JPH01117824A - Production of liposome - Google Patents

Abstract

PURPOSE:To obtain a liposome used as a carrier of various kind of physiological active substances in medicine field in simple treatment and good yield, by adding water to a liposome membrane ingredient substance in forming of uniform mixture of liposome membrane ingredient substance. CONSTITUTION:A liposome membrane ingredient substance is dissolved in a volatile organic solvent (e.g., chloroform). Then distilled or ion-exchanged water for hydrating the membrane ingredient is added to the membrane ingredient substance at an amount of 1-3ml per gram of the membrane ingredient substance. The organic solvent is removed while stirring not so as to separate water from the organic solvent to afford a hydrate of membrane ingredient substance. Water and aqueous solution of salts or saccharides are added to the hydrate and hydrate of the membrane ingredient substance is dispersed to provide the liposome. The membrane ingredient substance contains as a main ingredient a phospholipid such as phosphatidylcholine derived from natural material such as egg yellow or soybean or obtained by synthesis.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing liposomes. The liposomes of the invention are particularly useful in the pharmaceutical field.

[Prior Art] Liposomes are seen as promising in the medical field as carriers for various physiologically active substances and have been widely studied. The main reason for this is that liposome membrane components and membrane structures are similar to those of living cells, so they can be expected to have high affinity with living organisms and low toxicity.

Generally, many methods for preparing liposomes have been reported.

For example, the summary is P, 5zoka.

D. Papahadjopaulos, Ann.
Rev. Biophys.

Bioeng, 9.467 (1980). However, these methods have been difficult to apply to industrial production due to the small scale, complicated operations and conditions, and low yields.

In addition, as an industrial method for producing liposomes, JP-A-60-12
No. 127 method has been reported. In this method, the organic solvent in which the membrane component substance has been dissolved is removed, and an aqueous solution is added to it to disperse it. However, if the membrane component substance is simply stirred, the membrane component substance after the organic solvent has been removed will solidify or become almost invisible. Since the liposome loses its fluidity, even if an aqueous solution is added in this state, it is difficult to achieve a hydrated state, which has the disadvantage that it is difficult to obtain a liposome that retains a drug efficiently.

[Problems to be Solved by the Invention] An object of the present invention is to provide an industrial method for producing liposomes that solves the problems of the prior art described above.

[Means for Solving the Problems] In the thin film method, which has been widely known as a method for producing liposomes, it has been considered necessary to form a clean thin film of a film component substance on the inner wall of a flask or the like. However, recent research has shown that in order to form liposomes, it is not necessary to form a clean thin film; instead, it is necessary to hydrate the membrane components as long as they are mixed at the molecular level and do not form a single crystal. It has been found that liposomes with high retention efficiency can be obtained by dispersion.

However, the membrane component material obtained by removing the organic solvent from the membrane component material is in a state with almost no fluidity, and when it is made in large quantities, hydration cannot be achieved simply by adding an aqueous solution and stirring. There was a drawback that liposomes were difficult to form because the entire body did not swell without being promoted.

The inventor has discovered that by adding the amount of water necessary to form a hydrate to an organic solvent solution in which a membrane component substance is dissolved and removing the organic solvent while stirring, a uniform membrane component substance hydrate can be easily formed. The present invention has been completed based on the discovery that liposomes can be produced with good reproducibility by stirring the obtained liposomes with an aqueous solution.

The membrane component substances used in the present invention are phospholipids such as phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, and phosphatidylserine, which are derived from egg yolk, soybeans, and other natural materials, or are obtained solely by synthesis. or as a main component in combination. Further, sterols such as cholesterol and cholestanol may be added as membrane stabilizers, and phosphatidic acid, dicetyl phosphate, higher saturated fatty acids, etc. may be added as charged substances. Further, tocopherol or the like may be added as an antioxidant. The ratio of these membrane component substances is not particularly limited, but preferably 0 to 2 parts by weight of styrene and 0 to 0.2 parts by weight of charged substance per 1 part by weight of phospholipid.

Examples of volatile organic solvents that dissolve film component substances include chloroform, dichloromethane, ethanol, hexane, and the like. The amount used is not particularly limited as long as it completely dissolves the membrane component material, but per gram of membrane component material,
Approximately 2 to 12 m1 is appropriate.

Furthermore, as the water for hydrating the membrane component substances, water that does not contain ions or salts is preferably used. The appropriate amount to be used is about 1 to 3 ml per gram of membrane component material.

Further, as the aqueous solution in which the membrane component substance hydrate is dispersed, water, an aqueous solution of salts or sugars, etc. are preferably used. The usage ratio is 1 to 500 parts by weight of the membrane component material.
Parts by weight are parts by weight.

There are no particular limitations on the drug retained in the liposome preparation of the present invention, and various physiologically active substances can be used. In the case of water-soluble substances such as hemoglobin, superoxide dismutase, and urokinase, they are dissolved in an aqueous dispersion of a hydrated membrane component substance. In the case of a substance with strong affinity, it is preferable to mix it with the membrane component substance for production.

The method for producing liposomes of the present invention is suitably carried out by the procedure shown below.

The membrane component materials are first dissolved in a volatile organic solvent.

The appropriate amount is about 3 to 12 ml per gram of membrane component material. Then distilled or ion-exchanged water (
(approximately 1 to 3 ml per gram of membrane component material) is added, and the organic solvent is removed while stirring to such an extent that water and organic solvent do not separate. To remove the organic solvent, reduced pressure, blowing of an inert gas (argon gas, nitrogen gas, etc.), bubbling, etc. are used. The organic solvent that evaporates from the stirring vessel can be almost completely recovered by installing a trap cooled with liquid nitrogen, dry ice-methanol, etc., so the work can be carried out safely. This operation of removing the organic solvent to produce a hydrated membrane component material is preferably carried out at a temperature equal to or higher than the phase transition temperature (Tc) of the membrane component material.

Next, the membrane component substance hydrate thus obtained and the aqueous solution are vigorously stirred and dispersed. The amount of the aqueous solution is suitably 1 to 500 parts by weight per 1 part by weight of the membrane component material, and several types of aqueous solutions may be added in batches or in several batches. There are no particular restrictions on the stirrer, and devices commonly used for emulsification, such as a homo mixer and a paddle mixer, are suitable. Furthermore, in order to obtain a liposome preparation with a small particle size, it is desirable to further perform a dispersion treatment after stirring using a pressure emulsifier, an ultrasonic emulsifier, or a French press cell disrupter. The temperature during this stirring and dispersion is preferably higher than the phase transition temperature (Tc) of the membrane component substance. However, if the membrane component substance contains sterols, the phase transition of the membrane component as a whole becomes unclear, so that liposomes can be sufficiently produced even at an operating temperature of Tc or lower. This can be applied when liposomes retain physiologically active substances that are easily deactivated at high temperatures.

The particle size distribution of the liposome thus obtained may be controlled using a membrane filter, or unretained physiologically active substances may be removed by means of ultrafiltration, centrifugation, or gel filtration.

Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Fully hydrogenated egg yolk phosphatidylcholine 18.4g.

Weighed 59.3 g of cholesterol and 8.4 g of myristic acid, dissolved them in 400 ml of chloroform in an Ajihomo mixer (Tokushu Kika Kogyo type) container, added 400 ml of distilled water, and - while operating the Homo mixer and paddle mixer, Chloroform was removed by maintaining the temperature at 40° C. and reducing the pressure inside the container. At this time, chloroform was almost completely recovered by the trap cooled with dry ice and methanol. The membrane component substance hydrate thus obtained was white and cream-like. Next, the mixture was cooled while stirring with a paddle mixer, and the container was maintained at 5°C. A 30% (W/V) hemoglobin solution, previously cooled to 5°C, was added thereto for 60 minutes.
0 g was added, and vigorous stirring was performed using a paddle mixer and a homomixer. The operation was terminated when the whole was sufficiently uniform. To remove hemoglobin that was not retained in the liposomes, the resulting solution was diluted with 5 g of physiological saline.
Ultrafiltered with a membrane filter with a pore size of 0.1μ,
Ultrafiltration was repeated while diluting with physiological saline until no hemoglobin was detected in the Nito solution. 5 g of the thus obtained hemoglobin-retaining liposome suspension had a pink color, and when observed under an optical microscope, a uniform spherical structure with a particle size of approximately several μm was confirmed.

When the hemoglobin concentration of the liposome suspension was determined by the cyanmethemoglobin method and the amount of hemoglobin retained in the suspension was determined, the ratio (hemoglobin yield) to the amount of hemoglobin input during production was 28.8%.

Furthermore, the visible absorption spectrum of the suspension had a spectrum unique to oxyhemoglobin, and it was confirmed that hemoglobin was not denatured or oxidized during liposome production.

Example 2 Completely hydrogenated egg yolk lecithin (phospholipid 99% or more) 11
Weighed 5.0 g of cholesterol and 31.3 g of cholesterol, and added 5.5 g of dichloromethane in a homodisper (Tokushu Kika Kogyo type) container.
00 ml was added, and 350 ml of distilled water was added, and while stirring the liquid with a disper blade, the container was kept at 40° C. and the pressure inside the container was reduced with a vacuum pump to remove dichloromethane. At this time, a trap cooled with dry ice and methanol was installed between the reaction vessel and the vacuum pump, and dichloromethane was almost completely recovered. The membrane component substance hydrate thus obtained was white and cream-like. Next, the mixture was cooled while stirring with a disper, and the container was kept at 5°C.

30% (W/V) cooled to 5℃ in advance
Boo g of hemoglobin solution was added, and the disper was rotated at high speed to make the entire solution uniform. In order to produce smaller liposomes, 50 ml of the obtained liquid was taken out and treated with a French press cell disrupter (manufactured by Otake Seisakusho) at 5° C. and a pressure cuff of 00 kg/c−. Furthermore, in order to remove hemoglobin that was not retained in the liposomes of this solution (solution ■), 15RIM Tris buffer (pl + 7.4) was added.
Containing physiological saline (hereinafter abbreviated as Tris saline) 300m
After adding 1g, centrifuge at 5°C (1g0.000X g,
30 minutes). The obtained precipitate was immersed in Tris saline 1
Centrifugation and resuspension were repeated until no hemoglobin was detected in the centrifuged supernatant. The finally obtained liposome suspension had a pink color. Hemoglobin was quantified and the ratio of the amount of hemoglobin retained in the liposome suspension to the amount of hemoglobin in the solution (liquid ■) before centrifugation (hemoglobin retention rate) was determined.2
It was 6.4%. When the suspension was observed using an optical microscope, a small number of spherical particles of several μm and many particles of 1 μm or less with Brownian motion were observed.

In Examples 1 and 2 above, when distilled water was not added to the organic solvent solution of egg yolk phosphatidylcholine or lecithin, no liposomes were produced.

[Effects of the Invention] According to the present invention, the amount of water necessary for forming a hydrate is added to an organic solvent solution in which a membrane component substance is dissolved, and the organic solvent is removed while stirring, thereby easily forming a uniform membrane. A membrane component substance hydrate can be obtained, and liposomes can be produced by vigorously stirring this with an aqueous solution. In this way, liposomes can be produced in high yield with a simple operation of adding water during the formation of a homogeneous mixture of membrane component substances, so the present invention is an extremely excellent method for producing industrial liposomes. .

Claims (4)

[Claims] (1) Dissolve the liposome membrane component substance in a volatile organic solvent, add the amount of water necessary for forming a hydrate to the resulting solution,
A method for producing liposomes, which is characterized by removing an organic solvent while stirring to produce a hydrate of a membrane component substance, and adding an aqueous solution to the hydrate to disperse it. (2) The method for producing liposomes according to claim 1, wherein the aqueous solution contains a physiologically active substance. (3) The method for producing liposomes according to claim 2, wherein the physiologically active substance is hemoglobin. (4) The method for producing a liposome according to claim 1, wherein the liposome membrane component substance contains a physiologically active substance.