JP6462073B1 - Method for producing pH-sensitive liposomes - Google Patents

Abstract

The present invention stabilizes pH-sensitive liposomes that have been unstable and difficult to prepare, and enables pH-sensitive liposomes to be prepared by a simple method.
A diol, a trivalent or higher polyol, and a liposome membrane constituent are mixed under heating conditions to prepare a mixture solution in which the liposome membrane constituent is dissolved in a mixture of the diol and the polyol. Mixing the step, this mixture solution and a pre-warmed aqueous medium, homogenizing them, quenching the homogenized aqueous medium to produce liposomes, and recovering the produced liposomes And a process.
[Selection] Figure 1

Description

  The present invention relates to a method for producing pH-sensitive liposomes, and more particularly to a method for producing pH-sensitive liposomes using a mixture of a diol and a trivalent or higher polyol.

  Liposomes are attracting attention as carriers for delivering biologically active molecules into the cytoplasm. The main problem in drug delivery using liposomes is that the release of drugs encapsulated in lipid bilayers is slow after transfer into the cytoplasm because normal liposomes have low membrane fusion. In order to solve this, pH-sensitive liposomes have been developed that are stable under physiological conditions but are destabilized under acidic conditions after moving into the cytoplasm.

  There are various methods for producing liposomes, such as an ultrasonic method, an extrusion method, a French press method, a homogenization method, an ethanol injection method, and the like. There is a method in which a lipid component such as phospholipid dissolved in a solvent is added to an aqueous solution while stirring. In this method, alcohols such as methanol, ethanol, isopropyl alcohol, and butanol can be used as a water-miscible organic solvent. However, in order to maintain the lipid dissolution state, the lipid solution is heated and mixed with the aqueous solution. It was necessary to precisely control the temperature, the addition rate, or the stirring rate (see Patent Document 1).

  In addition, it has been reported that liposomes containing lecithin, cholesterol and triglyceride at a predetermined weight ratio can be formed only by stirring required for general mixing without performing steps such as homogeneous high-pressure treatment (see Patent Document 2). ).

JP-T-2006-517594 JP 2017-66059 A

However, since pH-sensitive liposomes are unstable compared to normal liposomes, their industrial production is difficult, and there is a need for a simple and stabilized method for producing pH-sensitive liposomes.
The present invention has been made to solve such a problem, and is to stabilize pH-sensitive liposomes that have been unstable and difficult to prepare so far, and enable the preparation of pH-sensitive liposomes by a simple method.

  The method for producing a pH-sensitive liposome according to one embodiment of the present invention comprises mixing a diol, a trivalent or higher polyol, and a liposome membrane component under heating conditions, and mixing the diol with the polyol. Preparing a mixture solution in which the liposome membrane constituents are dissolved, mixing the mixture solution with a pre-warmed aqueous medium, homogenizing them, quenching the homogenized aqueous medium, It includes a step of producing liposomes and a step of collecting the produced liposomes. The liposome membrane component contains an anionic substance and a zwitterionic substance in a predetermined ratio, and when the liposome is dispersed in an aqueous medium under the following pH conditions, the zeta potential is positive at a pH of 5 or less. Yes, it was negative at pH 8 or higher, and between pH 5 and 8 was shifted from positive to negative as the pH value increased.

  According to the method of the present invention, a stable pH-sensitive liposome is produced by a simple operation of mixing and dissolving a diol, a trivalent or higher polyol, and a liposome membrane constituent, and stirring and mixing with an aqueous medium. Can do.

It is a flowchart of the manufacturing method of the pH sensitive liposome concerning one Embodiment of this invention. It is a flowchart of the manufacturing method of the pH sensitive liposome concerning other embodiment of this invention. It is the pH profile of the zeta potential measured by dispersing the liposome produced in Examples 1 to 6 in an aqueous medium. 3 is a pH profile of zeta potential measured by dispersing liposomes produced in Comparative Examples 1 and 2 in an aqueous medium.

Hereinafter, preferred embodiments of the present invention will be described in the following order with reference to the drawings.
1. 1. Substances used for production of pH sensitive liposomes 2. Method for producing pH-sensitive liposomes pH sensitivity and its expression mechanism

[Substances used for production of pH-sensitive liposomes]
(Diol)
In the present embodiment, 1,2-alkanediol or 1,3-alkanediol is preferable as the diol that dissolves the liposome membrane constituent. As 1,2-alkanediol or 1,3-alkanediol, 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 and the like. 1,2-alkanediol or 1,3-alkanediol can be used alone or in combination of two or more. The 1,2-alkanediol or 1,3-alkanediol is preferably 1,2-propanediol and 1,3-butylene glycol. The amount of 1,2-alkanediol or 1,3-alkanediol used is not particularly limited as long as it can dissolve the liposome membrane constituents, but is preferably about 10 to 50 times the total mass of the liposome membrane constituents, preferably By using about 15 to 30 times, the produced pH-sensitive liposome can be stabilized.

(Polyol)
Polyols that dissolve liposome membrane components together with diols are trivalent or higher polyols such as trehalose, sucrose, sorbose, melezitose, glycerol, fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, glucose , Mannitol, xylitol, erythritol, threitol, sorbitol, raffinose and the like. Preferably, sorbitol and glycerol are used. Although these usage-amounts are not specifically limited as long as a liposome membrane component can be dissolved, it was produced by using about 10 to 50 times, preferably about 15 to 30 times the total mass of the liposome membrane component. pH sensitive liposomes can be stabilized.

(Aqueous medium)
In the present invention, the “aqueous medium” refers to an aqueous medium that does not contain an organic solvent and can disperse liposome membrane components, and is not particularly limited. For example, water, preferably distilled water for injection, physiological Tonic saline, ion-exchanged water, and an isotonic agent or a buffer solution may be added to these solutions. Alternatively, a physiologically active substance as a liposome-encapsulating substance may be included.

(Liposome membrane component)
Examples of the liposome membrane constituent include phospholipids and cholesterol, as well as anionic substances and amphoteric substances for imparting pH sensitivity to the liposomes. In the following, description will be given in order.

(1) Phospholipids Generally, phospholipids are amphipathic substances having a hydrophobic group composed of a long-chain alkyl group and a hydrophilic group composed of a phosphate group or the like in the molecule. Phospholipids include, for example, glycerophospholipids such as phosphatidylcholine (lecithin), phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol, sphingophospholipids such as sphingomyelin, natural or cardiolipin Synthetic diphosphatidyl phospholipids and derivatives thereof, and those obtained by hydrogenating them according to a conventional method (for example, hydrogenated soybean phosphatidylcholine (HSPC)) can be used. Among these, hydrogenated phospholipids such as HSPC, sphingomyelin and the like are preferable. The amount of phospholipid is usually 20 to 100 mol%, preferably 40 to 100 mol%, in the whole lipid constituting the liposome membrane. Moreover, the quantity of another film | membrane structural component is 0-80 mol% normally, Preferably it is 0-60 mol%.

(2) Cholesterol As a constituent component of the liposome membrane, in addition to the phospholipid, a stabilizer such as cholesterol that reduces membrane fluidity may be included as necessary. Examples include sterols that act as lipid membrane stabilizers, such as cholesterol, dihydrocholesterol, cholesterol esters, phytosterols, sitosterol, stigmasterol, campesterol, cholestanol, or lanosterol. It has also been shown that sterol derivatives such as 1-O-sterol glucoside, 1-O-sterol maltoside or 1-O-sterol galactoside are effective in stabilizing liposomes (JP-A-5-245357). Among these, cholesterol is particularly preferable.

(3) Anionic substances Anionic substances for imparting pH sensitivity to liposomes are diacylglycerol hemisuccinate, diacylglycerol hemimalonate, diacylglycerol hemiglutarate, diacylglycerol hemiadipate, diacylglycerol hemicyclohexane-1, Examples include 4-dicarboxylic acid, fatty acids such as oleic acid, myristic acid, palmitic acid, stearic acid, nervonic acid, behenic acid, but are not limited thereto. 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 degreeC-30 degreeC. The content ratio of the anionic substance with respect to the total amount of the liposome constituent components 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 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 the emulsified state in the aqueous medium containing the liposome membrane constituents, resulting in white turbidity, aggregation and precipitation, resulting in a heterogeneous liposome preparation.

(4) Amphoteric Substances As amphoteric substances for imparting pH sensitivity to liposomes, N-alkyl-N, N-dimethylamino acid betaines such as laurylbetaine (lauryldimethylaminoacetic acid betaine); cocamidopropyl betaine Fatty acid amide alkyl-N, N-dimethylamino acid betaines such as lauramidopropylbetaine; imidazoline type betaines such as sodium cocoamphoacetate and sodium lauroamphoacetate; alkylsulfobetaines such as alkyldimethyltaurine; sulfuric acids such as alkyldimethylaminoethanol sulfate Type betaine: Phosphoric acid type betaines such as alkyldimethylaminoethanol phosphates can be mentioned. The content ratio of the zwitterionic substance with respect to the total amount of the liposome constituent components is 5 to 20% by mass, preferably 7% by mass or more. On the other hand, the upper limit of the content is preferably 20% by mass, and preferably 15% by mass. When the content ratio of the zwitterionic substance exceeds 20% by mass, it is difficult to maintain the liposome (lipid bilayer) structure.

(5) Other additives The pH-sensitive liposome of the present invention can contain other additives as required. For example, antioxidants include tocopherol homologues, that is, vitamin E. The lipid derivative of the hydrophilic polymer that modifies the liposome surface is not particularly limited as long as it does not impair the structural stability of the liposome. For example, polyethylene glycol, dextran, pullulan, ficoll, polyvinyl alcohol, synthetic polyamino acid, amylose , Amylopectin, mannan, cyclodextrin, pectin, carrageenan, and derivatives thereof. Of these, polyethylene glycol and polyethylene glycol derivatives are desirable. The molecular weight of the lipid derivative of the hydrophilic polymer is preferably about 200 to 50,000, more preferably about 1000 to 10,000.

(Inclusion substance)
The pH-sensitive liposome of the present invention can encapsulate various water-soluble or fat-soluble target substances. The method for 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 at the time of liposome production. The water-soluble drug that has not been retained can be separated from the liposome that retains 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 diol and polyol to form a liposome by mixing the drug, for example, in the hydrophobic part of the bilayer vesicle. The substance can be retained.

[Method for producing pH-sensitive liposome]
Next, the manufacturing method of the pH sensitive liposome concerning this embodiment is demonstrated, referring FIG. Each of the following steps is a preferable example, and each step may be appropriately modified or a known manufacturing method may be further added. For example, in order to adjust the particle diameter of the liposome, an ultrasonic irradiation method, an extrusion method, a French press method, a homogenization method, or the like may be combined.

(Diol and polyol dissolution process)
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 the liposome membrane constituent can be uniformly dissolved, but is preferably 1: 5 to 5: 1, more preferably 1: 2 to 2: 1, and approximately 1: 1. Is most preferred. These mixing methods can be performed using an ultrasonic vibrator or the like in addition to manual rocking, stirring using a stirring bar and stirring blades. Moreover, the heating conditions at the time of mixing will not be restrict | limited especially if these mixtures are the temperature which melt | dissolves, However, 60 to 90 degreeC is preferable and 80 to 85 degreeC is more preferable.

  Although the heating method is not particularly limited, for example, in addition to a warm bath in which a container is placed in a bathtub containing warm water, a method of heating the container with a direct flame in a state where the mixture is placed in the container, The method of putting a container in an electric heater can be adopted.

(Liposome membrane component dissolution process)
Next, in step S02, a liposome membrane constituent is added to the homogenized mixture. And the mixture solution which melt | dissolved the added liposome membrane structural component in the mixture of diol and a 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 above mixture in order to increase the solubilization efficiency. At this time, the content of each component is not particularly limited as long as it is within the above-described range, but the content ratio of the anionic substance and the amphoteric substance is preferably within a range of 1: 1 to 1: 3. .

  Cholesterol, a kind of lipid, is usually difficult to dissolve in water, and it is difficult to adjust the concentration in the liposome membrane. However, the amount of cholesterol introduced into the liposome membrane can be easily adjusted by preliminarily coexisting phospholipids in a mixture of diol and polyol as in the present embodiment to dissolve cholesterol.

(Homogenization process)
In step S03, the mixture solution prepared in step S02 is mixed with the aqueous medium that has been heated in advance at 80 to 85 ° C. At this time, the amount of the aqueous medium added to the entire mixture must be adjusted so that the liposome membrane component is 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 cannot be agglomerated rapidly to form liposomes. Therefore, the amount of the aqueous medium added in this step is preferably set to a critical concentration at which the lipid component dissolved in the mixture solution of diol and polyol can be dissolved when mixed with the aqueous medium. For example, the amount is 2 to 6 times, preferably 3 to 5 times, and more preferably about 4 times the volume of the mixture solution prepared in step S02.

(Liposome production process)
In step S04, the aqueous medium in which the liposome membrane constituents are dissolved at 80 ° C. to 85 ° C. is rapidly cooled to near room temperature to generate liposomes. Although the cooling method is not particularly limited, for example, in addition to a method of putting a container in a bathtub containing cold water, a method of putting the container in a refrigerator or the like with a mixture in the container is adopted. it can. The cooling temperature is not limited as long as it is a temperature at which liposomes are generated. For example, when phosphatidylcholine and cholesterol are used as lipids, the cooling temperature is preferably 62 ° C. or lower. Further, it may be cooled to around room temperature. Moreover, as a cooling rate, 0.5 degree-C / min or more is preferable, Furthermore, 1 degree-C / min or more is preferable.

(Recovery process)
In step S05, liposomes present in the aqueous medium can be collected by any method such as filtration or decantation. In the embodiment shown in FIG. 1, the liposome membrane constituents are added in step S02 to the solution obtained by previously mixing and homogenizing the diol and polyol in step S01. However, the procedure is not necessarily limited to such a procedure. Not. For example, liposome membrane constituents are added and dissolved in a pre-warmed diol, and then polyol is added and homogenized. Conversely, the polyol and liposome membrane constituents are first mixed and dissolved. Then, a method of adding a diol and homogenizing it may be used. Therefore, as another embodiment of the present invention, as shown in FIG. 2, in step S11, a diol, a polyol, and a liposome membrane constituent are mixed in an arbitrary order, and finally a mixture of a diol and a polyol is obtained. The liposome membrane constituents may be dissolved. The processes after step S11 are the same as those in FIG.

[PH sensitivity and its expression mechanism]
When dispersed in various aqueous media having different pH conditions, the pH-sensitive liposome of this embodiment has a zeta potential that is positive at pH 5 or lower, negative at pH 8 or higher, and between pH 5 and 8. Thus, it has the property of shifting from plus to minus as the pH value increases.

  Here, the zeta potential used as an indicator of the charge state of liposome particles dispersed in an aqueous medium is defined as zero in a region that is sufficiently neutral from the particle and electrically neutral, and this zero point is defined as zero. It is defined as the potential of the “sliding surface” when measured as a reference. In the case of fine particles, if the absolute value of the zeta potential increases, the repulsive force between the particles becomes stronger and the stability of the particles becomes higher. Conversely, when the zeta potential is close to zero, the particles tend to aggregate. Therefore, the zeta potential is used as an index of the dispersion stability of dispersed particles (Fumio Kitahara, Kunio Furusawa, Masataka Ozaki, Hiroyuki Oshima, “Zeta Potential Zeta Potential: Physical Chemistry of Fine Particle Interface”, Scientist, 1995).

  Accordingly, the pH-sensitive liposome of the present embodiment exhibits a behavior in which the surface charge shifts from plus to minus as the pH value increases between pH 5 and 8, so that the pH of the liposome dispersion is 5 or less under acidic conditions. It is considered that the target substance is stably present in the state, and becomes unstable under pH conditions in which the pH of the liposome dispersion is 5 to 8 and the zeta potential becomes zero, causing membrane fusion and releasing the inclusion. A known method can be used as a method for measuring the zeta potential. For example, when an external electric field is applied to a system in which charged particles are dispersed, the particles migrate (move) toward the electrode, but the speed is proportional to the charge of the particles. The zeta potential can be measured by measuring. The electrophoretic light scattering measurement method is also called a laser Doppler method, and the zeta potential is obtained by observing scattered light from the migrating particles.

  The pH-responsive liposome of this embodiment has a positive zeta potential under an acidic pH environment and a negative zeta potential under a basic pH environment when dispersed in an aqueous medium. The pH response behavior can be shown. 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 lower, it is expected that applications such as a method for introducing these substances into cells will expand.

  The following examples are set forth to demonstrate and further illustrate one embodiment and aspect of the present invention and should not be construed as limiting the scope of the invention.

[Example 1]
10.0 g of sorbitol was added to 10.0 g of propane-1,2-diol, and the mixture was homogenized by heating and stirring with a general-purpose stirrer at 350 rpm at 80 to 85 ° C. 0.41 g of hydrogenated lecithin containing phosphatidylcholine, 0.09 g of cholesterol, 0.05 g of palmitic acid, and 0.1 g of lauryl dimethylaminoacetic acid betaine were added to this liquid which was heated and stirred, and dissolved in the same manner.

  To the mixture solution prepared above, purified water heated to 80 ° C. to 85 ° C. in advance was added to 100 g and mixed with stirring, followed by stirring with a general-purpose stirrer at 350 rpm for 1 to 2 hours. The heating was stopped, the mixture was quenched with stirring, cooled to about room temperature, and the produced liposomes were collected by filtration.

[Example 2]
10.0 g of glycerol was added to 10.0 g of propane-1,2-diol, and the mixture was homogenized by heating and stirring at 80 to 85 ° C. with a general-purpose stirrer at 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 this solution which was heated and stirred, and dissolved in the same manner.

  Purified water heated to 80 ° C. to 85 ° C. was added in advance to 100 g to the mixture solution prepared above and mixed with stirring, and stirred at 8,000 rpm with a homomixer for 1 to 2 hours. The heating was stopped, the mixture was quenched with stirring, cooled to about room temperature, and the produced liposomes were collected by filtration.

[Example 3]
10.0 g of glycerin was added to 5.0 g of propane-1,2-diol, and the mixture was homogenized by heating at 80 to 85 ° C. with a general-purpose stirrer at 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 this solution which was heated and stirred, and similarly dissolved by stirring. Purified water previously heated to 80 ° C. to 85 ° C. was added to the mixture solution prepared above 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 with a general-purpose stirrer at 600 rpm at 80 to 85 ° C. 0.41 g of hydrogenated lecithin containing phosphatidylcholine, 0.09 g of cholesterol, and 0.2 g of lauryldimethylaminoacetic acid betaine were added to this solution which was heated and stirred, and dissolved in the same manner. Purified water previously heated to 80 ° C. to 85 ° C. was added to the mixture solution prepared above 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 lauryl dimethylaminoacetic acid betaine were added to this liquid which was heated and stirred, and dissolved in the same manner. Purified water previously heated to 80 ° C. to 85 ° C. was added to the mixture solution prepared above 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 with 80 rpm to 85 ° C. with a general-purpose stirrer at 600 rpm. 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 to this liquid which was heated and stirred, and dissolved in the same manner. Purified water previously heated to 80 ° C. to 85 ° C. was added to the mixture solution prepared above 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 to 85 ° C. with a general-purpose stirrer 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 this solution which was heated and stirred, and dissolved in the same manner. Purified water heated to 80 ° C. to 85 ° C. was added in advance to 100 g to the mixture solution prepared above and mixed with stirring, and after 1 to 2 hours, an extruder treatment was performed.

[Comparative Example 2]
10.0 g of glycerin was added, and the mixture was heated and dissolved with a general-purpose stirrer 350 rpm at 80 ° C. to 85 ° C., and homogenized. 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 this solution which was heated and stirred, and similarly dissolved by stirring. To the mixture solution prepared above, purified water heated to 80 ° C. to 85 ° C. in advance was added to 100 g and mixed with stirring, followed by stirring with a general-purpose stirrer at 350 rpm for 1 to 2 hours. The heating was stopped, the mixture was quenched with stirring, cooled to about room temperature, and filtered.

[Comparative Example 3]
10.0 g of glycerin was added, and the mixture was heated and dissolved with a general-purpose stirrer 350 rpm at 80 ° C. to 85 ° C., and homogenized. To this liquid which was heated and stirred, 0.82 g of hydrogenated lecithin containing phosphatidylcholine, 0.18 g of cholesterol and 0.05 g of palmitic acid were added and dissolved in the same manner. Purified water heated to 80 ° C. to 85 ° C. was added in advance to 100 g to the mixture solution prepared above and mixed with stirring, followed by stirring at 8,000 rpm for 1 to 2 hours using a homomixer. Heating was stopped, quenched with stirring, and cooled to about room temperature. In this comparative example, reprecipitation of fats and oils was large (considered to be insufficient emulsifying power), and liposome preparation was impossible.

[3] Measurement of zeta potential The aqueous dispersions of liposomes prepared in the above examples and comparative examples were adjusted to various pHs using an aqueous potassium hydroxide solution and an aqueous phosphoric acid solution, and Malvern was maintained at 26 ° C under constant temperature conditions. The zeta potential was measured using a trade name Zeta Sizer Nano Series ZSP manufactured by the company. The results are shown in FIGS. As shown in FIG. 3, the liposomes prepared in Examples 1 to 6 all have a positive zeta potential in an aqueous medium having a pH of 5 or lower, and increase in pH value between pH 5.4 and pH 7.6. It was found that the zeta potential approached zero and shifted from positive to negative. When the pH became 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 showed a slightly positive zeta potential at pH 4 or lower, but is extremely unstable in this region because the absolute value of the zeta potential is small. it is conceivable that. Moreover, the liposome prepared in Comparative Example 2 showed a negative zeta potential at all pHs. Thus, it was found that the liposomes prepared in Examples 1 to 6 showed pH sensitivity and could exist stably because the absolute value of the zeta potential was large at pH 5 or lower.

Claims (2)

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, and propylene glycol , trehalose, sucrose, sorbose, melezitose, glycerol, fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, glucose, At least one trivalent or higher polyol selected from mannitol, xylitol, erythritol, threitol, sorbitol and raffinose and a liposome membrane component are mixed under heating conditions, and the diol and polio are mixed. A mixture of Le, a step of preparing a mixture solution of the liposome membrane constituents,
Mixing the mixture solution with a pre-warmed aqueous medium and homogenizing them;
Quenching the homogenized aqueous medium to produce liposomes;
Recovering the liposomes, and
The liposome membrane component includes an anionic substance that is a saturated fatty acid that is solid at room temperature and an amphoteric substance in a ratio of 1: 1 to 1: 3.
The zwitterionic substance is N-alkyl-N, N-dimethylamino acid betaine containing lauryldimethylaminoacetic acid betaine; fatty acid amidoalkyl-N, N-dimethylamino acid betaine containing cocamidopropyl betaine, lauramidopropyl betaine; The group consisting of sodium acetate, imidazoline-type betaine containing sodium lauroamphoacetate; alkylsulfobetaine containing alkyldimethyltaurine; sulfate-type betaine containing alkyldimethylaminoethanol sulfate; and phosphate-type betaine containing alkyldimethylaminoethanol phosphate At least one selected from
When the liposome is dispersed in an aqueous medium having the following pH conditions, the zeta potential of the liposome is positive at pH 5 or lower, negative at pH 8 or higher, and between pH 5 and pH 8. A method for producing a pH-sensitive liposome, characterized in that it shifts from plus to minus with an increase in the pH. A diol composed of 1,2-propanediol, 1,3-butylene glycol or a mixture thereof, a trivalent or higher valent polyol composed of sorbitol, glycerol or a mixture thereof, and a liposome membrane component are mixed under heating conditions. And preparing a mixture solution in which the liposome membrane constituents are dissolved in a mixture of the diol and polyol,
Mixing the mixture solution with a pre-warmed aqueous medium and homogenizing them;
Quenching the homogenized aqueous medium to produce liposomes;
Recovering the liposomes, and
The liposome membrane component comprises an anionic substance that is palmitic acid or stearic acid and an amphoteric substance in a ratio of 1: 1 to 1: 3,
The zwitterionic substance is at least one selected from N-alkyl-N, N-dimethylamino acid betaines including lauryldimethylaminoacetic acid betaine;
When the liposome is dispersed in an aqueous medium having the following pH conditions, the zeta potential of the liposome is positive at pH 5 or lower, negative at pH 8 or higher, and between pH 5 and pH 8. A method for producing a pH-sensitive liposome, characterized in that it shifts from plus to minus with an increase in the pH.

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