WO2011062255A1 - Procédé de production d'un liposome par émulsification en deux stades à l'aide d'un solvant organique mixte comme phase huileuse - Google Patents

Procédé de production d'un liposome par émulsification en deux stades à l'aide d'un solvant organique mixte comme phase huileuse 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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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%.

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Abstract

La présente invention concerne un procédé de production d'un liposome, qui comprend un stade d'émulsification en deux étapes et un stade d'élimination de solvant, et qui est supérieur en termes de taux d'encapsulation d'un médicament ou similaire, d'efficacité de formation de liposome univésiculaire et autres par comparaison avec ceux de procédés classiques. L'invention concerne spécifiquement un procédé de production d'un liposome, qui comprend : une étape primaire d'émulsification d'une phase aqueuse interne (W1) et d'une phase huileuse (O) avec un composant lipidique mixte (F1) pour applications liposomales afin de préparer une émulsion W1/O ; une étape d'émulsification secondaire permettant d'émulsifier l'émulsion W1/O et une phase aqueuse externe (W2) avec un composant lipidique mixte (F2) pour applications liposomales afin de préparer une émulsion W1/O/W2 ; et un stade d'élimination de solvant permettant d'éliminer le solvant organique contenu dans la phase huileuse (O) de l'émulsion W1/O/W2 pour former un liposome. Le procédé est caractérisé en ce que la phase huileuse (O) dans l'émulsion W1/O/W2 à soumettre au stade d'élimination de solvant est un solvant organique comprenant au moins deux solvants organiques.
PCT/JP2010/070657 2009-11-20 2010-11-19 Procédé de production d'un liposome par émulsification en deux stades à l'aide d'un solvant organique mixte comme phase huileuse WO2011062255A1 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
US10117833B2 (en) 2014-04-30 2018-11-06 Fujifilm Corporation Method for producing liposome
CN115969039A (zh) * 2022-12-15 2023-04-18 天津科技大学 一种基于w/g/w结构的益生菌微胶囊、制备方法和应用

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