WO2011062255A1 - Process for production of liposome through two-stage emulsification using mixed organic solvent as oily phase - Google Patents

Process for production of liposome through two-stage emulsification using mixed organic solvent as oily phase Download PDF

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WO2011062255A1
WO2011062255A1 PCT/JP2010/070657 JP2010070657W WO2011062255A1 WO 2011062255 A1 WO2011062255 A1 WO 2011062255A1 JP 2010070657 W JP2010070657 W JP 2010070657W WO 2011062255 A1 WO2011062255 A1 WO 2011062255A1
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liposome
emulsion
emulsification
organic solvent
solvent
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PCT/JP2010/070657
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French (fr)
Japanese (ja)
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武志 和田
武寿 磯田
康之 元杭
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コニカミノルタホールディングス株式会社
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    • 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
    • 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
    • A61K8/04—Dispersions; Emulsions
    • A61K8/06—Emulsions
    • A61K8/066—Multiple emulsions, e.g. water-in-oil-in-water
    • 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
    • A61K8/11—Encapsulated compositions
    • 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
    • A61K8/31—Hydrocarbons
    • 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
    • A61K8/31—Hydrocarbons
    • A61K8/315—Halogenated hydrocarbons
    • 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
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • 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
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/34—Alcohols
    • 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
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/35—Ketones, e.g. benzophenone
    • 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
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/37—Esters of carboxylic acids
    • 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
    • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
    • 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—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin

Definitions

  • the present invention relates to a method for producing liposomes used in the fields of pharmaceuticals, cosmetics, foods and the like including a two-stage emulsification step and a solvent removal step.
  • Liposomes are closed vesicles composed of a monolayer or a multi-layer lipid bilayer, and can retain water-soluble and hydrophobic drugs in the inner aqueous phase and the lipid bilayer, respectively.
  • Lipid lipid bilayer membranes are similar to biological membranes and are therefore highly safe in vivo.
  • pharmaceuticals for DDS Drug Delivery System
  • DDS Drug Delivery System
  • a W / O / W emulsion is prepared by a two-stage emulsification step, and then the oil phase (O) is removed to form liposomes (microencapsulation method or two-step method).
  • an emulsification method (Referred to as an emulsification method)
  • the oil phase (O) has conventionally been constituted by a single organic solvent.
  • Patent Document 1 (Example) describes an embodiment of a two-stage emulsification method using a microchannel using only hexane as an oil phase.
  • Patent Document 2 discloses a two-step process using “at least one organic solvent”, for example, “ether, hydrocarbon, halogenated hydrocarbon, halogenated ether, ester, chloroform, and combinations thereof” as the oil phase.
  • An emulsification method is described (claims 2, 30, paragraph [0054]).
  • the manufacturing method described in Patent Document 2 has “a plurality of non-concentric chambers having a film distributed as a continuous network structure throughout the whole”, and the weighted average diameter is usually 0.5 to It is intended for “multivesicular liposomes” (MVL) in the micrometer range of about 25 ⁇ m (claim 2, [0019] paragraph), and “liposome structure having a single aqueous chamber”.
  • MDL multivesicular liposomes
  • liposome production methods including a two-stage emulsification step and a solvent removal step
  • various means have been used (for example, in Patent Document 1, a step of freezing the aqueous phase of the W / O emulsion is performed), but there is room for improvement in producing better liposomes. It was left.
  • the present inventors have made a mixed organic solvent containing at least two kinds of organic solvents, such as hexane and dichloromethane, for example, as an oil phase (O) of a W1 / O emulsion and a W1 / O / W2 emulsion. It has been found that the dispersion stability of these emulsions can be dramatically improved compared to the case where only an organic solvent of a kind is used as an oil phase, and the encapsulation rate of liposome drugs and the formation efficiency of single cell liposomes can be improved. The present invention has been completed.
  • organic solvents such as hexane and dichloromethane
  • the method for producing liposomes according to the present invention comprises a primary emulsification step of emulsifying an inner aqueous phase (W1) and an oil phase (O) using a mixed lipid component (F1) for liposomes to prepare a W1 / O emulsion; A secondary emulsification step of emulsifying the W1 / O emulsion and the external aqueous phase (W2) using the mixed lipid component (F2) for liposomes to prepare a W1 / O / W2 emulsion, and an oil phase (from the W1 / O / W2 emulsion) And a solvent removal step of removing the organic solvent of O) to form liposomes, wherein the oil phase (O) of the W1 / O / W2 emulsion used in the solvent removal step is at least two types It is a mixed organic solvent containing the organic solvent.
  • the production method of the present invention is preferably for producing single cell liposomes.
  • “monocystic liposome” (ULV, synonymous with mononuclear liposome) refers to a liposome structure having a single inner aqueous phase, and usually has a volume average particle size in the range of about 20 to 500 nm.
  • multivesicular liposome refers to a liposome structure comprising a lipid membrane surrounding a plurality of non-concentric inner aqueous phases, and also referred to as “multilamellar liposome” (MLV ) refers to a liposome structure having a plurality of concentric membranes, such as “onion skin”, with a shell-like concentric aqueous compartment in between.
  • MUV multilamellar liposome
  • the characteristics of multivesicular liposomes and multilamellar liposomes are that the volume average particle diameter is in the micrometer range, usually 0.5 to 25 ⁇ m.
  • Examples of the mixed organic solvent include chloroform, cyclohexane, dichloromethane, hexane, t-butyl methyl ether, ethyl acetate, diethyl ether, ethyl formate, isopropyl acetate, methyl acetate, methyl ethyl ketone, pentane, acetonitrile, methanol, acetone, ethanol, Preference is given to at least two organic solvents selected from the group consisting of 2-propanol.
  • the mixed organic solvent contains a hydrocarbon organic solvent, for example, a solvent group consisting of hydrocarbons (solvent A group) and ethers, halogenated hydrocarbons, halogenated ethers, esters, alcohols. It is preferable to contain at least two types of organic solvents selected from at least one of each of a solvent group (solvent B group) consisting of ketone and acetonitrile.
  • the solvent group A preferably comprises, for example, pentane, hexane and cyclohexane
  • the solvent group B includes, for example, diethyl ether, t-butyl methyl ether, chloroform, dichloromethane, ethyl formate, methyl acetate, isopropyl acetate, methanol, ethanol More preferably, it consists of 2-propanol, methyl ethyl ketone and acetonitrile.
  • the volume ratio of the solvent A contained in the mixed organic solvent is preferably 50 to 99%.
  • the primary emulsification step is a step of performing an emulsification process using a mixed organic solvent prepared in advance, and / or during or after the W1 / O emulsion is being prepared.
  • a step of adding an organic solvent to the oil phase may be included.
  • a membrane emulsification method or a microchannel emulsification method is preferable
  • a membrane emulsification method, a microchannel emulsification method or a stirring emulsification method is preferable.
  • these membrane emulsification methods for example, a membrane emulsification method using an SPG membrane is preferable.
  • the primary emulsification step may be performed after adding a substance to be encapsulated in the liposome to the inner aqueous phase (W1) or the oil phase (O), and should be encapsulated in the liposome after the primary emulsification step. Substances may be added to the oil phase (O).
  • Such a method for producing a liposome of the present invention can also be used as a method for producing a liposome dispersion or a dry powder thereof.
  • the dispersion stability of those emulsions is increased, so that contact and aggregation between particles in the solvent removal step is reduced, An increase in particle diameter (formation of aggregates such as multivesicular liposomes and aggregation / coalescence of W1 / O emulsion in W1 / O / W2 emulsion) can be suppressed.
  • the membrane emulsification method or the microchannel emulsification method is used in the secondary emulsification step, the problem of clogging of the emulsified base material can be solved. As a result, it is possible to efficiently produce liposomes suitable for pharmaceutical use and the like that contain a large amount of fine single-vesicle liposomes having a high drug encapsulation rate and a narrow particle size distribution.
  • FIG. 1 is an observation photograph of the W1 / O emulsion prepared in Sample 1 with an optical microscope.
  • FIG. 2 is an observation photograph of the W1 / O emulsion prepared in Comparative Sample 1 with an optical microscope.
  • FIG. 3 is an observation photograph of the W1 / O emulsion prepared in Comparative Sample 2 with an optical microscope.
  • FIG. 4 is an observation photograph of the W1 / O emulsion prepared in Sample 5 with an optical microscope.
  • FIG. 5 is an observation photograph of the W1 / O emulsion prepared in Sample 6 with an optical microscope.
  • FIG. 6 is an observation photograph of the W1 / O emulsion prepared in Sample 7 with an optical microscope.
  • FIG. 1 is an observation photograph of the W1 / O emulsion prepared in Sample 1 with an optical microscope.
  • FIG. 2 is an observation photograph of the W1 / O emulsion prepared in Comparative Sample 1 with an optical microscope.
  • FIG. 3 is an observation photograph of the W1 / O e
  • FIG. 7 is an observation photograph of the W1 / O emulsion prepared in Sample 10 with an optical microscope.
  • FIG. 8 is an observation photograph of the W1 / O / W2 emulsion prepared in Example 1 with an optical microscope.
  • FIG. 9 is an observation photograph of the liposome prepared in Example 1 with an optical microscope.
  • FIG. 10 is an observation photograph of the liposome prepared in Comparative Example 2 with an optical microscope.
  • FIG. 11 is an observation photograph of the liposome prepared in Example 4 with an optical microscope.
  • FIG. 12 is an observation photograph of the liposome prepared in Comparative Example 5 with an optical microscope.
  • FIG. 13 is an observation photograph of the liposome prepared in Example 22 with an optical microscope.
  • the oil phase (O) of the W1 / O / W2 emulsion is made to contain at least two kinds of organic solvents (mixed organic solvent).
  • the compounding composition of the mixed organic solvent (type and ratio of the organic solvent) can be adjusted as appropriate.
  • the organic solvent that can be blended in the mixed organic solvent preferably has a boiling point in the range of 5 to 95 ° C., and is usually chloroform, cyclohexane, 1,2-dichloroethane, which has been usually used alone in the conventional primary emulsification step.
  • Water-insoluble such as benzene, dichloromethane, 1,2-dimethoxyethane, hexane, 1,1,2-trichloroethene, t-butyl methyl ether, ethyl acetate, diethyl ether, ethyl formate, isopropyl acetate, methyl acetate, methyl ethyl ketone, pentane
  • water-soluble organic solvents such as acetonitrile, methanol, acetone, ethanol, 2-propanol, and other ethers, hydrocarbons, and halogenated hydrocarbons, which have not been commonly used as emulsifying solvents, are used.
  • Halogenated ethers and esters are preferable.
  • chloroform, cyclohexane, dichloromethane, hexane, t-butyl methyl ether, ethyl acetate, diethyl ether, ethyl formate, isopropyl acetate, methyl acetate, methyl ethyl ketone, pentane, acetonitrile, methanol, acetone, ethanol, 2-propanol and the like are preferable.
  • Three or more organic solvents may be combined, but from the viewpoint of ease of setting conditions, a combination of two organic solvents is preferable. If necessary, various functional components may be added to the mixed organic solvent.
  • At least two kinds of organic solvents to be blended in the mixed organic solvent can be selected in consideration of the solubility of the phospholipids contained in the mixed lipid components (F1) and (F2). is there.
  • the solvents listed above W1 / O emulsions using phospholipids with high solubility of phospholipids alone have good dispersibility, but coalescence progresses and the emulsion particle size becomes coarse. There is a problem that the water phase and the oil phase are completely separated or mixed uniformly.
  • a W1 / O emulsion using only a solvent having low solubility of phospholipids such as hexane can obtain an emulsion having a uniform particle size in which aggregation between emulsions is very strong and dispersion stability is poor. It can be difficult.
  • a solvent having low solubility in phospholipid a solvent having such a property is referred to as “solvent A” in the present invention
  • a solvent having high solubility in phospholipid in the present invention, the solvent A solvent having such properties is referred to as “solvent B”)
  • solvent A solvent having such properties is referred to as “solvent B”
  • Such a solvent A is preferably a hydrocarbon, and more preferably at least one selected from pentane, hexane, and cyclohexane.
  • the solvent B is preferably at least one selected from ethers, halogenated hydrocarbons, halogenated ethers, esters, alcohols, ketones, and acetonitrile. Diethyl ether, t-butyl methyl ether, chloroform, dichloromethane More preferably, at least one selected from ethyl formate, methyl acetate, isopropyl acetate, methanol, ethanol, 2-propanol, methyl ethyl ketone and acetonitrile.
  • the combination of organic solvents is such that the solubility of mixed lipid components (phospholipids, etc.), compatibility between organic solvents, setting of conditions for the solvent removal step, etc. are preferable, so that the polarity of the organic solvent (hydrophilic (Hydrophobicity), boiling point, density (specific gravity), and the like can have predetermined relationships.
  • the boiling point of the good solvent is not necessarily lower than the boiling point of the poor solvent.
  • the mixed organic solvent those having the above solvent A as a main component are preferable because they are excellent in effects such as improving the monodispersibility of the obtained nano-sized W1 / O emulsion. It is more preferable that the volume ratio of the solvent A (the total amount when the solvent A is a plurality of types) is 50 to 99%.
  • the volume ratio thereof hexane: dichloromethane, the total is 100
  • the volume ratio thereof is preferably 60:40 to 90:10.
  • the mixed organic solvent composed of hexane and ethyl acetate preferably has a volume ratio of 50:50 to 90:10
  • the mixed organic solvent composed of hexane and ethanol preferably has a volume ratio of 70:30 to 99: 1.
  • the oil phase (O) of the W1 / O / W2 emulsion to be used in the solvent removal step only needs to contain at least two kinds of organic solvents.
  • the method for preparing such a W1 / O / W2 emulsion is not particularly limited, and an appropriate method can be designed according to the emulsification method used in the primary emulsification step and the secondary emulsification step.
  • a mixed organic solvent (O ′′) containing at least two kinds of organic solvents is prepared in advance, a primary emulsification treatment is performed using the mixed organic solvent, and a W1 / O ′′ emulsion is prepared.
  • a primary emulsification treatment is performed using the mixed organic solvent
  • a W1 / O ′′ emulsion is prepared.
  • perform primary emulsification using one organic solvent (O ′) and add another kind of organic solvent during or after the W1 / O ′ emulsion is being prepared A W1 / O "emulsion in which the oil phase is a mixed organic solvent is prepared, and the W1 / O" emulsion thus obtained is subjected to a secondary emulsification step to prepare W1 / O "/ W2.
  • a solvent for example, dichloromethane
  • a solvent having a lower solubility for example, the coalescence process of the W1 / O emulsion can be stopped and the particle size of the emulsion can be controlled.
  • adding a solvent with higher solubility can reduce the aggregation of the W1 / O emulsion and improve the dispersibility.
  • the solvent removal step is performed by performing a predetermined treatment in the middle of the secondary emulsification step. It may be a technique in which W1 / O "/ W2 is prepared before being subjected to the above.
  • Aqueous solvent (W1) ⁇ (W2) As the aqueous solvents (W1) and (W2), known general solvents can be used.
  • the aqueous solvent (W1) used in the primary emulsification step constitutes the aqueous phase (inner aqueous phase) of the W1 / O emulsion
  • the aqueous solvent (W2) used in the secondary emulsification step is the outer aqueous phase of the W1 / O / W2 emulsion.
  • Examples of the aqueous solvent include pure water, other solvents mixed with water as necessary, salts and saccharides for adjusting osmotic pressure, buffers for adjusting pH, and other functional components (for example, dispersion stabilizers). ) Or the like is used.
  • the pH of the inner aqueous phase (W1) is usually in the range of 2 to 10, and can be adjusted to a preferable range according to the mixed lipid component.
  • the mixed lipid component (F1) mainly constitutes the inner membrane of the lipid bilayer membrane of the liposome, and the mixed lipid component (F2) mainly constitutes the outer membrane.
  • the mixed lipid components (F1) and (F2) may have the same composition or different compositions.
  • composition of these mixed lipid components is not particularly limited, and various mixed lipid components used for the production of liposomes can be used.
  • phospholipids lecithin derived from animals and plants; phosphatidylcholine) , Phosphatidylserine (DPPS), phosphatidylglycerol (DPPG), phosphatidylinositol, phosphatidic acid or their fatty acid esters, glycerophospholipids; sphingophospholipids; derivatives thereof, etc.) and sterols that contribute to the stabilization of lipid membranes ( Cholesterol, phytosterol, ergosterol, derivatives of these, etc.), and glycolipids, glycols, aliphatic amines, long-chain fatty acids (oleic acid, stearic acid, palmitic acid, etc.) and other various functions To grant Objects may be blended.
  • neutral phospholipids such as dipalmitoyl phosphatidylcholine (DPPC) and dioleyl phosphatidylcholine (DOPC) are commonly used as the phospholipid.
  • DPPC dipalmitoyl phosphatidylcholine
  • DOPC dioleyl phosphatidylcholine
  • F2 a lipid component necessary for imparting functionality as a DDS, such as PEGylated phospholipid.
  • the blending ratio of the mixed lipid component may be appropriately adjusted according to the application while taking into consideration properties such as the stability of the lipid membrane and the behavior of the liposome in vivo.
  • substances to be encapsulated in liposomes are not particularly limited, and are known in the fields of pharmaceuticals, cosmetics, foods, etc. depending on the use of liposomes. Various substances can be used.
  • water-soluble drugs suitable for the present invention are those in which 1 g or 1 mL or more of drug dissolves in 100 mL of water, preferably 10 g or 10 mL or more, more preferably 100 g or 100 mL or more. It is.
  • the volume average particle diameter, CV, and dispersibility of the liposome by the drugs are not so much seen.
  • water-soluble drugs for medical use include, for example, contrast agents (nonionic iodine compounds for X-ray contrast, complexes composed of gadolinium and chelating agents for MRI contrast), anticancer agents, and the like.
  • contrast agents nonionic iodine compounds for X-ray contrast, complexes composed of gadolinium and chelating agents for MRI contrast
  • anticancer agents and the like.
  • RNA vaccines as antigens
  • diphtheria Japanese encephalitis, polio, rubella, mumps, hepatitis and other viruses as antigens
  • DNA or RNA vaccines etc.
  • pharmacologically active substances dyes / fluorescent dyes, chelating agents, stabilizers, storage And pharmaceutical aids such as pharmaceuticals.
  • water-soluble drugs are dissolved or suspended in advance in the inner aqueous phase (W1) of the primary emulsification step, and at the end of the solvent removal step.
  • W1 inner aqueous phase of the primary emulsification step
  • fat-soluble drugs are dissolved or suspended in advance in the oil phase (O) in the primary emulsification step according to (i) above, and an aqueous dispersion of empty liposomes as in (ii) above. Can be encapsulated in liposomes (in a lipid bilayer).
  • the outer aqueous phase of the secondary emulsification step may be blended with a dispersion stabilizer that can further contribute to the improvement of the encapsulation rate of drugs and the efficient formation of single-cell liposomes, if necessary. .
  • a protein emulsifier such as sodium caseinate is one of conventionally used dispersion stabilizers.
  • a dispersion stabilizer that does not form a self-assembled molecular aggregate (typically micelle) or a self-assembled molecular aggregate that has a volume average particle size of 10 nm or less (hereinafter referred to as “specific dispersion”).
  • the "stabilizer” is preferred because it is excellent in the effect as a dispersion stabilizer and can be easily separated and removed from the liposome dispersion liquid as necessary.
  • Typical specific dispersion stabilizers include proteins, polysaccharides, ionic surfactants and nonionic surfactants.
  • Polysaccharides are distributed throughout the outer aqueous phase (W2) because the orientation to the interface between the primary emulsion (W1 / O) and the outer aqueous phase (W2) is relatively small, and W1 / O / W2 do not join together. By doing so, the liposome is stabilized.
  • Proteins and nonionic surfactants are relatively highly oriented to the interface of the W1 / O / W2 emulsion and are stabilized by surrounding the emulsion like a protective colloid.
  • the dispersion stabilizer can suppress the destabilization due to such coalescence, and contributes to the improvement of the formation efficiency of single cell liposomes and the encapsulation rate of the drug.
  • the latter specific dispersion stabilizer oriented at the interface of the W1 / O / W2 emulsion can easily dissolve individual liposomes as the liposomes are formed as the solvent is removed. Contributes to the improvement of the formation efficiency and drug encapsulation rate.
  • the protein examples include gelatin (a soluble protein obtained by denaturing collagen by heating), albumin and trypsin.
  • Gelatin usually has a molecular weight distribution of several thousand to several million, but preferably has a weight average molecular weight of 1,000 to 100,000, for example.
  • Gelatin commercially available for medical use or food use can be used.
  • Albumin includes egg albumin (molecular weight about 45,000), serum albumin (molecular weight about 66,000 ... bovine serum albumin), milk albumin (molecular weight about 14,000 ... ⁇ -lactalbumin), etc. A dry desugared egg white is preferred.
  • polysaccharide examples include dextran, starch, glycogen, agarose, pectin, chitosan, sodium carboxymethylcellulose, xanthan gum, locust bean gum, guar gum, maltotriose, amylose, pullulan, heparin, dextrin, and the like. Is preferably from 1,000 to 100,000.
  • Examples of the ionic surfactant include sodium cholate and sodium deoxycholate.
  • nonionic surfactant examples include alkyl glycosides such as octyl glucoside, polyalkylene oxide compounds such as “Tween 80” (Tokyo Chemical Industry Co., Ltd., polyoxyethylene sorbitan monooleate, molecular weight 1309.68) and “Pluronic”.
  • F-68 "(BASF, polyoxyethylene (160) polyoxypropylene (30) glycol, number average molecular weight 9600), polyethylene glycols having a weight average molecular weight of 1000 to 100,000, and the like.
  • Polyethylene glycol (PEG) products are "Unilube” (Nippon Oil Co., Ltd.), GL4-400NP, GL4-800NP (Nippon Oil Corporation), PEG200,000 (Wako Pure Chemical Industries), Macrogol (Sanyo Chemical Industries Co., Ltd.) Company).
  • the amount of the specific dispersion stabilizer added to the outer aqueous phase may be adjusted within an appropriate range depending on the type. Even a substance that forms a self-assembled molecular aggregate (with a volume average particle size exceeding 10 nm) at a certain concentration can be used as a specific dispersion stabilizer if the addition amount is adjusted within a range not reaching the concentration. it can. Depending on the type of the specific dispersion stabilizer, if the concentration is too high, the measurement by the particle size distribution meter may be hindered. Therefore, it is preferable to adjust the concentration within a low range that does not cause such a hindrance.
  • the volume average particle size of the self-assembled molecular aggregate or the aggregate of the specific dispersion stabilizer is 1/10 or less of the volume average particle size of the liposome. Is preferable, and 1/100 or less is more preferable.
  • the weight average molecular weight of the specific dispersion stabilizer is preferably in the range of 1,000 to 100,000.
  • the method for producing liposomes of the present invention has the following primary emulsification step, secondary emulsification step and solvent removal step, and can be appropriately combined with other steps as necessary.
  • a known device / equipment or other appropriate means may be used.
  • the liposome production method of the present invention is naturally a method for producing a liposome dispersion, and further includes a dry powdering step. By this, it can also be set as the manufacturing method of the dry powder of a liposome.
  • the production method of the present invention is preferably for producing single cell liposomes.
  • a method for producing single-cell liposomes it is not intended that multivesicular liposomes should be present in the liposomes obtained by the production method, but a production method designed mainly for the purpose of producing single-cell liposomes If it is.
  • multivesicular liposomes may be relatively easily formed, but the method of the present invention should be applied even in such a situation where multivesicular liposomes may be mixed. It is possible to obtain effects such as improvement in the encapsulation rate.
  • the primary emulsification step is a step of preparing a W1 / O emulsion by emulsifying the organic solvent (O), the aqueous solvent (W1), and the mixed lipid component (F1).
  • a method for preparing the W1 / O emulsion known methods such as an ultrasonic emulsification method, a stirring emulsification method, a membrane emulsification method, a microchannel emulsification method, and a method using a high-pressure homogenizer can be applied. From the viewpoint of the fine particle diameter, ultrasonic emulsification or emulsification using a high-pressure homogenizer is preferable. In addition, when encapsulating a drug that is unstable with respect to heat or the like, a microchannel emulsification method having a small energy required for emulsification, or a membrane emulsification method using an SPG film is preferable.
  • a premix membrane emulsification method is prepared such that a W1 / O emulsion having a smaller particle size is prepared by passing through a membrane having a small pore size. It may be used.
  • the primary emulsification step is (i) prepared in advance according to the method of the emulsification treatment. And / or (ii) a step of further adding an organic solvent to the oil phase during or after the W1 / O emulsion is being prepared. Good.
  • the average particle size of the W1 / O emulsion, the ratio of the mixed lipid component (F1) added to the organic solvent (O), the volume ratio of the organic solvent (O) and the aqueous solvent (W1), and other operating conditions can be appropriately adjusted according to the emulsification method to be employed, taking into consideration the conditions of the subsequent secondary emulsification step and the aspect of the liposome to be finally prepared.
  • the ratio of the mixed lipid component (F1) is 1 to 50% by mass with respect to the organic solvent (O)
  • the volume ratio of the organic solvent (O) and the aqueous solvent (W1) is 100: 1 to 1: 2. is there.
  • a substance (water-soluble or fat-soluble drug) to be encapsulated in the liposome is added to the inner aqueous phase (W1) or the oil phase (O) and dissolved or dissolved. You may make it suspend. Moreover, when a chemical
  • drugs are added thereto, or liposomes once lyophilized and powdered are redispersed in an aqueous solvent. Even when drugs are added, the drugs can be encapsulated in liposomes.
  • the secondary emulsification step emulsifies the W1 / O emulsion and the external aqueous phase (W2) prepared in the primary emulsification step using the mixed lipid component (F2) for liposome, and W1 / O / It is a step of preparing a W2 emulsion.
  • a membrane emulsification method As a method for preparing the W1 / O / W2 emulsion in the secondary emulsification step, a membrane emulsification method, a microchannel emulsification method, a stirring emulsification method, a droplet method, a contact method, and the like are known. Method, microchannel emulsification method and stirring emulsification method are preferred.
  • a microchannel emulsification method and a membrane emulsification method using an SPG membrane are suitable. Since these emulsification methods do not require a large amount of energy for the emulsification process, it is possible to suppress the collapse of the droplets during the emulsification operation and the leakage of the encapsulated material from the droplets. Since it is discharged and carried without staying, contact, aggregation and coalescence of droplets can be reduced.
  • a W1 / O / W2 emulsion having a large particle size is prepared in advance, and then a W1 / O / W2 emulsion having a smaller particle size is prepared by passing through a membrane having a small pore size.
  • a mixed film emulsification method may be used.
  • the premix membrane emulsification method is preferable because it requires a small amount of energy, requires a large amount of treatment, and can speed up the preparation of liposomes.
  • the mixing mode (addition order, etc.) of the aqueous solvent (W2), W1 / O emulsion, mixed lipid component (F2), and dispersion stabilizer used as necessary is not particularly limited. Just choose.
  • F2 is mainly composed of a water-soluble lipid
  • such F2 (and a dispersion stabilizer as required) can be added to W2 in advance, and a W1 / O emulsion can be added thereto for emulsification.
  • the method of adding F2 after preparing a W1 / O / W2 emulsion or after the below-mentioned solvent removal process is also possible.
  • F2 is mainly composed of fat-soluble lipids
  • such F2 is added to the oil phase of the W1 / O emulsion in advance, and it is added to W2 to which a dispersion stabilizer is added as necessary.
  • An emulsification treatment can be performed.
  • the mixed lipid components (F1) and (F2) may have the same composition, surplus that could not be fully oriented at the W / O interface as F1 among the mixed lipid components added during the primary emulsification step.
  • the minute can be F2 to be oriented at the O / W interface of the secondary emulsification step.
  • the volume average particle diameter of the W1 / O / W2 emulsion, the ratio of the mixed lipid component (F2) added to the organic solvent (O) of the aqueous solvent (W2) to the W1 / O emulsion, W1 / O emulsion The volume ratio of the aqueous solvent (W2) and other operating conditions can be appropriately adjusted in consideration of the application of the liposome to be finally prepared.
  • an emulsification base material (microchannel substrate, SPG film, etc.) used in those methods ) Is preferably subjected to a surface treatment with a hydrophilic drug.
  • hydrophilic agent examples include a silica precursor monomer and a silane coupling agent having a hydrophilic group on the surface (typically, the hydrophilic group includes a polyol structure, a polyether structure, a polyamine structure, a tertiary amine. Having at least one of a structure and a quaternary ammonium structure).
  • the contact angle of the emulsified substrate with water in the air is preferably 0 to 50 °, more preferably 0 to 42 °, and still more preferably 0 to 35 °. can do.
  • a system in which an oil phase containing the W / O emulsion and an external aqueous phase are separated by sandwiching the emulsifying base material Is used.
  • the W / O emulsion enters the holes formed in the emulsified base material by the pressure difference provided between the oil phase and the external water phase, and permeates the emulsified base material by passing through the holes.
  • the W / O emulsion that has reached the outlet of the hole and is in contact with the outer aqueous phase then forms droplets that grow from the outlet of the holes toward the outer aqueous phase.
  • the droplet grows, the droplet gradually narrows in the vicinity of the outlet of the hole, and eventually the droplet is separated from the emulsified base material and dispersed in the outer water layer.
  • the phenomenon that the droplet made of the W / O emulsion is constricted and separated from the emulsified substrate is considered to be caused by the interaction such as the surface tension acting between the droplet, the outer water phase, and the emulsified substrate surface.
  • a layer composed of a W / O emulsion may be generated on the surface of the emulsified base material, and droplets may not be released to the outer aqueous phase.
  • the surface of the emulsified substrate in contact with the outer aqueous phase at least the surface of the emulsified substrate in contact with the outer aqueous phase, if sufficient hydrophilicity is imparted to the surface of the emulsified substrate at the outlet of the hole and its peripheral part.
  • the droplets made of the W / O emulsion are easily separated from the emulsified base material without expanding along the surface of the emulsified base material.
  • the hydrophilicity inside the pores in the emulsified substrate is also high, the interface between the outer aqueous phase and the droplets made of the W / O emulsion will easily reach the inside of the pores beyond the outlet of the pores. It is considered that the droplets composed of the O emulsion are easily constricted, and as a result, the droplets composed of the W / O emulsion are further easily separated from the emulsified substrate.
  • the solvent removal step comprises removing the organic solvent of the oil phase (O) from the W1 / O / W2 emulsion prepared by the secondary emulsification step, and consisting of mixed lipid components (F1) and (F2). This is a step of forming a liposome having a lipid bilayer. As the removal of the organic solvent proceeds, the hydration of the lipids constituting the liposome progresses, and the multivesicular liposomes are dissolved and dispersed into the single-cell liposome state, or the single cells from a position close to the interface of the W1 / O / W2 emulsion. It is considered that the liposomes are torn and formed. In the present invention, the fact that the oil phase contains at least two kinds of organic solvents is considered to have a favorable effect when liposomes are formed by such a mechanism.
  • Examples of the solvent removal method include a method of evaporating with an evaporator and a method of drying in liquid.
  • the in-liquid drying method is a method in which the organic solvent (O) contained in the W1 / O / W2 emulsion is evaporated and removed by collecting the W1 / O / W2 emulsion, transferring it to an open container and allowing it to stand or stir. is there.
  • the temperature condition and the reduced pressure condition may be appropriately adjusted according to the type of the organic solvent to be used and the like within a range in which water does not volatilize in accordance with a conventional method.
  • the temperature condition is preferably in the range of 0 to 60 ° C., more preferably 0 to 25 ° C.
  • the decompression condition is preferably set within the range of the saturated vapor pressure of the solvent to atmospheric pressure, and more preferably within the range of + 1% to 10% of the saturated vapor pressure of the solvent.
  • liposomes can be prepared even under conditions where the organic solvent bumps.
  • the oil phase (O) contains at least two kinds of organic solvents.
  • the organic solvent having a low saturated vapor pressure can be obtained under conditions (lower temperature and / or smaller reduced pressure) suitable for the organic solvent having a higher saturated vapor pressure. It is preferable to change the conditions step by step to the conditions (higher temperature and / or higher pressure reduction) adapted to the above, and finally remove all kinds of organic solvents.
  • Liposomes obtained by the two-stage emulsification method may contain a certain percentage of W1 / O / W2 emulsion-derived multivesicular liposomes.
  • stirring or decompression preferably combining them, It is more effective to do. It is important to perform decompression and / or stirring longer than the time when most of the solvent is removed, so that the hydration of the lipids constituting the liposome proceeds and the multivesicular liposomes are dissolved to form a single-vesicle liposome. It is thought that it will break.
  • the liposome particle size is adjusted to a desired range (for example, about 50 to 500 nm), and is formed as a secondary from the W1 / O / W2 emulsion.
  • Multivesicular liposomes can be separated into single-cell liposomes, a sizing process using a filter, a separation process that removes free drugs and dispersants in the external aqueous phase, and a liposome suitable for storage
  • Various processes that have also been used in the production of conventional liposomes such as a dry powdering process for redispersion in an aqueous solvent at the time of use, and a filter sterilization process only when the liposome particle size is sufficiently small. Can be mentioned.
  • the collection of liposomes and leakage of inclusions hardly occur. If multivesicular liposomes remain after such an operation, they can be collected and removed by a filter for particle removal. These steps may be provided after the solvent removal step and continuously performed after the solvent removal step.
  • the volume average particle diameter is preferably 50 to 1,000 nm. More preferably, it is 50 to 300 nm. Liposomes with such a size have little risk of occluding capillaries and can pass through gaps formed in blood vessels in the vicinity of cancer tissue, so they are convenient for use by being administered to the human body as pharmaceuticals. is there.
  • the encapsulation rate of the liposomes obtained in the examples and comparative examples described below was measured according to the following method.
  • the fluorescence intensity (F total ) of the entire liposome aqueous solution (3 mL) was measured with a spectrophotometer (U-3310, JASCO Corporation). Next, 30 ⁇ L of 0.01 M CoCl 2 Tris-HCl buffer was added, and the fluorescence intensity (F in ) in the liposome was measured by quenching the fluorescence of calcein leaked into the outer aqueous phase with Co 2+ . Furthermore, liposomes were prepared under the same conditions as the sample without adding calcein, and the fluorescence (F 1 ) emitted by the lipids themselves was measured.
  • Inclusion rate E (%) (F in ⁇ F l ) / (F total ⁇ F l ) ⁇ 100 (Method for measuring the encapsulation rate of siRNA)
  • the liposome solution was ultracentrifuged to separate the external solution and the liposome, and the amount of each siRNA was measured by HPLC to determine the siRNA encapsulation rate.
  • the obtained W1 / O emulsion was confirmed to be a monodispersed W / O emulsion having a volume average particle size of 200 nm and a CV value of 38%, and its dispersibility was good. After standing at room temperature for 3 hours, there was no change in the volume average particle diameter, CV value, and dispersibility. In addition, in the optical microscope observation, although gentle aggregation was confirmed, no change with time was observed.
  • the obtained W1 / O emulsion was confirmed to be a W / O emulsion having a volume average particle size of 160 nm and a CV value of 33%, and its dispersibility was good.
  • the CV value and dispersibility were not changed, but the volume average particle diameter was 400 nm and the coalescence was slightly advanced. Aggregation was not confirmed by observation with an optical microscope, and it was difficult to confirm a monodisperse emulsion, but after 3 hours, a fine emulsion was confirmed by uniting.
  • Comparative sample 1 The mixed organic solvent used was changed to hexane only, and the same operation as Sample 1 was performed.
  • the obtained W1 / O emulsion was confirmed to be a W / O emulsion having a volume average particle size of 250 nm and a CV value of 45%, but its dispersibility was poor and settled immediately after standing. After standing at room temperature for 3 hours, there was no change in the volume average particle diameter, CV value, and dispersibility. In the optical microscope observation, considerable aggregation was confirmed. Unlike the particle size distribution measurement, a non-uniform W / O emulsion was observed, but no change with time was observed (FIG. 2).
  • Comparative sample 2 The mixed organic solvent used was changed to dichloromethane only, and the same operation as Sample 1 was performed.
  • the obtained W1 / O emulsion was a W / O emulsion having a volume average particle size of 120 nm and a CV value of 34%.
  • the dispersibility was good, but the CV value / dispersibility was only allowed to stand at room temperature for 30 minutes. Although there was no change, the coalescence was very advanced when the volume average particle size was 1 ⁇ m or more. Although aggregation was not confirmed by observation with an optical microscope, it was confirmed that the emulsions were united and enlarged, and after 3 hours, a united emulsion of several ⁇ m was confirmed (FIG. 3).
  • Samples 5-13 The same operation as Sample 1 was performed except that the composition of the mixed organic solvent was changed as described in Table 1. The results of each evaluation are shown in Table 1.
  • Example 1 (Production of liposomes using mixed organic solvents)
  • a W1 / O / W2 emulsion was produced by the SPG membrane emulsification method.
  • a cylindrical SPG membrane having a diameter of 10 mm, a length of 20 mm, and a pore diameter of 10 ⁇ m was used for an SPG membrane emulsifying device (trade name “external pressure type micro kit” manufactured by SPG Techno Co.), and an external aqueous phase solution (W2) was used on the outlet side of the device.
  • a tris-hydrochloric acid buffer solution (pH 7.4, 50 mM) containing 0.1% Pluronic F68 is filled, and the W1 / O emulsion is supplied from the apparatus inlet side, so that W1: W2 becomes 1:40.
  • a W1 / O / W2 emulsion was prepared as described above. At this time, droplets were formed without any continuous outflow from the SPG film, and the emulsion was stable even after emulsification (FIG. 8).
  • the SPG film was used that was surface-modified with N- (3-triethoxysilylpropyl) gluconamide in advance to make it more hydrophilic.
  • the preparation method of the gluconamide-modified SPG membrane is as follows. A cylindrical SPG membrane having a diameter of 10 mm, a length of 20 mm and a pore diameter of 10 ⁇ m manufactured by SPG Techno was degreased with an organic solvent (such as ethanol), washed with ultrapure water and dried, and then diluted with 20 times the weight of ethanol. It was immersed in an N- (3-triethoxysilylpropyl) gluconamide solution (Gelest, catalog number SIT8189), reacted at 50 ° C. for 2 hours, and then heated and dried at 100 ° C. for 3 hours.
  • an organic solvent such as ethanol
  • the W1 / O / W2 emulsion obtained by the secondary emulsification step was transferred to a closed container and stirred for about 8 hours under a reduced pressure condition of 20 ° C. and 500 mbar, and then for about 8 hours under a reduced pressure condition of 20 ° C. and 180 mbar. Stir and volatilize the solvent stepwise.
  • the obtained liposome suspension was translucent yellow, and it was confirmed that calcein was contained in the particles.
  • the resulting liposome had a volume average particle size of 80 nm and a CV value of 39%.
  • the calcein encapsulation rate of the liposome was 84% (FIG. 9).
  • Example 2 Using the W1 / O emulsion obtained from Sample 1 as a dispersed phase, a W1 / O / W2 emulsion was produced by the stirring emulsification method with the same liquid composition as in Example 1. In the stirring emulsification, the W1 / O emulsion was supplied to a place where W2 was vigorously stirred with a stirrer to produce a W1 / O / W2 emulsion.
  • the organic solvent was removed by the same method as in Example 1 to obtain a suspension of fine liposome particles.
  • the obtained liposome had a volume average particle diameter of 85 nm and a CV value of 43%.
  • the calcein encapsulation rate of the liposome was 82%.
  • Example 3 A W1 / O / W2 emulsion was obtained in the same manner as in Example 2 using the W1 / O emulsion obtained from Sample 1 as a dispersed phase, and then premix membrane emulsification was performed using an SPG membrane emulsifier.
  • the pore diameter of the SPG membrane was 1 ⁇ m, and it was processed by pressurizing at 4 MPa with nitrogen gas to produce a W1 / O / W2 emulsion having a volume average particle size of about 0.8 ⁇ m.
  • the organic solvent was removed by the same method as in Example 1 to obtain a suspension of fine liposome particles.
  • the obtained liposome had a volume average particle diameter of 75 nm and a CV value of 37%.
  • the calcein encapsulation rate of the liposome was 81%.
  • Comparative Example 1 Using the W1 / O emulsion obtained by Comparative Sample 1 as a dispersed phase, an attempt was made to produce a W1 / O / W2 emulsion by the SPG membrane emulsification method in the same manner as in Example 1, but the emulsification stopped in a few minutes from the start of emulsification, W1 / O / W2 could not be prepared efficiently. This was due to clogging of the membrane by the W1 / O emulsion.
  • Comparative Example 2 Using the W1 / O emulsion obtained from Comparative Sample 1 as a dispersed phase, a W1 / O / W2 emulsion was produced by stirring emulsification in the same manner as in Example 2, and then the organic solvent was removed.
  • the volume average particle size of the liposomes was 170 nm, the CV value was> 50% and was not monodispersed. In observation with an optical microscope, a plurality of multivesicular liposomes were observed.
  • the calcein encapsulation rate of the liposome was 51% (FIG. 10).
  • Comparative Example 3 Using the W1 / O emulsion obtained from Comparative Sample 2 as a dispersed phase, an attempt was made to produce liposomes using the SPG membrane emulsification method for secondary emulsification as in Example 1. However, as the secondary emulsification process proceeds, SPG The film was clogged, and a predetermined amount of W1 / O / W2 emulsion could not be obtained.
  • Comparative Example 4 Using the W1 / O emulsion obtained from Comparative Sample 2 as a dispersed phase, liposomes were prepared using stirring emulsification for secondary emulsification in the same manner as in Example 2.
  • the volume average particle diameter of the obtained liposomes was considerably large as> 2 ⁇ m, and the CV value was> 50%.
  • the calcein encapsulation rate of the liposome was 67%.
  • Example 4 The same operation as that of Sample 1 was performed except that yolk lecithin “COATSOME NC-50” (NOF Corporation) was used instead of DPPC.
  • Liposomes were prepared in the same manner as in Example 1 except that the W1 / O emulsion of Sample 14 was used as the dispersed phase.
  • the obtained liposome had a volume average particle diameter of 185 nm and a CV value of 44%.
  • the calcein encapsulation rate of the liposome was 87%.
  • In the optical microscope observation unlike Example 1, it was confirmed that there were a plurality of multivesicular liposomes (FIG. 11).
  • Comparative Example 5 The same operation as that of Comparative Sample 1 was performed except that yolk lecithin “COATSOME NC-50” (NOF Corporation) was used instead of DPPC. The dispersibility of the obtained W1 / O emulsion (Comparative Sample 3) was poor and settled immediately after standing. Except for using the W1 / O emulsion obtained by Comparative Sample 3 as a dispersed phase, the same operation as in Example 2 was performed to obtain liposomes. The resulting liposome had a volume average particle size of 270 nm and a CV value of> 50%. The liposome had a calcein encapsulation rate of 45%. Observation with an optical microscope confirmed that there were a considerable number of multivesicular liposomes (FIG. 12).
  • Comparative Example 6 The same operation as in Example 2 was carried out except that the W1 / O emulsion obtained from Comparative Sample 3 was used as a dispersed phase, and SPG membrane emulsification was used instead of the stirring emulsification method, and W1 / O / W2 When trying to obtain an emulsion, the SPG film was clogged as in Comparative Example 1, and emulsification could not be performed.
  • Example 5 Liposomes were prepared in the same manner as in Example 1 except that 0.075 g of PEG-modified phospholipid (DSPE-PEG2000) “SUNBRIGHT DSPE-020CN” was added after preparing the W1 / O emulsion of Sample 1. The resulting liposome had a volume average particle size of 135 nm and a CV value of 41%. The calcein encapsulation rate of the liposome was 80%.
  • Examples 6-14 Using the W1 / O emulsions of Samples 5 to 13, liposomes were obtained in the same manner as in Example 1 except that the decompression conditions were set according to the solvent. Table 2 shows the physical properties of the obtained liposomes.
  • the dispersibility of the obtained W1 / O emulsion (Sample 15) was good, and when it was allowed to stand at room temperature for 3 hours, there was no change in the dispersibility.
  • Liposomes were prepared in the same manner as in Example 1, except that the W1 / O emulsion of Sample 15 was used to set the reduced pressure conditions according to the solvent.
  • the volume average particle size of the liposome was 100 nm ⁇ CV value was 43%, and the calcein encapsulation rate was 76%.
  • the W1 / O emulsion obtained in the middle was a monodispersed emulsion with high dispersion stability, and no change was observed in 3 hours.
  • the dispersibility of the obtained W1 / O emulsion (Sample 16) was good, and when it was allowed to stand at room temperature for 3 hours, there was no change in the dispersibility.
  • Liposomes were prepared in the same manner as in Example 1 except that the reduced pressure condition according to the solvent was set using the W1 / O emulsion of Sample 16.
  • the volume average particle diameter of the liposome was 75 nm ⁇ CV value was 41%, and the calcein encapsulation rate was 67%.
  • the W1 / O emulsion obtained in the middle was a monodispersed emulsion with high dispersion stability, and no change was observed in 3 hours.
  • the dispersibility of the obtained W1 / O emulsion (Sample 17) was good, and when it was allowed to stand at room temperature for 3 hours, there was no change in the dispersibility.
  • Liposomes were prepared in the same manner as in Example 1 except that the W1 / O emulsion of Sample 17 was used to set the reduced pressure conditions according to the solvent.
  • the volume average particle size of the liposome was 70 nm ⁇ CV value was 38%, and the calcein encapsulation rate was 71%.
  • the W1 / O emulsion obtained in the middle was a monodispersed emulsion with high dispersion stability, and no change was observed in 3 hours.
  • the dispersibility of the obtained W1 / O emulsion (Sample 18) was good, and when it was allowed to stand at room temperature for 3 hours, there was no change in the dispersibility.
  • Liposomes were prepared in the same manner as in Example 1, except that the W1 / O emulsion of Sample 18 was used to set a reduced pressure condition according to the solvent.
  • the volume average particle size of the liposome was 100 nm ⁇ CV value was 47%, and the calcein encapsulation rate was 61%.
  • the W1 / O emulsion obtained in the middle was a monodispersed emulsion with high dispersion stability, and no change was observed in 3 hours.
  • the dispersibility of the obtained W1 / O emulsion (Sample 19) was good, and when it was allowed to stand at room temperature for 3 hours, there was no change in the dispersibility.
  • Liposomes were prepared in the same manner as in Example 1 except that the reduced pressure conditions according to the solvent were set using the W1 / O emulsion of Sample 19.
  • the volume average particle diameter of the liposome was 95 nm ⁇ CV value was 41%, and the calcein encapsulation rate was 64%.
  • the W1 / O emulsion obtained in the middle was a monodispersed emulsion with high dispersion stability, and no change was observed in 3 hours.
  • the dispersibility of the obtained W1 / O emulsion (Sample 20) was good, and when it was allowed to stand at room temperature for 3 hours, there was no change in the dispersibility.
  • Liposomes were prepared in the same manner as in Example 1 except that the W1 / O emulsion of Sample 20 was used.
  • the volume average particle diameter of the liposome was 140 nm ⁇ CV value was 41%, and the calcein encapsulation rate was 75%.
  • the W1 / O emulsion obtained in the middle was a monodispersed emulsion with high dispersion stability, and no change was observed in 3 hours.
  • Example 21 The same operation as Sample 1 was performed except that 200 ⁇ g / mL siRNA (random sequence) was used instead of calcein.
  • Liposomes were prepared in the same manner as in Example 1 except that the W1 / O emulsion of Sample 21 was used.
  • the obtained liposome liquid was white and translucent, and the volume average particle diameter was 70 nm ⁇ CV value was 39%.
  • the siRNA encapsulation rate in the liposome was 80%.
  • the siRNA concentration as the liposome dispersion was calculated to be 5 ⁇ g / mL, the actually obtained siRNA concentration was 4.5 ⁇ g / mL.
  • Example 22 After performing primary emulsification by the ultrasonic emulsification method, except that 11.25 mL of hexane in which 1.5 g of DPPC and 0.25 g of DPPG were dissolved was used as the organic solvent phase (O), dichloromethane 3 .75 mL was added. The dispersibility of the obtained W1 / O emulsion (Sample 22) was good. Liposomes were prepared in the same manner as in Example 2 except that the W1 / O emulsion of Sample 22 was used. The obtained liposome had a volume average particle diameter of 140 nm and a CV value of 41%. The calcein encapsulation rate of the liposome was 83%.

Abstract

Disclosed is a process for producing a liposome, which comprises a two-stage emulsification step and a solvent removal step, and which is superior in the ratio of encapsulation of a drug or the like, the univesicular liposome formation efficiency and others compared with those of conventional processes. Specifically disclosed is a process for producing a liposome, which comprises: a primary emulsification step of emulsifying an inner aqueous phase (W1) and an oily phase (O) with a mixed lipid component (F1) for liposome applications to prepare a W1/O emulsion; a secondary emulsification step of emulsifying the W1/O emulsion and an outer aqueous phase (W2) with a mixed lipid component (F2) for liposome applications to prepare a W1/O/W2 emulsion; and a solvent removal step of removing an organic solvent contained in the oily phase (O) from the W1/O/W2 emulsion to form a liposome. The process is characterized in that the oily phase (O) in the W1/O/W2 emulsion to be subjected to the solvent removal step is a mixed organic solvent comprising at least two organic solvents.

Description

混合有機溶媒を油相として用いる二段階乳化によるリポソームの製造方法Method for producing liposome by two-stage emulsification using mixed organic solvent as oil phase
　本発明は、医薬品、化粧品、食品などの分野で用いられるリポソームの、二段階の乳化工程および溶媒除去工程を含む製造方法に関する。 The present invention relates to a method for producing liposomes used in the fields of pharmaceuticals, cosmetics, foods and the like including a two-stage emulsification step and a solvent removal step.
　リポソームは、単層または複数層の脂質二重膜からなる閉鎖小胞体であり、内水相および脂質二重膜内部にそれぞれ水溶性および疎水性の薬剤類を保持することができる。リポソームの脂質二重膜は生体膜に類似しているため生体内での安全性が高いことなどから、たとえばＤＤＳ（ドラック・デリバリー・システム）用の医薬品などの、各種用途が注目され、研究開発が進められている。 Liposomes are closed vesicles composed of a monolayer or a multi-layer lipid bilayer, and can retain water-soluble and hydrophobic drugs in the inner aqueous phase and the lipid bilayer, respectively. Lipid lipid bilayer membranes are similar to biological membranes and are therefore highly safe in vivo. For example, pharmaceuticals for DDS (Drug Delivery System) have been attracting attention and research and development. Is underway.
　リポソームの製造方法の一つとして、二段階の乳化工程によりＷ／Ｏ／Ｗエマルションを調製した後、その油相（Ｏ）を除去することによりリポソームを形成させる方法（マイクロカプセル化法ないし二段階乳化法と呼ばれる。）が知られている（非特許文献１）。このようなリポソームの製造方法において、従来、油相（Ｏ）は単一の有機溶媒により構成されていた。たとえば特許文献１（実施例）には、油相としてヘキサンのみを用いる、マイクロチャネルによる二段階乳化法の態様が記載されている。 As one of the methods for producing liposomes, a W / O / W emulsion is prepared by a two-stage emulsification step, and then the oil phase (O) is removed to form liposomes (microencapsulation method or two-step method). (Referred to as an emulsification method) (Non-Patent Document 1). In such a method for producing liposomes, the oil phase (O) has conventionally been constituted by a single organic solvent. For example, Patent Document 1 (Example) describes an embodiment of a two-stage emulsification method using a microchannel using only hexane as an oil phase.
　また、特許文献２には、「少なくとも１種の有機溶媒」、たとえば「エーテル、炭化水素、ハロゲン化炭化水素、ハロゲン化エーテル、エステル、クロロホルム、およびそれらの組合せ」を油相として使用する二段階乳化法が記載されている（請求項２，３０、［００５４］段落）。しかしながら、この特許文献２に記載された製造方法は、「全体にわたって連続した網目構造として分布する膜を持った複数の非同心円状のチャンバー」を有し、加重平均直径が通常は０．５～２５μｍ程度のマイクロメーターの範囲にある「多胞状リポソーム」（ＭＶＬ）を対象とするものであり（請求項２、［００１９］段落、）、「単一の水系チャンバーを有するリポソーム構造物」である、通常は平均直径の範囲が約２０～５００ｎｍである「単膜リポソームまたは小胞体」（ＵＬＶ）（［００１９］段落、本明細書で言う単胞リポソーム）を対象としたものではない。また、２種以上の有機溶媒を用いることが具体的にどのような技術的意義を有するのかについて、上記ＵＬＶを製造対象とする場合はもちろん、上記ＭＶＬを製造対象とする場合にも、全く記載も示唆もされていない。実施例としては、溶剤としてクロロホルム単独を用いて、二段階の撹拌乳化法により、ＭＶＬを調製したことが記載されているのみである（実施例１：［００７５］～［００７９］等）。 Patent Document 2 discloses a two-step process using “at least one organic solvent”, for example, “ether, hydrocarbon, halogenated hydrocarbon, halogenated ether, ester, chloroform, and combinations thereof” as the oil phase. An emulsification method is described (claims 2, 30, paragraph [0054]). However, the manufacturing method described in Patent Document 2 has “a plurality of non-concentric chambers having a film distributed as a continuous network structure throughout the whole”, and the weighted average diameter is usually 0.5 to It is intended for “multivesicular liposomes” (MVL) in the micrometer range of about 25 μm (claim 2, [0019] paragraph), and “liposome structure having a single aqueous chamber”. However, it is not intended for “unilamellar liposomes or endoplasmic reticulum” (ULV) ([0019] paragraph, single vesicle liposomes referred to herein), which typically has an average diameter in the range of about 20-500 nm. Further, the technical significance of the use of two or more organic solvents is completely described not only when the ULV is a production target but also when the MVL is a production target. There is no suggestion. Examples only describe that MVL was prepared by a two-step stirring emulsification method using chloroform alone as a solvent (Example 1: [0075] to [0079] etc.).
特許第４００９７３３号公報Japanese Patent No. 4009733 特許第３６７６９７６号公報Japanese Patent No. 3676976
　二段階の乳化工程および溶媒除去工程を含むリポソームの製造方法については、これまでも、薬剤類の内包率を向上させることやリポソームの粒径を所定の範囲に揃えることなどが課題とされており、そのために様々な手段が用いられてきたが（たとえば特許文献１では、Ｗ／Ｏエマルションの水相を凍結する工程が行われている）、より良好なリポソームを製造する上で改善の余地が残されていた。 Regarding liposome production methods including a two-stage emulsification step and a solvent removal step, it has been a challenge to improve the encapsulation rate of drugs and to make the particle size of liposomes within a predetermined range. For this purpose, various means have been used (for example, in Patent Document 1, a step of freezing the aqueous phase of the W / O emulsion is performed), but there is room for improvement in producing better liposomes. It was left.
　本発明は、薬剤類の内包率や単胞リポソームの形成効率などの点で従来よりも優れている、二段階の乳化工程および溶媒除去工程を含むリポソームの製造方法を提供することを課題とする。 It is an object of the present invention to provide a method for producing a liposome comprising a two-stage emulsification step and a solvent removal step, which is superior to conventional methods in terms of the encapsulation rate of drugs and the formation efficiency of single cell liposomes. .
　本発明者らは、たとえばヘキサンとジクロロメタンのような、少なくとも２種類の有機溶媒を含有する混合有機溶媒をＷ１／ＯエマルションおよびＷ１／Ｏ／Ｗ２エマルションの油相（Ｏ）とすることにより、１種類の有機溶媒のみを油相とする場合に比べて、それらのエマルションの分散安定性が飛躍的に高まり、リポソームの薬剤類の内包率や単胞リポソームの形成効率を向上させることができることを見出し、本発明を完成させるに至った。 The present inventors have made a mixed organic solvent containing at least two kinds of organic solvents, such as hexane and dichloromethane, for example, as an oil phase (O) of a W1 / O emulsion and a W1 / O / W2 emulsion. It has been found that the dispersion stability of these emulsions can be dramatically improved compared to the case where only an organic solvent of a kind is used as an oil phase, and the encapsulation rate of liposome drugs and the formation efficiency of single cell liposomes can be improved. The present invention has been completed.
　すなわち、本発明に係るリポソームの製造方法は、内水相（Ｗ１）および油相（Ｏ）をリポソーム用混合脂質成分（Ｆ１）を用いて乳化しＷ１／Ｏエマルションを調製する一次乳化工程と、Ｗ１／Ｏエマルションおよび外水相（Ｗ２）をリポソーム用混合脂質成分（Ｆ２）を用いて乳化しＷ１／Ｏ／Ｗ２エマルションを調製する二次乳化工程と、Ｗ１／Ｏ／Ｗ２エマルションから油相（Ｏ）の有機溶媒を除去してリポソームを形成させる溶媒除去工程とを含むリポソームの製造方法であって、溶媒除去工程に供されるＷ１／Ｏ／Ｗ２エマルションの油相（Ｏ）が少なくとも２種類の有機溶媒を含有する混合有機溶媒であることを特徴とする。 That is, the method for producing liposomes according to the present invention comprises a primary emulsification step of emulsifying an inner aqueous phase (W1) and an oil phase (O) using a mixed lipid component (F1) for liposomes to prepare a W1 / O emulsion; A secondary emulsification step of emulsifying the W1 / O emulsion and the external aqueous phase (W2) using the mixed lipid component (F2) for liposomes to prepare a W1 / O / W2 emulsion, and an oil phase (from the W1 / O / W2 emulsion) And a solvent removal step of removing the organic solvent of O) to form liposomes, wherein the oil phase (O) of the W1 / O / W2 emulsion used in the solvent removal step is at least two types It is a mixed organic solvent containing the organic solvent.
　本発明の製造方法は、好ましくは単胞リポソームを製造するためのものである。なお、本発明において、「単胞リポソーム」（ULV、単核リポソームと同義である）は、単一の内水相を有するリポソーム構造物を指し、通常は体積平均粒径の範囲が約２０～５００ｎｍである。これに対して、「多胞リポソーム」（MVL: multivesicular liposomes）は、複数の非同心円状の内水相を包囲する脂質膜を含んでなるリポソーム構造物を指し、また「多重膜リポソーム」（MLV）は、複数の「タマネギの皮」のような同心円状の膜を有し、その間に殻様の同心円状の水系コンパートメントがあるリポソーム構造物を指す。多胞リポソームおよび多重膜リポソームの特徴は、体積平均粒径がマイクロメーターの範囲であり、通常は０．５～２５μｍである。 The production method of the present invention is preferably for producing single cell liposomes. In the present invention, “monocystic liposome” (ULV, synonymous with mononuclear liposome) refers to a liposome structure having a single inner aqueous phase, and usually has a volume average particle size in the range of about 20 to 500 nm. On the other hand, “multivesicular liposome” (MVL: リ ポ ソ ー ム multivesicular liposomes) refers to a liposome structure comprising a lipid membrane surrounding a plurality of non-concentric inner aqueous phases, and also referred to as “multilamellar liposome” (MLV ) Refers to a liposome structure having a plurality of concentric membranes, such as “onion skin”, with a shell-like concentric aqueous compartment in between. The characteristics of multivesicular liposomes and multilamellar liposomes are that the volume average particle diameter is in the micrometer range, usually 0.5 to 25 μm.
　前記混合有機溶媒としては、たとえば、クロロホルム、シクロヘキサン、ジクロロメタン、ヘキサン、t‐ブチルメチルエーテル、酢酸エチル、ジエチルエーテル、ギ酸エチル、酢酸イソプロピル、酢酸メチル、メチルエチルケトン、ペンタン、アセトニトリル、メタノール、アセトン、エタノール、２‐プロパノールからなる群より選択される少なくとも２種類の有機溶媒が好ましい。 Examples of the mixed organic solvent include chloroform, cyclohexane, dichloromethane, hexane, t-butyl methyl ether, ethyl acetate, diethyl ether, ethyl formate, isopropyl acetate, methyl acetate, methyl ethyl ketone, pentane, acetonitrile, methanol, acetone, ethanol, Preference is given to at least two organic solvents selected from the group consisting of 2-propanol.
　本発明の一態様として、前記混合有機溶媒は炭化水素の有機溶媒を含有すること、たとえば、炭化水素からなる溶媒群（溶媒Ａ群）及びエーテル、ハロゲン化炭化水素、ハロゲン化エーテル、エステル、アルコール、ケトンおよびアセトニトリルからなる溶媒群（溶媒Ｂ群）のぞれぞれより少なくとも１種類ずつ選択される少なくとも２種類の有機溶媒を含有することが好ましい。前記溶媒Ａ群は、たとえばペンタン、ヘキサン及びシクロヘキサンからなることが好ましく、前記溶媒Ｂ群は、たとえばジエチルエーテル、ｔ－ブチルメチルエーテル、クロロホルム、ジクロロメタン、ギ酸エチル、酢酸メチル、酢酸イソプロピル、メタノール、エタノール、２－プロパノール、メチルエチルケトン及びアセトリニトリルからなることが更に好ましい。また、前記混合有機溶媒に含まれる前記溶媒Ａの体積比率は５０～９９％が好ましい。 As one aspect of the present invention, the mixed organic solvent contains a hydrocarbon organic solvent, for example, a solvent group consisting of hydrocarbons (solvent A group) and ethers, halogenated hydrocarbons, halogenated ethers, esters, alcohols. It is preferable to contain at least two types of organic solvents selected from at least one of each of a solvent group (solvent B group) consisting of ketone and acetonitrile. The solvent group A preferably comprises, for example, pentane, hexane and cyclohexane, and the solvent group B includes, for example, diethyl ether, t-butyl methyl ether, chloroform, dichloromethane, ethyl formate, methyl acetate, isopropyl acetate, methanol, ethanol More preferably, it consists of 2-propanol, methyl ethyl ketone and acetonitrile. The volume ratio of the solvent A contained in the mixed organic solvent is preferably 50 to 99%.
　より具体的な好ましい混合有機溶媒の態様としては、たとえば、ヘキサン：クロロホルム＝６０：４０～９０：１０、ヘキサン：酢酸エチル＝５０：５０～９０：１０、ヘキサン：エタノール＝７０：３０～９９：１（いずれも体積比、合計を１００とする）からなるものが挙げられる。 More specifically, preferred mixed organic solvent modes include, for example, hexane: chloroform = 60: 40 to 90:10, hexane: ethyl acetate = 50: 50 to 90:10, hexane: ethanol = 70: 30 to 99: 1 (both are volume ratios, the total is 100).
　前記混合有機溶媒を用いるために、前記一次乳化工程は、あらかじめ調製された混合有機溶媒を用いて乳化処理に行うステップ、および／またはＷ１／Ｏエマルションが調製されている途中または調製された後にさらに有機溶媒を油相に添加するステップを含むものとすることができる。 In order to use the mixed organic solvent, the primary emulsification step is a step of performing an emulsification process using a mixed organic solvent prepared in advance, and / or during or after the W1 / O emulsion is being prepared. A step of adding an organic solvent to the oil phase may be included.
　また、前記一次乳化工程における乳化法としてはたとえば膜乳化法またはマイクロチャネル乳化法が好ましく、前記二次乳化工程における乳化法としてはたとえば膜乳化法、マイクロチャネル乳化法または撹拌乳化法が好ましい。さらに、これらの膜乳化法としてはたとえばＳＰＧ膜を用いた膜乳化法が好ましい。 Further, as the emulsification method in the primary emulsification step, for example, a membrane emulsification method or a microchannel emulsification method is preferable, and as the emulsification method in the secondary emulsification step, for example, a membrane emulsification method, a microchannel emulsification method or a stirring emulsification method is preferable. Furthermore, as these membrane emulsification methods, for example, a membrane emulsification method using an SPG membrane is preferable.
　前記一次乳化工程は、さらにリポソームに内包させるべき物質を前記内水相（Ｗ１）または前記油相（Ｏ）に添加した上で行ってもよく、また、前記一次乳化工程後にリポソームに内包させるべき物質を前記油相（Ｏ）に添加してもよい。 The primary emulsification step may be performed after adding a substance to be encapsulated in the liposome to the inner aqueous phase (W1) or the oil phase (O), and should be encapsulated in the liposome after the primary emulsification step. Substances may be added to the oil phase (O).
　このような本発明のリポソームの製造方法は、リポソーム分散液またはその乾燥粉末の製造方法として利用することもできる。 Such a method for producing a liposome of the present invention can also be used as a method for producing a liposome dispersion or a dry powder thereof.
　混合有機溶媒をＷ１／Ｏエマルションおよび／またはＷ１／Ｏ／Ｗ２エマルションの油相とすることにより、それらのエマルションの分散安定性が高まるため、溶媒除去工程における粒子同士の接触および凝集を低減し、粒子径の増大（多胞リポソームなどの凝集塊の形成や、Ｗ１／Ｏ／Ｗ２エマルション内でのＷ１／Ｏエマルションの凝集・合一）を抑制することができる。また、二次乳化工程において膜乳化法やマイクロチャネル乳化法を用いる場合は、乳化基材の目詰まりの問題を解消することもできる。その結果、薬剤の内包率が高く、粒度分布の狭い微小な単胞リポソームを多く含む、医薬品用等として好適なリポソームを効率的に製造することができるようになる。 By making the mixed organic solvent into an oil phase of a W1 / O emulsion and / or a W1 / O / W2 emulsion, the dispersion stability of those emulsions is increased, so that contact and aggregation between particles in the solvent removal step is reduced, An increase in particle diameter (formation of aggregates such as multivesicular liposomes and aggregation / coalescence of W1 / O emulsion in W1 / O / W2 emulsion) can be suppressed. Further, when the membrane emulsification method or the microchannel emulsification method is used in the secondary emulsification step, the problem of clogging of the emulsified base material can be solved. As a result, it is possible to efficiently produce liposomes suitable for pharmaceutical use and the like that contain a large amount of fine single-vesicle liposomes having a high drug encapsulation rate and a narrow particle size distribution.
図１は試料１で調製したＷ１／Ｏエマルションの光学顕微鏡による観察写真である。FIG. 1 is an observation photograph of the W1 / O emulsion prepared in Sample 1 with an optical microscope. 図２は比較試料１で調製したＷ１／Ｏエマルションの光学顕微鏡による観察写真である。FIG. 2 is an observation photograph of the W1 / O emulsion prepared in Comparative Sample 1 with an optical microscope. 図３は比較試料２で調製したＷ１／Ｏエマルションの光学顕微鏡による観察写真である。FIG. 3 is an observation photograph of the W1 / O emulsion prepared in Comparative Sample 2 with an optical microscope. 図４は試料５で調製したＷ１／Ｏエマルションの光学顕微鏡による観察写真である。FIG. 4 is an observation photograph of the W1 / O emulsion prepared in Sample 5 with an optical microscope. 図５は試料６で調製したＷ１／Ｏエマルションの光学顕微鏡による観察写真である。FIG. 5 is an observation photograph of the W1 / O emulsion prepared in Sample 6 with an optical microscope. 図６は試料７で調製したＷ１／Ｏエマルションの光学顕微鏡による観察写真である。FIG. 6 is an observation photograph of the W1 / O emulsion prepared in Sample 7 with an optical microscope. 図７は試料１０で調製したＷ１／Ｏエマルションの光学顕微鏡による観察写真である。FIG. 7 is an observation photograph of the W1 / O emulsion prepared in Sample 10 with an optical microscope. 図８は実施例１で調製したＷ１／Ｏ／Ｗ２エマルションの光学顕微鏡による観察写真である。FIG. 8 is an observation photograph of the W1 / O / W2 emulsion prepared in Example 1 with an optical microscope. 図９は実施例１で調製したリポソームの光学顕微鏡による観察写真である。FIG. 9 is an observation photograph of the liposome prepared in Example 1 with an optical microscope. 図１０は比較例２で調製したリポソームの光学顕微鏡による観察写真である。FIG. 10 is an observation photograph of the liposome prepared in Comparative Example 2 with an optical microscope. 図１１は実施例４で調製したリポソームの光学顕微鏡による観察写真である。FIG. 11 is an observation photograph of the liposome prepared in Example 4 with an optical microscope. 図１２は比較例５で調製したリポソームの光学顕微鏡による観察写真である。FIG. 12 is an observation photograph of the liposome prepared in Comparative Example 5 with an optical microscope. 図１３は実施例２２で調製したリポソームの光学顕微鏡による観察写真である。FIG. 13 is an observation photograph of the liposome prepared in Example 22 with an optical microscope.
　－ リポソームの製造に用いる物質 －
　　・有機溶媒（Ｏ）
　本発明では、Ｗ１／Ｏ／Ｗ２エマルションの油相（Ｏ）が、少なくとも２種類の有機溶媒を含有するもの（混合有機溶媒）となるようにする。 -Substances used in the production of liposomes-
・ Organic solvent (O)
In the present invention, the oil phase (O) of the W1 / O / W2 emulsion is made to contain at least two kinds of organic solvents (mixed organic solvent).
　混合有機溶媒の配合組成（有機溶媒の種類および割合）は適宜調整することができる。混合有機溶媒に配合することのできる有機溶媒としては、沸点が５～９５℃の範囲であることが望ましく、従来の一次乳化工程で通常単独で用いられてきたクロロホルム、シクロヘキサン、１，２‐ジクロロエテン、ジクロロメタン、１，２‐ジメトキシエタン、ヘキサン、１，１，２‐トリクロロエテン、t‐ブチルメチルエーテル、酢酸エチル、ジエチルエーテル、ギ酸エチル、酢酸イソプロピル、酢酸メチル、メチルエチルケトン、ペンタンなどの非水溶性有機溶媒のほかに、従来は乳化の溶媒としてあまり一般的ではなかったアセトニトリル、メタノール、アセトン、エタノール、２‐プロパノールなどの水溶性有機溶媒や、上記以外のエーテル、炭化水素、ハロゲン化炭化水素、ハロゲン化エーテル、エステル類が挙げられる。なかでもクロロホルム、シクロヘキサン、ジクロロメタン、ヘキサン、t‐ブチルメチルエーテル、酢酸エチル、ジエチルエーテル、ギ酸エチル、酢酸イソプロピル、酢酸メチル、メチルエチルケトン、ペンタン、アセトニトリル、メタノール、アセトン、エタノール、２‐プロパノールなどが好ましい。３種以上の有機溶媒を組み合わせてもよいが、条件設定の容易さの観点からは２種の有機溶媒の組み合わせとすることが好ましい。必要に応じて、さらに各種の機能性成分を混合有機溶媒に添加してもよい。 The compounding composition of the mixed organic solvent (type and ratio of the organic solvent) can be adjusted as appropriate. The organic solvent that can be blended in the mixed organic solvent preferably has a boiling point in the range of 5 to 95 ° C., and is usually chloroform, cyclohexane, 1,2-dichloroethane, which has been usually used alone in the conventional primary emulsification step. Water-insoluble such as benzene, dichloromethane, 1,2-dimethoxyethane, hexane, 1,1,2-trichloroethene, t-butyl methyl ether, ethyl acetate, diethyl ether, ethyl formate, isopropyl acetate, methyl acetate, methyl ethyl ketone, pentane In addition to water-soluble organic solvents, water-soluble organic solvents such as acetonitrile, methanol, acetone, ethanol, 2-propanol, and other ethers, hydrocarbons, and halogenated hydrocarbons, which have not been commonly used as emulsifying solvents, are used. , Halogenated ethers and esters. Of these, chloroform, cyclohexane, dichloromethane, hexane, t-butyl methyl ether, ethyl acetate, diethyl ether, ethyl formate, isopropyl acetate, methyl acetate, methyl ethyl ketone, pentane, acetonitrile, methanol, acetone, ethanol, 2-propanol and the like are preferable. Three or more organic solvents may be combined, but from the viewpoint of ease of setting conditions, a combination of two organic solvents is preferable. If necessary, various functional components may be added to the mixed organic solvent.
　本発明の一つの側面において、混合有機溶媒に配合する少なくとも２種類の有機溶媒は、混合脂質成分（Ｆ１）および（Ｆ２）に含まれるリン脂質の溶解性を考慮しながら選択することも可能である。上で挙げた溶媒の中で、リン脂質の溶解性の高いクロロホルムやエタノールなどを単独で用いたＷ１／Ｏエマルションは、分散性は良いが合一が進みエマルション粒径が粗大になる、さらには水相と油相が完全に分離する、もしくは均一に混和してしまうなどの問題がある。一方で、ヘキサンなどのリン脂質の溶解性が低い溶媒のみを単独で用いたＷ１／Ｏエマルションは、エマルション同士の凝集が非常に強く分散安定性が悪い、均一な粒子径のエマルションを得ることが困難なことがある。これらの問題に対し、たとえば、リン脂質に対する溶解性が低い溶媒（本発明においてそのような性質を持つ溶媒を「溶媒Ａ」と称する。）とリン脂質に対する溶解性が高い溶媒（本発明においてそのような性質を持つ溶媒を「溶媒Ｂ」と称する。）とを含有する（好ましくはからなる）混合有機溶媒を用い、それらの比率を適宜調整することで、合一が進みにくく、且つ、凝集しないまたは凝集が強くないＷ１／Ｏエマルションを得ることができる。 In one aspect of the present invention, at least two kinds of organic solvents to be blended in the mixed organic solvent can be selected in consideration of the solubility of the phospholipids contained in the mixed lipid components (F1) and (F2). is there. Among the solvents listed above, W1 / O emulsions using phospholipids with high solubility of phospholipids alone have good dispersibility, but coalescence progresses and the emulsion particle size becomes coarse. There is a problem that the water phase and the oil phase are completely separated or mixed uniformly. On the other hand, a W1 / O emulsion using only a solvent having low solubility of phospholipids such as hexane can obtain an emulsion having a uniform particle size in which aggregation between emulsions is very strong and dispersion stability is poor. It can be difficult. To solve these problems, for example, a solvent having low solubility in phospholipid (a solvent having such a property is referred to as “solvent A” in the present invention) and a solvent having high solubility in phospholipid (in the present invention, the solvent A solvent having such properties is referred to as “solvent B”), and is preferably mixed, and the ratio is appropriately adjusted so that coalescence is difficult to proceed and aggregation is performed. W1 / O emulsion that does not or does not strongly aggregate can be obtained.
　このような溶媒Ａとしては、炭化水素が好ましく、ペンタン、ヘキサン、及びシクロヘキサンより選択される少なくとも１種類であることがより好ましい。また、溶媒Ｂとしては、エーテル、ハロゲン化炭化水素、ハロゲン化エーテル、エステル、アルコール、ケトンおよびアセトニトリルより選択される少なくとも１種類であることが好ましく、ジエチルエーテル、ｔ－ブチルメチルエーテル、クロロホルム、ジクロロメタン、ギ酸エチル、酢酸メチル、酢酸イソプロピル、メタノール、エタノール、２－プロパノール、メチルエチルケトン及びアセトリニトリルより選択される少なくとも１種類であることがより好ましい。 Such a solvent A is preferably a hydrocarbon, and more preferably at least one selected from pentane, hexane, and cyclohexane. The solvent B is preferably at least one selected from ethers, halogenated hydrocarbons, halogenated ethers, esters, alcohols, ketones, and acetonitrile. Diethyl ether, t-butyl methyl ether, chloroform, dichloromethane More preferably, at least one selected from ethyl formate, methyl acetate, isopropyl acetate, methanol, ethanol, 2-propanol, methyl ethyl ketone and acetonitrile.
　このように有機溶媒の組み合わせは、混合脂質成分（リン脂質等）の溶解性、有機溶媒同士の相溶性、溶媒除去工程の条件設定などが好ましいものとなるよう、有機溶媒の極性（親水性・疎水性）、沸点、密度（比重）などが所定の関係性を有するものとなるようにすることができる。なお、この際に必ずしも良溶媒の沸点は貧溶媒の沸点よりも低い必要はない。 In this way, the combination of organic solvents is such that the solubility of mixed lipid components (phospholipids, etc.), compatibility between organic solvents, setting of conditions for the solvent removal step, etc. are preferable, so that the polarity of the organic solvent (hydrophilic (Hydrophobicity), boiling point, density (specific gravity), and the like can have predetermined relationships. At this time, the boiling point of the good solvent is not necessarily lower than the boiling point of the poor solvent.
　混合有機溶媒の一つの態様として、上記の溶媒Ａを主成分とするものは、得られるナノサイズのＷ１／Ｏエマルションの単分散性を向上させるなどの効果に優れるため好ましく、混合有機溶媒中に含まれる溶媒Ａ（溶媒Ａが複数の種類からなるものである場合はその総量）の体積比率が５０～９９％であることがより好ましい。たとえば、ヘキサンおよびジクロロメタンからなる混合有機溶媒では、それらの体積比（ヘキサン：ジクロロメタン、合計を１００とする）は６０：４０～９０：１０が好ましい。その他、ヘキサンおよび酢酸エチルからなる混合有機溶媒では、５０：５０～９０：１０の体積比が好ましく、ヘキサンおよびエタノールからなる混合有機溶媒では、７０：３０～９９：１の体積比が好ましい。 As one aspect of the mixed organic solvent, those having the above solvent A as a main component are preferable because they are excellent in effects such as improving the monodispersibility of the obtained nano-sized W1 / O emulsion. It is more preferable that the volume ratio of the solvent A (the total amount when the solvent A is a plurality of types) is 50 to 99%. For example, in a mixed organic solvent composed of hexane and dichloromethane, the volume ratio thereof (hexane: dichloromethane, the total is 100) is preferably 60:40 to 90:10. In addition, the mixed organic solvent composed of hexane and ethyl acetate preferably has a volume ratio of 50:50 to 90:10, and the mixed organic solvent composed of hexane and ethanol preferably has a volume ratio of 70:30 to 99: 1.
　本発明では、溶媒除去工程に供されるＷ１／Ｏ／Ｗ２エマルションの油相（Ｏ）が少なくとも２種類の有機溶媒を含有する状態になっていればよい。そのようなＷ１／Ｏ／Ｗ２エマルションを調製する手法は特に限定されるものではなく、一次乳化工程および二次乳化工程で用いられる乳化方法に応じて適切な手法を設計することができる。 In the present invention, the oil phase (O) of the W1 / O / W2 emulsion to be used in the solvent removal step only needs to contain at least two kinds of organic solvents. The method for preparing such a W1 / O / W2 emulsion is not particularly limited, and an appropriate method can be designed according to the emulsification method used in the primary emulsification step and the secondary emulsification step.
　代表的には、（ｉ）あらかじめ少なくとも２種類の有機溶媒を含有する混合有機溶媒（Ｏ"）を調製しておき、その混合有機溶媒を用いて一次乳化処理を行い、Ｗ１／Ｏ"エマルションを調製するか、（ii）１種単独の有機溶媒（Ｏ'）を用いて一次乳化処理を行い、Ｗ１／Ｏ'エマルションが調製されている途中または調製された後に別の種類の有機溶媒を添加し、油相が混合有機溶媒となったＷ１／Ｏ"エマルションを調製し、そのようにして得られたＷ１／Ｏ"エマルションを二次乳化工程に供してＷ１／Ｏ"／Ｗ２を調製するような手法が挙げられる。上記（ii）の手法を用いる場合は、混合脂質成分の溶解性がより高い溶媒（たとえばジクロロメタン）を用いてＷ１／Ｏエマルションを調製したのち、溶解性がより低い溶媒（たとえばヘキサン）を添加するようにすることで、Ｗ１／Ｏエマルションの合一の過程を止めることができ、エマルションの粒径の制御を行うことが可能である。逆に、溶解性が低い溶媒でＷ１／Ｏエマルションを調製したのちに、溶解性がより高い溶媒を添加することで、Ｗ１／Ｏエマルションの凝集を減らし、分散性を向上させることができる。さらにこの場合には、リポソームを製造した際の当該リポソームの分散性向上の効果が顕著である。また、油相が３種類以上の有機溶媒を含有するものとする場合には、上記（ｉ）および（ii）を組み合わせたような手法、つまり、あらかじめ調製された少なくとも２種類の有機溶媒を含有する混合有機溶媒（Ｏ"）を用いてＷ１／Ｏ"エマルションを調製した上で、さらに別の種類の有機溶媒を添加するようにすることもできる。 Typically, (i) a mixed organic solvent (O ″) containing at least two kinds of organic solvents is prepared in advance, a primary emulsification treatment is performed using the mixed organic solvent, and a W1 / O ″ emulsion is prepared. Or (ii) perform primary emulsification using one organic solvent (O ′) and add another kind of organic solvent during or after the W1 / O ′ emulsion is being prepared A W1 / O "emulsion in which the oil phase is a mixed organic solvent is prepared, and the W1 / O" emulsion thus obtained is subjected to a secondary emulsification step to prepare W1 / O "/ W2. In the case of using the above method (ii), after preparing a W1 / O emulsion using a solvent (for example, dichloromethane) having a higher solubility of the mixed lipid component, a solvent having a lower solubility ( For example In addition, the coalescence process of the W1 / O emulsion can be stopped and the particle size of the emulsion can be controlled. After preparing the / O emulsion, adding a solvent with higher solubility can reduce the aggregation of the W1 / O emulsion and improve the dispersibility. The effect of improving the dispersibility of the liposome is remarkable, and when the oil phase contains three or more kinds of organic solvents, a method in which the above (i) and (ii) are combined, That is, a W1 / O "emulsion is prepared using a mixed organic solvent (O") containing at least two kinds of organic solvents prepared in advance, and another kind of organic solvent is added. It is also possible to.
　一方で、上記のような手法とは異なり、二次乳化工程に供されるのはＷ１／Ｏ'エマルションであっても、二次乳化工程の途中で所定の処理を行うことにより、溶媒除去工程に供される前にＷ１／Ｏ"／Ｗ２が調製されるような手法であってもよい。 On the other hand, unlike the method described above, even if it is a W1 / O ′ emulsion that is subjected to the secondary emulsification step, the solvent removal step is performed by performing a predetermined treatment in the middle of the secondary emulsification step. It may be a technique in which W1 / O "/ W2 is prepared before being subjected to the above.
　　・水性溶媒（Ｗ１）・（Ｗ２）
　水性溶媒（Ｗ１）および（Ｗ２）は公知の一般的なものを用いることができる。一次乳化工程で用いられる水性溶媒（Ｗ１）はＷ１／Ｏエマルションの水相（内水相）をなし、二次乳化工程で用いられる水性溶媒（Ｗ２）はＷ１／Ｏ／Ｗ２エマルションの外水相をなす。水性溶媒としては、たとえば純水に、必要に応じて水と混合する他の溶媒、浸透圧調整のための塩類・糖類、ｐＨ調整のための緩衝液、その他の機能性成分（たとえば分散安定剤）などが添加された水溶液が用いられる。 ・ Aqueous solvent (W1) ・ (W2)
As the aqueous solvents (W1) and (W2), known general solvents can be used. The aqueous solvent (W1) used in the primary emulsification step constitutes the aqueous phase (inner aqueous phase) of the W1 / O emulsion, and the aqueous solvent (W2) used in the secondary emulsification step is the outer aqueous phase of the W1 / O / W2 emulsion. Make. Examples of the aqueous solvent include pure water, other solvents mixed with water as necessary, salts and saccharides for adjusting osmotic pressure, buffers for adjusting pH, and other functional components (for example, dispersion stabilizers). ) Or the like is used.
　内水相（Ｗ１）のｐＨは通常２～１０の範囲であり、混合脂質成分に応じて好ましい範囲に調整することができる。 The pH of the inner aqueous phase (W1) is usually in the range of 2 to 10, and can be adjusted to a preferable range according to the mixed lipid component.
　　・混合脂質成分（Ｆ１）・（Ｆ２）
　混合脂質成分（Ｆ１）は主としてリポソームの脂質二重膜の内膜を構成し、混合脂質成分（Ｆ２）は主として外膜を構成する。混合脂質成分（Ｆ１）および（Ｆ２）は、同一の組成であっても、異なる組成であってもよい。 ・ Mixed lipid component (F1) ・ (F2)
The mixed lipid component (F1) mainly constitutes the inner membrane of the lipid bilayer membrane of the liposome, and the mixed lipid component (F2) mainly constitutes the outer membrane. The mixed lipid components (F1) and (F2) may have the same composition or different compositions.
　これらの混合脂質成分の配合組成は特に限定されるものではなく、リポソームの製造用に用いられる各種の混合脂質成分を用いることができるが、一般的には、リン脂質（動植物由来のレシチン；ホスファチジルコリン、ホスファチジルセリン（ＤＰＰＳ）、ホスファチジルグリセロール（ＤＰＰＧ）、ホスファチジルイノシトール、ホスファチジン酸またはそれらの脂肪酸エステルであるグリセロリン脂質；スフィンゴリン脂質；これらの誘導体等）と、脂質膜の安定化に寄与するステロール類（コレステロール、フィトステロール、エルゴステロール、これらの誘導体等）とを中心に構成され、さらに糖脂質、グリコール、脂肪族アミン、長鎖脂肪酸（オレイン酸、ステアリン酸、パルミチン酸等）、その他各種の機能性を賦与する化合物が配合されていてもよい。本発明では、上記リン脂質としてジパルミトイルホスファチジルコリン（ＤＰＰＣ）、ジオレイルホスファチジルコリン（ＤＯＰＣ）等の中性リン脂質が慣用される。また、Ｆ２にＰＥＧ化リン脂質などＤＤＳとしての機能性の付与に必要な脂質成分を配合することで、リポソーム表面の効率的な修飾が可能となる。混合脂質成分の配合比も、脂質膜の安定性やリポソームの生体内での挙動などの性状を考慮しながら、用途に応じて適切に調整すればよい。 The composition of these mixed lipid components is not particularly limited, and various mixed lipid components used for the production of liposomes can be used. Generally, however, phospholipids (lecithin derived from animals and plants; phosphatidylcholine) , Phosphatidylserine (DPPS), phosphatidylglycerol (DPPG), phosphatidylinositol, phosphatidic acid or their fatty acid esters, glycerophospholipids; sphingophospholipids; derivatives thereof, etc.) and sterols that contribute to the stabilization of lipid membranes ( Cholesterol, phytosterol, ergosterol, derivatives of these, etc.), and glycolipids, glycols, aliphatic amines, long-chain fatty acids (oleic acid, stearic acid, palmitic acid, etc.) and other various functions To grant Objects may be blended. In the present invention, neutral phospholipids such as dipalmitoyl phosphatidylcholine (DPPC) and dioleyl phosphatidylcholine (DOPC) are commonly used as the phospholipid. In addition, it is possible to efficiently modify the liposome surface by blending F2 with a lipid component necessary for imparting functionality as a DDS, such as PEGylated phospholipid. The blending ratio of the mixed lipid component may be appropriately adjusted according to the application while taking into consideration properties such as the stability of the lipid membrane and the behavior of the liposome in vivo.
　　・内包させるべき物質
　本発明において、リポソームに内包させるべき物質（薬剤類と総称する）は特に限定されるものではなく、リポソームの用途に応じて医薬品、化粧品、食品などの分野で知られている各種の物質を用いることができる。 -Substances to be encapsulated In the present invention, substances to be encapsulated in liposomes (collectively referred to as drugs) are not particularly limited, and are known in the fields of pharmaceuticals, cosmetics, foods, etc. depending on the use of liposomes. Various substances can be used.
　本発明に使用する水溶性薬剤類は高い水溶性をもつものほどより高い内包率のリポソームを得ることができる。特に本発明に適する水溶性薬剤類としては、水１００ｍＬに対し薬剤１ｇまたは１ｍＬ以上が溶解するものであり、好ましくは１０ｇまたは１０ｍＬ以上が溶解するもの、さらに好ましくは１００ｇまたは１００ｍＬ以上が溶解するものである。なお、薬剤類によるリポソームの体積平均粒径やＣＶ、分散性への影響はあまり見られない。 The more water-soluble drugs used in the present invention have higher water solubility, the higher the encapsulation rate of liposomes. In particular, water-soluble drugs suitable for the present invention are those in which 1 g or 1 mL or more of drug dissolves in 100 mL of water, preferably 10 g or 10 mL or more, more preferably 100 g or 100 mL or more. It is. In addition, the volume average particle diameter, CV, and dispersibility of the liposome by the drugs are not so much seen.
　薬剤類のうち医療用の水溶性のものとしては、たとえば、造影剤（Ｘ線造影用の非イオン性ヨード化合物、ＭＲＩ造影用のガドリニウムとキレート化剤とからなる錯体等）、抗がん剤（アドリアマイシン、ビラルビシン、ビンクリスチン、タキソール、シスプラチン、マイトマイシン、５－フルオロウラシル、イリノテカン、エストラサイト、エピルビシン、カルボプラチン、イントロン、ジェムザール、メソトレキセート、シタラビン、アイソボリン、テガフール、シスプラチン、エトポシド、トポテシン、ビラルビシン、ネダプラチン、シクロホスファミド、メルファラン、イホスファミド、テスパミン、ニムスチン、ラニムスチン、ダカルバチン、エノシタビン、フルダラビン、ペントスタチン、クラドリビン、ダウノマイシン、アクラルビシン、イビルビシン、アムルビシン、アクチノマイシン、タキソテール、トラスツブマブ、リツキシマブ、ゲムツズマブ、レンチナン、シゾフィラン、インターフェロン、インターロイキン、アスパラギナーゼ、ホスフェストロール、ブスルファン、ボルテゾミブ、アリムタ、ベバシズマブ、ネララビン、セツキシマブ等）、抗菌剤（マクロライド系抗生物質、ケトライド系抗生物質、セファロスポリン系抗生物質、オキサセフェム系抗生物質、ペニシリン系抗生物質、ベータラクタマーゼ配合剤、アミノグリコシド系抗生物質、テトラサイクリン系抗生物質、ホスホマイシン系抗生物質、カルバペネム系抗生物質、ペネム系抗生物質）、MRSA・VRE・PRSP感染症治療剤、ポリエン系抗真菌剤、ピリミジン系抗真菌剤、アゾール系抗真菌剤、キャンディン系抗真菌剤、ニューキノロン系合成抗菌剤、抗酸化性剤、抗炎症剤、血行促進剤、美白剤、肌荒れ防止剤、老化防止剤、発毛促進性剤、保湿剤、ホルモン剤、ビタミン類、核酸（ＤＮＡもしくはＲＮＡのセンス鎖もしくはアンチセンス鎖、プラスミド、ベクター、ｍＲＮＡ、ｓｉＲＮＡ等）、タンパク質（酵素、抗体、ペプチド等）、ワクチン製剤（破傷風などのトキソイドを抗原とするもの；ジフテリア、日本脳炎、ポリオ、風疹、おたふくかぜ、肝炎などのウイルスを抗原とするもの；DNAまたはRNAワクチン等）などの薬理的作用を有する物質や、色素・蛍光色素、キレート化剤、安定化剤、保存剤などの製薬助剤が挙げられる。 Examples of water-soluble drugs for medical use include, for example, contrast agents (nonionic iodine compounds for X-ray contrast, complexes composed of gadolinium and chelating agents for MRI contrast), anticancer agents, and the like. (Adriamycin, Viralbicin, Vincristine, Taxol, Cisplatin, Mitomycin, 5-Fluorouracil, Irinotecan, Estrasite, Epirubicin, Carboplatin, Intron, Gemzar, Methotrexate, Cytarabine, Isoborine, Tegafur, Cisplatin, Etoposide, Topotine Famide, melphalan, ifosfamide, tespam, nimustine, ranimustine, dacarbatin, enositabine, fludarabine, pentostatin, cladribine, daunomycin, a Ralubicin, ibirubicin, amrubicin, actinomycin, taxotere, trastuzumab, rituximab, gemtuzumab, lentinan, schizophyllan, interferon, interleukin, asparaginase, phosfestol, busulfan, bortezomib, alimta, bevacizumab, nemarabine Ride antibiotics, ketolide antibiotics, cephalosporin antibiotics, oxacephem antibiotics, penicillin antibiotics, beta-lactamase combinations, aminoglycoside antibiotics, tetracycline antibiotics, fosfomycin antibiotics, carbapenem antibiotics Antibiotics, penem antibiotics), MRSA / VRE / PRSP infection treatment, polyene antifungal, pyrimidine antifungal, azole antifungal , Candin antifungal agent, new quinolone synthetic antibacterial agent, antioxidant agent, anti-inflammatory agent, blood circulation promoter, whitening agent, rough skin preventive agent, anti-aging agent, hair growth promoting agent, moisturizer, hormone agent, Vitamins, nucleic acids (DNA or RNA sense or antisense strands, plasmids, vectors, mRNA, siRNA, etc.), proteins (enzymes, antibodies, peptides, etc.), vaccine preparations (toxoids such as tetanus etc. as antigens; diphtheria , Japanese encephalitis, polio, rubella, mumps, hepatitis and other viruses as antigens; DNA or RNA vaccines, etc.), pharmacologically active substances, dyes / fluorescent dyes, chelating agents, stabilizers, storage And pharmaceutical aids such as pharmaceuticals.
　なお、水溶性の薬剤類をリポソームに内包させる方法としては、（ｉ）一次乳化工程の内水相（Ｗ１）に水溶性薬剤類をあらかじめ溶解または懸濁させておき、溶媒除去工程終了時点で当該水溶性薬剤類を内包するリポソームが得られるようにする方法、および（ｉｉ）水溶性薬剤類を内包しない空のリポソームを製造した後に、その空のリポソームの水性分散液に水溶性薬剤類を添加するか、あるいは空のリポソームを一旦乾燥粉末化し、それを水性溶媒に再分散させる際に水溶性薬剤類を添加することにより、リポソームに水溶性薬剤類を取り込ませる方法があり、いずれを用いることもできる。また、脂溶性の薬剤類も、上記（ｉ）に準じて一次乳化工程の油相（Ｏ）にあらかじめ溶解または懸濁させておく方法、上記（ｉｉ）のように空のリポソームの水性分散液に添加する方法により、リポソーム（脂質二重膜中）に内包させることができる。 As a method of encapsulating water-soluble drugs in liposomes, (i) water-soluble drugs are dissolved or suspended in advance in the inner aqueous phase (W1) of the primary emulsification step, and at the end of the solvent removal step. A method for obtaining a liposome encapsulating the water-soluble drug, and (ii) producing an empty liposome that does not encapsulate the water-soluble drug, and then adding the water-soluble drug to the aqueous dispersion of the empty liposome. There are methods to add water-soluble drugs to liposomes by adding them, or emptying liposomes once into dry powder and adding water-soluble drugs when redispersing them in an aqueous solvent. You can also Also, fat-soluble drugs are dissolved or suspended in advance in the oil phase (O) in the primary emulsification step according to (i) above, and an aqueous dispersion of empty liposomes as in (ii) above. Can be encapsulated in liposomes (in a lipid bilayer).
　　・分散安定剤
　二次乳化工程の外水相には、必要に応じて、薬剤類の内包率の向上および単胞リポソームの効率的な形成にさらに寄与しうる分散安定剤を配合してもよい。 -Dispersion stabilizer The outer aqueous phase of the secondary emulsification step may be blended with a dispersion stabilizer that can further contribute to the improvement of the encapsulation rate of drugs and the efficient formation of single-cell liposomes, if necessary. .
　たとえば、カゼインナトリウムのようなタンパク質乳化剤（水溶性乳化剤）は、従来用いられている分散安定剤の一つである。 For example, a protein emulsifier (water-soluble emulsifier) such as sodium caseinate is one of conventionally used dispersion stabilizers.
　また、自己による分子集合体（典型的にはミセル）を形成しない分散安定剤または自己による分子集合体を形成するがその体積平均粒径が１０ｎｍ以下である分散安定剤（以下これらを「特定分散安定剤」とよぶ。）は、分散安定剤としての効果に優れるとともに、必要に応じてリポソーム分散液から容易に分離除去することができるため好適である。 Further, a dispersion stabilizer that does not form a self-assembled molecular aggregate (typically micelle) or a self-assembled molecular aggregate that has a volume average particle size of 10 nm or less (hereinafter referred to as “specific dispersion”). The "stabilizer" is preferred because it is excellent in the effect as a dispersion stabilizer and can be easily separated and removed from the liposome dispersion liquid as necessary.
　代表的な特定分散安定剤としては、タンパク質、多糖類、イオン性界面活性剤および非イオン性界面活性剤が挙げられる。多糖類は、一次乳化物（Ｗ１／Ｏ）と外水相（Ｗ２）の界面への配向性が比較的小さいため外水相（Ｗ２）全体に分布し、Ｗ１／Ｏ／Ｗ２同士が接合しないようにすることでリポソームを安定化する。タンパク質および非イオン性界面活性剤は、Ｗ１／Ｏ／Ｗ２エマルションの界面への配向性が比較的高く、保護コロイドのようにエマルションを取り囲むことで安定化する。Ｗ１／Ｏ／Ｗ２同士が合一して粒径が大きくなると、液中乾燥法等による溶媒除去が不均一になり内包薬剤が漏れ出しやすくなるなどリポソームが不安定化してしまうが、前者の特定分散安定剤はそのような合一による不安定化を抑制することができ、単胞リポソームの形成効率および薬剤の内包率の向上に寄与する。また、Ｗ１／Ｏ／Ｗ２エマルションの界面に配向する後者の特定分散安定剤は、溶媒の除去にともないリポソームが形成されてゆく際に個々のリポソームを解けやすくすることができ、やはり単胞リポソームの形成効率および薬剤の内包率の向上に寄与する。 Typical specific dispersion stabilizers include proteins, polysaccharides, ionic surfactants and nonionic surfactants. Polysaccharides are distributed throughout the outer aqueous phase (W2) because the orientation to the interface between the primary emulsion (W1 / O) and the outer aqueous phase (W2) is relatively small, and W1 / O / W2 do not join together. By doing so, the liposome is stabilized. Proteins and nonionic surfactants are relatively highly oriented to the interface of the W1 / O / W2 emulsion and are stabilized by surrounding the emulsion like a protective colloid. When W1 / O / W2 are united and the particle size is increased, the removal of the solvent by in-liquid drying method etc. becomes non-uniform and the liposomes become unstable, such as the inclusion drug easily leaking out. The dispersion stabilizer can suppress the destabilization due to such coalescence, and contributes to the improvement of the formation efficiency of single cell liposomes and the encapsulation rate of the drug. In addition, the latter specific dispersion stabilizer oriented at the interface of the W1 / O / W2 emulsion can easily dissolve individual liposomes as the liposomes are formed as the solvent is removed. Contributes to the improvement of the formation efficiency and drug encapsulation rate.
　上記タンパク質としては、ゼラチン（コラーゲンを加熱により変性させた可溶性のタンパク質）、アルブミンやトリプシンなどが挙げられる。ゼラチンは通常、数千～数百万の分子量分布を有するが、たとえば重量平均分子量が１,０００～１００,０００であるものが好ましい。医療用ないし食品用として市販されているゼラチンを用いることができる。アルブミンには、卵アルブミン（分子量約４５,０００）、血清アルブミン（分子量約６６,０００…ウシ血清アルブミン）、乳アルブミン（分子量約１４,０００…α－ラクトアルブミン）などが含まれ、たとえば卵アルブミンである乾燥脱糖卵白が好ましい。 Examples of the protein include gelatin (a soluble protein obtained by denaturing collagen by heating), albumin and trypsin. Gelatin usually has a molecular weight distribution of several thousand to several million, but preferably has a weight average molecular weight of 1,000 to 100,000, for example. Gelatin commercially available for medical use or food use can be used. Albumin includes egg albumin (molecular weight about 45,000), serum albumin (molecular weight about 66,000 ... bovine serum albumin), milk albumin (molecular weight about 14,000 ... α-lactalbumin), etc. A dry desugared egg white is preferred.
　上記多糖類としては、デキストラン、デンプン、グリコーゲン、アガロース、ペクチン、キトサン、カルボキシメチルセルロースナトリウム、キサンタンガム、ローカストビーンガム、グァーガム、マルトトリオース、アミロース、プルラン、ヘパリン、デキストリンなどが挙げられ、たとえば重量平均分子量が１,０００～１００,０００のデキストランが好ましい。 Examples of the polysaccharide include dextran, starch, glycogen, agarose, pectin, chitosan, sodium carboxymethylcellulose, xanthan gum, locust bean gum, guar gum, maltotriose, amylose, pullulan, heparin, dextrin, and the like. Is preferably from 1,000 to 100,000.
　上記イオン性界面活性剤としては、コール酸ナトリウム、デオキシコール酸ナトリウムなどが挙げられる。 Examples of the ionic surfactant include sodium cholate and sodium deoxycholate.
　　上記非イオン性界面活性剤としては、オクチルグルコシド等のアルキルグリコシド、ポリアルキレンオキサイド系の化合物、たとえば「Tween 80」（東京化成工業株式会社，ポリオキシエチレンソルビタンモノオレアート，分子量1309.68）や「Pluronic F-68」（BASF、ポリオキシエチレン（160)ポリオキシプロピレン(30)グリコール、数平均分子量9600）の製品や、重量平均分子量が1000～100000のポリエチレングリコール類などが挙げられる。ポリエチレングリコール(PEG)類は、製品として「ユニルーブ」（日油株式会社）、GL4-400NP、GL4-800NP（日油株式会社）、PEG200,000(和光純薬)、マクロゴール（三洋化成工業株式会社）などが挙げられる。 Examples of the nonionic surfactant include alkyl glycosides such as octyl glucoside, polyalkylene oxide compounds such as “Tween 80” (Tokyo Chemical Industry Co., Ltd., polyoxyethylene sorbitan monooleate, molecular weight 1309.68) and “Pluronic”. F-68 "(BASF, polyoxyethylene (160) polyoxypropylene (30) glycol, number average molecular weight 9600), polyethylene glycols having a weight average molecular weight of 1000 to 100,000, and the like. Polyethylene glycol (PEG) products are "Unilube" (Nippon Oil Co., Ltd.), GL4-400NP, GL4-800NP (Nippon Oil Corporation), PEG200,000 (Wako Pure Chemical Industries), Macrogol (Sanyo Chemical Industries Co., Ltd.) Company).
　特定分散安定剤の外水相への添加量（特定分散安定剤の濃度）は、種類に応じて適切な範囲で調整すればよい。ある濃度で自己による分子集合体（体積平均粒径が１０ｎｍを超えるもの）を形成する物質であっても、添加量をその濃度に達しない範囲で調節すれば、特定分散安定剤として用いることができる。特定分散安定剤の種類によっては、濃度が高すぎると粒度分布計による測定に支障をきたすこともあるので、そのような支障をきたさない低めの範囲で濃度を調整することが好ましい。 The amount of the specific dispersion stabilizer added to the outer aqueous phase (the concentration of the specific dispersion stabilizer) may be adjusted within an appropriate range depending on the type. Even a substance that forms a self-assembled molecular aggregate (with a volume average particle size exceeding 10 nm) at a certain concentration can be used as a specific dispersion stabilizer if the addition amount is adjusted within a range not reaching the concentration. it can. Depending on the type of the specific dispersion stabilizer, if the concentration is too high, the measurement by the particle size distribution meter may be hindered. Therefore, it is preferable to adjust the concentration within a low range that does not cause such a hindrance.
　リポソームと特定分散安定剤を濾過工程により分離することを考慮すれば、特定分散安定剤の自己による分子集合体ないしそれらの集合物の体積平均粒径はリポソームの体積平均粒径の１／１０以下が好ましく、１／１００以下がより好ましい。 Considering that the liposome and the specific dispersion stabilizer are separated by the filtration step, the volume average particle size of the self-assembled molecular aggregate or the aggregate of the specific dispersion stabilizer is 1/10 or less of the volume average particle size of the liposome. Is preferable, and 1/100 or less is more preferable.
　特定分散安定剤の分子量は、小さすぎると脂質膜中に混入しやすくなってリポソームの形成を阻害するおそれがあり、逆に大きすぎるとＷ１／Ｏ／Ｗ２エマルションの外水相中への分散や界面への配向の速度が遅れてリポソームの合一や多胞リポソームの形成につながるおそれがある。そのため、特定分散安定剤の重量平均分子量は１，０００～１００，０００の範囲内にあることが好ましい。 If the molecular weight of the specific dispersion stabilizer is too small, it tends to be mixed into the lipid membrane and inhibits the formation of liposomes. Conversely, if the molecular weight is too large, the dispersion of the W1 / O / W2 emulsion in the outer aqueous phase may occur. There is a possibility that the orientation speed to the interface is delayed, leading to the coalescence of liposomes and the formation of multivesicular liposomes. Therefore, the weight average molecular weight of the specific dispersion stabilizer is preferably in the range of 1,000 to 100,000.
　－ リポソームの製造方法 －
　本発明のリポソームの製造方法は、下記のような一次乳化工程、二次乳化工程および溶媒除去工程を有し、必要に応じてその他の工程を適宜組み合わせることができるものである。各工程を行うためには、公知の装置・機器類その他適切な手段を用いればよい。また、各工程の手段の選び方によっては連続的に一次乳化から溶媒除去工程までを行うことが可能である。なお、本発明のリポソームの製造方法は、最終的に水性溶媒（外水相）中でリポソームが形成されるものであるので、自ずとリポソームの分散液の製造方法となり、さらに乾燥粉末化工程を設けることによりリポソームの乾燥粉末の製造方法とすることもできる。 -Manufacturing method of liposome-
The method for producing liposomes of the present invention has the following primary emulsification step, secondary emulsification step and solvent removal step, and can be appropriately combined with other steps as necessary. In order to perform each step, a known device / equipment or other appropriate means may be used. In addition, depending on how to select means for each step, it is possible to continuously perform the steps from primary emulsification to solvent removal step. In addition, since the liposome is finally formed in an aqueous solvent (outer aqueous phase), the liposome production method of the present invention is naturally a method for producing a liposome dispersion, and further includes a dry powdering step. By this, it can also be set as the manufacturing method of the dry powder of a liposome.
　本発明の製造方法は、好ましくは単胞リポソームを製造するためのものである。単胞リポソームの製造方法といった場合、その製造方法により得られるリポソームの中に多胞リポソームが一切存在してはならないという趣旨ではなく、主として単胞リポソームを製造することを目的として設計された製造方法であればよい。混合脂質成分の配合組成などの条件によっては、多胞リポソームが比較的できやすい場合もあるが、そのように多胞リポソームが混在しうるような状況であっても本発明の方法を適用することが可能であり、内包率の向上等の効果が得られる。 The production method of the present invention is preferably for producing single cell liposomes. In the case of a method for producing single-cell liposomes, it is not intended that multivesicular liposomes should be present in the liposomes obtained by the production method, but a production method designed mainly for the purpose of producing single-cell liposomes If it is. Depending on conditions such as the composition of the mixed lipid component, multivesicular liposomes may be relatively easily formed, but the method of the present invention should be applied even in such a situation where multivesicular liposomes may be mixed. It is possible to obtain effects such as improvement in the encapsulation rate.
　（１）一次乳化工程
　一次乳化工程は、有機溶媒（Ｏ）、水性溶媒（Ｗ１）、および混合脂質成分（Ｆ１）を乳化してＷ１／Ｏエマルションを調製する工程である。 (1) Primary emulsification step The primary emulsification step is a step of preparing a W1 / O emulsion by emulsifying the organic solvent (O), the aqueous solvent (W1), and the mixed lipid component (F1).
　Ｗ１／Ｏエマルションの調製方法としては、超音波乳化法、撹拌乳化法、膜乳化法、マイクロチャネル乳化法、高圧ホモジナイザーを用いた方法など、公知の方法を適用することができる。微小粒径の観点からは超音波乳化や高圧ホモジナイザーを用いた乳化が好ましい。また、熱などに対して不安定な薬剤を封入する際には、乳化に必要なエネルギーの小さいマイクロチャネル乳化法、ＳＰＧ膜などを用いた膜乳化法が好ましい。また、あらかじめ撹拌乳化等で大きな粒径のＷ１／Ｏエマルションを調製した後に、孔径の小さな膜を通過させることでより小さな粒径のＷ１／Ｏエマルションを調製するような、プレミックス膜乳化法を用いてもよい。 As a method for preparing the W1 / O emulsion, known methods such as an ultrasonic emulsification method, a stirring emulsification method, a membrane emulsification method, a microchannel emulsification method, and a method using a high-pressure homogenizer can be applied. From the viewpoint of the fine particle diameter, ultrasonic emulsification or emulsification using a high-pressure homogenizer is preferable. In addition, when encapsulating a drug that is unstable with respect to heat or the like, a microchannel emulsification method having a small energy required for emulsification, or a membrane emulsification method using an SPG film is preferable. In addition, after preparing a W1 / O emulsion having a large particle size by stirring and emulsifying in advance, a premix membrane emulsification method is prepared such that a W1 / O emulsion having a smaller particle size is prepared by passing through a membrane having a small pore size. It may be used.
　油相に少なくとも２種類の有機溶媒を含有するＷ１／Ｏエマルションを、二次乳化工程に供するようにするためには、一次乳化工程は、乳化処理の方法に応じて、（ｉ）あらかじめ調製された混合有機溶媒を用いて乳化処理に行うステップ、および／または（ii）Ｗ１／Ｏエマルションが調製されている途中または調製された後に、さらに有機溶媒を油相に添加するステップ、を含むようにしてもよい。 In order to use a W1 / O emulsion containing at least two kinds of organic solvents in the oil phase for the secondary emulsification step, the primary emulsification step is (i) prepared in advance according to the method of the emulsification treatment. And / or (ii) a step of further adding an organic solvent to the oil phase during or after the W1 / O emulsion is being prepared. Good.
　一次乳化工程における、Ｗ１／Ｏエマルションの平均粒子径、有機溶媒（Ｏ）に添加する混合脂質成分（Ｆ１）の割合、有機溶媒（Ｏ）と水性溶媒（Ｗ１）の体積比、その他の操作条件は、続く二次乳化工程の条件や最終的に調製するリポソームの態様などを考慮しながら、採用する乳化方法に応じて適宜調整することができる。通常、混合脂質成分（Ｆ１）の割合は有機溶媒（Ｏ）に対して１～５０質量％であり、有機溶媒（Ｏ）と水性溶媒（Ｗ１）の体積比は１００：１～１：２である。 In the primary emulsification step, the average particle size of the W1 / O emulsion, the ratio of the mixed lipid component (F1) added to the organic solvent (O), the volume ratio of the organic solvent (O) and the aqueous solvent (W1), and other operating conditions Can be appropriately adjusted according to the emulsification method to be employed, taking into consideration the conditions of the subsequent secondary emulsification step and the aspect of the liposome to be finally prepared. Usually, the ratio of the mixed lipid component (F1) is 1 to 50% by mass with respect to the organic solvent (O), and the volume ratio of the organic solvent (O) and the aqueous solvent (W1) is 100: 1 to 1: 2. is there.
　一次乳化工程では、薬剤類をリポソームに内包させるために、リポソームに内包させるべき物質（水溶性または脂溶性の薬剤類）を内水相（Ｗ１）または油相（Ｏ）に添加し、溶解または懸濁させるようにしてもよい。また、薬剤が脂溶性である場合は、Ｗ１／Ｏエマルション調製後に油相（Ｏ）に添加してもよい。なお、このような方法以外にも、薬剤類を内包しない空のリポソームの分散液を調製した後にそこに薬剤類を添加するか、あるいは一旦凍結乾燥粉末化されたリポソームを水性溶媒に再分散させる際に薬剤類を添加しても、薬剤類をリポソームに内包させることができる。 In the primary emulsification step, in order to encapsulate the drug in the liposome, a substance (water-soluble or fat-soluble drug) to be encapsulated in the liposome is added to the inner aqueous phase (W1) or the oil phase (O) and dissolved or dissolved. You may make it suspend. Moreover, when a chemical | medical agent is fat-soluble, you may add to an oil phase (O) after W1 / O emulsion preparation. In addition to this method, after preparing a dispersion of empty liposomes that do not encapsulate drugs, drugs are added thereto, or liposomes once lyophilized and powdered are redispersed in an aqueous solvent. Even when drugs are added, the drugs can be encapsulated in liposomes.
　（２）二次乳化工程
　二次乳化工程は、一次乳化工程で調製されたＷ１／Ｏエマルションおよび外水相（Ｗ２）をリポソーム用混合脂質成分（Ｆ２）を用いて乳化し、Ｗ１／Ｏ／Ｗ２エマルションを調製する工程である。 (2) Secondary emulsification step The secondary emulsification step emulsifies the W1 / O emulsion and the external aqueous phase (W2) prepared in the primary emulsification step using the mixed lipid component (F2) for liposome, and W1 / O / It is a step of preparing a W2 emulsion.
　二次乳化工程でＷ１／Ｏ／Ｗ２エマルションを調製するための方法としては、膜乳化法、マイクロチャネル乳化法、撹拌乳化法、液滴法、接触法などが公知であり、本発明では膜乳化法、マイクロチャネル乳化法および撹拌乳化法が好ましい。 As a method for preparing the W1 / O / W2 emulsion in the secondary emulsification step, a membrane emulsification method, a microchannel emulsification method, a stirring emulsification method, a droplet method, a contact method, and the like are known. Method, microchannel emulsification method and stirring emulsification method are preferred.
　本発明の製造方法においては、マイクロチャネル乳化法およびＳＰＧ膜を用いた膜乳化法が好適である。これらの乳化法は、乳化処理に大きなエネルギーを必要としないため、乳化操作時の液滴の崩壊や液滴からの内包物質の漏出を抑えることができ、さらに、流下する連続相に液滴が放出されて滞留することなく運ばれてゆくため、液滴同士の接触、凝集・合一を低減することができる。また、膜乳化法としては、あらかじめ大きな粒径のＷ１／Ｏ／Ｗ２エマルションを調製した後に、孔径の小さな膜を通過させることでより小さな粒径のＷ１／Ｏ／Ｗ２エマルションを調製するようなプレミックス膜乳化法を用いてもよい。プレミックス膜乳化法は、必要とされるエネルギーが小さく、また処理量が多く、リポソームの調製を迅速化することができるため好適である。 In the production method of the present invention, a microchannel emulsification method and a membrane emulsification method using an SPG membrane are suitable. Since these emulsification methods do not require a large amount of energy for the emulsification process, it is possible to suppress the collapse of the droplets during the emulsification operation and the leakage of the encapsulated material from the droplets. Since it is discharged and carried without staying, contact, aggregation and coalescence of droplets can be reduced. As a membrane emulsification method, a W1 / O / W2 emulsion having a large particle size is prepared in advance, and then a W1 / O / W2 emulsion having a smaller particle size is prepared by passing through a membrane having a small pore size. A mixed film emulsification method may be used. The premix membrane emulsification method is preferable because it requires a small amount of energy, requires a large amount of treatment, and can speed up the preparation of liposomes.
　上記水性溶媒（Ｗ２）、Ｗ１／Ｏエマルション、混合脂質成分（Ｆ２）、および必要に応じて用いられる分散安定剤の混合態様（添加順序等）は特に限定されるものではなく、適切な態様を選択すればよい。たとえばＦ２が主として水溶性脂質からなる場合、あらかじめそのようなＦ２（および必要に応じて分散安定剤）をＷ２に添加しておき、それにＷ１／Ｏエマルションを添加して乳化処理を行うことができる。また、Ｗ１／Ｏ／Ｗ２エマルションを調製した後、または後述の溶媒除去工程の後にＦ２を添加する方法も可能である。一方、Ｆ２が主として脂溶性脂質からなる場合、あらかじめそのようなＦ２をＷ１／Ｏエマルションの油相に添加しておき、それを必要に応じて分散安定剤が添加されているＷ２に添加して乳化処理を行うことができる。なお、混合脂質成分（Ｆ１）および（Ｆ２）が同一の組成であってもよい場合は、一次乳化工程の際に添加した混合脂質成分のうちＦ１としてＷ／Ｏ界面に配向しきれなかった余剰分を、二次乳化工程のＯ／Ｗ界面に配向させるべきＦ２とすることができる。 The mixing mode (addition order, etc.) of the aqueous solvent (W2), W1 / O emulsion, mixed lipid component (F2), and dispersion stabilizer used as necessary is not particularly limited. Just choose. For example, when F2 is mainly composed of a water-soluble lipid, such F2 (and a dispersion stabilizer as required) can be added to W2 in advance, and a W1 / O emulsion can be added thereto for emulsification. . Moreover, the method of adding F2 after preparing a W1 / O / W2 emulsion or after the below-mentioned solvent removal process is also possible. On the other hand, when F2 is mainly composed of fat-soluble lipids, such F2 is added to the oil phase of the W1 / O emulsion in advance, and it is added to W2 to which a dispersion stabilizer is added as necessary. An emulsification treatment can be performed. In the case where the mixed lipid components (F1) and (F2) may have the same composition, surplus that could not be fully oriented at the W / O interface as F1 among the mixed lipid components added during the primary emulsification step. The minute can be F2 to be oriented at the O / W interface of the secondary emulsification step.
　二次乳化工程における、Ｗ１／Ｏ／Ｗ２エマルションの体積平均粒子径、水性溶媒（Ｗ２）ないしＷ１／Ｏエマルションの有機溶媒（Ｏ）に添加する混合脂質成分（Ｆ２）の割合、Ｗ１／Ｏエマルションと水性溶媒（Ｗ２）の体積比、その他の操作条件は、最終的に調製するリポソームの用途などを考慮しながら適宜調節することができる。 In the secondary emulsification step, the volume average particle diameter of the W1 / O / W2 emulsion, the ratio of the mixed lipid component (F2) added to the organic solvent (O) of the aqueous solvent (W2) to the W1 / O emulsion, W1 / O emulsion The volume ratio of the aqueous solvent (W2) and other operating conditions can be appropriately adjusted in consideration of the application of the liposome to be finally prepared.
　・乳化基材の親水化処理
　上述した二次乳化工程において、マイクロチャネル乳化法、ＳＰＧ膜などを用いた膜乳化法を行う場合、それらの方法で用いる乳化基材（マイクロチャネル基板、ＳＰＧ膜等）は、親水性薬剤による表面処理がなされたものであることが好ましい。 -Hydrophilization treatment of emulsified base material In the above-described secondary emulsification step, when performing a membrane emulsification method using a microchannel emulsification method, an SPG film, etc., an emulsification base material (microchannel substrate, SPG film, etc.) used in those methods ) Is preferably subjected to a surface treatment with a hydrophilic drug.
　上記親水性薬剤としては、たとえば、シリカ前駆体モノマー、および表面に親水性基を有するシランカップリング剤（典型的には、親水性基として、ポリオール構造、ポリエーテル構造、ポリアミン構造、３級アミン構造および４級アンモニウム構造のうち少なくとも１つを有するもの）が挙げられる。 Examples of the hydrophilic agent include a silica precursor monomer and a silane coupling agent having a hydrophilic group on the surface (typically, the hydrophilic group includes a polyol structure, a polyether structure, a polyamine structure, a tertiary amine. Having at least one of a structure and a quaternary ammonium structure).
　このような親水性薬剤を用いた処理により、乳化基材の空気中での水との接触角を、好ましくは０～５０°、より好ましくは０～４２°、さらに好ましくは０～３５°にすることができる。 By such treatment using a hydrophilic agent, the contact angle of the emulsified substrate with water in the air is preferably 0 to 50 °, more preferably 0 to 42 °, and still more preferably 0 to 35 °. can do.
　乳化基材を用いてＷ／ＯエマルションからＷ／Ｏ／Ｗエマルションを製造する一つの方法に、Ｗ／Ｏエマルションを含む油相と外水相とが乳化基材をはさんで隔離された系が用いられる。ここで、Ｗ／Ｏエマルションは、油相と外水相との間に設けられた圧力差によって、乳化基材に形成された孔に進入し、この孔を通過することによって乳化基材を透過する。孔の出口に達し外水相と接したＷ／Ｏエマルションは、その後液滴を形成し、この液滴が孔の出口から外水相に向けて成長する。そして、この液滴が成長するにつれて、この液滴が孔の出口付近で次第にくびれてゆき、しまいにはこの液滴が乳化基材から分離して外水層中に分散する。このようにＷ／Ｏエマルションからなる液滴がくびれて乳化基材から分離する現象は、液滴と外水相と乳化基材表面との間に働く表面張力などの相互作用によって生じると考えられる。 In one method for producing a W / O / W emulsion from a W / O emulsion using an emulsifying base material, a system in which an oil phase containing the W / O emulsion and an external aqueous phase are separated by sandwiching the emulsifying base material Is used. Here, the W / O emulsion enters the holes formed in the emulsified base material by the pressure difference provided between the oil phase and the external water phase, and permeates the emulsified base material by passing through the holes. To do. The W / O emulsion that has reached the outlet of the hole and is in contact with the outer aqueous phase then forms droplets that grow from the outlet of the holes toward the outer aqueous phase. As the droplet grows, the droplet gradually narrows in the vicinity of the outlet of the hole, and eventually the droplet is separated from the emulsified base material and dispersed in the outer water layer. Thus, the phenomenon that the droplet made of the W / O emulsion is constricted and separated from the emulsified substrate is considered to be caused by the interaction such as the surface tension acting between the droplet, the outer water phase, and the emulsified substrate surface. .
　ところが、従来公知のマイクロチャネル基板等の乳化基材を用いてＷ／Ｏ／Ｗエマルションを製造する場合、Ｗ／Ｏエマルションが連続流出するために液滴の粒径が大きくなり、所期の粒径を有するＷ／Ｏエマルションが分散したＷ／Ｏ／Ｗエマルションを作成できない場合があるという問題点がある。その理由として、外水相に接する部分の乳化基材表面における親水性が充分ではないために、Ｗ／Ｏエマルションからなる液滴が乳化基材表面に沿って拡大することが挙げられる。このような現象が生じると、外水相に放出される液滴の巨大化を招き、あるいは、互いに隣接し合う複数の孔の出口からの液滴が合体することにより外水相に接する部分の乳化基材表面にＷ／Ｏエマルションからなる層が生成し、外水相への液滴の放出が起こらなくなる場合がある。 However, when a W / O / W emulsion is produced using a conventionally known emulsifying base material such as a microchannel substrate, the particle size of the droplets increases because the W / O emulsion continuously flows out, and the desired particles There is a problem in that a W / O / W emulsion in which a W / O emulsion having a diameter is dispersed cannot be prepared. The reason for this is that droplets made of a W / O emulsion expand along the surface of the emulsified substrate because the hydrophilicity on the surface of the emulsified substrate in the portion in contact with the outer aqueous phase is not sufficient. When such a phenomenon occurs, the droplets discharged to the outer aqueous phase become enormous, or the droplets from the outlets of a plurality of holes adjacent to each other merge to form a portion in contact with the outer aqueous phase. A layer composed of a W / O emulsion may be generated on the surface of the emulsified base material, and droplets may not be released to the outer aqueous phase.
　ここで、外水相に接する部分の乳化基材表面、少なくとも、孔の出口およびその周辺部における乳化基材表面に充分な親水性を付与すれば、外水相に接する部分の乳化基材表面の全体が外水相に濡れることとなり、これによって、Ｗ／Ｏエマルションからなる液滴が乳化基材表面に沿って拡大することなく乳化基材から分離しやすくなる。さらに、乳化基材における孔の内部の親水性も高ければ、外水相とＷ／Ｏエマルションからなる液滴との界面が孔の出口を超えて孔の内部に達しやすくなり、これによりＷ／Ｏエマルションからなる液滴がくびれやすくなり、結果、Ｗ／Ｏエマルションからなる液滴が乳化基材からさらに分離しやすくなると考えられる。 Here, the surface of the emulsified substrate in contact with the outer aqueous phase, at least the surface of the emulsified substrate in contact with the outer aqueous phase, if sufficient hydrophilicity is imparted to the surface of the emulsified substrate at the outlet of the hole and its peripheral part. As a result, the droplets made of the W / O emulsion are easily separated from the emulsified base material without expanding along the surface of the emulsified base material. Furthermore, if the hydrophilicity inside the pores in the emulsified substrate is also high, the interface between the outer aqueous phase and the droplets made of the W / O emulsion will easily reach the inside of the pores beyond the outlet of the pores. It is considered that the droplets composed of the O emulsion are easily constricted, and as a result, the droplets composed of the W / O emulsion are further easily separated from the emulsified substrate.
　（３）溶媒除去工程
　溶媒除去工程は、二次乳化工程により調製されたＷ１／Ｏ／Ｗ２エマルションから油相（Ｏ）の有機溶媒を除去し、混合脂質成分（Ｆ１）および（Ｆ２）からなる脂質二重膜を有するリポソームを形成させる工程である。有機溶媒の除去の進行につれて、リポソームを構成する脂質の水和が進み、多胞リポソームが解けて単胞のリポソーム状態にばらけるか、またはＷ１／Ｏ／Ｗ２エマルションの界面に近い位置から単胞のリポソームがちぎれて形成されるものと考えられる。本発明において、油相が少なくとも２種類の有機溶媒を含有することは、そのようなメカニズムによりリポソームが形成される際に良好な影響を与えるものと考えられる。 (3) Solvent removal step The solvent removal step comprises removing the organic solvent of the oil phase (O) from the W1 / O / W2 emulsion prepared by the secondary emulsification step, and consisting of mixed lipid components (F1) and (F2). This is a step of forming a liposome having a lipid bilayer. As the removal of the organic solvent proceeds, the hydration of the lipids constituting the liposome progresses, and the multivesicular liposomes are dissolved and dispersed into the single-cell liposome state, or the single cells from a position close to the interface of the W1 / O / W2 emulsion. It is considered that the liposomes are torn and formed. In the present invention, the fact that the oil phase contains at least two kinds of organic solvents is considered to have a favorable effect when liposomes are formed by such a mechanism.
　溶媒除去の方法としては、たとえばエバポレータで蒸発させる方法や液中乾燥法が挙げられる。液中乾燥法は、Ｗ１／Ｏ／Ｗ２エマルションを回収し、開放容器内に移して静置あるいは撹拌することで、Ｗ１／Ｏ／Ｗ２エマルションに含まれる有機溶媒（Ｏ）を蒸発除去する方法である。 Examples of the solvent removal method include a method of evaporating with an evaporator and a method of drying in liquid. The in-liquid drying method is a method in which the organic solvent (O) contained in the W1 / O / W2 emulsion is evaporated and removed by collecting the W1 / O / W2 emulsion, transferring it to an open container and allowing it to stand or stir. is there.
　この際、さらに加温や減圧によって溶媒除去を促進することができる。温度条件や減圧条件は、常法に従って水が揮発し過ぎない範囲で、用いる有機溶媒の種類などに応じて適宜調整すればよい。温度条件は、たとえば０～６０℃の範囲が好ましく、０～２５℃がより好ましい。また、減圧条件は溶媒の飽和蒸気圧～大気圧の範囲内に設定されることが好ましく、溶媒の飽和蒸気圧の＋１％～１０％の範囲内に設定されることがより好ましい。なお、有機溶媒が突沸するような条件であってもリポソームの調製は可能である。また、溶媒除去の際には温度条件と減圧条件とを組み合わせてもよい。たとえば、熱に弱い薬剤を使用する際は、より低温側でかつ減圧条件で溶媒を除去することが好ましい。 At this time, the solvent removal can be further promoted by heating or decompression. The temperature condition and the reduced pressure condition may be appropriately adjusted according to the type of the organic solvent to be used and the like within a range in which water does not volatilize in accordance with a conventional method. For example, the temperature condition is preferably in the range of 0 to 60 ° C., more preferably 0 to 25 ° C. The decompression condition is preferably set within the range of the saturated vapor pressure of the solvent to atmospheric pressure, and more preferably within the range of + 1% to 10% of the saturated vapor pressure of the solvent. Note that liposomes can be prepared even under conditions where the organic solvent bumps. Moreover, you may combine temperature conditions and pressure reduction conditions in the case of solvent removal. For example, when using a heat-sensitive chemical, it is preferable to remove the solvent at a lower temperature and under reduced pressure.
　本発明では、油相（Ｏ）は少なくとも２種類の有機溶媒を含有するが、飽和蒸気圧の高い有機溶媒に合わせた条件（より低温および／またはより小さな減圧）から飽和蒸気圧の低い有機溶媒に合わせた条件（より高温および／またはより大きな減圧）に段階的に条件を変化させ、最終的に全ての種類の有機溶媒を除去するようにすることが好ましい。 In the present invention, the oil phase (O) contains at least two kinds of organic solvents. However, the organic solvent having a low saturated vapor pressure can be obtained under conditions (lower temperature and / or smaller reduced pressure) suitable for the organic solvent having a higher saturated vapor pressure. It is preferable to change the conditions step by step to the conditions (higher temperature and / or higher pressure reduction) adapted to the above, and finally remove all kinds of organic solvents.
　また、溶媒除去の際にはＷ１／Ｏ／Ｗ２エマルションを攪拌しなくともよいが、撹拌すればより均一に溶媒除去が進み、気液界面が広がることで溶媒除去に係る時間も短縮される。二次乳化工程において撹拌乳化法によりＷ１／Ｏ／Ｗ２エマルションを調製した場合は、その後さらに撹拌を継続して溶媒を除去するといったように、二次乳化工程と溶媒除去工程を連続的に行うこともできる。 Also, it is not necessary to stir the W1 / O / W2 emulsion when removing the solvent. However, if the stirring is performed, the removal of the solvent proceeds more uniformly, and the time required for removing the solvent is shortened by spreading the gas-liquid interface. When the W1 / O / W2 emulsion is prepared by the stirring emulsification method in the secondary emulsification step, the secondary emulsification step and the solvent removal step are continuously performed so that the stirring is then continued to remove the solvent. You can also.
　二段階乳化法によって得られるリポソームには、Ｗ１／Ｏ／Ｗ２エマルション由来の多胞リポソームがある程度の割合含まれることがあるが、これを減じるためにも、撹拌または減圧、好ましくはそれらを組み合わせて行うことがより効果的である。溶媒の大半が除去される時間より長く減圧および／または撹拌を行なうことが重要であり、それにより、リポソームを構成する脂質の水和が進み、多胞リポソームが解けて、単胞リポソームの状態にばらけると考えられる。 Liposomes obtained by the two-stage emulsification method may contain a certain percentage of W1 / O / W2 emulsion-derived multivesicular liposomes. To reduce this, stirring or decompression, preferably combining them, It is more effective to do. It is important to perform decompression and / or stirring longer than the time when most of the solvent is removed, so that the hydration of the lipids constituting the liposome proceeds and the multivesicular liposomes are dissolved to form a single-vesicle liposome. It is thought that it will break.
　（４）その他の工程
　必要に応じて用いられるその他の工程としては、リポソームの粒径を所望の範囲（たとえば５０～５００ｎｍ程度）に調整し、Ｗ１／Ｏ／Ｗ２エマルションから副次的に形成された多胞リポソームをばらして単胞リポソームにすることができる、フィルターを用いる整粒工程や、外水相中にある遊離した薬剤や分散剤を除く分離工程、リポソームを保管に適した形態にする（使用時に水性溶媒中に再分散させる）ための乾燥粉末化工程、リポソーム粒径が十分に小さいときに限るがろ過滅菌工程、など、従来のリポソームの製造にも用いられていた各種の工程が挙げられる。特に驚くべきことに、上記目的での整粒工程の操作において、フィルターでのリポソームの捕集や内包物の漏出はほとんど起こらない。このような操作をしても多胞リポソームが残った場合には、粒子除去用のフィルターにより捕集・除去することができる。これらの工程は、溶媒除去工程の後に設けられ、溶媒除去工程から引き続き連続的に行われるようにしてもよい。 (4) Other steps As other steps used as necessary, the liposome particle size is adjusted to a desired range (for example, about 50 to 500 nm), and is formed as a secondary from the W1 / O / W2 emulsion. Multivesicular liposomes can be separated into single-cell liposomes, a sizing process using a filter, a separation process that removes free drugs and dispersants in the external aqueous phase, and a liposome suitable for storage Various processes that have also been used in the production of conventional liposomes, such as a dry powdering process for redispersion in an aqueous solvent at the time of use, and a filter sterilization process only when the liposome particle size is sufficiently small. Can be mentioned. Particularly surprisingly, in the operation of the sizing process for the above purpose, the collection of liposomes and leakage of inclusions hardly occur. If multivesicular liposomes remain after such an operation, they can be collected and removed by a filter for particle removal. These steps may be provided after the solvent removal step and continuously performed after the solvent removal step.
　－ リポソームの形態および用途 －
　本発明の製造方法により最終的に製造されるリポソームの形態や用途は特に限定されるものではないが、たとえば医療用のリポソーム製剤として用いる場合は、体積平均粒径は好ましくは５０～１，０００ｎｍであり、より好ましくは５０～３００ｎｍである。このようなサイズのリポソームは、毛細血管を閉塞するおそれがほとんどなく、またがん組織近辺の血管にできる間隙を通過することもできるため、医薬品等として人体に投与されて使用する上で好都合である。 -Liposomes and uses-
The form and application of the liposome finally produced by the production method of the present invention are not particularly limited. However, when used as a liposome preparation for medical use, the volume average particle diameter is preferably 50 to 1,000 nm. More preferably, it is 50 to 300 nm. Liposomes with such a size have little risk of occluding capillaries and can pass through gaps formed in blood vessels in the vicinity of cancer tissue, so they are convenient for use by being administered to the human body as pharmaceuticals. is there.
　以下に述べる実施例および比較例で得られたリポソームの内包率は、下記の方法に従って測定した。 The encapsulation rate of the liposomes obtained in the examples and comparative examples described below was measured according to the following method.
　（カルセインの内包率の測定方法）
　リポソーム水溶液（３ｍＬ）全体の蛍光強度（Ｆtotal）を分光光度計（Ｕ－３３１０、日本分光株式会社）により測定した。次に０．０１Ｍ，ＣｏＣｌ2トリス塩酸緩衝液３０μＬを加えて外水相に漏出したカルセインの蛍光をＣｏ2+により消光することで、リポソーム内の蛍光強度（Ｆin）を測定した。さらに、カルセインを加えないでサンプルと同じ条件でリポソームを作製し、脂質自身が発する蛍光（Ｆl）を測定した。内包率は下記式より算出した；
　内包率Ｅ（％）　＝　(Ｆin－Ｆl)／(Ｆtotal－Ｆl)×１００
　（siRNAの内包率の測定方法）
　リポソーム液を超遠心分離して外液とリポソームを分離し、それぞれのｓｉＲＮＡ量をＨＰＬＣで測定することで、ｓｉＲＮＡの内包率を求めた。 (Measurement method of calcein inclusion rate)
The fluorescence intensity (F total ) of the entire liposome aqueous solution (3 mL) was measured with a spectrophotometer (U-3310, JASCO Corporation). Next, 30 μL of 0.01 M CoCl 2 Tris-HCl buffer was added, and the fluorescence intensity (F in ) in the liposome was measured by quenching the fluorescence of calcein leaked into the outer aqueous phase with Co 2+ . Furthermore, liposomes were prepared under the same conditions as the sample without adding calcein, and the fluorescence (F 1 ) emitted by the lipids themselves was measured. The inclusion rate was calculated from the following formula;
Inclusion rate E (%) = (F in −F l ) / (F total −F l ) × 100
(Method for measuring the encapsulation rate of siRNA)
The liposome solution was ultracentrifuged to separate the external solution and the liposome, and the amount of each siRNA was measured by HPLC to determine the siRNA encapsulation rate.
　（粒度分布の測定方法）
　以下に述べる各種試料として得られたＷ１／Ｏエマルションならびに実施例および比較例で得られたリポソームの体積平均粒径およびＣＶ値は、下記の方法に従って測定した。 (Measuring method of particle size distribution)
The volume average particle diameter and CV value of the W1 / O emulsions obtained as various samples described below and the liposomes obtained in Examples and Comparative Examples were measured according to the following methods.
　Ｗ１／Ｏエマルションをヘキサン／ジクロロメタン混合有機溶媒（体積比：１／１）で１０倍に希釈し、動的光散乱式ナノトラック粒度分析計（ＵＰＡ－ＥＸ１５０、日機装株式会社）を用いて粒度分布を測定し、これに基づき体積平均粒径およびＣＶ値（＝（標準偏差／体積平均粒径）×１００［％］）を算出した。また、リポソームの体積平均粒径は、同装置を用いて、作製したリポソーム懸濁液をそのまま測定した。 Dilute W1 / O emulsion 10 times with hexane / dichloromethane mixed organic solvent (volume ratio: 1/1) and use a dynamic light scattering nanotrack particle size analyzer (UPA-EX150, Nikkiso Co., Ltd.) Based on this, the volume average particle diameter and the CV value (= (standard deviation / volume average particle diameter) × 100 [%]) were calculated. Moreover, the volume average particle diameter of the liposome was measured as it was for the prepared liposome suspension using the same apparatus.
　（混合有機溶媒を用いた一次乳化工程によるＷ１／Ｏエマルションの製造）
　試料１
　ジパルミトイルホスファチジルコリン（ＤＰＰＣ）「COATSOME MC-6060」（日油株式会社）１．５ｇ、およびジパルミトイルホスファチジルグリセロール（ＤＰＰＧ）「COATSOME MG-6060LA」（日油株式会社）０．２５ｇを含む混合有機溶媒（ヘキサン／ジクロロメタン＝３／１）１５ｍＬを有機溶媒相（Ｏ）とし、カルセイン（０．４ｍＭ）を含むトリス－塩酸緩衝液（ｐＨ７．４、５０ｍＭ）５ｍＬを内水相用の水分散相（Ｗ１）とした。５０ｍＬのビーカーにこれらの混合液を入れ、φ２０ｍｍのプローブをセットした超音波分散装置（ＵＨ－６００Ｓ、株式会社エスエムテー）により、２０℃にて１５分間超音波を照射し乳化処理を行った。上記方法に従って測定したところ、この一次乳化工程で得られたＷ１／Ｏエマルションは体積平均粒径約１９０ｎｍ、ＣＶ値３３％の単分散Ｗ／Ｏエマルションであることが確認され、分散性は良好であった。室温下で３時間静置しても同様の体積平均粒径・ＣＶ値・分散性を示した。光学顕微鏡観察では、単分散なエマルションを確認することは困難であり、緩やかな凝集が若干確認されたが、時間による変化は見られなかった（図１）。 (Production of W1 / O emulsion by primary emulsification process using mixed organic solvent)
Sample 1
Mixed organic solvent containing 1.5 g of dipalmitoylphosphatidylcholine (DPPC) “COATSOME MC-6060” (NOF Corporation) and 0.25 g of dipalmitoylphosphatidylglycerol (DPPG) “COATSOME MG-6060LA” (NOF Corporation) (Hexane / dichloromethane = 3/1) 15 mL of the organic solvent phase (O), and 5 mL of tris-hydrochloric acid buffer (pH 7.4, 50 mM) containing calcein (0.4 mM) in the aqueous dispersion phase for the inner aqueous phase ( W1). These mixed liquids were put into a 50 mL beaker and subjected to an emulsification treatment by irradiating ultrasonic waves at 20 ° C. for 15 minutes with an ultrasonic dispersion apparatus (UH-600S, SMT Co., Ltd.) equipped with a φ20 mm probe. When measured according to the above method, it was confirmed that the W1 / O emulsion obtained in this primary emulsification step was a monodispersed W / O emulsion having a volume average particle size of about 190 nm and a CV value of 33%, and the dispersibility was good. there were. The same volume average particle diameter, CV value, and dispersibility were exhibited even after standing at room temperature for 3 hours. Observation with an optical microscope made it difficult to confirm a monodisperse emulsion, and although slight aggregation was confirmed, no change with time was observed (FIG. 1).
　試料２
　用いた混合有機溶媒をヘキサン／ジクロロメタン＝６／４に換え、試料１と同様の操作を行った。得られたＷ１／Ｏエマルションは体積平均粒径１７０ｎｍ、ＣＶ値３２％の単分散Ｗ／Ｏエマルションであることが確認され、その分散性は良好であった。室温下で３時間静置したところ体積平均粒径１９０ｎｍと僅かに合一が進んだが、ＣＶ値・分散性に変化は無かった。また、光学顕微鏡観察では、ほとんど凝集が確認されず、時間による変化は見られなかった。 Sample 2
The mixed organic solvent used was changed to hexane / dichloromethane = 6/4, and the same operation as in Sample 1 was performed. The obtained W1 / O emulsion was confirmed to be a monodispersed W / O emulsion having a volume average particle size of 170 nm and a CV value of 32%, and its dispersibility was good. When the mixture was allowed to stand at room temperature for 3 hours, the coalescence progressed slightly with a volume average particle size of 190 nm, but the CV value and dispersibility were not changed. Moreover, almost no aggregation was confirmed by observation with an optical microscope, and no change with time was observed.
　試料３
　用いた混合有機溶媒をヘキサン／ジクロロメタン＝９／１に換え、試料１と同様の操作を行った。得られたＷ１／Ｏエマルションは体積平均粒径２００ｎｍ、ＣＶ値３８％の単分散Ｗ／Ｏエマルションであることが確認され、その分散性は良好であった。室温下で３時間静置したところ体積平均粒径・ＣＶ値・分散性に変化は無かった。また、光学顕微鏡観察では、緩やかな凝集が確認されたが、時間による変化は見られなかった。 Sample 3
The mixed organic solvent used was changed to hexane / dichloromethane = 9/1, and the same operation as in Sample 1 was performed. The obtained W1 / O emulsion was confirmed to be a monodispersed W / O emulsion having a volume average particle size of 200 nm and a CV value of 38%, and its dispersibility was good. After standing at room temperature for 3 hours, there was no change in the volume average particle diameter, CV value, and dispersibility. In addition, in the optical microscope observation, although gentle aggregation was confirmed, no change with time was observed.
　試料４
　用いた混合有機溶媒をヘキサン／ジクロロメタン＝５／５に換え、試料１と同様の操作を行った。得られたＷ１／Ｏエマルションは体積平均粒径１６０ｎｍ、ＣＶ値３３％のＷ／Ｏエマルションであることが確認され、その分散性は良好であった。室温下で３時間静置したところＣＶ値・分散性に変化は無かったものの、体積平均粒径が４００ｎｍと合一がやや進んでいた。光学顕微鏡観察では凝集は確認されず、単分散のエマルションを確認することは困難であったが、３時間後には合一して微細なエマルションが確認された。 Sample 4
The mixed organic solvent used was changed to hexane / dichloromethane = 5/5, and the same operation as Sample 1 was performed. The obtained W1 / O emulsion was confirmed to be a W / O emulsion having a volume average particle size of 160 nm and a CV value of 33%, and its dispersibility was good. When the mixture was allowed to stand at room temperature for 3 hours, the CV value and dispersibility were not changed, but the volume average particle diameter was 400 nm and the coalescence was slightly advanced. Aggregation was not confirmed by observation with an optical microscope, and it was difficult to confirm a monodisperse emulsion, but after 3 hours, a fine emulsion was confirmed by uniting.
　比較試料１
　用いた混合有機溶媒をヘキサンのみに換え、試料１と同様の操作を行った。得られたＷ１／Ｏエマルションは体積平均粒径２５０ｎｍ、ＣＶ値４５％のＷ／Ｏエマルションであることが確認されたが、その分散性は悪く静置するとすぐに沈降した。室温下で３時間静置したところ体積平均粒径・ＣＶ値・分散性に変化は無かった。光学顕微鏡観察ではかなりの凝集が確認され、また、粒度分布測定とは異なり不均一なＷ／Ｏエマルションが観測されたが、時間による変化は見られなかった（図２）。 Comparative sample 1
The mixed organic solvent used was changed to hexane only, and the same operation as Sample 1 was performed. The obtained W1 / O emulsion was confirmed to be a W / O emulsion having a volume average particle size of 250 nm and a CV value of 45%, but its dispersibility was poor and settled immediately after standing. After standing at room temperature for 3 hours, there was no change in the volume average particle diameter, CV value, and dispersibility. In the optical microscope observation, considerable aggregation was confirmed. Unlike the particle size distribution measurement, a non-uniform W / O emulsion was observed, but no change with time was observed (FIG. 2).
　比較試料２
　用いた混合有機溶媒をジクロロメタンのみに換え、試料１と同様の操作を行った。得られたＷ１／Ｏエマルションは体積平均粒径１２０ｎｍ、ＣＶ値３４％のＷ／Ｏエマルションであり、その分散性は良好であったが、室温下で３０分間静置のみでＣＶ値・分散性に変化は無かったものの、体積平均粒径が１μｍ以上と合一が非常に進んでしまった。光学顕微鏡観察では凝集は確認されないものの、エマルションが合一して巨大化する様子が確認され、３時間後には合一した数μｍのエマルションが確認された（図３）。 Comparative sample 2
The mixed organic solvent used was changed to dichloromethane only, and the same operation as Sample 1 was performed. The obtained W1 / O emulsion was a W / O emulsion having a volume average particle size of 120 nm and a CV value of 34%. The dispersibility was good, but the CV value / dispersibility was only allowed to stand at room temperature for 30 minutes. Although there was no change, the coalescence was very advanced when the volume average particle size was 1 μm or more. Although aggregation was not confirmed by observation with an optical microscope, it was confirmed that the emulsions were united and enlarged, and after 3 hours, a united emulsion of several μm was confirmed (FIG. 3).
　試料５～１３
　表１に記載のように混合有機溶媒の組成を変更する以外は、試料１と同様の操作を行った。各評価の結果を表１に示す。 Samples 5-13
The same operation as Sample 1 was performed except that the composition of the mixed organic solvent was changed as described in Table 1. The results of each evaluation are shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　（混合有機溶媒を用いたリポソームの製造）
　実施例１
　（二次乳化工程によるＷ１／Ｏ／Ｗ２エマルションの製造）
　試料１により得られたＷ１／Ｏエマルションを分散相として、ＳＰＧ膜乳化法によるＷ１／Ｏ／Ｗ２エマルションの製造を行った。ＳＰＧ膜乳化装置（ＳＰＧテクノ社製、商品名「外圧式マイクロキット」）に直径１０ｍｍ、長さ２０ｍｍ、細孔径１０μｍの円筒形ＳＰＧ膜を用い、装置出口側に外水相溶液（Ｗ２）である０．１％のプルロニックＦ６８を含むトリス－塩酸緩衝液（ｐＨ７．４、５０ｍＭ）を満たしておき、装置入口側から上記Ｗ１／Ｏエマルションを供給して、Ｗ１：Ｗ２が１：４０となるようにＷ１／Ｏ／Ｗ２エマルションを製造した。このとき、ＳＰＧ膜より連続流出などは起こらずに液滴が形成され、乳化後もこのエマルションは安定であった（図８）。 (Production of liposomes using mixed organic solvents)
Example 1
(Production of W1 / O / W2 emulsion by secondary emulsification process)
Using the W1 / O emulsion obtained from Sample 1 as a dispersed phase, a W1 / O / W2 emulsion was produced by the SPG membrane emulsification method. A cylindrical SPG membrane having a diameter of 10 mm, a length of 20 mm, and a pore diameter of 10 μm was used for an SPG membrane emulsifying device (trade name “external pressure type micro kit” manufactured by SPG Techno Co.), and an external aqueous phase solution (W2) was used on the outlet side of the device. A tris-hydrochloric acid buffer solution (pH 7.4, 50 mM) containing 0.1% Pluronic F68 is filled, and the W1 / O emulsion is supplied from the apparatus inlet side, so that W1: W2 becomes 1:40. A W1 / O / W2 emulsion was prepared as described above. At this time, droplets were formed without any continuous outflow from the SPG film, and the emulsion was stable even after emulsification (FIG. 8).
　尚、ＳＰＧ膜は事前にＮ－（３－トリエトキシシリルプロピル）グルコンアミドで表面修飾し、より親水化させたものを用いた。グルコンアミド修飾ＳＰＧ膜の調製方法は次の通りである。ＳＰＧテクノ社製の直径１０ｍｍ、長さ２０ｍｍ、細孔径１０μｍの円筒形ＳＰＧ膜を有機溶媒（エタノールなど）により脱脂、超純水にて洗浄および乾燥させた後、２０倍重量のエタノールで希釈したＮ－（３－トリエトキシシリルプロピル）グルコンアミド液（Gelest社製：カタログ番号SIT8189）に浸漬させ、５０℃で２時間反応させ、その後、１００℃で３時間加熱乾燥させた。 In addition, the SPG film was used that was surface-modified with N- (3-triethoxysilylpropyl) gluconamide in advance to make it more hydrophilic. The preparation method of the gluconamide-modified SPG membrane is as follows. A cylindrical SPG membrane having a diameter of 10 mm, a length of 20 mm and a pore diameter of 10 μm manufactured by SPG Techno was degreased with an organic solvent (such as ethanol), washed with ultrapure water and dried, and then diluted with 20 times the weight of ethanol. It was immersed in an N- (3-triethoxysilylpropyl) gluconamide solution (Gelest, catalog number SIT8189), reacted at 50 ° C. for 2 hours, and then heated and dried at 100 ° C. for 3 hours.
　（有機溶媒相の除去によるリポソームの製造）
　上記二次乳化工程により得られたＷ１／Ｏ／Ｗ２エマルションを密閉容器に移し替え、２０℃・５００ｍｂａｒの減圧条件下で約８時間攪拌し、次いで２０℃・１８０ｍｂａｒの減圧条件下で約８時間撹拌し、段階的に溶媒を揮発させた。得られたリポソーム懸濁液は半透明の黄色であり、この粒子内にはカルセインが含まれていることが確認された。得られたリポソームの体積平均粒径は８０ｎｍであり、ＣＶ値は３９％であった。リポソームのカルセイン内包率は８４％であった（図９）。 (Production of liposomes by removal of organic solvent phase)
The W1 / O / W2 emulsion obtained by the secondary emulsification step was transferred to a closed container and stirred for about 8 hours under a reduced pressure condition of 20 ° C. and 500 mbar, and then for about 8 hours under a reduced pressure condition of 20 ° C. and 180 mbar. Stir and volatilize the solvent stepwise. The obtained liposome suspension was translucent yellow, and it was confirmed that calcein was contained in the particles. The resulting liposome had a volume average particle size of 80 nm and a CV value of 39%. The calcein encapsulation rate of the liposome was 84% (FIG. 9).
　なお、得られたリポソーム懸濁液中のヘキサン・ジクロロメタンの残留溶媒量をGC/MS（検出下限１０ｐｐｍ）を用いて測定したところ、ヘキサン・ジクロロメタンともに検出されず、リポソーム懸濁液中にほとんど残留していないことを確認した。 In addition, when the amount of residual solvent of hexane / dichloromethane in the obtained liposome suspension was measured using GC / MS (detection lower limit 10 ppm), neither hexane / dichloromethane was detected, and almost no residual in the liposome suspension. I confirmed that I did not.
　また、二次乳化工程における外水相溶液中の０．１％プルロニックＦ６８の代わりに、０．０１％プルロニックＦ６８や３％デキストラン（分子量１０万）、３％精製ゼラチン（株式会社ニッピ，ニッピ　ハイグレードゼラチンタイプAP）および０．１％コール酸ナトリウムをそれぞれ添加剤に用いても、同様に微細なリポソーム懸濁液を得ることができている。 Also, instead of 0.1% pluronic F68 in the outer aqueous phase solution in the secondary emulsification step, 0.01% pluronic F68, 3% dextran (molecular weight 100,000), 3% purified gelatin (Nippi Corporation, Nippi High) Even when each of grade gelatin type AP) and 0.1% sodium cholate is used as an additive, a fine liposome suspension can be obtained.
　実施例２
　試料１により得られたＷ１／Ｏエマルションを分散相として、実施例１と同様の液組成で撹拌乳化法によるＷ１／Ｏ／Ｗ２エマルションの製造を行った。撹拌乳化は、スターラーによりＷ２を強く撹拌しているところに、上記Ｗ１／Ｏエマルションを供給し、Ｗ１／Ｏ／Ｗ２エマルションを製造した。 Example 2
Using the W1 / O emulsion obtained from Sample 1 as a dispersed phase, a W1 / O / W2 emulsion was produced by the stirring emulsification method with the same liquid composition as in Example 1. In the stirring emulsification, the W1 / O emulsion was supplied to a place where W2 was vigorously stirred with a stirrer to produce a W1 / O / W2 emulsion.
　次いで実施例１と同様の方法で有機溶媒を除去し、微細なリポソーム粒子の懸濁液を得た。得られたリポソームの体積平均粒径は８５ｎｍであり、ＣＶ値は４３％であった。リポソームのカルセイン内包率は８２％であった。 Subsequently, the organic solvent was removed by the same method as in Example 1 to obtain a suspension of fine liposome particles. The obtained liposome had a volume average particle diameter of 85 nm and a CV value of 43%. The calcein encapsulation rate of the liposome was 82%.
　実施例３
　試料１により得られたＷ１／Ｏエマルションを分散相として、実施例２と同様の操作でＷ１／Ｏ／Ｗ２エマルションを得たうえで、ＳＰＧ膜乳化装置を用いたプレミックス膜乳化を行った。ＳＰＧ膜の細孔径は１μｍを用い、窒素ガスを用いて４ＭＰａで加圧して処理し、体積平均粒径０．８μｍ前後のＷ１／Ｏ／Ｗ２エマルションを製造した。 Example 3
A W1 / O / W2 emulsion was obtained in the same manner as in Example 2 using the W1 / O emulsion obtained from Sample 1 as a dispersed phase, and then premix membrane emulsification was performed using an SPG membrane emulsifier. The pore diameter of the SPG membrane was 1 μm, and it was processed by pressurizing at 4 MPa with nitrogen gas to produce a W1 / O / W2 emulsion having a volume average particle size of about 0.8 μm.
　次いで実施例１と同様の方法で有機溶媒を除去し、微細なリポソーム粒子の懸濁液を得た。得られたリポソームの体積平均粒径は７５ｎｍであり、ＣＶ値は３７％であった。リポソームのカルセイン内包率は８１％であった。 Subsequently, the organic solvent was removed by the same method as in Example 1 to obtain a suspension of fine liposome particles. The obtained liposome had a volume average particle diameter of 75 nm and a CV value of 37%. The calcein encapsulation rate of the liposome was 81%.
　比較例１
　比較試料１により得られたＷ１／Ｏエマルションを分散相として、実施例１と同様にＳＰＧ膜乳化法によるＷ１／Ｏ／Ｗ２エマルションの製造を試みたが、乳化開始から数分で乳化が止まり、Ｗ１／Ｏ／Ｗ２を効率的に調製できなかった。この原因は膜のＷ１／Ｏエマルションによる目詰まりであった。 Comparative Example 1
Using the W1 / O emulsion obtained by Comparative Sample 1 as a dispersed phase, an attempt was made to produce a W1 / O / W2 emulsion by the SPG membrane emulsification method in the same manner as in Example 1, but the emulsification stopped in a few minutes from the start of emulsification, W1 / O / W2 could not be prepared efficiently. This was due to clogging of the membrane by the W1 / O emulsion.
　比較例２
　比較試料１により得られたＷ１／Ｏエマルションを分散相として、実施例２と同様に撹拌乳化によるＷ１／Ｏ／Ｗ２エマルションの製造を行い、次いで有機溶媒の除去を行った。リポソームの体積平均粒径は１７０ｎｍであり、ＣＶ値は＞５０％で単分散ではなかった。光学顕微鏡での観察では、多胞リポソームが複数観察された。リポソームのカルセイン内包率は５１％であった（図１０）。 Comparative Example 2
Using the W1 / O emulsion obtained from Comparative Sample 1 as a dispersed phase, a W1 / O / W2 emulsion was produced by stirring emulsification in the same manner as in Example 2, and then the organic solvent was removed. The volume average particle size of the liposomes was 170 nm, the CV value was> 50% and was not monodispersed. In observation with an optical microscope, a plurality of multivesicular liposomes were observed. The calcein encapsulation rate of the liposome was 51% (FIG. 10).
　比較例３
　比較試料２により得られたＷ１／Ｏエマルションを分散相として、実施例１と同様に二次乳化にＳＰＧ膜乳化法を用いたリポソームの製造を試みたが、二次乳化の処理が進むにつれてＳＰＧ膜が目詰まりを起こしてしまい、所定量のＷ１／Ｏ／Ｗ２エマルションを得ることができなかった。 Comparative Example 3
Using the W1 / O emulsion obtained from Comparative Sample 2 as a dispersed phase, an attempt was made to produce liposomes using the SPG membrane emulsification method for secondary emulsification as in Example 1. However, as the secondary emulsification process proceeds, SPG The film was clogged, and a predetermined amount of W1 / O / W2 emulsion could not be obtained.
　比較例４
　比較試料２により得られたＷ１／Ｏエマルションを分散相として、実施例２と同様に二次乳化に撹拌乳化を用いたリポソームの製造を行った。得られたリポソームの体積平均粒径は＞２μｍとかなり粗大になっており、ＣＶ値は＞５０％であった。リポソームのカルセイン内包率は６７％であった。 Comparative Example 4
Using the W1 / O emulsion obtained from Comparative Sample 2 as a dispersed phase, liposomes were prepared using stirring emulsification for secondary emulsification in the same manner as in Example 2. The volume average particle diameter of the obtained liposomes was considerably large as> 2 μm, and the CV value was> 50%. The calcein encapsulation rate of the liposome was 67%.
　実施例４
　ＤＰＰＣに換えて卵黄レシチン「COATSOME NC-50」（日油株式会社）を用いたこと以外は試料１と同様の操作を行った。得られたＷ１／Ｏエマルション（試料１４）の分散性は良好であった。試料１４のＷ１／Ｏエマルションを分散相として用いることを除いては、実施例１と同様にリポソームを作製した。得られたリポソームの体積平均粒径は１８５ｎｍであり、ＣＶ値は４４％であった。リポソームのカルセイン内包率は８７％であった。光学顕微鏡観察では、実施例１とは異なり、多胞リポソームが複数あることを確認できた（図１１）。 Example 4
The same operation as that of Sample 1 was performed except that yolk lecithin “COATSOME NC-50” (NOF Corporation) was used instead of DPPC. The dispersibility of the obtained W1 / O emulsion (sample 14) was good. Liposomes were prepared in the same manner as in Example 1 except that the W1 / O emulsion of Sample 14 was used as the dispersed phase. The obtained liposome had a volume average particle diameter of 185 nm and a CV value of 44%. The calcein encapsulation rate of the liposome was 87%. In the optical microscope observation, unlike Example 1, it was confirmed that there were a plurality of multivesicular liposomes (FIG. 11).
　比較例５
　ＤＰＰＣに換えて卵黄レシチン「COATSOME NC-50」（日油株式会社）を用いたこと以外は比較試料１と同様の操作を行った。得られたＷ１／Ｏエマルション（比較試料３）のの分散性は悪く、静置するとすぐに沈降した。比較試料３により得られたＷ１／Ｏエマルションを分散相として用いることを除いては、実施例２と同様の操作を行い、リポソームを得た。得られたリポソームの体積平均粒径は２７０ｎｍであり、ＣＶ値は＞５０％であった。リポソームのカルセイン内包率は４５％であった。光学顕微鏡観察では、かなりの個数の多胞リポソームがあることを確認できた（図１２）。 Comparative Example 5
The same operation as that of Comparative Sample 1 was performed except that yolk lecithin “COATSOME NC-50” (NOF Corporation) was used instead of DPPC. The dispersibility of the obtained W1 / O emulsion (Comparative Sample 3) was poor and settled immediately after standing. Except for using the W1 / O emulsion obtained by Comparative Sample 3 as a dispersed phase, the same operation as in Example 2 was performed to obtain liposomes. The resulting liposome had a volume average particle size of 270 nm and a CV value of> 50%. The liposome had a calcein encapsulation rate of 45%. Observation with an optical microscope confirmed that there were a considerable number of multivesicular liposomes (FIG. 12).
　比較例６
　比較試料３により得られたＷ１／Ｏエマルションを分散相として用い、また撹拌乳化法の代わりにＳＰＧ膜乳化を用いることを除いては、実施例２と同様の操作を行い、Ｗ１／Ｏ／Ｗ２エマルションを得ようとしたところ、比較例１と同様にＳＰＧ膜が目詰まりし、乳化を行うことができなかった。 Comparative Example 6
The same operation as in Example 2 was carried out except that the W1 / O emulsion obtained from Comparative Sample 3 was used as a dispersed phase, and SPG membrane emulsification was used instead of the stirring emulsification method, and W1 / O / W2 When trying to obtain an emulsion, the SPG film was clogged as in Comparative Example 1, and emulsification could not be performed.
　実施例５
　試料１のＷ１／Ｏエマルションを調製した後に、ＰＥＧ修飾リン脂質（ＤＳＰＥ－ＰＥＧ２０００）「SUNBRIGHT DSPE-020CN」０．０７５ｇを添加することを除いては、実施例１と同様にリポソームを作製した。得られたリポソームの体積平均粒径は１３５ｎｍであり、ＣＶ値は４１％であった。リポソームのカルセイン内包率は８０％であった。 Example 5
Liposomes were prepared in the same manner as in Example 1 except that 0.075 g of PEG-modified phospholipid (DSPE-PEG2000) “SUNBRIGHT DSPE-020CN” was added after preparing the W1 / O emulsion of Sample 1. The resulting liposome had a volume average particle size of 135 nm and a CV value of 41%. The calcein encapsulation rate of the liposome was 80%.
　実施例６～１４
　試料５～試料１３のＷ１／Ｏエマルションを用いて、溶媒に合わせた減圧条件を設定した以外は実施例１と同様の操作を行い、リポソームを得た。得られたリポソームの物性を表２に示す。 Examples 6-14
Using the W1 / O emulsions of Samples 5 to 13, liposomes were obtained in the same manner as in Example 1 except that the decompression conditions were set according to the solvent. Table 2 shows the physical properties of the obtained liposomes.
　実施例１５
　用いた混合有機溶媒をヘキサン／ギ酸エチル＝８/２に換えたこと以外は、試料１と同様の操作を行った。得られたＷ１／Ｏエマルション（試料１５）の分散性は良好であり、室温下で３時間静置したところ分散性に変化はなかった。試料１５のＷ１／Ｏエマルションを用いて、溶媒に合わせた減圧条件を設定することを除いて、実施例１と同様にリポソームを作製した。リポソームの体積平均粒径は１００ｎｍ・ＣＶ値は４３％であり、カルセイン内包率は７６％であった。また、途中で得られるＷ１／Ｏエマルションは単分散なエマルションで分散安定性が高く、３時間での変化は見られなかった。 Example 15
The same operation as Sample 1 was carried out except that the mixed organic solvent used was changed to hexane / ethyl formate = 8/2. The dispersibility of the obtained W1 / O emulsion (Sample 15) was good, and when it was allowed to stand at room temperature for 3 hours, there was no change in the dispersibility. Liposomes were prepared in the same manner as in Example 1, except that the W1 / O emulsion of Sample 15 was used to set the reduced pressure conditions according to the solvent. The volume average particle size of the liposome was 100 nm · CV value was 43%, and the calcein encapsulation rate was 76%. In addition, the W1 / O emulsion obtained in the middle was a monodispersed emulsion with high dispersion stability, and no change was observed in 3 hours.
　実施例１６
　用いた混合有機溶媒をシクロヘキサン／酢酸イソプロピル＝７／３に換えたこと以外は、試料１と同様の操作を行った。得られたＷ１／Ｏエマルション（試料１６）の分散性は良好であり、室温下で３時間静置したところ分散性に変化はなかった。試料１６のＷ１／Ｏエマルションを用いて、溶媒に合わせた減圧条件を設定することを除いて、実施例１と同様にリポソームを作製した。リポソームの体積平均粒径は７５ｎｍ・ＣＶ値は４１％であり、カルセイン内包率は６７％であった。また、途中で得られるＷ１／Ｏエマルションは単分散なエマルションで分散安定性が高く、３時間での変化は見られなかった。 Example 16
The same operation as Sample 1 was performed except that the mixed organic solvent used was changed to cyclohexane / isopropyl acetate = 7/3. The dispersibility of the obtained W1 / O emulsion (Sample 16) was good, and when it was allowed to stand at room temperature for 3 hours, there was no change in the dispersibility. Liposomes were prepared in the same manner as in Example 1 except that the reduced pressure condition according to the solvent was set using the W1 / O emulsion of Sample 16. The volume average particle diameter of the liposome was 75 nm · CV value was 41%, and the calcein encapsulation rate was 67%. In addition, the W1 / O emulsion obtained in the middle was a monodispersed emulsion with high dispersion stability, and no change was observed in 3 hours.
　実施例１７
　用いた混合有機溶媒をヘキサン／酢酸メチル＝３／１に換えたこと以外は、試料１と同様の操作を行った。得られたＷ１／Ｏエマルション（試料１７）の分散性は良好であり、室温下で３時間静置したところ分散性に変化はなかった。試料１７のＷ１／Ｏエマルションを用いて、溶媒に合わせた減圧条件を設定することを除いて、実施例１と同様にリポソームを作製した。リポソームの体積平均粒径は７０ｎｍ・ＣＶ値は３８％であり、カルセイン内包率は７１％であった。また、途中で得られるＷ１／Ｏエマルションは単分散なエマルションで分散安定性が高く、３時間での変化は見られなかった。 Example 17
The same operation as Sample 1 was performed except that the mixed organic solvent used was changed to hexane / methyl acetate = 3/1. The dispersibility of the obtained W1 / O emulsion (Sample 17) was good, and when it was allowed to stand at room temperature for 3 hours, there was no change in the dispersibility. Liposomes were prepared in the same manner as in Example 1 except that the W1 / O emulsion of Sample 17 was used to set the reduced pressure conditions according to the solvent. The volume average particle size of the liposome was 70 nm · CV value was 38%, and the calcein encapsulation rate was 71%. In addition, the W1 / O emulsion obtained in the middle was a monodispersed emulsion with high dispersion stability, and no change was observed in 3 hours.
　実施例１８
　用いた混合有機溶媒をヘキサン／アセトニトリル＝５／１に換えたこと以外は、試料１と同様の操作を行った。得られたＷ１／Ｏエマルション（試料１８）の分散性は良好であり、室温下で３時間静置したところ分散性に変化はなかった。試料１８のＷ１／Ｏエマルションを用いて、溶媒に合わせた減圧条件を設定することを除いて、実施例１と同様にリポソームを作製した。リポソームの体積平均粒径は１００ｎｍ・ＣＶ値は４７％であり、カルセイン内包率は６１％であった。また、途中で得られるＷ１／Ｏエマルションは単分散なエマルションで分散安定性が高く、３時間での変化は見られなかった。 Example 18
The same operation as Sample 1 was performed except that the mixed organic solvent used was changed to hexane / acetonitrile = 5/1. The dispersibility of the obtained W1 / O emulsion (Sample 18) was good, and when it was allowed to stand at room temperature for 3 hours, there was no change in the dispersibility. Liposomes were prepared in the same manner as in Example 1, except that the W1 / O emulsion of Sample 18 was used to set a reduced pressure condition according to the solvent. The volume average particle size of the liposome was 100 nm · CV value was 47%, and the calcein encapsulation rate was 61%. In addition, the W1 / O emulsion obtained in the middle was a monodispersed emulsion with high dispersion stability, and no change was observed in 3 hours.
　実施例１９
　用いた混合有機溶媒をペンタン／メタノール＝９５／５に換えたこと以外は、試料１と同様の操作を行った。得られたＷ１／Ｏエマルション（試料１９）の分散性は良好であり、室温下で３時間静置したところ分散性に変化はなかった。試料１９のＷ１／Ｏエマルションを用いて、溶媒に合わせた減圧条件を設定することを除いて、実施例１と同様にリポソームを作製した。リポソームの体積平均粒径は９５ｎｍ・ＣＶ値は４１％であり、カルセイン内包率は６４％であった。また、途中で得られるＷ１／Ｏエマルションは単分散なエマルションで分散安定性が高く、３時間での変化は見られなかった。 Example 19
The same operation as Sample 1 was performed except that the mixed organic solvent used was changed to pentane / methanol = 95/5. The dispersibility of the obtained W1 / O emulsion (Sample 19) was good, and when it was allowed to stand at room temperature for 3 hours, there was no change in the dispersibility. Liposomes were prepared in the same manner as in Example 1 except that the reduced pressure conditions according to the solvent were set using the W1 / O emulsion of Sample 19. The volume average particle diameter of the liposome was 95 nm · CV value was 41%, and the calcein encapsulation rate was 64%. In addition, the W1 / O emulsion obtained in the middle was a monodispersed emulsion with high dispersion stability, and no change was observed in 3 hours.
　実施例２０
　用いた混合有機溶媒をヘキサン／アセトン＝８／２に換えたこと以外は、試料１と同様の操作を行った。得られたＷ１／Ｏエマルション（試料２０）の分散性は良好であり、室温下で３時間静置したところ分散性に変化はなかった。試料２０のＷ１／Ｏエマルションを用いたことを除いて、実施例１と同様にリポソームを作製した。リポソームの体積平均粒径は１４０ｎｍ・ＣＶ値は４１％であり、カルセイン内包率は７５％であった。また、途中で得られるＷ１／Ｏエマルションは単分散なエマルションで分散安定性が高く、３時間での変化は見られなかった。 Example 20
The same operation as Sample 1 was performed except that the mixed organic solvent used was changed to hexane / acetone = 8/2. The dispersibility of the obtained W1 / O emulsion (Sample 20) was good, and when it was allowed to stand at room temperature for 3 hours, there was no change in the dispersibility. Liposomes were prepared in the same manner as in Example 1 except that the W1 / O emulsion of Sample 20 was used. The volume average particle diameter of the liposome was 140 nm · CV value was 41%, and the calcein encapsulation rate was 75%. In addition, the W1 / O emulsion obtained in the middle was a monodispersed emulsion with high dispersion stability, and no change was observed in 3 hours.
　実施例２１
　カルセインに換えて２００μｇ／ｍＬのｓｉＲＮＡ（ランダム配列）を用いたこと以外は、試料１と同様の操作を行った。得られたＷ１／Ｏエマルション（試料２１）の分散性は良好であった。試料２１のＷ１／Ｏエマルションを用いたことを除いては、実施例１と同様にリポソームを作製した。得られたリポソーム液は白色の半透明であり、体積平均粒径は７０ｎｍ・ＣＶ値は３９％であった。リポソーム中のｓｉＲＮＡ内包率は８０％であった。調製は、リポソーム分散液としてのｓｉＲＮＡ濃度が計算上５μｇ／ｍＬとなるようにしたところ、実際に得られたｓｉＲＮＡ濃度は４．５μｇ／ｍＬであった。 Example 21
The same operation as Sample 1 was performed except that 200 μg / mL siRNA (random sequence) was used instead of calcein. The dispersibility of the obtained W1 / O emulsion (Sample 21) was good. Liposomes were prepared in the same manner as in Example 1 except that the W1 / O emulsion of Sample 21 was used. The obtained liposome liquid was white and translucent, and the volume average particle diameter was 70 nm · CV value was 39%. The siRNA encapsulation rate in the liposome was 80%. In the preparation, when the siRNA concentration as the liposome dispersion was calculated to be 5 μg / mL, the actually obtained siRNA concentration was 4.5 μg / mL.
　実施例２２
　ＤＰＰＣ１．５ｇおよびＤＰＰＧ０．２５ｇが溶解したヘキサン１１．２５ｍＬを有機溶媒相（Ｏ）としたこと以外は試料１と同様の操作を行い、超音波乳化法で一次乳化処理を行った後、ジクロロメタン３．７５ｍＬを添加した。得られたＷ１／Ｏエマルション（試料２２）の分散性は良好であった。試料２２のＷ１／Ｏエマルションを用いたことを除いては、実施例２と同様にリポソームを作製した。得られたリポソームの体積平均粒径は１４０ｎｍであり、ＣＶ値は４１％であった。また、リポソームのカルセイン内包率は８３％であった。 Example 22
After performing primary emulsification by the ultrasonic emulsification method, except that 11.25 mL of hexane in which 1.5 g of DPPC and 0.25 g of DPPG were dissolved was used as the organic solvent phase (O), dichloromethane 3 .75 mL was added. The dispersibility of the obtained W1 / O emulsion (Sample 22) was good. Liposomes were prepared in the same manner as in Example 2 except that the W1 / O emulsion of Sample 22 was used. The obtained liposome had a volume average particle diameter of 140 nm and a CV value of 41%. The calcein encapsulation rate of the liposome was 83%.
　以上のすべての実施例および比較例において得られたリポソームは単胞リポソームであることが顕微鏡により確認できた。 It was confirmed by a microscope that the liposomes obtained in all of the above Examples and Comparative Examples were single cell liposomes.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003

Claims (19)

  1. 　内水相（Ｗ１）および油相（Ｏ）をリポソーム用混合脂質成分（Ｆ１）を用いて乳化しＷ１／Ｏエマルションを調製する一次乳化工程と、
    　Ｗ１／Ｏエマルションおよび外水相（Ｗ２）をリポソーム用混合脂質成分（Ｆ２）を用いて乳化しＷ１／Ｏ／Ｗ２エマルションを調製する二次乳化工程と、
    　Ｗ１／Ｏ／Ｗ２エマルションから油相（Ｏ）の有機溶媒を除去してリポソームを形成させる溶媒除去工程とを含むリポソームの製造方法であって、
    　溶媒除去工程に供されるＷ１／Ｏ／Ｗ２エマルションの油相（Ｏ）が少なくとも２種類の有機溶媒を含有する混合有機溶媒であることを特徴とするリポソームの製造方法。     A primary emulsification step of emulsifying the inner aqueous phase (W1) and the oil phase (O) with the mixed lipid component for liposome (F1) to prepare a W1 / O emulsion;
    A secondary emulsification step of emulsifying the W1 / O emulsion and the outer aqueous phase (W2) with the mixed lipid component for liposome (F2) to prepare a W1 / O / W2 emulsion;
    A method for producing liposomes, comprising a solvent removal step of removing the organic solvent of the oil phase (O) from the W1 / O / W2 emulsion to form liposomes,
    A method for producing liposomes, wherein the oil phase (O) of the W1 / O / W2 emulsion subjected to the solvent removal step is a mixed organic solvent containing at least two kinds of organic solvents.
  2. 　前記リポソームが単胞リポソームである、請求項１に記載のリポソームの製造方法。 The method for producing a liposome according to claim 1, wherein the liposome is a single cell liposome.
  3. 　前記混合有機溶媒が、クロロホルム、シクロヘキサン、ジクロロメタン、ヘキサン、t‐ブチルメチルエーテル、酢酸エチル、ジエチルエーテル、ギ酸エチル、酢酸イソプロピル、酢酸メチル、メチルエチルケトン、ペンタン、アセトニトリル、メタノール、アセトン、エタノール、２‐プロパノールからなる群より選択される少なくとも２種類の有機溶媒を含有するものである、請求項１または２に記載のリポソームの製造方法。 The mixed organic solvent is chloroform, cyclohexane, dichloromethane, hexane, t-butyl methyl ether, ethyl acetate, diethyl ether, ethyl formate, isopropyl acetate, methyl acetate, methyl ethyl ketone, pentane, acetonitrile, methanol, acetone, ethanol, 2-propanol. The method for producing a liposome according to claim 1 or 2, comprising at least two types of organic solvents selected from the group consisting of:
  4. 　前記混合有機溶媒が、炭化水素の有機溶媒を含有する、請求項１または２に記載のリポソームの製造方法。 The method for producing a liposome according to claim 1 or 2, wherein the mixed organic solvent contains a hydrocarbon organic solvent.
  5. 　前記混合有機溶媒が、下記の溶媒Ａ群及び溶媒Ｂ群のそれぞれより少なくとも１種類ずつ選択される少なくとも２種類の有機溶媒を含有する、請求項１または２に記載のリポソームの製造方法。
    　溶媒Ａ群：炭化水素
    　溶媒Ｂ群：エーテル、ハロゲン化炭化水素、ハロゲン化エーテル、エステル、アルコール、ケトンおよびアセトニトリル     The method for producing a liposome according to claim 1 or 2, wherein the mixed organic solvent contains at least two kinds of organic solvents selected from at least one of each of the following solvent A group and solvent B group.
    Solvent group A: hydrocarbon Solvent group B: ether, halogenated hydrocarbon, halogenated ether, ester, alcohol, ketone and acetonitrile
  6. 　前記溶媒Ａ群が、ペンタン、ヘキサン及びシクロヘキサンからなる、請求項５に記載のリポソームの製造方法。 The method for producing a liposome according to claim 5, wherein the solvent group A comprises pentane, hexane and cyclohexane.
  7. 　前記溶媒Ｂ群が、ジエチルエーテル、ｔ－ブチルメチルエーテル、クロロホルム、ジクロロメタン、ギ酸エチル、酢酸メチル、酢酸イソプロピル、メタノール、エタノール、２－プロパノール、メチルエチルケトン及びアセトリニトリルからなる、請求項５または６に記載のリポソームの製造方法。 The solvent B group comprises diethyl ether, t-butyl methyl ether, chloroform, dichloromethane, ethyl formate, methyl acetate, isopropyl acetate, methanol, ethanol, 2-propanol, methyl ethyl ketone and acetonitrile. The manufacturing method of the liposome of description.
  8. 　前記混合有機溶媒に含まれる前記溶媒Ａの体積比率が５０～９９％である、請求項５～７のいずれかに記載のリポソームの製造方法。 The method for producing liposomes according to any one of claims 5 to 7, wherein the volume ratio of the solvent A contained in the mixed organic solvent is 50 to 99%.
  9. 　前記混合有機溶媒が、ヘキサン：クロロホルム＝６０：４０～９０：１０（体積比、合計を１００とする）からなるものである、請求項３に記載のリポソームの製造方法。 The method for producing liposomes according to claim 3, wherein the mixed organic solvent is composed of hexane: chloroform = 60: 40 to 90:10 (volume ratio, total is 100).
  10. 　前記混合有機溶媒が、ヘキサン：酢酸エチル＝５０：５０～９０：１０（体積比、合計を１００とする）からなるものである、請求項３に記載のリポソームの製造方法。 The method for producing a liposome according to claim 3, wherein the mixed organic solvent is composed of hexane: ethyl acetate = 50: 50 to 90:10 (volume ratio, total is 100).
  11. 　前記混合有機溶媒が、ヘキサン：エタノール＝７０：３０～９９：１（体積比、合計を１００とする）からなるものである、請求項３に記載のリポソームの製造方法。 The method for producing liposomes according to claim 3, wherein the mixed organic solvent is composed of hexane: ethanol = 70:30 to 99: 1 (volume ratio, total is 100).
  12. 　前記一次乳化工程が、あらかじめ調製された混合有機溶媒を用いて乳化処理に行うステップ、および／またはＷ１／Ｏエマルションが調製されている途中または調製された後にさらに有機溶媒を油相に添加するステップを含むものである、請求項１～１１のいずれかに記載のリポソームの製造方法。 The primary emulsification step is performed in an emulsification process using a mixed organic solvent prepared in advance, and / or the organic solvent is further added to the oil phase during or after the W1 / O emulsion is being prepared. The method for producing liposomes according to any one of claims 1 to 11, which comprises
  13. 　前記一次乳化工程における乳化法が膜乳化法またはマイクロチャネル乳化法である、請求項１～１２のいずれかに記載のリポソームの製造方法。 The method for producing a liposome according to any one of claims 1 to 12, wherein the emulsification method in the primary emulsification step is a membrane emulsification method or a microchannel emulsification method.
  14. 　前記二次乳化工程における乳化法が膜乳化法、マイクロチャネル乳化法または撹拌乳化法である、請求項１～１２のいずれかに記載のリポソームの製造方法。 The method for producing a liposome according to any one of claims 1 to 12, wherein the emulsification method in the secondary emulsification step is a membrane emulsification method, a microchannel emulsification method or a stirring emulsification method.
  15. 　前記膜乳化法がＳＰＧ膜を用いた膜乳化法である、請求項１３または１４に記載のリポソームの製造方法。 The method for producing a liposome according to claim 13 or 14, wherein the membrane emulsification method is a membrane emulsification method using an SPG membrane.
  16. 　前記一次乳化工程がさらにリポソームに内包させるべき物質を前記内水相（Ｗ１）または前記油相（Ｏ）に添加した上で行われる、請求項１～１５のいずれかに記載のリポソームの製造方法。 The method for producing a liposome according to any one of claims 1 to 15, wherein the primary emulsification step is further performed after adding a substance to be encapsulated in the liposome to the inner aqueous phase (W1) or the oil phase (O). .
  17. 　前記一次乳化工程後にリポソームに内包させるべき物質を前記油相（Ｏ）に添加する、請求項１～１５のいずれかに記載のリポソームの製造方法。 The method for producing a liposome according to any one of claims 1 to 15, wherein a substance to be encapsulated in the liposome after the primary emulsification step is added to the oil phase (O).
  18. 　請求項１～１７のいずれかに記載のリポソームの製造方法を使用する、リポソーム分散液またはその乾燥粉末の製造方法。 A method for producing a liposome dispersion or a dry powder thereof using the method for producing a liposome according to any one of claims 1 to 17.
  19. 　請求項１８に記載の製造方法により製造されたリポソーム分散液またはその乾燥粉末。 A liposome dispersion produced by the production method according to claim 18 or a dry powder thereof.
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