JP5067733B2 - Lipid membrane structure capable of delivering target substance into mitochondria - Google Patents

Description

  The present invention relates to a lipid membrane structure, particularly a lipid membrane structure capable of delivering a target substance into mitochondria.

  Conventionally, as a technique for introducing a protein into mitochondria, a vector capable of expressing a fusion protein of a peptide having an ability to migrate to mitochondria (for example, a mitochondrial targeting signal (MTS) such as Rat liver succinyl-CoA synthetase α) and a target protein Has been mainly employed to introduce a fusion protein into cells and to express the fusion protein in the cells (Non-patent Documents 1, 2, and 3). However, mitochondrial membrane permeation is very limited, and there is no method for delivering oligonucleotides, plasmid DNA, etc. into mitochondria.

  On the other hand, development of vectors and carriers for reliably delivering drugs, nucleic acids, peptides, proteins, sugars and the like to target sites has been actively conducted. For example, in gene therapy, viral vectors such as retroviruses, adenoviruses, and adeno-associated viruses have been developed as vectors for introducing a target gene into target cells. However, since viral vectors have problems such as difficulty in mass production, antigenicity, toxicity, etc., liposome vectors and peptide carriers with few such problems are attracting attention. Liposome vectors also have the advantage that directivity with respect to target sites can be improved by introducing functional molecules such as antibodies, proteins, and sugar chains on the surface thereof.

Recently, by modifying the surface of condensed DNA-encapsulated liposomes with stearylated octaarginine, it has been reported that the efficiency of introducing condensed DNA into cells has improved by 1000 times, and the efficiency of introducing condensed DNA-encapsulated liposomes into cells has improved by 100 times. (Non-Patent Document 4). In addition, it has been reported that liposomes whose surfaces are modified with stearylated octaarginine can be introduced into cells in an intact state (in an intact state) (Patent Document 1, Non-Patent Documents 4 and 5).
International Publication WO2005 / 032593 Pamphlet B. W. Kong et al., “Biochimica et Biophysica Acta”, 2003, 1625, 98-108. T.A. Tamura et al., “Biochemical and Biophysical Research Communications”, 1996, 222, 659-63. K. Diekert et al., “Proceedings of the National Academy of Sciences of the United States of America,” 1999, 96, 11552-7. Kogure et al., “Journal of Controlled Release”, 2004, 98, 317-323. Karil Ikrami et al., “YAKUGAKU ZASSHI”, 2004, 124, Suppl. 4, 113-116 pages

  An object of the present invention is to provide a lipid membrane structure capable of delivering a target substance into mitochondria and a phospholipid derivative useful for the preparation of the lipid membrane structure.

  In order to solve the above-mentioned problems, the lipid membrane structure of the present invention comprises a lipid membrane containing a membrane-fused lipid and having a membrane-permeable peptide (see claim 1). A lipid membrane containing a membrane-fusible lipid and having a membrane-permeable peptide can be efficiently fused with a mitochondrial membrane. Therefore, the lipid membrane structure holding the target substance to be delivered into the mitochondria (see claim 16) can release the target substance into the mitochondria by membrane fusion between the lipid membrane and the mitochondrial membrane, Thereby, the target substance can be delivered into the mitochondria. That is, the lipid membrane structure of the present invention can be used as a vector for intramitochondrial delivery of a target substance (see claim 17).

  In the lipid membrane structure of the present invention, the content of the membrane-fusible lipid is preferably 70% (molar ratio) or more of the total lipid amount contained in the lipid membrane (see claim 2). By setting the content of the membrane-fusible lipid in the above range, the fusion ability of the lipid membrane to the mitochondrial membrane can be improved.

  In the lipid membrane structure of the present invention, the membrane-fusible lipid is, for example, corn type lipid (see claim 3), and the corn type lipid is, for example, dioleoylphosphatidylethanolamine (claim 4). reference).

  In the lipid membrane structure of the present invention, the membrane-permeable peptide is, for example, a peptide having a membrane-permeable domain (see claim 5), and the membrane-permeable domain is preferably polyarginine (claim). 6), the polyarginine consists of, for example, 4 to 20 consecutive arginine residues (see claim 7). By using polyarginine as the membrane permeability domain, the ability of the lipid membrane to fuse with the mitochondrial membrane can be improved. Further, when the membrane permeability domain is polyarginine, the lipid membrane structure of the present invention can be transferred into the cell while maintaining its original form (in an intact state). Therefore, when the lipid membrane structure of the present invention is administered extracellularly, the lipid membrane structure of the present invention moves into the cell while retaining the target substance, and the target substance is mitochondria by membrane fusion between the lipid membrane and the mitochondrial membrane. Is released inside.

  In the lipid membrane structure of the present invention, the membrane-permeable peptide is preferably present on the surface of the lipid membrane (see claim 8). The presence of the membrane-permeable peptide on the surface of the lipid membrane can improve the fusion ability of the lipid membrane to the mitochondrial membrane.

  In the lipid membrane structure of the present invention, the lipid membrane preferably has a mitochondrial targeting signal (see claim 9). Thereby, the binding ability of the lipid membrane to the mitochondrial membrane can be improved.

In the lipid membrane structure of the present invention, the mitochondrial targeting signal is, for example, a peptide shown in the following (a) or (b) (see claim 10).
(A) a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 31 (b) one or more amino acids in the amino acid sequence of any of SEQ ID NOs: 1 to 31 are deleted, substituted, inserted or A peptide consisting of an added amino acid sequence, which has the ability to migrate to mitochondria

  In the lipid membrane structure of the present invention, the mitochondrial targeting signal is bound to a component of the lipid membrane, for example, directly or via a linker (see claim 11). In this case, it is preferable that the C terminus of the mitochondrial targeting signal is bound to a component of the lipid membrane (see claim 12). Thereby, the function of mitochondria targeting signal (mitochondrial migration ability) can be efficiently exhibited.

  In the lipid membrane structure of the present invention, the mitochondrial targeting signal is preferably present on the surface of the lipid membrane (see claim 13). Thereby, the binding ability of the lipid membrane to the mitochondrial membrane can be improved.

  In the lipid membrane structure of the present invention, the amount of the mitochondrial targeting signal is preferably 2 to 10% (molar ratio) of the total lipid amount contained in the lipid membrane (see claim 14). Thereby, the binding ability of the lipid membrane to the mitochondrial membrane can be improved.

  The lipid membrane structure of the present invention is preferably a liposome (see claim 15). When the lipid membrane structure of the present invention is a liposome, the target substance can be efficiently delivered into the mitochondria by encapsulating the target substance in the lipid membrane structure of the present invention.

  In order to solve the above problems, the present invention has the following formula: A—X—B [wherein A represents a residue of a mitochondrial targeting signal, X represents a direct bond or a linker, and B represents the remaining phospholipid. Represents a group. (See claim 18.) The phospholipid derivative of the present invention modifies a lipid membrane structure with a mitochondrial targeting signal (for example, mitochondrial targeting of the liposome surface) It is useful when modifying with a signal).

  In the phospholipid derivative of the present invention, the C terminus of the mitochondrial targeting signal is preferably bound to the phospholipid directly or via a linker (see claim 19). Thereby, the function of mitochondria targeting signal (mitochondrial migration ability) can be efficiently exhibited.

In the phospholipid derivative of the present invention, the mitochondrial targeting signal is, for example, a peptide shown in the following (a) or (b) (see claim 20).
(A) a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 31 (b) one or more amino acids in the amino acid sequence of any of SEQ ID NOs: 1 to 31 are deleted, substituted, inserted or A peptide consisting of an added amino acid sequence, which has the ability to migrate to mitochondria

  In the phospholipid derivative of the present invention, the linker is, for example, an amino acid residue, a residue of a linker peptide, or a residue of a crosslinker reagent (see claim 21).

In the phospholipid derivative of the present invention, for example, the phospholipid has an amino group, and the phospholipid is bound to the mitochondrial targeting signal or the linker via the amino group (see claim 22).
In the phospholipid derivative of the present invention, for example, the phospholipid is phosphatidylethanolamine (see claim 23).

  According to the present invention, a lipid membrane structure capable of delivering a target substance into mitochondria and a phospholipid derivative useful for the preparation of the lipid membrane structure are provided.

It is a partial sectional view showing typically an embodiment of a lipid membrane structure of the present invention. It is a figure which shows the binding activity (%) with respect to the mitochondrial membrane of an EPC type | system | group liposome. It is a figure which shows the binding activity (%) with respect to a DOPE-type liposome mitochondrial membrane. It is a figure which shows the fusion activity (FRET cancellation rate (%)) with respect to the mitochondrial membrane of an EPC-type liposome. It is a figure which shows the fusion activity (FRET cancellation rate (%)) with respect to a DOPE-type liposome mitochondrial membrane. It is a figure which shows the observation result by a confocal laser microscope. It is a figure which shows the result of a Western blot. It is a figure which shows the observation result by a confocal laser microscope. It is a figure which shows the observation result by a confocal laser microscope. It is a figure which shows the synthetic scheme of SMCC-DOPE. It is a figure which shows the result of HPLC. It is a figure which shows the result of FAB-MS. It is a figure which shows the synthetic scheme of MTS-DOPE. It is a figure which shows the result of HPLC. It is a figure which shows the result of MALDI-TOF-MS. It is a figure which shows the transfer activity to the mitochondria of a liposome. It is a figure which shows the observation result by a confocal laser microscope. It is a figure which shows the observation result by a confocal laser microscope.

Explanation of symbols

1a, 1b, 1c, 1d ... liposome 2a, 21-22b, 21-22c, 21-23d ... lipid membrane 3 ... target substance

  Hereinafter, the lipid membrane structure of the present invention will be described in detail. The “lipid membrane containing a membrane-fusible lipid and having a membrane-permeable peptide” is hereinafter referred to as “mitochondrial membrane-fusible lipid membrane”.

  As long as it has a mitochondrial membrane-fusible lipid membrane, the lipid membrane structure of the present invention includes liposomes, O / W emulsions, W / O / W emulsions, spherical micelles, string micelles, amorphous layered structures, etc. Among them, any structure may be used, but a liposome is preferable. When the lipid membrane structure of the present invention is a liposome, the target substance can be efficiently delivered into the mitochondria by encapsulating the target substance in the lipid membrane structure of the present invention.

  When the lipid membrane structure of the present invention is a liposome, it may be a multilamellar liposome (MLV), a small unilamellar vesicle (SUV), a large unilamellar vesicle (LUV), or a giant unilamellar (GUV). It may be a single membrane liposome.

  The number of mitochondrial membrane-fusible lipid membranes of the lipid membrane structure of the present invention is not particularly limited. When the lipid membrane structure of the present invention has a plurality of lipid membranes, all lipid membranes are fused with mitochondrial membranes. It may be a functional lipid membrane, or a part of the lipid membrane may be a mitochondrial membrane-fused lipid membrane.

  The size of the lipid membrane structure of the present invention is not particularly limited, but when the lipid membrane structure of the present invention is a liposome or an emulsion, the particle size is usually 50 nm to 5 μm, and in the case of a spherical micelle, the particle size is Is usually 5 to 100 nm, and in the case of string micelles or irregular layered structures, the thickness per layer is usually 5 to 10 nm, and it is preferable that a plurality of such layers are laminated.

  In the lipid membrane structure of the present invention, examples of components of the lipid membrane (whether or not it is a mitochondrial membrane-fused lipid membrane) include, for example, lipids, membrane stabilizers, antioxidants, charged substances, membranes Examples include proteins.

  The lipid is an essential constituent of the lipid membrane, and the amount of lipid contained in the lipid membrane is usually 70% (molar ratio) or more, preferably 75% (molar ratio) or more of the total amount of the substance constituting the lipid membrane, More preferably, it is 80% (molar ratio) or more. The upper limit of the amount of lipid contained in the lipid membrane is 100% of the total amount of substances constituting the lipid membrane.

Examples of lipids include phospholipids, glycolipids, sterols, saturated or unsaturated fatty acids exemplified below.
[Phospholipid]
Phosphatidylcholine (eg, dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, etc.), phosphatidylglycerol (eg, dioleoylphosphatidylglycerol, dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, Phosphatidylglycerol, distearoyl phosphatidylglycerol, etc.), phosphatidylethanolamine (eg dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidiethanol Phosphatidylserine, phosphatidylinositol, phosphatidic acid, cardiolipin, sphingomyelin, ceramide phosphorylethanolamine, ceramide phosphorylglycerol, ceramide phosphorylglycerol phosphate, 1,2-dimyristoyl-1,2-deoxyphosphatidylcholine, plasmalogen, egg yolk Lecithin, soybean lecithin, hydrogenated products thereof, etc.

[Glycolipid]
Glyceroglycolipid (for example, sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride), glycosphingolipid (for example, galactosyl cerebroside, lactosyl cerebroside, ganglioside) and the like.

[Sterol]
Animal-derived sterols (eg, cholesterol, cholesterol succinic acid, cholestanol, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol), plant-derived sterols (phytosterol) (eg, stigmasterol, sitosterol, campesterol, brassicasterol) , Sterols derived from microorganisms (eg, timosterol, ergosterol) and the like.

[Saturated or unsaturated fatty acid]
Saturated or unsaturated fatty acids having 12 to 20 carbon atoms such as palmitic acid, oleic acid, stearic acid, arachidonic acid, myristic acid and the like.

  A membrane stabilizer is an optional component of a lipid membrane that can be included to physically or chemically stabilize the lipid membrane or to regulate the fluidity of the lipid membrane. The amount of the membrane stabilizer contained is usually 30% (molar ratio) or less, preferably 25% (molar ratio) or less, more preferably 20% (molar ratio) or less of the total amount of substances constituting the lipid membrane. . Note that the lower limit of the content of the film stabilizer is zero.

  Examples of the film stabilizer include sterol, glycerin or fatty acid ester thereof. Specific examples of sterols are the same as those described above, and examples of glycerin fatty acid esters include triolein and trioctanoin.

  An antioxidant is an optional component of the lipid membrane that can be included to prevent oxidation of the lipid membrane, and the amount of antioxidant contained in the lipid membrane is the total amount of substances that make up the lipid membrane. Is usually 30% (molar ratio) or less, preferably 25% (molar ratio) or less, more preferably 20% (molar ratio) or less. In addition, the lower limit of the content of the antioxidant is 0.

  Examples of the antioxidant include tocopherol, propyl gallate, ascorbyl palmitate, butylated hydroxytoluene and the like.

  A charged substance is an optional component of the lipid membrane that can be included to impart positive charge or negative charge to the lipid membrane, and the amount of charged substance contained in the lipid membrane is the total amount of the lipid membrane. The amount is usually 30% (molar ratio) or less, preferably 25% (molar ratio) or less, more preferably 20% (molar ratio) or less. The lower limit value of the charged substance content is zero.

  Examples of the charged substance that imparts a positive charge include saturated or unsaturated aliphatic amines such as stearylamine and oleylamine; saturated or unsaturated cationic synthetic lipids such as dioleoyltrimethylammonium propane, and the like. Examples of the charged substance to be provided include dicetyl phosphate, cholesteryl hemisuccinate, phosphatidylserine, phosphatidylinositol, and phosphatidic acid.

  A membrane protein is an optional component of a lipid membrane that can be included to maintain the structure of the lipid membrane or to add functionality to the lipid membrane, and the amount of membrane protein contained in the lipid membrane Is usually 10% (molar ratio) or less, preferably 5% (molar ratio) or less, more preferably 2% (molar ratio) or less of the total amount of substances constituting the lipid membrane. Note that the lower limit of the content of membrane protein is zero.

  Examples of membrane proteins include membrane surface proteins and membrane integral proteins.

  In the lipid membrane structure of the present invention, the lipid constituting the lipid membrane (regardless of whether it is a mitochondrial membrane-fused lipid membrane), for example, blood retention function, temperature change sensitivity function, pH sensitivity function, etc. Lipid derivatives having can be used. Thereby, 1 type, or 2 or more types of functions among the said functions can be provided to a lipid membrane structure. By imparting a lipid retention function to the lipid membrane structure, the retention of the lipid membrane structure in blood can be improved, and the capture rate by reticuloendothelial tissues such as the liver and spleen can be reduced. . In addition, by providing the lipid membrane structure with a temperature change sensitive function and / or a pH sensitive function, it is possible to increase the releasability of the target substance retained in the lipid membrane structure.

  Examples of the blood-retaining lipid derivative that can impart a blood-retaining function include glycophorin, ganglioside GM1, phosphatidylinositol, ganglioside GM3, glucuronic acid derivative, glutamic acid derivative, polyglycerin phospholipid derivative, N- { Carbonyl-methoxypolyethylene glycol-2000} -1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N- {carbonyl-methoxypolyethylene glycol-5000} -1,2-dipalmitoyl-sn-glycero- 3-phosphoethanolamine, N- {carbonyl-methoxypolyethylene glycol-750} -1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N- {carbonyl-methoxypolyethylene group Cole-2000} -1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N- {carbonyl-methoxypolyethylene glycol-5000} -1,2-distearoyl-sn-glycero-3-phospho Examples include polyethylene glycol derivatives such as ethanolamine.

  Examples of the temperature change sensitive lipid derivative capable of imparting a temperature change sensitive function include dipalmitoyl phosphatidylcholine and the like, and examples of the pH sensitive lipid derivative capable of imparting a pH sensitive function include, for example, dioleoylphosphatidyl. Examples include ethanolamine.

The lipid membrane structure of the present invention can retain an antibody that specifically recognizes a cell containing a target mitochondria, an enzyme secreted by the cell, and the like. As the antibody, it is preferable to use a monoclonal antibody, and as the monoclonal antibody, one kind of monoclonal antibody having specificity for a single epitope may be used, or the specificity to various epitopes may be used. Two or more kinds of monoclonal antibodies possessed may be used in combination. As the antibody, either a monovalent antibody or a multivalent antibody may be used, and any of a natural type (intact) molecule or a fragment or derivative thereof may be used. For example, F (ab ′) ₂ , Fab ′, Fab, chimeric antibody or hybrid antibody having at least two antigens or epitope binding sites, or bispecific recombinant antibody such as quadrome, triome, interspecific hybrid antibody Anti-idiotype antibodies, and derivatives that have been chemically modified, processed, etc. can be used. In addition, by applying known cell fusion, hybridoma technology, antibody engineering, etc., an antibody obtained by synthesis or semi-synthetic technology, an antibody obtained by DNA recombination technology by applying conventional technology known from the viewpoint of antibody production, Alternatively, an antibody having neutralizing properties or an antibody having binding properties with respect to the target epitope can be used.

  The mitochondrial membrane-fusible lipid membrane contains a membrane-fusible lipid as a constituent component. The amount of the fusogenic lipid contained in the mitochondrial fusogenic lipid membrane is not particularly limited, but is usually 70% (molar ratio) or more of the total lipid amount contained in the mitochondrial fusogenic lipid membrane, preferably It is 75% (molar ratio) or more, more preferably 80% (molar ratio) or more. By setting the amount of the fusogenic lipid contained in the mitochondrial fusogenic lipid membrane within the above range, the fusion ability of the mitochondrial fusogenic lipid membrane to the mitochondrial membrane can be improved. Note that the upper limit of the amount of the fusogenic lipid contained in the mitochondrial fusogenic lipid membrane is 100% of the total amount of lipid contained in the mitochondrial fusogenic lipid membrane.

  The membrane-fusible lipid contained in the mitochondrial membrane-fusible lipid membrane is not particularly limited as long as it can be fused with the lipid membrane. The lipid membrane to which the membrane-fusible lipid can be fused is not particularly limited as long as it has a lipid bilayer structure, and examples thereof include biological membranes such as cell membranes and mitochondrial membranes, and artificial membranes such as liposome membranes. Examples of membrane-fusible lipids include corn type lipids such as dioleoylphosphatidylethanolamine. Lipids are roughly classified into three types, corn type, cylindrical type, inverted cone type, according to the proportion of polar groups and nonpolar groups. Of these, corn type lipids are: Hydrophobic group is a lipid that occupies a larger volume than hydrophilic group, and is also referred to as non-bilayer lipid (Naoto Okuni, Liposomes and Experimental Methods, pages 27-33, Hirokawa Shoten) . It is considered that the reverse micelle structure is formed in the lipid bilayer due to the cone-type lipid taking the reverse hexagonal structure in the lipid bilayer, and the formed reverse micelle structure is related to membrane fusion, membrane permeability, etc. ing.

  The mitochondrial fusogenic lipid membrane has a membrane permeable peptide. The membrane-permeable peptide is present in the mitochondrial membrane-fusible lipid membrane in a state capable of binding to the mitochondrial membrane, and is preferably present on the surface of the mitochondrial membrane-fusible lipid membrane. By allowing the membrane-permeable peptide to exist on the surface of the mitochondrial membrane-fusible lipid membrane, the fusion ability of the mitochondrial membrane-fusible lipid membrane to the mitochondrial membrane can be improved.

  When the membrane-permeable peptide is present on the surface of the mitochondrial membrane-fusible lipid membrane, it is sufficient that the membrane-permeable peptide exists at least on the outer surface of the mitochondrial membrane-fusible lipid membrane, and the membrane-permeable peptide exists on the inner surface. It may or may not exist.

  The membrane-permeable peptide that the mitochondrial membrane-fusible lipid membrane has is not particularly limited as long as it can permeate the lipid membrane. The lipid membrane through which the membrane-permeable peptide can permeate is not particularly limited as long as it has a lipid bilayer structure, and examples thereof include biological membranes such as cell membranes and mitochondrial membranes, and artificial membranes such as liposome membranes. Examples of the membrane permeable peptide include a peptide having a membrane permeability domain (PTD). Examples of the membrane-permeable domain include polyarginine, HIV-1 derived Tat (48-60) (GRKKRRQRRRPQ), HIV-1 derived Rev (34-50) (TRQARNRRRRRWRRQR), and the like. These membrane-permeable domains are rich in arginine residues and can interact electrostatically with the negatively charged mitochondrial membrane.

  The total number of amino acid residues constituting the peptide having a membrane permeable domain is not particularly limited, but is usually 4 to 50, preferably 6 to 45, and more preferably 7 to 40. The total number of amino acid residues constituting the membrane permeable domain is not particularly limited, but is usually 4 to 40, preferably 6 to 30, and more preferably 7 to 20. The polyarginine as a membrane-permeable domain is usually composed of 4 to 20, preferably 6 to 12, more preferably 7 to 10 consecutive arginine residues.

  The peptide having a membrane-permeable domain may consist of only the membrane-permeable domain, or may have an arbitrary amino acid sequence at the C-terminal and / or N-terminal of the membrane-permeable domain. The amino acid sequence added to the C-terminal and / or N-terminal of the membrane permeable domain is preferably an amino acid sequence having rigidity (for example, polyproline). Unlike polyethylene glycol (PEG), which has a soft and irregular shape, polyproline is linear and retains a certain degree of rigidity. The amino acid residue contained in the amino acid sequence added to the C-terminal and / or N-terminal of the membrane-permeable domain is preferably an amino acid residue other than acidic amino acids. This is because an acidic amino acid residue having a negative charge may electrostatically interact with a arginine residue having a positive charge and attenuate the effect of the arginine residue contained in the membrane-permeable domain.

  The amount of the membrane-permeable peptide present in the mitochondrial membrane-fusible lipid membrane is usually 4-20% (molar ratio), preferably 6-16% (molar ratio) of the total lipid amount contained in the mitochondrial membrane-fusible lipid membrane. More preferably, it is 8 to 12% (molar ratio). By setting the amount of the membrane-permeable peptide present in the mitochondrial membrane-fusible lipid membrane within the above range, the ability of the mitochondrial membrane-fusible lipid membrane to bind to the mitochondrial membrane can be improved. As a result, the membrane fusion between the mitochondrial membrane-fusible lipid membrane and the mitochondrial membrane can be efficiently induced.

  As an aspect of the presence of a membrane-permeable peptide in a mitochondrial membrane-fusible lipid membrane, for example, a membrane-permeable peptide is bound to a lipid membrane component (eg, a hydrophobic group, a hydrophobic compound, etc.), and the lipid membrane component Is retained in the mitochondrial membrane-fusible lipid membrane, and a part or all of the membrane-permeable peptide is exposed from the mitochondrial membrane-fusible lipid membrane.

  The lipid membrane component to which the membrane-permeable peptide binds is not particularly limited, and examples thereof include saturated or unsaturated fatty acid groups such as stearyl groups; cholesterol groups or derivatives thereof; phospholipids, glycolipids or sterols; long chains Aliphatic alcohols (for example, phosphatidylethanolamine, cholesterol and the like); polyoxypropylene alkyls; glycerin fatty acid esters and the like, among which a fatty acid group having 10 to 20 carbon atoms (for example, palmitoyl group, oleyl group, stearyl) Group, arachidoyl group and the like).

  The mitochondrial membrane fusogenic lipid membrane preferably has a mitochondrial targeting signal (MTS). As a result, the ability of the mitochondrial membrane-fusible lipid membrane to bind to the mitochondrial membrane can be improved, and the mitochondrial membrane-fusible lipid membrane and the mitochondrial membrane are triggered by the binding between the mitochondrial membrane-fusible lipid membrane and the mitochondrial membrane. Fusion can be induced efficiently.

  MTS is present in the mitochondrial membrane-fusible lipid membrane in a state where it can exert its ability to migrate to mitochondria, and is preferably present on the surface of the mitochondrial membrane-fusible lipid membrane (ie, exposed from the surface of the mitochondrial membrane-fusible lipid membrane). Present). By allowing MTS to be present on the surface of the mitochondrial membrane-fusible lipid membrane, the ability of the mitochondrial membrane-fusible lipid membrane to bind to the mitochondrial membrane can be improved. When MTS is present on the surface of the mitochondrial membrane-fusible lipid membrane, it is sufficient that MTS is present at least on the outer surface of the mitochondrial membrane-fusible lipid membrane, and MTS may or may not be present on the inner surface. May be.

  The mitochondrial fusogenic lipid membrane may have only one type of MTS, or may have two or more types of MTS. The amount of MTS contained in the mitochondrial membrane-fusible lipid membrane is not particularly limited, but is usually 2 to 10% (molar ratio), preferably 3 to 8%, of the total lipid amount contained in the mitochondrial membrane-fusible lipid membrane. (Molar ratio), more preferably 4 to 6% (molar ratio). By making the amount of MTS present in the mitochondrial membrane fusion lipid membrane within the above range, the binding ability of the mitochondrial membrane fusion lipid membrane to the mitochondrial membrane can be improved, and the binding between the mitochondrial membrane fusion lipid membrane and the mitochondrial membrane can be improved. As an opportunity, membrane fusion between a mitochondrial membrane-fusible lipid membrane and a mitochondrial membrane can be induced efficiently.

  MTS is a signal peptide, many are already known, and the kind thereof is not particularly limited. In the present invention, any peptide can be used as long as the MTS function is maintained, and a known MTS may be used as it is, or a known MTS is mutated (missing one or more amino acids). (Deletion, substitution, insertion or addition) may be used. MTS usually consists of 20 to 70 amino acid residues, and MTS includes those that have the ability to migrate to various regions in mitochondria such as outer mitochondrial membrane, inner mitochondrial membrane, mitochondrial intermembrane space, and mitochondrial matrix However, any MTS may be used in the present invention.

As MTS, the peptide shown to the following (a) or (b) is mentioned, for example.
(A) a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 31 (b) one or more amino acids in the amino acid sequence of any of SEQ ID NOs: 1 to 31 are deleted, substituted, inserted or A peptide consisting of an added amino acid sequence, which has the ability to migrate to mitochondria

  The peptide consisting of the amino acid sequence described in SEQ ID NO: 1 is one in which several residues on the C-terminal side of MTS of succinyl CoA synthetase derived from rat liver are deleted.

  The number of amino acids to be deleted, substituted, inserted or added in the amino acid sequence of MTS (for example, the amino acid sequences described in SEQ ID NOs: 1 to 31) is not particularly limited as long as the function of MTS is maintained. 1 to 3, preferably 1 to 2.

  The position of the amino acid to be deleted, substituted, inserted or added in the amino acid sequence of MTS (for example, the amino acid sequences described in SEQ ID NOs: 1 to 31) is not particularly limited as long as the function of MTS is maintained. The terminal side is preferable (that is, the amino acid sequence on the N-terminal side is conserved).

  After the protein with MTS is delivered to the mitochondria, MTS is cleaved from the protein, but it may not be clear exactly which region serves as MTS. In this case, a peptide containing a region located on the N-terminal side of the protein cleavage site can be used as the MTS. When the peptide used as MTS contains a region located on the C-terminal side of the protein cleavage site, the position of the amino acid to be deleted, substituted, inserted or added is C-terminal to the protein cleavage site. (In other words, the amino acid sequence on the N-terminal side of the protein cleavage site is conserved).

  The binding mode of MTS to the mitochondrial membrane-fusible lipid membrane is not particularly limited. For example, MTS is bound to a lipid membrane component directly or via a linker. Any part of the MTS may bind to the lipid membrane component, but preferably the C-terminus binds to the lipid membrane component. Thereby, the function of MTS (ability to migrate to mitochondria) can be exhibited efficiently.

  The lipid membrane constituent to which MTS binds directly or via a linker is not particularly limited, but is preferably a lipid, more preferably a phospholipid. When the lipid membrane component to which MTS binds directly or via a linker is a lipid, particularly a phospholipid, MTS can be stably and conveniently presented on the lipid membrane surface, and the MTS presented on the lipid membrane surface Easy to control density.

  Examples of lipids to which MTS binds directly or via a linker include saturated or unsaturated fatty acids having 10 to 20 carbon atoms (for example, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, etc.) And lipids having one or more residues (acyl groups), specifically, phosphatidylethanolamines, phosphatidylcholines, phosphatidylserines, phosphatidylinositols, phosphatidylglycerols, cardiolipins, sphingomyelin , Ceramide phosphorylethanolamines, ceramide phosphorylglycerols, ceramide phosphorylglycerol phosphates, deoxyphosphatidylcholines, plasmalogens and phosphatidic acids, and derivatives thereof. Among these, as described later, lipids having amino groups are preferably used. Examples of lipids having amino groups include dioleoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, Stearoylphosphatidylethanolamine, octadecenyloctadecanoylglycerophosphoethanolamine, dioleoylglycerophosphoethanolamine- (N-dodecanylamine), dioleoylglycerophosphoethanolamine- (N-hexanoylamine) ), Dipalmitoylglycerophosphoethanolamine- (N-dodecanylamine), dipalmitoylglycerophosphoethanolamine- (N-hexanoylamine), distearoylglycol Rophoethanolamine- [N-amino (polyethylene glycol) 2000], stearoyl-arachidonoyl-glycero [phospho-L-serine], octadecyl-acetyl-glycerophosphoethanolamine, alkathidylethanolamine, hydroxyalkathidyl Examples include ethanolamine, cyclic alkathidylethanolamine, cardarcaethidylethanolamine, and the like. The lipid includes a salt form. The type of salt is not particularly limited, but mineral salts such as hydrochloride and sulfate; organic acid salts such as oxalate and tartrate; alkali metal salts such as sodium salt and potassium salt; ammonium salt and the like Organic bases and the like.

  When MTS and lipid membrane constituents are directly bonded, the functional groups of MTS (including functional groups artificially introduced into MTS) and the functional groups of lipid membrane constituents (artificial to lipid membrane constituents) A functional group that has been introduced automatically). Thereby, MTS and a lipid membrane structural component couple | bond together through a covalent bond. Examples of combinations of functional groups that can form a covalent bond include amino groups / carboxyl groups, hydroxyl groups / carboxyl groups, maleimide groups / SH groups, and the like. The reaction between these functional groups can be performed according to a known method. it can.

  For example, MTS and a lipid membrane component having an amino group are directly bonded by reacting a carboxyl group at the C-terminus of MTS with an amino group of the lipid membrane component to form an amide bond. be able to. This reaction can be performed using an acid halide method, an active esterification method, or the like.

  In the acid halide method, MTS is treated with a halogenating agent in an inert solvent to form an acid halide, and then the obtained acid halide is reacted with an amino group of a lipid membrane component. Examples of the halogenating agent include thionyl halides such as thionyl chloride and thionyl bromide, sulfuryl halides such as sulfuryl chloride and sulfuryl bromide; phosphorus trihalides such as phosphorus trichloride, phosphorus tribromide and phosphorus triiodide. Phosphorus pentahalides such as phosphorus pentachloride, phosphorus pentabromide and phosphorus pentaiodide; phosphorus oxyhalides such as phosphorus oxychloride, phosphorus oxybromide and phosphorus oxyiodide can be used. . The reaction solvent may be appropriately determined and determined. For example, ethers such as diethyl ether, tetrahydrofuran and dioxane; amides such as dimethylformamide and dimethylacetamide; halogenated hydrocarbons such as dichloromethane and chloroform; acetonitrile and the like Nitriles; esters such as formic acid ethyl ester and acetic acid ethyl ester, mixed solvents thereof and the like can be used. The reaction temperature may be appropriately examined, but is usually 0 ° C. to the reflux temperature of the solvent, preferably room temperature to the reflux temperature of the solvent. In the reaction between the acid halide and the amino group of the phospholipid, an organic base such as triethylamine or pyridine may be added as necessary.

  In the active esterification method, a carboxyl group of MTS is reacted with an active esterifying agent in a solvent to form an active ester, and then reacted with an amino group of a lipid membrane constituent. Examples of the active esterifying agent include N-hydroxy compounds such as N-hydroxysuccinimide, 1-hydroxybenzotriazole, N-hydroxy-5-norbornene-2,3-dicarboximide; 1,1′- Diimidazoles such as oxalyldiimidazole and N, N′-carbonyldiimidazole; disulfides such as 2,2′-dipyridyldisulfide; succinic compounds such as N, N′-disuccinimidyl carbonate; Phosphinic chloride compounds such as N, N′-bis (2-oxo-3-oxazolidinyl) phosphinic chloride; N, N′-disuccinimidyl oxalate, N, N′-diphthalimide oxalate N, N′-bis (norbornenyl succinimidyl) oxalate, 1,1′-bis (benzo Use oxalate compounds such as zotriazolyl) oxalate, 1,1′-bis (6-chlorobenzotriazolyl) oxalate, 1,1′-bis (6-trifluoromethylbenzotriazolyl) oxalate, and the like. Can do. The reaction solvent may be appropriately determined and determined. For example, halogenated hydrocarbons such as methylene chloride and chloroform; ethers such as diethyl ether and tetrahydrofuran; amides such as dimethylformamide and dimethylacetamide; benzene, toluene, Aromatic hydrocarbons such as xylene; esters such as ethyl acetate; mixed solvents thereof and the like can be used. The reaction of the obtained active ester with the amino group of the lipid membrane component is carried out by using a condensing agent, for example, azodicarboxylic acid di-lower alkyl-triphenylphosphine such as diethyl-triphenylphosphine asodicarboxylate; N-ethyl-5 -N-lower alkyl-5-arylisoxazolium-3'-sulfonates such as phenylisoxazolium-3'-sulfonate; oxydiformates such as diethyloxydiformate; N, N'- N, N'-dicycloalkylcarbodiimides such as dicyclohexylcarbodiimide; diheteroaryl diselenides such as di-2-pyridyl diselenide; triarylphosphines such as triphenylphosphine; p-nitrobenzenesulfonyltriazolide Arylsulfonyl triazolides such as 2-chloro; 2-halo-1-lower alkylpyridinium halides such as 1-methylpyridinium iodide; diarylphosphoryl azides such as diphenylphosphoryl azide; imidazole derivatives such as N, N′-carbodiimidazole; 1-hydroxybenzotriazole and the like Benzotriazole derivatives; dicarboximide derivatives such as N-hydroxy-5-norbornene-2,3-dicarboximide; carbodiimide derivatives such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide; 1-propanephosphonic acid What is necessary is just to perform in presence of phosphonic acid cyclic anhydrides, such as cyclic anhydride. The reaction temperature may be appropriately examined, but is preferably around room temperature.

  When MTS and a lipid membrane component are bound via a linker, the functional group of the lipid membrane component (including a functional group artificially introduced into the lipid membrane component) and the functional group of the linker ( And a functional group that the MTS has (including a functional group that is artificially introduced to the MTS) and a functional group that the linker has (the linker contains a functional group that is artificially introduced to the linker). It contains a functional group introduced artificially). As a result, the lipid membrane component and the linker are bound via a covalent bond, and the MTS and the linker are bound via a covalent bond. Examples of combinations of functional groups that can form a covalent bond include amino groups / carboxyl groups, hydroxyl groups / carboxyl groups, maleimide groups / SH groups, and the like. The reaction between these functional groups can be performed according to a known method. it can. When MTS and a lipid membrane component are bound via a linker, the lipid membrane component and the linker may be reacted and then MTS may be reacted, or after the MTS and the linker are reacted, the lipid membrane Components may be reacted.

  Examples of the linker include amino acid residues, linker peptides, and polyvalent (for example, divalent) crosslinker reagents.

  The number of amino acid residues constituting the linker peptide is not particularly limited, but is usually 2 to 10, preferably 2 to 8. The amino acid sequence of the linker peptide may be any amino acid sequence, but the amino acid residues contained in the amino acid sequence of the linker peptide are preferably amino acid residues other than acidic amino acids. This is because an acidic amino acid residue having a negative charge may electrostatically interact with a arginine residue having a positive charge and attenuate the effect of the arginine residue contained in the membrane-permeable domain.

  The polyvalent crosslinker reagent is not particularly limited, and examples thereof include a divalent crosslinker reagent having a reaction directivity with an amino group and an SH group, and a reaction directivity with an amino group and an SH group. Examples of the divalent crosslinker reagent having a include the following compounds.

  The polyvalent crosslinker reagent can bind MTS and the lipid membrane constituent by reacting with a functional group contained in MTS and a functional group of the lipid membrane constituent, for example. Thus, the following formula (1): M-XL [where M represents a residue of MTS, X represents a residue of a crosslinker reagent, and L represents a residue of a lipid membrane component. ] The conjugate | bonded_body of MTS and a lipid membrane structural component represented by this is formed.

In addition, the multivalent cross-linker reagent can bind the linker peptide and the lipid membrane constituent by reacting with a functional group contained in the linker peptide and a functional group of the lipid membrane constituent, for example. And MTS and a lipid membrane structural component can be combined by amide-bonding the carboxyl group or amino group which the linker peptide couple | bonded with the lipid membrane structural component has, and the amino group or carboxyl group which MTS has. Thus, the following equation _{(2): M 1 -NH-} CO-Y 1 -X 1 -L 1 or the following formula _{(3): M 1 -CO-} NH-Y 1 -X 1 -L 1 [ where, M ₁ represents a residue of MTS, X ₁ represents a residue of a crosslinker reagent, Y ₁ represents a residue of a linker peptide, and L ₁ represents a residue of a lipid membrane component. ] The conjugate | bonded_body of MTS and a lipid membrane structural component represented by this is formed. Formulas (2) and (3) clearly show the binding mode in the conjugate, and “—NH—CO—” in Formula (2) has the amino group and linker peptide of MTS. It is an amide bond with a carboxyl group, and “—CO—NH—” in Formula (3) is an amide bond between a carboxyl group of MTS and an amino group of a linker peptide.

Examples of the divalent crosslinker reagent having reaction directivity with an amino group and an SH group include, for example, an SH group possessed by a cysteine residue contained in MTS (preferably a cysteine residue located at the C-terminus of MTS), and a lipid. By reacting with the amino group of the membrane component, MTS and the lipid membrane component can be combined. Thus, the following formula (4): M ₂ —S—X ₂ —NH—L ₂ [wherein M represents a residue of MTS, X ₂ represents a residue of a crosslinker reagent, and L ₂ represents a lipid membrane. Represents a constituent residue. ] The conjugate | bonded_body of MTS and a lipid membrane structural component represented by this is formed. Note that equation (4), which has clearly shows the binding modes in the coupling body, _"- S-X 2 -", the functional groups of the SH groups and cross linker reagent having the MTS (e.g. maleimide group) “—X ₂ —NH—” is a combination of a functional group (for example, —COOR [R represents a leaving group]) possessed by a crosslinker reagent and an amino group possessed by a lipid membrane component. Formed by reaction.

In addition, a divalent crosslinker reagent having reaction directivity with an amino group and an SH group, for example, reacts with an SH group possessed by a cysteine residue contained in the linker peptide and an amino group possessed by a lipid membrane component. Thus, the linker peptide and the lipid membrane constituent can be bound. And MTS and a lipid membrane structural component can be combined by amide-bonding the carboxyl group or amino group which the linker peptide couple | bonded with the lipid membrane structural component has, and the amino group or carboxyl group which MTS has. Thus, the following formula (5): M ₃ —NH—CO—Y ₃ —SX ₃ —NH—L ₃ or the following formula (6): M ₃ —CO—NH—Y ₃ —SX ₃ —NH -L ₃ [wherein M ₃ represents a residue of MTS, X ₃ represents a residue of a crosslinker reagent, Y ₃ represents a residue of a linker peptide, and L ₃ represents a residue of a lipid membrane component] Represents. ] The conjugate | bonded_body of MTS and a lipid membrane structural component represented by this is formed. Formulas (5) and (6) clearly show the binding mode in the conjugate, and “—NH—CO—” in Formula (5) has an amino group and linker peptide that MTS has. It is an amide bond with a carboxyl group, and “—CO—NH—” in the formula (6) is an amide bond between the carboxyl group of the MTS and the amino group of the linker peptide, and the formulas (5) and (6) “—S—X ₃ —” is formed by the reaction of the SH group of MTS and the functional group (eg, maleimide group) of the crosslinker reagent, and “—X ₃ —NH—” It is formed by a reaction between a functional group (for example, —COOR [R represents a leaving group]) and an amino group of a lipid membrane component.

  In the above reaction, the intended reaction may be efficiently performed by introducing a protecting group. For the introduction of the protecting group, for example, “Protective Groups in Organic Synthesis” (PGGMs Wuts and T. Green, 3rd edition 1999 Wiley, John & Sons) may be referred to.

  The lipid membrane structure of the present invention preferably retains a target substance to be delivered into mitochondria. The lipid membrane structure of the present invention includes, for example, the inside of the lipid membrane structure (for example, voids formed inside the lipid membrane structure), the surface of the lipid membrane, in the lipid membrane, in the lipid membrane layer, in the lipid membrane layer. The target substance can be held on the surface or the like. When the lipid membrane structure is a fine particle such as a liposome, for example, the target substance can be encapsulated inside the fine particle.

  The type of the target substance is not particularly limited and includes, for example, drugs, nucleic acids, peptides, proteins, sugars or complexes thereof, and can be appropriately selected according to the purpose of diagnosis, treatment, prevention, etc. it can. When the target substance is a substance for the purpose of diagnosis, treatment, prevention, etc. of a disease, the lipid membrane structure holding the target substance can be used as a component of a pharmaceutical composition. The “nucleic acid” includes analogs or derivatives thereof (for example, peptide nucleic acid (PNA), phosphorothioate DNA, etc.) in addition to DNA or RNA. Further, the nucleic acid may be either single-stranded or double-stranded, and may be either linear or circular.

  When the target substance is a gene, the lipid membrane structure preferably has a compound having a gene introduction function from the viewpoint of improving the efficiency of gene introduction into the cell. Examples of the compound having a gene transfer function include O, O′-N-didodecanoyl-N- (α-trimethylammonioacetyl) -diethanolamine chloride, O, O′-N-ditetradecanoyl-N- (α -Trimethylammonioacetyl) -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, nor [α-trimethylammonioacetyl] -didodecyl-D -Glutamate, dimethyl dioctadecyl ammonium 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, etc. These compounds having a gene transfer function are contained in the lipid membrane structure (for example, the lipid membrane structure). Voids formed therein, in the lipid membrane, in the lipid membrane surface, in the lipid membrane layer, or on the lipid membrane layer surface (bonded).

  The target substance is preferably held as an aggregate of the target substance in the lipid membrane structure (particularly the inside of the lipid membrane structure). Thereby, the target substance can be efficiently delivered into the mitochondria.

  The aggregate of the target substance may be composed of only the target substance, or may contain a substance other than the target substance (for example, a carrier that holds the target substance).

  When the target substance is positively charged, an aggregate of the target substance can be prepared by, for example, electrostatically binding the target substance and the anionic substance to form a complex. When the target substance is negatively charged, for example, an aggregate of the target substance can be prepared by electrostatically binding the target substance and the cationic substance to form a complex. When the target substance is not negatively or positively charged, the target substance and a predetermined carrier are combined in an appropriate manner (for example, physical adsorption, hydrophobic bond, chemical bond, etc.) to form a complex. Thus, an aggregate of the target substance can be prepared. When complexing, an aggregate of the target substance that is positively or negatively charged as a whole can be prepared by adjusting the mixing ratio of the target substance and the cationic substance or anionic substance.

  When the target substance is a nucleic acid, an aggregate of nucleic acids can be prepared by electrostatically binding the nucleic acid and the cationic substance to form a complex. By adjusting the mixing ratio of the nucleic acid and the cationic substance at the time of complexing, an aggregate of nucleic acids that are charged positively or negatively as a whole can be prepared.

  The cationic substance that can be used in preparing the aggregate of the target substance is not particularly limited as long as it is a substance having a cationic group in the molecule. Examples of the cationic substance include cationic lipids (for example, Lipofectamine (manufactured by Invitrogen)); polymers having a cationic group; polylysine, polyarginine, homopolymer of basic amino acids such as lysine and arginine copolymer, and the like. Polymers or copolymers or derivatives thereof (eg stearylated derivatives); polycationic polymers such as polyethyleneimine, poly (allylamine), poly (diallyldimethylammonium chloride), glucosamine; protamine or derivatives thereof (eg protamine sulfate); Although chitosan etc. are mentioned, Stearyl-ized polyarginine is especially preferable among these. The number of arginine residues constituting the polyarginine is usually 4 to 20, preferably 6 to 12, and more preferably 7 to 10. The number of cationic groups possessed by the cationic substance is not particularly limited, but is preferably 2 or more. The cationic group is not particularly limited as long as it can be positively charged. For example, an amino group; a monoalkylamino group such as a methylamino group or an ethylamino group; a dialkylamino group such as a dimethylamino group or a diethylamino group; Group; a guanidino group and the like.

  The anionic substance that can be used in preparing the aggregate of the target substance is not particularly limited as long as it is a substance having an anionic group in the molecule. Examples of anionic substances include anionic lipids; polymers having anionic groups; homopolymers or copolymers of acidic amino acids such as polyaspartic acid or derivatives thereof; xanthan gum, carboxyvinyl polymer, carboxymethylcellulose polystyrene sulfone. Polyanionic polymers such as acid salts, polysaccharides and carrageenans can be used. The number of anionic groups contained in the anionic substance is not particularly limited, but is preferably 2 or more. The anionic group is not particularly limited as long as it can be negatively charged. For example, a functional group having a terminal carboxyl group (for example, a succinic acid residue, a malonic acid residue, etc.), a phosphoric acid group, a sulfuric acid group, etc. Can be mentioned.

  The form of the lipid membrane structure of the present invention is not particularly limited, and examples thereof include a dried mixture form, a dispersed form in an aqueous solvent, a dried or frozen form, and the like. The membrane structure can be produced using a known method such as a hydration method, an ultrasonic treatment method, an ethanol injection method, an ether injection method, a reverse phase evaporation method, a surfactant method, or a freezing / thawing method. it can.

Hereinafter, although the manufacturing method of the lipid membrane structure of each form is demonstrated, the form of the lipid membrane structure of this invention and its manufacturing method are not limited to these.
For the lipid membrane structure in the form of a dried mixture, for example, all the components of the lipid membrane structure are once dissolved in an organic solvent such as chloroform and then vacuum-dried by an evaporator or spray-dried by a spray dryer. Can be manufactured by.

  A lipid membrane structure in a form dispersed in an aqueous solvent is added to a lipid membrane structure in the form of a dried mixture and then emulsified by an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like. Can be manufactured. A lipid membrane structure dispersed in an aqueous solvent can be produced by a method well known as a method for producing liposomes, such as a reverse phase evaporation method. When it is desired to control the size of the lipid membrane structure, a membrane filter having a uniform pore diameter may be used to perform extrusion (extrusion filtration) under high pressure.

  The composition of the aqueous solvent (dispersion medium) is not particularly limited. For example, a buffer solution such as a phosphate buffer solution, a citrate buffer solution, and a phosphate buffered physiological saline solution, a physiological saline solution, and a cell culture solution. And the like. These aqueous solvents (dispersion media) can stably disperse the lipid membrane structure, but in order to disperse the lipid membrane structure more stably, monosaccharides (eg, glucose, galactose, mannose, fructose, inositol) , Ribose, xylose, etc.), disaccharides (eg, lactose, sucrose, cellobiose, trehalose, maltose, etc.), trisaccharides (eg, raffinose, merezinose, etc.), polysaccharides (eg, cyclodextrin, etc.), sugar alcohols (eg, , Erythritol, xylitol, sorbitol, mannitol, maltitol, etc.) or an aqueous solution thereof; glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, poly Ji glycol, ethylene glycol monoalkyl ethers, diethylene glycol monoalkyl ethers, polyhydric alcohols or may be added an aqueous solution thereof such as 1,3-butylene glycol. In order to stably store a lipid membrane structure in a form dispersed in an aqueous solvent (dispersion medium) for a long period of time, from the viewpoint of physical stability (for example, to prevent aggregation), the aqueous solution (dispersion medium) It is preferable to eliminate the electrolyte as much as possible. From the viewpoint of chemical stability of the lipid, the pH of the aqueous solvent (dispersion medium) is set from weakly acidic to near neutral (pH 3.0 to 8.0), or dissolved oxygen is removed by nitrogen bubbling. It is preferable.

  A lipid membrane structure in a form obtained by drying or freezing a lipid membrane structure dispersed in an aqueous solvent is obtained by drying or freezing a lipid membrane structure dispersed in an aqueous solvent by a known method such as freeze drying or spray drying. Can be manufactured. When a lipid membrane structure dispersed in an aqueous solvent is once produced and then dried, the lipid membrane structure can be stored for a long period of time. Since the lipid mixture is well hydrated, there is an advantage that the target substance can be efficiently retained in the lipid membrane structure.

  When freeze-drying or spray-drying, for example, monosaccharides (for example, glucose, galactose, mannose, fructose, inositol, ribose, xylose, etc.), disaccharides (for example, lactose, sucrose, cellobiose) , Trehalose, maltose, etc.), trisaccharides (eg, raffinose, merezinose, etc.), polysaccharides (eg, cyclodextrin, etc.), sugar alcohols (eg, erythritol, xylitol, sorbitol, mannitol, maltitol, etc.) or sugars thereof By adding an aqueous solution, the lipid membrane structure can be stored stably for a long period of time. When freezing, for example, the sugar or an aqueous solution thereof; glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol By adding a polyhydric alcohol such as monoalkyl ether, diethylene glycol monoalkyl ether, 1,3-butylene glycol or an aqueous solution thereof, the lipid membrane structure can be stably stored for a long period of time. A combination of sugar and polyhydric alcohol may be added to the aqueous solvent (dispersion medium). The concentration of sugar or polyhydric alcohol in the aqueous solvent (dispersion medium) in which the lipid membrane structure is dispersed is not particularly limited, but the concentration of sugar is preferably 2 to 20% (W / V), and 5 to 10%. More preferably, it is (W / V). In addition, the concentration of the polyhydric alcohol is preferably 1 to 5% (W / V), and more preferably 2 to 2.5% (W / V). When a buffer solution is used as the aqueous solvent (dispersion medium), the concentration of the buffering agent is preferably 5 to 50 mM, and more preferably 10 to 20 mM. The concentration of the lipid membrane structure in the aqueous solvent (dispersion medium) is not particularly limited, but is preferably 0.1 mM to 500 mM in terms of the concentration of the total amount of lipid contained in the lipid membrane structure, and is 1 mM to 100 mM. More preferably it is.

  The lipid membrane structure holding the antibody can be produced by producing the lipid membrane structure, adding the antibody, and binding the antibody to the surface of the lipid membrane. In addition, the lipid membrane structure holding the antibody is prepared by adding a lipid derivative capable of reacting with the antibody and a mercapto group in the antibody after the production of the lipid membrane structure, and binding the antibody to the surface of the lipid membrane. Can be manufactured.

  The form of the pharmaceutical composition containing the lipid membrane structure holding the target substance is not particularly limited. For example, the form of a dried mixture, the form dispersed in an aqueous solvent, or the form dried or frozen can be used. The pharmaceutical composition of each form can be produced in the same manner as the lipid membrane structure of each form described above.

  A pharmaceutical composition in the form of a dried mixture is prepared by, for example, dissolving a component of a lipid membrane structure and a target substance once in an organic solvent such as chloroform to obtain a mixture and then drying or spraying the mixture under reduced pressure using an evaporator. It can manufacture by attaching | subjecting to spray drying with a dryer.

  As a method for producing a pharmaceutical composition in which a lipid membrane structure holding a target substance is dispersed in an aqueous solvent, a plurality of methods are known as follows, and the retention mode of the target substance in the lipid membrane structure is as follows. The production method can be appropriately selected according to the properties of the mixture.

[Production Method 1]
In the production method 1, the components of the lipid membrane structure and the target substance are once dissolved in an organic solvent such as chloroform to obtain a mixture, which is then subjected to reduced pressure drying using an evaporator or spray drying using a spray dryer. To produce a dry mixture containing the lipid membrane structure and the target substance, and then, after adding an aqueous solvent thereto, emulsification by an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, etc. Is a method for producing a pharmaceutical composition in which a lipid membrane structure holding a target substance is dispersed in an aqueous solvent. In order to control the size (particle diameter) of the lipid membrane structure, extrusion (extrusion filtration) may be performed under a high pressure using a membrane filter having a uniform pore diameter. In the production method 1, in order to produce a dry mixture containing a lipid membrane structure and a target substance, it is necessary to dissolve the constituent component of the lipid membrane structure and the target substance in an organic solvent. There is an advantage that the interaction between the target substance and the target substance can be maximized. That is, even when the lipid membrane structure has a layered structure, the target substance can enter even into the multilayer, and there is an advantage that the retention rate of the target substance in the lipid membrane structure can be improved. is there.

[Production Method 2]
Production method 2 retains the target substance by dissolving the constituent components of the lipid membrane structure once in an organic solvent and then emulsifying by adding an aqueous solvent containing the target substance to the dried product obtained by distilling off the organic solvent. This is a method for producing a pharmaceutical composition in which a lipid membrane structure is dispersed in an aqueous solvent. In order to control the size (particle diameter) of the lipid membrane structure, extrusion (extrusion filtration) may be performed under a high pressure using a membrane filter having a uniform pore diameter. Production method 2 is difficult to dissolve in an organic solvent, but can be applied to a target substance that can be dissolved in an aqueous solvent. The production method 2 has an advantage that the target substance can also be retained in the inner aqueous phase when the lipid membrane structure is a liposome.

[Production Method 3]
In the production method 3, a lipid membrane structure holding a target substance is added to a lipid membrane structure such as a liposome, emulsion, micelle, or layered structure dispersed in an aqueous solvent by adding an aqueous solvent containing the target substance. This is a method for producing a pharmaceutical composition dispersed in an aqueous solvent. Production method 3 can be applied to a water-soluble target substance. Since the production method 3 is a method of adding the target substance from the outside to the already produced lipid membrane structure, when the target substance is a polymer, the target substance cannot enter the lipid membrane structure, and the lipid There is a possibility that the existence mode existing (bonded) on the surface of the membrane structure is taken. When the lipid membrane structure is a liposome, a sandwich structure in which the target substance is sandwiched between liposome particles (generally called “complex” or “complex”) may be formed by the production method 3. Are known. In production method 3, since an aqueous dispersion of a lipid membrane structure alone is produced in advance, it is not necessary to consider the decomposition of the target substance during emulsification, and the size (particle diameter) can be easily controlled. Therefore, compared with the manufacturing method 1 and the manufacturing method 2, the pharmaceutical composition of the form in which the lipid membrane structure holding the target substance is dispersed in the aqueous solvent can be easily manufactured.

[Production Method 4]
In the production method 4, an aqueous solvent containing a target substance is added to a dried product obtained by drying a lipid membrane structure dispersed in an aqueous solvent, whereby the lipid membrane structure holding the target substance is converted into an aqueous solvent. A method for producing a pharmaceutical composition in a dispersed form. The production method 4 can be applied to a water-soluble target substance similarly to the production method 3. The difference between the production method 4 and the production method 3 resides in the manner of existence of the lipid membrane structure and the target substance. In the production method 4, the lipid membrane structure dispersed in an aqueous solvent is once produced and then dried. Since the dried product is produced, the lipid membrane structure is present in a solid state as a fragment of the lipid membrane at this stage. In order to make this lipid membrane fragment exist in a solid state, it is preferable to use a solvent in which sugar or an aqueous solution thereof, preferably sucrose or an aqueous solution thereof, or lactose or an aqueous solution thereof is added to an aqueous solvent as described above. . When an aqueous solvent containing the target substance is added, the lipid membrane fragments that existed in the solid state begin to hydrate quickly with the invasion of water, and the lipid membrane structure is reconstructed. A lipid membrane structure in a form retained within the structure is produced.

  In the production method 4, since an aqueous dispersion of the lipid membrane structure alone is produced in advance, it is not necessary to consider the decomposition of the target substance during emulsification, and the size (particle diameter) can be easily controlled. Therefore, compared with the manufacturing method 1 and the manufacturing method 2, the pharmaceutical composition of the form in which the lipid membrane structure holding the target substance is dispersed in the aqueous solvent can be easily manufactured. In addition, once freeze-dried or spray-dried, it is easy to guarantee the storage stability as a preparation (pharmaceutical composition), and the size (particle diameter) is the same even if the dried preparation is rehydrated with an aqueous solution of the target substance. There are advantages such that the target substance can easily be retained inside the lipid membrane structure even if it is a polymer target substance. Therefore, when the target substance is a polymer, in the production method 3, the target substance may not enter the lipid membrane structure and may be present on the surface of the lipid membrane structure. 4 differs greatly in this respect.

  As another method for producing a pharmaceutical composition in which a lipid membrane structure holding a target substance is dispersed in an aqueous solvent, a method well known as a method for producing liposomes, such as a reverse phase evaporation method, is used. Can be adopted. In order to control the size (particle diameter) of the lipid membrane structure, extrusion (extrusion filtration) may be performed under a high pressure using a membrane filter having a uniform pore diameter. Examples of the method for drying a dispersion in which a lipid membrane structure holding a target substance is dispersed in an aqueous solvent include freeze drying and spray drying. The aqueous solvent includes the above sugar or an aqueous solution thereof, preferably sucrose or It is preferable to use the aqueous solution, or lactose or a solvent to which the aqueous solution is added. As a method of freezing a dispersion liquid in which a lipid membrane structure holding a target substance is dispersed in an aqueous solvent, a normal freezing method may be mentioned. As the aqueous solvent, the sugar or an aqueous solution thereof, or the polyhydric alcohol or the aqueous solution thereof may be used. It is preferable to use a solvent to which an aqueous solution is added.

  When the aggregate of the target substance is positively charged as a whole, if an anionic lipid (acidic lipid) is used as the lipid constituting the lipid membrane, the aggregate of the target substance is retained and the lipid membrane is efficiently produced If the aggregate of the target substance is negatively charged as a whole, if the cationic lipid (basic lipid) is used as the lipid constituting the lipid membrane, the lipid in which the aggregate of the target substance is retained A membrane structure can be manufactured efficiently. Examples of the anionic lipid (acid lipid) include cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-succinylphosphatidylethanolamine (N-succinyl PE), phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, phosphatidylethylene glycol, cholesterol succinate Examples of cationic lipids (basic lipids) include dioctadecyldimethylammonium chloride (DODAC), N- (2,3-dioleyloxy) propyl-N, N, N- Trimethylammonium (N- (2,3-dioleoyloxy) propyl-N, N, N-trimethyla monido: DOTMA), didodecylammonium bromide (DDAB), 1,2-dioleoyloxy-3-trimethylammoniopropane (DOTAP), 3β-N- (N ′, N ′,-dimethyl-aminoethane) -carbamol cholesterol (3β-N- (N ′, N ′,-dimethyl-aminoethane) -carbamol cholesterol: DC-Chol), 1,2-dimyristoyloxypropyl -3-Dimethylhydroxyethylammonium (1,2-dimethylhydroxypropyl-3-dimethylhydroxylity ammonium: DMRIE), 2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanamium trifluoroacetate (2,3-dioyloxy-N- [ 2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanamine trifluoroacetate (DOSPA), O, O′-N-didodecanoyl-N- (α-trimethylammonioacetyl) -diethanolamine chloride, O, O′-N Ditetradecanoyl-N- (α-trimethylammonioacetyl) -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) Examples include aminomethane bromide, nor [α-trimethylammonioacetyl] -didodecyl-D-glutamate, dimethyldioctadecylammonium bromide, and the like.

  In the lipid membrane structure of the present invention, when the mitochondrial membrane-fusible lipid membrane binds to the mitochondrial membrane via a membrane-permeable peptide, this triggers membrane fusion between the mitochondrial membrane-fusible lipid membrane and the mitochondrial membrane. The target substance retained in the mitochondrial membrane-fusible lipid membrane is released into the mitochondria. As a result, the target substance is delivered into the mitochondria. Therefore, the lipid membrane structure of the present invention can be used as a vector for intramitochondrial delivery of a target substance.

  The mitochondria targeted by the lipid membrane structure of the present invention may be mitochondria isolated from cells or may be mitochondria present in cells. The biological species from which mitochondria targeted by the lipid membrane structure of the present invention are derived is not particularly limited, and examples thereof include animals, plants, microorganisms, etc., but animals are preferred, and mammals are more preferred. Examples of mammals include humans, monkeys, cows, sheep, goats, horses, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, and the like. The type of cells containing mitochondria targeted by the lipid membrane structure of the present invention is not particularly limited, and examples include somatic cells, germ cells, stem cells, or cultured cells thereof.

  When the mitochondria targeted by the lipid membrane structure of the present invention are mitochondria present in the cell, the lipid membrane structure of the present invention is the most of the lipid membrane structure in which the mitochondrial membrane-fused lipid membrane has migrated into the cell. It is preferable to be configured to be located in the outer layer. Thereby, the mitochondrial membrane-fusible lipid membrane can be efficiently membrane-fused with mitochondria present in the cell.

  As shown in FIG. 1 (a), a liposome which is an embodiment of a lipid membrane structure having a configuration in which a mitochondrial membrane-fusible lipid membrane is located in the outermost layer of a lipid membrane structure transferred into a cell. A single membrane liposome 1a comprising a lipid membrane 2a and a target substance 3 encapsulated inside the lipid membrane 2a, wherein the lipid membrane 2a is a mitochondrial membrane-fusible lipid membrane, and is formed on the surface of the lipid membrane 2a. Examples thereof include single membrane liposome 1a in which the existing membrane-permeable peptide has polyarginine as a membrane-permeable domain (preferably the membrane-permeable peptide is composed of polyarginine). Single-membrane liposome 1a can be transferred from the outside of the cell into the cell while maintaining its original form (in an intact state) via a membrane-permeable peptide present on the surface of lipid membrane 2a. After translocation into the cell, when the lipid membrane 2a binds to the mitochondrial membrane via the membrane-permeable peptide on its surface, this triggers membrane fusion between the lipid membrane 2a and the outer membrane of the mitochondria, The target substance 3 enclosed inside the lipid membrane 2a is released into the mitochondria.

  In the single-membrane liposome 1a, the lipid membrane 2a preferably has MTS on the surface. Thereby, the binding ability of the lipid membrane 2a to the mitochondrial membrane can be improved, and the membrane fusion between the lipid membrane 2a and the mitochondrial membrane can be efficiently induced by the binding between the lipid membrane 2a and the mitochondrial membrane. . Moreover, when the lipid membrane 2a has MTS on the surface, the liposome that has migrated into the cell can be selectively migrated to the mitochondria.

  As a liposome which is another embodiment of the lipid membrane structure having a configuration in which the mitochondrial membrane-fusible lipid membrane is located in the outermost layer of the lipid membrane structure transferred into the cell, as shown in FIG. A bilayer liposome 1b comprising a lipid membrane 21b, a lipid membrane 22b located outside the lipid membrane 21b, and a target substance 3 encapsulated inside the lipid membrane 21b, wherein the lipid membranes 21b and 22b Is a mitochondrial membrane-fusible lipid membrane, and the membrane-permeable peptide present on the surface of the lipid membrane 22b has polyarginine as the membrane-permeable domain (preferably the membrane-permeable peptide consists of polyarginine). Is mentioned. The bilamellar liposome 1b can be transferred from the outside of the cell into the cell while maintaining its original form (in an intact state) via the membrane-permeable peptide present on the surface of the lipid membrane 22b. After transfer into the cell, when the lipid membrane 22b binds to the mitochondrial outer membrane via a membrane-permeable peptide on its surface, this triggers membrane fusion between the lipid membrane 22b and the mitochondrial outer membrane. The liposomes cross the mitochondrial outer membrane. In the liposome that has passed through the mitochondrial outer membrane, the lipid membrane 22b disappears due to membrane fusion with the mitochondrial outer membrane, but the lipid membrane 21b is retained. After passing through the outer mitochondrial membrane, when the lipid membrane 21b binds to the inner mitochondrial membrane via a membrane-permeable peptide on its surface, the membrane fusion between the lipid membrane 21b and the inner mitochondrial membrane is triggered. The target substance 3 encapsulated inside the lipid membrane 21b is released into the mitochondria.

  In the bilamellar liposome 1b, the lipid membranes 21b and 22b preferably have MTS on the surface. As a result, the binding ability of the lipid membranes 21b and 22b to the mitochondrial membrane can be improved, and the membrane fusion between the lipid membranes 21b and 22b and the mitochondrial membrane is efficiently triggered by the binding between the lipid membranes 21b and 22b and the mitochondrial membrane. It can be induced well. Moreover, since the lipid membrane 22b has MTS on the surface, the liposome that has migrated into the cell can be selectively migrated to the mitochondria.

  As a liposome which is another embodiment of the lipid membrane structure having a configuration in which the mitochondrial membrane-fusible lipid membrane is located in the outermost layer of the lipid membrane structure transferred into the cell, as shown in FIG. And a bilayer liposome 1c comprising a lipid membrane 21c, a lipid membrane 22c located outside the lipid membrane 21c, and a target substance 3 enclosed inside the lipid membrane 21c, wherein the lipid membrane 21c is a mitochondrion An example of the membrane fusion lipid membrane is a bilamellar liposome 1c in which the lipid membrane 22c contains a membrane fusion lipid. Examples of the membrane-fusion lipid contained in the lipid membrane 22c include neutral lipids such as dioleoylphosphatidylethanolamine; anionic lipids (acid lipids) such as cholesterol succinic acid and cardiolipin. The amount of the fusogenic lipid contained is usually 50% (molar ratio) or more, preferably 60% (molar ratio) or more, more preferably 70% (molar ratio) or more of the total lipid content contained in the lipid membrane 22c. It is. The upper limit of the content of membrane-fusible lipid is 100%.

  The bilamellar liposome 1c can be transferred from the outside of the cell into the cell via endocytosis. The bilayer liposome 1c transferred into the cell via endocytosis is taken into the endosome, but can escape from the endosome by membrane fusion between the endosomal membrane and the lipid membrane 22c. In the liposome escaped from the endosome, the lipid membrane 22c disappears due to membrane fusion with the endosomal membrane, but the lipid membrane 21c is retained. After escape from the endosome, the target substance 3 encapsulated inside the lipid membrane 21c is released into the mitochondria in the same manner as in the case of the single membrane liposome 1a.

  The bilayer liposome 1c can be transferred from the outside of the cell into the cell through membrane fusion between the lipid membrane 22c and the cell membrane. In the liposome that has migrated into the cell, the lipid membrane 22c disappears due to membrane fusion with the cell membrane, but the lipid membrane 21c is retained. After moving into the cell, the target substance 3 encapsulated inside the lipid membrane 21c is released into the mitochondria as in the case of the single membrane liposome 1a.

  In the bilamellar liposome 1c, the amount of anionic lipid contained in the lipid membrane 22c is 15% (molar ratio) or more, preferably 20% (molar ratio) or more of the amount of membrane-fused lipid contained in the lipid membrane 22c. When the endosome changes to acidic (pH 5.5 to 6.5), or by bringing the bilayer liposome 1c into contact with cells under acidic conditions (pH 5.5 to 6.5). The lipid membrane 22c can be efficiently fused with the endosomal membrane or cell membrane.

  In the bilamellar liposome 1c, the lipid membrane 21c preferably has MTS on the surface. Thereby, the binding ability of the lipid membrane 21c to the mitochondrial membrane can be improved, and the membrane fusion between the lipid membrane 21c and the mitochondrial membrane can be efficiently induced by the binding between the lipid membrane 21c and the mitochondrial membrane. . Moreover, since the lipid membrane 21c has MTS on the surface, the liposome that has migrated into the cell can be selectively migrated to the mitochondria.

  As a liposome which is another embodiment of the lipid membrane structure having a configuration in which the mitochondrial membrane-fusible lipid membrane is located in the outermost layer of the lipid membrane structure transferred into the cell, as shown in FIG. 3, comprising a lipid membrane 21d, a lipid membrane 22d located outside the lipid membrane 21d, a lipid membrane 23d located outside the lipid membrane 22d, and a target substance 3 enclosed inside the lipid membrane 21d. Examples of the membrane liposome 1d include lipid membranes 21d and 22d that are mitochondrial membrane fusion lipid membranes, and lipid membrane 23d that contains membrane fusion lipids. The kind and amount of the fusogenic lipid contained in the lipid membrane 23d are the same as those of the lipid membrane 22c.

  Trilamellar liposome 1d can be transferred from the outside of the cell into the cell via endocytosis. The trilamellar liposome 1d that has migrated into the cell via endocytosis is taken into the endosome, but can escape from the endosome by the membrane fusion of the endosomal membrane and the lipid membrane 23d. In the liposome escaped from the endosome, the lipid membrane 23d disappears due to membrane fusion with the endosomal membrane, but the lipid membranes 21d and 22d are retained. After escape from the endosome, the target substance 3 encapsulated inside the lipid membrane 21d is released into the mitochondria in the same manner as in the case of the bilayer liposome 1b.

  In addition, the trilamellar liposome 1d can be transferred from the outside of the cell into the cell through membrane fusion between the lipid membrane 23d and the cell membrane. In the liposome that has migrated into the cell, the lipid membrane 23d disappears due to membrane fusion with the cell membrane, but the lipid membranes 21d and 22d are retained. After moving into the cell, the target substance 3 encapsulated inside the lipid membrane 21d is released into the mitochondria as in the case of the bilayer liposome 1b.

  In the triple membrane liposome 1d, the lipid membranes 21d and 22d preferably have MTS on the surface. As a result, the binding ability of the lipid membranes 21d and 22d to the mitochondrial membrane can be improved, and the membrane fusion between the lipid membranes 21d and 22d and the mitochondrial membrane is efficiently performed by the binding between the lipid membranes 21d and 22d and the mitochondrial membrane. It can be induced well. Moreover, since the lipid membrane 22d has MTS on the surface, the liposome that has migrated into the cell can be selectively migrated to the mitochondria.

  The lipid membrane structure of the present invention can be used both in vivo and in vitro. When the lipid membrane structure of the present invention is used in vivo, examples of the administration route include parenteral administration such as intravenous, intraperitoneal, subcutaneous, and nasal administration. It can be appropriately adjusted according to the type and amount of the target substance retained in the lipid membrane structure.

[Example 1] Screening for liposomes having binding activity and fusion activity to mitochondrial membrane (1) Preparation of EPC-based liposomes Selected from egg yolk phosphatidyl choline (EPC) and Tables 1 (i) to (viii) One type of lipid was dissolved in an organic solvent (chloroform) at a ratio of 9: 2 (molar ratio), and then added to a glass test tube, and the organic solvent was removed to form a thin film on the bottom of the glass test tube. A mitochondrial isolated buffer (hereinafter referred to as “MIB”) (250 mM sucrose, 2 mM Tris-Cl, the solvent is water) was added thereto to hydrate the lipid membrane, followed by sonication. EPC-based liposomes having an appropriate size (200 to 300 nm) were prepared (total lipid concentration: 0.5 to 0.55 mM). All of the prepared EPC-based liposomes are single membrane liposomes.

(2) Preparation of DOPE-based liposome 9: 2 (molar ratio) or 1 of dioleoyl phosphatidylethanolamine (DOPE) and one kind of lipid selected from Tables 1 (i) to (viii) After dissolving in an organic solvent (chloroform) at a ratio of 1 (molar ratio), DOPE-based liposomes having an appropriate size (200 to 300 nm) were prepared in the same manner as in (1) above (total lipid concentration: 0. 5-0.55 mM). In addition, all the prepared DOPE type liposomes are single membrane liposomes.

  When preparing EPC-type liposomes or DOPE-type liposomes, 1 mol% NBD (4-nitrobenzo-2-oxa-1,3-diazole) -labeled DOPE (excitation wavelength) is used for the liposome membrane of the liposome for binding activity measurement. (Ex) 470 nm, fluorescence wavelength (Em) 530 nm, manufactured by Molecular Probe), and the liposome membrane of the fusion activity measurement liposome is labeled with 1 mol% NBD-labeled DOPE and 0.5 mol% rhodamine (Rho). DOPE (excitation wavelength (Ex) 560 nm, fluorescence wavelength (Em) 590 nm) was contained.

(3) Modification of liposome surface with stearylated octaarginine (STR-R8) Stealylated octaarginine solution (stearylated octaarginine concentration: 2 mg / mL, solvent: water) to 100 μL of EPC-based liposome or DOPE-based liposome suspension 4.64 μL (in the case of EPC-based liposome, DOPE-based liposome [DOPE: X = 9: 2]) or 4.23 μL (in the case of DOPE-based liposome [DOPE: X = 1: 1]) is added and constant at room temperature By incubating for a period of time, stearyl-ized octaarginine was distributed on the liposome membrane (distribution amount of stearyl-ized octaarginine: 10 mol% of the total lipid amount). Thus, EPC-based liposomes or DOPE-based liposomes having octaarginine (arginine 8 polymer) on the surface were prepared.

  Hereinafter, EPC-based liposomes having no octaarginine on the surface are “STR-R8 non-modified EPC-based liposomes”, EPC-based liposomes having octaarginine on the surface are “STR-R8-modified EPC-based liposomes”, and octaarginine is present on the surface. The DOPE-based liposome not to be used is referred to as “STR-R8 unmodified DOPE-based liposome”, and the DOPE-based liposome having octaarginine on the surface is referred to as “STR-R8-modified DOPE-based liposome”.

(4) Measurement of binding activity The binding activity of liposomes to the mitochondrial membrane was evaluated as follows using mitochondria isolated from rat liver and fluorescently labeled liposomes containing 1 mol% NBD-labeled DOPE.
Mitochondria were isolated from rat liver and diluted to 1 mg / mL in MIB to prepare a mitochondrial solution. Samples were prepared by adding 10 μL of liposome suspension to 90 μL of mitochondrial solution. After preparing two samples and incubating each sample at 25 ° C for 30 minutes, one sample is stored in a light-refrigerated place as a _Fall sample, and the other sample is centrifuged (16000 × g, 10 minutes, 4 ° C). ) Thereafter, the supernatant (the fraction containing liposomes that did not bind to mitochondria) was removed, the precipitate (the fraction containing liposomes bound to mitochondria) was washed with MIB, and centrifuged (20400 × g, 2 Min., 4 ° C.) and re-separated (twice) and stored in the dark as a F sample. After adding an equal amount of 1% Triton to each sample and stirring, the fluorescence intensity was measured, and the binding activity (%) was calculated based on the following formula.

[In the formula, F represents the fluorescence intensity of the F sample (fluorescence wavelength: 530 nm), and F _all represents the fluorescence intensity of the F _all sample (fluorescence wavelength: 530 nm). ]

  FIG. 2 shows the binding activity (%) of the EPC liposome to the mitochondrial membrane, and FIG. 3 shows the binding activity (%) to the DOPE liposome mitochondrial membrane. In FIGS. 2 and 3, □ indicates STR-R8 unmodified liposome, and ■ indicates STR-R8 modified liposome. FIG. 3 (a) shows DOPE liposomes in which the mixing ratio of DOPE to one lipid selected from Tables 1 (i) to (viii) is 9: 2 (molar ratio). (B) shows a DOPE-based liposome in which the mixing ratio of DOPE and one kind of lipid selected from Tables 1 (i) to (viii) is 1: 1 (molar ratio).

  As shown in FIGS. 2 and 3, in the STR-R8 unmodified EPC-based liposome and the STR-R8 unmodified DOPE-based liposome, the binding activity to the mitochondrial membrane was not observed, but the STR-R8 modified EPC-based liposome and In the STR-R8 modified DOPE-based liposome, binding activity to the mitochondrial membrane was observed.

(5) Fusion activity measurement The fusion activity with respect to the mitochondrial membrane of a liposome was evaluated using FRET (Fluorescence Resonance Energy Transfer).
If a fluorescently labeled liposome containing 1 mol% NBD labeled DOPE and 0.5 mol% Rho labeled DOPE binds to mitochondria and does not fuse with the mitochondrial membrane, the fluorescently labeled liposome exists as it is. In this case, since NBD and Rho are very close to each other, FRET is induced and the fluorescence of NBD disappears. On the other hand, when the fluorescence-labeled liposome is fused with the mitochondrial membrane, NBD-labeled DOPE and Rho-labeled DOPE diffuse on the mitochondrial membrane, so that FRET is eliminated and NBD fluorescence is restored.

F sample was prepared by adding 10 μL of liposome suspension to 90 μL of mitochondrial solution and incubating at 25 ° C. for 30 minutes. The liposome suspension 10μL was added to MIB90myuL, incubated for 30 minutes at 25 ° C., to prepare _{F 0} sample. In addition, 10 μL of liposome suspension was added to 90 μL of 0.5% Triton, and incubated at 25 ° C. for 30 minutes to prepare an F _max sample. Measure the fluorescence spectrum (fluorescence wavelength 530 nm) emitted when each sample is irradiated with excitation light of 470 nm, calculate the FRET elimination rate (%) based on the following formula, and eliminate the FRET when the liposome is destroyed by Triton The fusion activity of liposomes on the mitochondrial membrane was evaluated at a rate of 100%.

[Wherein F represents the fluorescence intensity of the F sample (fluorescence wavelength of 530 nm), F ₀ represents the fluorescence intensity of the F ₀ sample (fluorescence wavelength of 530 nm), and F _max represents the fluorescence intensity of the F _max sample (fluorescence wavelength of 530 nm). Represents. ]

The F ₀ samples, FRET fluorescence of NBD for is induced quenching. In the F _max sample, the liposome is destroyed by Triton and the intermolecular distance between NBD and Rho is increased, so that FRET is eliminated and the fluorescence of NBD is restored. In the F sample, when the liposome is fused with the mitochondrial membrane, FRET is eliminated and the NBD fluorescence is restored.

  FIG. 4 shows the fusion activity (FRET elimination rate (%)) of the EPC-based liposome against the mitochondrial membrane, and FIG. 5 shows the fusion activity (FRET elimination rate (%)) of the DOPE-based liposome mitochondrial membrane. In FIGS. 4 and 5, □ indicates STR-R8 unmodified liposome, and ■ indicates STR-R8 modified liposome. FIG. 5 (a) shows DOPE liposomes in which the mixing ratio of DOPE and one kind of lipid selected from Tables 1 (i) to (viii) is 9: 2 (molar ratio). (B) shows a DOPE-based liposome in which the mixing ratio of DOPE and one kind of lipid selected from Tables 1 (i) to (viii) is 1: 1 (molar ratio).

  As shown in FIGS. 4 and 5, in the STR-R8 unmodified EPC-based liposome, the STR-R8-modified EPC-based liposome, and the STR-R8 unmodified DOPE-based liposome, almost no fusion activity to the mitochondrial membrane was observed. In the STR-R8-modified DOPE liposome, remarkable fusion activity with respect to the mitochondrial membrane was observed.

  From the above results, in order for the liposome membrane to fuse with the mitochondrial membrane, the binding of the liposome membrane to the mitochondrial membrane via octaarginine present on the surface of the liposome membrane, and the membrane-fused lipid DOPE contained in the liposome membrane It was clarified that fusion between the liposome membrane and the mitochondrial membrane is necessary. In addition, when the ratio of the amount of DOPE contained in the liposome membrane is reduced (see FIG. 4B), the fusion activity of the liposome membrane to the mitochondrial membrane has decreased, so the fusion activity of the liposome membrane to the mitochondrial membrane is It became clear that it varied depending on the ratio of the amount of DOPE contained in the liposome membrane.

[Example 2] Analysis of liposome kinetics in living cells It is confirmed that STR-R8-modified DOPE-based liposomes exhibit fusion activity for mitochondrial membranes in living cells and can introduce liposome-encapsulating substances into mitochondria. Therefore, STR-R8-modified DOPE liposomes were introduced into HeLa cells and the intracellular dynamics were observed. It is known that STR-R8-modified liposomes can be introduced into cells while maintaining their original form (in an intact state) (Karyl Ikrami et al., “YAKUGAKU ZASSHI”, 2004, Vol. 124, Suppl. 4, 113-116; Kogure et al., “Journal of Controlled Release”, 2004, Vol. 98, pages 317-323).

The medium of HeLa cells seeded in a 6 cm dish at a concentration of 4 × 10 ⁴ cells / mL on the previous day was replaced with a serum-free medium, and then a 10 mol% STR-R8-modified DOPE-based liposome (GFP protein (Green Fluorescent Protein) was added so that the final total lipid concentration was 13.75 μM, and the mixture was incubated at 37 ° C. in the presence of 5% CO ₂ for 1 hour. Thereafter, the medium was replaced with serum-supplemented medium, and further incubated at 37 ° C. for 1 hour. Prior to observation with a confocal laser microscope, 1 mM mitofluor (Molecular Probe) was added to the medium to a final concentration of 100 nM, incubated at 37 ° C. in the presence of 5% CO ₂ for 20 minutes to remove mitochondria. Stained. The intracellular kinetics of the liposomes were observed using a confocal laser microscope (LMS 510, Carl Zeiss Co., Ltd.), and the localization of mitochondria (red) and liposome encapsulated GFP (green) was confirmed (co-localization). Yellow if localized).

  The observation result by a confocal laser microscope is shown in FIG. FIG. 6 (a) shows the results when STR-R8-modified EPC-based liposomes (EPC: Chol = 9: 2) were used. FIG. 6 (b) shows STR-R8-modified DOPE-based liposomes (DOPE: SM). FIG. 6 (c) shows the results when STR-R8-modified DOPE-based liposomes (DOPE: PA = 9: 2) are used. “Transmitted light” is the cell image by transmitted light, “Mt” is the localization of mitochondria (red), “GFP” is the localization of GFP (green), “Superposition” is superimposed on the cell image by transmitted light The localization of mitochondria (red) and GFP (green) is shown.

  When STR-R8-modified EPC-based liposomes were used, co-localization (yellow) between mitochondria and the encapsulated substance GFP was not confirmed, but when STR-R8-modified DOPE-based liposomes were used, mitochondria Co-localization (yellow) with the encapsulated substance GFP was confirmed. This revealed that the STR-R8-modified DOPE liposome can introduce the encapsulated substance GFP into the mitochondria.

[Example 3] Protein delivery into mitochondria (1) Introduction of liposome into mitochondria In the same manner as in Example 1, a rat liver mitochondrial solution (mitochondrial protein 10 mg / mL) was prepared, and 1 mL was dispensed into tubes. In the same manner as in Example 1, 10 mol% STR-R8 modified EPC-based liposome (EPC: SM = 9: 2, EPC: PA = 9: 2) and 10 mol% STR-R8 modified DOPE-based liposome (DOPE: SM = 9: 2, DOPE: PA = 9: 2) (GFP (Green Fluorescent Protein) is encapsulated inside each liposome), and 100 μL of liposome solution (GFP 125 ng / μL, total lipid 0. 55 mM) was added to the rat liver mitochondrial solution, followed by incubation at 25 ° C. for 30 minutes. At this time, the same amount of GFP was added to the negative control. Thereafter, the mixture was centrifuged (16000 × g, 10 minutes, 4 ° C.), and the supernatant was removed. The precipitate was washed with IM (+) [IM (-BSA) (70 mM sucrose, 220 mM D-mannitol, 2.0 mM HEPES (pH 7.4)) added to 50 mL of 50 mg / mL BSA solution] and centrifuged. (20400 × g, 2 minutes, 4 ° C.), and the supernatant was removed. After repeating the same operation, the precipitate was resuspended in 100 μL of IM (+) to obtain a mitochondrial solution (mitochondrial protein 100 mg / mL).

(2) Mitochondrial fractionation 100 μL of the mitochondrial solution prepared in (1) was dispensed into a vial, and an equal amount of 1.25% digitonin solution (dissolved with digitonin in IM (+)) was added to the mitochondrial solution, The mitochondrial outer membrane was detached by gentle stirring at 4 ° C. for 15 minutes (JW Greenawarth. The isolation of outer and inner mitochondral membranes. Methods Enzymol 31: 310-23 (1974)). Thereafter, 600 μL of IM (+) was added to the reaction solution to stop the reaction. After completion of the reaction, the mitochondrial solution was centrifuged (10000 × g, 10 minutes, 4 ° C.), the supernatant was transferred to another tube, and the precipitate was washed with IM (+). Centrifugation was performed under the same conditions, and the supernatant was transferred to another tube. The obtained supernatants were combined and centrifuged (144000 × g, 10 minutes, 4 ° C.), and the resulting precipitate was resuspended in 100 μL of IM (+) to obtain an outer membrane (OM) fraction. . The supernatant was centrifuged again under the same conditions, and the resulting supernatant was used as an intermembrane space (IMS) fraction. The obtained sample was measured with BCA protein assay kit (PIERCE) using BSA as a standard substance in the presence of 1% SDS, and then diluted with IM (+) to 1 mg / mL. And stored at -80 ° C.

(3) Western blot The obtained sample and SDS-PAGE loading buffer (0.1M Tris, 4% SDS, 12% 2-mercaptoethanol, 20% glycerol, BPB (appropriate amount)) were mixed and denatured. 5 μL of the sample was applied to an SDS-polyacrylamide gel (15%) and run. The gel after electrophoresis was transferred to a PVDF membrane (Nippon Genetics) (BE-330, BIO CRAFT). After blocking the PVDF membrane after the transfer, an antibody reaction was performed to detect the target GFP band. A primary antibody (Anti-GFP, N-Terminal (SIGMA)), a secondary antibody (HRP-conjugated donkey anti-rabbit IgG antibody) were each diluted 1000-fold, and a band was detected by ECL + plus Western Blotting Detection System (Amersham). .

(4) Results The results are shown in FIG. In FIG. 7, “OM” indicates the result of the OM fraction, “IMS” indicates the result of the IMS fraction, (a) indicates the result of the negative control, and (b) indicates the STR-R8-modified EPC liposome (EPC). : SM = 9: 2), (c) is the result of STR-R8 modified EPC liposome (EPC: PA = 9: 2), (d) is the STR-R8 modified DOPE liposome (DOPE: SM = 9) : 2) As a result, (e) shows the result of STR-R8-modified DOPE-based liposome (DOPE: PA = 9: 2).

  As shown in FIG. 7, when STR-R8-modified EPC-based liposomes (EPC: SM = 9: 2, EPC: PA = 9: 2) were used, a GFP band of 33 kDa was observed in the OM fraction. Not observed in the IMS fraction. On the other hand, when STR-R8-modified DOPE-based liposomes (DOPE: SM = 9: 2, DOPE: PA = 9: 2) were used, a 33 kDa band of GFP was observed in both the OM fraction and the IMS fraction. . When the STR-R8 modified DOPE-based liposome is used, the 28 kDa band observed in the OM fraction and the IMS fraction may also be a GFP band. From this result, it became clear that according to the STR-R8 modified DOPE-based liposome, the encapsulated substance GFP can be introduced into the intermembrane space of mitochondria.

[Example 4] Evaluation of membrane fusion ability of liposomes to live cell mitochondria (1) Preparation of liposomes In the same manner as in Example 1, 10 mol% STR-R8 modified EPC-based liposomes (EPC: SM = 9: 2) and 10 mol% STR-R8 modified DOPE-based liposomes (DOPE: SM = 9: 2) were prepared. However, 0.5 mol% of the total lipid amount of NBD-DOPE (green) was contained in the lipid membrane. Moreover, 1 mg / mL Dextran cascade blue (Molecular Probe) (blue) was enclosed as an aqueous phase marker inside the liposome.

(2) Observation with confocal laser scanning microscope Cells were seeded at 4 × 10 ⁴ cells / mL in a 2.5 cm dish (2 mL culture solution) and cultured for 24 hours. After culturing, the culture solution was removed, 1 mL of serum-free medium (without antibiotics) was added, and 25 μL of liposome (lipid amount 0.55 mM) was added, followed by incubation (37 ° C., 1 hour, 5% CO _2. ). Next, the plate was washed 3 times with 1 mL PBS (−), replaced with a serum-supplemented medium, and incubated for 1 hour under the same conditions. Thereafter, mitochondria were stained with mitofluor (Molecular Probe) and observed with a confocal laser microscope (LMS 510, Carl Zeiss Co., Ltd.).

(3) Results The results are shown in FIGS. 8 shows the result of the STR-R8 modified EPC-based liposome (EPC: SM = 9: 2), and FIG. 9 shows the result of the STR-R8 modified DOPE-based liposome (DOPE: SM = 9: 2). Further, “transmitted light” is a cell image by transmitted light, “mitochondria (red)” is localized in mitochondria (red), “NBD-DOPE (green)” is localized in NBD-DOPE (green), “Dextran cascade” “blue (blue)” is the localization of the dextran cascade blue (blue), “superposition” is the mitochondrial (red), NBD-DOPE (green), and dextran cascade blue (blue) stations superimposed on the cell image by transmitted light. Indicates presence.

  When STR-R8-modified EPC-based liposomes were used, NBD-DOPE (green) and Dextran cascade blue (blue) co-localized (light blue) outside the mitochondria (red). This indicates that the STR-R8-modified EPC-based liposome can bind to the mitochondrial membrane, but cannot fuse with the mitochondrial membrane, and cannot introduce the encapsulated substance Dextran cascade blue into the mitochondria. On the other hand, when STR-R8-modified DOPE-based liposomes were used, dextran cascade blue (blue) was localized inside the mitochondria (red) alone. In addition, mitochondria (red) and dextran cascade blue (blue) co-localized (pink). From these results, it was shown that the STR-R8-modified DOPE-based liposome can bind to the mitochondrial membrane, can be fused with the mitochondrial membrane, and can introduce the encapsulated substance Dextran cascade blue into the mitochondria.

[Example 5] Synthesis of mitochondrial targeting (MTS) -lipid derivative Membrane derived from rat liver derived from succinyl-CoA synthetase (SEQ ID NO: 1, R. Majudardar and WA Bridger. Mitochondrial transcription and processing of the precursor) MTS-DOPE was synthesized as an MTS-lipid derivative for modification with the alpha-subunit of rat liver succinyl-CoA synthetase. Biochem Cell Biol 68: 292-9 (1990)). Although the results are not shown, it has been confirmed that succinyl-CoA synthetase-derived MTS (SEQ ID NO: 1) derived from rat liver exhibits the ability to migrate to human cell mitochondria.

  MTS-DOPE is synthesized by linking the SH group of the C-terminal Cys of MTS and the amino group of DOPE via a cross-linker reagent SMCC (4- (maleimidomethyl) -1-cyclohexanecarboxylic acid N-hydroxysuccinimide ester). 10 and 13). In addition, in FIG.10 and 13, the structure of DOPE is shown typically.

(1) Synthesis of SMCC-DOPE (FIG. 10)
200 μL of CHCl ₃ , 500 nmol of DOPE, 500 nmol of SMCC and 1 μmol of triethylamine were added to an Eppendorf tube, and the mixture was shaken at room temperature for 12 hours to react (reaction solution A). After completion of the reaction, it was freeze-dried, the solvent was removed, and it was dissolved again using 50 μL of methanol. It was confirmed by HPLC analysis that the desired product was obtained (FIG. 11). In HPLC analysis, A buffer [0.1% TFA in double-distilled water (DDW) solution] and B buffer [0.1% TFA in CH ₃ CN] were used as the mobile phase, and 30-70. Gradient was applied for 10 minutes with% B buffer, then 10 minutes with 90-100% B buffer, then 10 minutes with 100% B buffer. Furthermore, SMCC-DOPE was fractionated and subjected to mass spectrometry by FAB-MS (Fast Atom Bombardment Mass Spectrometry), and it was confirmed that the molecular weight also matched the target product (FIG. 12). In mass spectrometry by FAB-MS, JEOL JMS-HX100 was used as a measuring instrument, methanol was used as a solvent, and NBA (m-nitrobenzyl alcohol) was used as a matrix.

(2) Coupling between MTS and SMCC-DOPE (FIG. 13)
After the synthesis of SMCC-DOPE, add 100 μL (250 nmol) of half of the reaction solution A and 100 μL (125 nmol) of MTS in DMSO (degassed) to the Eppendorf tube, let stand at room temperature for 1 hour, and let it react. It was. After completion of the reaction, the solution was freeze-dried, the solvent was removed, and redissolved with 100 μL of methanol / DMSO (1: 3). Thereafter, HPLC purification was performed (FIG. 14). HPLC was performed in the same manner as described above. Mass analysis of the purified MTS-DOPE was performed by MALDI-TOF-MS (Matrix-Assisted Laser Deposition Ionization Time-Of-Flight Mass Spectrometry), and it was confirmed that the molecular weight was consistent with the target product (FIG. 15). In mass spectrometry by MALDI-TOF-MS, VOYAGER DEPRO was used as a measuring instrument, a CH ₃ CN solution of 10% TFA was used as a solvent, and CHCA was used as a matrix. From the results of mass spectrometry, the obtained product was designated as MTS-DOPE.

[Example 6] Evaluation of ability of MTS-modified liposomes to migrate to mitochondria (1) Preparation of liposomes After forming a lipid film containing 5 mol% MTS-DOPE in a test tube, the lipid film was hydrated and subjected to ultrasonic treatment. By performing (the detailed conditions are the same as in Example 1), STR-R8 non-modified MTS-modified EPC-based liposomes (EPC: Chol = 9: 2) were prepared. The “MTS modification” means that the liposome surface is modified with MTS. Further, in the same manner as in Example 1, 5 mol% STR-R8 modified MTS unmodified EPC-based liposome (DOPE: Chol = 9: 2), STR-R8 and MTS unmodified EPC-based liposome (EPC; DDAB = 9: 2, EPC: Chol = 9: 2).

(2) Preparation of homogenate solution After washing HeLa cells with MIB (+) [250 mM sucrose, 2 mM Tris-Cl, 1 mM EDTA, pH 7.4], MIB (+) was adjusted to 1.0 × 10 ⁷ cells / mL. ) (Hereinafter, operation on ice). Using a Dounce homogenizer, homogenization was performed 20 times to prepare a homogenate solution. The obtained sample was diluted with MIB (+) to 1 mg / mL after measuring the protein amount using BCA protein assay kit (PIERCE) with BSA as a standard substance in the presence of 1% SDS. And stored at -80 ° C.

(3) Evaluation of ability to migrate to mitochondria 60 μL liposome (containing 1 mol% NBD-DOPE) was added to 540 μL homogenate solution [1 μg / μL protein]. As a control, 60 μL MIB (+) added was prepared. After incubation at 25 ° C. for 30 minutes, 100 μL was dispensed [F _all ], and the rest was used for centrifugation. After centrifuging a 500 μL sample (700 × g, 10 minutes, 4 ° C.), 400 μL of the obtained supernatant was centrifuged (16000 × g, 10 minutes, 4 ° C.), and 400 μL of the resulting precipitate was added to MIB (+ [F _mt ].

  The same amount of 1% Triton solution (99 μL MIB (−), 1 μL Triton) was added to 100 μL of the sample, suspended, and adjusted to a final concentration of 0.5% Triton, followed by fluorescence intensity measurement (ex: 470 nm, em: 530 nm), and the ability of liposomes to migrate to mitochondria was evaluated based on the activity (%) to mitochondria calculated from the following formula.

Migration activity (%) = fluorescence intensity [F _mt ] of liposomes migrated to mitochondria / fluorescence intensity [F _al1 ] of all added liposomes × 100

  The results are shown in FIG. As shown in FIG. 16, it was revealed that the liposome can be imparted with the ability to selectively migrate to mitochondria by modifying the liposome surface with MTS.

[Example 7] Analysis of liposome dynamics in living cells (1) Preparation of liposomes After forming a lipid membrane in a test tube, a GFP solution (125 ng / μL, the same applies hereinafter) is added to hydrate the lipid membrane, and ultrasound is applied. By performing the treatment (detailed conditions are the same as in Example 1), STR-R8 and MTS-unmodified EPC-based liposomes (EPC: Chol = 9: 2) were prepared.

  STR-R8 is obtained by forming a lipid membrane containing 5 mol% MTS-DOPE in a test tube, adding a GFP solution to hydrate the lipid membrane, and performing ultrasonic treatment (detailed conditions are the same as in Example 1). Unmodified MTS-modified EPC-based liposomes (EPC: Chol = 9: 2) were prepared. The “MTS modification” means that the liposome surface is modified with MTS.

  After forming a lipid membrane containing 10 mol% STR-R8 in a test tube, the GFP solution was added to hydrate the lipid membrane and subjected to ultrasonic treatment (detailed conditions are the same as in Example 1). R8-modified MTS non-modified EPC-based liposomes (EPC: Chol = 9: 2) were prepared.

  After forming a lipid membrane containing 5 mol% MTS-DOPE in a test tube, the GFP solution is added to hydrate the lipid membrane, and sonication is performed (detailed conditions are the same as in Example 1) to modify the MTS. EPC-based liposomes (EPC: Chol = 9: 2) were formed. Thereafter, STR-R8 was added to the liposome solution so as to be 10 mol% of the total lipid amount, and STR-R8 and MTS-modified EPC-based liposomes (EPC: Chol = 9: 2) were prepared.

  In addition, GFP (Green Fluorescent Protein) is enclosed inside any liposome.

  In the same manner as in Example 2, liposomes were introduced into HeLa cells, the intracellular dynamics were observed using a confocal laser microscope, and localization of mitochondria (red) and liposome inclusion GFP (green) was confirmed. (Yellow when colocalized).

  The observation result by a confocal laser microscope is shown in FIGS. FIG. 17 (a) shows the results of the control (GFP solution added to the cells), FIG. 17 (b) shows the results of STR-R8 and MTS-unmodified EPC-based liposomes, and FIG. 17 (c) shows the results of STR- As a result of R8 non-modified MTS-modified EPC-based liposomes, FIG. 18 (a) shows the results of STR-R8-modified MTS-unmodified EPC-based liposomes, and FIG. 18 (b) shows the results of STR-R8 and MTS-modified EPC-based liposomes. Show. “Transmitted light” is the cell image by transmitted light, “Mitochondria” is the location of mitochondria (red), “GFP” is the localization of GFP (green), “Overlapping” is superimposed on the cell image by transmitted light The localization of mitochondria (red) and GFP (green) is shown.

  When STR-R8 and MTS-unmodified EPC-based liposomes and STR-R8 unmodified MTS-modified EPC-based liposomes were used, the localization of GFP (green) was not confirmed in the cells. From this, it was clarified that the liposome cannot be introduced into the cells only by modifying the liposome surface with MTS.

  When STR-R8-modified MTS non-modified EPC-based liposomes were used, localization of GFP (green) was confirmed in the cells, and localization of GFP (green) was confirmed around mitochondria (red). From this, it was clarified that the liposome can be introduced into the cell by modifying the liposome surface with STR-R8, and that the liposome migrated into the cell can bind to the mitochondrial membrane. However, co-localization (yellow) of mitochondria (red) and GFP (green) was not observed, so that the liposome could not be given fusion ability to the mitochondrial membrane only by modifying the liposome surface with STR-R8. Became clear.

  When STR-R8 and MTS-modified EPC-based liposomes were used, co-localization (yellow) of mitochondria (red) and GFP (green) was confirmed. The STR-R8 and MTS-modified EPC system, like the liposome STR-R8-modified MTS non-modified EPC-type liposomes, are considered not to have a fusion ability to the mitochondrial membrane, so that the mitochondrial (red) and GFP (green) Localization (yellow) means that the liposomes are in close proximity to the mitochondrial membrane. That is, it was revealed that the liposome can be bound in the state of being very close to the mitochondrial membrane by modifying the liposome surface with MTS. If liposomes can bind to the mitochondrial membrane in close proximity, it is likely to induce membrane fusion between the liposome membrane and the mitochondrial membrane. In addition, since GFP (green) was hardly observed in parts other than mitochondria (red) (cytoplasm and other organelles), the liposome surface was modified with MTS to selectively transfer liposomes to mitochondria. It became clear that the ability can be given.

Claims (6)

It comprises a lipid membrane containing dioleyl phosphatidylethanolamine and phosphatidic acid and / or sphingomyelin as a constituent lipid of the lipid membrane and having a polyarginine peptide consisting of continuous 4 to 20 arginine residues on the surface. A characteristic lipid membrane structure. The lipid according to claim 1, wherein the content of dioleyl phosphatidylethanolamine and phosphatidic acid and / or sphingomyelin is 70% (molar ratio) or more of the total lipid amount contained in the lipid membrane. Membrane structure. The lipid membrane structure according to claim 1 or 2, wherein the lipid membrane has a mitochondrial targeting peptide shown in the following (a) or (b) on the surface .
(A) A peptide comprising the amino acid sequence set forth in SEQ ID NO: 1.
(B) A peptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 1 . The lipid membrane structure according to claim 3, wherein the mitochondrial targeting peptide is bound to a component of the lipid membrane directly or via a linker . The lipid membrane structure according to claim 4, wherein the C-terminus of the mitochondrial targeting peptide is bound to a component of the lipid membrane. The lipid membrane structure according to any one of claims 3 to 5 , wherein the amount of the mitochondrial targeting peptide is 2 to 10% (molar ratio) of the total lipid amount contained in the lipid membrane.

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