilized nanoliposome of the present invention will be described, and the structure of the liposome of the present invention will be described in more detail.

  The composite-type solubilized nanoliposome of the present invention can be produced by a method including the following steps. The O / W-type solubilized nanovesicle emulsion (A) and the O / W-type solubilized nanoemulsion (B) in the following steps (1) and (2) can be referred to FIG.

(1) A step of preparing an O / W-type solubilized nanovesicle emulsion (A) containing a phospholipid and a nonionic surfactant;
(2) a step of preparing an O / W-type solubilized nanoemulsion (B) containing an oil-soluble component and a nonionic surfactant;
(3) A step of preparing the mixed solubilized nanoemulsion by mixing and homogenizing the O / W type solubilized nanovesicle emulsion (A) and the O / W type solubilized nanoemulsion (B);
(4) A step of heating and emulsifying the mixed solubilized nanoemulsion obtained in step (3) above the cloud point.

Step (1) The phospholipid used in the step of preparing the O / W-type solubilized nanovesicle emulsion (A) containing the phospholipid and the nonionic surfactant in the above step (1) is a carbon number of an acyl group. Is a hydrophobic phospholipid comprising any phosphatidylcholine selected from 12-22, preferably 14-22, more preferably 16-22. The phase transition temperature of such a phospholipid is 0 to 75 ° C, preferably 23 to 75 ° C, more preferably 41 to 75 ° C. Specifically, it is preferable to use a highly hydrophobic hydrogenated product, distearyl phosphatidylcholine (HSPC), which has been purified to a phosphatidylcholine content of 90% or more. Alternatively, synthesized L-3-distearyl phosphatidylcholine may be used. Commercially available phosphatidylcholine can also be used. For example, COATSOME NC 21 (manufactured by NOF Corporation with a PC purity of 90% or more), NIKKOL Resinol S 10EX (PC purity of 98% or more by Nikko Chemicals) and the like are preferably exemplified. In addition, COATSOME NC 21 (hydrogenated soybean phospholipid (HSPC)) can be used as the phospholipid forming the lipid bilayer membrane.

Nonionic surfactants used in the above step (1) are polyoxyethylene sterol ether, polyoxyethylene stearyl ether or its isoform, and polyoxyethylene polyoxypropylene alkyl ether, and their affinity (HLB Value) is one or more selected from surfactants having a value of 10.7 to 18.0. Specific nonionic surfactants in the present specification include polyoxyethylene sterol ether having affinity for cholesterol in a hydrophobic group and nonionic surfactants described in Pharmaceutical Additive Standard 1998. For example polyoxyethylene stearyl ether _{(St0 (EO) p H ·} p = 10~25) and its isoforms and polyoxyethylene polyoxypropylene alkyl ethers of the same ether type hydrophobic group has an affinity with the lipid groups of phospholipids (CeO (PO) _m (EO) _n H · m = 4, n = 10, 20). A commercially available nonionic surfactant can also be used as the nonionic surfactant. For example, NIKKOL DHC 30 HLB17.0 (manufactured by Nikko Chemicals), NIKKOL BS 20 HLB18.0 (manufactured by Nikko Chemicals), UNIOX CS 800 HLB10.7 (manufactured by NOF), EMALEX CS 30 HLB14.0 (manufactured by Nippon Emulsion), EMALEX 620 HLB13.0 (manufactured by Nippon Emulsion), EMALEX 1825 HLB13.0 (manufactured by Nippon Emulsion), NIKKOL PBC 34 HLB16.5 (manufactured by Nikko Chemicals) and the like can be used.

  In the above step (1), in the O / W-type solubilized nanovesicle emulsion (A), 0.5 to 5% by mass of phospholipid can be contained, and in order to prepare stable liposomes, 1 to 2 It is particularly preferable to include the mass%.

  The O / W type solubilized nanovesicle emulsion (A) preferably contains an isotonic agent. As the isotonizing agent, any one selected from glycerin, propylene glycol, 1,3-butyl glycol, maltitol, benzyl alcohol and the like can be used. In particular, concentrated glycerin based on Japanese Pharmacopoeia with a purity of 98% or more is preferable, and the addition amount is preferably about 1 to 3 times by weight with respect to phospholipid 1.

  In the step (1), the phospholipid and the nonionic surfactant are mixed and dissolved using a solvent. Here, a water-soluble medium or a water-soluble alcohol solvent can be used as the solvent. As such a solvent, a common one known per se can be used. As the water-soluble alcohol solvent, ethanol having a boiling point lower than that of water is preferably used. Examples of the water-soluble medium include distilled water, purified water, and pure water. The amount of the solvent used is not particularly limited as long as it is an amount that completely dissolves the phospholipid and the nonionic surfactant. In general, the amount used is about 1 to 2 g per 1 g of the phospholipid mixture.

  In the above step (1), when the bulk specific gravity of the phospholipid is large and the amount of the nonionic surfactant or glycerin used as the emulsifying dispersant is small, uniform melt mixing is difficult. In addition, there is a risk of lipid alteration and carbonization due to prolonged heating for homogeneous melt mixing. Therefore, as a solution to this problem, it is preferable to add a water-soluble lower alcohol, which is a homogeneous dissolution solvent, and uniformly dissolve while gradually raising the temperature. As the water-soluble lower alcohol used here, ethanol is preferred. After uniformly melting and mixing, by heating to the boiling point of ethanol (about 80 ° C.) or more, 95% or more of ethanol can be evaporated, and a transparent film can be produced. Purified water heated to 80 ° C. or higher in a separate bath can be added thereto and emulsified and dispersed by a phase inversion emulsification method. Although there is no restriction | limiting in particular as a temperature which melt | dissolves a phospholipid mixture, Preferably it is 40 degreeC or more, and in order to accelerate | stimulate uniform dissolution, it is preferable to stir or shake under 50-60 degreeC heating. By these methods, the O / W-type solubilized nanovesicle emulsion (A) in the above step (1) can be prepared.

  As an emulsification method in this step, a propeller is used without using a special type of emulsifying device such as a pressure type microfluidizer (manufactured by Microfluidics) or a thin film swirl type high speed homomixer (manufactured by Primix). Alternatively, it can be emulsified and dispersed by a phase inversion emulsification method at normal pressure using a stirring device such as a paddle (bar).

Step (2) The oil-soluble component used in the step of preparing the O / W-type solubilized nanoemulsion (B) containing the oil-soluble component and the nonionic surfactant in step (2) is a membrane stabilizer. And an oil-soluble component having functionality added and encapsulated in the liposome of the present invention.

  Among the oil-soluble components in the step (2), the oil-soluble component as a membrane stabilizer is a component that can be used in liposomes, for example, sterols such as cholesterol, dihydrocholesterol, cholesterol esters, phytosterols, sitosterol, cholesterol, Examples include lanosterol, 1-O-sterol glucoside, 1-0-sterol galactoside (Japanese Patent Laid-Open No. 5-245357), and cholesterol is particularly preferable. Specifically, commercially available sterols can be used. For example, cholesterol JSCI (manufactured by Nippon Seika Co., Ltd., cholesterol), GENEROL 122N (manufactured by Cognis Japan Co., Ltd., phytosterol) and the like can be used. In addition, tocopherol having an antioxidative action, ascorbic acid / oleic acid and the like as functional imparting agents may be used in combination.

  Of the oil-soluble components in the above step (2), the oil-soluble component of the function-imparting oil-soluble component is not particularly limited as long as it is an oil-soluble component encapsulated in liposomes. For example, squalane, pearl barley oil, jojoba oil , Olive oil, dimethyl silicon, octyl paramethoxycinnamate, octyl paraaminodimethylbenzoate, benzotriazole, benzophenone, etc. (JP 09-143031 A), poorly water-soluble and water-insoluble such as doxorubicin, cisplatin, paclitaxel, mitomycin C, Examples thereof include anticancer agents such as irinotecan (Japanese Patent Laid-Open No. 2012-55885). These components are applicable to uses such as foods, cosmetics, and pharmaceuticals.

  The concentration (mass / mass) of the oil-soluble component that combines the oil-soluble component as the membrane stabilizer in the above step (2) and the function-imparting oil-soluble component is set to 1 in terms of the concentration ratio with the phospholipid. Sometimes 0.05-0.3 is preferred. In particular, when the concentration ratio of cholesterol to phospholipid 1 is less than 0.05 or more than 0.3, the stability of the liposome is not so good.

The nonionic surfactant as an emulsifying dispersant for preparing the O / W type solubilized nanoemulsion (B) in the above step (2) is polyoxyethylene sterol ether, polyoxy, as in the step (1). Ethylene stearyl ether or its isoform, and polyoxyethylene polyoxypropylene alkyl ether, which is one or more selected from surfactants having an affinity (HLB value) of 10.7 to 18.0. Specific nonionic surfactants in the present specification include polyoxyethylene sterol ether having affinity for cholesterol in a hydrophobic group and nonionic surfactants described in Pharmaceutical Additive Standard 1998. For example polyoxyethylene stearyl ether _{(St0 (EO) p H ·} p = 10~25) and its isoforms and polyoxyethylene polyoxypropylene alkyl ethers of the same ether type hydrophobic group has an affinity with the lipid groups of phospholipids (CeO (PO) _m (EO) _n H · m = 4, n = 10, 20). A commercially available nonionic surfactant can also be used as the nonionic surfactant. For example, NIKKOL DHC 30 HLB17.0 (manufactured by Nikko Chemicals), NIKKOL BS 20 HLB18.0 (manufactured by Nikko Chemicals), UNIOX CS 800 HLB10.7 (manufactured by NOF), EMALEX CS 30 HLB14.0 (manufactured by Nippon Emulsion), EMALEX 620 HLB13.0 (manufactured by Nippon Emulsion), EMALEX 1825 HLB14.0 (manufactured by Nippon Emulsion), NIKKOL PBC 34 HLB16.5 (manufactured by Nikko Chemicals) and the like can be used.

  In the step (2), an oil-soluble component is mixed with a nonionic surfactant and emulsified and dispersed by a phase inversion temperature emulsification method to prepare an O / W type solubilized nanoemulsion (B). Also in this step, a propeller or paddle (bar) is used without using a pressure type microfluidizer (manufactured by Microfluidics) or a thin film swirl type high-speed homomixer (manufactured by Primix) which is a special emulsifier. ) And the like, and can be emulsified and dispersed by a phase inversion emulsification method at normal pressure.

Step (3) In the above step (3), the prepared O / W-type solubilized nanovesicle emulsion (A) and O / W-type solubilized nanoemulsion (B) are mixed and homogenized, and mixed solubilized nano Prepare an emulsion. The weight ratio of phospholipid, oil-soluble component and nonionic surfactant in the mixed solubilized nanoemulsion is 2: 0.02 to 1.2: 0.78 to 4.0 when phospholipid: oil-soluble component: nonionic surfactant is used. Preferably, it is 2: 0.1-1.1: 0.78-3.1.

Step (4) In the step (4), the mixed solubilized nanoemulsion obtained in the step (3) is heated to emulsification by heating above the cloud point. The emulsion is heated to the cloud point or higher, mixed and stirred to prepare a uniformly re-emulsified dispersion in which the O / W type solubilized nanovesicle emulsion (A) and the O / W type solubilized nanoemulsion (B) are composited. can do. Here, the cloud point refers to a temperature at which phase separation occurs due to a temperature change in a transparent or translucent liquid, resulting in an opaqueness. For example, when an aqueous solution of a nonionic surfactant is heated, the hydrogen bond between the polyether chain of the nonionic surfactant and water is broken, the solubility in water decreases and the aqueous solution becomes cloudy. Says the temperature. Examples of the cloud point or higher include 68 ° C. or higher, preferably 75 ° C. or higher. For example, the re-emulsified dispersion can be prepared by mixing and stirring at 80 ° C. or higher for 30 to 60 seconds. Thereafter, it is rapidly cooled to room temperature to stabilize the emulsified dispersion, and a composite-type solubilized nanoliposome in which the oil-soluble component is encapsulated in the hydrophobic portion of the solubilized nanovesicle emulsion can be obtained.

  The particle size of the composite-type solubilized nanoliposome produced by the above production method is 18 to 25 nm in average particle size as measured by a dynamic light scattering method. The obtained liposome may contain a hydrophilic substance in the inner aqueous phase and a hydrophobic substance in the bilayer membrane. The present invention also extends to the liposome. The hydrophobic substance contained in the bilayer membrane includes the oil-soluble component of the present invention, and further includes an oil-soluble component as a membrane stabilizer such as cholesterol, and a function-imparting oil-soluble component.

  The emulsion containing the composite type solubilized nanoliposome of the present invention can be referred to as the solubilized liposome emulsion of the present invention. According to the present invention, it is possible to obtain a solubilized liposome emulsion having one peak in the particle size frequency distribution graph and including a uniform composite solubilized liposome. The particle size distribution of the liposome contained in the solubilized liposome emulsion of the present invention is 10 to 80 nm, and preferably 15 to 80 nm.

  The liposome of the present invention can be used in various fields such as pharmaceutical field, cosmetic field and food field.

  In order to deepen the understanding of the present invention, the contents of the present invention will be specifically described with reference to the contents described in the drawings. However, it is apparent that the present invention is not limited to these examples. It is.

(Examples 1-6) Preparation of composite type solubilized nanoliposomes The composite type solubilized nanoliposomes of the present invention are prepared through the following steps (1) to (4). In Examples 1 to 6 below, composite-type solubilized nanoliposomes produced when various blending ratios of phospholipid, nonionic surfactant and oil-soluble component, and other components are shown.

(1) A step of preparing an O / W-type solubilized nanovesicle emulsion (A) containing a phospholipid and a nonionic surfactant;
(2) a step of preparing an O / W-type solubilized nanoemulsion (B) containing an oil-soluble component and a nonionic surfactant;
(3) A step of preparing the mixed solubilized nanoemulsion by mixing and homogenizing the O / W type solubilized nanovesicle emulsion (A) and the O / W type solubilized nanoemulsion (B);
(4) A step of heating and emulsifying the mixed solubilized nanoemulsion obtained in step (3) above the cloud point.

(1) Preparation of O / W-type solubilized nanovesicle emulsion (A) First, (A) -37 and (A) -35 were prepared as O / W-type solubilized nanovesicle emulsion (A). Hydrophobic phospholipid powder coatsome NC 21 (manufactured by NOF Co., Ltd., HSPC) as phospholipids, NIKKOL DHC 30 (manufactured by Nikko Chemicals Co., Ltd., polyoxyethylene cholesteryl ether), UNIOX CS 800 (non-ionic surfactant) NOF Co., Ltd. (polyoxyethylene cholesteryl ether) and EMALEX 1825 (Nihon Emulsion Co., Ltd., polyoxyethylene isostearyl ether), concentrated glycerin as an isotonic agent, ethanol as a solubilizer It was. These compounding amounts are shown in Table 1.

  Add the above phospholipids (coatsome NC 21), nonionic surfactant (NIKKOL DHC 30, UNIOX CS 800, EMALEX 1825) isotonic agent (concentrated glycerin) and solubilizer (ethanol) to 60 ° C. The mixture was stirred and dissolved uniformly. After dissolution, the mixture was further heated to 80 ° C. to evaporate 95% or more of ethanol to produce a slightly yellow transparent film. Purified water (45.52 g) heated to 80 ° C or higher in a separate bath was gradually added thereto, emulsified with a phase inversion temperature while stirring with a bar, then rapidly cooled to room temperature, and O / W-type solubilized nanovesicle emulsion (A ) Was prepared.

  As a result, (A) -37 is a semi-solubilized vesicle emulsion having a cumulant average particle size of 101.6 nm, a particle size distribution of 10 to 30 nm and two peaks of 70 to 1000 nm and a faint white appearance. there were. (A) -35 was a solubilized nanovesicle emulsion having a cumulant average particle size of 20.4 nm, a particle size distribution of one peak of 10 to 65 nm and a transparent appearance (see FIG. 3).

(2) Preparation of O / W Type Solubilized Nanoemulsion (B) Next, as O / W type solubilized nanoemulsion (B), (B) -3, (B) -4, (B) -5 and (B) -6 was prepared. As an oil-soluble component as a membrane stabilizer, cholesterol JSCI (manufactured by Nippon Seika Co., Ltd.), as a nonionic surfactant and NIKKOL DHC 30 (Nikko Chemicals Co., Ltd., epolyoxyethylene cholesteryl ether), UNIOX CS 800 ( (Nippon Oil Co., Ltd., epolyoxyethylene cholesteryl ether) was used. Table 2 shows the mixing ratio of each substance for preparing the O / W type solubilized nanoemulsion (B).

  Oil-soluble components (cholesterol JSCI) and nonionic surfactants (NIKKOL DHC 30, UNIOX CS 800, NIKKOL BS 20, NIKKOL PBC 34) as stabilizers above the proportions shown in Table 2 above the melting point of cholesterol. Dissolved uniformly. Purified water heated to 80 ° C. or higher in a separate bath was added thereto while gradually adding and stirring, and after emulsifying the phase inversion temperature, it was rapidly cooled to room temperature.

  As a result, (B) -3 was a solubilized nanoemulsion with a cumulant average particle size of 15.0 nm, a particle size distribution of one peak of 10 to 40 nm, and a transparent appearance. (B) -4 was a solubilized nanoemulsion with a cumulant average particle size of 25.6 nm, a particle size distribution of one peak of 15 to 70 nm, and a transparent appearance. (B) -5 had a cumulant average particle size of 84.6 nm, a particle size distribution of 35 to 85 nm and two peaks of 85 to 400 nm, and a slightly white semi-solubilized nanoemulsion in appearance. (B) -6 was a solubilized nanoemulsion having a cumulant average particle size of 12.0 nm, a particle size distribution of one peak of 5 to 24 nm, and a transparent appearance (see FIG. 4).

(3) Preparation of mixed solubilized nanoemulsion by mixing O / W type solubilized nanovesicle emulsion (A) and O / W type solubilized nanoemulsion (B) Tables 1 and 2 show Examples 1 to 6 Each emulsion shown was mixed in the combinations shown in Table 3. Details of the mixing will be described later.

(Example 1) Preparation of composite-type solubilized nanoliposome emulsion 1 Using the emulsions (A) -37 and (B) -5 shown in Tables 1 and 2, the composite-type solubilized nanoliposomes of Example 1 were prepared. Produced.

  First, the respective emulsions obtained in (A) -37 (50 g) and (B) -5 (12.5 g) and purified water (37.5 g) were used in a weight ratio of 4: 1 so that the total was 100 g: The mixture was uniformly mixed at room temperature at a ratio of 3. As a result, a mixed solubilized nanoemulsion having a pale white appearance and two peaks with a cumulant average particle size of 79.2 nm, particle size distributions of 15 to 55 nm and 55 to 700 nm was obtained.

  The obtained mixed solubilized nanoemulsion was heated above the cloud point of the emulsion (80 to 85 ° C.) and aged for 30 seconds to make (A) -37 and (B) -5 uniform. A solubilized emulsified dispersion was obtained. Thereafter, the emulsion was rapidly cooled to room temperature to prepare composite-type solubilized nanoliposomes. As a result, a composite-type solubilized nanoliposome with a single cumulant average particle size of 31.3 nm and a particle size distribution of 14 to 55 nm and a single peak was obtained.

  The composite-type solubilized nanoliposomes obtained above are heated up to the cloud point of the emulsion (80 to 85 ° C.), aged for 30 seconds, and then rapidly cooled to room temperature. A composite solubilized nanoliposome emulsion having a cumulant average particle size of 20.3 nm and a particle size distribution of 10 to 58 nm with one peak was obtained.

(Example 2) Production of composite-type solubilized nanoliposome emulsion 2 Using the emulsions (A) -37 and (B) -4 shown in Tables 1 and 2, the composite-type solubilized nanoliposome of Example 2 was prepared. Produced.

  First, each emulsion obtained in (A) -37 (50 g) and (B) -4 (12.5 g) and purified water (37.5 g) were mixed at a weight ratio of 4: 1 so that the total was 100 g. The mixture was uniformly mixed at room temperature at a ratio of 3. As a result, a mixed solubilized nanoemulsion having a single cumulant average particle size of 29.6 nm and a particle size distribution of 15 to 80 nm and a single peak was obtained.

  The obtained mixed solubilized nanoemulsion was heated to the cloud point of the emulsion or higher (80 to 85 ° C.) in the same manner as in Example 1, aged for 30 seconds, and then rapidly cooled to room temperature. As a result, a composite solubilized nanoliposome emulsion having a cumulant average particle size of 22.0 nm and a particle size distribution of 15 to 70 nm with one peak was obtained.

  Compared with (B) -5 in Example 1, (B) -4 increased the amount of nonionic active agent by 0.1 g, so that the appearance at the time of mixing was changed from semi-solubilization to solubilization, and the average particle size was fine. It became. In this example, stable liposomes having a uniform particle size distribution were obtained without repeating reheating. In addition, reducing the number of heating times can shorten the process and improve the risk of thermal degradation.

(Example 3) Production of composite-type solubilized nanoliposome emulsion 3 Using the emulsions (A) -37 and (B) -3 shown in Tables 1 and 2, the composite-type solubilized nanoliposome of Example 3 was prepared. Produced.

  First, each emulsion obtained in (A) -37 (50 g) and (B) -3 (12.5 g) and purified water (37.5 g) were mixed at a weight ratio of 4: 1 so that the total was 100 g: The mixture was uniformly mixed at room temperature at a ratio of 3. As a result, a mixed solubilized nanoemulsion with a single cumulant average particle size of 21.9 nm and a particle size distribution of 10 to 80 nm was obtained.

  The obtained mixed solubilized nanoemulsion was heated to the cloud point of the emulsion or higher (80 to 85 ° C.) in the same manner as in Example 1, aged for 30 seconds, and then rapidly cooled to room temperature. As a result, a composite solubilized nanoliposome emulsion having a cumulant average particle size of 19.5 nm and a particle size distribution of 10 to 60 nm with one peak was obtained.

  Compared with (B) -5 of Example 1, (B) -3 increased the amount of nonionic active agent by 0.2 g. As a result, stable liposomes having an average particle size smaller than that of the liposomes obtained in Examples 1 and 2 and high appearance transparency were obtained.

(Example 4) Preparation of composite-type solubilized nanoliposome emulsion 4 Using the emulsions (A) -37 and (B) -6 shown in Tables 1 and 2, the composite-type solubilized nanoliposomes of Example 4 were prepared. Produced. The emulsion of (B) -6 contains a function-imparting oil-soluble component in addition to cholesterol as an oil-soluble component.

  First, each emulsion obtained in (A) -37 (50 g) and (B) -6 (25 g) and purified water (25 g) were in a weight ratio of 2: 1 so that the total was 100 g. Uniform mixing at room temperature at a ratio of 1. As a result, a mixed solubilized nanoemulsion was obtained in which the cumulant average particle size was 75.1 nm, the particle size distribution was 18 to 65 nm, and the number of peaks of two peaks was 65 to 600 nm, and the appearance was slightly white solubilized.

  The obtained mixed solubilized nanoemulsion was heated to the cloud point of the emulsion or higher (80 to 85 ° C.) in the same manner as in Example 1, aged for 30 seconds, and then rapidly cooled to room temperature. As a result, a composite solubilized nanoliposome emulsion having a cumulant average particle size of 23.8 nm and a particle size distribution of 15 to 70 nm with one peak was obtained. Although a large amount of nonionic active agent is necessary in the blending ratio of this example, a functionalized solubilized nanoliposome emulsion was obtained.

(Example 5) Preparation of composite-type solubilized nanoliposome emulsion 5 Using the emulsions (A) -35 and (B) -3 shown in Tables 1 and 2, the composite-type solubilized nanoliposomes of Example 5 were prepared. Produced.

  First, each emulsion obtained in (A) -35 (50 g) and (B) -3 (12.5 g) and purified water (37.5 g) were mixed at a weight ratio of 4: 1 so that the total was 100 g. The mixture was uniformly mixed at room temperature at a ratio of 3. As a result, a mixed solubilized nanoemulsion with a single cumulant average particle size of 18.9 nm and a particle size distribution of 10 to 50 nm and a single peak was obtained.

  The obtained mixed solubilized nanoemulsion was heated to the cloud point of the emulsion or higher (80 to 85 ° C.) in the same manner as in Example 1, aged for 30 seconds, and then rapidly cooled to room temperature. As a result, a composite-type solubilized nanoliposome emulsion having a cumulant average particle size of 20.1 nm and a particle size distribution of 10 to 55 nm with one peak was obtained.

(Example 6) Preparation of composite-type solubilized nanoliposome emulsion 6 Using the emulsions (A) -35 and (B) -6 shown in Tables 1 and 2, the composite-type solubilized nanoliposomes of Example 6 were prepared. Produced.

  First, each emulsion obtained in (A) -35 (50 g) and (B) -6 (25 g) and purified water (25 g) are in a weight ratio of 2: 1 so that the total is 100 g. Uniform mixing at room temperature at a ratio of 1. As a result, a mixed solubilized nanoemulsion having a single cumulant average particle size of 24.9 nm and a particle size distribution of 14 to 60 nm and one peak was obtained.

  The obtained mixed solubilized nanoemulsion was heated to the cloud point of the emulsion or higher (80 to 85 ° C.) in the same manner as in Example 1, aged for 30 seconds, and then rapidly cooled to room temperature. As a result, a composite solubilized nanoliposome emulsion having a cumulant average particle size of 23.5 nm and a particle size distribution of 15 to 80 nm with one peak was obtained. In the blending ratio of this example, a large amount of nonionic active agent was required, but the average particle size of the liposome was further reduced in the functionalized solubilized nanoemulsion.

(Comparative example)
Without using the specific nonionic surfactant used in the emulsification step of the present invention. 2 g of hydrophobic phospholipid powder coatedsome NC 21 (manufactured by NOF Corporation, HSPC) alone was dissolved at a temperature higher than the phospholipid dissolution temperature (about 75 ° C.). Into this, 48 g of purified water at 80 ° C. or higher was gradually added and emulsified by phase inversion temperature while stirring. However, the appearance was clouded with aggregated sediment and suspended phospholipids, and the phospholipids could not be uniformly emulsified and dispersed in an aqueous solution, making it impossible to form liposomes.

(Experimental Example 1) Confirmation of Sustained Release Using water-soluble enzyme α-amylase (manufactured by Novozymes Japan), sustained release was confirmed when the liposome of the present invention was used.
Sample (a) A diluted enzyme 1% aqueous solution was prepared by diluting the enzyme with purified water.
Sample (b) To 50 g of liposomes of the solubilized vesicle emulsion of (A) -37 in the above Example, 50 g of an enzyme diluted solution obtained by diluting 1 g of the enzyme with 49 g of purified water was added, and the total amount was 100 g ( A mixed solution having a weight ratio of 1: 1) was prepared. The mixed solution was aged at the maximum temperature of the enzyme for 60 seconds by a remote loading method to prepare an enzyme-encapsulated liposome trapping the enzyme, and then rapidly cooled to room temperature.
Sample (c) 25 g of an enzyme diluted solution obtained by diluting 1 g of enzyme in 24 g of purified water to 75 g of liposomes of (A) -35 and (B) -6 (weight ratio = 2: 1) of Example 6 Was added to prepare a mixed solution with a total amount of 100 g. Enzyme-encapsulated liposomes with the enzyme trapped by the remote loading method under the same conditions as in sample (b) were prepared, and then rapidly cooled to room temperature.
Sample (d) 37.5 g of diluted enzyme solution obtained by diluting 12.5 g of enzyme into 36.5 g of purified water to 62.5 g of liposomes of (A) -35 and (B) -3 (weight ratio = 4: 1) of Example 5 Was added to prepare a mixed solution with a total amount of 100 g. Enzyme-encapsulated liposomes with the enzyme trapped by the remote loading method under the same conditions as in samples (b) and (c) were prepared, and then rapidly cooled to room temperature.

  About the prepared samples (a) to (d), the viscosity reduction due to starch degradation of the enzyme was measured by elapsed time, and the sustained release effect of the liposome of the present invention was confirmed. The O / W type solubilized nanoemulsion (B) alone could not encapsulate a water-soluble enzyme. For samples (b) to (d), the enzyme-encapsulated liposome was an aqueous solution adjusted to pH 6.5 to 7.0 with citric acid, a disodium citrate buffer, and L-arginine.

The enzyme activities of samples (a) to (d) were measured by the viscosity of the enzyme due to starch degradation. Prepare starch suspension by adding 98 g of purified water to 2 g of commercially available potato starch (manufactured by AEON Co., Ltd.), heat and stir at 80-90 ° C to gelatinize, then cool to room temperature, 0.02 g of the enzyme solution of each of the above samples (a) to (d) was added at room temperature to 5 g of the prepared starch 2% aqueous solution, and the degree of viscosity reduction due to starch degradation with the elapsed time (minutes) Was judged. Viscosity reduction measurement was performed by preparing a viscosity sample standard solution in increments of 100% to 10% of a 2% starch aqueous solution, visually judging the degree of viscosity reduction of the starch solution to which each sample was added, and is shown in FIG. As a result, the sample (b), sample (c) and sample (d) in which the enzyme was trapped had a slow decrease in viscosity of the aqueous starch solution compared to the enzyme enzyme single dilution sample (a), and the sustained release effect. Was recognized. The samples (c) and (d) of the composite type solubilized nanoliposomes prepared in Example 5 and Example 6 are more viscous than the sample (b) of the O / W type solubilized nanovesicle emulsion. The drop time is longer.

(Experimental example 2) Time stability test 1
For each of the composite-type solubilized nanoliposome prepared in Examples 1, 2, and 3, the changes in the average particle diameter and appearance of the liposomes after 6 months at 5 ° C. were observed to confirm the stability. As a result, almost no change in the average particle size was observed (Table 4).

(Experimental example 3) Time stability test 2
Each composite-type solubilized nanoliposome emulsion prepared in Examples 2 and 5 was subjected to a repeated heat resistance test at 80 ° C. and then rapidly cooled at room temperature to confirm the stability. The heating conditions are as follows.
Heating once: O / W-type solubilized nanovesicle emulsion (A) and O / W-type solubilized nanoemulsion (B) were mixed, heated at 80 ° C. for 30 seconds, and then rapidly cooled to 25 ° C.
Heating twice: The first heating and rapid cooling were repeated again under the same conditions.
Heating 3 times: The second heating and rapid cooling were repeated again under the same conditions.

As a result, there was almost no difference in the average particle size and particle size distribution of the liposomes (Table 5).

  As described above in detail, the composite solubilized nanoliposome of the present invention comprising a structure containing a nonionic surfactant, a phospholipid, and a hydrophobic substance is an SUV liposome having an average particle size of 18 to 25 nm and is stable. Excellent in properties. According to the method for producing a composite-type solubilized nanoliposome of the present invention, it is not necessary to use an organic solvent, and no special emulsifying device or granulating device is required. Since it is a technical method and does not use an organic solvent, it can be said to be a liposome with excellent safety.

  The liposome of the present invention is economically superior in terms of equipment and materials in the production process. Furthermore, the liposome of the present invention and the emulsion containing the liposome can be used in many fields such as pharmaceuticals, foods, and cosmetics, and the effect is great from this point.

Claims (18)

A method for producing a composite-type solubilized nanoliposome comprising the following steps:
(1) A step of preparing an O / W-type solubilized nanovesicle emulsion (A) containing a phospholipid and a nonionic surfactant;
(2) a step of preparing an O / W-type solubilized nanoemulsion (B) containing an oil-soluble component and a nonionic surfactant;
(3) A step of preparing the mixed solubilized nanoemulsion by mixing and homogenizing the O / W type solubilized nanovesicle emulsion (A) and the O / W type solubilized nanoemulsion (B);
(4) A step of heating and emulsifying the mixed solubilized nanoemulsion obtained in step (3) above the cloud point. The step (1) is a step of dissolving and uniformly mixing a phospholipid having a phase transition temperature of 0 to 75 ° C in a solution containing a nonionic surfactant, and emulsifying and dispersing the solution by a phase inversion temperature emulsification method. The manufacturing method as described. The manufacturing method of Claim 1 or 2 whose phospholipid of the said process (1) is phospholipid which is phosphatidylcholine whose carbon number of an acyl group is 12-22. The manufacturing method of any one of Claims 1-3 which contains 0.5-5 mass% of phospholipids in the O / W type | mold solubilized nanovesicle emulsion (A) as described in the said process (1). The manufacturing method of any one of Claims 1-4 which further contains a tonicity agent in the said process (1). The manufacturing method according to claim 1, further comprising ethanol in the step (1). The manufacturing method according to any one of claims 1 to 6, wherein the step (2) comprises a step of dissolving and mixing an oil-soluble component in a nonionic surfactant and then emulsifying and dispersing the same by a phase inversion temperature emulsification method. The manufacturing method of any one of Claims 1-7 in which the oil-soluble component of the said process (2) contains cholesterol at least. The nonionic surfactant used in the steps (1) and (2) is polyoxyethylene sterol ether, polyoxyethylene stearyl ether or its isoform, and polyoxyethylene polyoxypropylene alkyl ether, and the affinity thereof Any one or more kinds of nonionic surfactants selected from surfactants having a property (HLB value) of 10.7 to 18.0, each independently or in common, 2. The production method according to 1. A composite-type solubilized nanoliposome produced by the production method according to claim 1. The composite-type solubilized nanoliposome according to claim 10, wherein the particle size distribution of the liposome is 10 to 80 nm. A liposome having an average particle size of 18 to 25 nm, comprising a hydrophilic substance in the inner aqueous phase, and an oil-soluble component enclosed in the bilayer membrane. When the composite-type solubilized nanoliposomes contain at least a phospholipid, an oil-soluble component and a nonionic surfactant, and the phospholipid is 1 by weight, the oil-soluble component is contained in an amount of 0.01 to 0.6%. The composite-type solubilized nanoliposome according to any one of claims 10 to 12. When the composite-type solubilized nanoliposome contains at least a phospholipid, an oil-soluble component, and a nonionic surfactant, and the weight ratio of each component is phospholipid: oil-soluble component: nonionic surfactant, 2 The composite-type solubilized nanoliposome according to claim 13, which is 0.02 to 1.2: 0.78 to 4.0. The composite-type solubilized nanoliposome according to claim 13 or 14, wherein the oil-soluble component contained in the liposome contains at least cholesterol and further contains a function-imparting oil-soluble component. A solubilized liposome emulsion comprising the composite-type solubilized nanoliposome according to any one of claims 10 to 15. The solubilized liposome emulsion according to claim 16, wherein the particle size distribution of the liposome contained in the solubilized liposome emulsion is 10 to 80 nm. The solubilized liposome emulsion according to claim 16 or 17, wherein the particle size peak distribution of liposomes contained in the solubilized liposome emulsion consists of one peak.