JPS63119847A - Production for liposome - Google Patents

Abstract

PURPOSE:To produce the liposome having stability in vivo with good reproducibility by mixing an aqueous solution contg. a substance which is contained within the space of a membrane to the acyl glycerol solution of a substance to be contained in a membrane component substance and a membrane structural substance. CONSTITUTION:The substance to be contained in the membrane component substance and the membrane structural substance is dissolved to a small amount of acylglycerol and/or a higher fatty acid to form the acylglycerol solution. Said acylglycerol solution is mixed to a small amount of the aqueous solution which contains the substance to be contained within the space surrounded by the membrance of a microspore and has pH of 4-10 followed by emulsifying the obtd. solution at a temp. of 15-65 deg.C and in the atmosphere of nitrogen gas. The stable obtd. emulsion is added to a large volume of the prescribed aqueous solution so as to maintain pH of the aqueous solution to 4-10, while agitating it to produce liposome.

Description

Detailed Description of the Invention (a) Industrial Application Field The present invention relates to membrane component substances and substances to be included in the membrane structure solubilized in acylgly7ol and/or higher fatty acids as a solvent, and ribosomes. The present invention relates to a simple and innovative method for safely mass-producing ribosomes having medically and biologically favorable properties using only an aqueous solution containing a substance to be included in the internal space.

(b) Prior art In recent years, technology that utilizes ribosomes, which are closed vesicles made of double-layered lipid membranes, has gained importance in a wide range of fields including medicine, pharmacy, immunology, and biology. A lot of knowledge is being accumulated towards practical application.

In other words, due to its quasi-cellular properties, ribosomes not only serve as a model for biological membranes and are the subject of basic biophysical chemistry research, but also contain various substances within the aqueous layer or membrane structure inside the vesicle. For example, as a carrier for efficiently retaining and transporting various contrast agents, therapeutic drugs, or physiologically active substances.

It can be used effectively both in and outside the body.

Its breakthrough clinical application is expected.The problems that the O V1 invention aims to solve. The following problems have been pointed out.

■When preparing ribosomes, the purpose is to solubilize membrane constituent substances, which are more or less toxic to living systems.
Volatile organic solvents such as ether/L/, chloroform, ethanol, methanol, benzene, hexane, or
Octylgmu coside, cholic acid, tophytone x-io
However, in order to remove these chemical substances, various complicated steps are required, and their residue in the final product is unavoidable. It is difficult to say that it is optimal for practical use because it has problems with operating technology.

■Several methods have been reported to produce ribosomes themselves without using volatile organic solvents or surfactants, including freezing, drying, freeze-thawing, and ultracentrifugation.

Gelation under low pressure, heating that unavoidably denatures protein components, etc., restrictions on the α class of membrane component materials that can be used, difficulty in scaling up the manufacturing process, etc., requiring special equipment or complicated processes. In other words, there are fundamental problems with the technical principle itself, and further improvements are essential if we are to aim for large-scale practical application.

(2) The resulting ribosomes themselves have problems that must be resolved, such as non-uniformity in particle size and structure, fragility of the membrane structure, and low retention of encapsulated substances.

Under these circumstances, the present invention does not use volatile organic solvents or surfactants, which are problematic in production and practical use, and requires special equipment or complicated processes. Types of substances that can be used as membrane constituents under mild conditions that do not contain or cause denaturation of protein components, etc. due to factors that are disadvantageous to practical use such as freeze-drying, ultracentrifugation, and dialysis. By recreating the more natural state of biological membrane systems while minimizing restrictions, the retention rate of the encapsulated substances is high.

Moreover, it is an object of the present invention to provide a simple method that enables the preparation of stable ribosomes in large quantities with good reproducibility both inside and outside the body.

B) Means for Solving the Problems The present inventor has conducted intensive studies on a safe and simple method for mass-producing ribosomes having more preferable characteristics.9
The following knowledge has been obtained and the present invention has been completed.

■Certain types of neutral fat components and higher fatty acids, which are important components of biological systems but have not been given much attention in the past, have a very strong effect on many ribosomal membrane constituents, including phospholipids, in small amounts. It has the ability to be easily blended under mild conditions.

■An oily solution in which a membrane component substance is dissolved in a small amount of acylglycerol and/or higher fatty acid.

When mixed with a small amount of an aqueous solution and completely emulsified, a +''/6 type emulsion containing homogeneous spherical MI/I/ in which the water droplets are completely wrapped in a monolayer of polar lipid and stabilized is formed.

(2) When a large volume of an aqueous solution is added to raw emulsion and stirred, a small amount of hydrophobic components are incorporated into the hydrophobic portion of the membrane structure, forming a stable double-layer membrane ribosome. By selecting all condition factors such as the type and amount of membrane constituents, the order of their addition, stirring method, and temperature, and controlling them with a simple system, it is possible to control the properties and particle size of the ribosome. Kutoe can also give different ratio characteristics to the inner layer and outer layer of a double layer film.

■The solution containing double-layer membrane ribosomes does not contain any substances harmful to living organisms, so it can be used as a ribosome dispersion as is, but it is possible to use it as it is as a ribosome dispersion. If the ribosomes should be removed as much as possible and the particle size of the ribosomes should be highly uniform, this purpose can be achieved by passing through an ultrafiltration filter, and in addition, excess water solubility in the dispersion can be The concentration of substances (such as free drug) can be easily reduced by washing and filtration while adding fresh ribosome dispersion buffer that is isotonic with the aqueous solution inside the ribosomes. The ribosote dispersion liquid is harmless to living organisms and may also contain polyhydric alcohols, which are conventionally used as stabilizers for pharmaceutical preparations. In addition, filtration efficiency is improved if the inert gas is used during filtration.

The ribosomes prepared based on the findings of ■■ to ■■ have a very high retention rate of encapsulated substances and are stable.

■ Ribosomes prepared with high-density lipoproteins and/or ultra-high-density lipoproteins as one of their membrane components have the property of being efficiently taken up by macrophage-based phagocytes.

Particularly large amounts of cholesterol/L/f: Maco7age stored in cells is very well taken up by ribosomes, but tends to remain in cells for a relatively long time without being digested.

In general, monoacylglycerols, depending on how many of glycerol's three hydroxyl groups are esterified with fatty acids.

It is distinguished from diacylglycerol and triacylglycerol, and together they are collectively called neutral fat, acylglycerol, or glyceride. Acylglycerol is the most abundant lipid group in living organisms.
It plays an important role as a main component of storage lipids and as an element of various life activity systems.

The physicochemical properties vary greatly depending on the type and number of fatty acids. Although the triaS/A/gly7ols are all insoluble in water and are nonpolar and therefore cannot themselves form highly dispersed mce)vf, diacylglycerols and monoacylglycerols are Due to its presence, it has considerable polarity and can form M.μ. Long-chain monoacylglyceros have surfactant properties and are widely used as safe additives in the manufacturing process of pharmaceuticals and foods.

One of the features of the present invention is that polar or non-polar acylglycerol and/or higher fatty acids are used in a system that does not use any organic solvent or surfactant as a solubilizer for membrane component substances.

It is used as a primary solvent for various ribosomal membrane component substances and lipophilic substances to be included in the membrane structure, and in conjunction with its role, it is used as a solvent for the hydrophobic portion in the 0.2-layer membrane structure. As one of the groups of substances that should be introduced into
Alternatively, (1) it is also possible to selectively use (9) as one of the constituent components of the double layer film itself. It exists in that sense.

A ribosome preparation characterized by containing a higher fatty acid as one of the membrane component substances is disclosed in JP-A-60-155109. However, the preparation of the ribosome preparation is based on the conventional method of using a volatile organic solvent as a solubilizing agent for membrane component substances including fatty acids, and the existing method based thereon. .

Many fundamental problems with conventional ribosomes and their production methods have not yet been completely solved.

It is hard to say that it is practically preferable. On the other hand, there are no examples yet of using the acylglycerol and/or higher fatty acid compounds proposed by the present invention as a solvent for blending lipophilic substances that should primarily be included in membrane component substances and membrane structures. While the principle itself is completely new, it overcomes many of the fundamental problems associated with conventional ribosome production methods and ribosomes.

The types of acylglyceros and higher fatty acids used in the present invention are not particularly limited.

Each should be selected according to its purpose.

Furthermore, in order to prepare ribosomes under mild conditions as close as possible to biological systems, it is desirable to select a substance with a low melting point. The nine acylglycerols are triolein and triolein.

Palmitoleic acid is a fatty acid such as monoolein.

Oleic acid, linoleic acid, linolenic acid, etc. are preferred.

The basic ribosomal membrane component substances commonly used in the present invention include natural and synthetic or modified in some way, phospholipids, glycolipids, sterols, charged substances, and membrane antioxidants. However, there is no restriction on the type, and it can be selected depending on necessity or purpose.

For example, as the phospholipid, various types of lecithin, various types of phosph-1-tidyrucones, phosph-1-tidylinositol lvI phosph-1-di-ethanolamine, or sphinogomyelin, which completely stabilizes ribosomes, can be used alone or in combination. It is also possible to use small amounts of lysolecithin (lysophosphatidylcholine) to improve membrane permeability. Conversely, sterols such as cholesterol are important for reducing membrane permeability and stabilizing ribosomes. Cholesterol has a melting point of 1
The temperature is as high as 48° C., and it is difficult to incorporate it into the membrane using a ribosome preparation method that does not use an organic solvent, but it can be easily introduced using the method according to the present invention. As the substance imparting a negative charge, phosphatidylserine, which is a phospholipid, is generally preferred in terms of its properties and stability. However, phosphatidic acid.

Dicetyl phosphate or calciolibin.

Gangliosides and the like can also be used without difficulty.

In addition, a substance such as stearyl μamine may be used for the purpose of imparting a positive charge. Furthermore, it is also preferable to introduce α-toco7 yellow y or the like as an antioxidant for membrane component substances.

The type of substance that can be encapsulated in ribosomes prepared based on the ratio method proposed by the present invention is not particularly limited.

Generally, lipid-soluble substances can be introduced into the membrane, and water-soluble substances can be introduced into the vesicles at the time of ribosome preparation. For example, the present invention is characterized in that it targets biological tissues and their cells, Q molecules, and is used in vitro and/or in vivo. Radioactive or non-radioactive chemical substances for wound inspection, contrast agents (including those for nuclear magnetic resonance imaging diagnosis and nuclear medicine diagnosis, etc.), therapeutic drugs (anticancer drugs, etc.).

Antibiotics, other general drugs), physiologically active substances (proteins, polysaccharides, nucleic acids, lipids, vitamins, and other natural substances) include synthetic chemicals or quasi-natural products produced by cell/genetic engineering. In addition, ribosomes containing chemically modified products of the ribosomes and systems in which the ribosomes and various substances are chemically, physically, or functionally combined can be prepared for use in veterinary testing and diagnosis. or therapeutic reagents, preparations, and their oral or
A parenteral administration system or an applied system may be considered.

(e) Examples Next, the present invention will be further explained by specific examples, but these examples are not intended to limit the present invention in any way.

First of all, all 5 of the 9 inventions! The equipment used will be fully explained. FIG. 1 shows the device, and the main body of the device has a cylindrical shape and is formed by connecting the main parts 1 to 5 after sterilization. Reference numerals 1 to 4 are fixed main bodies, 4 is a reaction tank, and 2.3 is an installation part for an ultrafiltration filter and an auxiliary filter. Ultrafiltration liner/1/ter and auxiliary filter installation section 2
．． 3 is a connection part B2 to B to which a Teague for separating ribosome dispersion liquid and/or for inlet/output of inert gas is connected.
5 is provided. Further, 5 is a syringe type movable part for pressure adjustment, 6.7 is a constant temperature water circulation part for temperature control, 8 is a motor for driving a homogenizer, and 9 is a sharp knife of the homogenizer. The rotating shaft of the homogenizer is hollow, and a chain 10 for injecting an inert gas and liquid material or for separating a ribosome dispersion liquid is inserted. In addition, Bl is a cheap connection part for inlet/output of inert gas and liquid transfer or transfer, Wl to W4 are connection parts with a constant temperature device, B6.
B7 is a cheap connection for injecting inert gas and liquid materials or for preparative separation of ribosome dispersions.

Ribosomes were produced using the apparatus configured as described above at 45° C. under a nitrogen gas atmosphere as follows.

5.0g of monoolein and 5.0g of egg yolk lecithin.

Phosphatidylserine 3. og# dicetyl phosphate 0.5g + cholesterol-fi/2.0 g and a
- Tocophero - ~0.5gt - Added to reaction tank (4) B
The mixture was thoroughly stirred using a homogenizer (9) while being bubbled with N2 gas from No. 6 to obtain a homogeneous viscous oily liquid. Next, a 0.28M glucose-containing physiological saline solution (PH7,
0) 3 m! ! It was added from tl-87 and stirred at low speed for 10 minutes and at high speed for 5 minutes. 200 from B7 in that state
When rnJ glucose-free physiological saline was gradually added and the mixture was stirred at high speed for 5 minutes, a suspension of tallyboom while retaining glucose was obtained. Therefore.

In order to remove excess oily components from 81, the above physiological saline was further added from 87 while washing.

It was pressurized by the movable part 5 and passed through a 3.2 ton ultrafiltration filter made of polycarbonate. The obtained ribosome has a particle size of 1
．． It exhibits a uniform particle size of 0 μm and does not aggregate in physiological saline (it is stable for a long time at 4°C or room temperature in an M2 gas atmosphere).Secondly, it has a high glucose retention rate of 40.5%. Good friend.

(f) Effects of the Invention The method for producing ribosomes of the present invention is more preferable and superior to known methods in the following respects.

■We do not use volatile organic solvents, which pose many operational and medical/biological safety problems, or surfactants, which pose problems if they remain in the final product.

■When producing ribosomes, there is basically no need for frequent steps such as freeze-drying, ultracentrifugation, or dialysis.

■It is easy to hydrate the membrane component by blending the ribosomal membrane component with a small amount of acylglycero-μ and/or fatty acid as a solvent under very mild conditions before mixing it with an aqueous solution. It is. That is, according to the method of the present invention, the film component material can be heated to a temperature higher than the phase transition temperature ff (T(X)) in the fully hydrated lamellar phase (T( Since it can be hydrated, it can be applied without any problems even when membrane component substances or substances to be included in ribosomes include substances susceptible to thermal denaturation such as proteins.

■ By changing the mk of the membrane component substance used in the pre-bag to form an emulsion containing a stable monolayer membrane, ribosomes with different compositions of the inner and outer layers of the double-layer membrane can be obtained. Is possible.

■Cholesterol-μ, which has a high melting point and is difficult to introduce as a membrane component without using an organic solvent, or gold, a substance such as dicetyl phosphate, which does not have a liquid crystal phase metal by itself, can be easily incorporated into the membrane structure of ribosomes. It is possible.

■The method for producing ribosomes according to the present invention is extremely simple and has excellent reproducibility, and can be easily carried out on an industrial scale from two scales both technically and economically.■Ribosomes prepared according to the method of the present invention Overall, the retention rate and stability of the encapsulated material is higher than that of conventional ones.

[Brief explanation of the drawing]

FIG. 1 schematically shows a ribosome manufacturing apparatus according to the present invention, which allows ribosome manufacturing to be carried out quickly and efficiently. 2.3: Ultrafiltration filter and auxiliary filter installation section. 4: Reaction tank 5: Syringe type movable part for pressure adjustment. 6.7: Constant temperature water return section for temperature/summer control. Each of W1 to W4 is connected to a constant temperature device. 8: Homogenizer drive motor. 9: Homogenizer sharp knife. 10: Tube for injection of inert gas and liquid material or for preparative collection of ribosome dispersion. 15L Figure 1

Claims (7)

[Claims] (1) Membrane component substances primarily dissolved in a small amount of acylglycerol and/or higher fatty acids as a solvent and substances to be incorporated into the membrane structure, and substances to be incorporated into the internal space of the vesicle surrounded by the membrane. pH 4~ containing the substance to be
10 and emulsified in a nitrogen gas atmosphere at a temperature of 15 to 65°C. To the resulting stable emulsion, a large volume of the aqueous solution was further added to maintain the above pH, and by stirring, ribosomes were A method for producing ribosomes, which is characterized in that it enables the preparation of ribosomes. (2) characterized in that the acylglycerol is monoacylglycerol or diacylglycerol;
A ribosome manufacturing method according to claim 1. (3) The method for producing ribosomes according to claim 2, wherein the monoacylglycerol is monoolein, monopalmitin, monopalmitolein, or monostearin. (4) The method for producing ribosomes according to claim 1, wherein the acylglycerol is simple or mixed triacylglycerol. (5) Simple triacylglycerol is triolein,
5. The ribosome manufacturing method according to claim 4, wherein the ribosome is trilinolein, trimyristin, tripalmitin, or tristearin. (6) The ribosome manufacturing method according to any one of claims 1 to 5, wherein the higher fatty acid is a C_1_0 to C_2_0 saturated or unsaturated fatty acid. (7) The method for producing ribosomes according to claim 6, wherein the unsaturated fatty acid is palmitoleic acid, oleic acid, linoleic acid, linolenic acid, or arachidonic acid.