JP6275538B2 - Temperature, pH responsive copolymer, and liposome complex - Google Patents

Description

  The present invention relates to a copolymer having temperature and pH responsiveness, and a liposome complex in which this copolymer is combined with a liposome.

  A liposome refers to a monolayer of a lipid bilayer membrane composed of phospholipids or a capsule structure composed of a plurality of layers. The phospholipid molecule is shaped like a pine needle, and has two properties: the phosphate portion is hydrophilic and the fatty acid ester portion is hydrophobic. When released into water, the hydrophobic portion Spherical liposomes are formed by gathering on the inside and the hydrophilic portion facing outward. Liposomes can contain water-soluble medicinal ingredients in their hydrophilic parts and oil-soluble medicinal ingredients in their hydrophobic parts. Liposome lipid bilayer membranes contain an aqueous phase that contains a drug and releases the drug at the target lesion site, mainly in the field of medicine. Attention has been paid. Also, in the cosmetic field, in response to changes in the environment when applied to the skin, to release the active ingredient, it is expected to effectively be able to penetrate into the skin. In general, light, UV, humidity, pressure, etc. are known as control factors in the development of technologies to control environmental responsiveness. In addition to poor quantitativeness, these factors are cosmetics in terms of safety. Application to was difficult.

  Patent Document 1 proposes a temperature-sensitive liposome in which a polymer compound having a heat-responsive portion and a hydrophobic portion and polyethylene glycol are supported on a liposome membrane and release inclusions when heated to about 40 to 45 degrees. Yes. In Patent Document 2, the inclusion is held in an acidic pH environment containing a cationic amphiphilic molecule and at least one of an anionic amphiphilic molecule and an amphoteric amphiphilic molecule, and a basic pH A pH-responsive liposome that releases inclusions in the environment has been proposed.

JP 2006-306794 A JP 2010-209021 A

  The present invention relates to a copolymer that undergoes a hydrophilic-hydrophobic change at a specific temperature and pH, and a liposome complex that can release inclusions at a specific temperature and pH by complexing with the copolymer. It is an issue to provide.

Means for solving the above problems are as follows.
1. The repeating unit (a) derived from the monomer represented by the following general formula (1), the repeating unit (b) derived from the carboxyl group-containing monomer, and the monomer represented by the following general formula (2) A copolymer having a repeating unit (c) derived from

(In formula (1), R ₁ represents hydrogen or a methyl group, R ₂ represents an alkyl group having 1 to 4 carbon atoms, and n represents a natural number of 1 to 4)


(In Formula (2), R ₃ represents hydrogen or a methyl group, R ₄ represents an alkyl group having 8 to 20 carbon atoms or an alkenyl group having 8 to 20 carbon atoms, and m represents a natural number of 1 to 6). Represents.)
2. The repeating unit (a) is 40 to 89 mol%, the repeating unit (b) is 10 to 50 mol%, and the repeating unit (c) is 1 to 100 mol% with respect to the total 100 mol% of the repeating units (a) to (c). It is 10 mol%. The copolymer described in 1.
3. The carboxyl group-containing monomer is methacrylic acid. Or 2. The copolymer described in 1.
4). The weight average molecular weight is 1,000 to 1,000,000. ~ 3. The copolymer in any one of.
5.1. ~ 4. A liposome complex, wherein the copolymer according to any one of the above is supported on a lipid bilayer of a liposome.
6). 4. The weight ratio of the lipid bilayer membrane to the copolymer is 50:50 to 99: 1. The liposome complex according to 1.
7). 4. The lipid bilayer membrane contains hydrogenated phospholipid. Or 6. The liposome complex according to 1.
8). 4. The lipid bilayer membrane contains a sterol. ~ 7. The liposome complex according to any one of the above.
9. 4. It contains a medicinal component inside. ~ 8. The liposome complex according to any one of the above.
10.1. ~ 4. A skin external preparation, cosmetic, food, pharmaceutical, or biochemical reagent comprising the copolymer according to any one of the above and a liposome.

  Since the aqueous solution of the copolymer of the present invention undergoes a phase transition from hydrophilic to hydrophobic at a specific temperature and pH, and the transparent liquid changes to a suspension, it can be used as an environmentally responsive factor. . In particular, the liposome complex in which the copolymer is supported on the lipid bilayer is changed to hydrophobic at a specific temperature and pH, so that the lipid bilayer of the liposome is destroyed and the inclusion is changed. Can be released. Liposome complex, since it is possible to release the inclusion specific temperature, at pH, cosmetics, skin external agent, the agent can be used in biochemical reagents.

The figure which shows the transmittance | permeability of copolymer aqueous solution. The figure which shows the transmittance | permeability of copolymer aqueous solution. The figure which shows the endothermic peak of a copolymer. The figure which shows the pH responsiveness of a liposome complex. The figure which shows the pH responsiveness of a liposome complex. The figure which shows the temperature responsiveness of a liposome complex. The figure which shows the pH responsiveness of a liposome complex. Confocal laser microscope image of HeLa cells treated with the liposome complex of the present invention. Confocal laser microscope image of HeLa cells treated with the liposome complex of the comparative example. The figure which shows the temperature responsiveness of a liposome complex. The figure which shows the pH responsiveness of a liposome complex. The figure which shows the temperature responsiveness of a liposome complex. The figure which shows the pH responsiveness of a liposome complex. The figure which shows the temperature responsiveness of a hydrogenated phospholipid containing liposome complex. The figure which shows the temperature responsiveness of a phytosterol containing liposome complex.

"Copolymer"
The present invention is represented by the repeating unit (a) derived from the monomer represented by the following general formula (1), the repeating unit (b) derived from the carboxyl group-containing monomer, and the following general formula (2). It relates to a copolymer having a repeating unit (c) derived from a monomer.

(In formula (1), R ₁ represents hydrogen or a methyl group, R ₂ represents an alkyl group having 1 to 4 carbon atoms, and n represents a natural number of 1 to 4)

(In Formula (2), R ₃ represents hydrogen or a methyl group, R ₄ represents an alkyl group having 8 to 20 carbon atoms or an alkenyl group having 8 to 20 carbon atoms, and m represents a natural number of 1 to 6). Represents.)

The structure of the copolymer of the present invention is not particularly limited, and may be a block copolymer or a random copolymer, but a random copolymer is preferred.
In addition, the molecular weight of the copolymer of the present invention is not particularly limited, but usually a weight average molecular weight in the range of 1,000 to 1,000,000 is preferable. When the weight average molecular weight is less than 1,000, a lump at the time of aggregation is small and the lipid bilayer membrane of the liposome cannot be efficiently destroyed. When the weight average molecular weight is larger than 1,000,000, the viscosity becomes too high and the handleability is poor. Moreover, the solubility to water falls. The weight average molecular weight is more preferably 5,000 to 300,000, still more preferably 10,000 to 100,000, and most preferably 50,000 to 80,000.

The repeating unit (a) imparts temperature responsiveness to the copolymer. This is derived from the ethylene glycol structure in the repeating unit (a). The ethylene glycol structure is hydrated and stable with surrounding water molecules at low temperatures, but the water molecules that have been hydrated are lost and become hydrophobic at high temperatures, so that the copolymer aggregates. R _{2 in the} repeating unit (a) represents an alkyl group having 1 to 4 carbon atoms, and an alkyl group having 1 to 3 carbon atoms is more preferable because of its high solubility in water.

The repeating unit (b) has a carboxyl group and imparts pH responsiveness to the copolymer. Carboxyl group, a neutral aqueous solution, and the alkaline aqueous solution -COO deprotonated ^- since has become, more hydrophilic. In addition, since the electrostatic repulsion hardly causes aggregation, the copolymer is dissolved in water. On the other hand, in a weakly acidic aqueous solution, it is protonated to become -COOH, the hydrophilicity is lowered, and electrostatic repulsion is eliminated, so that the copolymer is aggregated.

Furthermore, in the copolymer of the present invention, the repeating unit (b) also affects the temperature responsiveness. Since the deprotonated carboxyl group (—COO ⁻ ) repels an unshared electron pair present in the oxygen atom in the ethylene glycol structure of the repeating units (a) and (c), the solubility in water is reduced. Improve. When the carboxyl group is protonated under acidic conditions, the —OH of the carboxyl group and the unshared electron pair present in the oxygen atom in the ethylene glycol structure of the repeating units (a) and (c) Since a hydrogen bond is formed between them, the copolymer tends to aggregate. When the repeating unit (b) is increased, the pH required for protonating the carboxyl group is lowered, so that the response point becomes higher temperature and more acidic.

  The carboxyl group-containing monomer can be used without any particular limitation. For example, acrylic acid, methacrylic acid, maleic acid, crotonic acid, 3-butenoic acid, 4-pentenoic acid, itaconic acid, 2-hexenoic acid, 3-hexenoic acid, 5-hexenoic acid, vinyl acetic acid, cinnamic acid, 3- Examples include allyloxypropionic acid, itaconic acid monoester, maleic acid monoester, maleic anhydride, fumaric acid, fumaric acid monoester, vinyl phthalate, and vinyl pyromellitic acid. Among these, crotonic acid, acrylic acid, methacrylic acid, itaconic acid, maleic acid, maleic acid monoester, fumaric acid, fumaric acid monoester, and 3-allyloxypropionic acid are preferable. These carboxyl group-containing monomers may be used alone or in combination of two or more.

Since the repeating unit (c) has a skeleton similar to the repeating unit (a), it imparts temperature responsiveness to the copolymer in the same manner as the repeating unit (a). Furthermore, the repeating units (c) is a lipophilic than the alkyl group having 1 to 4 carbon atoms of the repeating unit (a) _{(R 2),} alkyl group having 8 to 20 carbon atoms, or 8 to 20 carbon atoms, Having an alkenyl group (R ₄ ). The alkyl group having 8 to 20 carbon atoms or the alkenyl group represented by R ₄ may be linear or branched. Specific examples include an octyl group having 8 carbon atoms, a 2-ethylhexyl group, a lauryl group having 12 carbon atoms, a stearyl group having 18 carbon atoms, an isostearyl group, and an oleyl group, and is lipophilic in the lipid bilayer membrane of liposomes. An alkyl group having 12 to 18 carbon atoms is preferable because it easily enters the portion.

  Further, the copolymer of the present invention is derived from other copolymerizable ethylenically unsaturated monomers other than the repeating units (a) to (c) as long as the effects of the present invention are not inhibited. Repeating units may be included. Examples of such ethylenically unsaturated monomers include α-olefins such as propylene, n-butene, isobutylene, and 1-hexene; unsaturated monomers having an acrylate group; methacrylate groups. Unsaturated monomer having; acrylamide, N-methylacrylamide, N-ethylacrylamide, N, N-dimethylacrylamide, diacetoneacrylamide, acrylamidepropanesulfonic acid and its salt, acrylamidopropyldimethylamine and its salt (for example, quaternary salt) ); Methacrylamide, N-methylmethacrylamide, N-ethylmethacrylamide, methacrylamidepropanesulfonic acid and salts thereof, methacrylamidepropyldimethylamine and salts thereof (for example, quaternary salts); methyl vinyl ether, ethyl vinyl ester Vinyl ethers such as ter, n-propyl vinyl ether, i-propyl vinyl ether, n-butyl vinyl ether, i-butyl vinyl ether, t-butyl vinyl ether, dodecyl vinyl ether, stearyl vinyl ether, 2,3-diacetoxy-1-vinyloxypropane; acrylonitrile , Vinyl cyanides such as methacrylonitrile; vinyl halides such as vinyl chloride and vinyl fluoride; vinylidene halides such as vinylidene chloride and vinylidene fluoride; allyl acetate, 2,3-diacetoxy-1-allyloxy Allyl compounds such as propane and allyl chloride; esters of unsaturated dicarboxylic acids such as maleic acid, itaconic acid and fumaric acid; vinylsilane compounds such as vinyltrimethoxysilane; isopropenyl acetate and the like. I can get lost.

  The polymerization method of the copolymer of the present invention is not particularly limited, and solution polymerization, suspension polymerization, emulsion polymerization and the like can be used. Solution polymerization is preferred because the polymerization reaction can proceed in a homogeneous system. Solution polymerization is carried out in a conventional manner, and a predetermined amount of raw material monomers, a polymerization initiator, and a solvent are charged. For example, the solution concentration is about 30 to 70% by mass, and radical polymerization at a polymerization temperature of 50 to 120 ° C. . As the polymerization initiator, a polymerization initiator usually used in radical polymerization is used, and the polymerization initiator is used at a ratio of 0.1 to 20% by mass with respect to the total amount of raw material monomers charged. As the polymerization initiator, those having a half-life temperature suitable for the polymerization temperature are preferable. For example, dipropyl peroxydicarbonate (T10 (10-hour half-life temperature) = 40 ° C.), benzoyl peroxide (T10 = 74 ° C.) , Organic peroxides such as lauroyl peroxide (T10 = 62 ° C.), t-butylperoxyhexanoate (T10 = 72 ° C.), 2,2′-azobis (2,4-dimethylvaleronitrile) (T10 = 51 ° C.), 2,2′-azobis (isobutyronitrile) (T10 = 65 ° C.), and the like can be used.

  As the solvent used in the solution polymerization, a solvent that dissolves both the monomer and the polymer is preferably used. For example, ketone solvents such as acetone and methyl ethyl ketone, alcohol solvents such as ethanol, isopropanol, ethylene glycol, and propylene glycol, propylene glycol Examples thereof include glycol solvents such as monomethyl ether acetate, hydrocarbons such as aliphatic hydrocarbons and aromatic hydrocarbons. Among these, acetone, ethanol, and isopropanol are preferable because the solvent can be easily removed. Two or more kinds of solvents may be mixed and used.

  The copolymer of the present invention changes from hydrophilic to hydrophobic at a specific temperature and pH. The response point at which this change occurs can be controlled by the mol% of the repeating units (a) to (c). The aqueous solution of the copolymer of the present invention changes from a transparent liquid to a suspension due to a phase transition from hydrophilic to hydrophobic at a specific temperature and pH. It can be applied as a factor.

  In the copolymer of the present invention, the mol% of the repeating units (a) to (c) is not particularly limited. However, as will be described in detail below, the repeating units (a) to (c) per 100 mol%) of the repeating unit (a) is 40 to 89 mol%, the repeating units (b) is 10 to 50 mol%, it is preferable repeating unit (c) is 1 to 10 mol% . Since the repeating unit (b) is less than 10 mol% is low hydrophobicity, the force to break the lipid bilayer when complexed with liposomes is not sufficient. When there are more repeating units (b) than 50 mol%, hydrophilicity will become high and pH required for a copolymer to aggregate will become low too much. Furthermore, this molar ratio is suitable for causing a parent-hydrophobic change in the intracellular environment of a thermostat animal.

"Liposome complex"
A liposome complex can be formed from the copolymer of the present invention and the liposome. This is because the alkyl group having 8 to 20 carbon atoms or the alkenyl group having 8 to 20 carbon atoms (R ₄ ) of the repeating unit (c) of the copolymer enters the lipophilic portion in the lipid bilayer membrane of the liposome. This is because the copolymer functions as an anchor for anchoring and supporting the copolymer to the liposome.
Here, the liposome complex of the present invention means that a part of the copolymer is supported in the lipid bilayer of the liposome, and is not a mere mixture. Whether or not the copolymer is supported on the liposome can be determined by whether or not the liposome and the copolymer can be separated by gel filtration, ultracentrifugation, dialysis, or the like.

  If the copolymer is complexed with liposomes to form a liposome complex, the repeating unit (c) should have 1 to 10 mol% with respect to 100 mol% of the total of repeating units (a) to (c). It is particularly preferable to have 1 to 7 mol%. When the repeating unit (c) is less than 1 mol%, the liposome and the copolymer cannot sufficiently form a complex, and when it exceeds 10 mol%, the water solubility decreases and the copolymer dissolves in water. It becomes difficult to do. In terms of water solubility, 7 mol% or less is particularly excellent in solubility. The mol% of the repeating units (a) and (b) may be appropriately adjusted according to the condition showing responsiveness, but as described above, with respect to the total of 100 mol% of the repeating units (a) to (c). The repeating unit (b) is preferably 10 to 50 mol%.

  In the liposome complex of the present invention, when the copolymer undergoes a phase transition from hydrophilic to hydrophobic, the lipid bilayer is broken and the liposome inclusions are released. Therefore, the liposome complex of the present invention can be applied to external preparations for skin, cosmetics, foods, pharmaceuticals, biochemical reagents and the like.

The principle that the lipid bilayer of the liposome is destroyed is as follows.
In the aqueous dispersion of the liposome complex, the copolymer is stably present in water in a state of being supported on the liposome under the condition that the copolymer can maintain hydrophilicity. When the copolymer becomes hydrophobic by changing the temperature and / or pH of the aqueous dispersion, the copolymer aggregates in water. In the liposome complex, since the copolymer is supported on the liposome, the structural change of the copolymer occurs near the surface of the lipid bilayer of the liposome. Copolymer aggregates that are hydrophobic enter the lipophilic portion of the lipid bilayer and disrupt the lipid bilayer.

  In the liposome complex of the present invention, the copolymer can efficiently destroy the lipid bilayer of the liposome. On the other hand, in the copolymer not supported on the liposome, the hydrophobic copolymers coagulate with each other, and there are those that continue to disperse in water without attacking the lipid bilayer. Inferior ability to break lipid bilayer.

  In the present invention, the weight ratio of the phospholipid and the copolymer constituting the lipid bilayer membrane of the liposome is not particularly limited, but is usually in the range of 50:50 to 99: 1. Even if the weight ratio of the copolymer is more than 50, the ability to break the liposome is saturated and does not improve any further. When the polymerization ratio of the copolymer is less than 1, the liposome cannot be sufficiently broken. The compounding ratio of the phospholipid and the copolymer is more preferably 60:40 to 95: 5, and further preferably 70:30 to 90:10.

  As the phospholipid constituting the liposome of the present invention, an amphiphilic phospholipid which is usually used as a membrane lipid of the liposome can be used. Examples of such phospholipids include phospholipids such as phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, cardiolipin, sphingomyelin, soybean phosphatidylcholine, and egg yolk phosphatidylcholine. Examples of fatty acids constituting these phospholipids include myristic acid, palmitic acid, stearic acid, arachidonic acid, oleic acid, linoleic acid, and linolenic acid. These can be used alone or in combination of two or more. In particular, phosphatidylcholine and phosphatidylethanolamine are preferable.

  Here, the properties of the phospholipid differ depending on the fatty acid constituting the acyl group. For phosphatidylcholine, phosphatidylcholine rich in unsaturated fatty acids, such as egg yolk phosphatidylcholine and soybean phosphatidylcholine is pasty. Non-hydrogenated phospholipids having polyunsaturated fatty acid residues may be brownish and change odor when exposed to air or exposed to light. This is because unsaturated fatty acids are peroxidized by oxygen in the air. Hydrogenated phospholipids are produced by adding hydrogen at the phospholipid purification stage and changing unsaturated fatty acid residues to saturated fatty acid residues to prevent denaturation due to oxidation of phospholipids. It is solid.

  The phospholipid forming the liposome of the present invention may be a non-hydrogenated phospholipid, a hydrogenated phospholipid, or a combination thereof. Hydrogenated phospholipids can be packed more densely than non-hydrogenated phospholipids because fatty acids do not have double bonds and are flexible, and can form dense lipid bilayer membranes. The ratio of the non-hydrogenated phospholipid and the hydrogenated phospholipid can be used together without any particular limitation, but the molar ratio of the non-hydrogenated phospholipid to the hydrogenated phospholipid is usually 99: 1 to 1. : 99. Here, since the phospholipid derived from a natural product is a mixture of various compounds, the molar ratio was calculated using the molecular weight of the main compound.

The liposome of the present invention may have a sterol. It is known that sterol has an action of entering into a lipid bilayer and making the lipid bilayer an intermediate state that is neither a gel nor a liquid crystal (Reference: “Liposome” Nankodo). Sterols decrease membrane permeability and membrane fluidity for lipid bilayers composed of non-hydrogenated phospholipids, and eliminate phase transitions for lipid bilayers composed of hydrogenated phospholipids. , Has the effect of increasing membrane fluidity.
When preparing the liposome of the present invention, the molar ratio of non-hydrogenated phospholipid to sterol is preferably 99: 1 to 50:50, particularly preferably 99: 1 to 70:30. When the molar ratio of sterol is less than 1 or exceeds 50, the stability of the liposome is lowered. Here, as described above, since sterols derived from natural products are a mixture of various compounds, the molar ratio was calculated using the molecular weight of the main compound.

Examples of sterols used in the present invention include animal sterols, plant sterols (phytosterols), and fungal sterols. Examples of animal sterols include cholesterol, cholestanol, and 7-dehydrocholesterol. Examples of plant sterols (phytosterols) include sitosterol, stigmasterol, fucosterol, spinasterol, and brassicasterol. Moreover, the phytostanol which is a hydrogenated substance of a plant sterol is mentioned. Examples of fungal sterols include ergosterol.
A commercially available product can be used as the sterol used in the present invention. For example, phytosterol S (phytosterol) manufactured by Tama Seikagaku Co., Ltd., cholesterol JSCI (cholesterol) manufactured by Nippon Seika Co., Ltd., GENEROL 122N (phytosterol) manufactured by Cognis Japan Co., Ltd., marine cholesterol (cholesterol) manufactured by Nippon Suisan Co., Ltd. It is done.

  By adding hydrogenated phospholipid and / or phytosterol, the lipid bilayer membrane can be made more stable. That is, the lipid bilayer membrane in which these are blended can suppress unintentional leakage of inclusions and make the environmental responsiveness more sensitive.

  The liposome of the present invention can be produced by a known liposome production method. Known methods for producing liposomes include an extruder method, an ultrasonic method, a French press method and a high-pressure emulsification method. As a high-pressure emulsifier, T.M. K-Fillmix (thin film swirl type high speed homomixer), Microfluidics Microfluidizer (ultra-high pressure homogenizer), Yoshida Kikai Kogyo Co., Ltd. Nanovaita (Nanomizer), etc. can be used.

  As an example, a method for producing the liposome of the present invention by the extruder method will be described. The phospholipid and copolymer constituting the lipid bilayer of the liposome are dissolved in a suitable organic solvent such as chloroform, and the solution is placed in a container. Next, the organic solvent is removed using an evaporator, and a mixed thin film composed of a phospholipid and a copolymer is formed on the inner wall surface of the container. This mixed thin film is preferably further vacuum-dried for about 3 to 12 hours to completely remove the organic solvent. Next, a suitable solution such as a buffer solution is put into the container, and the mixture is peeled off from the inner wall surface of the container by vigorously stirring using an ultrasonic treatment or a vortex mixer to form liposomes. . By incorporating a medicinal ingredient into a solution such as a buffer solution, the medicinal ingredient can be included in the liposome.

The particle size of the liposome can be adjusted by passing the obtained dispersion through an extruder and appropriately setting the filter pore size. The particle size of the liposome of the present invention is not particularly limited, but is usually 0.05 to 100 μm. What is necessary is just to adjust the particle size of a liposome suitably according to the intended purpose.
The liposome of the present invention may be either a monolayer liposome composed of a single lipid bilayer membrane or a multilamellar liposome composed of a plurality of lipid bilayer membranes as long as it falls within the above particle size range. Since the lipophilic medicinal component is included in the lipid bilayer membrane, it is preferable to use multilamellar liposomes in order to include a large amount of the lipophilic medicinal component.

  From the liposome dispersion obtained as described above, the copolymer not supported on the lipid bilayer of the liposome, the medicinal component not included in the liposome, gel filtration, ultracentrifugation, dialysis It can be removed by methods. If the substance to be removed has a charge, ion exchange chromatography can be used.

  In the liposome produced by the method as described above, the copolymer may be supported on the outer surface of the liposome, or may be supported on both the outer surface and the inner surface of the liposome membrane. May be. When a mixed thin film composed of phospholipid and copolymer is formed as in the above production method, the copolymer is supported on both the outer surface and the inner surface of the liposome. In the above production method, a copolymer can be supported only on the outer surface of the lipid bilayer by forming a thin film consisting only of phospholipid and adding a copolymer after forming a liposome in advance.

  The medicinal component contained in the liposome is not particularly limited as long as it does not inhibit the production of the liposome of the present invention. Anticancer agent, plasmid, protein, enzyme, moisturizer, anti-inflammatory agent, vitamins, antioxidant, ultraviolet absorbers, blood circulation promoters, wound healing agents, antibacterial substances, skin activators, normal flora control agent, active oxygen scavengers can be used whitening agent. These medicinal ingredients may be either hydrophilic or lipophilic, and both can be included. The hydrophilic medicinal component is trapped in the hydrophilic portion of the liposome, and the lipophilic medicinal component is trapped in the lipophilic portion of the liposome.

  Since the liposome complex of the present invention can release inclusions under specific conditions, it can be used in so-called drug delivery systems, and it can be used as a skin external preparation, cosmetic, food, pharmaceutical, biochemical reagent. Etc. can be used.

<Test Example 1>
"Synthesis of copolymer 1"
"Example"
Into a 1 L reaction vessel equipped with a stirrer, a temperature sensor, a condenser and a nitrogen introduction tube, 400 g of isopropanol was charged, and the inside of the reaction vessel was purged with nitrogen through nitrogen gas, and then the temperature was raised to an internal temperature of 60 ° C. Then, 150 g of a monomer mixture mixed in mol% shown in Table 1 below and 1.5 g of dipropyl peroxydicarbonate were dropped over 2 hours, followed by polymerization reaction at 60 ° C. for 2 hours and at 80 ° C. for 1 hour. It was. Thereafter, the solvent was removed at 80 ° C. under reduced pressure to obtain copolymers of Examples 1 to 5 and Comparative Examples 1 to 3. The structural formula of the copolymer is shown in the following chemical formula 5.

  The weight average molecular weight (Mw) of the copolymer was calculated from the results of gel filtration chromatography (GPC). GPC was performed using a high performance liquid chromatograph (GPC column Shodex LF-804, eluent: tetrahydrofuran), and was calculated as polystyrene based Mw by calculating polystyrene as a standard substance from the chromatographic results.

"Evaluation of copolymer temperature and pH responsiveness"
1. Measurement of transmittance Each of the copolymers of Examples 1 to 5 and Comparative Examples 1 to 3 was dissolved in a 10 mM phosphate buffer aqueous solution (140 mM NaCl) so as to be 10 mg / mL. The copolymer solution was adjusted to various pHs using HCl and NaCl with stirring under ice cooling. After taking 2 mL of the copolymer solution in a screw cell and stirring and holding at 10 ° C. for 10 minutes, the transmittance was measured while heating at a heating rate of 2 ° C./min.
The measurement was performed using a UV / visible spectrophotometer (manufactured by JASCO Corporation, apparatus name: V-560) at a measurement wavelength of 500 nm. The temperature control was performed using a Beltier temperature controller (manufactured by JASCO Corporation, apparatus name: ETC-505T). At this time, the temperature at which the transmittance decreased to 95% was defined as the cloud point.
The measurement results are shown in FIGS.

Examples 1 to 5 showed no cloud point at neutrality of pH 7.4 at 10 to 80 ° C., regardless of the content of the repeating unit (b). Moreover, the pH which shows a cloud point became acidic, so that the content rate of the repeating unit (b) became high.
The carboxyl group is deprotonated and hydrophilic when neutral, and is protonated and hydrophobic when acidic. When the carboxyl group content is increased, the pH necessary for protonation of the carboxyl group is decreased. Therefore, it is considered that as the carboxyl group content increases, it becomes more acidic and exhibits a cloud point.

In addition, from the behavior at pH 6.0 of Examples 1 to 4 and pH 5.5 of Examples 2 to 5, the degree of decrease in transmittance due to temperature change may be moderated as the number of repeating units (b) increases. I understand.
This is because, as the number of carboxyl groups increases, the pH necessary for protonation of the carboxyl groups becomes more acidic, so that the carboxyl group (—COO ⁻ ) in the deprotonated state and the repeating units (a) and (c) It is presumed that this is due to repulsion with the oxygen atom in the ethylene glycol structure, preventing molecular aggregation.
That is, as the number of carboxyl groups increases, the response point at which the structure changes to hydrophobicity becomes more acidic at higher temperatures.

  In Comparative Example 1 consisting only of the repeating unit (a), the cloud point did not change to around 20 ° C. at pH 5.0 to 7.4. This is considered to be because the ethylene glycol structure was dehydrated at 20 ° C. and aggregates were formed by hydrophobic interaction. Moreover, since it did not have the repeating unit (b) which has pH responsiveness, the cloud point was constant irrespective of pH.

  Comparative Example 2 which is a copolymer of the repeating unit (a) (95 mol%) and the repeating unit (c) (5 mol%) does not have the repeating unit (b) having a hydrophilic carboxyl group, and is lipophilic. Since it has a repeating unit (c) having a long-chain alkyl group, it was insoluble in water.

  Comparative Example 3, which is a copolymer of the repeating unit (a) (80 mol%) and the repeating unit (b) (20 mol%), shows no cloud point at neutral pH 7.4 and has a pH of 6.0 to 5.0. As it became more acidic, the cloud point shifted to lower temperatures. As discussed in Examples 1 to 4 above, under neutral conditions, the carboxyl group is deprotonated and hydrated, but under acidic conditions it is protonated and loses charge, becomes hydrophobic, and aggregates. It is thought that it was because of promoting.

2. DSC Measurement The various pH copolymer solutions of Example 2 and Comparative Examples 1 and 3 prepared in “1. Transmittance Measurement” were degassed for 3 minutes.
Each copolymer solution was used for 630 μL each and held at 1 ° C. for 10 minutes, and then DSC measurement was performed. The measurement was performed while heating from 1 ° C. to 80 ° C. at a heating rate of 1 ° C./min. Nano DSC (manufactured by TA Instruments Japan Inc.) was used for the measurement.
Moreover, the dispersion liquid of the liposome complex complexed with the copolymer of Example 2 prepared in Example 7 of Test Example 2 below was prepared at various pHs, and DSC measurement was performed in the same manner.

The result of the endothermic peak in the temperature change is shown in FIG. The vertical axis of the graph represents the amount of heat absorbed, and only the graph of Comparative Example 1 has a scale that is four times larger than the others. In the graph of Example 2, EYPC indicates the measurement result of liposomes composed only of non-hydrogenated egg yolk phosphatidylcholine not complexed with the copolymer.
The results of transmittance measurement and DSC measurement are shown in Table 2.

  Comparative Example 1 showed a large endothermic peak around 20 ° C. at any pH. The peak temperature, since it substantially coincides with the above was measured by "1. transmittance measurement" cloud point temperature, it is understood that the endotherm dehydration at temperatures response. Further, heat of the repeating unit (a) per 1mol thought to be involved in dehydration from the heat absorption amount of heat was calculated to be 7～8KJ. This means the amount of heat when all the water molecules hydrated to the repeating unit (a) are dehydrated.

  Similarly in Example 2 and Comparative Example 3, the temperature of the endothermic peak at each pH almost coincided with the cloud point temperature. Further, no endothermic peak was exhibited at a pH at which no cloud point was exhibited. It can be seen that the amount of heat per 1 mol of the repeating unit (a) increases as it becomes acidic, and approaches the amount of heat of 7 to 8 kJ when completely dehydrated. In neutrality, the deprotonated carboxy group affects the ethylene glycol structure in the repeating unit (a) in a hydrated state even when the temperature rises. It is thought that dehydration was promoted by increasing the proportion of groups and interacting with the ethylene glycol structure as the temperature increased.

  Further, even when the copolymer of Example 2 was complexed with liposome, the temperature of the endothermic peak did not change greatly. The amount of heat per 1 mol of the repeating unit (a) increased nearly twice at pH 6.0 but slightly decreased at pH 5.5. This means that the copolymer is more easily dehydrated by being immobilized on the liposome. However, at pH 5.5, it is considered that the carboxy group hydrophobized by protonation was reduced because it was incorporated into the hydrophobic part of the lipid membrane of the liposome without acting on the ethylene glycol structure.

When the temperature / pH responsiveness of the copolymer was evaluated by transmittance measurement and DSC measurement, the copolymer solution showed a decrease in transmittance at a certain temperature or higher due to temperature rise, and showed an endothermic peak at that temperature. This showed that the copolymer was hydrophobized by dehydration to form aggregates. In addition, by changing the pH of the copolymer solution, a hydrophilic-hydrophobic change can be caused at different temperatures.
That is, the copolymer having the repeating units (a) to (c) of the present invention exhibits responsiveness to both temperature and pH. Further, depending on the content of the repeating unit (b), the hydrophilic-hydrophobic response It was confirmed that the temperature and pH indicating the pH can be controlled.

<Test Example 2>
“Creation of liposome complex 1”
"Example 6"
100 mg of non-hydrogenated egg yolk phosphatidylcholine (manufactured by NOF Corporation, trade name: COATSOME NC-50, hereinafter referred to as EYPC) was dissolved in 10 mL of chloroform. The solution was placed in a 1 mL flask and chloroform was removed by a rotary evaporator to form a thin film of EYPC on the inner wall surface of the flask.
The methanol solution of the copolymer of Example 1 (2 mg / mL) was added so that the weight ratio of EYPC to the copolymer was 80:20, methanol was removed by a rotary evaporator, and the EYPC and copolymer were added. A mixed thin film was formed. The mixed thin film formed on the inner wall surface of the flask was vacuum dried for 4 hours while adhering to the inner wall surface of the flask, thereby completely removing the solvent.

  Pyranine (manufactured by Tokyo Chemical Industry Co., Ltd.) 35 mM as a fluorescent substance, paraxylenebis (N-pyridinium bromide) as a quencher (manufactured by Invitrogen, hereinafter referred to as DPX) 50 mM, disodium hydrogen phosphate (manufactured by Kishida Chemical ) An aqueous solution (hereinafter referred to as a coloring solution) was prepared so as to be 25 mM. The pH of this color developing solution was 7.4.

Add the above coloring solution to 1 mL per 1.25 × 10 ⁻⁵ mol of lipid in the mixed thin film, and irradiate ultrasonic waves using a bath-type ultrasonic irradiation device. After peeling and dispersing, the pH was adjusted to 7.4 using NaOH and HCl. This dispersion was frozen in a −20 ° C. ethanol bath and subsequently thawed in a 25 ° C. water bath, and this freezing and thawing operation was performed five times.

  In addition, the amount of lipid in the above-mentioned mixed thin film was determined using phospholipid C test Wako (manufactured by Wako Pure Chemical Industries, Ltd.), choline oxidase / DAOS (Sodium N-Ethyl-N- (2-Hydroxy-3). -Sulfopropyl) -3,5-Dimethoxyyanline) method. Specifically, the liposome dispersion, the blank solution, and the standard solution supplied with Phospholipid C Test Wako are mixed with the coloring solution and incubated at 37 ° C. for 5 minutes, and the absorbance of each solution at a wavelength of 600 nm is ultraviolet / visible. spectrophotometer (manufactured by JASCO Corporation, apparatus name: V-560) was measured was used to determine the concentration of liposome dispersion from the obtained absorbance.

  By sandwiching a membrane with a pore size of 100 nm between the extruder and passing the dispersion of the mixed thin film after freezing and thawing operations 25 times, the particle size of the liposome complex contained in the solution was adjusted to 100 nm. Thereafter, Sepharose 4B was purified by gel filtration using gel and a phosphate buffer aqueous solution as a mobile phase to obtain a liposome complex. By this purification, pyranine, which is a fluorescent substance, and free copolymer can be removed from the outer aqueous phase.

"Examples 7 to 10"
A liposome complex was obtained in the same manner as in Example 5 except that the copolymers of Examples 2 to 5 were used.
"Examples 11 and 12"
A liposome complex was obtained in the same manner as in Examples 9 and 10 except that the weight ratio of EYPC to the copolymer was 70:30.

"Comparative Examples 4-6"
A liposome complex was obtained in the same manner as in Example 5 except that the copolymers of Comparative Examples 1 to 3 were used.
“Comparative Example 7”
Liposomes were obtained in the same manner as in Example 5 except that the copolymer was not contained.

"Evaluation of temperature and pH response of liposome complex"
The responsiveness to the temperature and pH of the liposome complexes of Examples 6 to 10 and Comparative Examples 4 to 7 was evaluated.

When the lipid bilayer of the liposome is destroyed, pyranine contained in the liposome is released into the outer aqueous phase. The released pyranine was excited with 416 nm light by the following method, and the emitted fluorescence was measured at 512 nm to evaluate the temperature / pH responsiveness of the liposome complex.
In the liposome lipid bilayer, the concentration of pyranin, which is a fluorescent substance, and DPX, which is a quencher, are both high, and the frequency of collision between excited pyranine and DPX is high. Fluorescence disappears due to excitation. When the lipid bilayer of the liposome is destroyed and pyranine and DPX are released into the outer aqueous phase, the concentration is decreased by dilution, and therefore the collision frequency between the excited pyranine and the quencher is low and the fluorescence is maintained. Therefore, it can be considered that all the fluorescence observed by the measurement method of the present invention is derived from pyranine in the external aqueous phase released from the liposome.

A phosphate buffer aqueous solution adjusted to each pH was added to the quartz cell and placed in a spectrofluorometer. After maintaining the temperature at each measurement condition temperature for about 3 minutes, a dispersion of a liposome complex encapsulating pyranin was added with a spectrofluorometer so that the lipid concentration in the quartz cell was 0.02 mM (final volume). 2.5 mL). The amount of pyranine released after incubation at a predetermined temperature for 10 minutes was examined. Finally, 25 μL of a 10% solution of Triton X-100 (manufactured by Kishida Chemical) was added to destroy the lipid bilayer of the liposome. The fluorescence intensity immediately after addition of the liposome complex when the pH at each temperature is 7.4 (F _0,7.4 ) is 0%, and the fluorescence intensity after addition of 10% Triton X-100 (F _{100,7 .4} ) was regarded as a 100% release amount, and the release rate of inclusions from the liposome complex was calculated by the following calculation formula.

The release rate Ft when pH = x was calculated from the value corrected to pH 7.4, where Ft = (measured value) × F _100,7.4 / F _{100, x} . The fluorescence intensity was measured using a spectrofluorometer (manufactured by JASCO Corporation, apparatus names: FP-6200, FP-8500) and a temperature controller (manufactured by JASCO Corporation, apparatus name: ETC-272T).

  FIGS. 4 and 5 show graphs of the release rates when Examples 6 to 10 and Comparative Examples 5 and 7 were changed in pH at each temperature. Moreover, the graph of the release rate when changing the temperature at pH 7.4 and 5.0 of Comparative Examples 4 and 6 is shown in FIG.

  First, Comparative Example 7, which was only liposomes that were not complexed with a copolymer, retained the inclusion even when the pH changed. Moreover, there was no change in the amount released even when the temperature increased. Therefore no temperature · pH-responsive to a liposome itself, lipid bilayer membranes themselves temperature, it was confirmed that not destroyed even by changing the pH.

  In Examples 6 to 10 using the liposome complex of the present invention, as the content of the repeating unit (b) increased, the release of inclusions was significantly promoted at pH 6.0 or lower. This is because the hydrophobicity of the copolymer becomes stronger as the repeating unit (b) increases, and the lipid bilayer membrane is easily broken. Examples 7 to 10 show responsiveness in the vicinity of pH 5 which is the pH value in the lysosome which is the body digestive organ, 33 to 43 ° C. which is the body temperature of the thermostat animal, and are particularly excellent for prescription to the thermostat animal. Yes. In addition, Example 6 also shows a response at a higher temperature and a lower pH, and the molar ratio of the copolymer may be adjusted according to the desired response point.

  0 mol% is the content of the repeating unit (b), Comparative Example the content of the repeating unit (c) is 5 mole% 5, although Comparative Examples 4, 6 and 7 were excellent in release properties, carried Compared to the example, it was greatly inferior. Comparative Example 5 is presumed to form a complex with the liposome because it has a repeating unit (c), but it does not have a repeating unit (b), so the hydrophilic-hydrophobic phase of the copolymer. The transition originates only from hydrated water with an ethylene glycol structure. Since only the ethylene glycol structure that has lost water of hydration has weak hydrophobicity, Comparative Example 5 is not able to sufficiently destroy the lipid bilayer membrane, and it is presumed that the release rate is inferior.

From FIG. 6, in Comparative Examples 4 and 6, no release of inclusion was observed in the range of 25 to 70 ° C. at both pH 7.4 and pH 5.0, and the same results as Comparative Example 7 were shown.
Since the copolymer used in Comparative Examples 4 and 6 does not have the repeating unit (c), the copolymer does not form a complex with the liposome. It is presumed that the lipid bilayer was not destroyed because the coalescence was removed.

The graph of the release rate when changing the pH at each temperature in Examples 9 to 12 is shown in FIG.
From Examples 9 to 12, it was confirmed that the weight ratio of phospholipid to copolymer was 70:30 and 80:20, and both had good pyranine releasing properties.

<Test Example 3>
“Release test in cells”
"Example 13"
100 mg of EYPC was dissolved in 10 mL of chloroform. This solution was put into a 1 mL flask, and DiIC18, which is a fluorescent substance, was added so as to be 0.1 mol% with respect to the total amount of phospholipid. Chloroform was removed by a rotary evaporator, and a thin film composed of EYPC and DiIC18 was formed on the inner wall surface of the flask.
A methanol solution of the copolymer of Example 5 (2.0 mg / mL) was added so that the weight ratio of EYPC to the copolymer was 80:20, and methanol was removed by a rotary evaporator to form a mixed thin film. did. The mixed thin film formed on the inner wall surface of the flask was vacuum dried for 4 hours while adhering to the inner wall surface of the flask, thereby completely removing the solvent.

1 mL of calcein (Sigma Aldrich) solution (63 mM, pH 7.4), which is a fluorescent substance, is added and irradiated with ultrasonic waves using a bath-type ultrasonic irradiation device, and the mixed thin film is peeled off from the inner wall surface of the flask. The mixed thin film was dispersed. The pH was adjusted to 7.4 using NaOH and HCl. This dispersion was frozen in an ethanol bath at −20 ° C. and subsequently thawed in a water bath at 25 ° C. This freezing and thawing operation was performed 5 times to obtain a liposome dispersion consisting of a mixed thin film.
A membrane with a pore size of 100 nm was sandwiched between the extruders and the liposome dispersion was passed 25 times to make the liposome particle size uniform at 100 nm. Thereafter, the mixture was centrifuged at 55,000 rpm for 120 minutes and purified by removing the supernatant.

HeLa cells, which are cells derived from human cervical cancer, are seeded so that there are 2 × 10 ⁵ cells per hole in the Matsunami glass bottom dish, and DMEM medium is used as a culture solution in a CO ₂ incubator with a CO ₂ concentration of 5%. Cultured overnight at 37 ° C.

The composition of the DMEM medium is as follows.
Dulbecco's modified Eagle medium (DMEM, Nissui Pharmaceutical Co., Ltd.) 9.5 mg / mL, benzylpenicillin potassium 0.1 mg / mL, streptomycin sulfate 0.1 mg / mL, sodium bicarbonate 20 mM, L-Glutamine 4 mM, fetal bovine Serum (MP biomedical, Inc.) 10%.

Then, after washing twice with a phosphate buffer aqueous solution and once with a phosphate buffer aqueous solution not containing calcium and magnesium (hereinafter referred to as PBS (−)), 1.0 mL of DMEM medium was added.
The liposome complex dispersion was added to a lipid concentration of 0.75 mM, PBS (-) was added to a total volume of 2 mL, and then incubated at 37 ° C. for 4 hours to allow the liposomes to be incorporated into the cells. It was. The liposomes that had not been taken into HeLa cells were removed by washing three times with an aqueous phosphate buffer solution. 2 mL of DMEM was added and left in an incubator for 8 hours.

<Comparative Example 8>
Except for using the liposome not complexed with the copolymer prepared in Comparative Example 7, the liposome without the copolymer was incorporated into HeLa cells in the same manner as in Example 13 above.

"Observation of intracellular dynamics with confocal laser microscope"
2 mL of DMEM (phenol red free) was added, and intracellular kinetics was observed 12 hours after adding the liposome dispersion to HeLa cells with a confocal laser microscope (Carl Zeiss, device name: LSM 5 EXCITER). The fluorescence of DiIC18 is observed in red, and the fluorescence is strong in the lipid bilayer, but weak in the aqueous phase. Calcein is observed with green fluorescence and is self-quenched at a concentration of more than 10 mM. Therefore, calcein does not emit fluorescence within the liposome, but emits fluorescence only when released into the outer aqueous phase.

  The confocal laser scanning microscope images after incubating for 12 hours in Example 13 and Comparative Example 8 are shown in FIGS.

  Cells treated with the liposomes of Example 13 showed very strong green fluorescence of calcein. Moreover, the red fluorescence of DiIC18 was weak.

In Example 13, liposomes are taken up by cells by endocytosis and migrate to endosomes. Since the liposome complex transferred to the endosome has a high pH responsiveness at 37 ° C., the lipid bilayer is broken in response to the weakly acidic pH in the endosome, and thus the fluorescence of DiIC18 is weak. In addition, calcein was emitted outside the liposomes and diluted in the external aqueous phase, so that calcein fluorescence was strongly emitted.
The copolymer of the present invention has an ethylene glycol structure with high biocompatibility, and liposomes having this copolymer in the vicinity of the surface have increased stability, and degradation by a degrading enzyme is suppressed. It is speculated that there is almost no degradation of the liposomes by lysosomes.

HeLa cells incorporating liposomes not complexed with the copolymer of Comparative Example 8 also showed green fluorescence, although inferior to Example 13. In addition, many yellow bright spots where green and red coloration occurred at the same location were observed.
Since the liposome of Comparative Example 8 does not have a copolymer, it does not exhibit responsiveness due to temperature and pH, but calcein is released into the cell and exhibits green fluorescence. Since the lysosome of Comparative Example 8 is not complexed with a copolymer having an ethylene glycol structure, it is presumed that the lysosome is easily degraded by a degrading enzyme, and the liposome itself is degraded to release inclusions. This is supported by the fact that the green fluorescence of Comparative Example 8 is local compared to Example 12, and that green and red are emitted from the same place and become yellow.

<Test Example 4>
"Synthesis of copolymer 2"
"Example 14"
A copolymer was obtained in the same manner as in Example 2 of Test Example 1 except that the repeating unit (a) was 78 mol% and the repeating unit (c) was 2 mol%.

<Test Example 5>
“Creation of liposome complex 2”
"Example 15"
A chloroform solution of non-hydrogenated egg yolk phosphatidylcholine was mixed with 20 mg of non-hydrogenated soybean phosphatidylcholine (manufactured by Lipoid, trade name: Phospholipon 90G, hereinafter referred to as Soybean PC) in a mixed solvent of chloroform and methanol (volume ratio 2: 1). As a color solution, a 0.01 mol / L PBS (-) (pH 7.4) solution in which 63 mM of calcein was dissolved was used as a coloring solution, and a solution of non-hydrogenated soybean phosphatidylcholine and a copolymer was used. A liposome complex was obtained in the same manner as in Example 7 of Test Example 2 except that the weight ratio was 90:10 and the gel used for purification by gel filtration was Shepadex G-50 (manufactured by Sigma Ardrich). .

"Example 16"
A liposome complex was obtained in the same manner as in Example 15 except that the copolymer obtained in Example 14 was used.

"Comparative Example 9"
A liposome complex was obtained in the same manner as in Example 15 except that the copolymer obtained in Comparative Example 3 was used.

"Evaluation of temperature and pH response of liposome complex"
The temperature and pH of the liposome complexes of Examples 15 and 16 and Comparative Example 9 were changed.
When the lipid bilayer of the liposome is destroyed, calcein contained in the liposome is released into the outer aqueous phase. The released calcein was excited with light at 490 nm by the following method, and the emitted fluorescence was measured at 520 nm to evaluate the temperature / pH responsiveness of the liposome complex.
As described in Test Example 3 above, calcein is self-quenched at a concentration exceeding 10 mM, and thus does not emit fluorescence in the liposome.

-Temperature responsiveness evaluation The phosphate buffer aqueous solution adjusted to pH 7.4 was added to the test tube. The liposome solution was added so that the lipid concentration in the test tube was 0.02 mM (final volume 4 mL). The measurement was in the range of 25 to 70 ° C., and the temperature was constantly raised. After incubating for 10 minutes, 1% NaOH aqueous solution was added to adjust the pH to 7.4, and then the amount of calcein released was examined (because calcein is quenched under weakly acidic conditions). Finally, 80 μL of 10% Triton X-100 was added to break the liposomes. The fluorescence intensity immediately after adding the liposome when the pH at each temperature is 7.4 to the buffer is 0% (F _0,7.4 ), and the fluorescence intensity when 10% Triton X-100 is added is 100%. _{Considering the} release amount (F _{100, 7.4} ), the release rate of the inclusion from the liposome was calculated by the following calculation formula.

The fluorescence intensity was measured using SPECTRA MAX GEMINI EM (manufactured by Molecular Device Japan).

-PH responsiveness evaluation The phosphate buffer aqueous solution adjusted to each pH was added to the test tube. The liposome solution was added so that the lipid concentration in the test tube was 0.02 mM, and then incubated at 35 ° C. for 30 minutes (final volume 4 mL). Thereafter, 1% NaOH aqueous solution was added to adjust the pH to 7.4. Thereafter, the amount of calcein released was examined. Finally, 8 μL of 10% Triton X-100 was added to break the liposomes. Thereafter, the release rate from the liposomes was calculated in the same manner as in the above temperature responsiveness.

  Examples 15 and 16, Comparative Example 9, in 10 the release rate at each temperature when the pH 7.4, showing the release rate at each pH at a temperature of 35 degrees in FIG. 11.

  Examples 15 and 16 released the inclusion well at a specific temperature and pH. The neutrality at pH 7.4 showed almost the same behavior, but at a temperature of 35 degrees, Example 15 having 5 mol% of the repeating unit (c) was more than Example 16 having 2 mol% of the repeating unit (c). The release rate was also high. This is presumed to be because the more repeating units (c), the easier the copolymer forms a complex with the liposome. In addition, the release rate of Example 15 is higher than that of Example 7 of Test Example 2 which is a complex using the same copolymer, but this is because of the difference in phospholipids forming liposomes, It seems to be derived from the difference in fluorescent materials.

Comparative Example 9, which is a liposome complex using a copolymer having no repeating unit (C), did not show a response to changes in temperature and pH. This is presumably because the copolymer having no repeating unit (C) is difficult to form a complex with the liposome, and the copolymer has been removed during the production process.
.

<Test Example 6>
“Creation of liposome complex 3”
"Examples 17 to 20"
A liposome complex was prepared in the same manner as in Example 15 except that the copolymers obtained in Examples 1 to 4 were used, respectively, and the weight ratio of non-hydrogenated soybean phosphatidylcholine to the copolymer was 90:10. Obtained.

"Comparative Example 10"
A liposome complex was obtained in the same manner as in Example 15 except that the copolymer obtained in Comparative Example 2 was used.

In the same manner as in Test Example 5, the temperature and pH responsiveness of the liposome complex were evaluated.
The release rate at each temperature when pH is 5.0 is shown in FIG. 12, and the release rate at each pH when temperature is 35 degrees is shown in FIG.

  In Examples 17 to 20 using the liposome complex of the present invention, all showed good responses to changes in temperature and pH. In Example 17 in which the repeating unit (b) was compounded with a copolymer of 10 mol%, the repeating unit (b) is less because the number of the repeating unit (b) is small and the hydrophobicity due to protonation of the carboxyl group is weak. Compared to Examples 18 and 19 complexed with copolymers of 20 mol% and 30 mol%, respectively, it is recognized that there are conditions where the release rate is inferior, but the content of the repeating unit (b) is As the number increased, the response point tended to shift to high temperature and low pH. It was shown that the response point can be adjusted by the molar ratio of the copolymer.

  Comparative Example 10, which was combined with a copolymer having a repeating unit (b) content of 0 mol%, hardly released inclusions. Some leakage was observed under a strong acid at 35 ° C., but since the copolymer used in Comparative Example 10 does not have a carboxyl group having pH responsiveness, the cause is unknown.

<Test Example 7>
"Preparation of liposome complex containing hydrogenated phospholipid"
"Example 21"
Assuming that the molecular weight of Soybean PC is 786.1, which is the molecular weight of dioleoylphosphatidylcholine, Soybean PC is composed of 90 mol% of SoyBean PC and a hydrogenated phospholipid having a molecular weight of 734.04 (manufactured by NOF Corporation). , Trade name: COATSOME MC-6060, hereinafter referred to as DPPC.) A hydrogenated phospholipid-containing liposome complex was obtained in the same manner as in Example 20 except that the mixture was 10 mol%.

In the same manner as in Test Example 5, the temperature responsiveness of the hydrogenated phospholipid-containing liposome complex was evaluated.
The evaluation results are shown in FIG. 14 together with the liposome complex obtained in Example 20.

In both Examples 20 and 21, no temperature responsiveness was exhibited at pH 7.4, and good temperature responsiveness was exhibited at pH 5.0. The release rate at low temperature was suppressed lower in Example 21 containing hydrogenated phospholipid than in Example 20 consisting only of non-hydrogenated phospholipid. This is presumably because the inclusion of hydrogenated phospholipids made the lipid bilayer membrane denser and reduced the leakage of inclusions.
<Test Example 8>
"Phytosterol-containing liposome complex"
"Example 22"
Example 20 except that Soybean PC was a mixture having a molar ratio of 80:20 with Soybean PC and phytosterol S (manufactured by Tama Seikagaku Co., Ltd.) assuming a molecular weight of 414.7 which is the molecular weight of β-sitosterol. In the same manner as above, a phytosterol-containing liposome complex was obtained.

In the same manner as in Test Example 5, the temperature responsiveness of the phytosterol-containing liposome complex was evaluated.
The evaluation results are shown in FIG. 15 together with the liposome complex obtained in Example 20.

  Similarly to Examples 20 and 21, Example 22 did not show temperature responsiveness at pH 7.4, but showed good temperature responsiveness at pH 5.0. Further, towards the Example 22 containing phytosterols, compared to Example 20 consisting of non-hydrogenated phospholipids only, release rate at low temperatures is suppressed low. This is because by containing phytosterols, membrane fluidity of the lipid bilayer membrane consisting of non-hydrogenated phospholipids, membrane permeability is presumed to be because decreased.

Claims (8)

The repeating unit (a) derived from the monomer represented by the following general formula (1), the repeating unit (b) derived from the carboxyl group-containing monomer, and the monomer represented by the following general formula (2) The repeating unit (a) is 40 to 89 mol% and the repeating unit (b) is 10 to 100 mol% of the total of the repeating units (a) to (c). A liposome complex , wherein a lipid bilayer-supporting copolymer having 50 mol% and a repeating unit (c) of 1 to 10 mol% is supported on a lipid bilayer of a liposome.
(In formula (1), R ₁ represents hydrogen or a methyl group, R ₂ represents an alkyl group having 1 to 4 carbon atoms, and n represents a natural number of 1 to 4)
(In Formula (2), R ₃ represents hydrogen or a methyl group, R ₄ represents an alkyl group having 8 to 20 carbon atoms, or an alkenyl group (C _n H _2n-1 ) having 8 to 20 carbon atoms, m represents a natural number of 1 to 6.) The liposome complex according to claim 1, wherein the carboxyl group-containing monomer is methacrylic acid . The liposome complex according to claim 1 or 2, wherein the copolymer has a weight average molecular weight of 1,000 to 1,000,000 . The liposome complex according to any one of claims 1 to 3, wherein a weight ratio of the lipid bilayer membrane to the copolymer is 50:50 to 99: 1. The liposome complex according to any one of claims 1 to 4, wherein the lipid bilayer membrane contains hydrogenated phospholipid. The liposome complex according to any one of claims 1 to 5, wherein the lipid bilayer contains a sterol. The liposomal complex according to any one of claims 1 to 6 , wherein the medicinal component is contained inside. The repeating unit (a) derived from the monomer represented by the following general formula (1), the repeating unit (b) derived from the carboxyl group-containing monomer, and the monomer represented by the following general formula (2) The repeating unit (a) is 40 to 89 mol% and the repeating unit (b) is 10 to 100 mol% of the total of the repeating units (a) to (c). A skin external preparation, cosmetic, food, pharmaceutical, or biochemical reagent containing a lipid bilayer membrane-supporting copolymer having 50 mol% and a repeating unit (c) of 1 to 10 mol% and a liposome.
(In formula (1), R ₁ represents hydrogen or a methyl group, R ₂ represents an alkyl group having 1 to 4 carbon atoms, and n represents a natural number of 1 to 4)
(In Formula (2), R ₃ represents hydrogen or a methyl group, R ₄ represents an alkyl group having 8 to 20 carbon atoms, or an alkenyl group (C _n H _2n-1 ) having 8 to 20 carbon atoms , m represents a natural number of 1 to 6.)