JP2019042680A - METHOD FOR PRODUCING pH-SENSITIVE LIPOSOME - Google Patents

Abstract

To stabilize a pH-sensitive liposome which has heretofore been unstable and difficult to prepare, and to enable preparation of the pH-sensitive liposome by a simple method.SOLUTION: Provided is a method for producing a pH-sensitive liposome, comprising the steps of: mixing a diol, a tri- or more valent polyol, and a liposome membrane component under a warm condition to prepare a mixed solution of the diol and polyol in which the liposome membrane component is dissolved; mixing and homogenizing the mixed solution and an aqueous medium warmed beforehand; quickly chilling the homogenized aqueous medium to generate a liposome; and recovering the liposome generated.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method of producing pH sensitive liposomes, and more particularly to a method of 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 major problem in drug delivery using liposomes is that the release of the drug encapsulated in the lipid bilayer membrane is slow after being transferred into the cytoplasm, since the normal liposome has low fusogenicity. In order to solve this, pH-sensitive liposomes have been developed which are stable under physiological conditions and then destabilized under acidic conditions after being translocated into the cytoplasm.

  Liposomes can be prepared by various methods such as ultrasonic method, extrusion method, French press method, homogenization method, ethanol injection method, etc. There is a method of injecting and adding a lipid component such as phospholipid dissolved in a solvent to an aqueous solution while stirring. In this method, alcohols such as methanol, ethanol, isopropyl alcohol and butanol can be used as the water-miscible organic solvent, but the lipid solution is added and mixed while heating the lipid solution to maintain the dissolved state of the lipid. It is necessary to precisely control the temperature, the addition rate or the stirring rate (see Patent Document 1).

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

Japanese Patent Application Publication No. 2006-517594 JP, 2017-66059, A

However, since pH sensitive liposomes are unstable compared to conventional liposomes, their industrial production is difficult, and a simple and stable method for producing pH sensitive liposomes is required.
The present invention has been made to solve such problems, and its object is to stabilize pH-sensitive liposomes which have hitherto been unstable and difficult to prepare, and to enable 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 and the polyol with the mixture. Preparing a mixture solution in which the liposome membrane components are dissolved, mixing the mixture solution with a preheated aqueous medium, homogenizing them, quenching the homogenized aqueous medium, The method is characterized by comprising the steps of producing a liposome and recovering the produced liposome. 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 at each of the following pH conditions, its zeta potential is positive at pH 5 or less It was negative at pH 8 or higher, and was allowed to shift from positive to negative with increasing pH value between pH 5-8.

  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 component, and stirring and mixing with an aqueous medium. Can.

It is a flowchart of the manufacturing method of pH sensitive liposome concerning one Embodiment of this invention. It is a flowchart of the manufacturing method of pH sensitive liposome concerning other embodiment of this invention. It is the pH profile of zeta potential which disperse | distributed and measured the liposome manufactured in Examples 1-6 in the aqueous medium. It is a pH profile of zeta potential measured by dispersing the 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. Substances used for the preparation of pH sensitive liposomes Method for producing pH sensitive liposome pH sensitivity and its expression mechanism

Substances used for the preparation of pH-sensitive liposomes
(Diol)
In the present embodiment, as the diol for dissolving the liposome membrane component, 1,2-alkanediol or 1,3-alkanediol is preferable. 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. The 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 the 1,2-alkanediol or 1,3-alkanediol to be used is not particularly limited as long as it can dissolve the liposome membrane component, but it is preferably about 10 to 50 times, preferably about 10 to 50 times the total mass of the liposome membrane component. By using about 15 to 30 times, the generated pH sensitive liposome can be stabilized.

(Polyol)
Polyols which dissolve the liposome membrane component together with the diol are polyols having a valence of 3 or more and, for example, 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, it is sorbitol and glycerol. The amount thereof used is not particularly limited as long as the liposome membrane component can be dissolved, but it is generated 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" is an aqueous medium which does not contain an organic solvent and is a medium capable of dispersing the liposome membrane component, and is not particularly limited. Target saline solution, ion exchange water, or an isotonic agent, a buffer solution, etc. may be added to these solutions. Alternatively, a physiologically active substance as a liposome inclusion substance may be included.

(Liposome membrane component)
Liposome membrane components include, in addition to phospholipids and cholesterol, anionic substances and amphoteric substances for imparting pH sensitivity to liposomes. The following will be described in order.

(1) Phospholipids Phospholipids are generally amphiphilic substances having a hydrophilic group composed of a hydrophobic group composed of a long chain alkyl group and a phosphate group etc. in the molecule. Examples of phospholipids include phosphatidylcholine (lecithin), phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, glycerophospholipids such as phosphatidylserine and phosphatidylinositol, sphingophospholipids such as sphingomyelin, natural or cardiolipin Synthetic diphosphatidyl type phospholipids and derivatives thereof, and those obtained by hydrogenating them in a conventional manner (for example, hydrogenated soybean phosphatidyl choline (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 membrane of the liposome. In addition, the amount of other membrane components is usually 0 to 80 mol%, preferably 0 to 60 mol%.

(2) Cholesterol As a component of the liposome membrane, in addition to the above-mentioned phospholipids, if necessary, a stabilizer such as cholesterol which reduces the membrane fluidity may be included. For example, sterols acting as lipid membrane stabilizers such as cholesterol, dihydrocholesterol, cholesterol ester, phytosterol, sitosterol, stigmasterol, campesterol, cholestanol, lanosterol and the like can be mentioned. Sterol derivatives such as 1-O-sterol glucoside, 1-O-sterol maltoside or 1-O-sterol galactoside have also been shown to be effective in stabilizing the liposome (Japanese Patent Laid-Open No. 5-245357). Among these, cholesterol is particularly preferred.

(3) Anionic substance The anionic substance for imparting pH sensitivity to the liposome is diacylglycerol hemisuccinate, diacylglycerol hemimalonate, diacylglycerol hemiglutarate, diacylglycerol hemiadipate, diacylglycerol hemicyclohexane-1, Examples thereof include, but are not limited to, 4-dicarboxylic acids and fatty acids such as oleic acid, myristic acid, palmitic acid, stearic acid, nervonic acid, behenic acid and the like. In particular, saturated fatty acids which are solid at normal temperature are preferable, and palmitic acid and stearic acid are particularly preferable. In the present specification, normal temperature refers to 10 ° C to 30 ° C. The content rate of the said anionic substance with respect to the total amount of a liposome structural component is 0-20 mass%, 2.5 mass% or more is preferable, and 5 mass% or more is more preferable. On the other hand, 20 mass% is good and, as for the upper limit of a content rate, 15 mass% is preferable. 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 component, and clouding, aggregation and precipitation occur to result in a heterogeneous liposome preparation.

(4) Zwitterionic substance As a zwitterionic substance for imparting pH sensitivity to the liposome, N-alkyl-N, N-dimethyl amino acid betaines such as lauryl betaine (lauryl dimethylaminoacetic acid betaine) and the like; cocamidopropyl betaine Fatty acid amide alkyl-N, N-dimethyl amino acid betaines such as lauramidopropyl betaine; imidazoline type betaines such as sodium cocoamphoacetate, sodium lauroamphoacetate; alkyl sulfobetaines such as alkyl dimethyl taurine; sulfuric acid such as alkyl dimethylamino ethanol sulfate ester And betaine-type betaines such as alkyldimethylaminoethanol phosphate ester. The content ratio of the above-mentioned zwitterionic substance to the total amount of liposome constituents is 5 to 20% by mass, preferably 7% by mass or more. On the other hand, 20 mass% is good and, as for the upper limit of a content rate, 15 mass% is preferable. When the content of the amphoteric substance exceeds 20% by mass, it is difficult to maintain the liposome (lipid bilayer membrane) structure.

(5) Other Additives The pH sensitive liposome of the present invention can contain other additives as needed. For example, as an antioxidant, tocopherol homologues, that is, vitamin E and the like can be mentioned. 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, and, for example, polyethylene glycol, dextran, pullulan, ficoll, polyvinyl alcohol, synthetic polyamino acid, amylose Amylopectin, mannan, cyclodextrin, pectin, carrageenan, and derivatives thereof. Among them, 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, and more preferably about 1000 to 10,000.

(Inclusion substance)
The pH sensitive liposome of the present invention can encapsulate various target substances of water solubility or lipid solubility. The method for causing the liposome to retain the target substance may be appropriately selected according to the type of the target substance and the like. 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 not retained can be separated from the liposome retaining the target substance by gel filtration, ultracentrifugation, ultrafiltration membrane treatment or the like. On the other hand, in the case of a lipid-soluble drug, for example, by forming a liposome by mixing the drug in a state in which the liposome membrane component is dissolved in a mixture of diol and polyol, for example, It can hold the substance.

[Method for producing pH-sensitive liposome]
Next, a method of producing the pH sensitive liposome according to the present embodiment will be described with reference to FIG. The following steps are preferable examples, and each step may be appropriately modified or a known manufacturing method may be further added. For example, in order to adjust the particle size of the liposome, an ultrasonic irradiation method, an extrusion method, a French press method, a homogenization method or the like may be combined.

(Dissolution process of diol and polyol)
In FIG. 1, in step S01, at least one diol and at least one polyol are mixed under heating conditions, and these are homogenized to prepare a mixture of diol and polyol. The mixing ratio of diol and polyol is not particularly limited as long as the liposome membrane component can be uniformly dissolved, but 1: 5-5: 1 is preferable, 1: 2-2: 1 is more preferable, and almost 1: 1 Is most preferred. These mixing methods can be performed using an ultrasonic vibrator or the like in addition to manual shaking, stirring using a stirrer, and stirring blades. Moreover, the heating condition at the time of mixing is not particularly limited as long as the mixture melts, but 60 ° C. to 90 ° C. is preferable, and 80 ° C. to 85 ° C. is more preferable.

  The heating method is not particularly limited, and for example, a method of heating the container by direct fire in a state in which the mixture is put in the container, in addition to a warm bath in which the container is put in a bath containing warm water. It is possible to adopt a method of putting the container in the electric heater or the like.

(Dissolution process of liposome membrane components)
Next, in step S02, a liposome membrane component is added to the above mixture in the homogenized state. Then, a mixture solution in which the added liposome membrane component is dissolved in a mixture of diol and polyol is prepared. Although each component such as phospholipid may be separately added and mixed, it is preferable to mix all liposome membrane components in advance and add them to the above mixture to increase the efficiency of solubilization. At this time, the content of each component is not particularly limited as long as it is within the above-mentioned range, but the content ratio of the anionic substance and the zwitterionic substance is preferably within the range of 1: 1 to 1: 3. .

  Cholesterol, which is a type 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 dissolving the cholesterol in advance by causing the mixture of the diol and the polyol to coexist with the phospholipid as in the present embodiment.

(Homogenization process)
In step S03, the mixture solution prepared in step S02 is mixed with an aqueous medium preheated to 80 ° C to 85 ° C. At this time, the addition amount of the aqueous medium to the whole of these mixtures must be adjusted so that the liposome membrane components have an appropriate concentration range for forming liposomes. When the amount of the aqueous medium is too large, the lipid component dissolved in the mixture of diol and polyol can not be rapidly aggregated to form a liposome. Therefore, the amount of the aqueous medium added in this step is preferably such that the lipid component dissolved in the mixture solution of diol and polyol has a critical concentration that can be dissolved when it is 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 formation process)
In step S04, the aqueous medium in which the liposome membrane component is dissolved is rapidly cooled to about room temperature at 80 ° C. to 85 ° C. to form a liposome. The cooling method is not particularly limited. For example, in addition to a method of placing the container in a bath containing cold water, a method of placing the container in a refrigerator or the like with the mixture in the container is adopted. it can. The cooling temperature is not limited as long as the liposome is generated. For example, when phosphatidyl choline and cholesterol are used as the lipid, the cooling temperature is preferably 62 ° C. or less. Further, it may be cooled to around room temperature. Further, the cooling rate is preferably 0.5 ° C./min or more, and more preferably 1 ° C./min or more.

(Recovery process)
In step S05, the liposomes present in the aqueous medium can be recovered by any method such as filtration or decantation. In the embodiment shown in FIG. 1, the liposome membrane component is added in step S02 to the solution in which the diol and the polyol are mixed and homogenized in advance in step S01, but the procedure is not necessarily limited. I will not. For example, a liposome membrane component is added to a preheated diol, dissolved, and then a polyol is added and homogenized, or, conversely, the polyol and the liposome membrane component are first mixed and dissolved. Then, the diol may be added and homogenized. 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 mixture of the diol and the polyol is obtained. Liposome membrane components may be dissolved. The process after step S11 is the same as that of FIG.

[PH sensitivity and its expression mechanism]
The pH sensitive liposome of the present embodiment, when dispersed in various aqueous media of different pH conditions, has a zeta potential that is positive at pH 5 or less, negative at pH 8 or more, and between pH 5 and 8. It has the property of shifting from positive to negative 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 defines the potential of a region sufficiently separated from the particle to be electrically neutral as zero, and this zero point is defined as It is defined as the potential of the "slip surface" when measured as a reference. In the case of fine particles, as the absolute value of the zeta potential increases, the repulsive force between particles becomes stronger and the stability of the particles becomes higher. Conversely, when the zeta potential approaches zero, the particles tend to aggregate. Therefore, the zeta potential is used as an indicator of the dispersion stability of dispersed particles (Kohoku original text, Furusawa Kunio, Ozaki Masataka, Oshima Hiroyuki, "Zeta Potential zeta potential: physical chemistry of fine particle interface", Scientific Co., Ltd., 1995).

  Therefore, the pH-sensitive liposome of the present embodiment exhibits a behavior in which the surface charge shifts from positive to negative with an increase in pH value between pH 5 and 8, so that the pH of the liposome dispersion is under acidic conditions of 5 or less. It is believed that the target substance is retained and stably present, and the liposome dispersion becomes unstable under a pH condition where the zeta potential becomes zero between 5 and 8, causing membrane fusion and releasing the inclusion. A known method can be used as a method of 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 since the velocity is proportional to the charge of the particles, the migration velocity of the particles is The zeta potential can be measured by measurement. Electrophoretic light scattering measurement is also called laser Doppler method, and the zeta potential is determined by observing the scattered light from the migrating particles.

  The pH-responsive liposome of the present embodiment, when dispersed in an aqueous medium, has a positive zeta potential in an acidic pH environment and has a negative zeta potential in a basic pH environment, which has not been conventionally used. It can show pH response behavior. 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 applications such as methods of introducing these substances into cells will be expanded.

  The following examples demonstrate and describe one aspect and aspect of the present invention for further illustration and should not be construed as limiting the scope of the present invention.

Example 1
10.0 g of sorbitol was added to 10.0 g of propane-1,2-diol, and the mixture was dissolved by heating at 80 ° C. to 85 ° C. with a general-purpose stirrer 350 rpm for homogenization. 0.41 g of hydrogenated lecithin containing phosphatidyl choline, 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 under heating and stirring, and the mixture was similarly stirred and dissolved.

  To the mixture solution prepared above, 100 g of purified water preheated to 80 ° C. to 85 ° C. was added and mixed while stirring, and stirred with a general-purpose stirrer 350 rpm for 1 to 2 hours. The heating was stopped, the reaction solution was rapidly cooled while stirring, cooled to about room temperature, and the formed liposome was collected by filtration.

Example 2
10.0 g of glycerin was added to 10.0 g of propane-1,2-diol, and the mixture was heated and stirred at 80 ° C. to 85 ° C. with a general-purpose stirrer 350 rpm for homogenization. 0.82 g of hydrogenated lecithin containing phosphatidyl choline, 0.18 g of cholesterol, 0.1 g of stearic acid, and 0.2 g of lauryl dimethylaminoacetic acid betaine were added to this solution under heating and stirring, and the mixture was similarly stirred and dissolved.

  100 g of purified water preheated to 80 ° C. to 85 ° C. was added to the mixture solution prepared above and mixed while stirring, and stirred at 8,000 rpm with a homomixer for 1 to 2 hours. The heating was stopped, the reaction solution was rapidly cooled while stirring, cooled to about room temperature, and the formed liposome was 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 heated and stirred at 80 ° C. to 85 ° C. with a general-purpose stirrer 500 rpm to homogenize. 0.41 g of hydrogenated lecithin containing phosphatidyl choline, 0.09 g of cholesterol, and 0.1 g of lauryl dimethylaminoacetic acid betaine were added to this solution under heating and stirring, and stirred and dissolved in the same manner. 100 g of purified water preheated to 80 ° C. to 85 ° C. was added to the mixture solution prepared above, mixed while 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 dissolved by heating at 80 ° C. to 85 ° C. with a general-purpose stirrer 600 rpm for homogenization. 0.41 g of hydrogenated lecithin containing phosphatidyl choline, 0.09 g of cholesterol, and 0.2 g of lauryl dimethylaminoacetic acid betaine were added to this solution under heating and stirring, and stirred and dissolved in the same manner. 100 g of purified water preheated to 80 ° C. to 85 ° C. was added to the mixture solution prepared above, mixed while 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 heated and stirred at 80 ° C. to 85 ° C. with a general-purpose stirrer 600 rpm for homogenization. 0.41 g of hydrogenated lecithin containing phosphatidyl choline, 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 under heating and stirring, and the mixture was similarly stirred and dissolved. 100 g of purified water preheated to 80 ° C. to 85 ° C. was added to the mixture solution prepared above, mixed while 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 dissolved by heating at 80 ° C. to 85 ° C. with a general-purpose stirrer 600 rpm for homogenization. 0.41 g of hydrogenated lecithin containing phosphatidyl choline, 0.09 g of cholesterol, 0.15 g of palmitic acid, and 0.3 g of lauryl dimethylaminoacetic acid betaine were added to this solution under heating and stirring, and the mixture was similarly stirred and dissolved. 100 g of purified water preheated to 80 ° C. to 85 ° C. was added to the mixture solution prepared above, mixed while 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 heated and stirred at 80 ° C. to 85 ° C. with a general-purpose stirrer 350 rpm to be homogenized. 0.41 g of hydrogenated lecithin containing phosphatidyl choline, 0.09 g of cholesterol, and 0.05 g of palmitic acid were added to this solution under heating and stirring, and the mixture was similarly stirred and dissolved. To the mixture solution prepared above, 100 g of purified water preheated to 80 ° C. to 85 ° C. was added and mixed while stirring, and after 1 to 2 hours, it was subjected to an extruder treatment.

Comparative Example 2
10.0 g of glycerin was added, and the mixture was heated, stirred and dissolved at 80 ° C. to 85 ° C. with a general-purpose stirrer 350 rpm, and homogenized. 0.82 g of hydrogenated lecithin containing phosphatidyl choline, 0.18 g of cholesterol, 0.05 g of palmitic acid, and 0.1 g of lauryl dimethylaminoacetic acid betaine were added to this solution under heating and stirring, and the mixture was similarly stirred and dissolved. To the mixture solution prepared above, 100 g of purified water preheated to 80 ° C. to 85 ° C. was added and mixed while stirring, and 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 filtered.

Comparative Example 3
10.0 g of glycerin was added, and the mixture was heated, stirred and dissolved at 80 ° C. to 85 ° C. with a general-purpose stirrer 350 rpm, and homogenized. 0.82 g of hydrogenated lecithin containing phosphatidyl choline, 0.18 g of cholesterol, and 0.05 g of palmitic acid were added to this solution under heating and stirring, and the mixture was similarly stirred and dissolved. 100 g of purified water preheated to 80 ° C. to 85 ° C. was added to the mixture solution prepared above and mixed while stirring, and 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, the reprecipitation of fats and oils was large (considered to be insufficient in the emulsifying ability), and the liposome could not be formed.

[3] Measurement of Zeta Potential The aqueous dispersions of the liposomes prepared in the above Examples and Comparative Examples are adjusted to various pHs using an aqueous solution of potassium hydroxide and an aqueous solution of phosphoric acid, and Malvern under constant temperature conditions of 26 ° C. The zeta potential was measured using Zetasizer Nano Series ZSP manufactured by Co. Ltd. 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 the aqueous medium of pH 5 or less, and increase with pH value between pH 5.4 and pH 7.6. It was found that the zeta potential approaches zero and shifts from positive to negative. 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 showed a slightly positive zeta potential at pH 4 or less, but it is extremely unstable in this region because the absolute value of the zeta potential is small. it is conceivable that. In addition, the liposome prepared in Comparative Example 2 showed a negative zeta potential at all pH. Thus, it was found that the liposomes prepared in Examples 1 to 6 exhibit pH sensitivity, and can stably exist because the absolute value of the zeta potential is large at pH 5 or less.

Claims (10)

Preparing a mixture solution in which a diol, a trivalent or higher polyol, and a liposome membrane component are mixed under heating conditions, and the liposome membrane component is dissolved in the mixture of the diol and the polyol;
Mixing the mixture solution with a preheated aqueous medium and homogenizing them;
Quenching the homogenized aqueous medium to form liposomes;
Recovering the liposome.
The liposome membrane component contains an anionic substance and a zwitterionic substance in a predetermined ratio,
When the liposome is dispersed in an aqueous medium at each of the following pH conditions, the zeta potential of the liposome is positive at pH 5 or less, negative at pH 8 or more, and pH 5 to 8 The method for producing pH-sensitive liposomes, characterized in that the transition from positive to negative with the increase of   The mixture solution is prepared by adding and stirring the liposome membrane component to a solution obtained by mixing and homogenizing the diol and the trivalent or higher polyol in advance under heating conditions. The manufacturing method of the pH sensitive liposome as described.   The said anionic substance is contained 0 to 20 mass% with respect to the whole liposome membrane component, and the said amphoteric substance is contained 5 to 20 mass% with respect to the whole liposome membrane component. The manufacturing method of the pH sensitive liposome as described in 2.   The method for producing a pH sensitive liposome according to any one of claims 1 to 3, wherein the anionic substance is contained in a ratio of 2.5 to 15% by mass with respect to the whole liposome membrane component.   The method for producing a pH-sensitive liposome according to claim 4, wherein a content ratio of the anionic substance to the zwitterionic substance is 1: 1 to 1: 3.   The method for producing a pH-sensitive liposome according to any one of claims 1 to 5, wherein the liposome membrane component further comprises phospholipid and cholesterol.   The method for producing a pH sensitive liposome according to any one of claims 1 to 6, wherein the anionic substance is a saturated fatty acid which is solid at normal temperature.   N-alkyl-N, N-dimethyl amino acid betaine containing lauryl betaine (lauryl dimethylaminoacetic acid betaine); fatty acid amide alkyl-N, N-dimethyl acid comprising cocamidopropyl betaine, lauramidopropyl betaine Amino acid betaine; imidazoline type betaine including sodium cocoamphoacetate, sodium lauroamphoacetate; alkyl sulfobetaine including alkyl dimethyl taurine; sulfuric acid type betaine including alkyl dimethylaminoethanol sulfate ester; and phosphoric acid type including alkyl dimethylaminoethanol phosphate ester The method for producing a pH sensitive liposome according to any one of claims 1 to 7, which is at least one selected from the group consisting of betaines.   The method for producing a pH sensitive liposome according to any one of claims 1 to 8, wherein the diol is propane-1,2-diol or 1,3-butylene glycol, and the polyol is sorbitol or glycerin.   The method for producing a pH-sensitive liposome according to claim 7, wherein the saturated fatty acid which is solid at normal temperature is palmitic acid or stearic acid.

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