WO2009131216A1

WO2009131216A1 - Lipid membrane structure modified with oligo(alkylene glycol)

Info

Publication number: WO2009131216A1
Application number: PCT/JP2009/058172
Authority: WO
Inventors: 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: 2008-04-25
Filing date: 2009-04-24
Publication date: 2009-10-29

Abstract

Disclosed is a lipid membrane structure comprising, as a membrane-constituting component, a lipid modified with an oligo(alkylene glycol) having a degree of polymerization of an alkylene group of 4 to 25. The lipid membrane structure has an excellent ability to release a substance enclosed therein in a target cell. Particularly, when a nucleic acid is enclosed in the lipid membrane structure, the level of expression/transfer of the nucleic acid in a target cell can be remarkably increased compared to that of a control lipid membrane structure. Therefore, the lipid membrane structure is particularly useful as a vector for gene therapy.

Description

Lipid membrane structure modified with oligoalkylene glycol

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.

As a means for specifically transporting a drug to the affected area, a method of encapsulating the drug in a lipid membrane structure such as a liposome and administering it has been proposed. In particular, a lipid membrane structure called a multifunctional envelope-type nanostructure (MEND: Multifunctional-envelope-type-nano-device, hereinafter referred to as “MEND” in this specification) is in a form that can be expressed in a cell. It can be used as a drug delivery system for selectively delivering a nucleic acid into a specific cell, and is known to be useful, for example, for gene therapy of tumors.

However, 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. In addition, 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.

As means for avoiding the above problem, 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). This means that 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. based on. Based on this idea, 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.

However, when the surface of the lipid membrane structure is modified with polyalkylene glycol, the retention in the blood is improved, but a new problem that the lipid membrane structure becomes difficult to be taken into the target cell may occur. Are known. As a result, in the targeting treatment in which a drug is encapsulated in a lipid membrane structure modified with polyalkylene glycol and delivered to a target cell, not only the expected therapeutic effect is obtained, but also due to the drug not delivered to the target cell. Problems such as side effects occurred.

As a means for solving such a problem, 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.

However, in the method described in 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.

JP 2007-99750 A

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.

(1) 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.

(2) The lipid membrane structure according to (1), wherein the lipid membrane is a single membrane.

(3) The lipid membrane structure according to (1) or (2), wherein the oligoalkylene glycol is oligoethylene glycol.

(4) The lipid membrane according to (3), wherein the 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.

(5) The 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 .

(6) 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).

(7) The lipid membrane structure according to any one of (1) to (6), wherein a nucleic acid is encapsulated.

(8) 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.

(9) The intracellular transcription promoter according to (8), wherein the lipid membrane is a single membrane.

(10) The intracellular transcription promoter according to (8) or (9), wherein the oligoalkylene glycol is oligoethylene glycol.

(11) The intracellular cell according to (10), wherein the 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.

(12) The intracellular transcription promotion according to any one of (8) to (11), wherein the lipid modified with oligoalkylene glycol is phospholipid, glycolipid, sterol, long-chain aliphatic alcohol or glycerin fatty acid ester Agent.

(13) 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).

(14) A method for producing a lipid single membrane structure in which a drug is encapsulated, wherein the drug is encapsulated using a lipid modified with an oligoalkylene glycol having an alkylene group polymerization degree of 4 to 25 as a component of the membrane. The manufacturing method described above.

(15) The production method according to (14), wherein the oligoalkylene glycol is oligoethylene glycol.

(16) The production method according to (15), wherein the 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. .

(17) The production method according to any one of (14) to (16), wherein the lipid modified with oligoalkylene glycol is a phospholipid, glycolipid, sterol, long-chain aliphatic alcohol or glycerin fatty acid ester. .

(18) 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.

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. 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. It is a graph which shows the translation efficiency obtained by dividing the expression level of a nucleic acid by the mRNA expression level of the whole transformed HeLa cell based on the measured value of real-time PCR. It is a photograph taken using a confocal laser microscope after adding a fluorescent substance SYTO24 to HeLa cells transformed with MEND encapsulating a rhodamine-labeled nucleic acid. This is a photograph taken using a confocal laser microscope after Hoechst 33342 was added to HeLa cells transformed with MEND in which a rhodamine-labeled nucleic acid was encapsulated and a lipid membrane was labeled with NBE to label the nucleus. 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. In the figure, 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, and 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. Hereinafter, 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). 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.

Examples of glycolipids include glycolipids such as cephalin, cerebroside, ceramide, sphingomyelin, ganglioside, and one or more of these can be used.

Examples of sterols 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.

As the long-chain fatty acid or long-chain aliphatic alcohol, 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.

Examples of glycerin fatty acid esters 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.

For example, when the lipid is phosphatidylethanolamine (PE), 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.

In addition, as a method of modifying a long chain fatty acid with OAG, 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.

Furthermore, as a method for modifying sterols with OAG, Bhattacharya et al. (Langmuir, 2001, Vol. 17, pp. 2067-2075) is known. That is, 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.

In addition to the above lipids or lipids modified with OAG, 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.

For example, 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.

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. Examples of the cationic lipid include dioctadecyldimethylammonium chloride (DODAC), N- (2,3-oleyloxy) propyl-N, N, N-trimethylammonium (N- (2,3-dioyloxy)). (propyl-N, N, N-trimethylammonium, DOTMA), didodecylammonium bromide (DDAB), 1,2-dioleoyloxy-3-trimethylammoniumpropane (1,2-dioleoyl-3-trimethylammonium ) 3β-N- (N ′, N′-dimethylaminoethane) carbamo Cholesterol (3β-N- (N ′, N ′,-dimethyl-aminoethane) -carbamol cholesterol, DC-Chol), 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium (1,2-dimethyltripropylpropyl-3 -Dimethylhydroxyethyl ammonium, DMRIE), 2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propaneammonium trifluoroacetate (2,3-dioyloxy-N- [2 (Sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanamine trifluorocet te, DOSPA), and the like.

When, for example, the polyarginine peptide is added to the lipid membrane structure of the present invention, the lipid membrane structure 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. Since the cationic lipid has cytotoxicity, it is preferable to reduce the amount of the cationic lipid contained in the lipid bilayer as much as possible from the viewpoint of reducing the cytotoxicity of the liposome of the present invention. 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).

The above-mentioned “non-cationic lipid” means a neutral lipid or an anionic lipid, and examples of the neutral lipid include diacylphosphatidylcholine, diacylphosphatidylethanolamine, cholesterol, ceramide, sphingomyelin, cephalin, and cerebroside. Examples of 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. In addition to lipids modified with OAG, lipids not modified with OAG or other than OAG Lipids modified with these substances may be included as components. For example, 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.

As will be described in detail later in the examples, 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. In particular, when 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. Accordingly, 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. Surprisingly, by using a lipid modified with OAG as a component of the membrane, the lipid membrane structure can be a closed vesicle composed of a single membrane.

In general, a lipid membrane structure composed of a single lipid membrane is prepared by repeatedly passing a filter of an appropriate size. However, OAG, that is, a lipid modified with an oligoalkylene glycol having an alkylene group polymerization degree of 4 to 25 is used. By using it as a component of the membrane, a lipid membrane structure composed of a lipid monolayer can be prepared without using a filter.

Accordingly, 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.

In addition, as long as 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. Can be produced. For example, in the case of the 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. .

In the above method, as the 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.

Also, a lipid membrane structure having a certain particle size distribution can be obtained by passing through a filter having a predetermined pore size.

Various 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. When 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. In addition, when 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. The term “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. For example, a cationic lipid (for example, Lipofectamine (Invitrogen)), a polymer having a cationic group, 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, and protamine sulfate.

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. For example, 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, and 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. As the dispersion solvent, for example, a buffer solution such as physiological saline, phosphate buffer, citrate buffer, and acetate buffer can be used. For example, 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. When used in vivo, 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. Moreover, 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.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention should not be construed as being limited to these examples.

<Example 1>
1) Preparation of 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.

Ingredients consisting of the following composition in a total lipid weight ratio were collected in a glass test tube, dissolved in chloroform by adding chloroform so that the total amount was 250 μL, and then the solvent was distilled off with a desiccator to obtain three types of lipids. A film was formed.

2) Preparation of Nucleic Acid-Protamine Aggregate Plasmid pEGFP-Luc (Clontech) encoding luciferase and protamine were mixed at 1: 0.67 (mass ratio) in HEPES buffer (10 mM HEPES / NaOH pH 7.4). Thus, an aggregate of about 80 nm was obtained.

3) Preparation of lipid membrane structure encapsulating nucleic acid To each of the three lipid membranes obtained in 1), 250 μL of the aggregate solution of 2) was added and hydrated for 10 minutes. This hydrate is subjected to ultrasonic treatment with a water tank type ultrasonic generator for 30 seconds to 1 minute, whereby aggregates are enclosed and octaarginine peptides having different contents of TEGChol (5 mol of total lipid amount). %) Were prepared.

4) Evaluation of MENDs 1 to 3 a) Measurement of membrane potential The aggregate prepared in 2), the size of MENDs 1 to 3 obtained in 3) and the membrane potential (Zeta potential) were measured using Zetasizer (Malvern). It was measured. The results are shown in the table.

From this measurement, it was confirmed that the membrane potential was reversed by the production of MEND, and the aggregate was encapsulated by the lipid membrane. In addition, it was confirmed that the size of the lipid membrane structure tends to monotonously decrease as the content of TEGChol increases.

b) Introduction into HeLa cells 4 × 10 ⁴ cells / well HeLa cells are seeded in a 24-well plate, MENDs 1 to 3 are added to each plasmid so that the plasmid DNA becomes 0.4 μg, and at 37 ° C. and 5% CO ₂ . After culturing for 6 hours, the cells were collected, and luciferase activity and protein were quantified.

As a result, an increase in luciferase activity was confirmed with an increase in the content of TEGCol, and it was confirmed that the luciferase activity in MEND3 was increased to about 120 times compared to MEND1 not containing TEGChol (FIG. 1). ).

<Example 2>
41.25 μL of 1 mM DOTAP, 55 μL of 1 mM DOPE, and 41.3 μL of 1 mM cholesterol (DOTAP: DOPE: cholesterol = 3: 4: 3) were dispensed into a glass test tube, and 112.5 μL of chloroform to a total volume of 250 μL. Were added and mixed, and the solvent was distilled off with a desiccator to form a lipid membrane (control). Further, 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.

250 μL of the nucleic acid-protamine aggregate solution 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.

Inoculate 4 × 10 ⁴ cells / well HeLa cells in a 24-well plate, add each of MEND4-7 to a plasmid DNA of 0.4 μg, and incubate at 37 ° C., 5% CO ₂ for 6 hours. Quantification of luciferase activity and protein was performed.

As a result, even in the lipid membrane structure not containing STR-R8, an increase in luciferase activity was confirmed with an increase in the content of TEGChol, and the luciferase activity in MEND6 was about 24 compared to the control not containing TEGChol. A double increase was confirmed (FIG. 2).

<Test 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 β-actin.

As a result, it was confirmed that the expression level of luciferase in HeLa cells was increased about 6-fold in the expression level of MEND3 with respect to MEND1 not containing TEGChol (FIG. 3).

In addition, the nuclear fraction was collected from the HeLa cells into which the gene had been introduced according to the method of Iwasa et al. (Literature BiochimｐｈBiophys Acta. 2006; 1758: 713-720), It was measured. As a result, in the nucleus of the transformed HeLa cell, an increase in the expression level comparable to that of the whole cell was confirmed (FIG. 4).

From this, it was speculated that the increase in the luciferase activity in the lipid membrane structure (MEND2, 3) of the present invention was caused by the increase in the expression level of the luciferase gene in the nucleus.

Further, when calculating the nuclear translocation efficiency of the nucleic acid obtained by dividing the nuclear translocation amount of the nucleic acid by the intracellular uptake amount based on the measurement value of the real-time PCR, there is no significant difference between MENDs 1 to 3. However, the post-nuclear translocation expression efficiency obtained by dividing the nucleic acid expression level by the nuclear translocation amount increased in TMENDs 2 and 3 (FIG. 6). Furthermore, the translation efficiency obtained by dividing the expression level of the nucleic acid by the mRNA expression level of the whole transformed HeLa cells was not significantly different between MENDs 1 to 3 (FIG. 7). From the above, it is presumed that the increase in nucleic acid expression by the lipid membrane structure of the present invention is due to the increase in the transcription amount of the nucleic acid.

<Test 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.

In addition, in 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.

<Test 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.

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. In 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. On the other hand, in 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. This is presumably because the nucleic acid encapsulated in the lipid membrane structure is efficiently detached from the lipid membrane in the cell. Therefore, TEGChol was shown to be a useful element for smoothly dissociating the drug encapsulated in the lipid membrane structure.

<Test Example 4>
When MEND1 and MEND3 prepared in Example 1 were observed with a SEM electron microscope, it was confirmed that most of MEND3 was in the form of a lipid monolayer (FIG. 10).

<Test 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.

An experiment was conducted in the same manner as in Example 1 except that each lipid was 40%, and a HeLa cell was prepared using MEND having lipids modified with each oligoethylene glycol as a component of the membrane and having stearyl octaarginine. Then, the gene was introduced, and luciferase activity and protein were quantified 6 hours later.

As a result of measuring the luciferase activity in this HeLa cell into which this gene was introduced (FIG. 11), it was modified with MEND composed of stearic acid modified with oligoethylene glycol having a polymerization degree of 4 and oligoethylene glycol having a polymerization degree of 4. There is almost no difference in the amount of luciferase expressed between the MENDs containing cholesterol as a constituent, and MEND having a degree of polymerization of 45 and MEND containing stearic acid modified with oligoethylene glycol having a degree of polymerization of 45. There was almost no difference in the luciferase expression level among MENDs composed of DSPE modified with oligoethylene glycol, confirming that the effect of modification with oligoethylene glycol does not depend on the lipid to be modified. It was done.

In addition, among MENDs using stearic acid modified with oligoethylene glycol having a degree of ethylene polymerization of 4, 10, 25, 45 and 55 as a constituent of the membrane, when the degree of polymerization is 25, luciferase activity increases. As can be seen, it was confirmed that when the degree of polymerization was 45, the luciferase activity was lower than that of unmodified MEND.

From this result, it was confirmed that an ethylene group polymerization number of 4 to 25 is preferable in order to increase the expression and transfer efficiency of nucleic acid.

Claims

A lipid membrane structure comprising a lipid modified with an oligoalkylene glycol having an alkylene group polymerization degree of 4 to 25 as a component of the membrane.
The lipid membrane structure according to claim 1, wherein the lipid membrane is a single membrane.
The lipid membrane structure according to claim 1 or 2, wherein the oligoalkylene glycol is oligoethylene glycol.
The lipid membrane structure according to claim 3, wherein the oligoethylene glycol is at least one selected from the group consisting of triethylene glycol, tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, heptaethylene glycol and octaethylene glycol.
The lipid membrane structure according to any one of claims 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, timosterol and ergosterol. 5. The lipid membrane structure according to 5.
The lipid membrane structure according to any one of claims 1 to 6, wherein a nucleic acid is encapsulated.
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.
The intracellular transcription promoter according to claim 8, wherein the lipid membrane is a single membrane.
The intracellular transcription promoter according to claim 8 or 9, wherein the oligoalkylene glycol is oligoethylene glycol.
The intracellular transcription promoter according to claim 10, wherein the oligoethylene glycol is at least one selected from the group consisting of triethylene glycol, tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, heptaethylene glycol and octaethylene glycol. .
The intracellular transcription promoter according to any one of claims 8 to 11, 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, timosterol and ergosterol. 12. The intracellular transcription promoter according to 12.
A method for producing a lipid single membrane structure in which a drug is encapsulated, comprising encapsulating a drug using a lipid modified with an oligoalkylene glycol having an alkylene group polymerization degree of 4 to 25 as a component of the membrane. The manufacturing method characterized by the above-mentioned.
The production method according to claim 14, wherein the oligoalkylene glycol is oligoethylene glycol.
The production method according to claim 15, wherein the oligoethylene glycol is at least one selected from the group consisting of triethylene glycol, tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, heptaethylene glycol and octaethylene glycol.
The production method according to any one of claims 14 to 16, 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, timosterol and ergosterol. 18. The production method according to 17.