WO2010110471A1 - Lipid membrane structure - Google Patents

Abstract

Provided are a lipid membrane structure that is for delivering substances into the cytoplasm of a target cell and has INF7 surface modifications; and a lipid membrane structure that is for delivering substances into the nucleus of a target cell, encapsulates the substance to be delivered on the interior thereof, and has a surface modified with three or more oligosaccharide compounds.

Description

Lipid membrane structure

The present invention relates to a lipid membrane structure capable of efficiently releasing substances such as nucleic acids into the cytoplasm of target cells such as tissues and organs.
The present invention also relates to a lipid membrane structure having excellent transport efficiency into the nucleus. More specifically, the present invention relates to a lipid membrane structure that is surface-modified with a sugar compound and has excellent nuclear transport properties.

A method of encapsulating a drug in a liposome that is a lipid membrane structure has been proposed as a means of specifically transporting the drug to the affected area. In particular, the effectiveness of liposomes encapsulating an antitumor agent has been reported in the field of malignant tumor treatment. In addition, as a lipid membrane structure that can be used for gene expression, a multifunctional envelope-type nano structure (MEND: Multifunctional envelope-type nano device; hereinafter, may be abbreviated as “MEND”. For example, Drug Delivery System. 22-2, pp. 115-122, 2007, etc.). This structure can be used as a drug delivery system for selectively delivering a gene or the like into a specific cell, and is known to be useful for, for example, tumor gene therapy.

As a means to deliver target substances such as drugs, nucleic acids, peptides, proteins, and sugars to specific sites such as target organs and tumor tissues using lipid membrane structures, the surface of lipid membrane structures is functional molecules. Many methods of modifying have been proposed. In general, lipid membrane structures are subject to opsonization due to adsorption of serum proteins while circulating in the blood, and are trapped by macrophages present in the liver and spleen. In order to avoid this problem, for example, a method of modifying the surface of the liposome membrane with a hydrophilic polymer such as polyalkylene glycol such as polyethylene glycol (PEG) has been widely used. Many proposals have been made (Japanese Patent Laid-Open Nos. 1-249717, 2-149512, 4-346918, 2004-10481, etc.). Tumor tissues generally have large gaps between tumor vascular endothelial cells, and therefore, it is possible to use a route that allows liposomes to leak through the gaps and selectively deliver liposomes to tumor tissues. Since the delivery rate of liposomes to tumor tissue can be increased, it is common to apply a polyalkylene glycol to a lipid membrane structure containing an antitumor agent.

Lipid membrane structures encapsulating drugs such as antitumor agents reach the target cells and are taken into cells by endocytosis and become encapsulated in endosomes. The received drug is released into the cytoplasm. However, lipid membrane structures surface-modified with polyalkylene glycols such as PEG are difficult to release from endosomes, and there is a problem that drugs such as antitumor agents cannot be efficiently released from the endosome into the cytoplasm. . For example, in order to enhance drug release from liposomes incorporated into endosomes, liposomes whose surface is modified with a peptide (GALA: Biochemistry, 26, pp. 2964-2972, 1987) have been proposed ( Biochemistry, 43, pp. 5618-5623, 2004), and lipid membrane structures such as liposomes surface-modified with polyalkylene glycols such as PEG cannot improve drug release even when surface modification of GALA is performed.

On the other hand, a glutamic acid-rich peptide (INF7) obtained by modifying a peptide (1-23) derived from influenza HA protein (HA2) is known, and by mixing it with liposomes, the lipid structure collapses and the encapsulated substance can be easily obtained. It has been reported to be released (Biochemistry, 46, pp. 13490-13504, 2007). In addition, a delivery system in which DNA-binding peptide (DBP) is bound to polyethylene glycol tetraacrylate (PEG-TA) and INF7 is further bound has been proposed, and it has been reported that gene delivery efficiency can be improved. (The Journal of Gene Medicine, 10, pp. 1131-1149, 2008).

As a means for transferring a lipid membrane structure encapsulating a target substance such as a nucleic acid into the nucleus of a target cell, for example, a liposome whose outer surface is modified with octaarginine (International Publication WO2005 / 32593; Journal) of Controlled Release, 98, pp. 317-323, 2004), bilamellar liposomes having lipid membranes modified with nuclear translocation peptides (International Publication WO2006 / 101201), and surface modification with monosaccharides such as galactose and mannose Liposomes (International Publication WO2007 / 102481) have been proposed. About the said liposome modified with monosaccharide, it is said that the gene expression efficiency was able to be improved in the test result in vitro. However, the above publication does not show the effect of these liposomes in an in vivo experimental system. For example, by intravenous administration, the liposome is transferred into the nucleus of a cell of a specific organ or tissue to improve gene expression efficiency. Experimental results on whether it can be done are not shown.

JP-A-1-249717 JP-A-2-149512 JP-A-4-346918 JP 2004-10482 A International Publication WO2005 / 32593 International Publication WO2006 / 101201 International Publication WO2007 / 102481

An object of the present invention is to provide a lipid membrane structure for efficiently releasing substances such as drugs and genes into the cytoplasm of target cells. More specifically, an object of the present invention is to provide a lipid membrane structure capable of efficiently releasing a substance encapsulated in the lipid membrane structure from the endosome incorporating the lipid membrane structure into the cytoplasm. is there.
Another object of the present invention is to provide means for efficiently transferring a lipid membrane structure containing a target substance such as a nucleic acid into the nucleus of a target cell.
Among the above problems, one of the particularly preferable problems is to efficiently transfer a lipid membrane structure encapsulating a nucleic acid containing a gene into the nucleus of a target organ or tissue cell such as the liver to achieve high gene expression activity. It is in providing the means for.

As a result of intensive studies to solve the above problems, the present inventors have found that the surface of a lipid membrane structure such as a liposome encapsulating a substance such as a drug or a gene is an endosome escape peptide (EEP). It has been found that substances such as drugs and genes can be released into the cytoplasm very efficiently by modification with INF7. It was also found that even when the surface of the lipid membrane structure is modified with a polyalkylene glycol such as PEG, the encapsulated drug or gene can be efficiently released into the cytoplasm. The present invention has been completed based on the above findings.

In addition, the present inventors have found that a lipid membrane structure whose surface is modified with an oligosaccharide compound of 3 or more sugars such as maltotriose is efficiently taken into the nucleus of a cell, and a nucleic acid containing a gene is used in the above method. It has been found that extremely high gene expression activity can be achieved by transferring to the nucleus. The present invention has been completed based on the above findings.

That is, according to the present invention, a lipid membrane structure for delivering a substance into the cytoplasm of a target cell, which is surface-modified with INF7, is provided.
According to a preferred embodiment of the present invention, the above lipid membrane structure wherein the lipid membrane structure is a liposome; the above lipid membrane structure surface-modified with a polyalkylene glycol, preferably polyethylene glycol (PEG); and polyarginine Provided is the above lipid membrane structure, preferably surface-modified with octaarginine.

According to a further preferred embodiment, the amount of surface modification with INF7 is in the range of 1 to 5 mol% with respect to the total amount of lipid membrane structure; surface modification with polyalkylene glycol, preferably PEG The above lipid membrane structure whose amount is in the range of 1 to 40 mol% with respect to the total lipid amount of the lipid membrane structure; the amount of surface modification with polyarginine, preferably octaarginine is the total lipid amount of the lipid membrane structure On the other hand, the above lipid membrane structure in the range of 0.8 to 20 mol% is provided. There is also provided the above lipid membrane structure surface-modified with a lipid-modified INF7, preferably a stearyl INF7, in which a lipid compound and INF7 are covalently bonded.

From another viewpoint, a lipid membrane structure for delivering a substance into the cytoplasm of a target cell, wherein the substance to be delivered is encapsulated, and the lipid membrane structure is surface-modified with INF7 Is provided. Preferably, the lipid membrane structure as described above, wherein the lipid membrane structure is a liposome; and the lipid membrane structure as described above, surface-modified with a polyalkylene glycol, preferably PEG, are provided by the present invention.

According to a preferred embodiment of the present invention, the substance to be delivered is a nucleic acid, for example, the above lipid membrane structure which is a functional nucleic acid such as a nucleic acid containing a gene or siRNA; and surface-modified with polyarginine, preferably octaarginine The above lipid membrane structure is provided. Further, the lipid membrane structure is a multifunctional envelope nanostructure (MEND); the multifunctional envelope nanostructure is encapsulated with a nucleic acid and a cationic polymer; The above lipid membrane structure which is a liposome whose surface is modified with polyarginine, preferably octaarginine and INF7; the above lipid membrane structure whose surface is a liposome further modified with polyalkylene glycol, preferably PEG; And the above lipid membrane structure wherein the cationic polymer is protamine. Also provided are the above lipid membrane structures used for gene expression; and the above lipid membrane structures used for gene therapy.
A pharmaceutical composition comprising this lipid membrane structure as an active ingredient, preferably a pharmaceutical composition comprising a nucleic acid as a substance to be delivered is also provided by the present invention.

From still another aspect, a method of releasing a substance into the cytoplasm of a target cell in the living body of a mammal including humans, the lipid membrane structure having a surface modified with INF7 and enclosing the substance to be delivered inside A method is provided comprising the step of administering a body to the animal. Examples of substances to be delivered include active pharmaceutical ingredients or nucleic acids. Preferably, the above method is provided wherein the lipid membrane structure is surface modified with a polyalkylene glycol, preferably PEG. This lipid membrane structure is taken up into the target cell by endocytosis and transferred into the cytoplasm after being encapsulated in the endosome, and then escapes from the endosome by the action of INF7 and efficiently releases the substance into the cytoplasm. be able to.

A preferred embodiment of the present invention is a method for expressing a gene in cells of mammals including humans, which is surface-modified with INF7, further surface-modified with PEG and polyarginine, preferably octaarginine, and the gene A method comprising the step of administering to an animal a lipid membrane structure encapsulating a nucleic acid comprising Preferably, the above lipid membrane structure in which a cationic polymer such as protamine is encapsulated together with a nucleic acid can be used. Further provided is the above method for use in gene therapy.

In addition, according to the present invention, there is provided a method for preventing and / or treating diseases of mammals including humans, which are surface-modified with INF7, preferably further polyalkylene glycol, more preferably with PEG, and pharmaceuticals. There is provided a method comprising the step of administering to an animal a lipid membrane structure having an active ingredient encapsulated therein. Examples of pharmaceutically active ingredients include antitumor agents and nucleic acids.

Furthermore, a method for preventing and / or treating diseases of mammals including humans, which is surface-modified with INF7, preferably further surface-modified with polyalkylene glycol, more preferably with PEG, and active pharmaceutical ingredients such as There is provided a method comprising the step of administering to an animal a lipid membrane structure encapsulating therein an antitumor agent or a nucleic acid containing a gene.

From another point of view, according to the present invention, there is provided a lipid membrane structure for delivering a substance into the nucleus of a target cell, the substance to be delivered is encapsulated therein, and an oligosaccharide compound having 3 or more sugars. A surface-modified lipid membrane structure is provided.
According to a preferred embodiment of the present invention, the above lipid membrane structure wherein the oligosaccharide compound is a trisaccharide compound; the above lipid membrane structure wherein the oligosaccharide compound is a glucose trimer; the oligosaccharide compound is maltotriose There is provided the above lipid membrane structure; and the above lipid membrane structure wherein the lipid membrane structure is a liposome.

According to a further preferred embodiment, the above lipid membrane structure surface-modified with GALA; the amount of surface modification with GALA is in the range of 0.1 to 4 mol% with respect to the total lipid amount of the lipid membrane structure. Lipid membrane structure: The above lipid membrane structure further modified with polyalkylene glycol, preferably polyethylene glycol (PEG); the amount of surface modification by PEG is 1 to 40 mol relative to the total lipid amount of the lipid membrane structure % Of the above lipid membrane structure; and the above lipid membrane structure wherein the target cell is a liver cell.

From another viewpoint, a lipid membrane structure for delivering a substance into the nucleus of a target cell, in which the substance to be delivered is encapsulated, and surface modification with an oligosaccharide compound of 3 or more sugars Provided lipid membrane structures are provided. According to a preferred embodiment of the present invention, the above lipid membrane structure wherein the oligosaccharide compound is a trisaccharide compound; the above lipid membrane structure wherein the oligosaccharide compound is a glucose trimer; the oligosaccharide compound is maltotriose There is provided the above lipid membrane structure; and the above lipid membrane structure, wherein the target cell is a liver cell.

According to a further preferred aspect, there is provided the above lipid membrane structure wherein the substance is a nucleic acid, for example, a functional nucleic acid such as a nucleic acid containing a gene or siRNA, and the lipid membrane structure is a multifunctional envelope nanostructure. The above-mentioned lipid membrane structure which is (MEND); the multifunctional envelope nanostructure is a liposome in which a nucleic acid and a cationic polymer are encapsulated and the surface is modified with an oligosaccharide compound having 3 or more sugars The above-mentioned lipid membrane structure; the multifunctional envelope nanostructure is a liposome in which a nucleic acid and a cationic polymer are encapsulated and the surface is modified with an oligosaccharide compound having three or more sugars, GALA, and polyethylene glycol And the above lipid membrane structure wherein the cationic polymer is protamine. In addition, the above lipid membrane structure used for gene expression in the liver; the above lipid membrane structure used in gene therapy; the above lipid membrane structure used in gene therapy of liver disease; and liver disease is diabetes, liver cancer or virus There is provided the above lipid membrane structure which is hepatitis hepatitis.
A pharmaceutical composition comprising the lipid membrane structure as an active ingredient, preferably a pharmaceutical composition comprising a nucleic acid as the substance is also provided by the present invention.

From still another aspect, a method for delivering a substance into the nucleus of a target tissue or organ cell in vivo in mammals including humans, wherein the substance is modified with an oligosaccharide compound having 3 or more sugars, preferably further GALA And a method comprising administering to said animal a lipid membrane structure that is surface-modified with polyalkylene glycol and encapsulating a substance to be delivered. Examples of substances to be delivered include active pharmaceutical ingredients or nucleic acids.

A preferred embodiment of the present invention is a method for expressing a gene in a cell nucleus of a target tissue or organ in a living body of a mammal, including a human, which is modified with an oligosaccharide compound having 3 or more sugars, preferably further GALA and poly There is provided a method comprising the step of administering to an animal a lipid membrane structure that is surface-modified with an alkylene glycol and encapsulating a substance to be delivered. Preferably, the above lipid membrane structure in which a cationic polymer such as protamine is encapsulated together with a nucleic acid can be used. Further, the above method for expressing a gene in liver cells; the above method used for gene therapy; the above method used for gene therapy of liver disease; the above method wherein the liver disease is diabetes, liver cancer or viral hepatitis And the above method wherein the dosage form is intravenous administration.

Further, according to the present invention, there is provided a method for preventing and / or treating diseases of mammals including humans, which are modified with an oligosaccharide compound having 3 or more sugars, preferably further surface-modified with GALA and polyalkylene glycol, A method comprising the step of administering to the animal a lipid membrane structure encapsulating a pharmaceutically active ingredient therein is provided.

As one embodiment of the present invention, there is provided a method for preventing and / or treating diseases of mammals including humans, which are modified with oligosaccharide compounds having 3 or more sugars, preferably further surface-modified with GALA and polyalkylene glycol. And a method comprising administering to the animal a lipid membrane structure having nucleic acid encapsulated therein. According to a preferred embodiment of this method, there is provided the above method wherein the disease is liver disease; the above method wherein the liver disease is diabetes, liver cancer or viral hepatitis; and the above method wherein the dosage form is intravenous administration Is done.

When the lipid membrane structure of the present invention is used, substances encapsulated from the lipid membrane structure can be efficiently released into the cytoplasm. In particular, a lipid membrane structure modified with a polyalkylene glycol such as PEG can also increase the release efficiency of the substance into the cytoplasm. Therefore, for example, it contains an active pharmaceutical ingredient such as an antitumor agent or a nucleic acid containing a gene. It is possible to increase the retention of the lipid membrane structure in the blood and improve the release efficiency of the active pharmaceutical ingredient from the lipid membrane structure into the cytoplasm.

In addition, when the lipid membrane structure of the present invention is used, a desired substance, preferably a nucleic acid can be efficiently delivered into the nucleus of a cell of a target tissue or organ. For example, when a nucleic acid containing a gene is delivered It becomes possible to highly express the gene in a nucleus such as a liver cell. For example, using a multifunctional envelope nanostructure surface-modified with maltotriose and GALA as the lipid membrane structure of the present invention, a nucleic acid containing a gene is delivered into the nucleus of a liver cell to express the gene. In the case of an in vivo test system, the gene expression efficiency can be increased by about 100 times compared to the case of no modification in the in vivo test system, and the gene expression is higher than that of currently available transfection reagents. Since efficiency can be achieved, the lipid membrane structure of the present invention enables extremely efficient gene therapy for diabetes, viral hepatitis, liver cancer, and the like.

It is the figure which showed the effect of INF7 on the transfection activity of a gene inclusion R8 modification liposome. The result of EPC / DOPC / Chol / R8 (0.4 μg pDNA / well) in HeLa cells 40,000 cells / well is shown. It is the figure which showed the effect of INF7 on the transfection activity of a gene inclusion R8 modification liposome. The result of EPC / DOPE / Chol / R8 (0.4 μg pDNA / well) in HeLa cells 40,000 cells / well is shown. It is the figure which showed the gene expression result in vivo by intravenous administration of R8-MEND and 3% of STR-INF7 modification R8-MEND. It is the figure which showed the result of having introduce | transduced STR-INF7 modification R8-MEND containing plasmid DNA in a cell, and observing DNA escape from an endosome. (A) shows R8-MEND, (b) shows the result of STR-INF7 / R8-MEND, red shows MEND; Rhod-pDNA, green shows endosome; PKH67 modification, yellow shows colocalization (arrow), The bar is 10 μm. It is the figure which showed that STR-INF7 modification R8-MEND escapes from an endosome without passing through membrane fusion with an endosome. (A) shows the result of non-membrane fusion R8-MEND, (b) shows the result of non-membrane fusion STR-INF7 / R8-MEND, blue shows the result of nuclear staining with Hoechst 33342, red shows the rhodamine-DOPE and Green indicates NBD-DOPE. It is the figure which showed the pH dependence in the membrane destruction activity of the INF7 modification liposome using a calcein release. It is the figure which showed the endosome escape promotion activity by INF7 in a PEG modification liposome. Red indicates MEND; Rhod-pDNA, green indicates endosome; PKH67 modification, bars are 10 μm. It is the figure which showed the effect of INF7 on the transfection activity of a gene inclusion R8 / PEG modification liposome. EPC / DOPC / Chol / R8-10% -MEND (+/− 10% DSPE-PEG 2000) was used as MEND, and HeLa cells (40,000 cells / well) were used as cells. It is the figure which showed the result of having evaluated the particle diameter and zeta potential of MEND containing GALA (2%) and maltotriose (5%), and the gene expression (luciferase activity) in a mouse liver. It is the figure which showed the result of having changed the content of maltotriose-PEG-lipid in the range of 0 to 15%, and having evaluated the gene expression activity. It is the figure which showed the result of having evaluated gene expression activity about sugars (cellotriose, mannose, galactose, and (beta) maltose) other than maltotriose. It is the figure which showed the result of having evaluated gene expression activity about sugars (alpha maltose and lactose) other than maltotriose. It is the figure which showed the result of having evaluated the liver transfer amount after administering MEND intravenously to a mouse | mouth. It is the figure which showed the result of having evaluated the amount of liver transfer after intravenously administering MEND modified with GALA and maltotriose to a mouse | mouth. It is the figure which showed the result of having compared the gene expression in the liver at the time of Lipoplex and Polyplex administration with MEND.

Examples of the lipid constituting the lipid membrane structure of the present invention for delivering a substance into the cytoplasm of a target cell include phospholipids, glycolipids, sterols, or saturated or unsaturated fatty acids.
Examples of the lipid constituting the lipid membrane structure of the present invention for delivering a substance into the nucleus of a target cell include phospholipids, sterols, saturated or unsaturated fatty acids, and the like.

Examples of phospholipids and phospholipid derivatives include phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, cardiolipin, sphingomyelin, ceramide phosphorylethanolamine, ceramide phosphorylglycerol, ceramide phosphorylglycerol phosphate, 1 , 2-Dimyristoyl-1,2-deoxyphosphatidylcholine, plasmalogen, phosphatidic acid, and the like, which can be used alone or in combination of two or more. Fatty acid residues in these phospholipids are not particularly limited, and examples thereof include saturated or unsaturated fatty acid residues having 12 to 20 carbon atoms. Specific examples include lauric acid, myristic acid, palmitic acid, stearin Mention may be made of acyl groups derived from fatty acids such as acids, oleic acid and linoleic acid. Moreover, phospholipids derived from natural products such as egg yolk lecithin and soybean lecithin can also be used.

Examples of the glycolipid include glyceroglycolipid (eg, sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride), sphingoglycolipid (eg, galactosyl cerebroside, lactosyl cerebroside, ganglioside) and the like. Can be mentioned.

Examples of sterols include animal-derived sterols (for example, cholesterol, cholesterol succinic acid, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol), plant-derived sterols (phytosterol) (for example, stigmasterol, sitosterol, campesterol, Brush castrol), sterols derived from microorganisms (for example, timosterol, ergosterol) and the like.
Examples of the saturated or unsaturated fatty acid include saturated or unsaturated fatty acids having 12 to 20 carbon atoms such as palmitic acid, oleic acid, stearic acid, arachidonic acid, and myristic acid.

The form of the lipid membrane structure is not particularly limited. For example, as a form dispersed in an aqueous solvent, single membrane liposome, multilamellar liposome, O / W emulsion, W / O / W emulsion, spherical micelle, string micelle Or an irregular layered structure. A preferred form of the lipid membrane structure of the present invention is a liposome. Hereinafter, although a liposome may be described as a preferred embodiment of the lipid membrane structure of the present invention, the lipid membrane structure of the present invention is not limited to liposomes.

The lipid membrane structure of the present invention for delivering a substance into the cytoplasm of a target cell is characterized in that its surface is modified with INF7. INF7 is a glutamic acid-rich peptide obtained by modifying a peptide (1-23) derived from influenza HA protein (HA2), and it is reported that the inclusion of a substance with liposomes causes the lipid structure to collapse and the encapsulated substance is easily released. (Biochemistry, 46, pp. 13490-13504, 2007), and a delivery system in which INF7 is bound to polyethylene glycol tetraacrylate (PEG-TA) has also been proposed (The Journal of Gene Medicine, 10, pp. 13-28). 1134-1149, 2008). A person skilled in the art can easily use INF7 in the present invention by referring to these publications. In this specification, the term “INF7” is used in Biochemistry, 46, pp. In addition to the peptide specified by the sequence described in Table 1 of 13490-13504, 2007, it consists of an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or added in the amino acid sequence of the peptide. In particular, modified peptides having the same properties as INF7 are also included. The term “INF7” herein should not be construed as limiting in any way. The disclosures of the above publications and the disclosures of all documents cited in this publication are incorporated herein by reference.

The method of modifying the lipid membrane structure with INF7 is not particularly limited, but generally, by constructing a lipid membrane structure using lipid-modified INF7 in which a lipid compound and INF7 are covalently bonded as a lipid membrane-constituting lipid, A lipid membrane structure surface-modified with INF7 can be easily produced. As the lipid-modified INF, for example, stearyl INF7 can be used, and this compound is disclosed in Futaki S et al. Biocongug. Chem. , 12 (6), pp. 1005-1011, 2001, and can be easily manufactured. The amount of surface modification by INF7 is not particularly limited, but is generally in the range of 1 to 5 mol% with respect to the total lipid amount of the lipid membrane structure, preferably about 3 to 5 mol% with respect to the total lipid amount. It is.

The type of oligosaccharide compound having 3 or more sugars used for surface modification of the lipid membrane structure of the present invention for delivering a substance into the nucleus of a target cell is not particularly limited. For example, about 3 to 10 sugars An oligosaccharide compound having units bonded thereto can be used, and preferably an oligosaccharide compound having about 3 to 6 sugar units bonded can be used.

More specifically, examples of the oligosaccharide compound include cellotriose (Cellotriose: β-D-glucopyranosyl- (1 → 4) -β-D-glucopyranosyl- (1 → 4) -D-glucose), chacotriose: α -L-rhamnopyranosyl- (1 → 2)-[α-L-rhamnopyranosyl- (1 → 4)]-D-glucose), gentianose (β-D-fructofuranosyl β-D-glucopyranosyl- (1 → 6) -α-D-glucopyranoside), isomaltotriose (α-D-glucopyranosyl- (1 → 6) -α-D-glucopyranosyl- (1 → 6) -D-glucose), isopanose (Isopanose) : Α-D-glucopyranosyl- (1 → 4)-[α- D-glucopyranosyl- (1 → 6)]-D-glucose), maltotriose (α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranosyl- (1 → 4) -D-glucose) Manninotriose: α-D-galactopyranosyl- (1 → 6) -α-D-galactopyranosyl- (1 → 6) -D-glucose), melezitose: α-D -Glucopyranosyl- (1 → 3) -β-D-fructofuranosyl = α-D-glucopyranoside), Panose (Panose: α-D-glucopyranosyl- (1 → 6) -α-D-glucopyranosyl- (1 → 4) -D-glucose), Plantose: α-D-galactopyranosyl- (1 → 6) -β-D-fruc Tofuranosyl = α-D-glucopyranoside), raffinose (β-D-fructofuranosyl = α-D-galactopyranosyl- (1 → 6) -α-D-glucopyranoside), soratriose: α- L-rhamnopyranosyl- (1 → 2)-[β-D-glucopyranosyl- (1 → 3)]-D-galactose), umbelliferose (β-D-fructofuranosyl = α-D-galactopyra Trisacyl compounds such as nosyl- (1 → 2) -α-D-galactopyranoside); Lycotetraose: β-D-glucopyranosyl- (1 → 2)-[β-D-xylopyranosyl- ( 1 → 3)]-β-D-glucopyranosyl- (1 → 4) -β-D-galactose), maltotetraau (Maltotetraose: α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranosyl- (1 → 4) -D-glucose), stachyose: β- A tetrasaccharide compound such as D-fructofuranosyl = α-D-galactopyranosyl- (1 → 6) -α-D-galactopyranosyl- (1 → 6) -α-D-glucopyranoside); malto Pentaose (Maltopentaose: α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranosyl- (1 → 4) -D-glucose), Verbascosse (Verbascose: β-D-fructofuranosyl = α-D-galactopyranosyl- (1 → 6) -α Pentasaccharide compounds such as D-galactopyranosyl- (1 → 6) -α-D-galactopyranosyl- (1 → 6) -α-D-glucopyranoside); maltohexaose (α-D- Glucopyranosyl- (1 → 4) -α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranosyl- Hexasaccharide compounds such as (1 → 4) -D-glucose) can be mentioned, but are not limited thereto.

Preferably, an oligosaccharide compound that is a trimer or hexamer of glucose can be used, and more preferably, an oligosaccharide compound that is a trimer or tetramer of glucose can be used. More specifically, isomaltotriose, isopanose, maltotriose, maltotetraose, maltopentaose, or maltohexaose can be preferably used, and among these, malto in which glucose is α1-4 bonded. More preferred is triose, maltotetraose, maltopentaose, or maltohexaose. Particularly preferred is maltotriose or maltotetraose, and most preferred is maltotriose.

The method of modifying the surface of the lipid membrane structure with an oligosaccharide compound is not particularly limited. For example, liposomes (International Publication WO2007 / 102481) in which the lipid membrane structure is modified with a monosaccharide such as galactose or mannose are known. Therefore, the surface modification method described in this publication can be adopted. The entire disclosures of the above publications are incorporated herein by reference. This method is a method in which a monosaccharide compound is bonded to a polyalkylene glycolated lipid to modify the surface of the lipid membrane structure. By this means, the surface of the lipid membrane structure can be simultaneously modified with polyalkylene glycol. preferable.

The blood retention of liposomes can be increased by modifying the surface of the lipid membrane structure of the present invention with a hydrophilic polymer such as polyalkylene glycol. This means is described, for example, in JP-A-1-249717, JP-A-2-149512, JP-A-4-346918, and JP-A-2004-10482. As the hydrophilic polymer, polyalkylene glycol is preferable. As polyalkylene glycol, for example, polyethylene glycol, polypropylene glycol, polytetramethylene glycol, polyhexamethylene glycol and the like can be used. The molecular weight of the polyalkylene glycol is, for example, about 300 to 10,000, preferably about 500 to 10,000, and more preferably about 1,000 to 5,000.

The surface modification of the lipid membrane structure with polyalkylene glycol can be easily performed by constructing a lipid membrane structure using, for example, a polyalkylene glycol-modified lipid as a lipid membrane constituent lipid. For example, when the modification with polyethylene glycol is performed, stearyl polyethylene glycol (for example, PEG45 stearate (STR-PEG45) or the like) can be used. In addition, N- [carbonyl-methoxypolyethyleneglycol-2000] -1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, n- [carbonyl-methoxypolyethyleneglycol-5000] -1,2-dipalmitoyl -Sn-glycero-3-phosphoethanolamine, N- [carbonyl-methoxypolyethylene glycol-750] -1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N- [carbonyl-methoxypolyethylene glycol -2000] -1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N- [carbonyl-methoxypolyethyleneglycol-5000] -1,2-distearoyl-sn-glycero-3-phosphoethanol Amine Can also be used such as polyethylene glycol derivatives of polyalkylene glycols of lipids is the it is not limited thereto.

For example, surface modification with a polyalkylene glycol and an oligosaccharide compound can be simultaneously achieved by bonding an oligosaccharide compound to stearyl-modified polyethylene glycol. However, the method for modifying the surface of the lipid membrane structure is not limited to the above-mentioned method. For example, a lipidated oligosaccharide compound such as a stearyl oligosaccharide compound is used as a constituent lipid of the lipid membrane structure. In some cases, surface modification may be performed. The amount of the surface modification of the lipid membrane structure by the oligosaccharide compound is not particularly limited. For example, it is about 1 to 30 mol%, preferably about 2 to 20 mol%, more preferably 5 to 10 mol%, based on the total lipid amount. Degree.

In the production of the lipid membrane structure of the present invention, examples of lipid derivatives for enhancing retention in blood include glycophorin, ganglioside GM1, phosphatidylinositol, ganglioside GM3, glucuronic acid derivatives, glutamic acid derivatives, polyglycerin phospholipid derivatives, and the like. Can also be used. In addition to polyalkylene glycols, dextran, pullulan, ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer as well as polyalkylene glycol Amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, carrageenan and the like can also be used for surface modification.

In addition, the lipid membrane structure of the present invention can be provided with any one function or two or more functions such as a temperature change sensitivity function, a membrane permeation function, a gene expression function, and a pH sensitivity function. In addition to adding retention in the blood, adding these functions as appropriate improves the retention in the blood of lipid membrane structures that contain active pharmaceutical ingredients such as antitumor agents and nucleic acids containing genes. In addition to reducing the capture rate by reticuloendothelial tissues such as the liver and spleen, it is possible to increase the efficiency of releasing active pharmaceutical ingredients from endosomes after endocytosis in target cells. In addition, after endocytosis in the target cell, the lipid membrane structure can be efficiently escaped from the endosome and transferred into the nucleus, and high gene expression activity can be achieved in the nucleus.

Examples of the temperature change sensitive lipid derivative capable of imparting a temperature change sensitive function include dipalmitoyl phosphatidylcholine and the like. Examples of the pH-sensitive lipid derivative capable of imparting a pH-sensitive function include dioleoylphosphatidylethanolamine.

In addition, the lipid membrane structure of the present invention can be modified with a substance such as an antibody that can specifically bind to a cell surface receptor or antigen, thereby improving the endocytosis efficiency. From the viewpoint of delivery efficiency, for example, it is preferable to arrange a monoclonal antibody against a biological component specifically expressed in a target tissue or organ on the surface of the lipid membrane structure. This technique is described in, for example, STEALTH LIPOSOME (page 233-244, issued by CRC Press, Inc., edited by Danilo Basic and Frank Martin). As a component of the lipid membrane structure, a lipid derivative capable of reacting with a mercapto group in a monoclonal antibody or a fragment thereof (for example, Fab fragment, F (ab ′) ₂ fragment, Fab ′ fragment, etc.) such as poly (ethylene glycol) ) -Α-distearoylphosphatidylethanolamine-ω-maleimide, α- [N- (1,2-distearoyl-sn-glycero-3-phosphoryl-ethyl) carbamyl) -ω- [3- [2- ( By including a lipid derivative having a maleimide structure such as 2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl) ethanecarboxamide] propyl} -poly (oxy-1,2-ethanedyl). Monoclonal antibodies can be bound to the membrane surface of lipid membrane structures

The surface of the lipid membrane structure of the present invention may be modified with polyarginine. As the polyarginine, octaarginine or the like can be used. By modifying the surface of a lipid membrane structure such as a liposome with a polyarginine such as octaarginine, the intracellular delivery efficiency of the target substance encapsulated in the liposome can be improved (Journal of Controlled Release, 98, pp. 199-111). 317-323, 2004; International Publication WO2005 / 32593). Modification of the lipid membrane structure surface with polyarginine can be easily performed by using, for example, a lipid-modified polyarginine such as stearylated octaarginine as a constituent lipid of the lipid membrane structure according to the method described in the above publication. Can be done. The disclosures of the above publications and the disclosures of all documents cited in this publication are incorporated herein by reference.

Furthermore, the lipid membrane structure of the present invention may be modified with MPC polymer and / or GALA. The MPC polymer is an MPC polymer obtained by polymerizing 2-methacryloyloxyethyl phosphorylcholine (MPC). Since this polymer has a molecular structure similar to that of a biological membrane, interaction with biological components such as proteins and blood cells is extremely small, and it has been shown to have excellent biocompatibility. As used herein, the term “MPC polymer” includes both MPC homopolymers and copolymers of MPC and other polymerization components.

MPC polymer can be easily obtained as a commercially available polymer. For example, as a registered trademark “LIPIDURE” from NOF Corporation, MPC homopolymer (CAS: 67881-99-6); MPC and butyl methacrylate copolymer (CAS: 125275-25-4); MPC, methacrylic Ternary copolymer of sodium acid and butyl methacrylate; binary copolymer of MPC and 2-hydroxy-3- (meth) acryloyloxypropyltrimethylammonium chloride; phospholipid polymer (LIPIDURE-S), etc. Can also be used in the present invention.

The type of MPC polymer used in the present invention is not particularly limited, but for example, a copolymer of MPC and a methacrylic acid ester such as butyl methacrylate, particularly a block copolymer can be preferably used. The production method of this copolymer is described in detail in Japanese Patent No. 2890316, and those skilled in the art can easily produce a desired copolymer by referring to this patent publication. The entire disclosure of this patent publication is incorporated herein by reference. In the present invention, it is preferable to use an MPC polymer having water solubility and a hydrophobic group. From such a viewpoint, it is produced using an acrylic ester or methacrylic ester having about 4 to 18 carbon atoms. MPC copolymers can be preferably used. As a copolymer of MPC and butyl methacrylate (BMA), for example, a copolymer having a molar ratio of MPC to BMA of 5: 5 (PMB50), a copolymer having a molar ratio of MPC to BMA of 3: 7 (PMB30), etc. are known. For example, Polymer Journal, 22, pp. It can be easily prepared according to the method described in 355-360, 1990, etc. (for example, a specific production method is described in JP-A-2007-314526). In the present invention, PMB50 can be particularly preferably used. The degree of polymerization and molecular weight of the MPC polymer are not particularly limited. For example, from the viewpoint of maintaining water solubility, the average molecular weight (weight average molecular weight) is about 5,000 to 300,000, preferably about 10,000 to 100,000. Polymers can be used.

The method of modifying the lipid membrane structure with the MPC polymer is not particularly limited. For example, the MPC polymer may be added to an aqueous dispersion of the lipid membrane structure such as a liposome and left at room temperature for several minutes to several hours. The amount of the MPC polymer added to the aqueous dispersion is not particularly limited, but depending on the amount of the MPC polymer to be modified, for example, a range of 0.01 to 1% by mass with respect to the total lipid amount of the lipid membrane structure The MPC polymer is preferably added in an amount of 0.1 to 10% by mass, more preferably about 0.1 to 3% by mass. By this operation, the MPC polymer is rapidly incorporated into the lipid component of the lipid membrane structure, and a lipid membrane structure whose surface is modified with the MPC polymer can be prepared. The amount of surface modification by the MPC polymer is not particularly limited, but is, for example, in the range of about 0.1 to 5% by mass with respect to the total lipid amount of the lipid membrane structure.

In order to efficiently escape the lipid membrane structure from the endosome into the cytoplasm, the surface of the lipid membrane structure of the present invention may be modified with GALA. GALA is Biochemistry, 26, pp. 2964-2972, 1987. For example, JP-A-2006-28030 discloses liposomes surface-modified with GALA. Therefore, according to the method described in the above publication, A surface-modified lipid membrane structure can be easily produced. In general, by preparing a lipid membrane structure using a GALA cholesterol derivative (Chol-GALA) as a lipid component, a lipid membrane structure surface-modified with GALA can be produced. The amount of surface modification by GALA is not particularly limited, but is, for example, about 0.01 to 10 mol%, preferably about 0.1 to 4 mol%, more preferably about 1 to 3 mol% with respect to the total lipid amount. .

In this specification, the term “GALA” includes deletion and substitution of one or several amino acids in the amino acid sequence of the peptide in addition to the peptide specified by SEQ ID NO: 1 in the sequence listing of JP-A-2006-28030. And / or a modified peptide consisting of an added amino acid sequence and having substantially the same properties as GALA (for example, properties capable of fusing lipid membranes under acidic conditions). The term “GALA” herein should not be construed as limiting in any way. Regarding the surface modification method of the lipid membrane structure by GALA and GALA, all the disclosures of JP-A-2006-28030 are included as disclosure of this specification by reference.

Lipid membrane structures of the present invention include sterols, membrane stabilizers such as glycerin or fatty acid esters thereof, antioxidants such as tocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene, charged substances, and One or two or more substances selected from the group consisting of membrane proteins and the like may be included. Examples of the charged substance imparting positive charge include saturated or unsaturated aliphatic amines such as stearylamine and oleylamine; saturated or unsaturated cationic synthetic lipids such as dioleoyltrimethylammoniumpropane; or cationic polymers. Examples of the charged substance that imparts a negative charge include dicetyl phosphate, cholesteryl hemisuccinate, phosphatidylserine, phosphatidylinositol, and phosphatidic acid. Examples of membrane proteins include membrane surface proteins and membrane integral proteins. The compounding amount of these substances is not particularly limited, and can be appropriately selected according to the purpose.

Envelope-type nanostructures (MEND) with added functionality are known, and can be suitably used as the lipid membrane structure of the present invention. For example, MEND has a structure in which a complex of a nucleic acid such as plasmid DNA and a cationic polymer such as protamine is used as a core, and the core is enclosed in a lipid envelope membrane in the form of a liposome. A peptide for adjusting pH responsiveness and membrane permeability can be arranged on the lipid envelope membrane of MEND as needed, and the outer surface of the lipid envelope membrane can be modified with alkylene glycol such as polyethylene glycol. it can. Inside the lipid envelope of MEND, condensed DNA and a cationic polymer are encapsulated, and designed so that gene expression can be achieved efficiently. The MEND that can be suitably used in the present invention includes a MEND or oligosaccharide linkage in which a complex of a plasmid DNA incorporating a desired gene and protamine is encapsulated, and the outer surface of the lipid envelope is modified with PEG and INF7 MEND modified with PEG is preferred. The modification with PEG preferably uses stearyl polyethylene glycol as a constituent lipid component. For MEND, see, for example, Drug Delivery System, 22-2, pp. References such as 115-122, 2007 can be referred to. The disclosures of the above publications and the disclosures of all documents cited in this review are hereby incorporated by reference.

The form of the lipid membrane structure is not particularly limited, and examples thereof include a form dispersed in an aqueous solvent (for example, water, physiological saline, phosphate buffered physiological saline, etc.) and a form obtained by lyophilizing this aqueous dispersion. It is done.

The method for producing the lipid membrane structure is not particularly limited, and any method available to those skilled in the art can be employed. For example, all the lipid components are dissolved in an organic solvent such as chloroform, and after forming a lipid film by drying under reduced pressure with an evaporator or spray drying with a spray dryer, the above mixture is dried with an aqueous solvent. And further emulsifying with an emulsifier such as a homogenizer, an ultrasonic emulsifier, or a high-pressure jet emulsifier. Moreover, it can manufacture also by the method well-known as a method of manufacturing a liposome, for example, a reverse phase evaporation method etc. When it is desired to control the size of the lipid membrane structure, extrusion (extrusion filtration) may be performed under high pressure using a membrane filter having a uniform pore size. The size of the lipid membrane structure in a dispersed state is not particularly limited. For example, in the case of liposome, the particle diameter is about 50 nm to 5 μm, preferably about 50 nm to 400 nm, preferably about 50 nm to 300 nm, About 250 nm is more preferable. The particle diameter can be measured, for example, by a DLS (dynamic light scattering) method.

The composition of the aqueous solvent (dispersion medium) is not particularly limited, and examples thereof include a buffer solution such as a phosphate buffer solution, a citrate buffer solution, and a phosphate buffered saline solution, a physiological saline solution, a medium for cell culture, and the like. Can do. These aqueous solvents (dispersion media) can stably disperse lipid membrane structures, but also glucose, galactose, mannose, fructose, inositol, ribose, xylose sugar monosaccharides, lactose, sucrose, cellobiose, trehalose. , Disaccharides such as maltose, trisaccharides such as raffinose and merezinose, polysaccharides such as cyclodextrin, sugars such as erythritol, xylitol, sorbitol, mannitol, maltitol (aqueous solutions), glycerin, diglycerin, poly Glycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol -Alkyl ether, 1,3-polyhydric alcohol (aqueous solution), such as butylene glycol and the like may be added. In order to stably store the lipid membrane structure dispersed in the aqueous solvent for a long period of time, it is desirable to eliminate the electrolyte in the aqueous solvent as much as possible from the viewpoint of physical stability such as aggregation suppression. In terms of chemical stability of the lipid, the pH of the aqueous solvent should be set from weakly acidic to near neutral (about pH 3.0 to 8.0), and / or dissolved oxygen should be removed by nitrogen bubbling or the like. Is desirable.

When the aqueous dispersion of the obtained lipid membrane structure is freeze-dried or spray-dried, for example, glucose, galactose, mannose, fructose, inositol, ribose, xylose sugar monosaccharide, lactose, sucrose, cellobiose, trehalose Stability can be improved by using disaccharides such as maltose, trisaccharides such as raffinose and merezinose, polysaccharides such as cyclodextrin, sugars such as erythritol, xylitol, sorbitol, mannitol, and maltitol (aqueous solutions) There is a case. When the aqueous dispersion is frozen, for example, the saccharides, glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether If a polyhydric alcohol (aqueous solution) such as diethylene glycol monoalkyl ether or 1,3-butylene glycol is used, the stability may be improved.

In the lipid membrane structure of the present invention, for example, a liposome, a substance to be delivered can be encapsulated in the target tissue or organ or the nucleus of the cell of the target tissue or organ. The type of substance to be encapsulated is not particularly limited, but in addition to any active pharmaceutical ingredients such as antitumor agents, anti-inflammatory agents, antibacterial agents, antiviral agents, saccharides, peptides, nucleic acids, low molecular compounds, metal compounds Any substance can be encapsulated. Examples of the nucleic acid include a nucleic acid containing a gene, and more specifically, for example, a gene incorporated in a plasmid, but are not limited to this specific embodiment. Needless to say, any gene can be used as the gene.

For example, using the lipid membrane structure of the present invention for delivering a substance into the cytoplasm of the target cell, the substance can be released into the cytoplasm of the target cell in the living body of a mammal including humans. The lipid membrane structure of the present invention is taken up into the target cell by endocytosis and transferred into the cytoplasm after being encapsulated in the endosome, and then escapes from the endosome by the action of INF7 to efficiently put the substance into the cytoplasm. Can be released. For example, according to this method, it is possible to prevent and / or treat diseases in mammals including humans. For example, a lipid membrane structure that is surface-modified with INF7, preferably further surface-modified with polyalkylene glycol and encapsulating a pharmaceutically active ingredient can be administered to an animal, and the drug is contained in the cytoplasm of the target cell. The active ingredient can be delivered efficiently. Examples of pharmaceutically active ingredients include, but are not limited to, antitumor agents and nucleic acids. In addition, in order to express a gene in cells of mammals including humans, a lipid that is surface-modified with INF7, more preferably surface-modified with PEG, octaarginine, etc., and encapsulating a nucleic acid containing the gene inside Membrane structures can be administered to animals. Preferably, the lipid membrane structure in which a cationic polymer such as protamine is encapsulated together with a nucleic acid can be used. For example, gene therapy can be performed by this method.

In the lipid membrane structure of the present invention for delivering a substance into the nucleus of the target cell, a nucleic acid can be preferably encapsulated. Hereinafter, as an example of the present invention, a case where a nucleic acid is encapsulated will be specifically described below, but the scope of the present invention is not limited to this specific embodiment. The 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 nucleic acid may contain a gene. The gene may be any of oligonucleotide, DNA, or RNA. In particular, a gene for introduction in vitro such as transformation, a gene that acts by expression in vivo, for example, normal for homologous recombination Examples include genes for gene therapy such as genes. Examples of therapeutic nucleic acids include genes encoding physiologically active substances such as antisense oligonucleotides, antisense DNAs, antisense RNAs, enzymes and cytokines, as well as nucleic acids having a function of regulating gene expression, such as siRNA. Functional nucleic acids including RNA and the like can also be used, and these are also included in the term nucleic acid herein. In this specification, the term “nucleic acid” should be interpreted in the broadest sense, and should not be interpreted in any way restrictive.

Further, when the nucleic acid is encapsulated in the lipid membrane structure of the present invention, a compound having a nucleic acid introduction function can also be added. Examples of such compounds include O, O′-N-didodecanoyl-N- (α-trimethylammonioacetyl) -diethanolamine chloride, O, O′-N-ditetradecanoyl-N- (α-trimethyl). Ammonioacetyl) -diethanolamine chloride, O, O′-N-dihexadecanoyl-N- (α-trimethylammonioacetyl) -diethanolamine chloride, O, O′-N-dioctadecenoyl-N— ( α-trimethylammonioacetyl) -diethanolamine chloride, O, O ′, O ″ -tridecanoyl-N- (ω-trimethylammoniodecanoyl) aminomethane bromide and N- [α-trimethylammonioacetyl] -didodecyl- D-glutamate, dimethyl dioctadecyl ammonium bromide, 2,3- Oleyloxy-N- [2- (sperminecarboxamido) ethyl) -N, N-dimethyl-1-propaneammonium trifluoroacetate, 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide, 3-β -[N- (N ', N'-dimethylaminoethane) carbamoyl] cholesterol and the like can be mentioned. These compounds having a nucleic acid introduction function may be arranged at any position of the membrane of the lipid membrane structure and / or filled in the lipid membrane structure.

A lipid membrane structure for delivering a substance into the cytoplasm of a target cell encapsulating a nucleic acid can be used as a carrier for delivering the nucleic acid to a target tissue or organ. For the purpose of gene expression, it is particularly preferable to use a nucleic acid containing a desired gene as the nucleic acid and use the above MEND. For example, by administering a lipid membrane structure encapsulating a nucleic acid containing a gene, preferably MEND, to mammals including humans, the desired gene is delivered to the cells of the target tissue or organ and efficiently expressed. Can do. The lipid membrane structure of the present invention can be efficiently taken out from the endosome and released into the cytoplasm after being taken up into the cells of the target tissue or organ by endocytosis. The administration method is not particularly limited, but parenteral administration is preferable, and intravenous administration is more preferable. When targeting the liver, intraportal administration can also be performed to increase delivery efficiency.

The lipid membrane structure for delivering a substance into the nucleus of the target cell encapsulating the nucleic acid can be used as a carrier for delivering the nucleic acid into the nucleus of the cell of the target tissue or organ. For the purpose of gene expression, it is particularly preferable to use a nucleic acid containing a desired gene as the nucleic acid and use the above MEND. For example, by administering a lipid membrane structure encapsulating a nucleic acid containing a gene, preferably MEND, to a mammal, including a human, the desired gene is delivered into the nucleus of a cell of a target tissue or organ and efficiently expressed. be able to. The lipid membrane structure of the present invention is incorporated into cells of a target tissue or organ by endocytosis, and then efficiently escapes from the endosome and moves into the nucleus, thereby efficiently expressing the gene in the nucleus. be able to. The target tissue or organ is not particularly limited, and gene delivery to an appropriate tissue or organ can be achieved according to the type of substance to be surface-modified. In particular, the liver is a preferred target organ. The administration method is not particularly limited, but parenteral administration is preferable, and intravenous administration is more preferable. In some cases, intraportal administration can also be performed to increase the efficiency of delivery to the liver.

Also, one or more active pharmaceutical ingredients can be encapsulated in the lipid membrane structure lipid. For example, an antiviral agent or an antitumor agent can be encapsulated. For example, an anti-hepatitis virus agent that is effective against viral hepatitis or an anti-tumor agent that is effective against liver cancer is encapsulated in the lipid membrane structure of the present invention, and viral hepatitis or liver cancer Can also be treated.

EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.
Example 1
A. Experimental Method 1) Preparation of Gene Encapsulated R8 / INF7 Modified Liposomes After making plasmid DNA and protamine a 10 mM HEPES (pH 7.4) solution, an equal amount of 0.1 mg / ml with respect to a 0.06 μg / ml protamine solution in vortex The ml plasmid DNA solution was gradually added dropwise to mix over time (+/− ratio 1). Furthermore, the protamine / DNA complex was prepared by allowing to stand at room temperature for 10 minutes.

Egg yolk-derived phosphatidylcholine (EPC) / dioleoylphosphatidylcholine (DOPC) / cholesterol (Chol) / stearyl octaarginine (STR-R8) or EPC / DOPE / Chol / stearyl INF7 (STR-INF7) (3: 4: 2: 1) A lipid film was prepared by dissolving 137.5 nmol of lipid having the following composition in chloroform and evaporating in a glass tube. The plasmid and protamine solutions prepared above were sonicated in a bath-type sonicator to encapsulate the plasmid DNA in liposomes.

When modifying INF7 later, the ethanol solution of STR-INF7 or INF7 (1 or 3 mol% lipid content) was added (Post-surface modification). When INF7 is modified at the lipid film stage (Pre-inclusion to the lipid film), STR-INF7 is added by evaporation after adding 1 or 3 mol% of STR-INF7 to lipid chloroform solution. Incorporated into lipid film.

2) Preparation of gene-encapsulated PEG / R8 / INF7-modified liposome An aggregated core composed of a gene and protamine was prepared in the same manner as described above. EPC / DOPC / Chol / phosphatidylethanolamine (DSPE) -PEG2000 (DSPE-PEG2000) / STR-R8 or EPC / DOPE / Chol / DSPE-PEG2000 / STR-INF7 (2: 4: 2: 1: 1)) A lipid film was prepared by dissolving 137.5 nmol of lipid having a lipid composition in chloroform and evaporating in a glass tube. The plasmid and protamine solutions prepared above were sonicated in a bath-type sonicator to encapsulate the plasmid DNA in liposomes. Further, STR-INF7 was incorporated into a lipid film by adding 1 or 3 mol% of STR-INF7 to the lipid chloroform solution and then evaporating.

3) Analysis of intracellular dynamics using confocal laser microscope (Estimation of endosome escape)
Plasmid DNA was rhodamine modified using the Miras Label IT® CX-Rhodamine Nucleic Acid Label Kit (Mirus Corp., Madison, Wis., USA). HeLa cells were cultured in a glass bottom dish for 24 hours. Cell membranes were stained with PKH67 green fluorescent cell linker (Sigma-Aldrich, St. Louis, MO). Rhodamine-modified plasmid DNA-encapsulated liposomes were added to the cell culture at a lipid concentration of 0.02 mM and incubated at 37 ° C. for 30 minutes. The cells were washed once with medium, and further incubated for 2.5 hours in medium supplemented with 10% FBS. The cells were observed with a confocal laser microscope (LSM510, Carl Zeiss Co. Ltd, Jena, Germany) using an oil immersion objective lens (Plan-Apochromat 63x / NA = 1.4).

4) Analysis of intracellular dynamics by confocal laser microscope (evaluation of membrane fusion)
STR-INF7-modified R8 liposomes were treated with 1 mol% NBD-DOPE (N- (7-nitrobenzo-2-oxa1,3-diazol-4-yl) -dioleylphosphatidylethanolamine, excitation wavelength 460 nm; fluorescence wavelength 534 nm) and 0. Double staining was performed with .5 mol% rhodamine-DOPE (rhodamine-dioleoylphosphatidylethanolamine, excitation wavelength 550 nm; fluorescence wavelength 590 nm). The labeled liposome was brought into contact with HeLa cells cultured on a glass bottom dish to a concentration of 0.08 mM lipid, and incubated for 1 hour in the absence of serum. After washing the cells, the cells were replaced with fresh medium and incubated for an additional hour. Ten minutes before observation, Hoechst 33342 was incubated at a concentration of 5 μg / ml to stain the nuclei. The cells were observed with a confocal laser microscope, and images were acquired. In order to observe membrane fusion in the cell, NBD was excited with a laser beam of 488 nm, and the fluorescence transmitted through the dichroic mirror of HFT488 was dispersed to 510 to 630 nm by the META function. The data are shown as relative values when the fluorescence intensity at 586 nm is 1.

5) Measurement of membrane destruction effect using calcein release as an index A lipid film composed of DOPC / dioleoylphosphatidylserine (DOPS) / Chol (4.75: 4.75: 0.5) was prepared and calcein solution (40 mM) Of calcein / 1 mM EDTA, pH 7.4). At this time, the lipid concentration was adjusted to 10 mM. After mixing with a vortex mixer, sonication was performed for 1 minute to encapsulate calcein in the liposome. The liposomes were subjected to molecular sieve chromatography (Bio-Gel (registered trademark) A-15m gel, medium from Bio-Rad Laboratories, CA, USA) equilibrated with PBS (pH 7.4), and the calcein-encapsulated liposomes were combined with unencapsulated calcein. separated.

R8 and R8 / INF7 labeled liposomes were incubated with the calcein-encapsulated liposomes in citrate / phosphate buffer (pH 5.5) or PBS (pH 7.4) at 37 ° C. for 30 minutes. In order to measure calcein release, 100 μl of sample was collected and fluorescence at 520 nm was measured with excitation light at 490 nm. The ratio (%) of calcein leaked as F and F _Triton before and after adding 10 μl of 0.5% Triton X-100 was determined by the following formula.
% Release = [(F × 100) / (F _Triton × 110)] × 100

6) In vivo gene introduction MEND encapsulating a plasmid DNA (7,037 bp) encoding a firefly luciferase gene was used to conduct an in vivo gene introduction experiment using ICR mice (5 weeks old, male). MEND is J. Control. Release, 98 (2), pp. 317-323, 2004; Biol. Chem. , 283, pp. Prepared according to the method described in 23450-23461, 2008. Under diethyl ether anesthesia, MEND (40 μg DNA / 350 μl, 10 mM HEPES (pH 7.4), 5% glucose) was administered from the mouse tail vein. Six hours after administration, the liver, lungs and spleen were removed and weighed. After chopping and mixing the tissue, 0.2 g of the liver (lung and spleen in total) was measured, and Lysis buffer (0.1% Triton X-100, 2 mM EDTA, 0.1 M Tris-HCl, pH 7.8). 1 ml was added and the tissue was homogenized in a lysis buffer and centrifuged at 13,000 rpm for 10 minutes at 4 ° C. Luciferase activity (RLU) and protein mass were measured for 20 μl of supernatant and calculated as RLU / mg protein.

B. Results 1) Effect of INF The effect of INF7 on the transfection activity of gene-encapsulated R8-modified liposomes was analyzed. The two lipid compositions used in this experiment had low membrane fusion ability, but both gene expression dramatically increased by INF7 modification. Further, INF7 having no stearyl group (Free INH7, not inserted into the lipid membrane) increased about 4 times, but higher when STR-INF7 capable of insertion into the membrane was used (10 to 50). X) Promotion of gene expression was observed. Furthermore, gene expression was further promoted with STR-INF7 introduced from the time of creation of the lipid film (pre-inclusion), and 70 to 140-fold enhancement was observed (FIG. 1).

2) In vivo gene expression After intravenous administration of R8-MEND and 3% STR-INF7-modified R8-MEND, a high gene expression activity of 100 times or more was observed in the liver and spleen by modification of INF7 (FIG. 3). ).

3) Analysis of intracellular kinetics The endosomal escape ability of R8-MEND modified with STR-INF7 was evaluated. With the R8 modification alone, the plasmid DNA (red) signal colocalizes with the cell membrane and endosome (PKH67 modification: green) and is displayed as yellow (arrow). However, the modification with STR-INF7 causes the signal to coexist with the cell membrane and endosome. In addition to the localized DNA (yellow), the proportion of DNA (red) that exists independently in the cytoplasm increased (FIG. 4). This revealed that endosome escape was promoted by INF7 modification.

In addition, it was analyzed by spectral imaging whether or not liposomes were membrane-fused in the cells. In this analysis, fluorescence energy transfer occurs between NBD and rhodamine on the lipid membrane. That is, fluorescence (about 530 nm) obtained when NBD is excited is absorbed by rhodamine and observed as fluorescence having a maximum value of 590 nm. When the liposome membrane undergoes membrane fusion within the cell, mixing of the lipid membrane occurs. As a result, the distance between the two fluorescences is separated, so that FRET is eliminated, and light at 530 nm, which is fluorescence derived from NBD, is observed. The FIG. 5 shows a relative fluorescence spectrum when the fluorescence intensity at 590 nm is 1. When FRET is eliminated, the fluorescence at 530 nm recovers to 0.2 or more, but in this experiment, the fluorescence at 530 nm was maintained at a low value regardless of the presence or absence of INF7 modification. From this result, it was suggested that the present liposome escapes from the endosome without membrane fusion.

4) Evaluation of pH dependence of membrane breaking activity of INF7-modified liposome using calcein release The membrane breaking activity when calcein-encapsulated liposomes, R8-liposomes and R8 / INF7 liposomes were incubated was measured. For R8 liposomes, no leakage of calcein was observed at pH 7.4 or pH 5.5. On the other hand, in the INF7 modified type, strong calcein release was observed at both pHs, and it was revealed that INF7 shows membrane-disrupting activity regardless of pH (FIG. 6).

5) Endosome escape promoting activity by INF7 in PEG-modified liposomes The endosome escape ability of R8 / PEG-MEND modified with STR-INF7 was evaluated. In the case of R8 / PEG modification alone, the signal of plasmid DNA (red) is colocalized with cell membrane and endosome (PKH67 modification: green) and observed in yellow (arrow), but by modifying STR-INF7, In addition to those co-localized (yellow), the proportion of plasmid DNA observed in red alone increased (FIG. 7). This revealed that endosome escape was promoted even when PEG was modified on the surface by INF7 modification.

Also, the influence of INF7 on the transfection activity of the gene-encapsulated R8 / PEG-modified liposome was analyzed. In the absence of PEG, INF7 promoted gene expression. In addition, this promotion effect was dependent on the modification density, and a stepwise gene promotion effect was observed by increasing the modification density from 1% to 3% (FIG. 8). In addition, the INF7 modification density-dependent gene expression promoting effect in the absence of PEG was also observed in PEG-modified liposomes.

Example 2: maltotriose _-PEG6-C 11 Synthesis of -SH

2- (2- (2- (2- (2- (2- (Undec-10-enyloxy) ethoxy) ethoxy) ethoxy) ethoxy) ethoxy) ethanol (1)
Hexaethylene glycol (15.0 g, 53.1 mmol) was added to tetrahydrofuran (THF, 55 ml) and NaH (60% in oil, 1.59 g, 39.7 mmol) was added. When the generation of hydrogen was stopped, 11-bromo-1-undecene (6.7 ml, d = 1.063 g / ml, 30.5 mmol) was added and reacted overnight at room temperature under a nitrogen atmosphere. After washing with chloroform / water and chloroform / saturated saline, the solvent was distilled off by drying over anhydrous sodium sulfate. Purification by silica gel chromatography (ethyl acetate: chloroform = 8: 2 → ethyl acetate: methanol = 97: 3) gave compound (1) (6.57 g, yield 49.6%).

Compound (1) (1.90 g, 4.38 mmol) and peracetyl maltotriose (2.90 g, 3.00 mmol) were dissolved in anhydrous dichloromethane (20 ml), stirred for 20 minutes, and then boron trifluoride-ethyl ether complex. (500 μl) was added and stirred at room temperature for 18 hours. The obtained reaction solution was diluted with dichloromethane (10 ml), washed with ice-cooled saturated aqueous sodium hydrogen carbonate solution (1 × 150 ml) and distilled water (1 × 150 ml), and then the organic layer was dried over sodium sulfate. . The solution was concentrated and the residue was purified by silica gel chromatography (chloroform: ethyl acetate = 3: 7) to obtain amorphous compound (2) (2.16 g, 1.61 mmol, 53.7%).

Compound (2) (1.24 g, 0.92 mmol) was dissolved in THF (16 ml), and thioacetic acid (0.4 ml, d = 1.07 g / ml, 5.62 mmol) was added and stirred. Azobisisobutyronitrile (AIBN, 76 mg, 0.46 mmol) subjected to recrystallization purification was added thereto, and the mixture was stirred at 60 ° C. for 1 hour under ultraviolet irradiation and then at 60 ° C. overnight. After washing with chloroform / water and chloroform / saturated saline, the solvent was distilled off with an evaporator and the residue was redissolved in chloroform. Purification was performed by Rica gel chromatography (n-hexane → chloroform: ethyl acetate = 3: 7) to obtain a transparent amorphous compound (3) (0.94 g, 0.68 mmol, 72.5%).

Compound (3) (0.30 g, 0.22 mmol) was dissolved in methanol (10 ml), and sodium methoxide (28% methanol solution, 45 μl) was added. After stirring at room temperature for 3 hours, DOWEX (50WX8-200 ion exchange resin) was added to neutralize the solution, the resin was filtered off, and the solvent was evaporated to obtain a white solid compound (4) (0 .20 g, 0.21 mmol, 95.1%).

Example 3
A. Method 1) Preparation of MEND Cationic lipid (N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA) or N- [1- (2,3- Dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTAP)) and a lipid envelope containing two lipids, cholesterol, encapsulated core particles obtained by condensing the gene with protamine to prepare MEND. . As the lipid composition of the base, cationic lipid and cholesterol were adjusted to 30:70 (molar ratio). A lipid solution (an ethanol solution of cationic lipid and cholesterol) was added to a glass test tube so that the total amount was 412.5 nmol / 250 μL, and then the solvent was distilled off by drying under reduced pressure with a desiccator. After 250 μL of chloroform was added to the test tube to dissolve the lipid again, it was dried under reduced pressure with a desiccator, and the solvent was removed again to prepare a lipid film.

Core particles were prepared by mixing plasmid DNA and protamine at a +/− ratio of 1. After making plasmid DNA and protamine 10 mM HEPES (pH 7.4) solution, gradually add 125 μl of protamine solution (0.201 mg / ml) to 125 μl of plasmid DNA solution (0.3 mg / ml) in vortex. And mixed over time. Furthermore, the core particle was prepared by leaving still at room temperature for 10 minutes. After adding 250 μl of the gene core solution to the test tube in which the lipid film was prepared, it was hydrated by allowing it to stand at room temperature for 15 minutes, and sonication was performed for about 1 minute to obtain MEND. The particle size of MEND was measured by dynamic light scattering (DLS).

2) Modification of functional elements to MEND lipid membrane When GALA or maltotriose is presented on the lipid envelope surface of MEND, GALA cholesterol derivative (Chol-GALA) or maltotriose lipid derivative (maltotriose- _PEG6-C 11 -SH) was used. Chol-GALA uses 2% significant amount of the total lipid amount (molar amount), maltotriose _-PEG6-C 11 -SH is used equal to 5% of total lipids amount (molar amount), both ethanol solution As a lipid film. In the case of using maltotriose _-PEG6-C 11 -SH is cationic lipids a lipid composition constituting the lipid membrane, cholesterol, maltotriose _-PEG6-C 11 -SH to 30: 65: 5 (molar ratio ).

3) Preparation of MEND for examination of maltotriose modification rate and evaluation of usefulness MEND lipid membrane was prepared so that the composition ratio of DOTMA and cholesterol was 30:70 (molar ratio), and the total lipid amount (molar) The amount of Chol-GALA was 2% equivalent. It is used here maltotriose _-PEG6-C 11 in addition to lactose -PEG4- cholesterol lactose lipid derivative as a comparative control of -SH, and _PEG6-C 11 -SH having no saccharide in the structure. The modification rate of sugar to MEND was adjusted by adding these lipids in an amount corresponding to 2.5% to 15% of the total lipid amount (molar amount). At this time, the total lipid content was unified by adjusting the cholesterol content. (E.g., the case of maltotriose _-PEG6-C 11 -SH containing 10%, DOTMA, was made to cholesterol, sugar compounds -PEG- lipid 30:60:10 (molar ratio).

4) Preparation of Lipoplex Lipoplex was prepared by mixing a liposome solution composed of DOTAP and a plasmid DNA solution according to the method described in the literature (Hum. Gene Ther., 8, pp. 1584-1594, 1997). . Further, the molar ratio was set to 12 by adjusting the amount of liposome with respect to plasmid DNA.

After adding a lipid solution (ethanol solution of DOTAP) to a glass test tube so that the total amount becomes 750 nmol / 250 μL, the solvent was distilled off by drying under reduced pressure with a desiccator. After 250 μL of chloroform was added to the test tube to dissolve the lipid again, it was dried under reduced pressure with a desiccator, and the solvent was removed again to prepare a lipid film. 250 μL of PBS (−) (pH 7.4) was added to the prepared lipid film, left to stand at room temperature for 15 minutes to hydrate, and then sonicated for about 1 minute to prepare liposomes. Lipoplex was prepared by adding the prepared liposome solution to the PBS (−) solution of plasmid DNA in vortex by dropping, and then allowing to stand at room temperature for 15 minutes. The particle size of Lipoplex was measured by dynamic light scattering (DLS).

5) Preparation of Polyplex Polyplex was prepared using in vivo jetPEI and galactose conjugate (PolyPlus), which are commercially available in vivo gene transfer reagents, according to the attached recommended protocol. NP ratio, which is a mixing ratio of plasmid DNA, in vivo jetPEI, and galactose conjugate, was set to 10 as a recommended value.
A 5% glucose solution of prepared in vivo jetPEI and galactose conjugate was added to a 5% glucose solution of plasmid DNA, mixed by vortexing, and then allowed to stand at room temperature for 15 minutes to prepare Polyplex. The particle size of Polyplex was measured by dynamic light scattering (DLS).

6) Animal experiment (Evaluation of gene expression)
Using an MEND encapsulating a plasmid DNA (7,037 bp) encoding a firefly luciferase gene, an in vivo gene introduction experiment was conducted using ICR mice (5 weeks old, male). Under diethyl ether anesthesia, MEND (40 μg DNA / 350 μL 10 mM HEPES (pH 7.4) 5% glucose) was administered from the mouse tail vein. Six hours after administration, the liver, lungs and spleen were removed and weighed. After chopping and mixing the tissue, 0.2 g of the liver (lungs and spleen in total) was measured, and Lysis buffer (0.1% Triton X-100, 2 mM EDTA, 0.1 M Tris-HCl, pH 7. 8) 1 mL was added, the tissue was homogenized in a lysis buffer, and centrifuged at 13,000 rpm at 4 ° C. for 10 minutes. Luciferase activity (RLU) and protein mass were measured for 20 μL of supernatant and calculated as RLU / mg protein.

7) Animal experiments (evaluation of liver migration)
The administration sample was prepared by adding glucose such that MEND labeled with [ ³ H] on the lipid membrane was 5% (w / v). Under ether anesthesia, administration was performed from the mouse tail vein under the conditions of 40 μg pDNA / 0.44 μmol lipid / 350 μL / mouse. After 6 hours from the administration, the mice were treated with ether anesthesia, followed by laparotomy, and the liver was removed. After thoroughly washing with physiological saline and measuring the weight, it was chopped and mixed well. 0.2 to 0.3 g of the minced liver was weighed into a plastic vial, 2 mL of Solene-350 was added, and the tissue was lysed by incubation at 50 ° C. overnight. To this solution, 10 mL of Honic fluor was added and mixed well, and then allowed to stand at 4 ° C. overnight, and the count of [ ³ H] was measured with a liquid scintillation counter. In addition, in order to evaluate the dose of [ ³ H], 10 mL of Honic fluor was added to 10 μL of the administered MEND sample, mixed well, and then allowed to stand at 4 ° C. overnight. Similarly, [ ³ H] The count of was measured.

B. Results 1) Enhancement of gene expression by modification of GALA and maltotriose on MEND lipid membrane MEND containing GALA (2%) and maltotriose (5%) was prepared, and the particle size / ζ potential of MEND and genes in mouse liver The results of evaluating the expression (luciferase activity) are shown in FIG. Gene expression was significantly increased by GALA modification (DOTAP-MEND: about 28 times, DOTMA-MEND: about 6 times), but gene expression was further increased by modification with maltotriose, and DOTAP- compared to unmodified MEND. The gene expression activity increased about 88 times in MEND and about 64 times in DOTMA-MEND.

2) Examination of maltotriose modification rate and evaluation of usefulness In order to evaluate the effect of maltotriose modification rate on gene expression activity and the effect of maltotriose, the content of maltotriose-PEG-lipid was 0-15. The gene expression activity of MEND containing GALA (2%) was evaluated in the range of%. For comparison, lactose-PEG-lipid, which is a lipid derivative of lactose, and PEG6-lipid having no sugar in the structure were used. The results are shown in FIG. In the maltotriose-modified MEND, the gene expression activity varied depending on the modification rate of maltotriose, and the highest gene expression activity was shown when 5% modification was performed. No increase in gene expression activity was observed with lactose-modified MEND. In addition, when PEG-lipid without maltotriose was used, the gene expression activity was hardly changed, and the increase in gene expression activity when maltotriose-PEG-lipid was used was due to maltotriose. It was suggested that this was due to the promotion of the nuclear transfer process. When other sugars were examined in the same manner, no increase in gene expression activity was observed for any of cellotriose, mannose, galactose, β-maltose, and α-maltose (FIGS. 11 and 12).

3) Evaluation of liver migration amount of MEND Liver migration amount after intravenous administration of MEND to mice was evaluated. Unmodified MEND labeled with [ ³ H] or MEND modified with GALA and maltotriose was administered from the tail vein of ICR mice (5 weeks old, male), and [ ³ H] in the liver 6 hours later. The liver migration amount was evaluated by measuring the count with a liquid scintillation counter ((FIG. 13). The liver migration amount (% ID / Liver) was calculated as the migration amount per organ with respect to the MEND dose. DOTMA / Cholesterol (3: 7), which is a lipid composition showing gene expression activity, was used as the basic lipid composition of MEND.

In all MENDs, 60-70% of the dose was accumulated in the liver, and the amount of liver transfer decreased by modifying MEND with GALA and maltotriose, but there was a significant difference. I couldn't. When the gene expression activity per migration amount was calculated by dividing the gene expression activity evaluated in the previous section by the liver migration amount of MEND, it was about 7 times by GALA modification to MEND, and about 85 by modification of GALA and maltotriose. Doubled (per liver transfer amount) gene expression activity increased (FIG. 14). Therefore, it was shown that GALA and maltotriose led to high gene expression in the liver by improving the intracellular kinetics of MEND.

4) Gene expression in the liver during administration of Lipoplex and Polyplex 1) Lipoplex, which is a complex of pDNA and cationic liposome, as a gene carrier widely used in in vivo gene delivery studies, and 2) Complex of pDNA and cationic polymer Comparative evaluation of gene transfer efficiency was performed using Polyplex, a body. The lipid composition and the molar ratio of Lipoplex used were based on literature. Polyplex was a commercially available in vivo gene transfer reagent, in vivo jetPEI, galactose conjugate (PolyPlus), and NP ratio was 10. As MEND, GALA / maltotriose-MEND having the highest gene expression activity in 1) was used. The results are shown in FIG.

The gene expression activity in the liver after intravenous administration of MEND was about 25 times higher than that of Lipoplex and Polyplex. In particular, Lipoplex had a gene expression activity of about 10 ⁴ RLU / mg protein even though the dose of pDNA was 60 μg and 1.5 times that of MEND. From the above results, it was shown that the lipid membrane structure of the present invention is a gene carrier capable of efficiently delivering a gene to the liver.

Claims (14)

1. A lipid membrane structure for delivering a substance into the cytoplasm of a target cell, the surface of which is modified with INF7.
2. The lipid membrane structure according to claim 1, wherein the lipid membrane structure is a liposome.
3. The lipid membrane structure according to claim 1 or 2, which is surface-modified with polyalkylene glycol.
4. The lipid membrane structure according to any one of claims 1 to 3, which is surface-modified with polyarginine.
5. The lipid membrane structure according to any one of claims 1 to 4, wherein a substance to be delivered is enclosed therein.
6. The lipid membrane structure according to claim 5, wherein the substance to be delivered is a nucleic acid.
7. A lipid membrane structure for delivering a substance into the nucleus of a target cell, wherein the substance to be delivered is encapsulated inside and is surface-modified with an oligosaccharide compound of three or more sugars.
8. The lipid membrane structure according to claim 7, wherein the oligosaccharide compound is a trisaccharide compound.
9. The lipid membrane structure according to claim 7, wherein the oligosaccharide compound is a glucose trimer.
10. The lipid membrane structure according to claim 7, wherein the oligosaccharide compound is maltotriose.
11. The lipid membrane structure according to any one of claims 7 to 10, wherein the lipid membrane structure is a liposome.
12. The lipid membrane structure according to any one of claims 7 to 11, which is surface-modified with GALA.
13. The lipid membrane structure according to any one of claims 7 to 12, wherein a substance to be delivered is enclosed inside.
14. The lipid membrane structure according to claim 13, wherein the substance to be delivered is a nucleic acid.

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