WO2009131216A1 - Structure de membrane lipidique modifiée par un oligo(alkylène glycol) - Google Patents

Structure de membrane lipidique modifiée par un oligo(alkylène glycol) Download PDF

Info

Publication number
WO2009131216A1
WO2009131216A1 PCT/JP2009/058172 JP2009058172W WO2009131216A1 WO 2009131216 A1 WO2009131216 A1 WO 2009131216A1 JP 2009058172 W JP2009058172 W JP 2009058172W WO 2009131216 A1 WO2009131216 A1 WO 2009131216A1
Authority
WO
WIPO (PCT)
Prior art keywords
glycol
lipid
membrane structure
lipid membrane
modified
Prior art date
Application number
PCT/JP2009/058172
Other languages
English (en)
Japanese (ja)
Inventor
秀吉 原島
英万 秋田
智也 増田
健太朗 小暮
崇 西尾
謙一 新倉
邦治 居城
Original Assignee
国立大学法人 北海道大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人 北海道大学 filed Critical 国立大学法人 北海道大学
Publication of WO2009131216A1 publication Critical patent/WO2009131216A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a lipid membrane structure comprising a lipid modified with an oligoalkylene glycol having an alkylene group polymerization degree of 4 to 25, and a lipid modified with an oligoalkylene glycol having an alkylene group polymerization degree of 4 to 25 And a method for producing an intracellular transcription promoter for nucleic acid encapsulated in a lipid membrane structure, and a lipid single membrane structure encapsulating a drug, wherein the alkylene group has a degree of polymerization of 4 to 25
  • the present invention relates to the above production method, wherein a drug is encapsulated using a lipid modified with glycol as a component of a membrane.
  • MEND Multifunctional-envelope-type-nano-device
  • lipid membrane structures such as liposomes and MEND have the problem of poor retention in blood when administered intravenously and are easily trapped in reticuloendothelial tissues such as the liver and spleen. ing.
  • these lipid membrane structures have also been pointed out as problems of leakage of inclusions and aggregation of lipid membrane structures. These problems have been a major obstacle in performing targeting therapy for delivering drugs, particularly nucleic acids, to target organs and cells using liposomes and MENDs.
  • Non-patent Document 1 means for modifying the surface of the lipid membrane structure with polyalkylene glycol such as polyethylene glycol (PEG) has been proposed (Non-patent Document 1).
  • PEG polyethylene glycol
  • the hydration layer of PEG covers the outer surface of the lipid membrane structure, so opsonization due to adsorption of serum proteins is suppressed, and as a result, phagocytosis by macrophages and uptake by reticuloendothelial tissue can be avoided.
  • a lipid membrane structure is prepared using a phospholipid modified with polyalkylene glycol, and a lipid membrane structure modified with polyalkylene glycol is actually produced.
  • Patent Document 1 discloses that a specific phospholipid modified with polyalkylene glycol or the like can be hydrolyzed by a matrix metalloprotease between a modified portion such as polyalkylene glycol and the phospholipid portion.
  • a lipid membrane structure using a peptide-mediated lipid is proposed.
  • the lipid membrane structure has a high retention in the blood due to the presence of a modification site such as polyalkylene glycol in the blood, while the lipid membrane structure reaches the target tumor tissue.
  • the oligopeptide part of the body is hydrolyzed by a matrix metalloprotease, and the modification site such as polyalkylene glycol is eliminated. As a result, it is efficiently taken up by the target tumor cell and the encapsulated drug is efficiently released.
  • Patent Document 1 it is necessary to synthesize a lipid in which an oligopeptide hydrolyzable by a matrix metalloprotease is interposed between a modifying portion such as polyalkylene glycol and a phospholipid portion, There is a problem that it can be used only for target cells expressing a matrix metalloprotease.
  • the present invention is a lipid membrane structure in which the drug delivery efficiency to a target cell, particularly the expression efficiency of a nucleic acid used for gene therapy or the like in a target cell is enhanced by an approach different from the technique according to Patent Document 1.
  • the purpose is to provide.
  • the present inventors have modified the lipid membrane structure with an oligoalkylene glycol having a specific degree of polymerization in place of the polyalkylene glycol that has been used for the purpose of enhancing hydrophilicity so far.
  • the inventors have found that the efficiency, in particular, the intracellular expression and transcription efficiency of nucleic acids can be dramatically increased, and the following inventions have been completed.
  • a lipid membrane structure comprising a lipid modified with an oligoalkylene glycol having an alkylene group polymerization degree of 4 to 25 as a constituent of the membrane.
  • oligoethylene glycol is one or more selected from the group consisting of triethylene glycol, tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, heptaethylene glycol and octaethylene glycol. Structure.
  • lipid membrane structure according to any one of (1) to (4), wherein the lipid modified with oligoalkylene glycol is phospholipid, glycolipid, sterol, long-chain aliphatic alcohol or glycerin fatty acid ester .
  • the sterol is one or more selected from the group consisting of cholesterol, cholesterol succinic acid, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol, stigmasterol, sitosterol, campesterol, brassicasterol, thymosterol and ergosterol.
  • the lipid membrane structure according to (5) is one or more selected from the group consisting of cholesterol, cholesterol succinic acid, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol, stigmasterol, sitosterol, campesterol, brassicasterol, thymosterol and ergosterol.
  • An intracellular transcription promoter for a nucleic acid encapsulated in a lipid membrane structure comprising a lipid modified with an oligoalkylene glycol having an alkylene group polymerization degree of 4 to 25.
  • oligoethylene glycol is one or more selected from the group consisting of triethylene glycol, tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, heptaethylene glycol and octaethylene glycol. Transfer accelerator.
  • the sterol is one or more selected from the group consisting of cholesterol, cholesterol succinic acid, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol, stigmasterol, sitosterol, campesterol, brassicasterol, thymosterol and ergosterol.
  • the intracellular transcription promoter according to (12).
  • oligoethylene glycol is one or more selected from the group consisting of triethylene glycol, tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, heptaethylene glycol and octaethylene glycol.
  • lipid modified with oligoalkylene glycol is a phospholipid, glycolipid, sterol, long-chain aliphatic alcohol or glycerin fatty acid ester.
  • the sterol is one or more selected from the group consisting of cholesterol, cholesterol succinic acid, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol, stigmasterol, sitosterol, campesterol, brassicasterol, thymosterol and ergosterol. (17) The manufacturing method as described.
  • the lipid membrane structure of the present invention is excellent in the ability to release inclusions in a target cell, and particularly when a nucleic acid is encapsulated, the amount of expression and transcription of the nucleic acid in the target cell is controlled. It is particularly useful as a vector for gene therapy.
  • FIG. 3 is a graph showing the effect of promoting the expression of TEGChol on nucleic acid (luciferase gene) in HeLa cells transformed with MENDs 1 to 3 prepared in Example 1.
  • FIG. It is a graph which shows the expression promotion effect with respect to the nucleic acid (luciferase gene) of TEGChol in the lipid membrane structure which does not contain stearyl octaarginine.
  • 2 is a graph showing the results of measuring the expression level of nucleic acid (luciferase gene) in the whole HeLa cells transformed with MENDs 1 to 3 prepared in Example 1 by real-time PCR.
  • FIG. 2 is a graph showing the results of measuring the expression level of nucleic acid (luciferase gene) in the nuclear fraction fractionated from HeLa cells transformed with MENDs 1 to 3 produced in Example 1 by real-time PCR. It is a graph which shows the nuclear transfer efficiency of the nucleic acid obtained by dividing the nuclear transfer amount of a nucleic acid by the amount of intracellular uptake based on the measured value of real-time PCR. It is a graph which shows the expression efficiency after a nuclear transfer obtained by dividing a nucleic acid expression level by a nuclear transfer amount based on the measured value of real-time PCR.
  • Example 2 is a SEM electron micrograph of MEND1 and MEND3 prepared in Example 1. It is a graph showing the influence of the ethylene polymerization degree with respect to the expression level of a nucleic acid.
  • Chol is MEND containing cholesterol modified with oligoethylene glycol in the membrane
  • STR is MEND containing stearic acid modified with oligoethylene glycol in the membrane
  • DSPE is distearoyl phosphatidyl ethanol modified with oligoethylene glycol.
  • Each of the MENDs containing an amine in the membrane is shown.
  • the present invention relates to a lipid membrane structure comprising a lipid modified with an oligoalkylene glycol having an alkylene group polymerization degree of 4 to 25 as a constituent component of the membrane.
  • the alkylene group in the present invention is an alkylene group having 1 to 10 carbon atoms, preferably 1 to 5 carbon atoms, such as a methylene group, an ethylene group, a propylene group, a butene group, or a pentene group, and particularly preferably ethylene. It is a group. That is, a preferred oligoalkylene glycol in the present invention is an oligoethylene glycol having an ethylene group polymerization degree of 4 to 25.
  • More preferable oligoalkylene glycol is one or more selected from the group consisting of tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, heptaethylene glycol and octaethylene glycol, and tetraethylene glycol is particularly preferable.
  • the oligoalkylene glycol having an alkylene group polymerization degree of 4 to 25 used in the present invention is represented as OAG.
  • the lipid modified with OAG may be any lipid that is generally used as a component of a lipid membrane structure, but is preferably a phospholipid, glycolipid, sterol, long-chain aliphatic alcohol, or glycerin fatty acid ester. .
  • Phospholipids include phosphatidylcholine (for example, dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, etc.), phosphatidylglycerol (for example, dioleoylphosphatidylglycerol, dilauroylphosphatidylglycerol, dimyristoyl).
  • phosphatidylcholine for example, dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoyl
  • phosphatidylglycerol for example, dioleoylphosphatidylglycerol, dilauroylphosphatidylglycerol, dimyristoyl.
  • Phosphatidylglycerol dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol
  • phosphatidylethanolamine eg dioleoylphosphatidylethanolamine (DOPE), dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmit Ylphosphatidylethanolamine, distearoylphosphatidiethanolamine), N-succinylphosphatidylethanolamine (N-succinyl PE), phosphatidylethylene glycol, phosphatidylserine, phosphatidylinositol, phosphatidic acid, cardiolipin, or hydrogenated products thereof, egg yolk, soybean Examples include natural lipids derived from other animals and plants (eg, egg yolk lecithin, soybean lecithin, etc.).
  • Said phospholipid is used as a main structural component of a lipid membrane structure.
  • the amount to be used is preferably 10 to 100% (molar ratio), more preferably 50 to 80% (molar ratio) as the amount of the lipid membrane structure relative to the total lipid. It is not limited.
  • glycolipids examples include glycolipids such as cephalin, cerebroside, ceramide, sphingomyelin, ganglioside, and one or more of these can be used.
  • sterols examples include sterols derived from animals such as cholesterol, cholesterol succinic acid, lanosterol, dihydrolanosterol, desmosterol and dihydrocholesterol, sterols (phytosterols) derived from plants such as stigmasterol, sitosterol, campesterol and brassicasterol, and timosterol. And sterols derived from microorganisms such as ergosterol. These sterols are generally used to physically or chemically stabilize lipid bilayers and to regulate membrane fluidity. The amount to be used is preferably 5 to 40% (molar ratio), more preferably 10 to 30% (molar ratio), based on the total lipid of the lipid membrane structure. It is not limited.
  • a fatty acid having 10 to 20 carbon atoms or an alcohol thereof can be used.
  • Preferred examples include palmitic acid, oleic acid, stearic acid, arachidonic acid, myristic acid, lauric acid, arachidic acid, linoleic acid, palmitoyl acid, oleyl alcohol, stearyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, linolyl alcohol Etc.
  • the amount to be used is preferably 5 to 40% (molar ratio), more preferably 10 to 30% (molar ratio), based on the total lipid of the lipid membrane structure. It is not limited.
  • glycerin fatty acid esters examples include monoacyl glycerides, diacyl glycerides, and triacyl glycerides.
  • the amount to be used is preferably 5 to 40% (molar ratio), more preferably 10 to 30% (molar ratio), based on the total lipid of the lipid membrane structure. It is not limited.
  • Lipids generally have a chemically activatable functional group such as a carbonyl group, an amino group or a hydroxyl group. Between these appropriate functional groups and functional groups on OAG or functional groups introduced into OAG. Through the chemical reaction, lipids modified with OAG can be prepared.
  • a chemically activatable functional group such as a carbonyl group, an amino group or a hydroxyl group.
  • PE phosphatidylethanolamine
  • the method of Martin et al. (Biochem. Biophys. Acta, 1992, 1113, 171-199) is known. That is, PE modified with OAG can be prepared by converting a hydroxyl group of OAG into a carboxylic acid and performing a condensation reaction between the carboxyl group and the amino group of PE.
  • an amino group is introduced into OAG, and a condensation reaction is performed between this amino group and a carboxyl group of the long chain fatty acid, thereby modifying the long chain modified with OAG.
  • Fatty acids can be prepared.
  • sterols modified with OAG can be prepared by activating the hydroxyl group of sterols with a tosyl group or the like and using the SN2 reaction with the hydroxyl group of OAG in an organic solvent.
  • the lipid membrane of the lipid membrane structure of the present invention includes tocopherol, propyl gallate, ascorbyl palmitate, butylated hydroxytoluene and other antioxidants, stearylamine, Charged substances that impart positive charges such as oleylamine, charged substances that impart negative charges such as dicetyl phosphate, membrane surface proteins, membrane proteins such as integral membrane proteins can be included, and the content is appropriate Can be adjusted.
  • cell membrane permeable peptides such as polyarginine peptides disclosed in International Patent Application Publication No. WO2005 / 032593 pamphlet, pH-responsive membrane fusion such as GALA peptides disclosed in International Patent Application Publication No. WO2005 / 032593 pamphlet Peptides, which can add functions to other lipid membrane structures, are used in the lipid membrane structure of the present invention according to the mode, amount used, production method, etc. described in each patent document. May be.
  • the lipid membrane structure When transferring the lipid membrane structure of the present invention into cells by endocytosis, the lipid membrane structure preferably contains a cationic lipid as a constituent component of the membrane.
  • the cationic lipid include dioctadecyldimethylammonium chloride (DODAC), N- (2,3-oleyloxy) propyl-N, N, N-trimethylammonium (N- (2,3-dioyloxy)).
  • DODAC dioctadecyldimethylammonium chloride
  • N- (2,3-oleyloxy) propyl-N N, N-trimethylammonium (N- (2,3-dioyloxy)).
  • the lipid membrane structure of the present invention moves into the cell by macropinocytosis, and the cationic lipid is converted into a lipid. It is not always necessary to be included in the membrane structure. That is, when the polyarginine peptide is added, the lipid membrane of the lipid membrane structure of the present invention may be composed of either a cationic lipid or a non-cationic lipid, or may be composed of both. Alternatively, oligoethylene glycol may be used by binding to these lipids.
  • the ratio of the cationic lipid to the total lipid constituting is preferably 0 to 40% (molar ratio), and more preferably 0 to 20% (molar ratio).
  • non-cationic lipid means a neutral lipid or an anionic lipid
  • examples of the neutral lipid include diacylphosphatidylcholine, diacylphosphatidylethanolamine, cholesterol, ceramide, sphingomyelin, cephalin, and cerebroside.
  • anionic lipids include, for example, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-succinylphosphatidylethanolamine (N-succinyl PE), phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, phosphatidylethylene glycol And cholesterol succinic acid.
  • the lipid membrane constituting the lipid membrane structure of the present invention may contain a plurality of types of lipids modified with OAG.
  • lipids not modified with OAG or other than OAG Lipids modified with these substances may be included as components.
  • a phospholipid modified with OAG and another phospholipid modified with OAG a phospholipid and sterol modified with OAG, a sterol and phospholipid modified with OAG, and a sterol and phospholipid modified with OAG
  • a combination of lipids such as a sterol modified with a functional peptide can be arbitrarily selected, and may further contain various components usable for the preparation of a functional peptide and other lipid membrane structures.
  • a preferred combination in the present invention is a lipid membrane structure in which sterols and phospholipids modified with OAG are used as constituent lipids of the lipid membrane and further modified with polyarginine peptide.
  • the lipid membrane structure of the present invention containing a lipid modified with OAG as a constituent component of the membrane effectively allows the substance enclosed therein to efficiently contain the substance in the cell, particularly in the nucleus. Can be released.
  • the substance enclosed inside is a nucleic acid
  • the expression and transcription efficiency of the nucleic acid in the cell, particularly in the nucleus can be dramatically increased. Therefore, lipids modified with OAG can be used as intracellular transcription promoters for nucleic acids encapsulated in lipid membrane structures.
  • the present invention provides an intracellular transcription promoter for nucleic acid encapsulated in a lipid membrane structure, which comprises a lipid modified with OAG, that is, an oligoalkylene glycol having an alkylene group polymerization degree of 4 to 25.
  • OAG is as described above.
  • the preferred form of the lipid membrane structure of the present invention is a closed vesicle composed of a single membrane.
  • the lipid membrane structure can be a closed vesicle composed of a single membrane.
  • a lipid membrane structure composed of a single lipid membrane is prepared by repeatedly passing a filter of an appropriate size.
  • OAG that is, a lipid modified with an oligoalkylene glycol having an alkylene group polymerization degree of 4 to 25 is used.
  • a lipid membrane structure composed of a lipid monolayer can be prepared without using a filter.
  • the present invention provides a method for producing a lipid single membrane structure in which a drug is encapsulated, and uses OAG, that is, a lipid modified with an oligoalkylene glycol having an alkylene group polymerization degree of 4 to 25 as a component of the membrane. And providing the above-described production method.
  • OAG is as described above.
  • the lipid membrane structure of the present invention is in the form of a closed vesicle composed of a single membrane, it is any one of SUV (small ilamela ic vesicle), LUV (large il unilamella vesicle), GUV (giant uniella vesicle), etc. Also good. Accordingly, the size of the lipid membrane structure of the present invention is not particularly limited, but it is preferably 50 to 800 nm in diameter, and more preferably 80 to 150 nm in diameter.
  • the lipid membrane structure of the present invention can be obtained by using known methods such as a hydration method, an ultrasonic treatment method, an ethanol injection method, an ether injection method, a reverse phase evaporation method, a surfactant method, and a freezing / thawing method.
  • a hydration method lipids modified with OAG, other lipids and optional components contained in the lipid membrane described above are dissolved in an organic solvent, and then the organic membrane is removed by evaporation to remove the lipid membrane. After being obtained, the lipid membrane is hydrated, stirred or sonicated to produce a lipid membrane structure containing a lipid modified with OAG as a constituent of the membrane.
  • the lipid membrane structure of the present invention having a polyarginine peptide or GALA peptide is obtained by dissolving a lipid modified with OAG and other lipids in an organic solvent, and then evaporating and removing the organic solvent to obtain a lipid membrane.
  • Liposomes can be produced by hydrating the lipid membrane, stirring or sonicating, and then adding the polypeptide to the external solution of the liposome to introduce these peptides onto the surface of the liposome. .
  • organic solvent for example, hydrocarbons such as pentane, hexane, heptane and cyclohexane, halogenated hydrocarbons such as methylene chloride and chloroform, aromatic hydrocarbons such as benzene and toluene, methanol, ethanol Lower alcohols such as methyl acetate, esters such as methyl acetate and ethyl acetate, ketones such as acetone and the like can be used alone or in combination of two or more.
  • hydrocarbons such as pentane, hexane, heptane and cyclohexane
  • halogenated hydrocarbons such as methylene chloride and chloroform
  • aromatic hydrocarbons such as benzene and toluene
  • methanol ethanol
  • Lower alcohols such as methyl acetate, esters such as methyl acetate and ethyl acetate, ketones such as acetone and the like can be used alone or in combination
  • lipid membrane structure having a certain particle size distribution can be obtained by passing through a filter having a predetermined pore size.
  • physiologically active substances such as drugs, nucleic acids, peptides, proteins, sugars or complexes thereof can be encapsulated in the lipid membrane structure of the present invention, which is appropriately selected according to the purpose of diagnosis, treatment, etc. be able to.
  • the physiologically active substance is water-soluble
  • the aqueous phase inside the lipid membrane structure can be obtained by adding the physiologically active substance to an aqueous solvent used for hydrating the lipid membrane in the production of the lipid membrane structure.
  • a physiologically active substance can be encapsulated.
  • the physiologically active substance is fat-soluble, the physiologically active substance is encapsulated in the membrane of the lipid membrane structure by adding the physiologically active substance to the organic solvent used in the production of the lipid membrane structure. Can do.
  • the lipid membrane structure of the present invention is useful for transferring a complex of a nucleic acid and a cationic substance to the cytoplasm and nucleus.
  • cationic substance as used herein means a substance having a cationic group in the molecule, and means a substance that can form a complex with a nucleic acid by electrostatic interaction.
  • the kind of the cationic substance is not particularly limited as long as it can form a complex with the nucleic acid.
  • a cationic lipid for example, Lipofectamine (Invitrogen)
  • a polymer having a cationic group for example, Lipofectamine (Invitrogen)
  • a polymer having a cationic group for example, Lipofectamine (Invitrogen)
  • a polymer having a cationic group for example, Lipofectamine (Invitrogen)
  • polylysine, polyarginine And a homopolymer or copolymer of a basic amino acid such as a copolymer of lysine and arginine or a derivative thereof (for example, stearylated derivative)
  • a polycationic polymer such as polyethyleneimine
  • protamine sulfate for example, Lipofectamine (Invitrogen)
  • a polymer having a cationic group for example, polylysine, polyarginine And a homopolymer or copolymer of a basic amino acid such as a copolymer of lys
  • the number of arginine residues constituting polyarginine is usually 4 to 20, preferably 6 to 12, and more preferably 7 to 10.
  • the number of cationic groups possessed by the cationic substance is not particularly limited, but is preferably 2 or more.
  • the cationic group is not particularly limited as long as it can be positively charged.
  • a monoalkylamino group such as an amino group, a methylamino group, and an ethylamino group
  • a dialkylamino group such as a dimethylamino group and a diethylamino group
  • an imino group Group guanidino group and the like.
  • the complex of the nucleic acid and the cationic substance has a positive charge or a negative charge as a whole depending on the composition ratio thereof, so that the above complex is formed inside the liposome by electrostatic interaction with a non-cationic lipid or a cationic lipid.
  • the body can be sealed efficiently.
  • the lipid membrane structure of the present invention can be used in the state of a dispersion, for example.
  • a dispersion solvent for example, a buffer solution such as physiological saline, phosphate buffer, citrate buffer, and acetate buffer can be used.
  • additives such as sugars, polyhydric alcohols, water-soluble polymers, nonionic surfactants, antioxidants, pH regulators, hydration accelerators may be added to the dispersion.
  • the lipid membrane structure of the present invention can also be used in a state where the dispersion is dried (for example, freeze-dried, spray-dried, etc.). The dried lipid membrane structure can be made into a dispersion by adding a buffer solution such as physiological saline, phosphate buffer, citrate buffer, or acetate buffer.
  • the lipid membrane structure of the present invention can be used both in vivo and in vitro.
  • examples of the administration route include parenteral administration such as intravenous, intraperitoneal, subcutaneous, and nasal administration.
  • the dose and the number of doses can be appropriately adjusted according to the type and amount of the drug encapsulated in the lipid membrane structure.
  • Such a lipid membrane structure can exhibit intracellular migration in a wide temperature range of 0 to 40 ° C. (an effective temperature range is 4 to 37 ° C.). Can be set.
  • the lipid membrane structure of the present invention can be used as an intracellular delivery vector or a nuclear delivery vector of a target substance.
  • the biological species from which the cell to which the target substance is to be delivered is not particularly limited and may be any animal, plant, microorganism, etc., but is preferably an animal, more preferably a mammal. preferable. Examples of mammals include humans, monkeys, cows, sheep, goats, horses, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, and the like.
  • the kind of cell which should deliver a target substance is not specifically limited, For example, a somatic cell, a germ cell, a stem cell, or these cultured cells etc. are mentioned.
  • lipid membrane By reacting 10 g of cholesterol and 10 g of p-toluenesulfonic acid in a dry pyridine solution, the hydroxy group of cholesterol was derived into p-toluenesulfonic acid ester. By reacting 1 g of this esterified product and 2 g of tetraethylene glycol in 1,4-dioxane, cholesterol modified with tetraethylene glycol (hereinafter referred to as TEGChol) was produced. Further, an octaarginine peptide (STR-R8) modified with a stearyl group was prepared according to the method described in the pamphlet of Japanese Patent Application Publication No. WO2005 / 032593.
  • lipid membrane (control).
  • three types of lipid membranes were prepared in the same manner as described above except that 10%, 20%, and 30% of the cholesterol having the above composition were replaced with TEGChol.
  • a plasmid pEGFP-Luc (Clontech) encoding luciferase and protamine are mixed at a ratio of 1: 0.67 (mass ratio) in a HEPES buffer (10 mM HEPES / NaOH pH 7.4), so that an about 80 nm Aggregates were obtained.
  • nucleic acid-protamine aggregate solution 250 ⁇ L was added to each lipid membrane and hydrated for 10 minutes.
  • the hydrate was subjected to ultrasonic treatment with a water tank type ultrasonic generator for 30 seconds to 1 minute, whereby four types of MENDs with different aggregates and different TEGChol contents were produced.
  • Example 1 The expression level of the luciferase gene in HeLa cells transfected with MEND obtained in Example 1, 4) b) was determined using a real-time PCR measurement kit SYBR Green Realtime PCR Master Mix (TOYOBO) according to the manual of the kit. It was measured. The expression level was a value corrected by dividing the copy number of luciferase by the copy number of ⁇ -actin.
  • TOYOBO real-time PCR measurement kit SYBR Green Realtime PCR Master Mix
  • Example 2 The plasmid pEGFP-Luc of Example 1 2) was labeled with rhodamine using the same procedure as in Example 1 except that it was labeled with rhodamine using Label IT CX-Rhodamine reagent (Mirus) according to the manual of the same kit.
  • the gene was introduced into HeLa cells using MENDs 7 to 9 in which the nucleic acid aggregates were encapsulated.
  • FIG. 8 shows the result of observation using a confocal laser microscope after adding the fluorescent substance SYTO24 to the HeLa cells. As a result, it was confirmed that the expression site of the nucleic acid introduced into the cell was localized in the cell nucleus.
  • MEND8 (20%) and MEND9 (40%) shown in FIG. 8 the nucleic acid introduced into the cells (labeled with rhodamine and detected as red) is aggregated in one place in the cell without being unevenly distributed. It was confirmed to be scattered throughout the cell. This means that the MEND according to the present invention has good dispersibility in cells.
  • Example 3 The plasmid pEGFP-Luc of Example 1 2) was labeled with rhodamine using Label IT CX-Rhodamine reagent (Mirus) according to the manual of the kit, and further labeled with fluorescent substance NBD-labeled DOPE (Avanti). Otherwise, the same procedure as in Example 1 was performed, and the gene was introduced into HeLa cells using MENDs 10 to 12 in which a rhodamine-labeled nucleic acid aggregate was encapsulated and the lipid membrane was labeled with NBD.
  • Label IT CX-Rhodamine reagent Mirus
  • NBD-labeled DOPE Advanti
  • FIG. 9 shows the result of observation using a confocal laser microscope after adding a fluorescent substance Hoechst 33342 to the HeLa cells to label nuclei.
  • MEND10 the label of the nucleic acid and the lipid showed almost the same localization in the cell, and was detected as yellow, which is a mixed color of red indicating rhodamine labeling and green indicating NBD labeling.
  • MEND11 and 12 it was confirmed that the nucleic acid (rhodamine-labeled and detected as red) was localized independently of lipid (NBD-labeled and detected as green) as the TEGChol content increased. It was done.
  • TEGChol was shown to be a useful element for smoothly dissociating the drug encapsulated in the lipid membrane structure.
  • Example 5 Using oligoethylene glycol having an ethylene polymerization degree of 4, 10, 25, 45 and 50, stearic acid modified with each oligoethylene glycol was prepared in the same manner as in Example 1). Similarly, cholesterol modified with oligoethylene glycol having an ethylene polymerization degree of 4 and DSPE (distearoylphosphatidylethanolamine) modified with oligoethylene glycol having an ethylene polymerization degree of 45 were prepared.
  • DSPE disearoylphosphatidylethanolamine

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Dispersion Chemistry (AREA)
  • Medicinal Preparation (AREA)

Abstract

L'invention porte sur une structure de membrane lipidique comprenant, en tant que composant constituant la membrane, un lipide modifié par un oligo(alkylène glycol) ayant un degré de polymérisation d'un groupe alkylène de 4 à 25. La structure de membrane lipidique a une excellente aptitude à libérer une substance renfermée dans celle-ci dans une cellule cible. En particulier, lorsqu'un acide nucléique est enfermé dans la structure de membrane lipidique, le taux d'expression/de transfert de l'acide nucléique dans une cellule cible peut être augmenté de manière remarquable par comparaison à celui d'une structure de membrane lipidique témoin. Par conséquent, la structure de membrane lipidique est particulièrement utile en tant que vecteur pour une thérapie génique.
PCT/JP2009/058172 2008-04-25 2009-04-24 Structure de membrane lipidique modifiée par un oligo(alkylène glycol) WO2009131216A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2008115388 2008-04-25
JP2008-115388 2008-04-25

Publications (1)

Publication Number Publication Date
WO2009131216A1 true WO2009131216A1 (fr) 2009-10-29

Family

ID=41216942

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2009/058172 WO2009131216A1 (fr) 2008-04-25 2009-04-24 Structure de membrane lipidique modifiée par un oligo(alkylène glycol)

Country Status (1)

Country Link
WO (1) WO2009131216A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011135905A1 (fr) * 2010-04-28 2011-11-03 国立大学法人北海道大学 Structure de membrane lipidique

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4971803A (en) * 1985-04-08 1990-11-20 California Institute Of Technology Lamellar vesicles formed of cholesterol derivatives
WO2007102481A1 (fr) * 2006-03-07 2007-09-13 National University Corporation Hokkaido University Vecteur pour le transport nucléaire d'une substance

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4971803A (en) * 1985-04-08 1990-11-20 California Institute Of Technology Lamellar vesicles formed of cholesterol derivatives
WO2007102481A1 (fr) * 2006-03-07 2007-09-13 National University Corporation Hokkaido University Vecteur pour le transport nucléaire d'une substance

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ALLEN, T.M. ET AL., BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1066, 1991, pages 29 - 36 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011135905A1 (fr) * 2010-04-28 2011-11-03 国立大学法人北海道大学 Structure de membrane lipidique
US20130195962A1 (en) * 2010-04-28 2013-08-01 National University Corporation Hokkaido University Lipid membrane structure
JP5787323B2 (ja) * 2010-04-28 2015-09-30 国立大学法人北海道大学 脂質膜構造体

Similar Documents

Publication Publication Date Title
Maurer et al. Developments in liposomal drug delivery systems
L Arias et al. Lipid-based drug delivery systems for cancer treatment
US8658204B2 (en) Transpulmonary liposome for controlling drug arrival
US20090305409A1 (en) Liposome Capable of Effective Delivery of Given Substance Into Nucleus
US9617562B2 (en) Nonviral targeted nanoparticle system for gene transfer and drug delivery
JPWO2008105178A1 (ja) リポソーム用生体成分抵抗性増強剤及びこれにより修飾されたリポソーム
CA2652475A1 (fr) Liposome ayant une membrane lipidique contenant un composant cellulaire bacterien
WO2013140643A1 (fr) Support pour l'administration intracellulaire d'une protéine fonctionnelle
JP5787323B2 (ja) 脂質膜構造体
JP6238366B2 (ja) 非極性溶媒に分散性を有する細菌菌体成分を内封する脂質膜構造体およびその製造方法
CN100566810C (zh) 用脂质膜被覆粒子的方法
WO2009131216A1 (fr) Structure de membrane lipidique modifiée par un oligo(alkylène glycol)
JP2007210953A (ja) ｐＨ応答性分子集合体
JP2006238839A (ja) 核酸の細胞内送達効率又は細胞内発現効率を向上させた組成物
JP2006167521A (ja) Ｓｕｖ型リポソームの膜融合を利用した遺伝子等の新規封入技術
JP2007166946A (ja) 標的遺伝子の発現を抑制するための組成物
JP2014114267A (ja) 脂質膜構造体に対して肝臓血管内皮細胞への移行能を付与および／または増強するペプチド、肝臓血管内皮細胞への移行能を有するまたは肝臓血管内皮細胞への移行能が増強された脂質膜構造体、ならびに脂質膜構造体に対して肝臓血管内皮細胞への移行能を付与および／または増強する剤
Crommelin et al. Liposomes
JP2010126505A (ja) 核酸の放出性に優れたリポソームベクター
Wu et al. Lipid-based nanoparticulate drug delivery systems
WO2012117971A1 (fr) Structure à membrane lipidique, procédé de production de structure à membrane lipidique et procédé pour l'encapsulation d'une molécule de substance cible à l'aide d'une seule membrane lipidique
JP2009286709A (ja) ゴルジ装置ターゲティング用リポソーム

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09735542

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09735542

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP