WO2007102481A1 - Vecteur pour le transport nucléaire d'une substance - Google Patents

Vecteur pour le transport nucléaire d'une substance Download PDF

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Publication number
WO2007102481A1
WO2007102481A1 PCT/JP2007/054260 JP2007054260W WO2007102481A1 WO 2007102481 A1 WO2007102481 A1 WO 2007102481A1 JP 2007054260 W JP2007054260 W JP 2007054260W WO 2007102481 A1 WO2007102481 A1 WO 2007102481A1
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WIPO (PCT)
Prior art keywords
sugar
lipid membrane
ribosome
lipid
membrane
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PCT/JP2007/054260
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English (en)
Japanese (ja)
Inventor
Tomoya Masuda
Hidetaka Akita
Kentaro Kogure
Takashi Nishio
Kenichi Niikura
Kuniharu Ijiro
Hideyoshi Harashima
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National University Corporation Hokkaido University
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Priority to JP2008503854A priority Critical patent/JPWO2007102481A1/ja
Publication of WO2007102481A1 publication Critical patent/WO2007102481A1/fr

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    • 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
    • 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
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • 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 nuclear delivery vector for a target substance.
  • vectors have been actively developed to reliably deliver target substances such as drugs and nucleic acids to target sites.
  • viral vectors such as retroviruses, adenoviruses, and adeno-associated viruses have been developed as vectors for introducing a target gene into target cells.
  • viral vectors have problems such as difficulty in mass production, antigenicity and toxicity, liposomal vectors with few such problems are attracting attention.
  • Ribosome vectors have the advantage of being able to improve the directivity to the target site by introducing functional molecules such as antibodies, proteins, sugar chains, etc. on the surface! .
  • ribosomes in which a hydrophilic polymer for example, polyalkylene glycol such as polyethylene glycol
  • Patent Document 1 Patent Document 2, Patent Document 3, and Patent Document 4
  • a substance capable of binding to a receptor or an antigen present on the surface of the cell membrane on the outer surface of the ribosome membrane for example, transferrin, insulin, folic acid, hyaluronic acid, antibody or fragment thereof, sugar chain
  • Patent Document 3 and Patent Document 4 a substance capable of binding to a receptor or an antigen present on the surface of the cell membrane on the outer surface of the ribosome membrane
  • transferrin, insulin, folic acid, hyaluronic acid, antibody or fragment thereof, sugar chain for example, transferrin, insulin, folic acid, hyaluronic acid, antibody or fragment thereof, sugar chain
  • Patent Document 1 JP-A-1-249717
  • Patent Document 2 JP-A-2-149512
  • Patent Document 3 Japanese Patent Laid-Open No. 4-346918
  • Patent Document 4 Japanese Patent Laid-Open No. 2004-10481
  • Patent Document 5 International Publication WO2005Z032593 Pamphlet
  • Non-patent literature l Kogure, K. et al., Journal of Controlled Release, 2004, No. 98, p.317
  • An object of the present invention is to provide a nuclear delivery vector of a target substance and a composition containing the same.
  • the present invention provides the following vectors and compositions.
  • a vector for nuclear delivery of a target substance having a ribosomal force comprising a lipid membrane having a sugar on its surface.
  • the amount of the membrane stabilizer contained in the lipid membrane is 0% of the total amount of the lipid membrane components.
  • the target substance to be delivered into the nucleus is encapsulated inside the ribosome.
  • a pharmaceutical composition comprising the vector according to (8) above, which is a target substance capable of exerting a therapeutic effect and a Z or preventive effect on a predetermined disease by being delivered into the nucleus.
  • the pharmaceutical composition encapsulated in the ribosome.
  • (11) The gene expression composition comprising the vector according to (8), wherein the gene to be expressed in the nucleus is enclosed in the ribosome. .
  • a vector for delivery of a target substance into a nucleus and a composition containing the same are provided.
  • lipid membrane having a sugar on its surface may be hereinafter referred to as “sugar-modified lipid membrane”.
  • the ribosome is a closed vesicle having a sugar-modified lipid membrane, it is a multilamellar ribosome (MLV), and SUV (small unilamellar vesicle), LUV (large unilamellar vesicle), GUV ( It may be a single membrane ribosome such as giant unilamellar vesicle).
  • MLV multilamellar ribosome
  • SUV small unilamellar vesicle
  • LUV large unilamellar vesicle
  • GUV It may be a single membrane ribosome such as giant unilamellar vesicle.
  • the ribosome When the ribosome is a multilamellar ribosome, the ribosome comprises one or more lipid membranes inside and / or outside the sugar-modified lipid membrane.
  • the lipid membrane located inside and / or outside the sugar-modified lipid membrane has sugar on the surface as long as the functions expected of the lipid membrane (eg, cell membrane fusion ability, endosome membrane fusion ability, etc.) are retained. It may or may not have sugar on the surface. If the ribosome is a single membrane ribosome, the ribosome comprises only a sugar-modified lipid membrane.
  • the size of the ribosome is not particularly limited, but is usually 30 to 700 nm in diameter, preferably 30 to 500 nm in diameter, and more preferably 30 to 300 nm in diameter.
  • the components constituting the lipid membrane are not particularly limited as long as they do not inhibit the formation of the lipid bilayer. Quality, membrane stabilizer, antioxidant, charged substance and the like.
  • Lipid is an essential component of the lipid membrane, and its content is usually 70% of the total amount of lipid membrane components.
  • ⁇ 100% preferably 80 ⁇ : LOO% (molar ratio), more preferably 90 ⁇ : LOO% (molar ratio).
  • Lipids include, for example, phosphatidylcholine (eg, dioleoyl phosphatidylcholine, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dinormi Toylphosphatidylcholine, distearoylphosphatidylcholine, etc.), phosphatidylglycerol (eg, dioleoylphosphatidylglycerol, dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol) Phosphatidylethanolamine (eg, dioleoylphosphatidylethanolamine, dilauroyl phosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidyl
  • galactosyl celeb oral side, latatosyl celeb oral side, gandarioside sterols derived from animals (e.g. Nore, Desmostero Monore, Dihydrocholesterol), plant-derived sterols (phytosterols) (eg, stigmasterol, sitosterol, campesterol, brassicasterol), microorganism-derived sterols (eg Chimosuteroru, ergosterol) sterols like; palmitic acid, Orein acid, stearic acid, Arakidon acid, saturated or unsaturated fatty acid (number of carbon atoms such as myristic acid and the like for example, 12 to 20) is.
  • animals e.g. Nore, Desmostero Monore, Dihydrocholesterol
  • plant-derived sterols eg, stigmasterol, sitosterol, campesterol, brassicasterol
  • microorganism-derived sterols eg Chimosuteroru,
  • a membrane stabilizer is an optional component of a lipid membrane that can be added to physically or chemically stabilize the lipid membrane or to adjust the fluidity of the lipid membrane, and its content is a component of the lipid membrane. Is generally 0 to 70% (molar ratio), preferably 0 to 30% (molar ratio), and more preferably 0 to 10% (molar ratio). Particularly in the sugar-modified lipid membrane, the content of the membrane stabilizer is preferably 0 to 60% (molar ratio) of the total amount of lipid membrane constituents. It is most preferably 0 to 10% (molar ratio).
  • the disintegration property of the sugar-modified lipid membrane can be improved by drawing the sugar bound to the nuclear pore complex into the nucleus. Improves the efficiency of intranuclear delivery of the target substance enclosed in the ribosome through the nuclear pore It can be done.
  • Examples of the film stabilizer include sterol, glycerin or fatty acid ester thereof.
  • Specific examples of the sterol are the same as those described above, and examples of the glycerin fatty acid ester include triolein and trioctanoin.
  • the antioxidant is an optional component of the lipid membrane that can be added to prevent lipid membrane acidification, and its content is usually 0-30% (molar ratio) of the total amount of the lipid membrane components, Preferably it is 0-20% (molar ratio), More preferably, it is 0-10% (molar ratio).
  • antioxidants examples include tocopherol, propyl gallate, ascorbyl palmitate, butylated hydroxytoluene and the like.
  • the charged substance is an optional component of the lipid membrane that can be added to impart a positive charge or negative charge to the lipid membrane, and its content is usually 0-30% (molar ratio) of the total amount of the lipid membrane constituents. It is preferably 0 to 20% (molar ratio), more preferably 0 to 10% (molar ratio).
  • Examples of the charged substance imparting a positive charge include saturated or unsaturated aliphatic amines such as stearylamine and oleylamine; saturated or unsaturated cationic synthetic lipids such as dioleoyltrimethylammonium propane and the like.
  • Examples of the charged substance that imparts a negative charge include dicetyl phosphate, cholesteryl hemisuccinate, phosphatidylserine, phosphatidylinositol, and phosphatidic acid.
  • Lipids constituting a lipid membrane include lipids having a membrane fusion function, a blood retention function, a temperature change sensitivity function, a pH sensitivity function, etc. May use lipid derivatives. As a result, one or more of the above functions can be imparted to the ribosome. By imparting a membrane fusion function to ribosomes, ribosomes can be fused with cell membranes, endosomal membranes, and the like.
  • the retention of ribosomes in blood can be improved, and the capture rate by reticuloendothelial tissues such as the liver and spleen can be reduced.
  • the ribosome By giving the ribosome a temperature change sensitive function and a Z or PH sensitive function, the release of the target substance encapsulated in the ribosome can be enhanced.
  • Lipids capable of imparting a membrane fusion function include, for example, neutral lipids such as dioleoylphosphatidylethanolamine; cholesterol succinic acid, strength Examples include key lipids (acidic lipids) such as ludiolipin.
  • the ribosome is a multilamellar liposome and the ribosome is provided with a lipid membrane other than the sugar-modified lipid membrane for the purpose of fusing with a cell membrane, endosome membrane, etc., the lipid membrane contains a membrane-fusible lipid. be able to.
  • the membrane-fusible lipid contained in the lipid membrane is not particularly limited as long as it can be fused with the cell membrane and the endosomal membrane.
  • the amount of fusogenic lipid contained in the lipid membrane is not particularly limited, but is usually 40% (molar ratio) or more, preferably 70% (molar ratio) or more of the total lipid content contained in the lipid membrane. More preferably, it is 90% (molar ratio) or more.
  • the upper limit of the amount of fusogenic lipid contained in the lipid membrane is 100% of the total amount of lipid contained in the lipid membrane.
  • Examples of the blood-retaining lipid derivative capable of imparting the blood-retaining function include, for example, glycophorin, gandarioside GM1, phosphatidylinositol, gandarioside GM3, glucuronic acid derivative, glutamic acid derivative, and polyglycerin phospholipid derivative.
  • Examples of the temperature change sensitive lipid derivative capable of imparting a temperature change sensitive function include dipalmitoylphosphatidylcholine and the like.
  • Examples of the pH sensitive lipid derivative capable of imparting a pH sensitive function include, for example, di And oleoylphosphatidylethanolamine.
  • the sugar-modified lipid membrane may have one type of sugar on the surface, or two or more types of sugar on the surface. You may have.
  • the amount of sugar on the surface of the sugar-modified lipid membrane is usually 0.5 to 65% (molar ratio), preferably 1 to 60% (molar ratio), more preferably the total amount of lipid contained in the sugar-modified lipid film. Is 5-50% (molar ratio).
  • the total number of sugar residues constituting the sugar on the surface of the sugar-modified lipid membrane is not particularly limited, but is usually 1 to 20, preferably 1 to 5, and more preferably 1.
  • the sugar type is not particularly limited, and examples thereof include monosaccharides, amino sugars, deoxy sugars, disaccharides, trisaccharides, oligosaccharides, etc. Of these, monosaccharides and amino sugars are preferred.
  • Examples of monosaccharides include mannose, galactose, glucose, fucose, and fructose. Among these, mannose, galactose, glucose, and fucose are preferable.
  • the amount of mannose on the surface of the sugar-modified lipid membrane is preferably 5 to 60% (molar ratio) of the total lipid contained in the sugar-modified lipid membrane. More preferably, it is% (molar ratio).
  • the sugar is galactose, it is preferable that the amount of extra-gallium on the surface of the sugar-modified lipid membrane is 1 to 35% (molar ratio) of the total lipid content in the sugar-modified lipid membrane.
  • the amount of mannose or galactose on the surface of the sugar-modified lipid membrane is ⁇ 15% (molar ratio).
  • amino sugars examples include N-acetylyldarcosamine, N-acetylylgalatatosamine, N-acetylmethylmannosamine, etc. Among them, N-acetylyldarcosamine is preferable.
  • the sugar is N-acetyl darcosamine
  • the amount of N-acetyl darcosamine on the surface of the sugar-modified lipid membrane is 1-30% (molar ratio) of the total lipid content in the sugar-modified lipid membrane. It is more preferable that it is 5 to 15% (molar ratio).
  • the presence of sugar in the sugar-modified lipid membrane is not particularly limited as long as the sugar can also expose the surface force of the lipid membrane, and the sugar is directly bound to the lipid membrane constituents to form the surface of the lipid membrane. It may be present and may be present on the surface of the lipid membrane via a hydrophilic polymer (i.e., by binding of a lipophilic polymer with sugar introduced to the lipid membrane component) It is preferably present on the surface of the lipid membrane via a hydrophilic polymer. Thereby, the monodispersity of the ribosome can be improved.
  • the ratio of the two can be adjusted as appropriate.
  • the ratio is usually 1% (molar ratio) or more, preferably 5% (molar ratio) or more, more preferably 10% (molar ratio) or more of the total amount of sugars on the surface of the sugar-modified lipid membrane.
  • the sugar may be bonded to the end of the main chain of the hydrophilic polymer, or on the side of the hydrophilic polymer. Although it may be bonded to the end of the chain, it is preferably bonded to the end of the main chain of the hydrophilic polymer. Further, the end of the main chain of the hydrophilic polymer may be bonded to the lipid membrane constituent, or the end of the side chain of the hydrophilic polymer may be bonded to the lipid membrane constituent 1, and so on. However, it is preferable that the end of the main chain of the hydrophilic polymer is bonded to the lipid membrane constituent. That is, it is preferable that sugar is introduced into one end of the main chain of the hydrophilic polymer, and the other end of the main chain of the hydrophilic polymer is bonded to the lipid membrane component.
  • the lipid membrane component to which the sugar is bound directly or via a hydrophilic polymer is not particularly limited, but is preferably a phospholipid, sterol or fatty acid which is preferably a lipid or membrane stabilizer. More preferably it is. Saccharides bind directly or through hydrophilic polymers When the lipid membrane constituents are lipids or membrane stabilizers, especially phospholipids, sterols or fatty acids, sugars can be presented on the lipid membrane surface in an extremely stable and simple manner, and are also presented on the lipid membrane surface. Control the density of sugar.
  • hydrophilic polymers into which sugar is introduced include polyalkylene glycols (eg, polyethylene glycol, polypropylene glycol, polytetramethylene glycol, polyhexamethylene glycol, etc.), dextran, pullulan, ficoll, and polybule.
  • polyalkylene glycols eg, polyethylene glycol, polypropylene glycol, polytetramethylene glycol, polyhexamethylene glycol, etc.
  • dextran pullulan
  • ficoll and polybule.
  • Alcohol styrene Maleic anhydride alternating copolymer
  • dibule ether Maleic anhydride alternating copolymer
  • Polyethylene glycol is preferred, with polyalkylene glycol being preferred.
  • hydrophilic polymer is a polyalkylene glycol
  • its molecular weight is usually 500 to 5000, preferably ⁇ 1000 to 3000, and more preferably ⁇ 2000 to 3000.
  • the hydrophilic polymer includes methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sbutyl group, tbutyl group, n-pentyl group, isopentyl group, tpentyl group.
  • a linear or branched alkyl group having 1 to 5 carbon atoms such as a group or neopentyl group; methoxy group, ethoxy group, n propoxy group, isopropoxy group, n butoxy group, isobutoxy group, s butoxy group, t
  • a linear or branched alkoxy group having 1 to 5 carbon atoms such as a butoxy group; a substituent such as a hydroxyl group, a carbo group, an alkoxy carbo group, or a cyan group; Moyo.
  • the lipid membrane constituent component and the hydrophilic polymer include a functional group possessed by the lipid membrane constituent component (including a functional group artificially introduced into the lipid membrane constituent component) and a functional group possessed by the hydrophilic polymer ( It contains a functional group artificially introduced into the hydrophilic polymer, and can be bonded via a covalent bond formed by reaction with.
  • Examples of combinations of functional groups that can form a covalent bond include amino group Z carboxyl group, amino group Z halogenated acyl group, amino group ZN hydroxysuccinimide ester group, amino group Z benzotriazole carbonate group, amino group Group z aldehyde group, thiol group z maleimide group, thiol group z berylsulfone group, hydroxyl group Z carboxyl group and the like, and the reaction between these functional groups can be carried out according to a known method.
  • the hydrophilic polymer and sugar include a functional group possessed by the hydrophilic polymer (including a functional group artificially introduced into the hydrophilic polymer) and a functional group possessed by the sugar (artificially introduced into the sugar. Can be linked via a covalent bond formed by a reaction with a functional group. Examples of combinations of functional groups that can form a shared bond include amino groups, amino groups and hydroxyl groups, amino groups and thiol groups, and the reaction between these functional groups can be performed according to a known method. .
  • the sugar and the lipid membrane constituent component include a functional group possessed by the sugar (including a functional group artificially introduced into the sugar) and a functional group possessed by the lipid membrane constituent component (artificial to the lipid membrane constituent component).
  • a functional group possessed by the sugar including a functional group artificially introduced into the sugar
  • a functional group possessed by the lipid membrane constituent component artificial to the lipid membrane constituent component.
  • the hydrophilic polymer introduced with a saccharide is, for example, the following formula: P—X—S [wherein P represents a residue of the hydrophilic polymer, X represents a direct bond or a linking group, and S Represents a sugar residue. ].
  • the linking group represented by X for example, a divalent hydrocarbon group which may have a hetero atom; —O—; —S—; —NH—; —COO—; —SS— -NHCO-; -NHCONH-; -SO- and the like.
  • hetero atom examples include a nitrogen atom, an oxygen atom, a halogen atom (fluorine atom, chlorine atom, bromine atom, etc.), a sulfur atom, and the like.
  • divalent hydrocarbon group which may have a heteroatom include, for example, an alkylene group such as an ethylene group, a trimethylene group, and a propylene group; —SCH CH COO—; —OCH
  • the sugar may be bound to the lipid membrane constituent or the hydrophilic polymer via V, a substituent of the offset carbon, or the lipid membrane constituent via the substituent of the carbon at the 1-position. Or it is preferable to bind with a hydrophilic polymer.
  • the target substance to be delivered into the nucleus can be encapsulated inside the ribosome.
  • the type of the target substance is not particularly limited, and examples thereof include drugs, nucleic acids, peptides, proteins, sugars, or complexes thereof, depending on the purpose of diagnosis, treatment, prevention, etc. It can be selected appropriately.
  • the target substance is a substance for the purpose of diagnosis, treatment, prevention or the like of a disease
  • the ribosome holding the target substance can be used as a component of a pharmaceutical composition.
  • Nucleic acid includes DNA or RNA, and analogs or derivatives thereof (for example, peptide nucleic acid (PNA), phosphorothioate DNA, etc.).
  • the nucleic acid may be either single-stranded or double-stranded, and may be either linear or circular.
  • the target substance is preferably encapsulated in the ribosome as an aggregate of the target substance.
  • the target substance can be efficiently delivered into the nucleus.
  • the aggregate of the target substance may be composed only of the target substance, or may contain a substance other than the target substance (for example, a carrier that holds the target substance)!
  • an aggregate of the target substance is prepared by electrostatically binding the target substance and the ionic substance to form a composite.
  • an aggregate of the target substance can be prepared by electrostatically binding the target substance and the cationic substance to form a composite.
  • the target substance and a predetermined carrier are combined in an appropriate manner (for example, physical adsorption, hydrophobic bond, chemical bond, etc.) to form a complex.
  • an aggregate of the target substance can be prepared.
  • complexing an aggregate of the target substance that is positively or negatively charged as a whole can be prepared by adjusting the mixing ratio of the target substance to the cationic substance or the anionic substance.
  • an aggregate of nucleic acids can be prepared by electrostatically binding the nucleic acid and the cationic substance to form a complex.
  • an aggregate of nucleic acids that are positively or negatively charged as a whole can be prepared.
  • the cationic substance that can be used when preparing the aggregate of the target substance is not particularly limited as long as it is a substance having a cationic group in the molecule.
  • the cationic substance include cationic lipids (for example, Lipofectamine (manufactured by Invitrogen)); macromolecules having a cationic group; A polymer or copolymer or a derivative thereof (e.g. Polycationic polymers such as polyethyleneimine, poly (arylamine), poly (diallyldimethylammonium chloride), darcosamine; protamine or its derivatives (for example, protamine sulfate); Of these, stearyl lyin arginine is particularly preferred.
  • the number of arginine residues constituting the 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.
  • an amino group; a monoalkylamino group such as a methylamino group or an ethylamino group; a dialkylamino group such as a dimethylamino group or a jetylamino group; an imino group; -Dino group etc. are mentioned.
  • the anionic substance that can be used in preparing the aggregate of the target substance is not particularly limited as long as it is a substance having an anionic group in the molecule.
  • the ionic substance include ionic lipids; polymers having ionic groups; homopolymers or copolymers of acidic amino acids such as polyaspartic acid or derivatives thereof; xanthan gum, carboxybi Diar polymers, carboxymethylcellulose polystyrene sulfonates, polysaccharides, polyarion polymers such as carrageenan, and the like can be used.
  • the terionic group is not particularly limited as long as it can be negatively charged, for example, a functional group having a terminal carboxyl group (for example, a succinic acid residue, a malonic acid residue, etc.), a phosphoric acid group, a sulfuric acid group, etc. Is mentioned.
  • a ribosome equipped with a sugar-modified lipid membrane the sugar existing on the surface of the sugar-modified lipid membrane binds to the nuclear pore complex, and the sugar bound to the nuclear pore complex becomes intranuclear.
  • the target substance encapsulated in the ribosome is released into the nucleus through the nuclear pore, and as a result, the target substance is delivered into the nucleus.
  • a ribosome provided with a sugar-modified lipid membrane can be used as a vector for delivering a target substance into the nucleus.
  • the nucleus targeted by the ribosome may be a nucleus separated from cell force or a nucleus existing in a cell.
  • the species from which the nucleus targeted by the ribosome is derived is not particularly limited. For example, animals such as animals, plants, and microorganisms are preferred. Mammals are more preferred. Examples of mammals include humans, monkeys, mice, hidges, sheep, horses, pigs, rabbits, dogs, cats, rats, mice, guinea pigs and the like.
  • the type of cell containing the nucleus targeted by the liposome is not particularly limited, and examples include somatic cells, germ cells, stem cells, or cultured cells thereof.
  • the ribosome is configured such that the sugar-modified lipid membrane is located in the outermost layer of the ribosome transferred into the cell.
  • the ribosome having a configuration in which the sugar-modified lipid membrane is located in the outermost layer of the ribosome transferred into the cell, for example, as shown in Fig. 1 (a), lipid membrane 2a and A single membrane ribosome la comprising the target substance 3 encapsulated inside the lipid membrane 2a, the lipid membrane 2a having a peptide containing a plurality of arginine residues continuous with sugar on the surface Single membrane ribosome la.
  • the “peptide containing a plurality of consecutive arginine residues” will be described in detail below.
  • Single-membrane ribosome la can be transferred from the outside of the cell to the inside of the cell through the peptide existing on the surface of the lipid membrane 2a while maintaining its original form (in an intact state).
  • the sugar present on the surface of the lipid membrane 2a binds to the nuclear pore complex, and the sugar bound to the nuclear pore complex is extracted into the nucleus, thereby disrupting the lipid membrane 2a.
  • the target substance 3 enclosed inside the lipid membrane 2a is released into the nucleus through the nuclear membrane pore.
  • a lipid membrane 21b As another embodiment of the ribosome having a configuration in which the sugar-modified lipid membrane is located in the outermost layer of the ribosome transferred into the cell, as shown in Fig. 1 (b), a lipid membrane 21b, A bilayer ribosome comprising a lipid membrane 22b located outside the lipid membrane 21b and a target substance 3 encapsulated inside the lipid membrane 21b, the lipid membrane 21b having a sugar on its surface, An example is bilayer ribosome lb, where 22b contains a membrane-fusible lipid.
  • Examples of the membrane-fused lipid contained in the lipid membrane 22b include neutral lipids such as dioleoylphosphatidylethanolamine; and cation lipids (acid lipids) such as cholesterol succinic acid and cardiolipin.
  • the amount of membrane-fusible lipid contained in membrane 22b is usually 40% (molar ratio) or more, preferably 70% (molar ratio) or more, more preferably 90% of the total lipid content contained in lipid membrane 22b. (Molar ratio) or more.
  • the upper limit of the content of membrane-fusible lipid is 100%.
  • the bilayer ribosome lb transferred into the cell via endocytosis is taken up into the endsome, but the endosomal force can also escape by membrane fusion between the endosomal membrane and the lipid membrane 22b.
  • the lipid membrane 22b disappears due to membrane fusion with the endosomal membrane, but the lipid membrane 21b is retained.
  • the target substance 3 encapsulated inside the lipid membrane 21b is released into the nucleus in the same manner as in the case of the single membrane ribosome la.
  • the bilayer ribosome lb can be transferred from the outside of the cell into the cell via membrane fusion between the lipid membrane 22b and the cell membrane.
  • the lipid membrane 22b disappears due to membrane fusion with the cell membrane, but the lipid membrane 21b is retained.
  • the target substance 3 encapsulated inside the lipid membrane 21b is released into the nucleus in the same manner as in the case of the single membrane ribosome la.
  • the amount of cation lipid contained in the lipid membrane 22b is 15% (molar ratio) or more, preferably 20% of the amount of membrane-fused lipid contained in the lipid membrane 22c. (Molar ratio) or higher, the endosomal interior changes to acidic (pH 5.5-6.5) or under acidic conditions (pH 5.5-6.5) , The lipid membrane 22b can be efficiently fused with the endosomal membrane or cell membrane.
  • the number of consecutive arginine residues is not particularly limited as long as it is a plurality, but usually 4 to 20, preferably 6 to 12 and more preferably 7 to: LO.
  • the total number of amino acid residues constituting the peptide is not particularly limited, but is usually 4 to 35, preferably 6 to 30, and more preferably 7 to 23.
  • the peptide can include any amino acid sequence at the C-terminus and the Z- or N-terminus of a plurality of consecutive arginine residues, but it is preferable that only the arginine residue can be used.
  • the amino acid sequence added to the C-terminal or N-terminal of a plurality of consecutive arginine residues is preferably an amino acid sequence having rigidity (for example, polyproline). Unlike polyethylene glycol (PEG), which is soft and irregularly shaped, polyproline is linear and retains a certain degree of rigidity. Also included in the amino acid sequence added to the C-terminal or N-terminal of multiple consecutive arginine residues.
  • the amino acid residue is preferably an amino acid residue other than acidic amino acids. This is because an acidic amino acid residue having a negative charge may electrostatically interact with an arginine residue having a positive charge to attenuate the effect of the arginine residue.
  • the lower limit of the amount of peptide present on the surface of the lipid membrane is usually 0.1% (molar ratio), preferably 1% (molar ratio), more preferably 2 with respect to the total lipid constituting the lipid membrane.
  • the upper limit is usually 30% (molar ratio), preferably 25% (molar ratio), more preferably 20% (molar ratio) with respect to the total lipid constituting the lipid membrane. is there.
  • a ribosome having a peptide containing a plurality of continuous arginine residues on its surface can migrate into the cell via the peptide present on the surface (see WO2005Z03 2593).
  • the amount of peptide present on the surface of the ribosome is less than 2% (molar ratio), preferably less than 1.5% (molar ratio), more preferably 1% (molar ratio) relative to the total lipid constituting the lipid membrane. If it is less than that, the ribosome can be transferred into the cell or nucleus mainly via endocytosis (see WO2005Z032593).
  • the lower limit of the amount of peptide at this time is usually 0.1% (molar ratio), preferably 0.5% (molar ratio), more preferably 0.7% (total ratio) with respect to the total lipid constituting the lipid membrane. Molar ratio).
  • the amount of peptide present on the surface of the ribosome is 2% (molar ratio) or more, preferably 3% (molar ratio) or more, more preferably 4% (molar ratio) with respect to the total lipid constituting the lipid membrane.
  • the ribosome can be transferred into the cell or nucleus mainly via macropinocytosis (see WO 2005Z032593).
  • the upper limit of the peptide amount at this time is usually 30% (molar ratio), preferably 25% (molar ratio), more preferably 20% (molar ratio) with respect to the total lipid constituting the lipid membrane.
  • macropinocytosis extracellular substances are taken into the cell as a fraction called macropinosome, and unlike macrosomes, macropinosomes do not fuse with lysosomes, so macropinosome inclusions avoid degradation by lysosomes. can do. Therefore, when the ribosome moves into the cell via macropinocytosis, the target substance encapsulated in the ribosome can be efficiently delivered into the cell.
  • the lipid membrane When the intracellular pathway of the ribosome depends on endocytosis, the lipid membrane must contain a cationic lipid as its main component, but a peptide containing a plurality of consecutive arginine residues on the surface.
  • the ribosome translocation pathway has Since it does not depend only on cis, it is not necessary that the lipid membrane contains a cationic lipid. That is, the lipid membrane of the ribosome may be composed of either a cationic lipid or a non-power thione lipid, or may be composed of both.
  • the ratio of cationic lipid to the total lipid composing the lipid membrane is preferably 0 to 40% (molar ratio). 0 to 20% (molar ratio) More preferably,
  • the presence of the peptide containing a plurality of consecutive arginine residues is not particularly limited as long as the peptide is exposed to the surface force of the liposome, but the peptide is modified with a hydrophobic group or a hydrophobic compound.
  • a hydrophobic group or a hydrophobic compound is inserted into the lipid bilayer, and the peptide is exposed from the lipid bilayer.
  • the lipid bilayer consists of a hydrophilic part and a hydrophobic part, and a hydrophobic group or hydrophobic compound can be inserted into the lipid bilayer by hydrophobic bonding with the hydrophobic part of the lipid bilayer.
  • the hydrophobic group or hydrophobic compound is not particularly limited as long as it can be inserted into the lipid bilayer.
  • the hydrophobic group include a saturated or unsaturated fatty acid group such as a stearyl group, a cholesterol group, or a derivative thereof.
  • a fatty acid group having 10 to 20 carbon atoms for example, a palmitoyl group, an oleyl group).
  • Stearyl group, arachidoyl group, etc. are preferable.
  • hydrophobic compounds include phospholipids, glycolipids or sterols exemplified above, long chain aliphatic alcohols (eg, phosphatidylethanolamine, cholesterol, etc.), polyoxypropylene alkyls, glycerin fatty acids. Examples include esters.
  • Ribosomes can be prepared using known methods such as hydration, sonication, ethanol injection, ether injection, reverse phase evaporation, surfactant method, and freezing and thawing. You can. Moreover, by passing the ribosome through a filter having a predetermined pore size, a ribosome having a certain particle size distribution can be obtained. Further, according to a known method, the multilamellar ribosome force can be converted into a single membrane ribosome, and the single membrane ribosome force can also be converted into a multilamellar liposome. [0073] A composition containing a ribosome provided with a sugar-modified lipid membrane can be used for various applications.
  • the target substance is encapsulated inside the ribosome.
  • a target substance capable of exerting a therapeutic effect or a preventive effect on a predetermined disease by being delivered into the nucleus for example, antisense DNA, siRNA, etc. capable of suppressing the expression of a disease-causing gene
  • a gene to be expressed in the nucleus is encapsulated inside the ribosome.
  • the dosage forms of various compositions are not particularly limited, and examples thereof include ribosome dispersions.
  • a buffer solution such as physiological saline, phosphate buffer solution, quenen buffer solution, and acetate buffer solution can be used.
  • additives such as sugars, polyhydric alcohols, water-soluble polymers, nonionic surfactants, antioxidants, pH adjusters, hydration accelerators may be added to the dispersion.
  • Other dosage forms of the composition include dried products of ribosome dispersions (eg, freeze-dried products, spray-dried products, etc.). The dried product can be used as a ribosome dispersion by adding a buffer solution such as physiological saline, phosphate buffer, queen buffer, or acetate buffer.
  • compositions When the composition is administered to a living body, examples of the route of administration include parenteral administration such as intravenous, intraperitoneal, subcutaneous, nasal administration, etc. It can be appropriately adjusted according to the kind, amount, etc. of the target substance enclosed.
  • FIG. 1 is a diagram schematically showing the structure of a ribosome.
  • FIG. 2 (a) is a graph showing the amount of gene expression 3 hours after transfection of gene-encapsulated sugar-modified ribosome, and (b) is 6 hours after transfection of gene-encapsulated sugar-modified ribosome. It is a figure which shows the gene expression level.
  • FIG. 3 (a) is a graph showing the amount of gene expression after 3 hours of transfection of a gene-encapsulated sugar-modified ribosome, and (b) is 6 hours after transfection of the gene-encapsulated sugar-modified ribosome. It is a figure which shows the gene expression level.
  • FIG. 4 (a) shows gene development 3 hours after gene-encapsulated sugar-modified ribosome transfection. (B) is a diagram showing the gene expression level 6 hours after transfection of the gene-encapsulated sugar-modified ribosome.
  • FIG. 5 (a) is a graph showing the amount of gene expression 3 hours after transfection of gene-encapsulated sugar-modified ribosome, and (b) is 6 hours after transfection of gene-encapsulated sugar-modified ribosome. It is a figure which shows the gene expression level.
  • FIG. 6 is a graph showing the relationship between the amount of gene expression 6 hours after transfection of a gene-encapsulated sugar-modified ribosome and the amount of cholesterol contained in the lipid membrane.
  • FIG. 7 is a graph showing the gene expression level 6 hours after transfection of a gene-encapsulated sugar-modified ribosome in the presence of an excessive amount of sugar.
  • FIG. 8 is a photograph showing that a gene-encapsulated sugar-modified ribosome is transferred into the nucleus. Blue indicates the nucleus, red indicates the gene, and green indicates the vector-derived lipid.
  • the DNA used is a luciferase gene and a 7037 bp full-length plasmid DNA with a CMV promoter upstream (the pcDNA3.1 plasmid with the CMV promoter incorporated with the luciferase gene), and polycation is protamine sulfate.
  • DNA and polycation are each dissolved in 10 mM HEPES buffer (pH 7.4), and DNA solution (0.1 mgZmL) and polycation solution (0.07 mg / mL) are mixed at room temperature under vortexing. Aggregated.
  • lipid membrane consists of dioleoy 1 phosphatidyl ethanolamine (DOPE) and cholesterol (GlcNAc-PEG- Choi) bound to N-acetylethylcolacamine-introduced polyethylene glycol 2: 1 or 9: 1 (M
  • DOPE dioleoy 1 phosphatidyl ethanolamine
  • GlcNAc-PEG- Choi cholesterol bound to N-acetylethylcolacamine-introduced polyethylene glycol 2: 1 or 9: 1
  • M octaarginine
  • STR—R8 solution lipid 5 mol% was added to the mixture and formed by removing the solvent in a glass test tube. The final lipid concentration after hydration was 0.55 mM.
  • GlcNAc PEG Choi was synthesized according to the following scheme.
  • Dissolved and activated molecular sieves (4 A) (about 2 g) are stirred and added to this. 3,4,5 —tri—0—Acety to 1—thiopheny to 2—N—Troc—glucosamine 240 mg Subsequently, 145 mg of N-hydroxy succinimide and 10 ⁇ L of trifluoromethanesulfonic acid (purity 98%, manufactured by Wako Pure Chemical Industries, Ltd.) were added to initiate the reaction. The reaction was carried out for about 3 hours under inert and water-free conditions, and the resulting reaction solution was diluted with chloroform and then washed with a saturated aqueous solution of sodium thiosulfate and then with water.
  • GlcNAc-PEG- Choi was used in place of Choi, and ribosomes with octarginine on the surface of the lipid membrane but no N-acetyl darcosamine were prepared (particle size: 220 nm, Zeta potential: +30 mV).
  • the lipid membrane was not modified with stearyloctaarginine, and ribosomes with N-acetyl darcosamine on the surface of the lipid membrane but no octaarginine were prepared (particle size: 190 nm, zeta potential: -20mV).
  • GlcNAc- PEG- Choi is used instead of Choi, and the lipid membrane is not modified with stearyloctaarginine, and N-acetylethyldarcosamine and octarginine are present on the lipid membrane surface.
  • Ribosomes were prepared (particle size: 200 nm, zeta potential: 10 mV).
  • the cells were synchronized to G1 phase by incubation for 18 hours at 37 ° C, 5% CO. afterwards,
  • the cells were supplemented with ribosomes (DNAlOpgZcell) and incubated at 37 ° C for 3 hours to transfect the cells with ribosomes. After 3 and 6 hours of transfection, the cells were washed, lysed with 75 ⁇ L of reporter lys s buffer (Promega), and luciferase atse reagent (Promega) 50 ⁇ L into 20 ⁇ L of cell lysate. The luciferase activity was measured with a luminometer (Luminescencer-PSN). In addition, the amount of protein in the cell lysate was measured using a BCA protein assembly kit (PIERCE).
  • Fig. 2 (a) The gene expression level after 3 hours of transfection is shown in Fig. 2 (a), and the gene expression level after 6 hours of transfection is shown in Fig. 2 (b).
  • Fig. 2 (b) As shown in Fig. 2 (a), after 3 hours of transfection, the surface of the lipid membrane has neither N-acetylyl darcosamine nor octarginine, but the ribosome has octarginine on the surface of the lipid membrane. Ribosomes that do not have N-acetylyldarcosamine, and ribosomes that have N-acetylyldarcosamine on the surface of the lipid membrane but not octaarginine did not show any gene expression regardless of the lipid composition.
  • N-acetyltilcosamine was recognized by cell surface receptor recognition. It does not function as an element that promotes cellular uptake of ribosomes. It is thought that it functions as an element that promotes nuclear translocation of ribosomes after ribosomes have been taken into cells by the function of octaarginine.
  • the ribosome When sugars present on the surface of the membrane are bound to the nuclear pore complex, the ribosome is translocated to the nucleus, and the sugar bound to the nuclear pore complex is extracted into the nucleus, thereby disrupting the lipid membrane.
  • the target substance encapsulated inside the ribosome is considered to be released into the nucleus through the nuclear pore.
  • the nuclear translocation of ribosomes is thought to mainly involve the binding of sugars to the nuclear pore complex, and the intranuclear delivery of ribosome-encapsulated substances is mainly due to the sugar bound to the nuclear pore complex. It is considered that the lipid membrane is disrupted by drawing into the nucleus, and the lipid membrane disintegration is improved by reducing the amount of membrane stabilizer (eg, cholesterol) contained in the lipid membrane.
  • membrane stabilizer eg, cholesterol
  • DOPE, cholesterol (Choi), and cholesterol-conjugated polyethylene glycol (Sugar—PEG—Choi) 5: 5: 0, 5: 4: 1, 5: 3: 2, 5: 2: 3 , 5: 1: 4 or 5: 0: 5 (molar ratio), dissolved in black mouth form and mixed (total lipid content 55 nmol), except that a lipid membrane was formed to form the same as in Example 1.
  • the procedure was performed to prepare a liposome having a sugar (sugar binds to the lipid membrane via polyethylene glycol) and octaarginine on the surface of the lipid membrane (particle size: 220 nm, zeta potential: +10 mV).
  • N-acetylyldarcosamine GlcNAc
  • Man mannose
  • galatatoose Gal
  • Fig. 3 (a), Fig. 4 (a) or Fig. 5 (a) shows the gene expression level after 3 hours of transfection
  • Fig. 3 (b) and Fig. 4 (6) show the gene expression level after 6 hours of transfection. It is shown in b) or Fig. 5 (b).
  • a stearyl Locta arginine (STR-R8) solution (5 mol% of lipid) was added, and the solvent was removed in a glass test tube to form a lipid membrane.
  • Example 1 After the DPC suspension prepared in Example 1 was added to this lipid membrane and incubated for 15 minutes, it was sonicated in an ultrasonic bath (125 W, Branson Ultrasonics) for about 1 minute to obtain the above sugar And ribosomes with octaarginine were prepared. In addition, a control ribosome was prepared using cholesterol instead of cholesterol to which sugar-introduced polyethylene glycol was bound.
  • an ultrasonic bath 125 W, Branson Ultrasonics
  • the cell cycle was synchronized with the G phase. If not, 18 hours before transfer
  • the medium was replaced with a new one, and the cells were cultured at 37 ° C in a 5% CO environment for 18 hours.
  • Rhodamine modified DPC was prepared. Except that this rhodamine-modified DPC was used and NBD—DOPE (AVANTI POLAR LIPIDS) was added to the black mouth form solution so as to be 1% mol of the total lipid amount, all by the method shown in Example 4, Prepared sugar-modified ribosomes with lipids labeled with NB D and encapsulating rhodamine-modified DPC
  • HeLa cells were seeded in a 35 mm glass bottom dish (Iwaki) 48 hours prior to transfection so that 5 x 10 4 cells / 2 mL DMEM (containing 10% serum) Zdish was obtained at 37 ° C. Culturing was performed in a 5% CO environment. 2.5 mM hydrate 18 hours before transfer
  • the cell cycle was synchronized with G phase by interculturing.

Abstract

L'invention concerne un vecteur destiné au transport nucléaire d'une substance. Elle concerne également une composition contenant ce vecteur. Un liposome unilamellaire (1a) comprend une membrane lipidique (2a) et une substance (3) renfermée dans la membrane lipidique (2a), un sucre et un peptide comprenant deux ou plusieurs résidus acides aminés contigus étant introduits à la surface de la membrane lipidique (2a). Le liposome (1a) peut être transporté de l'extérieur d'une cellule à l'intérieur de la cellule par le peptide présent sur la surface de la membrane lipidique (2a). Après que le liposome (1a) a été transporté dans la cellule, le sucre à la surface de la membrane lipidique (2a) se lie à un complexe de pores nucléaires. Le sucre lié au complexe de pores nucléaires est entraîné dans le noyau de la cellule, provoquant la désagrégation de la membrane lipidique (2a), et la substance (3) renfermée dans la membrane lipidique (2a) peut ainsi être libérée à l'intérieur du noyau par le biais des pores nucléaires.
PCT/JP2007/054260 2006-03-07 2007-03-06 Vecteur pour le transport nucléaire d'une substance WO2007102481A1 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009131216A1 (fr) * 2008-04-25 2009-10-29 国立大学法人 北海道大学 Structure de membrane lipidique modifiée par un oligo(alkylène glycol)
WO2010110471A1 (fr) * 2009-03-23 2010-09-30 国立大学法人北海道大学 Structure membranaire lipidique
JP2011500011A (ja) * 2007-10-10 2011-01-06 アンスティテュ、ナショナル、ド、ラ、サント、エ、ド、ラ、ルシェルシュ、メディカル（アンセルム） 癌治療用の組合せ製品
JP2011506432A (ja) * 2007-12-10 2011-03-03 エピターゲット・アーエス 非ラメラ形成脂質を含む音響感受性薬物送達粒子
WO2011132713A1 (fr) 2010-04-21 2011-10-27 国立大学法人北海道大学 Structure membranaire lipidique ayant une transférabilité nucléaire
WO2018230710A1 (fr) 2017-06-15 2018-12-20 国立大学法人北海道大学 Structure de membrane lipidique pour administration intracellulaire d'arnsi
US10182987B2 (en) 2014-05-20 2019-01-22 National University Corporation Hokkaido University Lipid membrane structure for intracellular delivery of siRNA
WO2020241679A1 (fr) 2019-05-30 2020-12-03 国立大学法人北海道大学 Nanoparticule lipidique
WO2022071582A1 (fr) 2020-10-02 2022-04-07 国立大学法人北海道大学 Nanoparticule lipidique
WO2022244844A1 (fr) 2021-05-21 2022-11-24 国立大学法人北海道大学 Nanoparticules lipidiques

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005032593A1 (fr) * 2003-10-01 2005-04-14 Japan Science And Technology Agency Liposome a polyarginine modifiee pouvant etre transfere dans un noyau
WO2006101201A1 (fr) * 2005-03-24 2006-09-28 National University Corporation Hokkaido University Liposome capable de liberer efficacement une substance donnee dans le noyau
WO2007037444A1 (fr) * 2005-09-30 2007-04-05 National University Corporation Hokkaido University Vecteur destiné à la délivrance d'une substance cible dans un noyau ou une cellule

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005032593A1 (fr) * 2003-10-01 2005-04-14 Japan Science And Technology Agency Liposome a polyarginine modifiee pouvant etre transfere dans un noyau
WO2006101201A1 (fr) * 2005-03-24 2006-09-28 National University Corporation Hokkaido University Liposome capable de liberer efficacement une substance donnee dans le noyau
WO2007037444A1 (fr) * 2005-09-30 2007-04-05 National University Corporation Hokkaido University Vecteur destiné à la délivrance d'une substance cible dans un noyau ou une cellule

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BARDONNET P.-L. ET AL.: "Cholesteryl oligoethyleneglycol glycosides: fluidizing effect of their embedment into phospholipid bilayers", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 329, no. 4, 2005, pages 1186 - 1192, XP004784858 *
DUVERGER E. ET AL.: "Nuclear import of glycoconjugates is distinct from the classical NLS pathway", J. CELL SCI., vol. 108, no. PART 4, 1995, pages 1325 - 1332, XP003017804 *
FAJAC I. ET AL.: "Sugar-mediated uptake of glycosylated polylysines and gene transfer into normal and cystic fibrosis airway epithelial cells", HUM. GENE THER., vol. 10, no. 3, 1999, pages 395 - 406, XP000982499 *
KOGURE K. ET AL.: "Development of a non-viral multifunctional envelope-type nano device by a novel lipid film hydration method", J. CONTROL RELEASE, vol. 98, no. 2, 2004, pages 317 - 323, XP004521720 *
NISHIKAWA M. ET AL.: "Glycosylated cationic liposomes for carbohydrate receptor-mediated gene transfer", METHODS ENZYMOL., vol. 373, 2003, pages 384 - 399, XP003017802 *
WANG J.L. ET AL.: "Lectins in the cell nucleus", GLYCOBIOLOGY, vol. 1, no. 3, 1991, pages 243 - 252, XP003017803 *

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JP2015091826A (ja) * 2007-10-10 2015-05-14 アンスティテュ、ナショナル、ド、ラ、サント、エ、ド、ラ、ルシェルシュ、メディカル（アンセルム）Ｉｎｓｔｉｔｕｔ Ｎａｔｉｏｎａｌ Ｄｅ Ｌａ Ｓａｎｔｅ Ｅｔ Ｄｅ Ｌａ Ｒｅｃｈｅｒｃｈｅ Ｍｅｄｉｃａｌ （Ｉｎｓｅｒｍ） 癌治療用の組合せ製品
US9364494B2 (en) 2007-10-10 2016-06-14 Institut National De La Sante Et De La Recherche Medicale (Inserm) Somatostatin 2 receptor (sst2), deoxycytidine kinase (dck) and uridine monophosphate kinase (umk) pancreatic gene therapy
JP2011500011A (ja) * 2007-10-10 2011-01-06 アンスティテュ、ナショナル、ド、ラ、サント、エ、ド、ラ、ルシェルシュ、メディカル（アンセルム） 癌治療用の組合せ製品
JP2011506432A (ja) * 2007-12-10 2011-03-03 エピターゲット・アーエス 非ラメラ形成脂質を含む音響感受性薬物送達粒子
WO2009131216A1 (fr) * 2008-04-25 2009-10-29 国立大学法人 北海道大学 Structure de membrane lipidique modifiée par un oligo(alkylène glycol)
WO2010110471A1 (fr) * 2009-03-23 2010-09-30 国立大学法人北海道大学 Structure membranaire lipidique
WO2011132713A1 (fr) 2010-04-21 2011-10-27 国立大学法人北海道大学 Structure membranaire lipidique ayant une transférabilité nucléaire
US8981044B2 (en) 2010-04-21 2015-03-17 National University Corporation Hokkaido University Lipid membrane structure having intranuclear migrating property
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