WO2015098907A1 - Lipid membrane structure having intranuclear localization property - Google Patents

Abstract

A lipid membrane structure for delivering a substance into the nucleus of a cell. The lipid membrane structure can deliver a nucleic acid into the nucleus of an immunocyte such as a dendritic cell with high efficiency. In the lipid membrane structure, a lipid membrane is modified with (a) a polypeptide which comprises the amino acid sequence represented by SEQ ID NO: 1 and/or (b) a polypeptide which comprises an amino acid sequence produced by the deletion and/or substitution and/or insertion of one or several amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 and which has an activity of promoting the migration of the lipid membrane structure into the nucleus of a cell.

Description

Lipid membrane structure with nuclear translocation

The present invention relates to a lipid membrane structure having nuclear translocation properties. More specifically, the present invention relates to a lipid membrane structure such as a liposome that can be easily transferred into the nucleus of immune cells, particularly the nucleus of dendritic cells.

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 nanostructure (MEND: Multifunctionallopeenvelope-type nano 以下 device; hereinafter, it may be abbreviated as “MEND”. For example, Drug Delivery System , 22-2, 115pp.115-122, 2007, etc.) have been proposed. 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, polypeptides, and sugars to specific sites such as target organs and tumor tissues using lipid membrane structures, the surface of lipid membrane structures is a functional molecule. A number of methods of modifying with have been proposed. 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. In order to enhance drug release from liposomes incorporated into endosomes, liposomes modified with peptides (GALA: Biochemistry, 26, pp.2964-2972, 1987) (Biochemistry, 43, pp.5618- 5623, 2004) and MEND (Japanese Patent Laid-Open No. 2006-28030) have been proposed.

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; WO Journal of Controlled Release, 98, pp.317-323, 2004), bilamellar liposomes with lipid membranes modified with nuclear translocation peptides (International Publication WO2006 / 101201), surface modification with monosaccharides such as galactose and mannose Liposomes (International Publication WO2007 / 102481) have been proposed. Multi-lipid membrane structures (T-MEND) modified with monosaccharides showed fusion properties with lipid membranes and nuclear membranes, and it was reported that gene expression efficiency could be improved in the in-vitro test results.

On the other hand, if a nucleic acid encoding an antigenic protein can be introduced into the nucleus of an immune cell, particularly a dendritic cell having an antigen-presenting effect, the protein transcribed and translated from the nucleic acid in the dendritic cell can The living body can acquire immunity to the protein. From such a viewpoint, there is a need for a technique for efficiently delivering a nucleic acid into the nucleus of immune cells such as dendritic cells.

However, the efficiency of nucleic acid introduction when introducing a nucleic acid into the nucleus of a dendritic cell using a lipid membrane structure such as MEND described above is sufficiently higher than that of other cells such as tumor cells and liver parenchymal cells. is not. Before the introduced nucleic acid is finally expressed in the nucleus, it must undergo various intracellular kinetic processes such as intracellular uptake, endosomal escape, nuclear translocation, and nuclear transcription. In non-dividing cells such as dendritic cells, a nuclear membrane consisting of two membranes always exists intact, and this nuclear membrane is presumed to impede the ability of lipid membrane structures to translocate into the nucleus. Therefore, in order to deliver nucleic acids to the nuclei of dendritic cells using lipid membrane structures, how to break through the above-mentioned processes, particularly the barrier by the nuclear membrane composed of two membranes, is a very important issue. It becomes.

A 27 amino acid residue polypeptide called KALA peptide is known, and it has been reported that it can form a complex with plasmid DNA using its own cationic charge (Biochemistry, 36, pp.3008). -3017, 1997). It has been reported that this KALA peptide can promote the translocation of lipid membrane structures into the nucleus by modifying the surface of lipid membrane structures such as liposomes (International Publication WO 2011/132713).

International Publication WO2005 / 32593 International Publication WO2006 / 101201 International Publication WO2007 / 102481 JP 2006-28030 A International publication WO 2011/132713

An object of the present invention is to provide a means for efficiently delivering a nucleic acid into the nucleus of an immune cell, particularly a dendritic cell having antigen-presenting ability. More specifically, it is an object of the present invention to provide a lipid membrane structure capable of efficiently delivering a nucleic acid into the nucleus of immune cells such as dendritic cells.

In order to solve the above problems, the present inventors have improved MEND (international publication WO2005 / 32593) modified with octaarginine polypeptide, which is a functional polypeptide capable of enhancing nuclear translocation ability, and endosome escape ability. Although the nucleic acid was encapsulated in MEND modified with GALA peptide, which is a functional polypeptide to be imparted (Japanese Patent Laid-Open No. 2006-28030), and introduced into dendritic cells, polypeptide expression from the nucleic acid was almost I was not able to admit. In addition, we tried to introduce nucleic acid into dendritic cells by encapsulating nucleic acid in a multi-lipid membrane structure (T-MEND) having a lipid membrane that is fusogenic with endosomes and a nuclear membrane that is fusogenic with nuclear membranes. Although the expression of the polypeptide from the introduced nucleic acid was slightly promoted in the nucleus, no presentation of the polypeptide on the surface of the dendritic cell was observed.

As a result of further studies, the present inventors have found that when the lipid membrane of a lipid membrane structure such as MEND encapsulating nucleic acid is modified with KALA peptide, the efficiency of nucleic acid introduction into the nucleus of immune cells such as dendritic cells is remarkable. (International publication WO 2011/132713). However, the lipid membrane structure modified with the KALA peptide has found a new problem that gene expression and interleukin 6 (IL-6) production decrease in the presence of serum. Therefore, intensive research was conducted to improve the above lipid membrane structure in order to efficiently enhance the ability to translocate into the nucleus in vivo. As a result, when the lipid membrane structure is modified with the following peptide (I) containing a partial peptide sequence of the KALA peptide, the efficiency of nucleic acid introduction into the nucleus of immune cells such as dendritic cells is significantly increased in vivo. I found it. In addition, it has been found that when a specific lipid having a tertiary amine and a disulfide bond is used as the lipid component of the lipid structure, a remarkably high nucleic acid introduction efficiency can be achieved. 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 nucleus of a cell, wherein the lipid membrane is the following polypeptide (I):
A polypeptide having 8 to 26 amino acid residues and comprising 2 to 4 repeating units of tetrapeptide: Lys-Ara-Leu-Ara in the peptide chain in succession (however, the repeating unit Lys may be Arg in any one or more of them, and / or Ara sandwiched between Lys and Leu in any one of the repeating units may be His. )
A lipid membrane structure modified with is provided.

According to a preferred embodiment of the present invention, the polypeptide (I) comprises two consecutive repeating units of Lys-Ara-Leu-Ara, or 2 consecutive repeating units of Arg-Ara-Leu-Ara. The above-mentioned lipid membrane structure, which is a polypeptide comprising Trp-Glu-Ara-Lys-Leu-Ara or Trp-Glu-Ara-Arg-Leu at the N-terminus of the two consecutive repeating units The above lipid membrane structure, which is a polypeptide to which the sequence of -Ara is bound; the polypeptide is Trp-Glu-Ara-Lys-Leu-Ara-Lys-Ara-Leu-Ara-Lys-Ara-Leu-Ara or Trp Provided is the above lipid membrane structure which is -Glu-Ara-Arg-Leu-Ara-Arg-Ara-Leu-Ara-Arg-Ara-Leu-Ara.

According to a preferred embodiment of the above lipid membrane structure, the above lipid membrane structure comprising a tertiary amine and a lipid having a disulfide bond as a lipid component of the lipid membrane structure; the above lipid wherein the lipid membrane structure is a liposome The above lipid membrane structure wherein the cell is an immune cell, preferably a dendritic cell; and the above polypeptide (I) is modified with a hydrophobic group, preferably a stearyl group or a cholesteryl group, The above lipid membrane structure in which a hydrophobic group is inserted into a lipid membrane; a polypeptide containing a plurality of consecutive arginine residues, preferably a polypeptide containing 4 to 20 consecutive arginine residues, more preferably A polypeptide comprising only 4 to 20 consecutive arginine residues, particularly preferably the above lipid membrane structure having octaarginine on its surface; polyalkylene The above lipid membrane structure having a polyalkylene glycol condensed with glycol or phospholipid, preferably polyethylene glycol (PEG) on its surface; the ratio of the cationic lipid to the total lipid constituting the lipid bilayer is 0 to 40% (mole) The above lipid membrane structure is provided.

Also, from another viewpoint, any of the above lipid membrane structures in which a substance to be delivered is enclosed is provided. According to a preferred embodiment of the present invention, the substance to be delivered is a nucleic acid, for example, any of the above lipid membrane structures, which is a functional nucleic acid such as a nucleic acid containing a gene or siRNA; Lipid membrane structure; the lipid membrane structure described above, wherein the DNA is DNA bound to a vector DNA not containing CpG; the DNA is bound to a vector DNA not containing CpG, and the DNA is free of CpG A lipid membrane structure as described above; the lipid membrane structure as described above, wherein the substance to be delivered is a nucleic acid encoding an antigen polypeptide to be presented on the surface of immune cells, preferably on the surface of dendritic cells; Any of the lipid membrane structures described above, which is a sex envelope nanostructure (MEND); any of the lipid membrane structures described above, in which a nucleic acid and a cationic polymer, preferably protamine are encapsulated.

Also, the lipid membrane structure described above used for introducing a nucleic acid encoding an antigen polypeptide to be presented on the surface of an immune cell, preferably a dendritic cell, into the nucleus of the cell; for immunotherapy against the antigen polypeptide The above lipid membrane structure for use; provided is the above lipid membrane structure wherein the antigen polypeptide is a surface polypeptide specific for cancer cells. 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, the present invention provides a method for delivering a nucleic acid in the nucleus of a cell, preferably in the nucleus of an immune cell, more preferably in the nucleus of a dendritic cell in the body of a mammal, including a human, A method comprising the step of administering to the animal the above lipid membrane structure encapsulated therein; a method of presenting an antigenic polypeptide on the surface of immune cells, preferably dendritic cells, in the body of mammals including humans. A method comprising the step of administering to the animal the above lipid membrane structure encapsulating a nucleic acid encoding the antigen polypeptide.

Also, a method for obtaining immunity to an antigen polypeptide by presenting the antigen polypeptide on the surface of an immune cell, preferably a dendritic cell, in a living body of a mammal including humans, which encodes the antigen polypeptide A method comprising the step of administering to the animal the above lipid membrane structure having nucleic acid encapsulated therein; and a cancer cell-specific surface on the surface of an immune cell, preferably a dendritic cell, in a mammal including human. An immunotherapy of a malignant tumor in which a surface polypeptide is presented to acquire immunity to the polypeptide, the method comprising the step of administering to the animal the above lipid membrane structure in which a nucleic acid encoding the polypeptide is encapsulated A method of including is provided.

Furthermore, according to the present invention, the polypeptide (I) used for promoting the migration of a lipid membrane structure into the nucleus of a cell, preferably into the nucleus of an immune cell, more preferably into the nucleus of a dendritic cell, Provided.

The lipid membrane structure provided by the present invention can efficiently migrate into the nucleus of any cell such as immune cells including dendritic cells, and efficiently transfer substances such as nucleic acids encapsulated in the nucleus. The polypeptide encoded by the nucleic acid can be expressed upon release. When a nucleic acid encoding a polypeptide is introduced into the nucleus of a dendritic cell using the lipid membrane structure of the present invention, the polypeptide transcribed and translated from the nucleic acid is presented on the surface of the dendritic cell, Can acquire immunity to the polypeptide, so that effective immunotherapy can be performed against the desired polypeptide. The lipid membrane structure provided by the present invention can be efficiently transferred into the nucleus of a cell even in the presence of serum and release a substance such as a nucleic acid encapsulated in the nucleus. Can be preferably used.

In addition, the lipid membrane structure provided by the present invention itself can exert an adjuvant action on dendritic cells and can promote the production of various cytokines. By administering dendritic cells transformed with a lipid membrane structure to be treated, tumor exacerbation and proliferation can be remarkably suppressed regardless of the presence or absence of an adjuvant. Can enhance cytotoxic activity in vivo. Furthermore, there is a feature that gene expression efficiency is remarkably improved by removing the CpG sequence from the vector part in the DNA to be encapsulated and, if necessary, removing the CpG sequence from the DNA encoding the protein to be expressed. .

It is the figure which showed the result of having evaluated the gene expression activity in a mouse | mouth bone marrow derived dendritic cell using MEND modified with various polypeptide (I). It is the figure which showed the result of having evaluated the cytokine production ability in a mouse | mouth bone marrow derived dendritic cell using MEND modified with various polypeptide (I). FIG. 3 is a graph showing the In-vivo CTL activity of the short-KALA3-modified MEND prepared in Example 2.

Examples of the lipid constituting the lipid membrane structure of the present invention include phospholipids, glycolipids, sterols, and saturated or unsaturated fatty acids.
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, and these 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 glycolipids include glyceroglycolipids (for example, sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride), glycosphingolipids (for example, galactosyl cerebroside, lactosyl cerebroside, ganglioside) and the like. Can be mentioned.

Examples of sterols include animal-derived sterols (e.g., cholesterol, cholesterol succinic acid, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol), plant-derived sterols (phytosterols) (e.g., stigmasterol, sitosterol, campesterol, Brush casterol), microorganism-derived sterols (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.

In the lipid structure of the present invention, a lipid having a tertiary amine and a disulfide bond is preferably used as the lipid component. Such lipids are disclosed in, for example, International Publication 2013/73480, and have a tertiary amine and disulfide bond in the molecule, and thus have a pH responsiveness and a membrane destabilizing action in a reducing environment. Are known. The compound represented by the formula (1) disclosed in the above international publication is preferable as a constituent lipid of the lipid structure of the present invention. In the compound represented by the formula (1), as a side chain, for example, lipids (PalmM) bound with myristic acid, lipids (PamlA) bound with retinoic acid, and lipids (PalmE) bound with tocopherol (PalmE) Among these lipids, PalmE is a neutral lipid and can be preferably used in the present invention because it can achieve gene expression even in the presence of serum. PalmE is compound B-2-5 in Table 1 of paragraph [0056] of International Publication 2013/73480. All of the above internationally disclosed disclosures are incorporated herein by reference. The amount of the lipid used is not particularly limited, but can be generally about 5 to 50% by weight based on the total lipid constituting the lipid structure. In addition, application examples of these Palms are known (Immunity, 21 (4), pp.527-538, 2004; Int. Immunol., 21 (4), pp.361-377, 2009; Blood, 115 (10), pp.1958-1968, 2010) and appropriate publications can be selected according to the purpose with reference to these publications.

The form of the lipid membrane structure is not particularly limited. For example, as a form dispersed in an aqueous solvent, a single membrane liposome, a multilamellar liposome, an O / W emulsion, a W / O / W emulsion, a spherical micelle, a 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 is a lipid membrane structure for delivering a substance into the nucleus of a cell, wherein the lipid membrane has an activity of promoting the transfer of the lipid membrane structure into the nucleus of the cell. It is modified with peptide (I).

Polypeptide (I) is a polypeptide having 8 to 26 amino acid residues, and includes 2 to 4 consecutive repeating units of tetrapeptide: Lys-Ara-Leu-Ara in the peptide chain However, Lys may be Arg in any one or more of the repeating units, and / or Ara sandwiched between Lys and Leu in any one of the repeating units May be His.

Conventionally, it is a polypeptide consisting of 27 amino acid residues obtained by removing 3 amino acids from the C-terminus of a known polypeptide consisting of 30 amino acid residues (hereinafter referred to as `` KALA peptide '' in this specification). `` Biochemistry, 36, pp.3008-3017, 1997) can be used to modify the surface of the lipid membrane structure to promote the transfer of the lipid membrane structure into the cell nucleus. It is known (International Publication WO / 2011/132713). All of the above international disclosures are incorporated herein by reference. The polypeptide (I) used in the present invention is a polypeptide created by focusing on the repeating unit of Lys-Ara-Leu-Ara contained in the KALA peptide and clarifying the function of the repeating unit. It is.

The activity of promoting the transfer of the polypeptide (I) into the nucleus of the lipid membrane structure can be confirmed, for example, by evaluating the activity of promoting the ability of dendritic cells to move into the nucleus. For example, a dendritic cell is used as a target cell, and a nucleic acid that can be expressed in its nucleus, for example, a nucleic acid that encodes a polypeptide linked downstream of a promoter operable in the nucleus of the dendritic cell is contained therein. In the retained lipid membrane structure, a lipid membrane structure in which the lipid membrane is modified with polypeptide (I) (hereinafter referred to as `` modified lipid membrane structure '') and an unmodified lipid membrane structure (hereinafter referred to as `` non- Is the expression level of the marker polypeptide in the cells introduced with the modified lipid membrane structure increased in the cells introduced with the unmodified lipid membrane structure? What is necessary is just to evaluate. When an increase in expression level is observed, the polypeptide can be used as the polypeptide (I). One or a combination of two or more of the above polypeptides (I) can be used.

The polypeptide (I) can be biologically prepared using host cells by various gene recombination techniques available to those skilled in the art. The polypeptide (I) may be produced by an organic chemical method using a peptide synthesis reaction such as a solid phase synthesis method available to those skilled in the art. Alternatively, automatic synthesis may be performed using a peptide synthesizer.

The polypeptide (I) contains 2 to 4 repeating units of tetrapeptide: Lys-Ara-Leu-Ara in the polypeptide chain in succession. For example, a polypeptide comprising two consecutive repeating units is preferred. Lys may be Arg in any one or more of the above repeating units, and Lys may be Arg in all the repeating units in the polypeptide chain. Further, Ara sandwiched between Lys and Leu may be His in any one of the repeating units.

As the polypeptide (I), for example, a polypeptide containing two consecutive repeating units of Lys-Ara-Leu-Ara or a polypeptide containing two consecutive repeating units of Arg-Ara-Leu-Ara is preferable. More preferred is a polypeptide in which the sequence of Trp-Glu-Ara-Lys-Leu-Ara or Trp-Glu-Ara-Arg-Leu-Ara is bound to the N-terminus of the two consecutive repeating units.

Specific examples of the polypeptide (I) preferably used in the present invention are shown below, but the polypeptide (I) usable in the present invention is not limited to the following examples (in the following examples, an amino acid is a single letter). Notation).
short-KALA1: WEAKLAKALAKALAKHLAKALA (SEQ ID NO: 2 in the sequence listing)
short-KALA2: WEAKLAKALAKALAKHLA (SEQ ID NO: 3 in the sequence listing)
short-KALA3: WEAKLAKALAKALA (SEQ ID NO: 4 in the sequence listing)
short-KALA-4U: KALAKALAKALAKALA (SEQ ID NO: 6 in the sequence listing)
short-KALA-3U: KALAKALAKALA (SEQ ID NO: 7 in the sequence listing)
short-KALA-2U: KALAKALA (SEQ ID NO: 8 in the sequence listing)
short-RALA3: WEARLARALARALA (SEQ ID NO: 9 in the sequence listing)

The means for immobilizing the polypeptide (I) to the lipid membrane of the lipid membrane structure is not particularly limited.For example, the polypeptide (I) is modified with a hydrophobic group such as a stearyl group or a cholesteryl group, and the hydrophobicity By preparing the lipid membrane structure so that the group is buried in the lipid membrane of the lipid membrane structure, the lipid membrane modification can be easily performed. Any hydrophobic compound residue can be used as the hydrophobic group. When a liposome having a lipid multilayer is used as the lipid membrane structure, the lipid membrane modification with the polypeptide (I) may be performed on the inner lipid membrane in addition to the outer lipid membrane.

The lipid membrane structure of the present invention can be used to deliver a substance into the nucleus of a cell, but the type of cell is not particularly limited, and the type of substance to be delivered and the purpose of substance delivery into the nucleus Depending on the situation, appropriate cells can be targeted. Preferred examples of the cell to be targeted include immune cells, and among the immune cells, antigen-presenting cells can be preferably used. For example, antigen-presenting cells such as macrophages, dendritic cells, and B cells are preferred, and dendritic cells are particularly preferred.

In order to promote the translocation of the lipid membrane structure of the present invention into the nucleus, for example, the lipid membrane structure can be surface-modified with an oligosaccharide compound having 3 or more sugars. The type of oligosaccharide compound having 3 or more sugars is not particularly limited. For example, an oligosaccharide compound having about 3 to about 10 sugar units bound thereto can be used, and preferably about 3 to about 6 sugar units. Bound oligosaccharide compounds 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 (Gentianose: β-D-fructofuranosyl β-D-glucopyranosyl- (1 → 6) -α-D-glucopyranoside), isomaltotriose (Isomaltotriose: α-D-glucopyranosyl- (1 → 6) -α-D-glucopyranosyl- (1 → 6) -D-glucose), isopanose : Α-D-glucopyranosyl- (1 → 4)-[α-D-glucopyranosyl- (1 → 6)]-D-glucose), maltotriose (Maltotriose: α-D-glucopyranosyl- (1 → 4)- α-D-Glucopyranosyl- (1 → 4) -D-glucose), Manninotriose: α-D-galactopyranosyl- (1 → 6) -α-D-galactopyrano -(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), planteose (Planteose: α-D-galactopyranosyl- (1 → 6) -β- D-fructofuranosyl = α-D-glucopyranoside), raffinose (β-D-fructofuranosyl = α-D-galactopyranosyl- (1 → 6) -α-D-glucopyranoside), soratriose (Solatriose: α-L-rhamnopyranosyl- (1 → 2)-[β-D-glucopyranosyl- (1 → 3)]-D-galactose), Umbelliferose: β-D-fructofuranosyl = α Trisaccharide compounds such as -D-galactopyranosyl- (1 → 2) -α-D-galactopyranoside); Lycotetraose: β-D-glucopyranosyl- (1 → 2)-[β- D-Xylopyranosyl- (1 → 3)]-β-D-glucopyranosyl- (1 → 4) -β-D-galactose), maltotetraose: α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranosyl- (1 → 4) -α-D-Glucopyranosyl- (1 → 4) -D-glucose), Stachyose (β-D-fructofuranosyl = α-D-galactopyranosyl- (1 → 6) -α- Tetrasaccharide compounds such as D-galactopyranosyl- (1 → 6) -α-D-glucopyranoside); maltopentaose: altα-D-glucopyranosyl- (1 → 4) -α-D-glucopyranosyl- ( 1 → 4) -α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranosyl- (1 → 4) -D-glucose), Verbascose: β-D-fructofuranosyl = α -D-galactopyranosyl- (1 → 6) -α-D-galactopyranosyl- (1 → 6) -α-D-galactopyranosyl- (1 → 6) -α-D-glucopyranoside) 5 sugar compounds such as maltohexaose: α-D-glucopyranosyl- (1 → 4) -α-D- (Lucopyranosyl- (1 → 4) -α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranosyl- (1 → 4) -α-D-glucopyranosyl- (1 → 4) -D-glucose) A hexasaccharide compound may be mentioned, but is 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, maltohexaose, etc. can be suitably 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 amount of 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% with respect to the total amount of lipid. Degree.

The method of modifying the surface of the lipid membrane structure with an oligosaccharide compound is not particularly limited. For example, liposomes whose surfaces are modified with monosaccharides such as galactose and mannose (International Publication WO2007 / 102481) 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.

By modifying the surface of the lipid membrane structure with a hydrophilic polymer such as polyalkylene glycol, the blood retention of liposomes can be enhanced. This means is described in, for example, Japanese Patent Application Laid-Open No. 1-249717, Japanese Patent Application Laid-Open No. 2-149512, Japanese Patent Application Laid-Open No. 4-346918, Japanese Patent Application Laid-Open No. 2004-10481, and the like. 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, in the case of modification with polyethylene glycol, stearyl polyethylene glycol (for example, PEG45 stearate (STR-PEG45) or the like) can be used. Others, 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-methoxypolyethylene glycol-5000} -1,2-distearoyl-sn-glycero-3-phosphoethanol Polyethylene glycol derivatives such as amines can also be used, but polyalkylene glycolated lipids are not limited to these.

For example, the surface modification of the lipid membrane structure can be performed using the polypeptide (I) modified with polyalkylene glycol. For example, the above-mentioned polypeptide (I) modified with a polyalkylene glycol condensed with an appropriate phospholipid, such as stearyl polyethylene glycol, can be used. For example, it is preferable to use a polypeptide obtained by condensing polyalkylene glycol with an N-terminal or C-terminal cysteine (Cys) residue. According to this embodiment, the lipid membrane modification with the polyalkylene glycol and the polypeptide (I) can be simultaneously achieved.

In addition, surface modification with the polyalkylene glycol and the oligosaccharide compound can be simultaneously achieved by bonding the oligosaccharide compound to the polyalkylene glycol. However, the method of surface-modifying the lipid membrane structure with a polyalkylene glycol or oligosaccharide compound is not limited to the above-mentioned method. For example, a lipidated compound such as stearyl polyalkylene glycol or oligosaccharide compound is used. In some cases, surface modification can be performed by using as a constituent lipid of the lipid membrane structure.

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, etc. Can also be used. In addition to polyalkylene glycol, dextran, pullulan, ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer as well as polyalkylene glycol are used as hydrophilic polymers to enhance blood retention. Amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, carrageenan and the like can also be used for surface modification.

In order to efficiently escape the lipid membrane structure from the endosome into the cytoplasm, the lipid membrane of the lipid membrane structure of the present invention may be modified with GALA. For example, since JP-A-2006-28030 discloses a liposome surface-modified with GALA, a lipid membrane structure surface-modified with GALA can be easily produced according to the method described in the above-mentioned publication. Can do. 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 surface modification amount 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%, based on the total lipid amount.

In the present 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, the entire disclosure of JP-A-2006-28030 is included as a disclosure of the present specification by reference.

The surface of the lipid membrane structure of the present invention can be modified with an MPC polymer. 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 polypeptides and blood cells is extremely small, and it has been shown to have excellent biocompatibility. In the present specification, the term “MPC polymer” includes both a homopolymer of MPC and a copolymer of MPC and other polymerization components.

∙ MPC polymers can be easily obtained from commercially available polymers. For example, MPC homopolymer (CAS: 67881-99-6); MPC and butyl methacrylate copolymer (CAS: 125275-25-4); MPC, methacrylic as a registered trademark “LIPIDURE” from NOF Corporation 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 acid ester or methacrylic acid 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 with a molar ratio of MPC and BMA of 5: 5 (PMB50) and a copolymer with a molar ratio of MPC and BMA of 3: 7 (PMB30) are known. For example, it can be easily prepared according to the method described in Polymer Journal, 22, pp.355-360, な ど 1990, etc. (for example, a specific description of the production method is described in JP-A-2007-314526). is there). 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, a polymer having an average molecular weight (weight average molecular weight) of about 5,000 to 300,000, preferably about 10,000 to 100,000 can be used from the viewpoint of maintaining water solubility.

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 a lipid membrane structure such as a liposome and allowed to stand 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, in the range of 0.01 to 1% by mass with respect to the total lipid amount of the lipid membrane structure, preferably May be added in an amount of about 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.

The lipid membrane structure of the present invention comprises a sterol or a membrane stabilizer such as glycerin or a fatty acid ester thereof, an antioxidant such as tocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene, a charged substance, and a membrane. One or two or more substances selected from the group consisting of polypeptides 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 the membrane polypeptide include a membrane superficial polypeptide or an integral membrane polypeptide. The compounding amount of these substances is not particularly limited, and can be appropriately selected according to the purpose.

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. By appropriately adding these functions, for example, the retention of a lipid membrane structure containing a nucleic acid containing a gene in blood is improved, and the capture rate by reticuloendothelial tissues such as the liver and spleen is reduced. At the same time, 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 that can impart 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 capable of specifically binding to a cell surface receptor or antigen, thereby improving the substance delivery efficiency into the cell nucleus. can do. For example, it is preferable to arrange a monoclonal antibody against a biological component specifically expressed in the target tissue or organ on the surface of the lipid membrane structure. This technique is described in, for example, STEALTH LIPOSOME (pages 233-244, issued by CRC Press, Inc., edited by Danilo Lasic 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 incorporating a lipid derivative having a maleimide structure such as 2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl) ethanecarboxamido] propyl} -poly (oxy-1,2-ethanedyl) The monoclonal antibody can be bound to the membrane surface of the lipid membrane structure.

The surface of the lipid membrane structure of the present invention may be modified with a polypeptide containing a plurality of continuous arginine residues (hereinafter referred to as “polyarginine”). The polyarginine is preferably a polypeptide containing 4 to 20 consecutive arginine residues, more preferably a polypeptide consisting of only 4 to 20 consecutive arginine residues, particularly preferably octaarginine. Can do. By modifying the surface of a lipid membrane structure such as a liposome with polyarginine such as octaarginine, the intracellular delivery efficiency of the target substance encapsulated in the liposome can be improved (Journal of Controlled Release, ase98, pp. 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 surface of the lipid membrane structure of the present invention may be modified with INF7. INF7 is a glutamic acid-rich peptide obtained by modifying a peptide (1-23) derived from influenza HA polypeptide (HA2) .When mixed with liposomes, the lipid structure collapses and the encapsulated substance is easily released. (Biochemistry, 46, pp.13490-13504, 2007) and a delivery system in which INF7 is conjugated to polyethylene glycol tetraacrylate (PEG-TA) has also been proposed (The Journal Gene Medicine, 10, pp .1134-1149, 2008). Those skilled in the art can easily use INF7 in the present invention by referring to these publications. In this specification, the term “INF7” includes one or several amino acids in the amino acid sequence of the above peptide in addition to the peptides specified by the sequences described in Table 1 of Biochemistry, 46, pp.13490-13504, 2007. A modified peptide consisting of an amino acid sequence in which is deleted, substituted and / or added and having substantially the same properties as INF7 is 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 in general, by constructing a lipid membrane structure using lipid-modified INF7 in which a lipid compound and INF7 are covalently bound 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, stearylated INF7 can be used, and this compound is described in Futaki, S. et al., Biocongug. Chem., 12 (6), pp.1005-1011, 2001. It can be easily manufactured according to the method. 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 content of the lipid membrane structure, preferably about 3 to 5 mol% with respect to the total lipid content. It is.

Envelope-type nanostructures (MEND) with multi-functionality are known and can be suitably used as the lipid membrane structure of the present invention. MEND, for example, has a structure in which a core is a complex of a nucleic acid such as plasmid DNA and a cationic polymer such as protamine, and the core is enclosed in a lipid envelope membrane in the form of a liposome. MEND lipid envelope membranes can be equipped with peptides to adjust pH responsiveness and membrane permeability as needed, and the outer surface of lipid envelope membranes can be modified with alkylene glycols such as polyethylene glycol. it can. Inside the lipid envelope of MEND, condensed DNA and cationic polymer are encapsulated, and designed to achieve efficient gene expression. As the MEND that can be suitably used in the present invention, a MEND in which a complex of plasmid DNA incorporating a desired gene and protamine is encapsulated inside and the outer surface of the lipid envelope is modified with oligosaccharide-conjugated PEG is preferable. The modification with oligosaccharide-linked PEG preferably uses stearyl polyethylene glycol to which the above-described polypeptide (I) is bound as a constituent lipid component. For MEND, for example, reviews such as DrugDDelivery System, 22-2, pp.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. 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 the dispersed state is not particularly limited. For example, in the case of liposomes, the particle diameter is about 50 to 5 μm, preferably about 50 to 400 nm, and about 50 to 300 nm. Is preferable, and about 150 to 250 nm is more preferable. The particle diameter can be measured, for example, by the 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 monoa Polyhydric alcohols (aqueous solutions) such as alkyl ether and 1,3-butylene glycol 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 addition, from the viewpoint of chemical stability of the lipid, it is desirable to set the pH of the aqueous solvent from weakly acidic to near neutral (about pH 3.0 to 8.0) and / or to remove dissolved oxygen by nitrogen bubbling or the like. .

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, stability may be improved.

In the lipid membrane structure of the present invention, for example, a liposome, a substance to be delivered into the nucleus of a cell of a target tissue or organ can be encapsulated. 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, sugars, peptides, nucleic acids, low molecular weight compounds, metals Any substance such as a compound 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. As an example of the present invention, the 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.

In the lipid membrane structure of the present invention, a nucleic acid can be preferably encapsulated. 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 siRNAs. Functional nucleic acids including RNA and the like can also be used, and these are also included in the term nucleic acid in the present specification. In this specification, the term “nucleic acid” should be interpreted in the broadest sense, and should not be interpreted in a limited way in any sense. For example, when DNA is used as the nucleic acid, for example, the gene DNA to be expressed can be bound to the vector DNA and encapsulated in the lipid membrane structure, but in order to achieve higher gene expression efficiency, the vector It is preferred that the DNA does not contain a CpG sequence, and in addition it may be more preferred that the gene DNA to be expressed does not contain a CpG sequence. It is preferable that an enhancer and / or a promoter is bound to the vector.

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, dimethyldioctadecylammonium bromide, 2,3-dioleyloxy-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. 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.

For example, a lipid membrane structure encapsulating a nucleic acid can be used as a carrier for delivering the nucleic acid into the nucleus of a cell of 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 into the nucleus of the cells of the target tissue or organ and efficiently expressed. be able to. The administration method is not particularly limited, but parenteral administration is preferable, and intravenous administration is more preferable. When the lipid membrane structure of the present invention is used as a medicine, for example, a medicine in the form of a pharmaceutical composition can be prepared and administered together with an appropriate formulation additive.

When a nucleic acid is transferred into the nucleus of an immune cell, preferably a dendritic cell, using the lipid membrane structure of the present invention, a substance such as a nucleic acid encapsulated in the nucleus is efficiently released and encoded by the nucleic acid. Polypeptides can be expressed. When a nucleic acid encoding a polypeptide is introduced into the nucleus of a dendritic cell using the lipid membrane structure of the present invention, the polypeptide transcribed and translated from the nucleic acid is presented on the surface of the dendritic cell, Can acquire immunity to the polypeptide, so that effective immunotherapy can be performed against the desired polypeptide. This aspect is a particularly preferable aspect in the present invention.

The lipid membrane structure provided by the present invention itself can exert an adjuvant action on dendritic cells and can promote the production of various cytokines. Therefore, immunotherapy can be performed very efficiently by using the lipid membrane structure of the present invention. In addition, by administering dendritic cells transformed with the lipid membrane structure provided by the present invention to mammals including humans, it is possible to remarkably suppress malignant tumor exacerbation and proliferation regardless of the presence or absence of adjuvants. It is possible to enhance the cytotoxic activity in vivo by encapsulating and administering the antigen protein, so that it is a highly effective preventive vaccine for malignant tumors or as an active ingredient of a medicament for the treatment of malignant tumors. Can be used.

EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example. For the production of MEND and the evaluation of gene expression activity, the methods described in International Publication WO 2011/132713 can be referred to.
Example 1
(1) Preparation of short-KALA peptide-modified MEND Various plasmid DNAs were dissolved in 10 mM HEPES aqueous solution (pH 5.3) so as to be 0.15 mg / mL. A compact body of protamine and pDNA was prepared by adding dropwise 100 μL of Protamine solution of 0.106 mg / mL (10 mM HEPES aqueous solution (pH 5.3)) to 100 μL of pDNA solution while stirring little by little (N / P ratio). 1.1).

5 mM PalmE 19.8μL, 5 mM DOPE 26.4 μL, 5 mM Cholesterol 19.8 μL and 1 mM DMG-PEG2000 9.9μL were mixed in a tube, and PalmE: DOPE: Cholesterol: DMG-PEG2000 = 30: 30: 30: 30 did. Ethanol was added so that the total amount was 200 μL. While stirring the lipid solution, 200 μL of compaction body was quickly added, and this mixed solution was quickly added to 1600 μL of 10 mM HEPES solution (pH 5.3). Furthermore, after diluting twice with 10 mM HEPES aqueous solution (pH 5.3), concentrating using a centrifugal ultrafiltration device with MWCO 100,000, diluting with 4 mL of 100 mM HEPES buffer, and then centrifugal ultrafiltration with MWCO 100,000. Concentration was performed with a filtration device. The solution was diluted with 10 μmM HEPES buffer to a lipid concentration of 0.66 μm. Stearylated KALA (STR-KALA) (1 mg / mL aqueous solution) synthesized by the method described in JP-A-2003-343857, and various stearylated short-KALA (STR-short-KALA) (1 mg / mL) The solution was added to 3 mol% of the total lipid and allowed to stand at room temperature for 10 minutes. The sequence of each peptide subjected to the test is shown. In either case, the N-terminus is stearylated.

original KALA: WEAKLAKALAKALAKHLAKALAKALKA (SEQ ID NO: 1)
short-KALA1: WEAKLAKALAKALAKHLAKALA (SEQ ID NO: 2)
short-KALA2: WEAKLAKALAKALAKHLA (SEQ ID NO: 3)
short-KALA3: WEAKLAKALAKALA (SEQ ID NO: 4)
short-KALA4: WEAKLAKALA (SEQ ID NO: 5)
short-KALA-4U: KALAKALAKALAKALA (SEQ ID NO: 6)
short-KALA-3U: KALAKALAKALA (SEQ ID NO: 7)
short-KALA-2U: KALAKALA (SEQ ID NO: 8)
short-RALA3: WEARLARALARALA (SEQ ID NO: 9)

(2) Isolation and induction of mouse bone marrow-derived dendritic cells (BMDC) Femur and tibia from C57BL / 6 mice (6-8 weeks old) with RPMI1640 medium and PBS 20 mL each added to a sterile petri dish and cervical dislocation Was removed, lightly disinfected with 70% ethanol, and then immersed in PBS. Both ends of the bone were cut, and bone marrow cells were pushed into the medium with a 1 mL syringe (26G needle) containing the medium. The cell suspension was transferred to a 50 mL conical tube through a 40 μm cell strainer (FALCON). After centrifugation (450 g, 4 ° C, 5 min), the supernatant is removed and the cells are dispersed by tapping. Then, 1 mL of ACK Lysing Buffer (Lonza, Walkersville, MD) is added, mixed, and mixed at room temperature for 3-5 Let stand for a minute. After adding 10 mL of the medium, the supernatant was removed by centrifugation, and further washed twice with 10 mL of the medium. Next, the cells were suspended in 10 mL of medium, added to a 10 cm cell culture dish (FALCON), and cultured at 37 ° C. under 5% CO ₂ for 4 hours or more. Lightly pipetted to collect only floating cells in a 50 mL conical tube, centrifuged, removed the supernatant, suspended in 10 mL of medium and counted. Suspend in medium to 1 × 10 ⁶ cells / mL, add GM-CSF (final concentration 10 ng / mL), seed 1 mL each on a 24 well plate (Corning, NY), 37 ° C, 5% Cultivation was performed for 2 days under CO ₂ conditions. After 2 days and 4 days, cell clumps were left and floating cells were removed, and then 1 mL of fresh GM-CSF-containing RPMI1640 medium was added. Suspended and weakly adherent cells 6-8 days after the start of culture in the presence of GM-CSF were used as immature dendritic cells in the experiment. The purity was 85 to 90% in the evaluation using the CD11c antibody (PE anti-mouse CD11c, Clone: N418, BioLegend).

(3) Evaluation of gene expression activity in BMDC BMDC induced by the method shown in (2) above was seeded on a 24-well plate at 4 × 10 ⁵ cells / well. The prepared MEND was diluted with RPMI 1640 (without serum) so that the amount of pDNA was 0.4 μg / well, and prepared to 500 μL. A MEND solution was applied to each well and incubated in a 37 ° C., 5% CO ₂ environment. After 3 hr, RPMI 1640 (with serum) 500 μL was added to each well, and 21 hr later, luciferase activity was measured. Regarding the measurement of luciferase activity, the floating cell group was recovered together with the medium, centrifuged (4 ° C., 500 g, 5 min), and recovered by removing the supernatant. Reporter Lysis Buffer (1x) was added to the cells remaining in each well and the previously collected floating cells to make a total of 75 μL, then pipetted back to each well and frozen at −80 ° C. . After thawing the sample frozen at −80 ° C., the cells were removed using a cell scraper and collected in an Eppendorf tube. The collected cell lysate was centrifuged at 15,000 rpm, 4 ° C. for 2 min, and 50 μL of the supernatant was recovered. Luciferase activity measurement (RLU / mL) was measured using the obtained supernatant. Furthermore, protein quantification (mg / mL) by BCA method was performed, and luciferase activity (RLU / mg protein) per unit protein amount was calculated.

(4) Cytokine production ability evaluation in BMDC It seed | inoculated so that it might become 4 * 10 < ⁵ > cells / well to the 24 well plate induced | guided | derived by the method shown to said (2). The prepared MEND was diluted with RPMI 1640 (without serum) so that the amount of pDNA was 0.4 μg / well, and prepared to 500 μL. A MEND solution was applied to each well and incubated in a 37 ° C., 5% CO ₂ environment. Three hours later, 500 μL of RPMI 1640 (with serum) was added to each well. After further 21 hr, the supernatant of each well was collected, centrifuged (4 ° C., 500 g, 5 min), and the supernatant was frozen at −80 ° C. The IL-6 concentration in the supernatant was quantified using Quantikine IL-6 ELISA kit (R & D systems).

(5) Results FIG. 1 shows gene expression activity when a gene encoding luciferase is introduced. Although the expression is maintained even if the sequence is deleted sequentially from the C-terminus of the original KALA peptide (leftmost: 27 residues, SEQ ID NO: 1 in the sequence listing), the highest gene expression is obtained in short-KALA3 It was. On the other hand, it was clarified that the gene expression decreased dramatically when further cut and shortened to Short-KALA4 (SEQ ID NO: 5 in the sequence listing). Moreover, even when this lysine (K) of Short-KALA3 was substituted with arginine (R) (Short-RALA3), the same level of activity was maintained. In addition, focusing on the peptide sequence of short-KALA3, since the C-terminus has a sequence in which KALA is repeated twice, KALA (Lys-Ara-Arg-Ara) is an important sequence as a repeated sequence. Therefore, peptides with repeated KALA sequences were designed (Short-KALA-2U to Short-KALA-4U). As a result, high gene expression was maintained in any peptide sequence, but among them, the highest gene expression activity was maintained in Short-KALA3.

The same results as in Fig. 1 were obtained at the cytokine production level (Fig. 2). When the original KALA sequence was trimmed, the highest cytokine production was obtained in short-KALA3. On the other hand, as with gene expression, it was revealed that expression was dramatically reduced in Short-KALA4. Short-KALA3 in which lysine (K) was changed to arginine (R) also maintained the same level of cytokine production. In addition, the highest cytokine acidity was maintained in Short-KALA-3U even when the KALA sequence was repeated.

Example 2
(1) Preparation of short-KALA3 peptide-modified MEND Short-KALA3 peptide-modified MEND was prepared in the same manner as in Example 1.
Various plasmid DNAs were dissolved in 10 mM HEPES aqueous solution (pH 5.3) so as to be 0.15 mg / mL. A compact body of protamine and pDNA was prepared by adding dropwise 100 μL of Protamine solution of 0.106 mg / mL (10 mM HEPES aqueous solution (pH 5.3)) to 100 μL of pDNA solution while stirring little by little (N / P ratio). 1.1).

5 mM PalmE 19.8 μL, 5 mM DOPE 26.4 μL, 5 mM Cholesterol 19.8 μL, and 1 mM DMG-PEG2000 9.9 μL were mixed in a tube, and PalmE: DOPE: Cholesterol: DMG-PEG2000 = 30: 40: 30: 30 did. Ethanol was added so that the total volume was 200 μL. While stirring this lipid solution, 200 μL of compaction body was quickly added, and this mixed solution was quickly added to 1600 μL of 10 mM HEPES solution (pH 5.3). Furthermore, after diluting twice with 10 mM HEPES aqueous solution (pH 5.3), concentrating using a centrifugal ultrafiltration device with MWCO 100,000, diluting with 4 mL of 100 mM HEPES buffer, and then centrifugal ultrafiltration with MWCO 100,000. Concentration was performed with a filtration device. Further, after dilution and concentration in the same manner with 4 mL of 10 mM HEPES buffer, a PalmE MEND suspension was prepared with 10 mM HEPES buffer so that the lipid concentration was 0.66 mM.
When preparing short-KALA3-modified MEND, add stearyl-ized short-KALA3 (STR-short-KALA3) (1 mg / mL) to 0.25 mol% of the total lipid, and leave it at room temperature for 10 minutes. did.

(2) In vivo CTL (cytotoxic T lymphocyte) activity of short-KALA3 modified MEND C57BL / 6 mice (female, 6 weeks old) with short-KALA3 modification encapsulated in pCpGfree-OVA plasmid DNA prepared in (1) PalmE MEND was intraperitoneally administered so that it would be 2.4 μg and 1.2 μg in terms of plasmid DNA (26G needle). One week after immunization, target cells prepared by the following method were administered. First, the spleen was removed from naive mice dislocated from the cervical spine, loosened in a petri dish containing 5 to 7.5 mL of RPMI1640 medium, collected using a 5 mL syringe, and then transferred to a 50 mL conical tube through a cell strainer. .

After centrifugation (1600-1700 rpm, 4 ° C, 5 min), the supernatant was removed, suspended in 1 mL (/ spleen) of ACK Lysing buffer, and hemolyzed by incubation at room temperature for 5 minutes. After 5-fold dilution with medium, the supernatant was removed, and after washing with 10 mL of medium, the suspension was suspended in 30 mL, counted, and transferred to two 50 mL conical tubes through a cell strainer. Each was centrifuged and the supernatant removed, and then resuspended in the medium (10 ⁷ cells / mL).

OVA _257-264 peptide (1 mM, final concentration 5 μM) was added as a model antigen to one of the two, and the cell suspension was incubated at 37 ° C. under 5% CO ₂ for 60 minutes. Cell groups pulsed with OVA _257-264 peptide after washing with 10 mL of medium and 10 mL of PBS are _treated with 50 μM CFSE (Molecular probe) (CFSE ^High ), and cells not pulsed are 0.5 μM CFSE (CFES ^Low ) Was suspended in PBS containing 3 × 10 ⁷ cells / mL and incubated at 37 ° C. for 10 minutes. After washing twice with 10 mL of RPMI1640 medium and twice with 10 mL of PBS, it was finally suspended in PBS (5 × 10 ⁷ cells / mL). Two types of cells stained at different concentrations just before administration were mixed together and administered to the immunized mouse via the tail vein (1 × 10 ⁷ cells / 200 μL / mouse, 27G needle).

Twenty hours after the administration of the target cells, the spleen was removed and the aggregate was loosened, and a uniform cell suspension was made through a nylon mesh. This cell suspension was centrifuged at 1600 rpm, 4 ° C., 3 min. After removing the supernatant, 1 mL of ACK Lysing buffer (/ spleen) was added, and the mixture was allowed to stand at room temperature for 5 minutes. 9 mL of FACS buffer was added and centrifuged at 1600 rpm, 4 ° C., 3 min. After removing the supernatant, 5 mL of FACS buffer was added and centrifuged again. After removing the supernatant, it was suspended in 5 mL of FACS buffer. The prepared cell suspension was transferred to a FACS tube through a nylon mesh, and the number of CFSE positive cells was measured with a flow cytometer. For cells stained at a low concentration (CFSE ^Low ), 7,500 cells were analyzed. CTL activity was calculated by comparing the number of cells in CFSE ^High and CFSE ^Low . The error between experiments was corrected by the CFSE ^High / CFSE ^Low ratio in naive mice.

As a result, the CTL activity (% Lysis) in the intraperitoneal administration group of short-KALA3 modified PalmEENDMEND was short-KALA3 unmodified PalmE MEND at both 2.4 μg and 1.2 μg of PalmE MEND dose in terms of plasmid DNA. A value higher than the CTL activity of the administration group was obtained (FIG. 3). This revealed that CTL activity was improved by modifying short-KALA3 to PalmE MEND.

The lipid membrane structure provided by the present invention can efficiently migrate into the nucleus of any cell such as immune cells including dendritic cells, and efficiently transfer substances such as nucleic acids encapsulated in the nucleus. Since it can be released to express the polypeptide encoded by the nucleic acid, and it can also exert an efficient adjuvant effect, effective immunotherapy can be performed on the desired polypeptide. There is a feature. In particular, the lipid membrane structure of the present invention can be efficiently transferred into the nucleus of a cell in vivo, and a substance such as a nucleic acid encapsulated in the nucleus can be efficiently released. Can be suitably used.

Claims (19)

1. A lipid membrane structure for delivering a substance into the nucleus of a cell, the lipid membrane comprising the following polypeptide (I):
   A polypeptide having 8 to 26 amino acid residues and comprising 2 to 4 repeating units of tetrapeptide: Lys-Ara-Leu-Ara in the peptide chain in succession (however, the repeating unit Lys may be Arg in any one or more of them, and / or Ara sandwiched between Lys and Leu in any one of the repeating units may be His. )
   Lipid membrane structure modified with
2. 2. The polypeptide (I) is a polypeptide containing two consecutive repeating units of Lys-Ara-Leu-Ara or two consecutive repeating units of Arg-Ara-Leu-Ara. The lipid membrane structure described in 1.
3. A polypeptide in which the sequence of Trp-Glu-Ara-Lys-Leu-Ara or Trp-Glu-Ara-Arg-Leu-Ara is bound to the N-terminus of two consecutive repeating units according to claim 2 The lipid membrane structure according to claim 2, which is a peptide.
4. The polypeptide is Trp-Glu-Ara-Lys-Leu-Ara-Lys-Ara-Leu-Ara-Lys-Ara-Leu-Ara or Trp-Glu-Ara-Arg-Leu-Ara-Arg-Ara-Leu-Ara The lipid membrane structure according to claim 1, which is -Arg-Ara-Leu-Ara.
5. The lipid membrane structure according to any one of claims 1 to 4, comprising a lipid having a tertiary amine and a disulfide bond as a lipid component of the lipid membrane structure.
6. The lipid membrane structure according to any one of claims 1 to 5, wherein the lipid membrane structure is a liposome.
7. The lipid membrane structure according to any one of claims 1 to 6, wherein the cell is an immune cell.
8. The lipid membrane structure according to any one of claims 1 to 7, wherein the polypeptide (I) is modified with a hydrophobic group, and the hydrophobic group is inserted into the lipid membrane.
9. The lipid membrane structure according to any one of claims 1 to 8, which has a polypeptide containing a plurality of continuous arginine residues on its surface.
10. The lipid membrane structure according to any one of claims 1 to 9, having a polyalkylene glycol on the surface.
11. The lipid membrane structure according to any one of claims 1 to 10, which is used for introducing a nucleic acid encoding an antigen polypeptide to be presented on the surface of an immune cell into the nucleus of the cell.
12. The lipid membrane structure according to any one of claims 1 to 10, wherein a substance to be delivered is encapsulated therein.
13. The lipid membrane structure according to claim 12, wherein a nucleic acid and a cationic polymer are encapsulated therein.
14. The lipid membrane structure according to claim 12 or 13, wherein the substance to be delivered is a nucleic acid encoding an antigen polypeptide to be presented on the surface of immune cells.
15. The lipid membrane structure according to claim 14 for use in immunotherapy against the antigen polypeptide.
16. The lipid membrane structure according to claim 13 or 14, wherein the nucleic acid is DNA, and the DNA does not contain CpG.
17. A pharmaceutical composition comprising the lipid membrane structure according to any one of claims 12 to 16 as an active ingredient.
18. The pharmaceutical composition according to claim 17, which is used for prevention and / or treatment of a malignant tumor.
19. A polypeptide used for promoting the transfer of a lipid membrane structure into the nucleus of a cell, comprising the following polypeptide (I):
    A polypeptide having 8 to 26 amino acid residues and comprising 2 to 4 repeating units of tetrapeptide: Lys-Ara-Leu-Ara in the peptide chain in succession (however, the repeating unit Lys may be Arg in any one or more of them, and / or Ara sandwiched between Lys and Leu in any one of the repeating units may be His. )

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