Patent Application
20080241917
The present invention relates to a vector for delivering a target substance into a nucleus or a cell.
Vectors and carriers for reliably delivering drugs, nucleic acids, peptides, proteins, sugars, and the like to target sites are being actively developed. For example, in the field of gene therapy, viral vectors such as retroviral vectors, adenoviral vectors, and adeno-associated viral vectors have been developed as vectors for introducing desired genes into target cells. However, such viral vectors have various problems such as difficulty in mass production, antigenicity, and toxicity. For this reason, liposomal vectors and peptide carriers having fewer problems than viral vectors are receiving attention. In addition, liposomal vectors have an advantage that their directionality toward target sites can be improved by introducing functional molecules such as antibodies, proteins, and sugar chains to their surfaces.
Nuclear membrane permeability is a very significant bottleneck in development of effective vectors for use in gene therapy. Transport of substances between the cytoplasm and the nucleus occurs through nuclear pore complexes present in the nuclear membrane, and therefore low-molecular weight proteins and ions can permeate the nuclear membrane by free diffusion through nuclear pore complexes. However, polymers cannot freely permeate the nuclear membrane because of their size. For this reason, it is difficult to efficiently deliver genes into nuclei simply by directly introducing the genes into cells.
Meanwhile, in recent years, it has been reported that by modifying the surface of a liposome entrapping condensed DNA with stearylated octaarginine, the cellular entry efficiency of condensed DNA is increased 1000-fold and the cellular entry efficiency of the liposome entrapping condensed DNA is increased 100-fold (see Non-patent Document 1). Further, it has been also reported that a liposome having a surface modified with stearylated octaarginine can enter a cell with its original form being kept intact (see Non-patent Documents 1 and 2).
Non-patent Document 1: Kogure et al., “Journal of Controlled Release”, vol. 98, pp. 317-323, 2004
Non-patent Document 2: Khalil Ikramy et al., “YAKUGAKU ZASSHI”, vol. 124, suppl. 4, pp. 113-116, 2004
An object of the present invention is to provide a vector for delivering a target substance into a nucleus or a cell.
In order to achieve the above object, the present invention is directed to a first vector for delivering a target substance into a nucleus, which includes a lipid membrane structure having a first lipid membrane containing an anionic lipid. The first lipid membrane of the first vector is bound to and fused with a nuclear membrane so that a target substance contained in the first vector is efficiently transferred into a nucleus.
Further, in order to achieve the above object, the present invention is also directed to a second vector for delivering a target substance into a cell, which includes a lipid membrane structure having a second lipid membrane containing an anionic lipid. The second lipid membrane of the second vector can be efficiently bound to and fused with an endosomal membrane, a macropinosomal membrane, or the like. Therefore, after the second vector is introduced into a cell as an endosomal fraction, a macropinosomal fraction, or the like via a pathway such as endocytosis, macropinocytosis, or the like, the second lipid membrane of the second vector is bound to and fused with an endosomal membrane, a macropinosomal membrane, or the like so that a target substance contained in the second vector escapes from an endosome, a macropinosome, or the like and is efficiently transferred into the cell.
Further, in order to achieve the above object, the present invention is also directed to a third vector for delivering a target substance into a nucleus, which includes a lipid membrane structure having a first lipid membrane containing an anionic lipid and a second lipid membrane containing an anionic lipid and provided outside the first lipid membrane. The first lipid membrane of the third vector can be efficiently bound to and fused with a nuclear membrane and the second lipid membrane of the third vector can be efficiently bound to and fused with an endosomal membrane, a macropinosomal membrane, or the like. Therefore, after the third vector is introduced into a cell as an endosomal fraction, a macropinosomal fraction, or the like via a pathway such as endocytosis, macropinocytosis, or the like, the second lipid membrane of the third vector is bound to and fused with an endosomal membrane, a macropinosomal membrane, or the like so that a target substance contained in the third vector escapes from an endosome, a macropinosome, or the like and is efficiently transferred into the cell. Then, the first lipid membrane of the third vector is bound to and fused with a nuclear membrane so that the target substance contained in the third vector is efficiently transferred into a nucleus.
The anionic lipid contained in the first lipid membrane of the first or third vector is preferably cholesteryl hemisuccinate, phosphatidic acid, or cardiolipin. This makes it possible to improve the ability of the first lipid membrane to bind to and fuse with a nuclear membrane.
The amount of the anionic lipid contained in the first lipid membrane of the first or third vector is preferably 20 to 80% (mole ratio) of the total amount of lipids contained in the first lipid membrane. This makes it possible to improve the ability of the first lipid membrane to bind to and fuse with a nuclear membrane.
The anionic lipid contained in the first lipid membrane of the first or third vector is preferably phosphatidic acid or cardiolipin. In this case, the amount of the anionic lipid contained in the first lipid membrane is preferably 40 to 60% (mole ratio) of the total amount of lipids contained in the first lipid membrane. This makes it possible to improve the ability of the first lipid membrane to bind to and fuse with a nuclear membrane.
The first lipid membrane of the first or third vector preferably contains dioleoylphosphatidyl ethanolamine. This makes it possible to improve the ability of the first lipid membrane to bind to and fuse with a nuclear membrane.
The amount of dioleoylphosphatidyl ethanolamine contained in the first lipid membrane of the first or third vector is preferably 20 to 80% (mole ratio) of the total amount of lipids contained in the first lipid membrane. This makes it possible to improve the ability of the first lipid membrane to bind to and fuse with a nuclear membrane.
The first lipid membrane of the first or third vector preferably has a membrane-permeable peptide. This makes it possible to improve the ability of the first lipid membrane to bind to and fuse with a nuclear membrane.
The membrane-permeable peptide of the first or third vector is a peptide having, for example, a protein transduction domain. The protein transduction domain is preferably polyarginine, and the polyarginine is preferably composed of 4 to 20 consecutive arginine residues. By allowing the first lipid membrane to have such polyarginine as a protein transduction domain, it is possible to improve the ability of the first lipid membrane to bind to and fuse with a nuclear membrane. Further, in a case where the first lipid membrane provides the surface of the vector and has polyarginine as a protein transduction domain, it is possible to allow the vector to be transferred into a cell mainly via macropinocytosis by adjusting the amount of polyarginine present on the surface of the vector. In macropinocytosis, an extracellular substance is introduced into a cell as a fraction called “macropinosome”. Such a macropinosome is different from an endosome in that it is not fused with a lysosome, and therefore it is possible to avoid decomposition of a substance entrapped in a macropinosome by a lysosome. For this reason, when the vector is transferred into a cell mainly via macropinocytosis, a target substance contained in the vector is efficiently delivered into a cell.
The membrane-permeable peptide of the first or third vector is preferably present on the surface of the first lipid membrane. By allowing the membrane-permeable peptide to be present on the surface of the first lipid membrane, it is possible to improve the ability of the first lipid membrane to bind to and fuse with a nuclear membrane. Further, in a case where the first lipid membrane provides the surface of the vector and polyarginine is present as a protein transduction domain on the surface of the first lipid membrane, the vector is transferred into a cell mainly via macropinocytosis so that a target substance contained in the vector is efficiently delivered into the cell.
In a case where the first lipid membrane of the first or third vector has a membrane-permeable peptide, the anionic lipid contained in the first lipid membrane is preferably cholesteryl hemisuccinate, phosphatidic acid, or phosphatidylserine. By allowing the first lipid membrane to have such an anionic lipid and a membrane-permeable peptide, it is possible to improve the ability of the first lipid membrane to bind to and fuse with a nuclear membrane.
The anionic lipid contained in the second lipid membrane of the second or third vector is preferably phosphatidic acid, cardiolipin, dialkyl phosphate, or diacyl phosphate. This makes it possible to improve the ability of the second lipid membrane to bind to and fuse with an endosomal membrane or a macropinosomal membrane.
The amount of the anionic lipid contained in the second lipid membrane of the second or third vector is preferably 10 to 90% (mole ratio) of the total amount of lipids contained in the second lipid membrane. This makes it possible to improve the ability of the second lipid membrane to bind to and fuse with an endosomal membrane or a macropinosomal membrane.
The second lipid membrane of the second or third vector preferably contains dioleoylphosphatidyl ethanolamine. This makes it possible to improve the ability of the second lipid membrane to bind to and fuse with an endosomal membrane or a macropinosomal membrane.
The amount of dioleoylphosphatidyl ethanolamine contained in the second lipid membrane of the second or third vector is preferably 10 to 90% (mole ratio) of the total amount of lipids contained in the second lipid membrane. This makes it possible to improve the ability of the second lipid membrane to bind to and fuse with an endosomal membrane or a macropinosomal membrane.
The second lipid membrane of the second or third vector preferably has a membrane-permeable peptide. This makes it possible to improve the ability of the second lipid membrane to bind to and fuse with an endosomal membrane or a macropinosomal membrane.
The membrane-permeable peptide of the second or third vector is a peptide having, for example, a protein transduction domain. The protein transduction domain is preferably polyarginine, and the polyarginine is preferably composed of 4 to 20 consecutive arginine residues. By allowing the second lipid membrane to have polyarginine as a protein transduction domain, it is possible to improve the ability of the second lipid membrane to bind to and fuse with an endosomal membrane or a macropinosomal membrane. Further, in a case where the second lipid membrane provides the surface of the vector and has polyarginine as a protein transduction domain, it is possible to allow the vector to be transferred into a cell mainly via macropinocytosis by adjusting the amount of polyarginine present on the surface of the vector. In macropinocytosis, an extracellular substance is introduced into a cell as a fraction called “macropinosome”. Such a macropinosome is different from an endosome in that it is not fused with a lysosome, and therefore it is possible to avoid decomposition of a substance entrapped in a macropinosome by a lysosome. For this reason, when the vector is transferred into a cell mainly via macropinocytosis, a target substance contained in the vector is efficiently delivered into a cell.
The membrane-permeable peptide of the second or third vector is preferably present on the surface of the second lipid membrane. By allowing the membrane-permeable peptide to be present on the surface of the second lipid membrane, it is possible to improve the ability of the second lipid membrane to bind to and fuse with an endosomal membrane or a macropinosomal membrane. Further, in a case where the second lipid membrane constitutes the surface of the vector and polyarginine is present as a protein transduction domain on the surface of the second lipid membrane, the vector is transferred into a cell mainly via macropinocytosis so that a target substance contained in the vector is efficiently delivered into the cell.
The lipid membrane structure of the first, second, or third vector is preferably a liposome. In a case where the lipid membrane structure is a liposome, a target substance is entrapped in the lipid membrane structure, thereby making it possible to efficiently deliver the target substance into a cell or a nucleus.
According to the present invention, it is possible to provide a vector for delivering a target substance into a nucleus or a cell.
Hereinbelow, a vector according to the present invention will be described in detail.
A first vector is a vector for delivering a target substance into a nucleus, which includes a lipid membrane structure having a first lipid membrane containing an anionic lipid. A second vector is a vector for delivering a target substance into a cell, which includes a lipid membrane structure having a second lipid membrane containing an anionic lipid. A third vector is a vector for delivering a target substance into a nucleus, which includes a lipid membrane structure having a first lipid membrane containing an anionic lipid and a second lipid membrane containing an anionic lipid and provided outside the first lipid membrane.
The lipid membrane structure may be any one of a liposome, an O/W-type emulsion, a W/O/W-type emulsion, a spherical micelle, a wormlike micelle, a layered structure having no regular shape, and the like, but is preferably a liposome. In a case where the lipid membrane structure is a liposome, a target substance is entrapped in the lipid membrane structure, thereby making it possible to efficiently deliver the target substance into a cell.
In a case where the lipid membrane structure is a liposome, the liposome may be either a multilamellar vesicle (MLV) or a unilamellar liposome such as SUV (small unilamellar vesicle), LUV (large unilamellar vesicle), or GUV (giant unilamellar vesicle), but is preferably a multilamellar vesicle. By allowing the liposome to have two or more first lipid membranes, it is possible for the liposome to permeate two nuclear membranes (outer membrane and inner membrane). Further, by allowing the liposome to have an anionic lipid-containing second lipid membrane provided outside the first lipid membrane, it is possible for the liposome to permeate an endosomal membrane or a macropinosomal membrane.
The number of first lipid membranes of the lipid membrane structure is not particularly limited, but is usually 1 to 5, preferably 1 to 3, more preferably 2. The number of second lipid membranes of the lipid membrane structure is not particularly limited, but is usually 1 to 5, preferably 1 to 3, more preferably 1. The lipid membrane structure may also have a lipid membrane other than the first and second lipid membranes.
The size of the lipid membrane structure is not particularly limited. In a case where the lipid membrane structure is a liposome or an emulsion, the particle size thereof is usually in the range of 50 nm to 5 μm. In a case where the lipid membrane structure is a spherical micelle, the particle size thereof is usually in the range of 5 to 100 nm. In a case where the lipid membrane structure is a wormlike micelle or a layered structure having no regular shape, the thickness of one layer thereof is usually in the range of 5 to 10 nm, and two or more such layers are preferably stacked.
Examples of components of the lipid membranes (including the first lipid membrane, the second lipid membrane, and a lipid membrane other than the first and second lipid membranes) of the lipid membrane structure include lipids, membrane stabilizers, antioxidants, charged substances, and membrane proteins.
Lipid is an essential component of the lipid membrane. The amount of lipid contained in the lipid membrane is usually 70% (mole ratio) or more, preferably 75% (mole ratio) or more, more preferably 80% (mole ratio) or more of the total amount of substances constituting the lipid membrane. It is to be noted that the upper limit value of the amount of lipid contained in the lipid membrane is 100% of the total amount of substances constituting the lipid membrane.
Examples of lipid include the following phospholipids, glycolipids, sterols, and saturated and unsaturated fatty acids.
<Phospholipids>
Examples of phospholipids include phosphatidylcholines (e.g., dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine), phosphatidylglycerols (e.g., dioleoylphosphatidylglycerol, dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, and distearoylphosphatidylglycerol), phosphatidyl ethanolamines (e.g., dilauroylphosphatidyl ethanolamine, dimyristoylphosphatidyl ethanolamine, dipalmitoylphosphatidyl ethanolamine, and distearoylphosphatidyl ethanolamine), 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, hydrogenation products thereof, and the like.
<Glycolipids>
Examples of glycolipids include glyceroglycolipids (e.g., sulfoxyribosylglyceride, diglycosyldiglyceride, digalactosyldiglyceride, galactosyldiglyceride, and glycosyldiglyceride), sphingoglycolipids (e.g., galactosylcerebroside, lactosylcerebroside, and ganglioside), and the like.
<Sterols>
Examples of sterols include animal-derived sterols (e.g., cholesterol, cholesterol succinic acid, cholestanol, lanosterol, dihydrolanosterol, desmosterol, and dihydrocholesterol), plant-derived sterols (phytosterols) (e.g., stigmasterol, sitosterol, campesterol, and brassicasterol), microbially-derived sterols (e.g., zymosterol and ergosterol), and the like.
<Saturated and Unsaturated Fatty Acids>
Examples of saturated and unsaturated fatty acids include saturated and 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 a component optionally added to the lipid membrane to physically or chemically stabilize the lipid membrane or to control the fluidity of the lipid membrane. The amount of the membrane stabilizer contained in the lipid membrane is usually 30% (mole ratio) or less, preferably 25% (mole ratio) or less, more preferably 20% (mole ratio) or less of the total amount of substances constituting the lipid membrane. It is to be noted that the lower limit value of the amount of the membrane stabilizer contained in the lipid membrane is 0.
Examples of the membrane stabilizer include sterols, glycerin and fatty acid esters thereof, and the like. Specific examples of the sterols include the same sterols as mentioned above. Examples of the glycerin fatty acid esters include triolein, trioctanoin, and the like.
An antioxidant is a component optionally added to the lipid membrane to prevent oxidation of the lipid membrane. The amount of the antioxidant contained in the lipid membrane is usually 30% (mole ratio) or less, preferably 25% (mole ratio) or less, more preferably 20% (mole ratio) or less of the total amount of substances constituting the lipid membrane. It is to be noted that the lower limit value of the amount of the antioxidant contained in the lipid membrane is 0.
Examples of the antioxidant include tocopherol, propyl gallate, ascorbyl palmitate, butylated hydroxytoluene, and the like.
A charged substance is a component optionally added to the lipid membrane to give a positive electric charge or a negative electric charge to the lipid membrane. The amount of the charged substance contained in the lipid membrane is usually 30% (mole ratio) or less, preferably 25% (mole ratio) or less, more preferably 20% (mole ratio) or less of the total amount of substances constituting the lipid membrane. It is to be noted that the lower limit value of the amount of the charged substance contained in the lipid membrane is 0.
Examples of the charged substance for giving a positive electric charge to the lipid membrane include: saturated and unsaturated aliphatic amines such as stearylamine, oleylamine, and the like; saturated and unsaturated cationic synthetic lipids such as dioleoyltrimethylammoniumpropane and the like; and the like. Examples of the charged substance for giving a negative electric charge to the lipid membrane include dicetylphosphate, cholesteryl hemisuccinate, phosphatidylserine, phosphatidylinositol, and phosphatidic acid.
A membrane protein is a component optionally added to the lipid membrane to maintain the structure of the lipid membrane and give functionality to the lipid membrane. The amount of the membrane protein contained in the lipid membrane is usually 10% (mole ratio) or less, preferably 5% (mole ratio) or less, more preferably 2% (mole ratio) or less of the total amount of substances constituting the lipid membrane. It is to be noted that the lower limit value of the amount of the membrane protein contained in the lipid membrane is 0.
Examples of the membrane protein include peripheral membrane proteins, integral membrane proteins, and the like.
As the lipid constituting the lipid membrane of the lipid membrane structure, for example, a lipid derivative having blood retentive function, temperature change-sensitive function, pH-sensitive function, or the like can be used. By doing so, it is possible to give one or two or more of these functions to the lipid membrane structure. By giving blood retentive function to the lipid membrane structure, it is possible to improve the ability of the lipid membrane structure to remain in the blood, thereby reducing the rate of capture of the lipid membrane structures by reticuloendothelial systems such as liver, spleen, and the like. Further, by giving temperature change-sensitive function and/or pH-sensitive function to the lipid membrane structure, it is possible to enhance the releasability of a target substance retained in the lipid membrane structure.
Examples of the blood retentive lipid derivative which can give blood retentive function to the lipid membrane structure include glycophorin, ganglioside GM1, phosphatidylinositol, ganglioside GM3, glucuronic acid derivatives, glutamic acid derivatives, polyglycerin phospholipid derivatives, and polyethylene glycol derivatives such as 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 glycol-2000}-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-{carbonyl-methoxypolyethylene glycol-5000}-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and the like.
Examples of the temperature change-sensitive lipid derivative which can give temperature change sensitive function to the lipid membrane structure include dipalmitoylphosphatidylcholine and the like. Examples of the pH-sensitive lipid derivative which can give pH-sensitive function to the lipid membrane structure include dioleoylphosphatidyl ethanolamine and the like.
The lipid membrane structure can have an antibody which can specifically recognize a cell containing a target nucleus, an enzyme secreted by the cell, or the like. Such an antibody is preferably a monoclonal antibody. The monoclonal antibody may be one kind of monoclonal antibody having specificity for a single epitope or a combination of two or more kinds of monoclonal antibodies having specificity for various epitopes. Further, the antibody may be either a monovalent antibody or a multivalent antibody, and a naturally occurring type (intact) molecule or a fragment or derivative thereof may be used. For example, F(ab′)2, Fab′, Fab, a chimeric antibody or hybrid antibody having at least two antigen- or epitope-binding sites, a bispecific recombinant antibody such as quadrome or triome, an interspecific hybrid antibody, an anti-idiotypic antibody, or a derivative thereof obtained by chemically modifying or processing any one of these antibodies may be used. Further, an antibody synthetically or semisynthetically obtained using known cell fusion techniques, known hybridoma techniques, or antibody engineering techniques, an antibody obtained by DNA recombinant techniques using conventional techniques known from the viewpoint of producing antibodies, an antibody having a neutralization or binding property for a target epitope, or the like may be used.
The first or second lipid membrane contains an anionic lipid as a component thereof. The amount of the anionic lipid contained in the first lipid membrane is not particularly limited, but is usually 20 to 80% (mole ratio), preferably 40 to 60% (mole ratio), more preferably 45 to 55% (mole ratio) of the total amount of lipids contained in the first lipid membrane. By setting the amount of the anionic lipid contained in the first lipid membrane to a value within the above range, it is possible to improve the ability of the first lipid membrane to bind to and fuse with a nuclear membrane. The amount of the anionic lipid contained in the second lipid membrane is not particularly limited, but is usually 10 to 90% (mole ratio), preferably 10 to 50% (mole ratio), more preferably 10 to 30% (mole ratio) of the total amount of lipids contained in the second lipid membrane. By setting the amount of the anionic lipid contained in the second lipid membrane to a value within the above range, it is possible to improve the ability of the second lipid membrane to bind to and fuse with an endosomal membrane or a macropinosomal membrane.
The anionic lipid contained in the first or second lipid membrane is not particularly limited, and examples thereof include cholesteryl hemisuccinate, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and cardiolipin. The anionic lipid contained in the first lipid membrane is preferably choresteryl hemisuccinate, phosphatidic acid, or cardiolipin, more preferably phosphatidic acid or cardiolipin. The anionic lipid contained in the second lipid membrane is preferably cholesteryl hemisuccinate, phosphatidic acid, cardiolipin, dialkyl phosphate, or diacyl phosphate, more preferably phosphatidic acid, cardiolipin, dialkyl phosphate, or diacyl phosphate. In a case where the anionic lipid contained in the first lipid membrane is phosphatidic acid or cardiolipin, the amount of the anionic lipid contained in the first lipid membrane is preferably 40 to 60% (mole ratio) (particularly preferably, 45 to 55% (mole ratio)) of the total amount of lipids contained in the first lipid membrane, thereby making it possible to significantly improve the ability of the first lipid membrane to bind to and fuse with a nuclear membrane. In a case where the anionic lipid contained in the second lipid membrane is phosphatidic acid, cardiolipin, dialkyl phosphate, or diacyl phosphate, the amount of the anionic lipid contained in the second lipid membrane is preferably 10 to 50% (mole ratio) (particularly preferably, 10 to 30% (mole ratio)) of the total amount of lipids contained in the second lipid membrane, thereby making it possible to significantly improve the ability of the second lipid membrane to bind to and fuse with an endosomal membrane or a macropinosomal membrane.
The first or second lipid membrane preferably contains dioleoylphosphatidyl ethanolamine. By allowing the first lipid membrane to contain dioleoylphosphatidyl ethanolamine, it is possible to improve the ability of the first lipid membrane to bind to and fuse with a nuclear membrane. By allowing the second lipid membrane to contain dioleoylphosphatidyl ethanolamine, it is possible to improve the ability of the second lipid membrane to bind to and fuse with an endosomal membrane or a macropinosomal membrane. The amount of dioleoylphosphatidyl ethanolamine contained in the first lipid membrane is not particularly limited, but is usually 20 to 80% (mole ratio), preferably 40 to 60% (mole ratio), more preferably 45 to 55% (mole ratio) of the total amount of lipids contained in the first lipid membrane. By setting the amount of dioleoylphosphatidyl ethanolamine contained in the first lipid membrane to a value within the above range, it is possible to improve the ability of the first lipid membrane to bind to and fuse with a nuclear membrane. The amount of dioleoylphosphatidyl ethanolamine contained in the second lipid membrane is not particularly limited, but is usually 10 to 90% (mole ratio), preferably 50 to 90% (mole ratio), more preferably 70 to 90% (mole ratio) of the total amount of lipids contained in the second lipid membrane. By setting the amount of dioleoylphosphatidyl ethanolamine contained in the second lipid membrane to a value within the above range, it is possible to improve the ability of the second lipid membrane to bind to and fuse with an endosomal membrane or a macropinosomal membrane It is to be noted that lipids are broadly classified into three types: cone-shaped type, cylinder-shaped type, and inverted cone-shaped type according to their proportion between polar groups and nonpolar groups contained therein. Among them, a cone-shaped lipid such as dioleoylphosphatidyl ethanolamine is a lipid in which hydrophobic groups occupy a larger volume than hydrophilic groups, and is also referred to as “nonbilayer lipid” (see, N. Oku, “Preparation and Experimentation of Liposome”, pp. 27-33, Hirokawa-Shoten Ltd.). It is considered that a cone-shaped lipid has an inverted hexagonal structure in a lipid bilayer and therefore an inverted micelle structure is formed in the lipid bilayer, and the thus formed inverted micelle structure is involved in membrane fusion, membrane permeation, and the like.
The first or second lipid membrane preferably has a membrane-permeable peptide. The membrane-permeable peptide is present in the first or second lipid membrane in a state where it can bind to a nuclear membrane. Preferably, the membrane-permeable peptide is present on the surface of the first or second lipid membrane. By allowing the membrane-permeable peptide to be present on the surface of the first lipid membrane, it is possible to improve the ability of the first lipid membrane to bind to and fuse with a nuclear membrane. By allowing the membrane-permeable peptide to be present on the surface of the second lipid membrane, it is possible to improve the ability of the second lipid membrane to bind to and fuse with an endosomal membrane or a macropinosomal membrane.
In a case where the membrane-permeable peptide is present on the surface of the first or second lipid membrane, the membrane-permeable peptide is present on at least the external surface of the first or second lipid membrane, that is, it is not always necessary for the membrane-permeable peptide to be present on the internal surface of the first or second lipid membrane. In a case where the first lipid membrane has a membrane-permeable peptide, the anionic lipid contained in the first lipid membrane is preferably cholesteryl hemisuccinate, phosphatidic acid, or phosphatidylserine. By allowing the first lipid membrane to have a membrane-permeable peptide as well as such an anionic lipid, it is possible to improve the ability of the first lipid membrane to bind to and fuse with a nuclear membrane.
The membrane-permeable peptide contained in the first or second lipid membrane is not particularly limited as long as it can permeate a lipid membrane. Such a 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 biomembranes such as cell membranes and nuclear membranes and artificial membranes such as liposomal membranes. Examples of the membrane-permeable peptide include peptides having a protein transduction domain (PTD). Examples of the protein transduction domain include polyarginine, HIV-1-derived Tat (48-60) (GRKKRRQRRRPPQ), and HIV-1-derived Rev (34-50) (TRQARRNRRRRWRERQR). Such protein transduction domains are rich in arginine residues, and can therefore electrostatically interact with a negatively-charged nuclear membrane.
The total number of amino acid residues constituting the peptide having a protein transduction domain is not particularly limited, but is usually 4 to 40, preferably 6 to 30, more preferably 7 to 20. The total number of amino acid residues constituting the protein transduction domain is not particularly limited, but is usually 4 to 40, preferably 6 to 30, more preferably 7 to 20. The number of consecutive arginine residues constituting polyarginine as a protein transduction domain is usually 4 to 20, preferably 6 to 12, more preferably 7 to 10.
The peptide having a protein transduction domain may be composed of only a protein transduction domain, or may have any amino acid sequence added to the C-terminal and/or the N-terminal of a protein transduction domain. The amino acid sequence added to the C-terminal and/or the N-terminal of a protein transduction domain is preferably an amino acid sequence (e.g., polyproline) having rigidity. Unlike polyethylene glycol (PEG) which is soft and has no regular shape, polyproline is straight and maintains a certain level of rigidity. The amino acid residues contained in the amino acid sequence added to the C-terminal and/or the N-terminal of a protein transduction domain are preferably amino acid residues other than acidic amino acid residues. This is because there is a possibility that negatively-charged acidic amino acid residues electrostatically interact with positively-charged arginine residues and therefore the effect of the arginine residues contained in a protein transduction domain is reduced.
The amount of the membrane-permeable peptide present in the first lipid membrane is usually 2 to 20% (mole ratio), preferably 3 to 15% (mole ratio), more preferably 4 to 10% (mole ratio) of the total amount of lipids contained in the lipid membrane. By setting the amount of the membrane-permeable peptide present in the first lipid membrane to a value within the above range, it is possible to improve the ability of the first lipid membrane to bind to a nuclear membrane, thereby making it possible to efficiently bind the first lipid membrane to a nuclear membrane and therefore to efficiently induce membrane fusion between the first lipid membrane and the nuclear membrane.
The amount of the membrane-permeable peptide present in the second lipid membrane is usually 2 to 20% (mole ratio), preferably 3 to 15% (mole ratio), more preferably 4 to 10% (mole ratio) of the total amount of lipids contained in the lipid membrane. By setting the amount of the membrane-permeable peptide present in the second lipid membrane to a value within the above range, it is possible to improve the ability of the second lipid membrane to bind to an endosomal membrane or a macropinosomal membrane, thereby making it possible to efficiently bind the second lipid membrane to an endosomal membrane or a macropinosomal membrane and therefore to efficiently induce membrane fusion between the second lipid membrane and the endosomal membrane or the macropinosomal membrane.
An example of the aspect of the membrane-permeable peptide present in the first or second lipid membrane includes an aspect in which the membrane-permeable peptide binds to a lipid membrane constituent (e.g., a hydrophobic group, a hydrophobic compound), retained in the lipid membrane, so as to be partially or entirely exposed to the outside of the lipid membrane.
The lipid membrane constituent, to which the membrane-permeable peptide is to be bound, is not particularly limited, and examples thereof include: saturated and unsaturated fatty acid groups such as a stearyl group; cholesterol groups and derivatives thereof; phospholipids, glycolipids, and sterols; long-chain aliphatic alcohols (e.g., phosphatidyl ethanolamine, cholesterol); polyoxypropylene alkyls; and glycerin fatty acid esters. Among them, fatty acid groups having 10 to 20 carbon atoms (e.g., a palmitoyl group, an oleyl group, a stearyl group, an arachidoyl group) are particularly preferred.
The first, second, or third vector can retain a target substance to be delivered into a nucleus or a cell. For example, the first, second, or third vector can retain a target substance in an internal space (e.g., in a void formed in the lipid membrane structure), on the surface of a lipid membrane, in a lipid membrane, in a lipid membrane layer, or on the surface of a lipid membrane layer of the lipid membrane structure. In a case where the lipid membrane structure is a microparticle such as a liposome or the like, the target substance can be entrapped in the microparticle.
The type of target substance is not particularly limited, and examples of the target substance include drugs, nucleic acids, peptides, proteins, sugars, and complexes of two or more of them. The target substance can be appropriately selected according to the intended use such as diagnosis, treatment, or prevention of diseases. In a case where the lipid membrane structure retains a target substance intended to be used for diagnosis, treatment, or prevention of diseases, such a lipid membrane structure can be used as a constituent of a pharmaceutical composition. It is to be noted that “nucleic acids” include not only DNA and RNA but also analogues and derivatives thereof (e.g., peptide nucleic acids (PNAs), phosphorothioate DNA). The nucleic acid may be either single-stranded or double-stranded, and may be either linear or cyclic.
In a case where the target substance is a gene, the lipid membrane structure preferably has a compound having gene transfer function from the viewpoint of improving the efficiency of introducing a gene into a cell. Examples of such a compound having gene transfer function include O,O′—N-didodecanoyl-N-(α-trimethylammonioacetyl)-diethanol amine 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)aminome thane bromide, nor[α-trimethylammonioacetyl]-didodecyl-D-glutamate, dimethyldioctadecylammonium bromide, 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethy 1-1-propaneammonium trifluoroacetate, 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide, and 3-β-[n-(N′,N′-dimethylaminoethane)carbamoyl]cholesterol. These compounds having gene transfer function can be present in (bind to) an internal space (e.g., in a void formed in the lipid membrane structure), in a lipid membrane, on the surface of a lipid membrane, in a lipid membrane layer, or on the surface of a lipid membrane layer of the lipid membrane structure.
The target substance is preferably retained in the lipid membrane structure (particularly preferably, in the internal space of the lipid membrane structure) in the form of an aggregate of the target substance. This makes it possible to efficiently deliver the target substance into a nucleus.
The aggregate of the target substance may be composed of only the target substance or may contain a substance other than the target substance (e.g., a carrier for holding the target substance).
In a case where the target substance is positively charged, an aggregate of the target substance can be prepared by, for example, electrostatically binding the target substance to an anionic substance to form a complex. In a case where the target substance is negatively charged, an aggregate of the target substance can be prepared by, for example, electrostatically binding the target substance to a cationic substance to form a complex. In a case where the target substance is charged neither positively nor negatively, an aggregate of the target substance can be prepared by binding the target substance to a predetermined carrier in an appropriate manner (e.g., physical adsorption, hydrophobic bonding, chemical bonding) to form a complex. By adjusting the mixing ratio between the target substance and the cationic substance or the anionic substance in forming a complex, it is possible to prepare an aggregate of the target substance positively or negatively charged as a whole.
In a case where the target substance is nucleic acid, an aggregate of nucleic acid can be prepared by electrostatically binding nucleic acid to a cationic substance to form a complex. By adjusting the mixing ratio between the nucleic acid and the cationic substance in forming a complex, it is possible to prepare an aggregate of nucleic acid positively or negatively charged as a whole.
The cationic substance used for preparing an aggregate of the target substance is not particularly limited as long as it has a cationic group in its molecule. Examples of such a cationic substance include: cationic lipids (e.g., Lipofectamine (manufactured by Invitrogen)); cationic group-containing polymers; homopolymers and copolymers of basic amino acids such as polylysine, polyarginine, and copolymers of lysine and arginine, and derivatives thereof (e.g., stearylated derivatives); polycationic polymers such as polyethyleneimine, poly(allylamine), poly(diallyldimethylammonium chloride), and glucosamine; protamine and derivatives thereof (e.g., protamine sulfate); and chitosan. Among these cationic substances, stearylated polyarginine is particularly preferred. The number of arginine residues constituting polyarginine is usually 4 to 20, preferably 6 to 12, more preferably 7 to 10. The number of cationic groups contained in 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, and examples thereof include an amino group, monoalkylamino groups such as a methylamino group and an ethylamino group; dialkylamino groups such as a dimethylamino group and a diethylamino group; an imino group; and a guanidino group.
The anionic substance used for preparing an aggregate of the target substance is not particularly limited as long as it has an anionic group in its molecule. Examples of such an anionic substance include: anionic lipids; anionic group-containing polymers; homopolymers and copolymers of acidic amino acids such as polyaspartic acid, and derivatives thereof; and polyanionic polymers such as xanthan gum, carboxyvinyl polymers, carboxymethyl cellulose polystyrene sulfonate, polysaccharide, and carrageenan. 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, and examples thereof include functional groups having a terminal carboxyl group (e.g., a succinic acid residue, a malonic acid residue), a phosphate group, and a sulfate group.
The form of the lipid membrane structure is not particularly limited, and examples thereof include a form of dry mixture, a form of dispersion in an aqueous solvent, and a dried or frozen form thereof. These lipid membrane structures in such various forms can be produced using known methods such as hydration, ultrasonic treatment, ethanol injection, ether injection, reverse-phase evaporation, a surfactant method, freezing and thawing, and the like.
Hereinbelow, methods for producing lipid membrane structures in various forms will be described, but the form and production method of the lipid membrane structure according to the present invention are not limited thereto.
The lipid membrane structure in the form of dry mixture can be produced by, for example, once dissolving all the components of the lipid membrane structure in an organic solvent such as chloroform and then by subjecting the thus obtained solution to drying under reduced pressure using an evaporator or to spray drying using a spray drier.
The lipid membrane structure in the form of dispersion in an aqueous solvent can be produced by adding the lipid membrane structure in the form of dry mixture to an aqueous solvent and then emulsifying the thus obtained mixture using an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like. Alternatively, the lipid membrane structure in the form of dispersion in an aqueous solvent can also be produced by a method well-known as a method for producing a liposome, such as a reverse-phase evaporation method. In a case where it is desired that the size of the lipid membrane structure is controlled, extrusion can be performed under high pressure using a membrane filter or the like having pores uniform in pore size.
The composition of the aqueous solvent (dispersion medium) is not particularly limited, and examples of the aqueous solvent include: buffer solutions such as phosphate buffer, citrate buffer, and phosphate buffered saline; normal saline; culture media for cell culture; and the like. The lipid membrane structures can be stably dispersed in such an aqueous solvent (dispersion medium), but in order to further stably disperse the lipid membrane structures, a sugar or an aqueous solution thereof, a polyhydric alcohol or an aqueous solution thereof, or the like may be added to the aqueous solvent. Examples of the sugar include; monosaccharides (e.g., glucose, galactose, mannose, fructose, inositol, ribose, and xylose); disaccharides (e.g., lactose, sucrose, cellobiose, trehalose, and maltose); trisaccharides (e.g., raffinose and melezitose); polysaccharides (e.g., cyclodextrin); sugar alcohols (e.g., erythritol, xylitol, sorbitol, mannitol, and maltitol); and the like. Examples of the polyhydric alcohol include glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, 1,3-butylene glycol, and the like. In order to stably store the lipid membrane structures dispersed in an aqueous solvent (dispersion medium) for a long period of time, it is preferred that electrolytes in the aqueous solvent (dispersion medium) are minimized from the viewpoint of physical stability (e.g., from the viewpoint of preventing aggregation). Further, from the viewpoint of chemical stability of lipid, it is preferred that the pH of the aqueous solvent (dispersion medium) is adjusted to lie within a range from weakly acidic to around neutral (pH 3.0 to 8.0) or dissolved oxygen is removed by nitrogen bubbling.
The lipid membrane structure in a dried or frozen form of the lipid membrane structure dispersed in an aqueous solvent can be produced by drying or freezing the lipid membrane structures dispersed in an aqueous solvent by a well-known method such as freeze drying or spray drying. In a case where the lipid membrane structures dispersed in an aqueous solvent are once produced and then dried, there is an advantage that it becomes possible to store the lipid membrane structures for a long period of time. In addition, in a case where an aqueous solution containing a medicinally active ingredient is added to the dried lipid membrane structures, there is also an advantage that it is possible to efficiently hydrate the lipid mixture and therefore to allow the lipid membrane structures to efficiently retain the medicinally active ingredient.
In a case where the lipid membrane structures dispersed in an aqueous solvent are dried by freeze drying or spray drying, a sugar or an aqueous solution thereof may be added to the aqueous solvent (dispersion medium) to stably store the lipid membrane structures for a long period of time. Examples of the sugar include: monosaccharides (e.g., glucose, galactose, mannose, fructose, inositol, ribose, and xylose); disaccharides (e.g., lactose, sucrose, cellobiose, trehalose, and maltose); trisaccharides (e.g., raffinose and melezitose); polysaccharides (e.g., cyclodextrin); sugar alcohols (e.g., erythritol, xylitol, sorbitol, mannitol, and maltitol); and the like. Further, in a case where the lipid membrane structures dispersed in an aqueous solvent are frozen, for example, the above-mentioned sugar or an aqueous solution thereof or a polyhydric alcohol or an aqueous solution thereof may be added to the aqueous solvent (dispersion medium) to stably store the lipid membrane structures for a long period of time. Examples of the polyhydric alcohol include glycerin, diglycerin, polyglycerin, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, 1,3-butylene glycol, and the like. It is to be noted that both the sugar and the polyhydric alcohol may be added to the aqueous solvent (dispersion medium). The concentration of the sugar or the polyhydric alcohol in the aqueous solvent (dispersion medium) in which the lipid membrane structures are dispersed is not particularly limited, but the concentration of the sugar is preferably 2 to 20% (W/V), more preferably 5 to 10% (W/V), and the concentration of the polyhydric alcohol is preferably 1 to 5% (W/V), more preferably 2 to 2.5% (W/V). In a case where a buffer solution is used as the aqueous solvent (dispersion medium), the concentration of a buffering agent in the buffer solution is preferably 5 to 50 mM, more preferably 10 to 20 mM. The concentration of the lipid membrane structures in the aqueous solvent (dispersion medium) is not particularly limited. However, when expressed as a concentration of the total amount of lipids contained in the lipid membrane structures, the concentration of the lipid membrane structures in the aqueous solvent is preferably 0.1 to 500 mM, more preferably 1 to 100 mM.
The lipid membrane structure retaining an antibody can be produced by producing a lipid membrane structure and then adding an antibody to bind the antibody to the surface of a lipid membrane of the lipid membrane structure. Alternatively, the lipid membrane structure retaining an antibody can also be produced by producing a lipid membrane structure and then adding an antibody and a lipid derivative which can react with a mercapto group contained in the antibody to bind the antibody to the surface of a lipid membrane of the lipid membrane structure.
The form of a pharmaceutical composition containing the lipid membrane structures retaining a target substance is not particularly limited, and examples thereof include a form of dry mixture, a form of dispersion in an aqueous solvent, and a dried or frozen form thereof, and the like. These pharmaceutical compositions in such various forms can be produced in the same manner as the above-described lipid membrane structures in various forms.
The pharmaceutical composition in the form of dry mixture can be produced by, for example, once dissolving the components of the lipid membrane structure and a target substance in an organic solvent such as chloroform to obtain a mixture and then subjecting the mixture to drying under reduced pressure using an evaporator or to spray drying using a spray drier.
The pharmaceutical composition in a form that the lipid membrane structures retaining a target substance are dispersed in an aqueous solvent can be produced by any one of the following various known methods, which can be appropriately selected according to the form of a target substance retained in the lipid membrane structure, properties of a mixture, etc.
<Production Method 1>
A production method 1 is a method for producing a pharmaceutical composition in a form that the lipid membrane structures retaining a target substance are dispersed in an aqueous solvent, which includes once dissolving the components of the lipid membrane structure and a target substance in an organic solvent such as chloroform to obtain a mixture, subjecting the mixture to drying under reduced pressure using an evaporator or to spray drying using a spray drier to produce a dry mixture containing the lipid membrane structures and the target substance, and adding an aqueous solvent to the dry mixture to carry out emulsification using an emulsifier such as a homogenizer, an ultrasonic emulsifier, a high-pressure jet emulsifier, or the like. In a case where it is desired that the size (particle size) of the lipid membrane structure is controlled, extrusion can be performed under high pressure using a membrane filter having pores uniform in pore size. In the case of using the production method 1, it is necessary to dissolve the components of the lipid membrane structure and a target substance in an organic solvent to produce a dry mixture containing the lipid membrane structures and the target substance, but there is an advantage that it is possible to make the most of the interaction between the components of the lipid membrane structure and the target substance. That is, the production method 1 has an advantage that even in a case where the lipid membrane structure has a layered structure, the target substance can enter the inside of multiple layers of the layered structure and therefore the retention ratio of the target substance in the lipid membrane structure can be improved.
<Production Method 2>
A production method 2 is a method for producing a pharmaceutical composition in a form that the lipid membrane structures retaining a target substance are dispersed in an aqueous solvent, which includes once dissolving the components of the lipid membrane structure in an organic solvent, removing the organic solvent by evaporation to obtain dried matter, and adding an aqueous solvent containing a target substance to the dried matter to carry out emulsification. In a case where it is desired that the size (particle size) of the lipid membrane structure is controlled, extrusion can be performed under high pressure using a membrane filter having pores uniform in pore size. The production method 2 can be used for a target substance which is hard to be dissolved in an organic solvent but can be dissolved in an aqueous solvent. Further, the production method 2 has an advantage that when the lipid membrane structure is a liposome, the target substance can be retained also in the internal aqueous portion of the liposome.
<Production Method 3>
A production method 3 is a method for producing a pharmaceutical composition in a form that the lipid membrane structures retaining a target substance are dispersed in an aqueous solvent, which includes further adding an aqueous solvent containing a target substance to lipid membrane structures such as liposomes, emulsions, micelles, layered structures or the like dispersed in an aqueous solvent. The production method 3 can be used for a water-soluble target substance. As described above, the production method 3 is a method in which a target substance is externally added to lipid membrane structures which have been already produced, and therefore there is a possibility that when the target substance is a polymer, the target substance cannot enter the inside of the lipid membrane structures so that the target substance exists on (binds to) the surface of the lipid membrane structures. It is known that in a case where liposomes are used as the lipid membrane structures, use of the production method 3 results in the formation of a sandwich structure (generally referred to as a “complex”) in which a target substance is sandwiched between liposome particles. In the case of using the production method 3, an aqueous dispersion liquid, in which only lipid membrane structures are dispersed, is previously prepared, and therefore it is not necessary to take decomposition of a target substance during emulsification into consideration and it is easy to control the size (particle size) of the lipid membrane structure. Therefore, as compared to the production methods 1 and 2, the production method 3 makes it possible to more easily produce a pharmaceutical composition in a form in which the lipid membrane structures retaining a target substance are dispersed in an aqueous solvent.
<Production Method 4>
A production method 4 is a method for producing a pharmaceutical composition in a form that lipid membrane structures retaining a target substance are dispersed in an aqueous solvent, which includes adding an aqueous solvent containing a target substance to a dried product obtained by drying lipid membrane structures dispersed in an aqueous solvent. As in the case of the production method 3, the production method 4 can be used for a water-soluble target substance. A difference between the production method 3 and the production method 4 is a mode of presence of the lipid membrane structures and the target substance. According to the production method 4, lipid membrane structures dispersed in an aqueous solvent are first produced and then dried to produce a dried product, and therefore at this stage, the lipid membrane structures are present in a solid state as fragments of lipid membranes. In order to allow the fragments of lipid membranes to be present in a solid state, as descried above, a solvent obtained by adding a sugar or an aqueous solution thereof to an aqueous solvent is preferably used, and a solvent obtained by adding sucrose or an aqueous solution thereof to an aqueous solvent or a solvent obtained by adding lactose or an aqueous solution thereof to an aqueous solvent is more preferably used. When an aqueous solvent containing a target substance is added, water enters the fragments of lipid membranes present in a solid state to quickly hydrate them, and then the lipid membrane structures are reconstructed. As a result, lipid membrane structures retaining the target substance therein are produced.
According to the production method 4, an aqueous dispersion in which only the lipid membrane structures are dispersed is previously prepared, and therefore decomposition of the target substance during emulsification need not be taken into consideration and the size (particle size) of the lipid membrane structure can be easily controlled. For this reason, as compared to the production methods 1 and 2, the production method 4 makes it possible to more easily produce a pharmaceutical composition in a form that lipid membrane structures retaining a target substance are dispersed in an aqueous solvent. In addition, the production method 4 also has advantages that storage stability of an obtained pharmaceutical preparation (pharmaceutical composition) can be easily secured because freeze drying or spray drying is once carried out; lipid membrane structures having their original size (particle size) can be obtained even when the dried pharmaceutical preparation is rehydrated with an aqueous solution of a target substance; and a target substance can be easily retained inside the lipid membrane structures even when the target substance is a polymer. In a case where the target substance is a polymer, the use of the production method 3 makes a possibility that the target substance cannot enter the inside of the lipid membrane structures and therefore binds to the surface of the lipid membrane structures. The production method 4 is significantly different from the production method 3 in this point.
As another method for producing a pharmaceutical composition in a form that lipid membrane structures retaining a target substance are dispersed in an aqueous solvent, a method well known as a method for producing a liposome, for example, a reverse-phase evaporation method can be used. In a case where it is desired that the size (particle size) of the lipid membrane structure is controlled, extrusion can be performed under high pressure using a membrane filter having pores uniform in pore size.
Examples of a method for drying a dispersion liquid, in which lipid membrane structures retaining a target substance are dispersed in an aqueous solvent, include freeze drying and spray drying. As the aqueous solvent, a solvent obtained by adding the above-mentioned sugar or an aqueous solution thereof to an aqueous solvent is preferably used, and a solvent obtained by adding sucrose or an aqueous solution thereof to an aqueous solvent or a solvent obtained by adding lactose or an aqueous solution thereof to an aqueous solvent is more preferably used. An example of a method for freezing a dispersion liquid, in which lipid membrane structures retaining a target substance are dispersed in an aqueous solvent, includes a freezing method usually used. As the aqueous solvent, a solvent obtained by adding the above-mentioned sugar or an aqueous solution thereof to an aqueous solvent or a solvent obtained by adding the above-mentioned polyhydric alcohol or an aqueous solution thereof to an aqueous solvent is preferably used.
When the first lipid membrane of the first vector is bound to a nuclear membrane, membrane fusion between the first lipid membrane and the nuclear membrane is induced so that a target substance retained in the lipid membrane structure is released into a nucleus, and as a result the target substance is delivered into the nucleus. For this reason, the first or third vector can be used as a vector for delivering a target substance into a nucleus.
When the second vector is introduced into a cell as an endosomal fraction, a macropinosomal fraction, or the like via a pathway such as endocytosis, macropinocytosis, or the like and then the second lipid membrane of the second vector is bound to an endosomal membrane or a macropinosomal membrane, membrane fusion between the second lipid membrane and the endosomal membrane or the macropinosomal membrane is induced so that a target substance retained in the lipid membrane structure escapes from an endosome, a macropinosome, or the like and is then released into a cell, and as a result the target substance is delivered into the cell. For this reason, the second vector can be used as a vector for delivering a target substance into a cell.
When the third vector is introduced into a cell as an endosomal fraction, a macropinosomal fraction, or the like via a pathway such as endocytosis, macropinocytosis, or the like and then the second lipid membrane of the third vector is bound to an endosomal membrane or a macropinosomal membrane, membrane fusion between the second lipid membrane and the endosomal membrane or the macropinosomal membrane is induced so that a target substance retained in the lipid membrane structure escapes from an endosome, a macropinosome, or the like and is then released into a cell. Then, when the first lipid membrane of the third vector is bound to a nuclear membrane, membrane fusion between the first lipid membrane and the nuclear membrane is induced so that the target substance retained in the lipid membrane structure is released into a nucleus, and as a result the target substance is delivered into the nucleus. For this reason, the third vector can be used as a vector for delivering a target substance into a nucleus.
A nucleus as a target for the first or third vector may be either one separated from a cell or one present in a cell. The kind of organism whose nucleus is a target for the first or third vector is not particularly limited, and examples of the organism include animals, plants, microorganisms, and the like. However, among these organisms, animals are preferred, and mammals are more preferred. Examples of the mammals include humans, monkeys, cows, sheep, goats, horses, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, and the like. The kind of cell containing a nucleus as a target for the first or third vector is not particularly limited, and examples of such a cell include somatic cells, germ cells, stem cells, and cultured cells thereof.
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These vectors described above can be used both in vivo and in vitro. In a case where any one of these vectors is used in vivo, it can be administered by a parenteral route such as an intravenous, intraperitoneal, subcutaneous, or nasal route. The dose and number of times of administration can be appropriately controlled according to the kind or amount of target substance entrapped in the lipid membrane structure according to the present invention.
Abbreviations used in the present examples are as follows.
BCA: bovine serum albumin
CHEMS: cholesteryl hemisuccinate
DMSO: dimethyl sulfoxide
DNA: deoxyribonucleic acid
DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
DOTAP: 1,2-dioleoyl-3-trimethylammonium propane
DTT: dithiothreitol
D′MEM: dulbecco's modified eagle medium
EDTA: ethylenediamine N′,N′,N′,N′-tetraacetic acid
EGTA: ethylene glycol-bis(β-aminoethyl ether)-N′,N′,N′,N′-tetraacetic acid
EPC: egg yolk L-α-phosphatidylcholine
GFP: green fluorescence protein
HBSS: Hank's balanced salt solution
HEPES: 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid
LB: luria-bertani's broth
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)
NLS: nuclear localization signal
NP-40: nonionic detergent
PA: 3-sn-phosphatidic acid
PBS: phosphate-buffered saline
PC: phosphatidylcholine
PS: 3-sn-phosphatidyl-L-serine
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)
STR-R8: stearylated octaarginine
rpm: round per minute
Manufacturers of reagents and materials used for experiments are as follows.
BCA protein assay kit (PIERCE)
GM1 (prepared in our laboratory)
Protamine sulfate (CALBIOCHEM)
Rho-R8 (obtained from Dr. S. Futaki, Kyoto University) pEGFPluc (prepared in our laboratory)
Some reagents were prepared in the following manner.
PBS: 4.9 g of Dalbecco PBS(−) (Nissui Pharmaceutical Co., Ltd.) was dissolved in 500 mL of DDW and was then autoclaved.
LB+kan: 10 g of Bacto tryptone (Wako Pure Chemical Industries), 5 g of Yeast extract (Wako Pure Chemical Industries), and 10 g of NaCl (SIGMA) were dissolved in DDW so that the total volume of the solution was 1 L, and the solution was autoclaved. Then, kanamysine was added to the solution so that the concentration of kanamysine was 20 mg/mL.
HEPES buffer (10 mM): 119 mg of HEPES (Wako Pure Chemical Industries) was dissolved in 50 mL of DDW to obtain a solution, and the pH of the solution was adjusted to 7.4 with 1N NaOH. Then, the solution was sterilized by filtration.
HEPES buffer 5% glucose (10 mM, pH 5.5): 119 mg of HEPES (Wako Pure Chemical Industries) and 2.5 g of D-glucose (katayama Chemical Industries) were dissolved in 50 mL of DDW to obtain a solution, and the pH of the solution was adjusted to 7.4 with 1N NaOH. Then, the solution was sterilized by filtration in a clean bench.
Import buffer (×10): 4.77 g of HEPES (Wako Pure Chemical Industries), 10.8 g of KOAc (Wako), 0.25 g of NaOAc (Wako), 0.43 g of MgOAc (SIGMA), and 0.23 g of EGTA.4Na were dissolved in DDW to obtain a solution, and the pH of the solution was adjusted to 7.3 with KOH. Then, DDW was added to the solution so that the total volume of the solution was 100 mL. 200 mM DTT was added to the solution just before use to dilute the solution 10-fold, and the diluted solution was sterilized by filtration.
200 mM DTT: 154.25 mg of DTT (nacalai tesque) was dissolved in DTT so that the total volume of the solution was 5 mL.
IGEPAL buffer: 0.5% (W/V) NP-40 (SIGMA), 10 mM NaCl, 3 mM MgCl2, and 10 mM Tris-HCl were mixed, and the pH of the mixture was adjusted to 7.4.
HBSS: 4.0 g of NaCl (SIGMA), 0.2 g of KCl, 0.07 g of CaCl2, 0.1 g of MgSO4.7H2O, KH2PO4, 0.5 g of glucose, and 0.18 g of NaHCO3 were dissolved in DDW, and the pH of the solution was adjusted to 7.5 with NaOH. Then, DDW was added to the solution so that the total volume of the solution was 500 mL.
1. Establishment of Method for Isolating Nuclei and Experimental System for Assaying Ability of Lipids to Bind to Nuclear Membranes
1-1. Introduction
The ability of lipids to bind to nuclear membranes is an essential factor to evaluate their ability to fuse with nuclear membranes. Therefore, in the first step of screening of lipids to find those having the ability to fuse with nuclear membranes, the ability of lipids to bind to nuclear membranes was evaluated. Since it has been reported that lipids such as PA and PS are fused with nuclear membrane (R. Lawaczeck.: Intraction of negatively charged liposomes with nuclear membranes: adsorption, lipid mixing and lysis of the vesicles. Biochem. Biophys. Acta. 903 (1987) 123-131), attention was focused on charged lipids, especially negatively-charged lipids in the following examples. Further, since it has been reported that a sugar chain has an affinity for lectin present in a nucleus, lipids having a sugar chain in their structures were also subjected to the screening. Lipids present in the nuclear membrane were also subjected to the screening. Lipids contained in the nuclear membrane are as follows: Cardiolipin 4%, PE 13%, PC 55%, PI 10%, PS 3%, PA 2%, lysoglycerophospholipid 3%, and SM 0.5%.
1-2. Establishment of Method for Isolating Nuclei
1-2-1. Cell Culture
(1) Preparation of Hela Cells
A stock of Hela cells was mixed with and dissolved in 20 mL of D′MEM (containing 10% inactivated FCS) previously placed in a 50 mL conical tube. The conical tube was centrifuged at 1000 rpm at 25° C. for 5 minutes. Then, the supernatant was removed from the conical tube, and 10 mL of D′MEM was added to the conical tube to suspend the cells. The cells were placed in a 10 cm dish and were then cultured in a CO2 incubator at 37° C.
(2) Subculture of Cells
The D′MEM in the 10 cm dish was removed by an aspirator, and then the cells were washed with 5 mL of PBS(−). Then, the PBS(−) was removed, and 2 mL of PBS(−) and 200 μL of a 0.5% trypsin/5 μL EDTA solution were added to the dish, and the cells were incubated in a 5% CO2 incubator at 37° C. for 5 minutes. Then, the cells were peeled off from the bottom surface of the dish. 8 mL of D′MEM was added to the dish to suspend the cells, and the thus obtained cell suspension was transferred into a 50 mL conical tube and was then centrifuged at 1000 rpm at 25° C. for minutes. Then, the supernatant was removed from the conical tube, and 10 mL of D′MEM was added to the conical tube to suspend the cells. The number of cells contained in the cell suspension was counted, and the cell suspension was diluted with D′MEM to an appropriate concentration, and the cells were placed in a cm dish. It is to be noted that in a case where cell culture was continued for two or more days until the next subculture, the D′MEM was replaced with fresh one every two days.
(3) Cell Counting
The cells were suspended in D′MEM to prepare a cell suspension, and 10 μL of the cell suspension was placed in a 1.5 mL sample tube. Then, 10 μL of a 0.3% trypan blue solution was added to the cell suspension, and they were thoroughly mixed with a pipette. Then, 10 μL of the thus obtained solution was placed onto a hemocytometer to count the number of cells.
(4) Preparation of Stock of Cells
Seventy percent confluent cells were used. The D′MEM in the 10 cm dish was removed by an aspirator, and the cells were washed with 5 mL of PBS(−). Then, the PBS(−) was removed from the dish, and then 2 mL of PBS(−) and 200 μL of a 0.5% trypsin/5 μL EDTA solution were added to the dish, and the cells were incubated in a 5% CO2 incubator at 37° C. for 5 minutes. Then, the cells were peeled off from the bottom surface of the dish. 8 mL of D′MEM was added to the dish to suspend the cells, and the thus obtained cell suspension was transferred into a 50 mL conical tube and was then centrifuged at 1000 rpm at 25° C. for 5 minutes. Then, the supernatant was removed from the conical tube, and then 10 mL of D′MEM was added to the conical tube to suspend the cells to obtain a cell suspension. The number of cells contained in the cell suspension was counted. The cell suspension was again centrifuged at 1000 rpm at 25° C. for 5 minutes, and then the supernatant was removed. A solution obtained by mixing D′MEM and DMSO at a ratio of 9:1 was added to the cells so that the cell suspension had a desired cell concentration. The cell suspension was suspended and then dispensed in 1.5 mL sample tubes with cap so that the volume of the cell suspension per sample tube was 1 mL. Then, the sample tubes were placed in a container filled with isopropanol, and frozen in a freezer kept at −80° C. for 4 or more hours, and then stored in a cell storage tank filled with liquid nitrogen.
1-2-2. Method for Isolating Nuclei
(1) Protocol Ideas
(a) Protocol Using IGEPAL Buffer
6×105 cells/dish of Hela cells were placed in a 6 cm dish one day before. IGEPAL buffer and PBS were returned to room temperature before use. The cells were washed with PBS(−) twice, and then 500 μL of PBS(−) was added to the cells. The cells were collected using a cell scraper and transferred into a 1.5 mL sample tube, and were then centrifuged at 1000 rpm at 4° C. for 5 minutes. The supernatant was removed by an aspirator, and then 500 μL of IGEPAL buffer was added to the pellet, and the IGEPAL buffer and the pellet were mixed with a pipette. The thus obtained suspension was centrifuged at 1400 g at 4° C. for 5 minutes. The supernatant was removed by an aspirator, and then 500 μL of IGEPAL buffer was again added to the pellet, and the pellet and the IGEPAL buffer were mixed with a pipette. The thus obtained suspension was centrifuged at 1400 g at 4° C. for 5 minutes. The thus obtained isolated nuclei were suspended in Import buffer.
(b) Protocol Using HBSS (J. Hasbold.: Flow Cytometric Cell Division Tracking Using Nuclei. Cytometry 40 (2000) 230-237)
An appropriate number of Hela cells were placed in a 10 cm dish two days before. An HBSS/0.1% BSA solution was placed on ice before the experiment. The cells were washed with PBS(−)/0.1% BSA/0.1% NaN3 twice, and then 500 μL of PBS(−)/0.1% BSA/0.1% NaN3 was added to the cells. The cells were collected using a cell scraper and transferred into a 1.5 mL sample tube, and were then centrifuged at 1000 rpm at 4° C. for 5 minutes. The supernatant was removed by an aspirator, and then 100 μL of HBSS/0.1% BSA was added to the pellet and the thus obtained suspension was mixed by tapping the tube. Then, 100 μL of an HBSS/0.1% BSA/0.2% NP-40 solution was further added to the suspension, and the suspension was incubated on ice for 5 minutes. Then, 800 μL of HBSS/0.1% BSA was added to the suspension, and the suspension was centrifuged at 10,000 rpm at 4° C. for 30 seconds. The supernatant was removed by an aspirator, and 1 mL of HBSS/0.02% NP-40 was added to the pellet to suspend the pellet. The thus obtained suspension was centrifuged at 10,000 rpm at 4° C. for 30 seconds. The supernatant was removed by an aspirator, and then 1 mL of HBSS/0.02% NP-40 was again added to the pellet to suspend the pellet. The thus obtained suspension was centrifuged at 10,000 rpm at 4° C. for 30 seconds. The supernatant was removed by an aspirator. The thus obtained isolated nuclei were suspended in Import buffer.
(c) Nuclear Staining with Hoechst
10 μL of Hoechst (1 mg/l mL) diluted 50-fold with buffer and 10 μL of a sample obtained by diluting the suspension of isolated nuclei to an appropriate concentration were mixed, and the mixture was incubated at room temperature for 10 minutes. Then, the mixture was placed onto a hemocytometer used also for cell counting, and the number of nuclei was counted using a fluorescent microscope.
(2) Consideration
In the case of the protocol using IGEPAL buffer, nuclei were aggregated during operation and therefore the nuclei were not suspended in IGEPAL buffer at all. It can be considered that this was mainly caused by the following factors: NP-40 contained in IGEPAL buffer promoted aggregation of cells; and the centrifugal speed was too high. Based on the consideration, the conditions of the second centrifugation were changed from 1400 g, 4° C., and 5 minutes to 2000 rpm, 4° C., and 5 minutes. However, after all, aggregation of nuclei occurred and a particular change was not observed. Based on the result, 500 μL of IGEPAL buffer to be added for the second time was changed to buffer not containing NP-40. As a result, aggregation of nuclei still occurred but the nuclei were suspended in the buffer to some extent. On the other hand, in the case of the protocol using HBSS, aggregation of nuclei did not occur and therefore nuclei were easily suspended in HBSS. 2×106 cells were placed in a dish one day before, and then nuclei were extracted from these cells and labeled with Hoechst to count the number of the nuclei. As a result, the number of the nuclei was 1.329×106. The extraction efficiency was calculated from the concentration of cells placed in a dish and was found to be 69.5%. Further, there is a possibility that a centrifugal speed of 10,000 rpm is too high and therefore not only nuclei but also other fractions are precipitated. In order to investigate the possibility, the conditions of centrifugation were changed to 1,000 rpm, 4° C., and 5 minutes, and as a result the resulting pellet was too loose. For this reason, the idea of changing the conditions of centrifugation was abandoned because there was a fear that not only the supernatant but also nuclei were removed by an aspirator. Based on the result, the conditions of centrifugation were changed to 1400 g, 4° C., and 1 minute. As a result, the resulting pellet had an appropriate hardness and was easily suspended in buffer, and fractions other than nuclei were not observed by a microscope. Therefore, these conditions were employed in the following experiments. It is to be noted that the number of cells placed in a dish for subculture two days before was decided to be 2.5×106 cells/dish, and the number of cells placed in a dish for subculture three days before was decided to be 1.5×106 cells/dish.
(3) Determination of Protocol for Extracting Nuclei
Hela cells of appropriate concentration were placed in a 10 cm dish two days before. An HBSS/0.1% BSA solution was placed on ice before experiment. The cells were washed with PBS(−)/0.1% BSA/0.1% NaN3 twice, and then 500 μL of PBS(−)/0.1% BSA/0.1% NaN3 was added to the cells. Then, the cells were collected using a cell scraper and transferred into a 1.5 mL sample tube, and were then centrifuged at 1000 rpm at 4° C. for 5 minutes. The supernatant was removed by an aspirator, and then 100 μL of HBSS/0.1% BSA was added to the pellet, and the thus obtained suspension was mixed by tapping the tube. Then, 100 μL of an HBSS/0.1% BSA/0.2% NP-40 solution was further added to the suspension, and the suspension was incubated on ice for 5 minutes. Then, 800 μL of HBSS/0.1% BSA was added to the suspension, and the suspension was centrifuged at 1400 g at 4° C. for 1 minute. The supernatant was removed by an aspirator, and then 1 mL of HBSS/0.02% NP-40 was added to the pellet to suspend the pellet. The thus obtained suspension was centrifuged at 1400 g at 4° C. for 1 minute. The supernatant was removed by an aspirator, and then 1 mL of HBSS/0.02% NP-40 was again added to the pellet to suspend the pellet. The thus obtained suspension was centrifuged at 1400 g at 4° C. for 1 minute. The supernatant was removed by an aspirator, and the thus obtained isolated nuclei were suspended in Import buffer.
1-3. Establishment of Experimental System for Assaying Binding of Liposomes to Nuclear Membranes (Binding Assay)
1-3-1. Preparation of Membrane-Labeled Liposomes
(1) Lipid:Chol=2:1
A 10 mM lipid stock and 10 mM Chol were mixed in a mole ratio of 2:1 to obtain a mixture, and a CHCl3 solution containing NBD-DOPE in an amount of 1 mol % of the total lipid concentration was added to the mixture. Then, CHCl3 was further added to the mixture and the mixture was dried in an atmosphere of nitrogen gas to prepare lipid film. Import buffer as an internal aqueous phase was added to the lipid film so that the total lipid concentration became 2 mM to hydrate the lipid film. Then, ultrasonic treatment was carried out to prepare liposomes.
(2) Lipid:DOPE=2:3
Liposomes were prepared in the same manner as in 1-3-1 (1) except that a 10 mM lipid stock and 10 mM DOPE were mixed in a mole ratio of 2:1 to obtain a mixture, and a CHCl3 solution containing NBD-DOPE in an amount of 1 mol % of the total lipid concentration was added thereto.
1-3-2. Experimental Method of Binding Assay
Nuclei were isolated by the method described in paragraph 1-2-2 and were then stained with Hoechst to count the number of the nuclei. 20 μL of each Probe Lip was added to about 5.0×105 nuclei placed in a 1.5 mL sample tube, and Import buffer was added to the sample tube so that the total volume of a sample contained in the sample tube was 200 μL. Likewise, 20 μL of Probe Lip was added to 180 μL of a 0.5% Triton solution to prepare a sample for measuring the maximum fluorescence intensity of liposomes. These samples were incubated at 37° C. for 1 hour and then centrifuged at 1400 g at 4° C. for 1 minute, and then each of the pellets was washed twice. Then, the pellet was suspended in 100 μL of a 0.5% Triton solution, and the fluorescence intensity of the suspension was measured at 530 nm. The binding rate of liposomes to nuclear membranes was calculated by the following formula: binding rate (%)=(fluorescence intensity of liposomes bound to nuclei)/(maximum fluorescence intensity of liposomes used)×100. It is to be noted that the number of nuclei used was corrected to 5.0×105.
1-4. Screening of Lipids to Find Those Having Ability to Bind to Nuclear Membranes (Experimental Results)
1-4-1. Evaluation of Influence of Electric Charge of Lipids on their Ability to Bind to Nuclear Membranes
The influence of electric charge of lipids on their ability to bind to nuclear membranes was examined using the following lipids: EPC(±), SM(±), DOTAP(+), PA(−), PS(−), PI(−), Cardiolipin(−), PG(−), and CHEMS (−).
Various liposomes were prepared using each of these lipids and Chol or DOPE in combination, and then the binding rates of these liposomes to nuclear membranes were determined. The results are shown in
1-4-2. Evaluation of Ability of Other Cell Functional Lipids to Bind to Nuclear Membranes
In order to evaluate the influence of factors other than electric charge on the ability of lipids to bind to nuclear membranes, an experiment was carried out using GM1 that is a lipid present in nuclear membranes and involved in physiological functions, cerebroside that is a lipid containing a sugar residue expected to interact with lectin present in a nucleus, etc. Lipids and surfactants used are as follows: GM1, DGDG, Galactocerebroside, Glucocerebroside, Lactocerebroside, and Octylglucoside.
Various liposomes were prepared using each of these lipids and EPC/Chol or EPC/DOPE in combination, and the binding rates of these liposomes to nuclear membranes were determined. The results are shown in
1-5. Summary
The above experiments were carried out to establish a method for isolating nuclei and an experimental method for assaying the ability of lipids to bind to nuclear membranes. The experimental results suggest that lipids need to have electric charge to bind to nuclear membranes of isolated nuclei. Further, it can be considered that the action of lipids involved in physiological functions in nuclear membranes and the interaction with sugar residues do not occur in test tubes containing only isolated nuclei because Importin, ATP, etc. involved in nuclear entry are not present in the test tubes. For this reason, electrically-charged lipids were used in the following experiments.
2. Establishment of Experimental System for Verifying Ability of Lipids to Fuse with Nuclear Membranes Using FRET
2-1. Introduction
FRET is a phenomenon observed during the interaction between the excitation wavelength of one of two fluorescent materials adjacent to each other and the fluorescence wavelength of the other fluorescent material. More specifically, FRET is a phenomenon in which, when two fluorescent materials are adjacent to each other and the wavelength of fluorescence emitted by exciting one of the two fluorescent materials (A) is close to the excitation wavelength of the other fluorescent material (B), the fluorescent material (B) is excited by the fluorescence wavelength to emit fluorescence and therefore it becomes impossible to observe the fluorescence of the fluorescent material (A). In this research, liposomes containing two kinds of fluorescent materials, NBD and Rho were prepared. When the liposomes are not fused with nuclear membranes, fluorescence energy derived from NBD is transferred to Rho, and therefore the fluorescence of NBD is suppressed but fluorescence with a wavelength of 590 nm that is the fluorescence wavelength of Rho is increased. On the other hand, when the liposomes are fused with nuclear membranes, the distance between the two fluorescent materials is increased so that the transfer of energy to Rho is canceled and therefore fluorescence with a wavelength of 530 nm that is the fluorescence wavelength of NBD is recovered. Based on the principles, fluorescence-labeled liposomes were prepared using the electrically-charged lipids which had been used in the experiments described in Chapter 1, and the ability of the liposomes to fuse with nuclear membranes was evaluated.
2-2. Verification of Ability of Lipids to Fuse with Nuclear Membranes Using FRET
2-2-1. Method for Quantitatively Determining the Amount of Phospholipid
In order to equalize the concentration of isolated nuclei and the concentration of liposomes, the amount of phospholipid contained in nuclei was measured using a kit, Phospholipid C-Test Wako according to the operation manuals included in the kit.
2-2-2. Examination of Conditions of Fluorescent Lipids
In order to observe the cancellation of FRET most clearly, optimum conditions of fluorescent lipids were examined by changing the concentrations of the fluorescent lipids. Various Probe Lips were prepared by compositions as shown in Table 1.
(1) Preparation of Non-probe Lips (pH 7.4 or pH 4.7) 6.65 mg (8.65 μmol) of EPC and 1.7 mg (4.35 μmol) of Chol were prepared and dissolved in 2.5 mL of CHCl3 to obtain a solution. Then, the solution was stirred using a vortex mixer, and 1 mL of the solution was placed into each of two glass test tubes. The solution in the glass test tubes was dried in an atmosphere of nitrogen gas to prepare lipid film. Then, 1 mL of PBS(−) was added to each of the two glass test tubes to hydrate the lipid film for 10 minutes. Then, ultrasonic treatment was carried out to prepare liposomes. Then, the liposome suspension was diluted 2.5-fold with PBS(−) (pH 7.4 or 4.7) (final total lipid concentration: 2 mM).
(2) Preparation of Probe Lips
30 μL of 10 mM DOPE and 20 μL of 10 mM CHEMS were placed in a glass test tube (lipid concentration: 0.5 μmol), and then x mol % of NBD-DOPE and y mol % of Rho-DOPE (see Table 1) were added to the test tube, and then CHCl3 was further added to the test tube. The thus obtained mixture was dried in an atmosphere of nitrogen gas to prepare lipid film. Then, 250 μL of PBS(−) (pH 7.4) was added to the lipid film to hydrate the lipid film for 10 minutes. Then, ultrasonic treatment was carried out to prepare liposomes.
(3) Analysis of FRET
In a 1.5 mL sample tube, the 2 mM Probe Lip and 2 mM Non-probe Lip prepared above were mixed in a ratio of 1:9 to prepare a sample. Further, the Probe Lip and PBS were mixed in a ratio of 1:9 to prepare a FRET control, and the Probe Lip and 0.5% Triton (pH 7.4) were mixed in a ratio of 1:9 to prepare a control for measuring the maximum fluorescence intensity of liposomes. These samples were incubated at 37° C. for 1 hour. Then, the pH of each of the samples was adjusted with 1N HCl, and the fluorescence intensity of each of the samples was measured at ex 470 nm and em 530 nm and 590 nm. The Transfer Efficiency of each sample was calculated by substituting the fluorescence intensities at 530 nm of the sample and controls into the following formula: TF=(F−F0)/(Fmax−F0)×100, where TF represents Transfer Efficiency, F represents the fluorescence intensity of the sample, F0 represents the fluorescence intensity of the blank, and Fmax represents the fluorescence intensity of the control obtained by breaking the liposomes by Triton having a final concentration of 0.5%.
The results are shown in Table 2. DOPE/CHEMS are lipids which are pH-dependently fused with membranes, and therefore it can be considered that membrane fusion hardly occurs at pH 4.7 but occurs at pH 7.4. As can be seen from Table 2, the difference between TF at pH 4.7 and TF at pH 7.4 is the largest when NBD and Rho are added in a ratio of 1:0.5, and at this time, TF at pH 7.4 is the largest. From the result, it can be considered that the cancellation of FRET is most clearly observed under the condition. Based on the results, this concentration was employed, respectively, in the following experiments.
2-2-3. Preparation of Membrane-Labeled Liposomes
(1) Lipid:Chol=2:1
A 10 mM lipid stock and 10 mM Chol were mixed in a mole ratio of 2:1 to obtain a mixture, and then a CHCl3 solution containing NBD-DOPE in an amount of 1 mol % of the total lipid concentration and a CHCl3 solution containing Rho-DOPE in an amount of 0.5 mol % of the total lipid concentration were added thereto, and then CHCl3 was further added thereto. The thus obtained mixture was dried in an atmosphere of nitrogen gas to prepare lipid film. Then, Import buffer as an inner aqueous phase was added to the lipid film so that the total lipid concentration became 2 mM to hydrate the lipid film. Then, ultrasonic treatment was carried out to prepare liposomes.
(2) Lipid:DOPE=2:3
Liposomes were prepared in the same manner as in 2-2-3 (1) except that a 10 mM lipid stock and 10 mM DOPE were mixed in a mole ratio of 2:1, and then a CHCl3 solution containing NBD-DOPE in an amount of 1 mol % of the total lipid concentration and a CHCl3 solution containing Rho-DOPE in an amount of 0.5 mol % of the total lipid concentration were added thereto.
2-2-4. FRET
The amount of phospholipid contained in nuclei isolated by the method described in Section 1-2-2 was measured to determine the amount of PC contained in the nuclei. It has been reported that the amount of PC contained in the nuclear membrane is 55%. Based on the report, the total concentration of lipids in the nuclei was determined. Then, the suspension of the isolated nuclei was diluted to have the same total lipid concentration (2 mM) as the Probe Lip. The Probe Lip prepared by the method described in Section 2-2-3 and the 2 mM suspension of isolated nuclei were mixed in a ratio of Probe Lip:nuclei=1:9 in a 1.5 mL sample tube, and the thus obtained mixture was incubated at 37° C. for 1 hour. A control was prepared by mixing Probe Lip and Import buffer in a ratio of 1:9 and incubating the mixture at 37° C. for 1 hour. A control for measuring the maximum fluorescence intensity of the liposomes was prepared by mixing Probe Lip and 0.5% Triton in a ratio of 1:9 and incubating the mixture at 37° C. for 1 hour. After the completion of incubation, each sample was diluted to 0.1 mM to measure a fluorescence intensity (excitation wavelength: 470 nm, absorption wavelength: 530 nm, 590 nm). The fluorescence intensity of the 2 mM sample was determined by calculation based on the measured fluorescence intensity of the 0.1 mM sample, and TF was calculated by substituting the fluorescence intensities at 530 nm of the sample and controls into the formula shown in Section 2-2-2.
2-3 Screening of Lipids to Find Those Having Ability to Fuse with Nuclear Membranes (Experimental Results)
FRET analysis was performed using EPC and lipids which had been found to have ability to bind to nuclear membranes by the experiment results described in Section 1-4. Lipids used in this experiment are as follows: EPC(±), SM(±), DOTAP(+), PA(−), PS(−), PI(−), Cardiolipin(−), PG(−), and CHEMS (−).
Various liposomes were prepared using these lipids by the method described in Section 2-2-3, and TFs of these liposomes were measured. The results are shown in
2-4. Summary
The above experiments were carried out to establish an experimental system for verifying the ability of lipids to fuse with nuclear membranes using FRET. From the experimental results, it has been confirmed that electrically-charged lipids not only bind to nuclear membranes but also fuse with nuclear membranes. However, it has been suggested that there is a possibility that liposomes are broken during incubation, and therefore it is necessary to devise a way to carry out incubation. Further, it can be considered that it is important to further study lipid composition such as a mixing ratio of DOPE or Chol to improve the ability of liposomes to fuse with membranes.
3. Evaluation of Affinity Between STR-R8-Modified Liposomes and Nuclear Membranes
3-1. Introduction
STR-R8 (CH3(CH2)16CONHRRRRRRRR) is a very strongly basic peptide. It has been reported that a STR-R8-modified liposome is introduced into a cell by macropinocytosis and then delivered into a nucleus with its lipid membrane structure being kept intact (S. Futaki, “Membrane permeable basic peptides”, PROTEIN, NUCLEIC ACID, AND ENZYME, Vol. 47 (2002), 1415-1419). Further, by adding STR-R8 to a liposome solution, it is possible to easily modify liposomes with STR-R8 to allow the liposomes to have positive electric charge, thereby improving the ability of the liposomes to bind to nuclear membranes. Therefore, it can be considered that an effective gene vector can be constructed by using a lipid which can keep its ability to fuse with nuclear membranes even when it is modified with STR-R8 and which can have an improved ability to bind to nuclear membranes by modification with STR-R8. In order to examine the influence of modification of liposomes with STR-R8 on their affinity for nuclear membranes and the correlation between the ability of liposomes modified with STR-R8 to bind to nuclear membranes and their ability to fuse with nuclear membranes, the following experiments were performed. It is to be noted that liposomes were prepared by mixing a lipid and DOPE (Chol) in a ratio of 9:2 so that the liposomes were easily modified with STR-R8.
3-2. Evaluation of Affinity Between STR-R8-Modified Liposomes and Nuclear Membranes
3-2-1. Preparation of Membrane-Labeled Liposomes
(1) Preparation of Liposomes for Binding Assay
(a) Lipid:Chol=9:2
A 1 mM lipid stock and a 1 mM Chol stock were mixed in a mole ratio of 9:2, and then a CHCl3 solution containing NBD-DOPE in an amount of 1 mol % of the total lipid concentration was added thereto, and then CHCl3 was further added thereto. The thus obtained mixture was dried in an atmosphere of nitrogen gas to prepare lipid film. Then, Import buffer as an inner aqueous phase was added to the lipid film so that the total lipid concentration became 0.55 mM to hydrate the lipid film. Then, ultrasonic treatment was carried out to prepare liposomes. In the case of modifying the liposomes with STR-R8, 5 mol % of STR-R8 was added to the liposome solution obtained by ultrasonic treatment, and then they were mixed with a pipette and the mixture was left stand at room temperature for 30 minutes.
(b) Lipid:DOPE=2:9
Liposomes and STR-R8-modified liposomes were prepared in the same manner as in Section 3-2-1 (1)(a) except that a 1 mM lipid stock and a 1 mM DOPE stock were mixed in a mole ratio of 2:9, and a CHCl3 solution containing NBD-DOPE in an amount of 1 mol % of the total lipid concentration was added thereto.
(2) Preparation of Liposomes Used for FRET Analysis Lipid:Chol=9:2
A 1 mM lipid stock and a 1 mM Chol stock were mixed in a mole ratio of 9:2, and then a CHCl3 solution containing NBD-DOPE in an amount of 1 mol % of the total lipid concentration and a CHCl3 solution containing Rho-DOPE in an amount of 0.5 mol % of the total lipid concentration were added thereto, and then CHCl3 was further added thereto. Then, the thus obtained mixture was dried in an atmosphere of nitrogen gas to prepare lipid film. Then, Import buffer as an internal aqueous phase was added to the lipid film so that the total lipid concentration became 0.55 mM to hydrate the lipid film. Then, ultrasonic treatment was carried out to prepare liposomes. In the case of modifying the liposomes with STR-R8, 5 mol % of STR-R8 was added to the liposome solution obtained by ultrasonic treatment, and then they were mixed with a pipette and the mixture was left stand at room temperature for 30 minutes.
(b) Lipid:DOPE=2:9
Liposomes and STR-R8-modified liposomes were prepared in the same manner as in Section 3-2-1 (2)(a) except that a 1 mM lipid stock and a 1 mM DOPE stock were mixed in a mole ratio of 2:9, and a CHCl3 solution containing NBD-DOPE in an amount of 1 mol % of the total lipid concentration and a CHCl3 solution containing Rho-DOPE in an amount of 0.5 mol % of the total lipid concentration were added thereto.
3-2-2. Binding Assay
A binding assay was performed for liposomes (STR-R8+/−) using lipids which had showed high binding rates in the experiment described in Section 1-4-1. The binding assay was performed by the method described in Section 1-3-2. The results are shown in
3-2-3. FRET Analysis
FRET analysis was performed for the same lipids used in the experiment described in Section 3-2-2 by the method described in Section 2-2-4. The results (TFs) are shown in
As shown in
Then, analysis was performed to examine the correlation between the ability of liposomes to bind to nuclear membranes and their ability to fuse with nuclear membranes by using liposomes prepared by mixing a lipid and Chol in a ratio of 2:1 which had showed a particularly low ability to fuse with nuclear membranes in the experiments described above, liposomes prepared by mixing a lipid and DOPE in a ratio of 2:9 which had showed a high ability to fuse with nuclear membranes in the experiments described above, and liposomes obtained by modifying the liposomes (lipid:DOPE=2:9) with STR-R8. The results are shown in
3-3. Visualization of Affinity Between STR-R8-Modified Liposomes and Nuclear Membranes
3-3-1. Preparation of Membrane-Labeled Liposomes Entrapping GFP
A 1 mM lipid stock and a 1 mM DOPE stock were mixed in a mole ratio of 9:2, and then a CHCl3 solution containing Rho-DOPE in an amount of 0.5 mol % of the total lipid concentration was added thereto, and then CHCl3 was further added thereto. The thus obtained mixture was dried in an atmosphere of nitrogen gas to prepare lipid film. Then, GFP was diluted with 10 mM HEPES to 0.125 mg/mL, and the HEPES solution of GFP was added as an internal aqueous phase to the lipid film so that the total lipid concentration became 0.55 mM to hydrate the lipid film. Then, ultrasonic treatment was carried out to prepare liposomes. Then, 5 mol % of STR-R8 was added to the liposome solution obtained by ultrasonic treatment, and they were mixed with a pipette and the mixture was left stand at room temperature for 30 minutes.
3-3-2. Analysis of Affinity Between Liposomes and Nuclear Membrane by Confocal Laser Microscope
(1) Operational Method
An experiment was carried out using various liposomes having the following different lipid compositions.
EPC/Chol/STR-R8, DOPE/CHEMS/STR-R8, DOPE/PA/STR-R8, DOPE/PS/STR-R8
These liposomes were prepared by the method described in Section 3-3-1. Nuclei were isolated by the method described in Section 1-2-2, and were then mixed with Hoechst in equal proportions, and then the mixture was incubated at room temperature for 10 minutes to stain the nuclei. The number of the nuclei was counted, and then the amount of phospholipid contained in the nuclei was measured in the same manner as in Section 2-2-4 to make the suspension have the same lipid concentration as the liposome suspension. The liposome suspension and the suspension of the isolated nuclei were mixed in a ratio of liposome:nuclei=9:2, and then the mixture was incubated at 37° C. for 1 hour and then centrifuged at 1400 g at 4° C. for 1 minute. The pellet was washed with Import buffer twice, and then suspended in an appropriated amount of Import buffer. An appropriate amount of the suspension was dropped onto a glass slide, covered with a cover glass, and the cover glass was fixed to the glass slide with nail polish. The thus prepared glass slide was observed by CLSM.
(2) Observation Result
The observation results are shown in
3-4. Summary
It has been found that by modifying lipids with STR-R8, some lipids can have an improved ability to bind to nuclear membranes without significantly changing their ability to fuse with nuclear membranes, and some lipids can have not only an improved ability to bind to nuclear membranes but also an improved ability to fuse with nuclear membranes. Contrary to expectations, it has been found that neutral lipids can also have an improved ability to bind to and fuse with nuclear membranes by modifying them with STR-R8. Further, visualization of membrane fusion was achieved by a confocal microscope image, thereby significantly increasing the possibility of gene delivery by membrane fusion with nuclear membranes.
1. Screening Using FRET
1-1. Preparation of Membrane-Labeled Liposomes
(1) DOPE:Lipid=9:2
1 mM DOPE and a 1 mM lipid stock were mixed in a mole ratio of 9:2, and a CHCl3 solution containing NBD-DOPE in an amount of 1 mol % of the total lipid concentration and a CHCl3 solution containing Rho-DOPE in an amount of 0.5 mol % of the total lipid concentration were added thereto, and CHCl3 was further added thereto. The thus obtained mixture was dried in an atmosphere of nitrogen gas to prepare lipid film. Then, 10 mM HEPES buffer as an internal aqueous phase was added to the lipid film so that the total lipid concentration became 0.55 mM to hydrate the lipid film. Then, ultrasonic treatment was carried out to prepare liposomes.
(2) DOPE:Lipid=5:5
1 mM DOPE and a 1 mM lipid stock were mixed in a mole ratio of 5:5, and a CHCl3 solution containing NBD-DOPE in an amount of 1 mol % of the total lipid concentration and a CHCl3 solution containing Rho-DOPE in an amount of 0.5 mol % of the total lipid concentration were added thereto, and CHCl3 was further added thereto. The thus obtained mixture was dried in an atmosphere of nitrogen gas to prepare lipid film. Then, 10 mM HEPES buffer as an internal aqueous phase was added to the lipid film so that the total lipid concentration became 0.55 mM to hydrate the lipid film. Then, ultrasonic treatment was carried out to prepare liposomes.
1-2. Evaluation of Ability of Liposomes to Fuse with Nuclear Membranes Using FRET
Nuclei were isolated from Hela cells, and the amount of phospholipid contained in the nuclei was measured to determine the amount of PC contained in the nuclei. It has been reported that the amount of PC contained in the nuclear membrane is 55%, and therefore the total lipid concentration of the nuclei was determined on the assumption that the amount of PC contained in the nuclear membrane was 55%, and the suspension of the isolated nuclei was diluted so that the total lipid concentration was 0.13 mM (that is a quarter of Example 1).
A Probe Lip prepared by the method described in Section 1-1 and the suspension of the isolated nuclei were mixed in a ratio of Probe Lip:nuclei=1:9 in a 1.5 mL sample tube, and the thus obtained mixture was incubated at 37° C. for 30 minutes. A control was prepared by mixing the Probe Lip and Import buffer in a ratio of 1:9 and incubating the mixture at 37° C. for 30 minutes. A control for measuring a maximum fluorescence intensity was prepared by mixing the Probe Lip and 0.5% Triton in a ratio of 1:9 and incubating the mixture at 37° C. for 30 minutes. After the completion of incubation, the fluorescence intensity of each of the samples (excitation wavelength: 470 nm, absorption wavelength: 530 nm, 590 nm) was measured to obtain a spectrum. Based on the measured fluorescence intensity at 530 nm, TF was calculated in the same manner as in Example 1.
1-1-3. Result
Various liposomes were prepared using the following lipids by the method described in Section 1-1, and an experiment was carried out using the liposomes. The results (TF) are shown in
EPC(±), SM(±), DOTAP(+), PA(−), PS(−), PI(−), Cardiolipin(−), PG(−), CHEMS (−)
As shown in
As a result, it has been found that the negatively-charged lipids have an affinity for nuclear membranes. In addition, it has also be found that PA and CL are lipids having a particularly high affinity for nuclear membranes.
2. Visualization of Lipids Having Ability to Fuse with Nuclear Membranes by Confocal Laser Microscope
2-1. Preparation of Membrane-Labeled Liposomes Entrapping GFP
A 1 mM lipid stock and a 1 mM DOPE stock were mixed in a specified mole ratio, and a CHCl3 solution containing Rho-DOPE in an amount of 0.5 mol % of the total lipid concentration was added thereto, and then CHCl3 was further added thereto. The thus obtained mixture was dried in an atmosphere of nitrogen gas to prepare lipid film. Then, GFP was diluted with 10 mM HEPES to 0.125 mg/mL, and the HEPES solution of GFP was added as an internal aqueous phase to the lipid film so that the total lipid concentration became 0.55 mM to hydrate the lipid membrane. Then, ultrasonic treatment was carried out to prepare liposomes.
2-2. Analysis by Confocal Laser Microscope
A liposome was prepared by mixing DOPE and PA (membrane-fusible lipids) in a ratio of 5:5, and a liposome was prepared by mixing DOPE and CL (membrane-fusible lipids) in a ratio of 5:5. Further, as controls, a liposome was prepared by mixing EPC, Chol, and STR-R8 in a ratio of 9:2:0.55 (bindability: ◯, fusibility: x), and a liposome was prepared by mixing EPC and CHEMS in a ratio of 5:5 (bindability: x, fusibility: x). It is to be noted that all the liposomes were prepared by the method described in Section 2-1. Further, isolated nuclei were mixed with Hoechst in equal proportions, and the mixture was incubated at room temperature for 10 minutes to stain the nuclei. The amount of phospholipid contained in the nuclei was measured to make the suspension have the same total lipid concentration as the liposome suspension. The liposome suspension and the suspension of the isolated nuclei were mixed in a ratio of liposomes:nuclei=9:2, and the mixture was incubated at 37° C. for 1 hour and then centrifuged at 1400 g at 4° C. for 1 minute. The pellet was washed with Import buffer twice, and was then suspended in an appropriate amount of Import buffer. An appropriate amount of the suspension was dropped onto a glass slide and was covered with a cover glass, and then the cover glass was fixed to the glass slide using nail polish. The thus prepared glass slide was observed with a confocal laser microscope. The results are shown in
As shown in
In a case where a liposome containing two kinds of fluorescent pigments (NBD and Rhodamine) in its lipid membrane is present alone and the fluorescence spectrum of one of the fluorescent pigments and the excitation spectrum of the other fluorescent pigment are overlapped, the fluorescence of NBD is not usually observed even when NBD is irradiated with light having the excitation wavelength of NBD (this is because when NBD and Rhodamine coexist adjacent to each other, the fluorescence energy of NBD is used to excite Rhodamine; and this phenomenon is called “FRET” (Fluorescence Resonance Energy Transfer). When such a liposome is fused with another lipid membrane (e.g., cell membrane), the liposomal lipid is diluted with the lipid membrane as a receiver so that the distance between NBD and Rhodamine contained in the liposomal membrane is increased, and therefore a phenomenon called FRET is cancelled. Utilizing such a phenomenon, screening of lipids was performed to find lipids (endosomal escape-promoting lipids) having high ability to fuse with cell membranes (endosomal membranes). It is to be noted that in this research, a pH at which liposomes and cells are brought into contact with each other is adjusted to 6.5 that is the initial pH inside the endosome to reproduce endosomal environments, and membrane fusion between liposomes and endosomal membranes is quantitatively evaluated by utilizing FRET.
1. Method
(1) Preparation of Liposomes
Lipid film doubly labeled with NBD-DOPE and Rhodamine-DOPE was prepared in a test tube, and HEPES-Glucose (5%) was added to the lipid film to sufficiently hydrate the lipid film. Then, the hydrated lipid film was ultrasonically treated with a bath-type sonicator to prepare liposomes. If necessary, STR-R8 was added (in an amount of 5, 10, or 20 mol % of the amount of lipids). A final liposome concentration was 0.55 mM, the amount of NBD-DOPE was 1% of the total amount of lipids, and the amount of Rhodamine-DOPE was 0.5% of the total amount of lipids. After the preparation of the liposomes, the particle size and Z-potential thereof were measured. It is to be noted that the lipid compositions of various liposomes are shown in
(2) Preparation of Sample and Measurement of Fluorescence Intensity
Before 24 hours from the experiment, 5×105 NIH3T3 cells per well were plated in a 6-well plate, and were incubated for 24 hours. After the completion of 24 h-incubation, DMEM was removed and then the cells were washed with DMEM (Serum Free). Then, 800 μL of DMEM (pH 6.5, Serum Free) and 200 μL of liposomes were added per well to incubate the cells for 1 hour (Cell suspension:Liposome=4:1). After 1 hour, the solvent and liposome solution not fused with cell membranes were removed, and then the cells were washed with PBS. The cells were collected in trypsin, and the cell suspension was centrifuged at 10,000 rpm at 4° C. for 5 minutes, and then the supernatant was removed. The remaining cells were suspended in 100 μL of HEPES-(5%)Glucose, and the thus obtained cell suspension was divided into two. To one of the cell suspensions, 50 μL of HEPES-(5%)Glucose was further added, and the fluorescence intensity of the cell suspension was defined as F′. To the other cell suspension, 50 μL of Triton was added to lyse the cells (final Triton concentration: 0.5%), and the fluorescence intensity of the cell suspension was defined as F′max. The fluorescence intensity of a blank (Buffer:Liposome=80 μL:20 μL) was defined as F0, and the fluorescence intensity of a control obtained by breaking liposomes with Triton (final concentration: 0.5%) (0.5% Triton:Liposome=80 μL:20 μL) was defined as Fmax. The amount of protein contained in each of the samples for measuring the fluorescence intensities F′ and F′max was measured using a BCA TM Protein Assay Kit (PIERCE), and then the amount of protein contained in each of the sample tubes and the amount of protein contained in one well of the 6-well plate were calculated from the absorbances of the samples for measuring F′ and F′max.
(3) Analysis of Fluorescence Intensity
The fluorescence intensities of the samples were measured at 530 nm that is the fluorescence wavelength of NBD. By substituting the measured fluorescence intensity data of F′, F0, F′max, and Fmax into the following formulas to determine Transfer Efficiency (TF) and Binding Efficiency (BF). The amount of protein contained in the sample for measuring F′ was defined as P(F′), the amount of protein contained in the sample for measuring F′max was defined as P(F′max), and the amount of protein contained in one well was defined as P(total). The fluorescence intensity of a sample containing all the cells collected was determined by calculation and was defined as F′ collected (a), but the liposome concentration of the sample was condensed 10-fold as compared to the concentration of liposomes applied when the cells had been collected, and therefore F′ collected was decreased by a factor of ten for correction and the corrected F′ collected was defined as F′ recovered (b). The fluorescence intensity of a sample obtained by breaking liposomes bound to all the cells with 0.5% Triton was determined by calculation and defined as F′max collected (c), but the liposome concentration of the sample was condensed 10-fold as compared to the concentration of liposomes applied when the cells had been collected, and therefore the F′max collected was decreased by a factor of ten for correction and the corrected F′max was defined as F′max recovered (d). Then, BE(e) and TF ((f), (g)) were calculated using the following formulas, and the efficiency of delivering the vector into the cytoplasm by membrane fusion was calculated from BE and TF and was defined as endosomal escape efficiency (h).
(4) Transfection Assay
pDNA was condensed with Poly-L-Lysine to prepare liposomes entrapping condensed DNA (final lipid concentration: 0.55 mM). The surface of the liposomes was modified with an appropriate amount of STR-R8. The lipid compositions of various liposomes are shown in
2. Result and Consideration
(1) Screening of Lipids to Find Endosomal Membrane Fusion-Promoting Lipids Based on Endosomal Escape Efficiency
Endosomal escape efficiency was compared between various liposomes constituted by different lipids and containing two kinds of fluorescent pigments and STR-R8 (for the purpose of selecting a macropinocytosis pathway) (
(1) Preparation of Membrane-Labeled MLA Entrapping GFP
10 mM DOPE and a 10 mM lipid stock were mixed in a mole ratio of 5:5 in a glass test tube, and then a CHCl3 solution containing Rho-DOPE in an amount of 1 mol % of the total lipid concentration was added thereto, and then CHCl3 was further added thereto. Then, the thus obtained mixture was dried in an atmosphere of nitrogen gas to prepare lipid film. Then, GFP was diluted with 10 mM HEPES Buffer to 0.125 mg/mL, and the HEPES solution of GFP was added as an internal aqueous phase so that the total lipid concentration became 0.55 mM to hydrate the lipid membrane. Then, ultrasonic treatment was carried out to prepare MLVs.
(2) Preparation of Membrane-Labeled SUV Entrapping GFP
10 mM DOPE and a 10 mM lipid stock were mixed in a mole ratio of 5:5 in a glass test tube, and then a CHCl3 solution containing Rho-DOPE in an amount of 1 mol % of the total lipid concentration was added thereto, and then CHCl3 was further added thereto. Then, the thus obtained mixture was dried in an atmosphere of nitrogen gas to prepare lipid film. Then, GFP was diluted with 10 mM HEPES Buffer to 0.125 mg/mL, and the HEPES solution of GFP was added as an internal aqueous phase to the lipid film so that the total lipid concentration became 0.55 mM to hydrate the lipid membrane for 10 minutes. Then, ultrasonic treatment was carried out using a probe-type sonicator for 10 minutes to prepare SUVs.
(3) Analysis Using Confocal Laser Microscope
MLV and SUV were prepared by the method described in (1) and the method described in (2), respectively, using DOPE/PA (5:5) as a fusible lipid. Further, MLV and SUV were prepared by the method described in (1) and the method described in (2), respectively, using DOPE/CL (5:5) as a fusible lipid. Then, isolated nuclei were mixed Hoechst in equal proportions, and then the mixture was incubated at room temperature for 10 minutes to stain the nuclei. Then, the liposomes and the nuclei were mixed in a ratio of 9:2 (w/w), and the mixture was incubated at 37° C. for 1 hour, and was then centrifuged at 1400×g at 4° C. for 30 seconds. The thus obtained pellet was washed with Import buffer twice, and was then suspended in an appropriate amount of Import Buffer. An appropriate amount of the thus obtained suspension was dropped onto a glass slide and covered with a cover glass, and the cover glass was fixed to the glass slide using nail polish. The thus prepared glass slide was observed by a confocal laser microscope.
The observation results are shown in
MLV is a vector having a multilamellar structure. In either of the case using the MLV containing PA and the case using the MLV containing CL, the lipid (red) was localized around the nuclei, but the GFP (green) entrapped in the MLVs was dispersed inside the nuclei. On the other hand, in the case of the SUVs having a unilamellar structure, the lipid (red) was localized on the surface of the nuclear membranes and the GFP (green) entrapped in the SUVs was localized alone along the outline of the nuclei. This result suggests that the SUVs having only one membrane could not permeate the bilamellar structure of the nuclei and were therefore trapped in the gap between two nuclear membranes, whereas the MLVs permeated the bilamellar structure of the nuclei stepwise by membrane fusion and then the GFP entrapped in the MLVs was released into the nuclei.
(1) Preparation of DNA Core
Plasmid DNA (pEGFP/Luc) and Protamine were diluted with HEPES Buffer, and then the plasmid DNA (0.1 mg/mL) was dropped into the Protamine (0.1 mg/mL) under the vortex condition (weight ratio=1:1.5, N/P ratio=2:2) to prepare a DNA core.
(2) Preparation of Liposomes
1 mM DOPE and a 1 mM lipid stock were mixed in a predetermined mole ratio in a glass test tube, and then a CHCl3 solution was further added thereto, and then the thus obtained mixture was dried in an atmosphere of nitrogen gas to prepare lipid film. The pDNA/Protamine core prepared by the method described in the above (1) was added as an internal aqueous phase to the lipid film so that the total lipid concentration became 0.55 mM to hydrate the lipid film. Then, ultrasonic treatment was carried out to prepare liposomes. Then, STR-R8 (2 mg/mL) was added to allow the liposomes to be positively charged, and the liposomes and the STR-R8 were mixed with a pipette, and then the mixture was incubated at room temperature for 30 minutes.
In this way, a liposome A containing DOPE/CHEMS (9:2) and STR-R8 (5%), a liposome B containing DOPE/CHEMS (5:5) and STR-R8 (5%), a liposome C containing EPC/CHEMS (9:2) and STR-R8 (5%), a liposome D containing DOPE/PA (5:5) and STR-R8 (20%), a liposome E containing DOPE/CL (5:5) and STR-R8 (20%), and a liposome F containing DOPE/PA (7:2) and STR-R8 (10%) were prepared. It is to be noted that these liposomes A to F are sometimes generically called “MENDs”. MENDs are liposomes having one or two or more lipid membranes.
(3) Preparation of Liposome-Liposomes
(a) Inner Lipid Membranes Containing Dope/Pa (5:5) and Outer Lipid Membranes Containing Dope/Pa (7:2)
10 mM DOPE and 10 mM PA were mixed in a mole ratio of 5:5 in a glass test tube, and then CHCl3 was added thereto. Then, the thus obtained mixture was dried in an atmosphere of nitrogen gas to prepare lipid film. Then, 10 mM HEPES buffer was added as an internal aqueous phase to the lipid film so that the total lipid concentration became 0.55 mM to hydrate the lipid film for 10 minutes. Then, ultrasonic treatment was carried out using a probe type-sonicator for 10 minutes to prepare SUVs. Then, the SUVs containing DOPE/PA (5:5) and the pDNA/protamine core prepared by the method described in the above (1) were mixed in a ratio of 2:1 to prepare bilamellar liposomes. To the bilamellar liposome solution, STR-R8 (2 mg/mL) was added in an amount of 20% of the total lipid concentration, and then the mixture was incubated at room temperature for 30 minutes.
Then, SUVs containing DOPE/PA (7:2) and the bilamellar liposomes were mixed in a ratio of 2:1 to prepare tetralamellar liposomes. To the tetralamellar liposome solution, STR-R8 (2 mg/mL) was added in an amount of 10% of the total lipid concentration, and the mixture was incubated at room temperature for 30 minutes.
In this way, a tetralamellar liposome A having two inner lipid membranes containing DOPE/PA (5:5) and two outer lipid membranes containing DOPE/PA (7:2) was prepared.
(b) Inner Lipid Membranes Containing Dope/CL (5:5) and Outer Lipid Membranes Containing Dope/Pa (7:2)
10 mM DOPE and 10 mM CL were mixed in a mole ratio of 5:5 in a glass test tube, and then CHCl3 was added thereto. Then, the thus obtained mixture was dried in an atmosphere of nitrogen gas to prepare lipid film. Then, 10 mM HEPES buffer was added as an internal aqueous phase to the lipid film so that the total lipid concentration became 0.55 mM to hydrate the lipid film for 10 minutes. Then, ultrasonic treatment was carried out using a probe type-sonicator for 10 minutes to prepare SUVs.
Then, the SUVs containing DOPE/PA (5:5) and the pDNA/protamine core prepared by the method described in the above (1) were mixed in a ratio of 2:1 to prepare bilamellar liposomes. To the bilamellar liposome solution, STR-R8 (2 mg/mL) was added in an amount of 20% of the total lipid concentration, and then the mixture was incubated at room temperature for 30 minutes.
Then, SUVs containing DOPE/PA (7:2) and the bilamellar liposomes were mixed in a ratio of 2:1 to prepare tetralamellar liposomes. To the tetralamellar liposome solution, STR-R8 (2 mg/mL) was added in an amount of 10% of the total lipid concentration, and the mixture was incubated at room temperature for 30 minutes.
In this way, a tetralamellar liposome B having two inner lipid membranes containing DOPE/CL (5:5) and two outer lipid membranes containing DOPE/PA (7:2) was prepared.
(c) Inner Lipid Membranes Containing Dope/Pa (5:5) (DOPE/CL=5:5) and Outer Lipid Membranes Containing EPC/CHEMS (9:2)
SUVs containing DOPE/PA (5:5) (DOPE/CL=5:5) were prepared by the method described above. Then, 10 mM EPC and 10 mM PA (CL) were mixed in a mole ratio of 9:2 in a glass test tube, and then CHCl3 was added thereto. Then, the thus obtained mixture was dried in an atmosphere of nitrogen gas to prepare lipid film. Then, 10 mM HEPES buffer was added as an internal aqueous phase to the lipid film so that the total lipid concentration became 0.55 mM to hydrate the lipid film for 10 minutes. Then, ultrasonic treatment was carried out using a probe type-sonicator for 10 minutes to prepare SUVs.
Then, the SUVs containing DOPE/PA (5:5) (DOPE/CL=5:5) and the pDNA/protamine core prepared by the method described in the above (1) were mixed in a ratio of 2:1 to prepare bilamellar liposomes. To the bilamellar liposome solution, STR-R8 (2 mg/mL) was added in an amount of 20% of the total lipid concentration, and then the mixture was incubated at room temperature for 30 minutes.
Then, SUVs containing EPC/CHEMS (9:2) and the bilamellar liposomes were mixed in a ratio of 2:1, and an appropriate amount of 1N HCl was added thereto to induce membrane fusion to prepare tetralamellar liposomes. To the tetralamellar liposome solution, STR-R8 (2 mg/mL) was added in an amount of 5% of the total lipid concentration, and the mixture was incubated at room temperature for 30 minutes.
In this way, a tetralamellar liposome C having two inner lipid membranes containing DOPE/PA (5:5) and two outer lipid membranes containing EPC/CHEMS (9:2) and a tetralamellar liposome D having two inner lipid membranes containing DOPE/CL (5:5) and two outer lipid membranes containing EPC/CHEMS (9:2) were prepared.
(d) Inner Lipid Membranes Containing EPC/CHEMS (9:2) and Outer Lipid Membranes Containing DOPE/PA (7:2) (EPC/CHEMS=9:2)
SUVs containing EPC/CHEMS (9:2) and SUVs containing DOPE/PA (7:2) were prepared by the method described above. Then, the SUVs containing EPC/CHEMS (9:2) and the pDNA/Protamine core prepared by the method described in the above (1) were mixed in a ratio of 2:1, and then an appropriate amount of 1N HCl was added thereto to induce membrane fusion to prepare bilamellar liposomes. To the bilamellar liposome solution, STR-R8 (2 mg/mL) was added in an amount of 5% of the total lipid concentration, and then the mixture was incubated at room temperature for 30 minutes.
Then, the SUVs containing DOPE/PA (7:2) or the SUVs containing EPC/CHEMS (9:2) and the bilamellar liposomes were mixed in a ratio of 2:1 to prepare tetralamellar liposomes. To the tetralamellar liposome solution, STR-R8 (2 mg/mL) was added in an amount of 5% of the total lipid concentration, and the mixture was incubated at room temperature for 30 minutes.
In this way, a tetralamellar liposome E having two inner lipid membranes containing EPC/CHEMS (9:2) and two outer lipid membranes containing DOPE/PA (7:2) and a tetralamellar liposome F having two inner lipid membranes containing EPC/CHEMS (9:2) and two outer lipid membranes containing EPC/CHEMS (9:2) were prepared. It is to be noted that these tetralamellar liposomes A to F are sometimes generically called “T-MENDs”.
(4) Transfection
JAWSII cells (8.0×104 cells) were placed in a 24 well-plate, and then were cultured for 2 days. Then, a sample corresponding to 0.4 μg of DNA was diluted with A-MEM not containing antibiotic and FCS so that the total volume of the sample was 250 μL, and the dilute sample was added to the cells. Naked DNA and HEPES Buffer were diluted in the same manner to prepared Negative controls, and these negative controls were added to the cells and the cells were cultured in a 5% CO2 incubator at 37° C. After 3 hours from addition of the sample, 500 μL of α-MEM containing 10% FCS was added to the cells to again culture the cells in the 5% CO2 incubator at 37° C. The cells were collected after 24 hours from addition of the sample, and luciferase activity and the amount of protein were measured. It is to be noted that in this experiment, JAWSII cells that are nondividing cells of immune system (mouse myeloid dendritic cells) were used for the purpose of reducing the influence of cell division on gene expression.
(5) Evaluation of Gene Expression
The measurement results of the efficiency of gene expression using the MENDs are shown in
As compared to the cases of DOPE/CHEMS (liposomes A and B) and EPC/CHEMS (liposome C), a significant increase in gene expression efficiency was shown in the cases of DOPE/PA (liposome D) and DOPE/CL (liposome E).
The measurement results of the efficiency of gene expression using the T-MENDs are shown in
The inner membranes and outer membranes of T-MENDs were changed to those containing EPC/CHEMS (9:2) having a low affinity for nuclear membranes or cell membranes (see
| Number | Date | Country | Kind |
|---|---|---|---|
| 2005-289411 | Sep 2005 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2006/319601 | 9/29/2006 | WO | 00 | 3/28/2008 |
