JPH06246150A - Production of liposome - Google Patents

Abstract

PURPOSE:To produce a large amt. of liposome by depositing a membrane forming lipid on a porous material and bringing the surface of the material into contact with an aq. soln. CONSTITUTION:A membrane forming lipid alone or its soln. in an org. solvent is injected into a device packed with a porous material to sufficiently impregnate the material, and the excess lipid is discharged outside the system. An inert gas such as nitrogen is passed through the device or the device is evacuated to remove the solvent, and the lipid is dried to solid. After the lipid is sufficiently dried, an aq. soln. is supplied and brought into contact with the dried lipid, and a dispersion of liposome in water is obtained. The dispersion is forcedly passed through the porous material repeately or vibrated by an ultrasonic wave or vibrator, hence the swelling of lipid and the separation of lipid from the porous material are promoted, and a large amt. of uniform liposome is efficiently produced.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing liposomes.

[0002]

2. Description of the Related Art Liposomes, which are closed vesicles of lipids,
As a biomembrane model, it is possible to retain lipophilic substances and hydrophilic substances inside, and its application to drug carriers and sensors is being studied.

The vortexing method, which is a typical method for producing liposomes, is a method in which a thin film of the lipid constituting the membrane is formed on the glass surface, and then an aqueous solution is added to swell and hydrate the thin film, and mechanical vibration is applied ( Methods Member.Biol., 1,1 (1
974)).

[0004]

However, although this method is very effective in the laboratory, it cannot obtain a uniform particle size and is not suitable for industrial mass production.

An object of the present invention is to provide a method for producing a large amount of liposomes having a uniform particle size.

[0006]

Therefore, the present inventors have
As a result of repeated intensive studies to solve the above problems, it was found that the above problems can be solved by attaching the membrane-constituting lipid to the porous material and then contacting the surface of the porous material with the aqueous solution. The present invention has been completed.

As the porous material, for example, a porous sintered body such as metal, glass or ceramic can be used. The pore diameter or void of the porous material is 0.15
1000 μm, especially 10 to 100 μm are suitable. In addition, the thickness of the porous material is preferably 5 to 100 mm in consideration of the retention rate of the lipid constituting the membrane and the pressure.

Membrane constituent lipids include phospholipids such as egg yolk lecithin, soybean lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and sphingomyelin, sucrose fatty acid ester, trehalose fatty acid. One or more of sugar fatty acid esters such as ester and rhamnose fatty acid ester, or a mixture thereof can be used.

In addition to the above, as membrane constituents, sterols such as cholesterol as a membrane stabilizer, phosphatidic acid as a charged substance, dicetyl phosphate,
Ganglioside, stearylamine and the like may be added, and tocopherol and the like may be added as an antioxidant.

The composition ratio of these membrane-constituting substances is not particularly limited, but 1 part by weight of phospholipid and 0 of sterols.
˜0.5 parts by weight, 0 to 0.1 parts by weight of the charged substance, and 0 to 0.1 parts by weight of the antioxidant are preferably added.

The aqueous solution passes through a porous material, and swells and disperses the dried membrane-constituting lipid on the surface thereof. Water alone, physiological saline, various buffer solutions, or an aqueous solution in which saccharides are dissolved is used. Can be used.

Liposomes, which are closed endoplasmic reticulum of lipids, can be used as a biomembrane model and can retain lipophilic substances and hydrophilic substances inside, and are typical as substances to be embedded in liposomes. There is. Drugs include hydrophilic drugs and lipophilic drugs, but in the case of hydrophilic drugs, they are dissolved in the above aqueous solution and, if necessary, added to a hydrophilic solvent such as methanol, ethanol, acetone, dimethylsulfoxide (DMSO) and dioxane. Used by dissolving. Further, in the case of a lipophilic drug, it may be dissolved in the membrane-constituting lipid, and if necessary, an organic solvent such as ethanol, acetone, chloroform, ether or hexane may be added.

The liposome of the present invention is produced by injecting a membrane-constituting lipid, alone or dissolved in a solvent, into a device equipped with a porous material so that the porous material is sufficiently impregnated and adhered, and excess lipid is removed from the system. Remove. At this time, the membrane-constituting lipid is passed through the porous material several times in order to adhere the membrane-constituting lipid well to the surface of the porous material, if necessary. Then, an inert gas such as nitrogen gas is passed through or a vacuum is applied to remove water and the organic solvent, and the lipid constituting the membrane is dried. Then, after the membrane-constituting lipid is sufficiently dried, an aqueous solution is supplied and brought into contact (swelling) with the dried membrane-constituting lipid to obtain an aqueous solution in which liposomes are dispersed. At this time, the porous material is forcibly repeatedly passed by a pump or the like, or vibration is applied by an ultrasonic wave or a vibrator to promote swelling of the lipid constituting the membrane by the aqueous solution and release of the membrane constituent from the porous material. It can be promoted to efficiently produce liposomes.

[0014]

INDUSTRIAL APPLICABILITY According to the method for producing liposomes of the present invention, uniform liposomes can be produced in large quantities.

[0015]

【Example】

Example 1 5 ml of chloroform solvent containing 20 mg / ml egg yolk lecithin and 3.8 mg / ml cholesterol membrane-constituting lipid was sandwiched with a glass sintered body (pore diameter 40 to 100 μm) having a diameter of 30 mm and a thickness of 5 mm. The membrane-constituting lipid was supplied to the filtration device and passed through the porous material several times to sufficiently attach the membrane-constituting lipid to the porous material. Then, nitrogen gas was blown at room temperature to remove chloroform, and the lipid constituting the membrane was dried on the porous material. 4 ml of phosphate buffered saline having a pH of 7.4 swelled the membrane-constituting lipid attached to the partially porous material, and the remaining phosphate buffered saline was added to obtain a liposome dispersion liquid. The diameter of the obtained liposomes was measured by a particle size distribution meter (Nicomp NOF Liposome Co., Ltd.). As a result, the average particle size was 2.5 μm and the particle size distribution was 45%.

Example 2 In accordance with Example 1, a glass sintered body (having a pore size of 40 to 100) was prepared.
μm), 20 mg / ml egg yolk lecithin, 3.
5 ml of a chloroform solution containing a membrane-constituting lipid of 8 mg / ml cholesterol was supplied, and the membrane-constituting lipid was adhered to a glass sintered body according to Example 1. Brilliant blue 0.17 mg / ml as a sample of hydrophilic substances
After contacting (swelling) the porous material to which the membrane-constituting lipid was attached with 1 ml of an aqueous solution in which was dissolved, 3 ml of phosphate buffered saline having a pH of 7.4 was added, and ultrasonic waves were further applied to obtain liposomes. When the obtained liposomes were measured according to Example 1, the average particle size of the liposomes was 3.0 μm, and the particle size distribution was 36%. The aqueous solution obtained by ultrafiltration of the liposome dispersion was compared with a standard solution by a spectrophotometer to measure the retention rate of brilliant blue, which was 15%.

Example 3 1 ml of an ethanol solution containing 0.17 mg / ml of brilliant blue was added to 5 ml of a chloroform solvent of a lipid constituting the membrane according to Example 1, and then adhered to a glass sintered body and dried. Then, phosphate buffered saline 4 with pH 7.4
After adding ml, the mixture was vibrated with a vibrator to obtain liposomes. The particle size of the obtained liposomes was measured according to Example 1 to be 2.0 μm on average, and the particle size distribution was 33.
%Met. The aqueous solution obtained by ultrafiltration of the liposome dispersion was compared with a standard solution by a spectrophotometer to measure the retention rate of brilliant blue, which was 63%. As a result, it was found that even if the hydrophilic substance has a low retention rate, the retention rate increases due to the liposome formation by the production method of the present invention.

Example 4 A filtration device sandwiching a sintered glass body (pore diameter 40 to 100 μm) of a porous material having a diameter of 300 mm and a thickness of 7 mm,
500 ml of a chloroform solution containing a membrane-constituting lipid of 20 mg / ml egg yolk lecithin and 3.8 mg / ml cholesterol was supplied, and the membrane-constituting lipid was attached to the porous material. Then, nitrogen gas was blown at room temperature to remove chloroform, and the lipid constituting the membrane was dried on the porous material. The membrane-constituting lipid attached to the porous material was expanded with a part of 400 ml of phosphate buffered saline having a pH of 7.4, and the remaining phosphate buffered saline was added to obtain a liposome dispersion liquid. When the obtained liposomes were measured according to Example 1, the average particle size of the liposomes was 3.0 μm and the particle size distribution was 3
It was 6%. As a result, it was found that the treatment amount could be easily increased by increasing the diameter and thickness of the porous material in the production method of the present invention.

Example 5 A liposome dispersion liquid was prepared in the same manner as in Example 4 except that the porous material having a diameter of 300 mm and a thickness of 7 mm mounted on a filtration device was a sintered body of glass (pore diameter 30 to 200 μm). . When the diameter of the liposome was measured according to Example 1, the average was 6.0 μm, and the particle size distribution was 35%. Further, the obtained liposome was subjected to gel filtration and freeze-dried, and the retention rate of the lipophilic dye Sudan III was measured according to Example 2 to be 92%. As a result, it was found that the treatment amount can be easily increased by increasing the diameter and thickness of the porous material in the production method of the present invention, and the retention rate of the lipophilic substance contained therein is also increased.

Example 6 23 mg / ml dipalmitoylphosphatidylcholine (DPPC), 3.8 mg / ml dipalmitoylphosphatidylserine (DPPS), 2 mg / ml cholesterol, and dye as lipophilic sample 5 ml of a chloroform solution containing 1.7 mg / ml of Sudan III was supplied to a filtration device equipped with a ceramic sintered body (pore size: 10 to 50 μm) having a diameter of 30 mm and a thickness of 3 mm, and the membrane-constituting lipid was used as a porous material. Attached. Less than,
A liposome dispersion was obtained in the same manner as in Example 1. The average diameter of the liposome was 0.8 μm, and the particle size distribution was 28%.
Further, the obtained liposome aqueous solution was subjected to gel filtration and freeze-dried. 0.1 g of the solution was dissolved in 20 ml of xylene, and the retention rate of Sudan III was measured by a spectrophotometer as compared with the standard solution. As a result, if ceramic is used as the porous material in the production method of the present invention, the retention rate of the lipophilic substance to be encapsulated is increased, and
It was found that the particle size can be made small and uniform.

Example 7 A liposome dispersion liquid was prepared in the same manner as in Example 2 except that a sintered body (pore diameter 30 to 200 μm) of iron having a diameter of 30 mm and a thickness of 3 mm and equipped with a filtration device was used. did. The average diameter of the liposome was 5.0 μm, and the particle size distribution was 35%. The dispersion liquid of the obtained liposomes was subjected to gel filtration and freeze-drying, and the Sudan retention rate was measured according to Example 2 to find that it was 91%.

Comparative Example 1 Egg yolk lecithin 20 mg cholesterol 3.8 mg 5
The mixture was placed in a 0 ml eggplant-shaped flask, and 2 ml of chloroform was added and dissolved. Then, chloroform was removed under reduced pressure using a rotary evaporator, 1 ml of physiological saline was added thereto, and the flask was shaken for 30 minutes to peel off and disperse the film adhering to the inner wall of the flask, and then to form the dispersion liquid. Ultrasonic processor (Nippon Seiki, NS200-
Type 2) for 20 minutes. To this, 6 ml of physiological saline was added to obtain a liposome dispersion liquid. The average particle size of the obtained liposomes was 3.5 μm, and the particle size distribution was 45%. As described above, in the conventional method, the treatment amount is very small because the surface area of the eggplant-shaped flask determines the treatment amount, and the width of the particle size distribution is large because the liposome is peeled off from the membrane prepared in a large area. Is large, and a large amount of uniform products cannot be obtained.

Claims (1)

[Claims] 1. A method for producing liposomes, wherein a membrane-constituting lipid is attached to a porous material and then the surface of the porous material is contacted with an aqueous solution.