JPH0326166B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a composition for vesicles, and more particularly to a composition for vesicles that can prepare vesicles that are stable over a long period of time. It is already well known that phospholipids, particularly lecithin, which are important constituents of biological membranes, form double-membrane hollow endoplasmic reticulum called liposomes in water. Liposomes are hollow lipid-layered spheres with a structure that closely resembles that of red blood cells, including the ability to contain various chemical substances within their lumens. It plays an important role in research. Furthermore, in recent years, liposomes have attracted attention as in vivo drug carriers. In other words, liposomes, which can contain various chemical substances in their lumens, can be considered a type of capsule, and when a drug is administered inside the liposome, the metabolism of the drug in the body is suppressed and it remains in the body for a long time. (e.g., FEBS Letters, Vol. 36, No. 3,
292, 1973), drug side effects, such as allergic reactions, may be suppressed (e.g., FEBS Letters, 45
Vol. 1, p. 71, 1974), and the distribution of drugs to various organs may change (e.g. Eur. J. Biochem.
47, p. 179, 1974).
As described above, liposomes exhibit a variety of excellent properties as in-vivo drug carriers, but among them, the property of being able to change the organ distribution of drugs has the potential to cause drugs to act selectively on diseased organs. This is attracting attention as a targeting effect by so-called liposomes. For example, anticancer drugs act not only on cancer cells but also on healthy normal cells, so they often have side effects. However, it would be extremely useful if the anticancer drug could be administered selectively to cancer tissues by encasing it in liposomes and administering it. It is thought that. In fact, such attempts have sometimes yielded good results (for example, Japanese Cancer Society Abstracts, p. 8, 1976). In addition, an appropriate conductive substance such as an alkylamine is embedded in the double membrane part, a photoreducing substance such as cupric ions is added to the internal liquid of the liposome, and a photooxidizing substance such as ascorbic acid is added to the external liquid of the liposome. By irradiating it with light, it is possible to concentrate specific ions from the external fluid into the internal fluid depending on the properties of the conductive substance embedded in the double membrane. For example, it is possible to concentrate specific ions from seawater into the internal fluid. It is possible to extract elemental ions and turn them into resources. In this way, liposomes bring a revolutionary new method for drug administration, etc. However, the phospholipids that can form liposomes are biologically derived substances, so their chemical structures are extremely limited, making it inconvenient to impart various functions. It also had drawbacks such as relatively poor chemical stability. Therefore, in order to eliminate this limitation of phospholipid liposomes, research has recently been conducted on forming liposome-type endoplasmic reticulum, that is, vesicles, using synthetic surfactants as raw materials, and to date, several types of surfactants have the ability to form vesicles. It has been confirmed that there is. Similar to liposomes, such surfactant vesicles also have a double membrane hollow vesicle structure, and can be applied to the above-mentioned drug carriers. However, these surfactant vesicles are obtained by dispersing only the surfactant in water and irradiating it with ultrasonic waves, for example, and the vesicle structure is unstable and cannot be stored for a long time. The current situation is that it cannot be tolerated. For example, even if vesicles are obtained using the method described above for the dialkyldimethylammonium salt used in the present invention, the vesicle structure breaks down within a few days, and the solution becomes cloudy or gel-like. Therefore, in order to utilize surfactant vesicles as drug carriers or for other purposes, it is essential to improve this stability, and there is currently a strong demand for technology for this purpose. In view of the current situation, the present inventors conducted intensive research to obtain stable vesicles over a long period of time, and as a result, found that certain quaternary ammonium salts, certain surfactants, and The present invention was completed based on the discovery that vesicles obtained from a system containing water can exist stably for a long period of time. That is, the present invention contains the following two components (A) and (B), and the weight ratio of component (A) and component (B) is 100:1 to 100:
100 of the composition for vesicles. (A) General formula () In the formula, each symbol means the following. R ₁ and R ₂ : Hydrocarbon group having 10 to 24 carbon atoms R ₃ and R ₄ : Hydrocarbon group having 1 to 4 carbon atoms, hydroxy hydrocarbon group, or benzyl group X : Becomes a counter ion to the quaternary ammonium ion Double-stranded quaternary ammonium salt (B) represented by an anion, one or more of the following formulas (a) to (q) R ₆ O (AO) _o H (b) HO (AO) _p (C ₃ H ₆ O) _q (AO) _r H (d) R ₆ COO (AO) _o R ₁₀ (g) R ₁₅ NH (A) _t COOM (p) In the formula, each symbol represents the following. R ₅ : Hydrocarbon group having 1 to 4 carbon atoms, hydroxy hydrocarbon group or benzyl group R ₆ : Hydrocarbon group having 8 to 36 carbon atoms R ₇ : Hydrocarbon group having 5 to 23 carbon atoms R ₈ , R ₉ and R ₁₃ : Hydrogen atom or acyl group having 6 to 24 carbon atoms (however, if one molecule contains both R ₇ and R ₈ , at least one of them is a hydrogen atom) R ₁₁ and R ₁₂ : Hydrogen atom or carbon Hydrocarbon group having 6 to 24 carbon atoms (at least one of them is a hydrogen atom) R ₁₃ : Hydrocarbon group having 9 to 23 carbon atoms R ₁₄ : Hydrocarbon group having 1 to 24 carbon atoms, hydroxy hydrocarbon group, or benzyl Groups R ₁₅ , R ₁₆ and R ₁₇ : Hydrocarbon group having 1 to 24 carbon atoms A: Alkylene group having 2 to 4 carbon atoms M: alkali metal ion, monoethanol ammonium ion, diethanol ammonium ion or triethanol ammonium ion l, m and n: 0 or an integer of 1 to 150 p, q and r: an integer of 1 to 150 t: an integer of 1 to 4 X: a surfactant selected from the group of compounds represented by the same as above. It is already known that the double-stranded quaternary ammonium salt, component (A), forms vesicles, for example, as disclosed in JP-A-53-134784;
Academic journal J.Am.Chem.Soc., volume 99, page 3860,
(1977) J. Colloid Interface Sci., Vol. 65, No. 1,
155 pages, (1978) J.Am.Chem.Soc., vol. 101, 4030
Page, (1979), etc. However, it is completely unknown that vesicles can be kept stable for a long period of time by combining component (B) with component (A), and this was revealed for the first time through research by the present inventors. It is something that Specific examples of the component (A) of the present invention include didecyldimethylammonium salt, didodecyldimethylammonium salt, ditetradecyldimethylammonium salt, dihexadecyldimethylammonium salt, dioctadecyldimethylammonium salt, dieicosyldimethylammonium salt, salt, didecyl
N-hydroxyethyl-N-methylammonium salt, didodecyl-N-hydroxyethyl-N-methylammonium salt, ditetradecyl-N-hydroxyethyl-N-methylammonium salt, dihexadecyl-N-hydroxyethyl-N-methylammonium salt, Examples include dioctadecyl-N-hydroxyethyl-N-methylammonium salt, dieicosyl-N-hydroxyethyl-N-methylammonium salt, and the like. Further, the counter ion of component (A) is not particularly limited, but halogen ions, methyl sulfate ions, and ethyl sulfate ions are preferred. In addition, the surfactant as component (B) of the present invention is thought to have the effect of keeping vesicles stable, and among these, (a) is a cationic surfactant, and (b) ~
(m) is a nonionic surfactant, (n) to (q)
belong to amphoteric surfactants.
Among these components (B), ethylene oxide is preferable as the alkylene oxide added to the nonionic surfactants (b) to (m). In the composition for vesicles of the present invention, the blending weight ratio of component (A) and component (B) is important;
It is necessary that the ratio of the components be in the range of 100:1 to 100:100. If the blending weight ratio is out of this range, vesicles will not be produced, or even if vesicles are produced, they will be unstable. To produce the composition for vesicles of the present invention, according to a known method, component (A) and component (B) are dissolved in a solvent that can dissolve both components, and the mixture is stirred to be homogeneous.
The solvent may then be removed. In order to obtain vesicles from the thus obtained composition for vesicles of the present invention, the composition for vesicles may be suspended in water and irradiated with ultrasound. However, this method is not limited to this method, for example, a method of dissolving the composition for vesicles in a water-soluble solvent such as ethanol, and strongly injecting this aqueous solution into water, or solubilizing it with a water-soluble surfactant, A vesicle solution can then be obtained by using a method in which the surfactant is removed by dialysis. The vesicle solution prepared by the composition for vesicles of the present invention preferably has a concentration of 1 to 50% by weight (hereinafter simply expressed as %), more preferably 5 to 30% by weight.
%. When the vesicle concentration of the solution exceeds 50%, the viscosity becomes too high, which may cause problems in the vesicle preparation process and during the use of the vesicles. Furthermore, when the concentration is less than 1%, there is no problem in preparation and use, but the cost increases in terms of transport costs and containers for the vesicle solution, which is not economical. The most reliable method currently known for identifying vesicles in a vesicle solution prepared in this manner is electron microscopy using negative staining. The negative staining method is a method in which the electron density of the hydrophilic group portion of a surfactant, etc. that can form vesicles is increased using phosphotungstic acid or uranyl acetate, and that portion is dyed black. In the present invention, vesicle formation was observed using this method. A vesicle solution containing vesicles is transparent and has good fluidity. On the other hand, when the compound does not form a vesicle but forms a multilayered structure, the solution is gel-like, cloudy, and has extremely poor fluidity.
Therefore, in a vesicle stability test (described later), for example, a vesicle solution is prepared using an ultrasonic method,
The stability of the composition for vesicles can be determined by observing the transparency and fluidity of the solution over time. In addition, nuclear magnetic resonance (NMR) is known as an auxiliary and simple method for confirming vesicles. That is, the relaxation time or absorption line width of ¹ H- or ¹³ C-NMR differs greatly between vesicles and non-vesicles. In other words, in the case of vesicles, the relaxation time is long and the absorption linewidth is narrow and sharp, whereas in the case of non-vesicles, the relaxation time is short and the absorption linewidth is wide. however
With the NMR method, it is not possible to directly confirm the presence of a double membrane structure, which is a characteristic of vesicles, and in order to strictly confirm the presence of vesicles, observation using the above-mentioned electron microscope should be performed. EXAMPLES Next, the present invention will be explained in more detail with reference to examples, but the present invention is not limited to these examples. Example 1 Compositions for vesicles were prepared from the double-stranded quaternary ammonium salts shown in Table 1 and various surfactants, and the formation of vesicles and their stability were investigated. The results are shown in Table-1. [Preparation of composition for vesicles] 10 g of a double-stranded quaternary ammonium salt dissolved in chloroform and 1 g of a surfactant were mixed and stirred until uniform. Next, chloroform was removed using an evaporator to obtain a powder of a composition for vesicles. [Generation of vesicles and confirmation thereof] For 10 g of powder of the obtained composition for vesicles,
When 90 g of water was added and stirred, a viscous and cloudy gel-like composition was obtained. This gel composition was maintained at 60° C. and irradiated with 100 W, 25 KHz ultrasonic waves for about 1 hour.
The obtained solution was observed with an electron microscope to confirm the formation of vesicles. In addition, the solution in which vesicles were generated was almost transparent and had good fluidity. [Vesicle stability test] The vesicle solution obtained as above was stored in a constant temperature bath at 20°C for 3 months, and the state after 3 months was compared with the state immediately after production in terms of transparency and fluidity. The stability was evaluated. The evaluation criteria for vesicle generation and its stability are as follows. A: The state after storage is completely unchanged from immediately after production, and the vesicle structure is completely maintained. B: The viscosity is only slightly increased compared to immediately after production, and the vesicle structure is almost completely maintained. C: Immediately after production, the solution showed significant thickening and cloudiness, and almost no vesicle structure was present. D: Immediately after production, the solution was completely gelled and became cloudy, and no vesicle structure was observed. *: Vesicles do not form even after ultrasonic irradiation.

【table】

[Table] ‖ ｜
O CH ₃
(distearylaminimide)
Comparative Example 1 Double-stranded quaternary ammonium salt shown in Table-1
10 g was dissolved in 90 g of water, and vesicles were produced according to the method of Example 1. This was stored in a constant temperature bath at 20°C for 3 months, and its condition after storage was examined. Vesicles were formed in all 21 double-stranded quaternary ammonium salts, but after 3 months of storage, the solution became gel-like and cloudy, and the vesicle structure was lost. Example 2 A composition for vesicles was prepared according to the method of Example 1 using dioctadecyldimethylammonium chloride and the surfactant shown in Table 2, and the stability of vesicles obtained from the composition was investigated. Ta.
The results are shown in Table-2.

[Table] Example 3 A total of 12 types of double-stranded quaternary ammonium salts shown in Table 3 were combined with the three types of surfactants used in Example 1, and the methods of Example 1 were followed. A total of 36 types of vesicle compositions were prepared.
The stability of vesicles obtained from the composition was investigated. The results are shown in Table-3.

[Table] Example 4 Dioctadecyldimethylammonium chloride and the surfactant used in Example 1 were mixed at the blending ratio shown in Table 4, and a total of 39 types of vesicle compositions were prepared according to the method of Example 1. I prepared something. Regarding this composition, vesicle formation and its stability were investigated in the same manner as in Example 1. The results are shown in Table 4.

【table】

【table】

Claims (1)

[Scope of Claims] 1. A composition for vesicles containing the following two components (A) and (B), wherein the weight ratio of component (A) to component (B) is 100:1 to 100:100. (A) General formula () In the formula, each symbol means the following. R ₁ and R ₂ : Hydrocarbon group having 10 to 24 carbon atoms R ₃ and R ₄ : Hydrocarbon group having 1 to 4 carbon atoms or hydroxy hydrocarbon group or benzyl group X: Serves as a counter ion for quaternary ammonium ion Double-stranded quaternary ammonium salt (B) represented by an anion, one or more of the following formulas (a) to (q) R ₆ O (AO) _o H (b) HO (AO) _p (C ₃ H ₆ O) _q (AO) _r H (d) R ₆ COO (AO) _o R ₁₀ (g) R ₁₅ NH (A) _t COOM (p) In the formula, each symbol represents the following. R ₅ : Hydrocarbon group having 1 to 4 carbon atoms, hydroxy hydrocarbon group or benzyl group R ₆ : Hydrocarbon group having 8 to 36 carbon atoms R ₇ : Hydrocarbon group having 5 to 23 carbon atoms R ₈ , R ₉ and R ₁₀ : Hydrogen atom or acyl group having 6 to 24 carbon atoms (however, if one molecule contains both R ₇ and R ₈ , at least one of them is a hydrogen atom) R ₁₁ and R ₁₂ : Hydrogen atom or carbon Hydrocarbon group having 6 to 24 carbon atoms (at least one of them is a hydrogen atom) R ₁₃ : Hydrocarbon group having 9 to 23 carbon atoms R ₁₄ : Hydrocarbon group having 1 to 24 carbon atoms, hydroxy hydrocarbon group, or benzyl Groups R ₁₅ , R ₁₆ and R ₁₇ : Hydrocarbon group having 1 to 24 carbon atoms A: Alkylene group having 2 to 4 carbon atoms M: alkali metal ion, monoethanol ammonium ion, diethanol ammonium ion or triethanol ammonium ion l, m and n: 0 or an integer of 1 to 150 p, q and r: an integer of 1 to 150 t: an integer of 1 to 4 X: a surfactant selected from the group of compounds represented by the same as above. 2. The composition for vesicles according to claim 1, wherein the counter ion X in component (A) and component (B) is a halogen ion, a methyl sulfate ion, or an ethyl sulfate ion.