JP3471909B2 - Stabilization method of amphiphilic compound film - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for stabilizing an amphipathic compound film. When the amphipathic compound membrane is coated with a specific water-soluble polymer according to the method of the present invention, a stable liposome or flat membrane that can be used as a pharmaceutical carrier or a carrier for immobilizing membrane proteins is formed.

[0002]

2. Description of the Related Art Regarding amphipathic compounds, liposomes can be easily dispersed by ultrasonic dispersion in water, planar bilayers can be easily cast by water, and monolayers can be easily spread by spreading at the gas-liquid interface. It is known to be formed in the form of a drug, and because of its ease of handling, it has been widely studied as a capsule for sustained drug release, a carrier for immobilizing a membrane protein for a sensor, and the like.

However, the amphipathic compound film thus formed is often unstable, and the film itself aggregates, fuses,
Further, there is a problem that the formation of precipitates and the precipitation of insoluble substances occur in a relatively short time.

To solve this problem, as a method for stabilizing an amphipathic compound film, J. Am. Chem. Soc., 1988, 11
0, 2495, J. Am. Chem. Soc., 1982, 104, 791-795 and JP-A-62-205092, a lipid having a polymerizable group was synthesized as an amphipathic compound, from which a liposome or a planar membrane was synthesized. Are polymerized after being prepared. Makromol. Chem.
In Rapid Commun., 1984, 5, 345, acidic phospholipid as an amphiphilic compound adsorbs basic polymer by ionic bond.

In JP-A-62-283986, an amphipathic compound having a special structure is used.
No. 9-204198 uses a phospholipid modified with a polyoxyethylene group as an amphipathic compound.

Further, in JP-A-58-49311 and JP-A-61-72721, an amphipathic compound film is coated with a fatty acid ester of a saccharide, and J. Am. Chem. Soc.
In 993, 115, 3812, the amphipathic compound film is coated with an N-isopropylacrylamide to N-octadecylacrylamide copolymer.

[0007]

However, in the method,
When polymerizing an amphipathic compound, it is necessary to generate active species such as radicals and ions, so it cannot be used when a substance having a reactive group such as a protein is used, and when a flat film is used. However, there is a problem in that the flat film causes pores to cause leakage or release of the contents due to contraction due to polymerization.

The method of adsorbing a polymer by an ionic bond like the method of (1) does not cause problems such as shrinkage due to polymerization, but it requires the use of a large amount of amphipathic compound having a charge, so that the liposome or protein It cannot be used with a preferable lipid composition as an immobilizing membrane carrier.

[0009] In the method using an amphipathic compound having a special structure or a phospholipid modified with a polyoxyethylene group like the method described in (1), it is difficult to separate and purify the amphipathic compound, and at the same time, it cannot be separated from a living body or a protein. It is not preferable because the affinity is lowered.

In the method of coating with a fatty acid ester of a saccharide like the method of (1), there is no problem of shrinkage due to polymerization, and various lipids can be freely selected as an amphipathic compound. It is difficult to obtain a compound in which only the target portion in the inside is esterified, and a mixture of the target compound and a by-product is obtained, so that sufficient performance cannot be obtained.

In the method of coating with N-isopropylacrylamide to N-octadecylacrylamide copolymer as in the other method, although the amphipathic compound film can be easily produced, when the content is added to liposomes. Is
The amphipathic compound film becomes unstable and accelerates leakage of the contents.

As described above, in the conventional methods, there has been no effective method for stabilizing the amphipathic compound film. INDUSTRIAL APPLICABILITY The present invention not only allows amphipathic compounds to be freely selected and does not need to be used in a large amount, is easy to manufacture, has excellent affinity with living organisms and proteins, and does not easily cause aggregation or fusion. An object of the present invention is to provide a method for stabilizing an amphipathic compound film, which can obtain a stable amphipathic compound film with little leakage or release of substances.

[0013]

In view of the above situation, the present inventor has coated an amphipathic compound film with a water-soluble polymer having a long-chain linear alkyl acrylamide unit and an N-substituted acrylamide unit as constituents. Then, they have found that the above problems can be solved, and have achieved the present invention.

That is, the method for stabilizing an amphipathic compound film of the present invention is to use an amphipathic compound film represented by the following general formulas (I) and (II).
A molecular weight of 10,000 or more containing a unit represented by
It is characterized by being coated with 5 million water-soluble polymers.

[0015]

[Chemical 3]

[0016]

[Chemical 4] (In the formula, R ¹ represents an alkyl group or an alkenyl group having 12 to 22 carbon atoms, and R ² and R ³ may be the same or different and represent hydrogen or a linear alkyl group having 1 to 6 carbon atoms. However, R ² and R ³ are not both hydrogen atoms.
Represents a number from 0.001 to 0.08, and m is 0.99.
Represents a number from 9 to 0.50. The present invention will be described in detail below. <Water-Soluble Polymer> The water-soluble polymer used in the present invention contains the units represented by the above general formulas (I) and (II) as constituent components.

In the general formula (I), R ¹ has 12 to 12 carbon atoms.
22 represents an alkyl group or an alkenyl group, preferably an alkyl group or an alkenyl group having 14 to 22 carbon atoms,
More preferably, it is a linear alkyl group or a linear alkenyl group having 14 to 22 carbon atoms, and particularly preferably 14, 16 carbon atoms.
It is a linear alkyl group or oleyl group selected from the group of 18, 20, and 22. Further, 1 represents a number in the range of 0.001 to 0.08, but preferably in the range of 0.003 to 0.06.

On the other hand, in the general formula (II), R ² and R ³
May be the same or different and are hydrogen or have 1 to 6 carbon atoms.
Of the formula (1), R ² and R ³ are not both hydrogen atoms. R ² and R ³ preferably have 1 carbon atom
Is a straight-chain alkyl group of 4 to 4, more preferably a methyl group. Further, m represents a number in the range of 0.999 to 0.50, preferably 0.995 to 0.50, and more preferably 0.995 to 0.60.

In the water-soluble polymer of the present invention, the monomer corresponding to the unit of the general formula (I) or (II) is
Mention may be made of the corresponding N-substituted acrylamides. These are known compounds and can be produced, for example, by reacting acrylic acid with a corresponding alkylamine or alkenylamine.

The water-soluble polymer of the present invention may be composed only of the units represented by the general formulas (I) and (II), or may contain other copolymerizable monomer units. The copolymerizable monomer is not particularly limited as long as the resulting copolymer is water-soluble, and examples thereof include hydrophobic monomers such as styrene and acrylonitrile; nonionic water-soluble monomers such as acrylamide and methacrylamide; acrylic acid; Examples thereof include ionic monomers such as vinyl pyridine and methacryloyloxyethyl trimethyl ammonium chloride.

The water-soluble polymer of the present invention may be produced by any method, for example, photopolymerization, radiation polymerization, radical polymerization, anion polymerization and the like. Of these, radical polymerization is preferred. In the case of radical polymerization,
A peroxide, an azo compound or the like can be used as an initiator. Above all, it is preferable to use an azo compound.
The polymerization temperature is usually in the range of 20 to 90 ° C, preferably 30 to 80 ° C. The polymerization may be carried out in an organic solvent or emulsion polymerization.

The weight average molecular weight of the water-soluble polymer of the present invention is usually 10,000 to 5,000,000, preferably 30,000 to 20.
It is 0,000, and more preferably 50,000 to 2,000,000. Specific examples of the above water-soluble polymer include octadecyl acrylamide-N, N-dimethyl acrylamide copolymer, hexadecyl acrylamide-N, N-dimethyl acrylamide copolymer, tetradecyl acrylamide-
N, N-dimethylacrylamide copolymer, oleylacrylamide to N, N-dimethylacrylamide copolymer, 3,7,11,15-tetramethyl-2-hexadeceneacrylamide to N, N-dimethylacrylamide copolymer, octadecyl Acrylamide-N, N-diethylacrylamide copolymer, hexadecylacrylamide-N, N-diethylacrylamide copolymer, tetradecylacrylamide-N, N-diethylacrylamide copolymer, oleylacrylamide-N, N-diethylacrylamide copolymer Polymer, 3,7,11,15-tetramethyl-2-hexadecene acrylamide to N, N
-Diethyl acrylamide copolymer, octadecyl acrylamide-N, N-dibutyl acrylamide copolymer, hexadecyl acrylamide-N, N-dibutyl acrylamide copolymer, tetradecyl acrylamide-
N, N-dibutyl acrylamide copolymer, oleyl acrylamide to N, N-dibutyl acrylamide copolymer, 3,7,11,15-tetramethyl-2-hexadecene acrylamide to N, N-dibutyl acrylamide copolymer, and the like, Preferable examples include octadecyl acrylamide-N, N-dimethyl acrylamide copolymer and hexadecyl acrylamide-N, N-dimethyl acrylamide copolymer. <Amphipathic Compound> The amphipathic compound used in the present invention may be any compound capable of forming a liposome, a planar bilayer membrane, a monolayer or the like. Such amphipathic compounds are preferably phospholipids, more preferably natural phospholipids. <Method for coating amphipathic compound film> The coating of the amphipathic compound film with the water-soluble polymer of the present invention is achieved by bringing the water-soluble polymer aqueous solution into contact with the aqueous solution forming the amphipathic compound. It For example, in the case of liposomes,
An aqueous solution of a water-soluble polymer may be added after forming liposomes, or when a lipid is used as an amphipathic compound, an aqueous solution to which a water-soluble polymer is added in advance may be used when forming liposomes.

In the case of a flat film, it is achieved by adding a water-soluble polymer aqueous solution after forming the flat film. In the case of a Langmuir-Projet film, it is preferable to form the film on a subphase containing a water-soluble polymer solution. When the amphipathic compound is dispersed in water such as liposomes, the addition amount of the water-soluble polymer is usually in the range of 0.001 to 20% by weight, preferably 0. It is in the range of 003 to 10% by weight. In the case of a flat membrane or Langmuir-Projet membrane, the concentration of the water-soluble polymer is usually 0.001 to 250 mg / l, preferably 0.01 to 150 mg / l.

[0024]

EXAMPLES The method of the present invention will be described more specifically below with reference to examples. The parts are parts by weight.

[0025]

EXAMPLE 1 13.48 g of octadecylamine, 8.4 ml of triethylamine and di-tert-butyl-p were placed in a 200 ml four-necked flask equipped with a nitrogen introducing tube and a cooling tube.
-With cresol 34mg, methylene chloride 150m
It was dissolved in 1. Under reflux, acrylic acid chloride 4.5m
1 was added dropwise over 25 minutes. After refluxing for 2 hours, the mixture was transferred to a separating funnel, and the organic layer was washed with 100 ml of 0.1N hydrochloric acid and 100 ml of 0.1N sodium chloride in this order. After the solvent was distilled off, the residue was purified by silica gel chromatography using chloroform / methanol = 20/1 (volume / volume) as a developing solvent. The effluent was crystallized with acetone to give 12.5 g of octadecyl acrylamide.

The obtained octadecyl acrylamide 0.
A mixture of 3 parts and 9.6 parts of N, N-dimethylacrylamide was dissolved in 40 parts of dioxane, and a solution of 2,2′-azobisisobutyronitrile in dioxane (10 mg / g).
After adding 1.7 parts and purging with nitrogen, the mixture was kept in a sealed tube at 60 ° C. for 8 hours. According to the bromine titration of the resulting octadecyl acrylamide (l = 0.01 in the formula (I)) to N, N-dimethylacrylamide (m = 0.99 in the formula (II)) copolymer, it 98.1% of the heavy bonds were converted. The copolymer was precipitated with diethyl ether and dried under vacuum. The weight average molecular weight of the copolymer determined by light scattering was 300,000. This was dissolved in water to a concentration of 1 mg / ml to obtain a copolymer solution.

Dissolve 4 mg of dipalmitoylphosphatidylglycerol and 16 mg of soybean phosphatidylcholine (Type IV-S manufactured by Sigma) in 2 ml of chloroform,
A thin film was formed by distilling off the solvent under reduced pressure. This was vacuum dried for 6 hours to completely remove chloroform. To this, 4.7 g of water was added, stirred with a vortex mixer, and passed through an extruder to prepare liposomes, and the particle size was adjusted. In this case, stirring with a vortex mixer forms multilamellar liposomes having a particle size of 400 to 4000 nm. The multilamellar liposomes have a wide particle size distribution and a large particle size, so that they easily precipitate. Then, when this is passed through an extruder, it becomes a unilamellar liposome having a particle size of 100 nm and does not precipitate.

The weight average particle size of the thus obtained liposomes by light scattering was 130 nm. 0.4% by weight of this liposome solution based on lipid
The copolymer solution was added so that Next, the calcium chloride concentration of the liposome aqueous solution was adjusted to 20 mM by adding a 200 mM calcium chloride aqueous solution. After standing for 30 minutes, the weight average particle size was measured.
It was 100 nm.

[0029]

Example 2 2.3% by weight of the copolymer solution with respect to the lipid
The same method as in Example 1 was performed, except that The weight average particle size of the obtained liposomes was 144 nm.

[0030]

Comparative Example 1 The same method as in Example 1 was carried out except that water was added instead of the copolymer solution. The weight average particle size of the obtained liposomes was 614 nm.

[0031]

[Example 3] 10 mg of soybean phosphatidylcholine (Type IV-S manufactured by Sigma) was dissolved in 2 ml of chloroform,
A thin film was formed by distilling off the solvent under reduced pressure. This was vacuum dried for 6 hours to completely remove chloroform. 0.1M phosphate buffer (pH 7.1) 1.
After adding 5 g and stirring with a vortex mixer, the liposome was produced by passing through an extruder and the particle size was adjusted. 20 mM 5
(6) -Carboxyfluorescein solution (0.6 ml) was added and the mixture was allowed to stand overnight for encapsulation. Sephadex G
The liposome fraction was collected by a -25M column to remove unencapsulated carboxyfluoncein, and the sample solution was prepared. A portion of this was taken, the liposome was completely solubilized with Triton X-100 (surfactant, manufactured by Rohm & Haas), and the retained carboxyfluorescein was quantified with a spectrophotometer as a reference value. For the rest of the sample solution,
The copolymer solution was added to the lipid in an amount of 1.5% by weight, and the mixture was allowed to stand for 30 minutes. Triton X-100 was added to this to a predetermined concentration, and after standing for 30 minutes, the carboxyfluorescein released on the Sephadex G-25M column was removed, and the liposome was solubilized in the same manner as in the case of the standard value, The retained carboxyfluorescein was quantified spectrophotometrically. The release rate was calculated with a standard value of 100. The results are shown in FIG. 1 (where CF is carboxyfluorescein).

[0032]

Comparative Example 2 The same method as in Example 3 was carried out except that the same amount of water was added instead of the copolymer. The results are shown in Fig. 1.

It can be seen from FIG. 1 that the amphipathic compound film of Example 3 has a lower release rate of carboxyfluorescein than the film of Comparative Example 2 and is stabilized.

[0034]

According to the method of the present invention, the amphipathic compound can be freely selected, and it is not necessary to use it in a large amount, the production is easy, the affinity with living organisms and proteins is excellent, and the aggregation,
It is possible to obtain a stable amphipathic compound film in which not only fusion does not easily occur but leakage or release of the content is small.

[Brief description of drawings]

FIG. 1 is a graph showing a release rate of carboxyfluorescein (CF) in an amphipathic compound film produced in Example 3 and Comparative Example 2.

Claims (3)

(57) [Claims] 1. An amphipathic compound film is coated with a water-soluble polymer having a molecular weight of 10,000 to 5,000,000 and containing units represented by the following general formulas (I) and (II) as constituent components. Method for stabilizing an amphipathic compound film. [Chemical 1] [Chemical 2] (In the formula, R ¹ represents an alkyl group or an alkenyl group having 12 to 22 carbon atoms, and R ² and R ³ may be the same or different and represent hydrogen or a linear alkyl group having 1 to 6 carbon atoms. However, R ² and R ³ are not both hydrogen atoms.
Represents a number from 0.001 to 0.08, and m is 0.99.
Represents a number from 9 to 0.50. ) 2. In the general formulas (I) and (II), R ¹ is a linear alkyl group or a linear alkenyl group having 14 to 22 carbon atoms, and R ² and R ³ are both methyl groups. The method for stabilizing an amphipathic compound film according to claim 1, which is characterized in that. 3. The method for stabilizing an amphipathic compound membrane according to claim 1, wherein the amphipathic compound is a phospholipid.