WO2019045097A1 - Liposomes sensibles au ph et leur procédé de production - Google Patents

Liposomes sensibles au ph et leur procédé de production Download PDF

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WO2019045097A1
WO2019045097A1 PCT/JP2018/032611 JP2018032611W WO2019045097A1 WO 2019045097 A1 WO2019045097 A1 WO 2019045097A1 JP 2018032611 W JP2018032611 W JP 2018032611W WO 2019045097 A1 WO2019045097 A1 WO 2019045097A1
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liposome
sensitive
betaine
membrane component
substance
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那波　慶彦
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Ichimaru Pharcos Co Ltd
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Ichimaru Pharcos Co Ltd
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Priority claimed from JP2017169356A external-priority patent/JP6399530B1/ja
Priority claimed from JP2017169359A external-priority patent/JP6462073B1/ja
Application filed by Ichimaru Pharcos Co Ltd filed Critical Ichimaru Pharcos Co Ltd
Priority to CN201880059659.0A priority Critical patent/CN111132753B/zh
Priority to KR1020207008501A priority patent/KR20200050982A/ko
Publication of WO2019045097A1 publication Critical patent/WO2019045097A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/14—Liposomes; Vesicles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/55—Phosphorus compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/63—Steroids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/04—Making microcapsules or microballoons by physical processes, e.g. drying, spraying

Definitions

  • the present invention relates to a pH sensitive liposome and a method for producing 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 producing the same About.
  • 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.
  • pH-sensitive liposomes have been developed which are stable under physiological conditions and then destabilized under acidic conditions after being translocated into the cytoplasm.
  • phosphatidyl ethanolamine type phospholipids transfer aggregate structure in response to pH and release inclusions in an acidic environment (pH 5 or less) (For example, refer to nonpatent literature 1).
  • a liposome containing as a constituent lipid a cationic amphiphilic molecule and at least one of an anionic amphiphilic molecule and a zwitterionic amphiphilic molecule is dispersed in an aqueous medium, under acidic pH environment
  • the liposome has a positive zeta potential, and in a basic pH environment, the liposome has a negative zeta potential, and the zeta potential increases as the pH of the dispersion increases. It has been reported that the change from plus to minus releases the target substance held (see, for example, Patent Document 1).
  • Liposomes can be prepared by various methods such as ultrasonic method, extrusion method, French press method, homogenization method, ethanol injection method, etc.
  • a lipid component such as phospholipid dissolved in a solvent
  • 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 2).
  • Patent No. 5588619 gazette Japanese Patent Application Publication No. 2006-517594 JP, 2017-66059, A
  • pH-sensitive liposomes are generally unstable, it is necessary to set the pH value at which the zeta potential is zero in an aqueous medium to a desired range, that is, the fusogenicity of the liposome is acidic. There is a problem that it is difficult to set arbitrarily within a wide range up to basicity, and it can not meet the potential applications of pH sensitive liposomes.
  • pH sensitive liposomes are unstable compared to normal 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 by adjusting the content ratio of specific components constituting the liposome, the zeta potential of the liposome can be obtained under an arbitrary pH condition within a predetermined range.
  • An object of the present invention is to provide a pH sensitive liposome which can be transferred from positive to negative.
  • Another object of the present invention 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 pH sensitive liposome comprises phospholipid, steroids, anionic substance, and zwitterionic substance as a liposome membrane component, relative to the whole liposome membrane component. It is characterized in that it contains 2.5 to 15% by mass of an anionic substance and 5 to 20% by mass of an amphoteric substance. And when it disperses in the aqueous medium of 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 increases in pH value between pH 5 and 8. Together with the move from plus to minus.
  • the method for producing a pH-sensitive liposome 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 at least a zwitterionic substance, 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, and negative at pH 8 or more, Then, it was made to shift from positive to negative with increasing pH value between pH 5-8.
  • a pH sensitive liposome which can shift the zeta potential of the liposome from positive to negative under a wide range of arbitrary pH conditions can be provided.
  • stable pH-sensitive liposomes can be 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.
  • composition and composition of pH sensitive liposomes Method for producing pH sensitive liposome pH sensitivity and its expression mechanism Shape, application or usage
  • a diol capable of dissolving the liposome membrane component is preferably a 1,2-alkanediol or a 1,3-alkanediol.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • a physiologically active substance as a liposome inclusion substance may be included.
  • Liposome membrane component 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.
  • 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.
  • HSPC hydrogenated soybean phosphatidyl choline
  • hydrogenated phospholipids such as HSPC, sphingomyelin and the like are preferable.
  • the amount of phospholipid is usually 20% by mass or more, preferably 40% by mass or more, based on the whole liposome membrane component.
  • the amount of other liposome membrane components is usually 80% by mass or less, preferably 60% by mass or less.
  • Steroids include all steroids having perhydrocyclopentanophenanthrene, such as sterols, bile acids, provitamin D, steroid hormones and the like. Among them, sterols are preferably used. Sterols include, for example, sterols acting as lipid membrane stabilizers such as cholesterol, dihydrocholesterol, cholesterol ester, phytosterol, sitosterol, stigmasterol, campesterol, cholestanol, lanosterol and the like.
  • sterol derivatives such as 1-O-sterol glucoside, 1-O-sterol maltoside or 1-O-sterol galactoside are effective for stabilizing the liposome (Japanese Patent Laid-Open No. 5-245357).
  • cholesterol is particularly preferred.
  • the content of the steroids is not particularly limited, but is preferably 0.01% by mass or more, more preferably 0.05% by mass or more, and more preferably 0.1% by mass or more based on the entire components of the liposome. Is more preferred. 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 for molecular assemblies. The steroids may be used singly or in combination of two or more.
  • 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.
  • normal temperature means 10 ° C. to 30 ° C.
  • the content ratio of the above-mentioned anionic substance to the total amount of 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, 20 mass% is good and, as for the upper limit of a content rate, 15 mass% is preferable.
  • 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.
  • N-alkyl-N, N-dimethyl amino acid betaines such as lauryl betaine (lauryl dimethylaminoacetic acid betaine); 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; alkylsulfobetaines 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.
  • the pH sensitive liposome of the present invention can contain other additives as needed.
  • 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 1,000 to 10,000.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 to 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.
  • 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.
  • step S03 the mixture solution prepared in step S02 is mixed with an aqueous medium preheated to 80 ° C. to 85 ° C.
  • 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.
  • 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.
  • 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.
  • the amount is 2 to 6 times, preferably 3 to 5 times, more preferably about 4 times the volume of the mixture solution prepared in step S02.
  • the aqueous medium in which the liposome membrane component is dissolved is rapidly cooled to around room temperature at 80 ° C. to 85 ° C. to form a liposome.
  • the cooling method is not particularly limited.
  • 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.
  • the cooling temperature is preferably 62 ° C. or less. Further, it may be cooled to around room temperature.
  • the cooling rate is preferably 0.5 ° C./min or more, and more preferably 1 ° C./min or more.
  • the liposomes present in the aqueous medium can be recovered by any method such as filtration or decantation.
  • 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.
  • 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.
  • 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.
  • the pH sensitive liposome of the present embodiment when dispersed in various aqueous media having different pH conditions, has a positive zeta potential at pH 5 or lower, negative at pH 8 or higher, and pH 5 to 8 It has the property of shifting from positive to negative as the pH value increases.
  • 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.
  • the zeta potential 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.
  • 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).
  • 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 acidic condition of 5 or less. It is considered 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.
  • 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-sensitive liposome of the present embodiment When dispersed in an aqueous medium, the pH-sensitive liposome of the present embodiment has a positive zeta potential in an acidic pH environment and a negative pH such as having a negative zeta potential in a basic pH environment. It can show response behavior. In recent years, studies have been conducted to introduce negatively charged substances such as genes and nucleic acid derivatives into cells.
  • the pH-sensitive liposome of the present embodiment has a positive surface charge under acidic conditions of pH 5 or less, and thus can adsorb these substances, and releases these substances under weak to neutral pH 5 to 8 conditions. It can be used to introduce it into cells.
  • the shape is also not particularly limited, and may be a multilamellar liposome or unilamellar liposome having a particle diameter of 100 nm to 10 ⁇ m. It is also possible to adjust the particle size of the pH-sensitive liposome of the present embodiment as appropriate depending on its use. For example, for the purpose of in vivo administration, it is preferable to adjust the particle size to 200 nm or less.
  • the specific particle diameter adjustment method can adjust the particle diameter by passing through a filter with a small pore diameter using an extruder. It is said that unilamellar liposomes having a small particle size of about 100 nm or less are uniform in size and thermodynamically stable, and have good skin permeability even when used as a cosmetic It is said.
  • 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.
  • Example 1 10.0 g of sorbitol was added to 10.0 g of propane-1,2-diol and dissolved by heating at 80 ° C. to 85 ° C. using 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.
  • 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.
  • 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 for homogenization. 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 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 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 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 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 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 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 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 heated and stirred 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, 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 dissolved by heating 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, 0.05 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. 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 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 dissolved by heating 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, 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 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.
  • 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.
  • the liposome prepared in Comparative Example 2 showed a negative zeta potential at all pH.
  • the liposomes prepared in Examples 1 to 6 exhibit pH sensitivity and can be stably present because the absolute value of the zeta potential is large at pH 5 or less.

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Abstract

Ce procédé de production de liposomes sensibles au pH comprend : une étape dans laquelle un diol, un polyol ayant une valence de 3 ou plus et un composant constituant une membrane de liposome sont mélangés les uns avec les autres dans des conditions de chauffage, préparant ainsi une solution de mélange dans laquelle le composant constituant une membrane de liposome est dissous dans un mélange du diol et du polyol; une étape dans laquelle cette solution de mélange et un milieu aqueux, qui a été chauffé à l'avance, sont mélangés l'un à l'autre et homogénéisés; une étape dans laquelle le milieu aqueux homogénéisé est rapidement refroidi, produisant ainsi des liposomes; et une étape dans laquelle les liposomes produits sont collectés. Le procédé selon la présente invention stabilise des liposomes sensibles au pH, qui ont été instables et donc difficiles à préparer dans le passé, et est ainsi apte à préparer des liposomes sensibles au pH par un processus simple.
PCT/JP2018/032611 2017-09-04 2018-09-03 Liposomes sensibles au ph et leur procédé de production Ceased WO2019045097A1 (fr)

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