JPH0649145B2 - Method for producing bilayer vesicles - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a bilayer vesicle. More specifically, it relates to a method for producing a bilayer vesicle by combining a quaternary ammonium salt having a long-chain monoalkyl group and an n-higher alcohol.

[Prior Art] Conventionally, phospholipids such as lecithin, which is a constituent of biological membranes, have been known as substances that form bilayer vesicles. That is, it was found that by subjecting phospholipids to an appropriate treatment, vesicles of lipid bilayers called liposomes were formed, and these vesicles were studied from their structure as a model similar to cells or erythrocytes, It plays an important role in biomembrane research.

More recently, it has been found that synthetic cationic surfactants, for example, cationic surfactants having a long chain dialkyl such as dioctadecyldimethylammonium salt and octadecylhexadecyldimethylammonium salt also form liposome-like vesicles. (JP-A-53-134784).

The endoplasmic reticulum formed by such a substance is a monolayer or multilayer bilayer sphere, and various chemical substances can be embedded in the lumen, so that the endoplasmic reticulum can be promoted as a capsule such as a drug. it can. For example, enzymes, hormones, vitamins, carcinostatics, antipyretics, antibiotics, local and general anesthetics, bacteria, viruses, nucleic acids, chromosomes, proteins, chelating agents, etc. can be accurately returned to the target site to exert its effect. it can.

Further, since the synthetic cationic surfactant is sometimes used as a softening agent for fibers or a rinsing base for hair, it can be expected to be used as a softening agent or rinsing agent in which a drug or the like is embedded.

[Problems to be Solved by the Invention] However, conventionally known phospholipids such as egg yolk lecithin (EYL) and distearyl dimethyl ammonium salts (D
The liposome-like endoplasmic reticulum formed of a cationic active agent having a dialkyl such as S) has a gel-liquid crystal phase transition of the molecule in the membrane at a low temperature (EYL≈-4 ° C, DS≈40 ° C). There has been a problem that the active substance such as a drug embedded in the endoplasmic reticulum phase leaks to the outer phase due to the fluidity of the molecules in the membrane at room temperature or slightly higher than room temperature. In addition, it was also very difficult to freely change the gel-liquid crystal phase transition temperature for the purpose of controlling the leakage temperature of the active substance in the internal phase, etc. Furthermore, when a cationic active agent having a dialkyl is used as a rinse agent or the like, the amount used is limited due to the problem of irritation to the eye mucous membrane, etc., and as a result, the embedded amount of the active substance in the liposomal vesicles decreases. There was a flaw.

[Means for Solving the Problems] In view of these circumstances, the present inventors have conducted extensive studies in order to solve the above-mentioned drawbacks, and as a result, have found that a quaternary ammonium salt having a long-chain monoalkyl and n- By combining with a higher alcohol and performing treatments such as high temperature stirring and ultrasonic irradiation in water, the gel-liquid crystal phase transition temperature of the molecule in the bilayer becomes the phase transition temperature of the endoplasmic reticulum formed by the dialkylammonium salt. In comparison, it is highly stable under temperature conditions that can be reached with changes in the four seasons, and the gel-liquid crystal phase transition temperature can be freely changed according to the application, and the amount of cationic activator is dialkyl type. Based on this finding, we found that liposome-like vesicles, i.e., bilayer vesicles, were obtained even with the same amount of As a result, the present invention has been completed.

That is, the present invention provides the compound represented by the general formula (I) (In the formula, R ₁ is an alkyl group having 10 to 22 carbon atoms, R ₂ and R ₃ are alkyl groups having 1 to ₃ carbon atoms, and R ₄ is 1 carbon atom.
~ 3 alkyl group or benzyl group, X represents a chlorine or bromine atom. ) One or more quaternary ammonium salts represented by the formula: and a general formula (II) R ₅ —OH (II) (in the formula, R ₅ represents an alkyl group having 12 to 22 carbon atoms). A method for producing a bilayer vesicle, which comprises stirring or mechanically vibrating an aqueous solution containing one or more n-higher alcohols represented by the formula above the phase transition temperature of the aqueous solution. is there.

Next, the configuration of the present invention will be described.

Examples of the quaternary ammonium represented by the general formula (I) used in the present invention include, for example, dodecyltrimethylammonium chloride, myristyltrimethylammonium chloride, cetyltrimethylammonium chloride, stearyltrimethylammonium chloride, aralkyltrimethylammonium chloride, Behenyltrimethylammonium chloride, myristyldimethylethylammonium chloride, cetyldimethylethylammonium chloride, stearyldimethylethylammonium chloride, aralkyldimethylethylammonium chloride,
Behenyldimethylethylammonium chloride, myristyldiethylmethylammonium chloride, cetyldiethylmethylammonium chloride, stearyldiethylmethylammonium chloride, aralkyldiethylmethylammonium chloride, behenyldiethylmethylammonium chloride, benzyldimethylmyristylammonium chloride, benzyldimethylcetylammonium chloride, benzyl Examples thereof include dimethylstearyl ammonium chloride, benzyldimethylbehenylammonium chloride, benzylmethylethylcetylammonium chloride, benzylmethylethylstearylammonium chloride and the corresponding bromide.

In carrying out the present invention, one or two of these are used.
At least one species is selected. The blending amount is selected in the range of 0.1 to 15% by weight (hereinafter, referred to as%) in the total amount of the above aqueous solution, and preferably 0.1 to 10%.

Examples of the n-higher alcohol represented by the general formula (II) used in the present invention include dodecyl alcohol,
Examples thereof include cetyl alcohol, stearyl alcohol, behenyl alcohol and the like. 1 or 2 from these
At least one species is selected.

The bilayer vesicle of the present invention comprises one or more quaternary ammonium salts described above and one of higher alcohols.
It is produced by stirring or mechanically vibrating an aqueous solution containing one or two or more species at a phase transition temperature of the aqueous solution or higher.

The amount of n-higher alcohol is 1.0 or more (weight ratio; the same applies below) to 1 of quaternary ammonium salt, but when the concentration is high, the viscosity of the system is increased and the quaternary ammonium salt cannot be combined. Quaternary ammonium salt: n-
Higher alcohol = 1: 15 or less is preferable, and 1: 1 to 1:10 (weight ratio) gives the best result.

The stirrer used in the present invention may be anything as long as it can sufficiently mix the quaternary ammonium salt, the n-higher alcohol and other compounds in warm water, and examples thereof include a propeller stirrer, a homomixer, and Mt Donggarlin. The stirring condition is preferably 0.5 to 5 hours.

Examples of the mechanical vibration used in the present invention include ultrasonic irradiation, the frequency is 20 to 50 kHz, and the irradiation time is 30 to 90 °.
C is preferred for 0.5 to 2 hours.

The aqueous solution containing the quaternary ammonium salt and the n-higher alcohol according to the present invention each have an inherent phase transition temperature, and the bilayer vesicles are obtained by stirring or applying ultrasonic vibration at the temperature or higher. To form. After forming the endoplasmic reticulum, even if the temperature is further raised, the endoplasmic reticulum membrane becomes weak but the endoplasmic reticulum structure is maintained. As a matter of course,
As it cools to below the phase transition temperature, the endoplasmic reticulum membrane becomes stronger, and the substance embedded in the endoplasmic reticulum becomes less likely to leak out.

The phase transition temperature varies depending on the type of quaternary ammonium salt, the type of higher alcohol and their combination,
Changes between 90 ° C. [R ₁ of formula _(I), the general formula R ₅ in _(II)] alkyl chain longer the, and as to combine long each other in phase transition temperature tends to increase.

The bilayer vesicle of the present invention, in addition to the above essential components,
Moisturizers, salts, nonionic surfactants, oils, fragrances, dyes, preservatives and the like are added as necessary. Of course, they must be used under qualitative and quantitative conditions that do not impair the object of the present invention.

The bilayer vesicle according to the present invention is extremely stable in a wide temperature range because the gel-liquid crystal phase transition of the molecule in the film occurs at a high temperature as compared with the vesicle formed by the conventional dialkyl type cationic activator. Further, since it is possible to change the kind and the compounding ratio of the n-higher alcohol to the quaternary ammonium salt having a long-chain monoalkyl, or the combination of two or more n-higher alcohols, it is possible to change the drug from the endoplasmic reticulum phase. It is very convenient for encapsulation of active substances, which has the property of being advantageous cosmetically and as a drug, since the leakage temperature can be freely controlled. That is, as substances having advantageous properties as cosmetics, wetting agents, cooling agents, hair nourishing agents,
Water-soluble coloring agents, anti-dandruff agents, bleaching agents, etc., and substances that are pharmaceutically active include the aforementioned enzymes, hormones, vitamins, anticancer agents, antipyretics, antibiotics, anesthetics, bacteria, viruses, nucleic acids, Chromosomes, proteins, chelating agents and the like can be mentioned.

Further, since the bilayer vesicle according to the present invention is combined with n-higher alcohol as compared with the vesicle formed by a dialkyl-type cationic activator, even when the amount of the activator is the same, irritation to the eye mucosa or the like is caused. Since it is weak and has a large amount of endoplasmic reticulum formed, it has advantages such as a large amount of embedded drug and the like.

Next, for further understanding of the present invention, examples will be given to explain in more detail. The present invention is not limited to this. In the examples, "%" means "% by weight".

EXAMPLES AND EFFECTS OF THE INVENTION Example 1 5% Stearyl trimethyl ammonium chloride at 80 ° C
To the aqueous solution, add 10% of melted cetyl alcohol at the same temperature and stir the propeller at 300 rpm for 1 hour.
Cooling to ° C gives a milky dispersion. Observation of this milky dispersion with an electron microscope revealed that the diameter was 0.1-
A multi-layered endoplasmic reticulum of 1 μm was found, and it was confirmed that it had a bilayer membrane structure because the thickness of one layer was about 6 nm. In addition, as a comparison, FIG. 2 shows an electron micrograph of a 5% suspension of distearyldimethylammonium chloride, which has been conventionally known to form liposome-like vesicles, and shows that the structure is very similar to that of the vesicles of the present invention. I understand.

Here, FIGS. 1 and 2 are photographs taken by a transmission electron microscope of a sample diluted 10-fold, added with an appropriate amount of a 2% uranyl acetate solution, and naturally dried (referred to as a negative staining method).

Example 2 and Comparative Example 1 Sodium chloride was dissolved in an aqueous solution of 2% behenyltrimethylammonium chloride at 80 ° C to form a 0.2M sodium chloride solution, and 3% of behenyl alcohol melted at the same temperature was used.
And added in the same manner as in Example 1 to prepare an endoplasmic reticulum suspension in the same manner as in Example 1.

Transfer 100 ml of this suspension to a dialysis pack with a diameter of 1 cm and
Dialyzed against pure water. The state of dialysis was monitored by electrical conductivity. When the pure water of 1 was changed 6 times, the electric conductivity became the same as that of pure water, and the outflow of ions was no longer observed by dialysis. Further, as Comparative Example 1, dialysis was performed on the cooled milky dispersion of Example 1 to which sodium chloride was added, and as a result, no ion outflow was observed after 6 exchanges of pure water.

After ultrasonic irradiation of the dialyzed suspensions of the two types of endoplasmic reticulum, the electrical conductivity was measured. The value of Comparative Example 1 in which sodium chloride was added after cooling was as low as that of pure water. However, in Example 2 in which sodium chloride was added before cooling, the conductivity was higher than that of pure water.

This means that in Example 2 in which sodium chloride was added at 80 ° C. and then cooled, sodium chloride was encapsulated in the endoplasmic reticulum, and subsequent ultrasonic irradiation reconstructed the bilayer membrane structure of the endoplasmic reticulum shell. It shows that a part of the saline solution in the lumen of the endoplasmic reticulum was transferred to the outer aqueous phase.

Example 3, Comparative Example 2 A suspension of vesicles to which sodium chloride was added at 80 ° C of Example 2 and 0.2M sodium chloride in which sodium chloride was dissolved in an aqueous solution of 2% distearyldimethylammonium chloride at 80 ° C. Stir the aqueous solution with a propeller at 300 rpm for 1 hour at 25 ° C.
10 of the ER suspensions obtained by cooling to 10 (Comparative Example 2)
0 ml was dialyzed in the same manner as in Example 2, and when the outflow of ions was not observed, the temperature stability was compared by the electric conductivity.

The results are shown in Table-1.

Each value shows a relative value when the electric conductivity at 80 ° C. in Example 2 is 1.0, and the larger value means that the membrane is in a molten state and the sodium chloride from the endoplasmic reticulum phase to the outer phase is increased. Indicates that it becomes easy to move.

As is clear from Table 1, in Comparative Example 2, the film starts to melt at 40 ° C, whereas in Example 2, the film is extremely stable up to around 70 ° C, and the electric conductivity at 80 ° C is high. Since the relative value of the degree is Example 2: Comparative example 2 = 1.0: 0.8, it can be seen that the sodium chloride inclusion amount in Example 2 is larger.

Example 4, Comparative Example 3 A sample (Example 4) in which 15 mg of stearyltrimethylammonium chloride, 22 mg of cetyl alcohol and 23 mg of tocopherol acetate were uniformly dissolved in a 200 ml eggplant-shaped flask (Example 4) and 25 mg of distearyldimethylammonium chloride ( Stearyl trimethylammonium chloride (15 mg; same mole) and tocopherol acetate (23 mg) were uniformly dissolved in chloroform (2 ml) (Comparative Example 3). The solvent was removed under reduced pressure using a rotary evaporator, and the inner wall of the flask was milky white. Form a film. Next, 100 ml of purified water was added, and by vigorous shaking, the thin film formed on the inner wall of the flask was completely dispersed in water to form vesicles, which was used as a vesicle dispersion liquid.

The two vesicle dispersions were ultrafiltered using a 2 μm Millipore filter, and the amount of tocopherol acetate in the filtrate was quantified by liquid chromatography. The amount of tocopherol acetate embedded in the vesicles was In Example 4, 22.8 mg was 99% of the initial blending amount, and in Comparative Example 3, 11.0 mg was 48% of the initial blending amount.

[Brief description of drawings]

FIG. 1 is a transmission electron micrograph showing the particle structure of the bilayer vesicle obtained in the present invention, and FIG. 2 is a bilayer vesicle obtained with an active agent that has been known to form liposome-like vesicles. 2 is a transmission electron micrograph showing the particle structure of

Claims (8)

[Claims] 1. A general formula (I) (In the formula, R ₁ is an alkyl group having 10 to 22 carbon atoms, R ₂ and R ₃ are alkyl groups having 1 to ₃ carbon atoms, and R ₄ is 1 carbon atom.
~ 3 alkyl group or benzyl group, × represents a chlorine or bromine atom. ) One or more quaternary ammonium salts represented by the formula: and a general formula (II) R ₅ —OH (II) (in the formula, R ₅ represents an alkyl group having 12 to 22 carbon atoms). To an aqueous solution containing one or more higher alcohols represented by the formula (1) or two or more quaternary ammonium salts represented by the above general formula (I) and a general formula It varies from 30 ° C to 90 ° C depending on the kind or combination of one or more higher alcohols represented by (II). ] The method for producing a bilayer vesicle characterized by applying stirring or mechanical vibration as described above. 2. The method for producing a bilayer vesicle according to claim 1, wherein the content of the quaternary ammonium salt is 0.1 to 10% by weight based on the total amount of the aqueous solution. 3. The method according to any one of claims 1 and 2, wherein the ratio of the quaternary ammonium salt to the higher alcohol is 1: 1 (weight ratio) to 1:10 (weight ratio). Method for producing bilayer vesicles of. 4. The method for producing a bilayer vesicle according to any one of claims 1 to 3, wherein the stirrer is a propeller stirrer, a homomixer or Mt'n'garlin. 5. The method for producing a bilayer vesicle according to claim 4, wherein the stirring is carried out for 0.5 to 5 hours. 6. The method for producing a bilayer vesicle according to any one of claims 1 to 3, wherein the mechanical vibration is irradiation with ultrasonic waves. 7. The method for producing a bilayer vesicle according to claim 6, wherein the ultrasonic wave is 20 to 50 KHz. 8. The method according to claim 6, wherein the ultrasonic irradiation is performed for 0.5 to 2 hours.