JPS6012127A - Preparing method of liposome - Google Patents

Abstract

PURPOSE:To prepare uniform liposome good in a chemical agent holding rate, by a method wherein the componential substance of a liposome membrane is mixed with a volatile org. solvent and, after the org. solvent is removed under stirring, the obtained uniform mixture is dispersed in an aqueous solution. CONSTITUTION:About 0.1-8pts.wt. of a volatile org. solvent is added to and mixed with 1pts.wt. of the componential substance of a liposome membrane (phosphatidyl choline) and the org. solvent is removed under stirring or kneading to form a uniform mixture. In this case, a membrane stabilizer (cholesterol), a charging substance (diacetyl phosphate) and an oxidation inhibitor (alpha-tocopherol) may be added to the membrane componential substance if necessary. Subsequently, an aqueous solvent (water or a physiological saline solution) is added to the uniform mixture in a ratio of 10-1,000pts.wt. to 1pts.wt. of the membrane componential substance to swell liposome at the phase transition temp. of lipid and the resulting mixture is further finely dispersed to prepare liposome.

Description

[Detailed description of the invention] The present invention relates to an industrial method for producing liposomes.

Liposomes, which are closed vesicles of lipids, have originally been widely used as a biological membrane model, but recently they have been used for drug delivery.
Various applications have been made for this purpose. The types of liposomes are broadly divided into multi-lamellar liposomes (MLV).
), large unilamellar liposomes (LUV), and small unilamellar liposomes (BUY), and various preparation methods have already been reported [Annual Rev. Engineering, vol. 9, p. 467 ('i9Mo year)]. However, all of these are just methods at the laboratory level on a test tube or eggplant-shaped Kolben scale, and they are not manufacturing methods that can lead to immediate industrial production. As an industrial method for producing liposomes, a method has recently been reported in Japanese Patent Application Laid-Open No. 171915/1985, but this method uses two types of organic solvents in which membrane component substances are dissolved, namely, a hydrophobic one and an aqueous solvent. It is a method of injecting soluble substances into an aqueous solution, and a specific combination of organic solvents must be used;
Bilaminar liposomes, mainly made of phospholipids, tend to form.

There are disadvantages such as the tendency to form ribosonims with unrestricted particle size and the difficulty in efficiently retaining drugs within liposomes, so it cannot be said that industrial production of liposomes has become possible. Therefore, despite the clinical application research of liposomes being carried out by many researchers, one of the major reasons why liposome preparations have not been commercialized is the difficulty of industrial production. It's not too much to say.

The inventors were concerned about these circumstances and conducted extensive research into an industrial method for producing liposomes.

The most widely known portic swing (Vort)
exing ) law [Journal of Morcuso
Biology, vol. 18, p. 288 (1965)], it is not necessary to form a clean thin film of phospholipids on a glass wall in the production of liposomes, but simply to form a homogeneous mixture of membrane component substances. We discovered that by swelling this in an aqueous solvent to form a hydrated liquid crystal and stirring thoroughly, it was possible to produce a ribosome that was surprisingly uniform and had good drug retention efficiency with good reproducibility. The invention was completed.

That is, according to the present invention, a homogeneous mixture is produced by mixing ordinary membrane component substances constituting liposomes in a small amount of volatile organic solvent, and then removing the organic solvent while stirring or kneading with a stirrer. However, a large amount of liposomes can be produced by adding an aqueous solvent to the mixture, causing it to swell at a temperature above the phase transition temperature (Tc) of the lipid, and further finely dispersing it using a stirrer.

One example of the membrane component material used in the present invention is Evaphos 7.
γtidylcholine, phosftetidylethanolamine, phosphatidylserine, phosphatidylinositol, lysophosftetidylcholine, sphingomyelin, egg yolk lecithin.

In addition to phospholipids such as soybean lecithin, one or a mixture of two or more of glycolipids, dialkyl type synthetic surfactants, etc. are mainly used. In addition, sterols such as cholesterol and cholestanol are used as membrane stabilizers, dicetyl phosphate, phosph-T thidic acid, ganglioside, stearylamine, etc. are added as charged substances, and α-tocopherol etc. are added as antioxidants. may be added to form a membrane component substance. Although the ratio of the membrane component substances of these liposomes should not be limited in any way, it is preferable that
It is suitable to add about 0 to 2 parts by weight of sterols and about 0.1 parts by weight of charged substances.

Chloroform, ether, ethanol, etc. are suitable as the volatile organic solvent with which the membrane component substances are mixed. These organic solvents can be used alone or in combination, but
When used in combination, it is desirable that they are mutually miscible. What is the usage ratio of these organic solvents to the membrane component materials?

It is preferably 0.1 to 8 parts by weight per part by weight of the membrane component material.

In addition, water, water,
Physiological saline, buffer solutions, aqueous saccharide solutions, mixtures thereof, etc. are preferably used, and the appropriate ratio of use to the membrane component material is about 10 to 1000 parts by weight per 1 part by weight of the membrane component material.

The drug retained in the liposome preparation of the present invention is not particularly limited, but includes anticancer drugs typified by cytosin arabinoside and methotrexate.

Antibiotics such as penicillin G, insulin, inter-7epn, proteins such as glucoamylase, polysaccharides such as dextran, pNum, and RN.
In addition to nucleic acids such as A, vitamins such as vitamin A, general drugs such as sodium salicylate are used. These drugs are used after being dissolved in an aqueous solvent, but
It is more efficient to mix membrane-compatible drugs such as clophyll, gramicidin S, vitamin A, etc. in an organic solvent together with membrane constituent substances.

In order to industrially produce liposomes based on the present invention, the following procedure may be used.

First, a predetermined amount of the membrane component material and, if necessary, a membrane-compatible drug is mixed with a small amount of a volatile organic solvent (approximately 0.1 to 8 parts by weight per 1 part by weight of the membrane component material). This is the first point in which the present invention differs from the portic swing method of Pangam et al. In other words, the portic swing method produces a clean thin film (lipid) of film component substances.
Since it is required to form a film (1ipid film) on a glass wall surface, it is essential that the film component material be completely dissolved in an organic solvent. Therefore, many companies use organic solvents, and the ratio of the organic solvent to the membrane component material is 80 to 100 parts by weight per 1 part by weight of the membrane component material. In the present invention, the volatile organic solvent is simply added to prepare a homogeneous mixture in which membrane component substances (for example, lipids, cholesterols, and charged substances) are molecularly dispersed. Therefore, the amount of organic solvent used does not need to be very large in the portic swing method; it is sufficient to have the minimum amount required to allow the membrane component substances to be freely mixed or solvated and mixed with each other at the molecular level.
There are no limitations on appearance. In other words, whether it is in the form of a clear liquid or paste, an opaque paste or solid, or a mixture of these, if the membrane component substances are freely mixed or solvated at the molecular level, appearance does not matter at all. do not. In general, membrane component materials can be mixed with volatile organic solvents relatively easily, but only after heating.

It is even more efficient to use means such as stirring or kneading.

From the solvate of the membrane component substance thus obtained.

While stirring or kneading with a stirrer, the organic solvent is completely removed to prepare a homogeneous mixture. The term "homogeneous mixture" as used herein refers to a semi-solid material in the form of glass or paste, in which membrane component substances and the like are molecularly dispersed. This is the second major difference from the portik swing method. In other words, in the portic swing method, it is required to form a clean thin film of film component substances on the glass wall surface, but this is not necessarily necessary in the present invention; it is only necessary that the film component substances, etc. are molecularly dispersed with each other. , appearance is not an issue at all.

In order to completely remove the organic solvent, pressure reduction, inert gas such as nitrogen gas, pressure feeding of cleaning dry air, etc. may be used. In this case, it goes without saying that heating is more efficient. Furthermore, when pumping inert gas or cleaning dry air, etc., the gas jetting port may be on the surface of the organic solvent, but bubbling directly immersed in the organic solvent is more efficient. Stirring may be performed at the same time. The volatile organic solvent removed from the reaction vessel can be recovered by using a trap cooled with liquid nitrogen, acetone-dry ice, etc., and since it can be almost completely recovered, there is no problem in terms of safety.

The thus obtained homogeneous mixture of membrane component substances, etc. may be collected as is, subjected to treatment such as nitrogen substitution, and stored at temperatures below -20°C, or may be subjected to a secondary operation, i.e., by adding an aqueous solvent to swell it and hydrate it. The operation for manufacturing the liquid crystal may be continued.

This operation of adding an aqueous solvent to cause swelling proceeds quickly by heating to a temperature higher than the phase transition temperature ('I'e) of the lipid.

Once sufficiently swollen, a liposome preparation can be produced by sufficiently dispersing it using an emulsifying device commonly used for emulsification, such as a homogenizer or a propeller mixer. At this time, it goes without saying that heating above Tc is more efficient. It is also preferable to perform bubbling with an inert gas during and/or after the swelling and dispersion operation.

Note that this swelling and dispersion operation will be more efficient if it is performed above the phase transition temperature (Tα) of the membrane component material as a powder crystal. It is efficient even when it comes to operation.

In addition, in order to increase the retention rate of a drug in liposomes within the same formulation, the prescribed amount of the drug to be retained is dissolved in a small amount of aqueous solvent, and this is first added to a homogeneous mixture of membrane component substances to swell and disperse. Finally, the remaining aqueous solvent may be added to dilute.

To produce liposome preparations with even smaller particle sizes, it is better to use ultrasonic emulsifiers, emulsifiers, high-pressure emulsifiers, etc., and to make the diameter even more uniform, ultrafiltration membrane methods such as polycarbonate membrane filters can be used. It is also possible to control the particle size distribution.

In this way, a large amount of drug-retaining suposome preparations of uniform particle size can be obtained with good reproducibility. This liposome preparation may be used as it is, or it may be used after separating and removing the drug not retained in the liposomes by means such as dialysis, gel filtration, or centrifugation.

EXAMPLES Next, the present invention will be illustrated by Examples, but these Examples are not intended to limit the present invention in any way.

Example 1 Fully hydrogenated purified egg yolk lecithin (IV-1, 99% phospholipid)
After dissolving in chloroform 100°C at Tc -45 to 60°C, Tmax -52°C).

While stirring with a paddle mixer, nitrogen gas was supplied to remove the solvent. At this time, the tiger cooled with liquid nitrogen,
The solvent was almost completely recovered using a vacuum cleaner. The temperature inside the Ajihomo mixer at this time was between 50 and 60°C.

To the thus obtained dry paste-like homogeneous mixture,
Two portions of a 0.28M glucose aqueous solution previously kept at 60°C were added to sufficiently swell the mixture. Temperature 50-60
When the mixture was sufficiently stirred using a homomixer and a paddle mixer while the mixture was kept at a temperature between 0.degree. C. and returned to room temperature, a milky white liposome suspension retaining glucose was obtained.

Take 0.5 - of this liposome suspension and use Sephadex G.
Glucose not retained in the liposomes was separated and removed by gel filtration (1 cmφXIQcm, physiological saline) using -50. Next, add glucose to the liposome fraction according to a conventional method.

When extracted into the aqueous layer by oil/water partition and quantitatively determined, the retention rate was 81.4%.

In addition, the liposome fraction obtained by gel filtration was analyzed using an optical microscope (
When observed using a wide-field microscope, the diameter of each particle was 1 to several μm.
It had a uniform spherical shape.

Example 2 Partially hydrogenated purified egg yolk lecithin (IV-20, IJ lipid 9
9% or more, Tc=5-50℃, Tmax-8rc)2
B, 09. After weighing out 7.7 g of cholestesil and 2.2 g of dicetyl phos7ate, they were dissolved in 100% chloroform in an Ajihomo mixer.

The solvent was removed while bubbling with nitrogen gas.

Thereafter, in the same manner as in Example 1, a 0.5% sodium salicylate physiological saline solution 2 was used instead of the glucose aqueous solution, and a milky white liposome suspension retaining sodium salicylate was obtained.

Take 0.5 m of this liquid and apply gel filtration (
5°C) to determine the retention rate.

It was 27.1%.

Example 8 In the same manner as in Example 1, glucose aqueous solution 1 was used instead of 0.
% Dextran T40 saline solution was prepared using 21%. However, the solvent was removed under reduced pressure using a rotary vacuum pump, and stirring after addition of the aqueous solvent was performed using a homodisper and a paddle mixer.

A milky white liposome suspension containing dextran T40 was thus obtained.

Take this liposome suspension 1-, and
Gel filtration using -4B (2*2C, mφX42cm,
Physiological saline) and dextran 'I'40 not retained in the liposomes were separated and removed.

Next, dextran T40□ in the liposome fraction was extracted into the aqueous layer by oil/water partitioning according to a conventional method and quantified, and the retention rate was 10.4%.

Example 4 Fully hydrogenated purified egg yolk lecithin 2B, 09. 7.4 g of cholesterol * 1.12 g of stearylamine was weighed out and dissolved in 100% of chloroform in an Ajihomo mixer, and the solvent was removed while bubbling with nitrogen. At this time, the solvent was almost completely recovered using a trap cooled with liquid nitrogen. At this time, the temperature inside the Ajihomo mixer was 5
The temperature was between 0 and 60°C.

To the dried paste-like homogeneous mixture obtained,
21 of a 1% dextran T40 saline solution kept at 60°C in advance was added.

It was sufficiently swollen. The mixture was thoroughly stirred using a homodisper and a paddle mixer while maintaining the temperature between 50 and 60°C, and the temperature was returned to room temperature. A milky white liposome suspension containing dextran 'I''40 was obtained.

1 gnt of this liquid was taken and subjected to gel filtration in the same manner as in Example 8 to determine the retention rate, which was 11.2%.
was added as a highly concentrated physiological saline solution and kneaded with the membrane component material, followed by the addition of physiological saline and stirring. That is, a solution was prepared by dissolving 20 grams of dextran T40 in 260 grams of physiological saline.

This was heated to 60°C in advance, and this and the homogeneous mixture were sufficiently kneaded using a homodisper at around 60°C to obtain a milky white paste. Next, physiological saline 174, which had been kept warm at 55°C, was added to this paste.
0- was added, thoroughly stirred using a homodisper and a paddle mixer, and returned to room temperature, a milky white liposome suspension containing dextran T40 was obtained.

This solution 1- was taken and subjected to gel filtration in the same manner as in Example 8, and the retention rate was determined to be 26.7%.

Example 6 280 g of fully hydrogenated purified egg yolk lecithin, 155 g of cholesterol, and 22 g of dicetyl phosphate were weighed out and completely dissolved in 1000 g of chloroform in an Ajihomo mixer. The temperature inside the Ajihomo mixer is 50-60℃
The solvent was removed by bubbling with nitrogen gas while leaving the solution in between. At this time, the solvent was almost completely recovered using a trap cooled with liquid nitrogen.

A 0.28 M glucose aqueous solution Sat, previously kept at 60'C, was added to the thus obtained dried paste-like homogeneous mixture and allowed to swell sufficiently. temperature to 50
When the mixture was sufficiently stirred using a homomixer and a paddle mixer while being maintained at a temperature between -60°C and returned to room temperature, a milky white liposome suspension retaining glucose was obtained.

0.5 gLt of this liquid was taken and subjected to gel filtration in the same manner as in Example 1 to determine the retention rate, which was 82.5%.

Example 7 The same formulation as in Example 1 was used, except that the membrane component material was swollen with less glue than in Example 1. That is, after a predetermined amount of membrane component material was sufficiently swollen in 49 mj of chloroform, the solvent was removed by nitrogen gas bubbling. Thereafter, a milky white liposome suspension retaining glucose was obtained in the same manner as in Example 1 using 0.28M glucose aqueous solution 21.

A sample of 0.5% of this liquid was subjected to gel filtration in the same manner as in Example 1, and the retention rate was determined to be 14.3%.

Furthermore, when the liposome fraction obtained by gel filtration was observed using a wide-field optical microscope, the average particle size was around lpm1.
Relatively large ones were also seen here and there. This thick one had an onion-like structure.

Example 8 A product was prepared using the same formulation as in Example 2 and the same preparation method as in Example 7 using two 0.5% sodium salicylate physiological saline solutions.

A milky white liposome suspension containing sodium salicylate was thus obtained.

0.5 gnt of this liquid was taken and subjected to gel filtration in the same manner as in Example 2 to determine the retention rate, which was 18.9%.

Claims (1)

[Claims] After mixing the liposome membrane component with the amount of volatile organic solvent necessary for solvation, remove the organic solvent while stirring or kneading to create a homogeneous mixture, and add an aqueous solution to the mixture to disperse it. A method for producing liposomes characterized by