KR20200050982A - pH-sensitive liposomes and preparation method thereof - Google Patents

Abstract

The method for producing a pH-sensitive liposome includes a step of mixing a diol, a polyol having a valence of 3 or more, and a liposome membrane component under warm conditions, and preparing a mixture solution in which the liposome membrane component is dissolved in a mixture of diol and polyol. It includes a process of mixing the solution with a pre-warmed aqueous medium to homogenize them, a process of quenching the homogenized aqueous medium to produce liposomes, and a process of recovering the produced liposomes. By the method of the present invention, pH-sensitive liposomes, which have been difficult to prepare due to instability, can be stabilized, and pH-sensitive liposomes can be prepared by a simple method.

Description

pH-sensitive liposomes and preparation method thereof

The present invention relates to a pH-sensitive liposome and a method for manufacturing the same, and more particularly, to a pH-sensitive liposome obtained by adding and dissolving a membrane component of a liposome to a mixture of a diol and a trivalent or higher polyol, and a method for manufacturing the same .

<Cross reference of related applications>

This application claims priority based on Japanese Patent Application Nos. 2017-169356 and Japanese Patent Application Nos. 2017-169359 filed in Japan on September 4, 2017. It is used in the specification. In addition, all the patents, patent applications, and contents described in the documents cited herein are incorporated herein by reference.

Liposomes have been noted as carriers for delivery of biologically active molecules into the cytoplasm. The main problem in the delivery of drugs using liposomes is that since the membrane fusion is low in normal liposomes, the release of the drug contained in the lipid bimolecular membrane after migration into the cytoplasm is slow. To solve this, a pH-sensitive liposome has been developed that is stable under physiological conditions and destabilizes under acidic conditions after migration into the cytoplasm. For example, in liposomes using phosphatidylethanolamine-type phospholipids, it has been reported that phosphatidylethanolamine-type phospholipids undergo a shift in aggregate structure in response to pH and release inclusions in an acidic environment (pH 5 or less) (e.g. Patent Document 1).

On the other hand, when a liposome containing a cationic amphiphilic molecule, and at least one kind of anionic amphiphilic molecule and at least one cationic amphiphilic molecule as a constituent lipid is dispersed in an aqueous medium, the liposome is under an acidic pH environment. The target substance retained if it has a positive zeta potential and, in an alkaline pH environment, the liposome has a negative zeta potential, and the zeta potential changes from plus to minus as the pH of the dispersion increases. It has been reported that this is released (for example, see Patent Document 1).

In the preparation of liposomes, there are various methods such as an ultrasonic method, an extrusion method, a French press method, a homogenization method, an ethanol injection method, etc., but in the industrial production method of a typical liposome, There is a method of injecting and adding a lipid component such as phospholipid dissolved in a water miscible organic solvent into an aqueous solution while stirring. This method can use alcohols such as methanol, ethanol, isopropyl alcohol, butanol as a water-miscible organic solvent, but it is necessary to add and mix the lipid solution in an aqueous solution while heating the lipid solution to maintain the dissolved state of the lipid. It was necessary to precisely control the addition rate or the stirring rate (see Patent Document 2).

In addition, it has been reported that liposomes containing lecithin, cholesterol, and triglycerides in a predetermined weight ratio can be formed only by agitation required for general mixing without performing a process such as homogeneous high pressure treatment (see Patent Document 3).

Japanese Patent No. 5588619 Japanese Patent Publication 2006-517594 Japanese Patent Publication No. 2017-66059

 Duzgunes N, Straubinger RM, Baldwin PA, Friend DS, Papahadjopoulos D. Proton-induced fusion of oleic acid-phosphatidylethanolamine liposomes. Biochemistry. 1985 24 (13): 3091-3098.

However, since these pH-sensitive liposomes are generally unstable, setting the pH value at which the zeta potential in the aqueous medium becomes 0 to a desired range, i.e., the membrane fusion properties of the liposomes range from acidic to alkaline. It was difficult to set arbitrarily in, and there was a problem that it could not meet expectations for potential uses of the pH-sensitive liposomes. Moreover, since pH-sensitive liposomes are unstable compared to normal liposomes, industrial production thereof is difficult, and there is a need for a method for producing a simple and stabilized pH-sensitive liposome.

The present invention has been made to solve such a problem, and the zeta potential of liposomes can be shifted from plus to minus under any pH condition within a predetermined range by adjusting the content ratio of specific components constituting the liposome. In providing a pH-sensitive liposome. In addition, it is to stabilize the pH-sensitive liposomes, which have been difficult to prepare because they have been unstable so far, and to enable the preparation of pH-sensitive liposomes by a simple method.

The pH-sensitive liposome according to one embodiment of the present invention includes phospholipids, steroids, anionic substances, and both ionic substances as liposomal membrane components, and anionic substances for all of the liposomal membrane components. It is characterized by containing 2.5 to 15% by mass, and 5 to 20% by mass of both ionic substances. Then, when dispersed in an aqueous medium under each of the following pH conditions, the zeta potential of the liposome is positive at pH 5 or lower, negative at pH 8 or higher, and positive to negative with an increase in pH between pH 5-8. To be implemented.

In the method for producing a pH-sensitive liposome according to another embodiment of the present invention, a diol, a polyol having a valence of 3 or more, and a liposome membrane component are mixed under heating conditions to dissolve the liposome membrane component in a mixture of the diol and polyol. A process of preparing a mixture solution, a process of mixing the mixture solution and a previously warmed aqueous medium to homogenize them, a process of quenching the homogenized aqueous medium to produce liposomes, and a process of recovering the resulting liposomes It is characterized by. The liposome membrane component contains at least zwitterionic substances, and when the liposomes are dispersed in an aqueous medium under each pH condition below, the zeta potential is positive at pH 5 or lower, negative at pH 8 or higher, and It was made to shift from plus to minus with increasing pH value between 5 and 8.

According to the present invention, it is possible to provide a pH-sensitive liposome capable of shifting the zeta potential of liposomes from positive to negative under a wide range of arbitrary pH conditions by adjusting the content ratio of specific components constituting the liposome. . Moreover, stable pH-sensitive liposomes can be produced by a simple operation of mixing and dissolving a diol, a polyol having a trivalent or higher value, and a component of the liposome membrane, stirring and mixing with an aqueous medium.

1 is a flowchart of a method for producing a pH-sensitive liposome according to an embodiment of the present invention.
2 is a flowchart of a method for producing a pH-sensitive liposome according to another embodiment of the present invention.
3 is a pH profile of the zeta potential measured by dispersing the liposomes prepared in Examples 1 to 6 in an aqueous medium.
4 is a pH profile of the zeta potential measured by dispersing the liposomes prepared in Comparative Examples 1 and 2 in an aqueous medium.

Hereinafter, a very suitable embodiment of the present invention will be described in the following order with reference to the drawings.

1. Components and composition of pH-sensitive liposomes

2. Method for preparing pH-sensitive liposomes

3. pH sensitivity and expression mechanism

4. Shape, use or method of use

[Components and Composition of pH-Sensitive Liposomes]

(Dior)

In the present embodiment, 1, 2-alkanediol or 1,3-alkanediol is preferable as the diol dissolving the liposome membrane constituent. Examples of 1, 2-alkanediol or 1, 3-alkanediol are 1, 2-propanediol, 1, 2-butanediol, 1, 2-pentanediol, 1, 2-octanediol, 1, 2-hexanediol, 1, 2-decanediol, 1, 3-butylene glycol, 1, 3-propanediol, propylene glycol, etc. are mentioned. 1, 2-alkanediol or 1, 3-alkanediol can use 1 type, or 2 or more types together. The 1, 2-alkanediol or 1, 3-alkanediol is preferably 1, 2-propanediol and 1, 3-butylene glycol. The amount of the 1, 2-alkanediol or 1, 3-alkanediol is not particularly limited as long as it can dissolve the liposome membrane constituents, but is about 10 to 50 times the total mass of the liposome membrane constituents, preferably 15 By using ~ 30 times, the resulting pH-sensitive liposomes can be stabilized.

(Polyol)

The polyol that dissolves the liposome membrane component together with the diol is a triol or higher polyol, for example, trehalose, sucrose, sorbose, melezitose, glycerol, fructose, mannose, maltose, lactose, arabinose, xylose , Ribose, rhamnose, galactose, glucose, mannitol, xylitol, erythritol, thritol, sorbitol, raffinose, and the like. Preferred are sorbitol and glycerol. The amount of these used is not particularly limited as long as it can dissolve the liposome membrane constituents, but the pH-sensitive liposomes produced by using about 10 to 50 times, preferably 15 to 30 times the total mass of the liposome membrane constituents Can stabilize.

(Aqueous medium)

In the present invention, "aqueous medium" is an aqueous medium that does not contain an organic solvent, and refers to a medium capable of dispersing the liposome membrane constituents, but is not particularly limited, for example, water, very suitably distilled water for injection, You may add an isotonic agent, a buffer, etc. to physiological saline, ion-exchange water, or these solutions. Alternatively, a physiologically active substance as a liposomal inclusion substance may be included.

(Liposome membrane component)

Examples of the liposome membrane component include phospholipids and cholesterol, as well as anionic substances and zwitterionic substances for imparting pH sensitivity to liposomes. Hereinafter, it demonstrates in order.

(1) Phospholipid

Phospholipids are both amphiphilic substances that generally have a hydrophobic group consisting of a long chain alkyl group and a hydrophilic group consisting of a phosphoric acid group and the like in the molecule. Phospholipids include, for example, natural or synthetic phospholipids such as phosphatidylcholine (lecithin), phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, sphingosphospholipids such as sphingomyelin, and cardiolipin. Diphosphatidyl-based phospholipids and derivatives thereof, and those obtained by hydrogenation according to a conventional method (for example, hydrogenated soybean phosphatidylcholine (HSPC)) can be used. Among these, hydrogenated phospholipids such as HSPC and sphingomyelin are preferred. The amount of the phospholipid is usually 20% by mass or more, and preferably 40% by mass or more, among the total components of the liposome membrane. In addition, the amount of other liposome membrane constituents is usually 80% by mass or less, and preferably 60% by mass or less.

(2) Steroids

As a component of the liposome membrane, steroids may be further included in addition to the phospholipids. As steroids, all steroids having perhydrocyclopentanophenanthrene such as sterol, bile acid, provitamin D, and steroid hormones can be mentioned. Among them, it is preferable to use sterols. Examples of sterols include sterols that act as lipid membrane stabilizers, such as cholesterol, dihydrocholesterol, cholesterol ester, phytosterol, sitosterol, stigmasterol, campesterol, cholesterol, or lanosterol. . In addition, a sterol derivative such as 1-O-sterol glucoside, 1-O-sterol maltoside or 1-O-sterol galactoside has also been shown to be effective in stabilizing liposomes (Japanese Patent Publication No. 1993-245357) ). Of these, cholesterol is particularly preferred.

The content of steroids is not particularly limited, but is preferably 0.01% by mass or more, more preferably 0.05% by mass or more, and even more preferably 0.1% by mass or more with respect to the entire component of the liposome. Moreover, 30 mass% or less is preferable, 10 mass% or less is more preferable, and 5 mass% or less is more preferable. Steroids can act as stabilizers of molecular aggregates. Steroids may be used alone or in combination of two or more.

(3) anionic substances

Anionic substances for imparting pH sensitivity to liposomes include diacylglycerol hemisuccinate, diacylglycerol hemimalonate, diacylglycerol hemiglutarate, diacylglycerol hemiadipate, diacylglycerol hemicyclohexane-1 , 4-dicarboxylic acid, fatty acid, for example, oleic acid, myristic acid, palmitic acid, stearic acid, nerbonic acid, behenic acid and the like, but are not limited to these. In particular, saturated fatty acids that are solid at room temperature are preferred, and palmitic acid and stearic acid are particularly preferred. In this specification, normal temperature means 10 ° C to 30 ° C. The content ratio of the anionic substance to the total amount of the liposome constituents is 0 to 20% by mass, preferably 2.5% by mass or more, and more preferably 5% by mass or more. On the other hand, the upper limit of the content ratio is preferably 20% by mass, and preferably 15% by mass. When the content ratio of the anionic substance exceeds 20%, it is difficult to maintain an emulsified state in an aqueous medium containing a liposome membrane component, and it becomes a heterogeneous liposome preparation due to clouding, aggregation or precipitation.

(4) zwitterionic material

Examples of zwitterionic substances for imparting pH sensitivity to liposomes include N-alkyl-N, N-dimethylamino acid betaine such as lauryl betaine (lauryl dimethylamino acetate betaine); cocamidopropyl betaine and lauramidopropyl Fatty acid amidoalkyl-N, N-dimethylamino acid betaine such as betaine; imidazoline-type betaine such as sodium cocoamphoacetate and sodium lauroamphoacetate; alkylsulfobetaine such as alkyldimethyltaurine; alkyldimethylamino Sulfate type betaine, such as ethanol sulfate ester; Phosphoric acid type betaine, such as alkyl dimethylaminoethanol phosphate ester, etc. are mentioned. The proportion of the amphoteric component relative to the total amount of the liposome constituents is 5 to 20% by mass, preferably 7% by mass or more. On the other hand, the upper limit of the content ratio is preferably 20% by mass, and preferably 15% by mass. It is difficult to maintain a liposome (lipid bilayer) structure when the content ratio of both ionic substances exceeds 20% by mass.

(5) Other additives

The pH-sensitive liposome of the present invention may contain other additives as necessary. For example, as an antioxidant, a tocopherol homologue, namely vitamin E, etc. are mentioned. In addition, the lipid derivative of the hydrophilic polymer that modifies the surface of the liposome is not particularly limited as long as it does not impair the structural stability of the liposome. For example, polyethylene glycol, dextran, pullulan, picol, polyvinyl alcohol, synthesis Polyamino acids, amylose, amylopectin, mannan, cyclodextrin, pectin, carrageenan, and derivatives thereof. Among them, polyethylene glycol and polyethylene glycol derivatives are preferred. The molecular weight of the lipid derivative of the hydrophilic polymer is preferably about 200 to 50,000, more preferably about 10 to 10,000.

(Inclusion material)

The pH-sensitive liposome of the present invention can contain various target substances that are water-soluble or oil-soluble. The method of retaining the target substance in the liposome may be appropriately selected according to the type of the target substance. For example, when the target substance is a water-soluble drug, it can be prepared by dissolving the drug in an aqueous medium during liposome preparation. Water-soluble drugs that are not retained can be separated from liposomes carrying the target substance by gel filtration, ultracentrifugation, or ultrafiltration membrane treatment. On the other hand, in the case of a fat-soluble drug, the liposome membrane component is dissolved in a mixture of diols and polyols, and the drug is mixed to form liposomes, for example, the desired substance can be retained in the hydrophobic portion of the bimolecular membrane vesicle.

[Method for producing pH-sensitive liposomes]

Next, a method for producing a pH-sensitive liposome according to the present embodiment will be described with reference to FIG. 1. Moreover, the following each process is a preferable example, You may add a deformation | transformation to each process suitably, or you may add a well-known manufacturing method. For example, an ultrasonic irradiation method, an extrusion method, a French press method, a homogenization method, or the like may be combined to adjust the particle diameter of the liposome.

(Dissolution process of diol and polyol)

In Fig. 1, in step S01, at least one diol and at least one polyol are mixed under warming conditions, and these are homogenized to prepare a mixture of diol and polyol. The mixing ratio of the diol and the polyol is not particularly limited as long as it can dissolve the liposome membrane constituents uniformly, but 1: 5 to 5: 1 is preferable, 1: 2 to 2: 1 is more preferable, and almost 1: 1 is most preferred. These mixing methods can be performed by using an ultrasonic vibrator or the like in addition to stirring using manual shaking, agitator, and stirring blade. Moreover, the heating conditions at the time of mixing are not particularly limited as long as the temperature at which these mixtures are melted is preferably 60 ° C to 90 ° C, and more preferably 80 ° C to 85 ° C.

The heating method is not particularly limited, for example, in addition to a warm bath in which the vessel is placed in a hot water bath, a method of heating the vessel directly with a mixture in the vessel, a method of placing the vessel in a heater, etc. Can be.

(Process for dissolving liposome membrane components)

Next, in step S02, the liposome membrane component is added to the homogenized mixture. Then, a mixture solution in which the added liposome membrane component is dissolved in a mixture of diol and polyol is prepared. Each component such as phospholipid may be added and mixed separately, but it is preferable to mix all the liposome membrane components in advance and add them to the mixture to increase the solubilization efficiency. In this case, the content of each component is not particularly limited as long as it is within the above-described range, but it is preferable that the content ratio of the anionic substance and the biionic substance is in the range of 1: 1 to 1: 3.

Cholesterol, a type of lipid, is usually difficult to dissolve in water, making it difficult to prepare a concentration in the liposome membrane. However, as in the present embodiment, phospholipids coexist in a mixture of diols and polyols in advance to dissolve cholesterol, so that the amount of cholesterol introduced into the liposome membrane can be easily adjusted.

(Homogenization process)

In step S03, the mixture solution prepared in step S02 and the aqueous medium previously heated to 80 ° C to 85 ° C are mixed. At this time, the amount of the aqueous medium added to all of these mixtures should be adjusted so that the liposome membrane constituents are in an appropriate concentration range for forming liposomes. If the amount of the aqueous medium is too large, the lipid component dissolved in the mixture of diol and polyol suddenly aggregates and cannot form liposomes. Therefore, it is preferable that the amount of the aqueous medium added in this step is such that the lipid component dissolved in the mixture solution of the diol and polyol becomes a critical concentration that can be dissolved when mixed with the aqueous medium. For example, 2 to 6 times, preferably 3 to 5 times, more preferably about 4 times the amount of the mixture solution prepared in step S02.

(Liposome production process)

In step S04, the liposome is produced by rapidly cooling the aqueous medium in which the liposome membrane constituents are dissolved at about 80 ° C to 85 ° C to near room temperature. Although the cooling method is not particularly limited, for example, a method of putting the container in a refrigerator or the like in a state in which the mixture is placed in the container may be employed, in addition to a method of placing the container in a bath filled with cold water. The cooling temperature is not limited as long as the temperature at which liposomes are produced. For example, when phosphatidylcholine and cholesterol are used for the lipid, the cooling temperature is preferably 62 ° C or lower. Moreover, you may cool to room temperature vicinity. Moreover, 0.5 degreeC / min or more is preferable as cooling rate, and 1 degreeC / min or more is preferable.

(Recovery process)

In step S05, the liposomes present in the aqueous medium can be recovered by any method such as filtration or decantation. In addition, in the embodiment shown in FIG. 1, the component of the liposome membrane is added in step S02 to the solution homogenized by mixing the diol and the polyol in step S01 in advance, but is not necessarily limited to this procedure. For example, a method for homogenizing a diol by adding and dissolving a liposomal membrane component in a pre-warmed diol, or, conversely, first mixing and dissolving a polyol and a liposomal membrane component, and then adding a diol It may be a homogenizing method. Therefore, as another embodiment of the present invention, as shown in Fig. 2, in step S11, the diol, the polyol, and the liposome membrane component are mixed in any order, and finally, the liposome membrane component is dissolved in the mixture of the diol and the polyol. You may do it. The process after step S11 is the same as in FIG. 1.

[PH sensitivity and expression mechanism]

When the pH-sensitive liposome of the present embodiment is dispersed in various aqueous media having different pH conditions, its zeta potential is positive at pH 5 or lower, negative at pH 8 or higher, and an increase in pH between pH 5-8 and Together, it has the property of shifting from plus to minus.

Here, the zeta potential used as an index of the charged state of the liposome particles dispersed in the aqueous medium is defined as the potential of an electrically neutral region sufficiently far away from the particle and is measured based on this zero point. It is defined as the potential of (滑 面). In the case of fine particles, when the absolute value of the zeta potential increases, the repulsive force between particles becomes stronger, and the stability of the particles increases. Conversely, when the zeta potential approaches zero, the particles tend to aggregate. Thus, the zeta potential is used as an index of dispersion stability of dispersed particles (Fumio Kitahara, Kunio Furusawa, Masataka Ozaki, Hiroyuki Oshima, `` Zeta Potential Zeta Potential: Physical Chemistry of Particulate Interfaces, '' Scientificist, 1995) .

Therefore, the pH-sensitive liposome of the present embodiment exhibits the behavior that the surface charge shifts from plus to minus with an increase in the pH value between pH 5 and 8, so that the target substance is used under acidic conditions where the pH of the liposome dispersion is 5 or less. It is believed that it retains and remains stable, and the pH of the liposome dispersion becomes unstable at pH conditions where the zeta potential becomes 0 between 5 and 8, causing membrane fusion and releasing inclusions. As a method of measuring the zeta potential, a known method can be used. For example, when an electric field is applied from the outside to a system in which charged particles are dispersed, the particles move (move) toward the electrode. Since the velocity is proportional to the charge of the particles, the zeta is measured by measuring the moving velocity of the particles. Potential can be measured. The electrophoretic light scattering measurement method is called an alias laser Doppler method, and the zeta potential is obtained by observing the scattered light from the moving particles.

[Shape, purpose or method of use]

When the pH-sensitive liposome of the present embodiment is dispersed in an aqueous medium, it can exhibit an unprecedented pH response behavior of having a positive zeta potential under an acidic pH environment and a negative zeta potential under an alkaline pH environment. In recent years, studies have been conducted to introduce negatively charged substances such as genes and nucleic acid derivatives into cells. Since the pH-sensitive liposome of the present embodiment has a positive surface charge under acidic conditions of pH 5 or less, these substances can be adsorbed, and these substances can be released under neutral conditions from weak acidity of pH 5 to 8 and introduced into cells. Can be used. In addition, there is no particular limitation on the shape, and a multi-layered liposome having a particle diameter of 100 nm to 10 µm or a single-layered liposome may be used. The pH-sensitive liposome of the present embodiment can also be appropriately adjusted in particle size according to its use. For example, for the purpose of administration in vivo, it is preferable to adjust the particle size to 200 nm or less. As a specific method of adjusting the particle diameter, the particle diameter can be adjusted by passing through a filter having a small hole diameter using an extruder. Single-layered liposomes having a particle size of about 100 nm or less have a uniform size and are thermodynamically stable, and are said to have good skin permeability even when used as a cosmetic.

When the pH-responsive liposome of the present embodiment is dispersed in an aqueous medium, it can exhibit an unprecedented pH response behavior that has a positive zeta potential under an acidic pH environment and a negative zeta potential under an alkaline pH environment. In recent years, studies have been conducted to introduce negatively charged substances such as genes and nucleic acid derivatives into cells. Since the pH-responsive liposome of the present embodiment has a positive surface charge under acidic conditions of pH 5 or less, it is expected that the uses such as a method of introducing these substances into cells will be expanded.

Example

The following examples are provided to demonstrate and exemplify one aspect and aspect of the present invention, and should not be interpreted as limiting the scope of the present invention.

[Example 1]

10.0 g of sorbitol was added to 10.0 g of propane-1 and 2-diol, and the mixture was homogenized by heating and stirring at 80 ° C to 85 ° C with a general-purpose stirrer 350 rpm. 0.41 g of hydrogenated lecithin containing phosphatidylcholine, 0.09 g of cholesterol, 0.05 g of palmitic acid, and 0.1 g of lauryldimethylaminoacetic acid betaine were added to the solution under warm stirring, and the mixture was stirred and dissolved similarly.

To the mixture solution prepared above, 80 g to 85 ° C. of purified water previously heated to 100 g was added and mixed while stirring, and the mixture was stirred with a general-purpose stirrer 350 rpm for 1 to 2 hours. The heating was stopped, quenched while stirring, cooled to about room temperature, and the resulting liposomes were filtered and recovered.

[Example 2]

10.0 g of glycerin was added to 10.0 g of propane-1 and 2-diol, and the mixture was homogenized by heating and stirring at 80 ° C to 85 ° C with a general-purpose stirrer 350 rpm. 0.82 g of hydrogenated lecithin containing phosphatidylcholine, 0.18 g of cholesterol, 0.1 g of stearic acid, and 0.2 g of lauryldimethylaminoacetic acid betaine were added to the solution under warm stirring, and the mixture was stirred and dissolved similarly.

To the mixture solution prepared above, 80 ° C to 85 ° C of purified water previously heated to 100 g was added and mixed while stirring, and the mixture was stirred at 8,000 rpm with a homomixer for 1 to 2 hours. The heating was stopped, quenched while stirring, cooled to about room temperature, and the resulting liposomes were filtered and recovered.

[Example 3]

10.0 g of glycerin was added to 5.0 g of propane-1 and 2-diol, and the mixture was homogenized by heating and stirring at 80 ° C to 85 ° C with a general-purpose stirrer of 500 rpm. 0.41 g of hydrogenated lecithin containing phosphatidylcholine, 0.09 g of cholesterol, and 0.1 g of lauryldimethylaminoacetic acid betaine were added to the solution under heating and stirring, and the mixture was stirred and dissolved similarly. To the mixture solution prepared above, 80 ° C to 85 ° C of purified water previously heated was added to 100 g, mixed with stirring, and treated with an extruder.

[Example 4]

10.0 g of sorbitol was added to 30.0 g of propane-1, 2-diol, and the mixture was homogenized by heating and stirring at 80 ° C to 85 ° C with a universal stirrer at 600 rpm. 0.41 g of hydrogenated lecithin containing phosphatidylcholine, 0.09 g of cholesterol, and 0.2 g of lauryldimethylaminoacetic acid betaine were added to the solution under warm stirring, and the mixture was stirred and dissolved similarly. To the mixture solution prepared above, 80 ° C to 85 ° C of purified water previously heated was added to 100 g, mixed with stirring, and treated with an extruder.

[Example 5]

10.0 g of glycerin is added to 10.0 g of 1, 3-butylene glycol, and the mixture is homogenized by heating and stirring at 80 ° C to 85 ° C with a general-purpose stirrer 600 rpm. 0.41 g of hydrogenated lecithin containing phosphatidylcholine, 0.09 g of cholesterol, 0.05 g of palmitic acid, and 0.1 g of lauryldimethylaminoacetic acid betaine were added to the solution under warm stirring, and the mixture was stirred and dissolved similarly. To the mixture solution prepared above, 80 ° C to 85 ° C of purified water previously heated was added to 100 g, mixed with stirring, and treated with an extruder.

[Example 6]

10.0 g of sorbitol was added to 30.0 g of 1, 3-butylene glycol, and the mixture was homogenized by heating and stirring at 80 ° C. to 85 ° C. with a general-purpose stirrer of 600 rpm. To the solution under warm stirring, 0.41 g of hydrogenated lecithin containing phosphatidylcholine, 0.09 g of cholesterol, 0.15 g of palmitic acid, and 0.3 g of lauryldimethylaminoacetic acid betaine were added and dissolved under stirring. To the mixture solution prepared above, 80 ° C to 85 ° C of purified water previously heated was added to 100 g, mixed with stirring, and treated with an extruder.

[Comparative Example 1]

15.0 g of glycerin was added to 5.0 g of 1, 3-butylene glycol, and the mixture was homogenized by heating and stirring at 80 ° C to 85 ° C with a general-purpose stirrer of 350 rpm. 0.41 g of hydrogenated lecithin containing phosphatidylcholine, 0.09 g of cholesterol, and 0.05 g of palmitic acid were added to the solution under warm stirring, and the mixture was stirred and dissolved in the same manner. To the mixture solution prepared above, 80 g to 85 ° C. of purified water previously heated to 100 g was added and stirred while mixing, and after 1 to 2 hours, extruder treatment was performed.

[Comparative Example 2]

10.0 g of glycerin was added and homogenized by heating and stirring at 80 ° C to 85 ° C with a general-purpose stirrer 350 rpm. 0.82 g of hydrogenated lecithin containing phosphatidylcholine, 0.18 g of cholesterol, 0.05 g of palmitic acid, and 0.1 g of lauryldimethylaminoacetic acid betaine were added to the solution under warm stirring, and the mixture was stirred and dissolved. To the mixture solution prepared above, 80 g to 85 ° C. of purified water previously heated to 100 g was added and mixed while stirring, and the mixture was stirred with a general-purpose stirrer 350 rpm for 1 to 2 hours. The heating was stopped, quenched while stirring, cooled to room temperature, and filtered.

[Comparative Example 3]

10.0 g of glycerin was added and homogenized by heating and stirring at 80 ° C to 85 ° C with a general-purpose stirrer 350 rpm. 0.82 g of hydrogenated lecithin containing phosphatidylcholine, 0.18 g of cholesterol, and 0.05 g of palmitic acid were added to the solution under heating and stirring, and the mixture was stirred and dissolved in the same manner. To the mixture solution prepared above, 80 g to 85 ° C. of purified water previously heated to 100 g was added and mixed while stirring, and the mixture was stirred at 8,000 rpm using a homomixer for 1 to 2 hours. The heating was stopped, quenched while stirring, and cooled to about room temperature. In this comparative example, a large amount of re-precipitation of the fats and oils (which is thought to be insufficient emulsifying power) made liposomes incapable.

[3] Measurement of zeta potential

The aqueous dispersion of the liposomes prepared in the above Examples and Comparative Examples was adjusted to various pHs using an aqueous potassium hydroxide solution and an aqueous phosphoric acid solution, and the zeta potential was changed using the Malta brand name Zeta Sizer Nano Series ZSP under constant temperature conditions at 26 ° C. It was measured. The results are shown in FIGS. 3 and 4. As shown in Fig. 3, the liposomes prepared in Examples 1 to 6 all had a positive zeta potential in an aqueous medium of pH 5 or lower, and the zeta potential became close to zero with an increase in pH between pH 5.4 and pH 7.6. It was found that the transition from minus to. When the pH was higher than this range, the zeta potential changed to a negative value. On the other hand, as shown in Fig. 4, the liposome prepared in Comparative Example 1 exhibited a slightly positive zeta potential at pH 4 or lower, but is considered to be very unstable in this region because the absolute value of the zeta potential is small. In addition, the liposomes prepared in Comparative Example 2 exhibited a negative zeta potential at all pHs. As described above, it was found that the liposomes prepared in Examples 1 to 6 exhibited pH sensitivity and were able to stably exist because the absolute value of the zeta potential was large at pH 5 or less.

Claims (15)

As a pH-sensitive liposome comprising phospholipids, steroids, anionic substances, and zwitterionic substances as liposome membrane components,
The liposome contains 2.5 to 15% by mass of the anionic substance, and 5 to 20% by mass of the amphoteric substance relative to the entire component of the liposome membrane.
When the liposomes are dispersed in an aqueous medium of each of the following pH conditions, the zeta potential of the liposomes is positive at pH 5 or lower, negative at pH 8 or higher, and at positive with an increase in pH between pH 5-8. A pH-sensitive liposome characterized by shifting to minus. According to claim 1,
The pH-sensitive liposome, characterized in that the content ratio of the anionic substance and the both ionic substances is 1: 1 to 1: 3. The method according to claim 1 or 2,
A pH-sensitive liposome, wherein the anionic substance is a saturated fatty acid that is solid at room temperature. The method according to any one of claims 1 to 3,
N-alkyl-N, N-dimethylamino acid betaine, wherein the zwitterionic material includes lauryl betaine (lauryl dimethylamino acetate betaine); fatty acid ami containing cocamidopropyl betaine and lauramidopropyl betaine Doalkyl-N, N-dimethylamino acid betaine; imidazoline-type betaine containing sodium cocoamphoacetate, sodium lauroamphoacetate; alkylsulfobetaine containing alkyldimethyltaurine; alkyldimethylaminoethanol sulfate ester A pH-sensitive liposome comprising at least one selected from the group consisting of sulfuric acid-containing betaine; and phosphoric acid-type betaine containing an alkyldimethylaminoethanol phosphate ester. According to claim 3,
A pH-sensitive liposome characterized in that the saturated fatty acid solid at room temperature is palmitic acid or stearic acid. A step of mixing a diol, a polyol having a valence of 3 or more, and a liposome membrane component under warm conditions, and preparing a mixture solution in which the liposome membrane component is dissolved in a mixture of the diol and the polyol;
Mixing the mixture solution and a pre-warmed aqueous medium to homogenize them,
Quenching the homogenized aqueous medium to produce liposomes,
It includes a process for recovering the liposomes,
The liposome membrane component comprises at least zwitterionic material,
When the liposomes are dispersed in an aqueous medium of each of the following pH conditions, the zeta potential of the liposomes is positive at pH 5 or lower, negative at pH 8 or higher, and at positive with an increase in pH between pH 5-8. A method for producing a pH-sensitive liposome characterized by shifting to minus. The method of claim 6,
The mixture solution is a method for producing a pH-sensitive liposome, characterized in that the diol and the polyhydric polyol are mixed under a pre-heating condition to homogenize the solution, and the components of the liposome membrane are added and stirred. The method of claim 6 or 7,
Method for producing a pH-sensitive liposome, characterized in that the zwitterionic material is contained 5 to 20% by mass relative to the total component of the liposome membrane. The method according to any one of claims 6 to 8,
Method of producing a pH-sensitive liposome, characterized in that it comprises an anionic substance in a ratio of 2.5 to 15% by mass relative to the total component of the liposome membrane. The method of claim 9,
Method for producing a pH-sensitive liposome, characterized in that the content ratio of the anionic material and the amphoteric material is 1: 1 to 1: 3. The method according to any one of claims 6 to 10,
Method of producing a pH-sensitive liposome, characterized in that the liposome membrane component further comprises phospholipids and cholesterol. The method according to any one of claims 9 to 11,
Method of producing a pH-sensitive liposome, characterized in that the anionic substance is a saturated fatty acid that is solid at room temperature. The method according to any one of claims 6 to 12,
N-alkyl-N, N-dimethylamino acid betaine, wherein the zwitterionic material includes lauryl betaine (lauryl dimethylamino acetate betaine); fatty acid ami containing cocamidopropyl betaine and lauramidopropyl betaine Doalkyl-N, N-dimethylamino acid betaine; imidazoline-type betaine containing sodium cocoamphoacetate, sodium lauroamphoacetate; alkylsulfobetaine containing alkyldimethyltaurine; alkyldimethylaminoethanol sulfate ester A method for producing a pH-sensitive liposome comprising at least one selected from the group consisting of sulfuric acid-containing betaine; and phosphoric acid-type betaine containing an alkyldimethylaminoethanol phosphate ester. The method according to any one of claims 6 to 13,
The diol is propane-1, 2-diol, 1, 3-butylene glycol or a mixture thereof, and the polyol is a method for producing a pH-sensitive liposome, characterized in that sorbitol, glycerin or a mixture thereof. The method of claim 12,
Method of producing a pH-sensitive liposome, characterized in that the saturated fatty acid solid at room temperature is palmitic acid or stearic acid.

Images