JPWO2012117971A1 - Lipid membrane structure, method for producing lipid membrane structure, and method for encapsulating one target substance in one lipid membrane - Google Patents

Abstract

PROBLEM TO BE SOLVED: To easily manufacture a lipid membrane structure having a fine and uniform particle size, a lipid membrane structure in which the number of substances to be encapsulated (target substance) and the number of lipid membranes are controlled, and a method for producing the same And a method for encapsulating one target substance with one lipid membrane. SOLUTION: A lipid membrane structure having a lipid membrane modified with a cationic surfactant as a single membrane or as an outermost membrane of a plurality of membranes. According to the lipid membrane structure and the method for producing the same according to the present invention, a fine and uniform lipid membrane structure, and a lipid membrane structure in which the number of substances to be encapsulated (target substance) and the number of lipid membranes are controlled are obtained. be able to.

Description

  The present invention relates to a lipid membrane structure, a method for producing a lipid membrane structure, and a method for encapsulating one target substance in a single lipid membrane, and more specifically, a lipid obtained by modifying a cationic surfactant The present invention relates to a lipid membrane structure having a single membrane or a plurality of outermost membranes, a method for producing the lipid membrane structure, and a method for encapsulating one target substance with a single lipid membrane.

  A vector for reliably delivering a substance such as a protein, a drug, or a nucleic acid to a target site of a living individual has been actively developed. Vectors developed so far are roughly classified into viral vectors and non-viral vectors. Viral vectors are generally excellent in gene transfer ability to introduce the target gene into the nucleus of the target cell, but there are problems such as difficulty in mass production, antigenicity, and toxicity. As a few non-viral vectors, lipid membrane structures represented by liposomes have attracted attention.

  Liposomes are lipid membrane structures having a lipid membrane composed of artificially prepared lipid bilayers as a basic component, and have the ability to encapsulate various substances and send them into target cells. In addition to the advantage that liposomes can improve the directivity to the target site by introducing functional molecules such as antibodies, proteins, sugar chains, etc. on the surface, liposomes can be decomposed in vivo. It has the advantage of being protected from action and metabolic action, and the advantage of preventing the substance from acting at a site other than the target (side effects). So far, for example, a liposome having a peptide containing a plurality of continuous arginine residues on its surface and having a nuclear translocation ability has been developed (Patent Document 1).

  Further, Stabilized plasmid-lipid particles (SPLP) have been developed as lipid membrane structures encapsulating genes (Non-patent Document 1). SPLP is produced by incubating a gene in a buffer solution containing a positively charged lipid and a nonionic surfactant, and then dialysis using the buffer solution. Is the body.

International Publication WO2005 / 032593 Pamphlet

Wheeler J.M. J. et al. Et al., Gene Ther. , Vol. 6, No. 2, pp. 271-281, 1999

  In general, in order to efficiently deliver particles to a disease site such as cancer or inflammation, the particle diameter is preferably about 100 nm to 150 nm (Liu D. et al., Biochim Biophys Acta., Vol. 1104, No. 1). 95-101, 1992; Akinc A. et al., Mol.Ther., 17, No. 5, 872-879, 2009). However, as shown in the upper part of FIG. 1, the liposome described in Patent Document 1 is easily produced by a method (hydration method) in which a lipid film is prepared and then hydrated and stirred or sonicated. However, since it is difficult to control the number of substances (target substances) to be encapsulated and the number of lipid membranes, the average particle diameter becomes excessively about 300 nm, which can be efficiently delivered to the diseased site. It is difficult.

  The SPLP described in Non-Patent Document 1 is so small that it can be efficiently delivered to a disease site, but it takes a long time for the dialysis operation and changes in slight conditions such as pH of the buffer solution. This makes it difficult to put it to practical use because the efficiency of entrapment of the gene in the lipid membrane structure is remarkably reduced (Jeffs LB, Pharm Res., Vol. 22, No. 3, pp. 362-72). 2005).

  The present invention has been made in order to solve such problems, and can be easily produced and includes a lipid membrane structure having a minute and uniform particle diameter and a substance to be encapsulated (target substance). It is an object to provide a lipid membrane structure in which the number and the number of lipid membranes are controlled, a method for producing the same, and a method for encapsulating one target substance with one lipid membrane.

  As a result of earnest research, the present inventors have a fine and uniform particle size by performing a hydration method using a lipid film modified with a cationic surfactant, as shown in the lower part of FIG. The ability to obtain liposomes in which the number of liposomes and substances to be encapsulated (target substances) and the number of lipid membranes are controlled, the ability to encapsulate one target substance with a single lipid membrane, and cationic surface activity The inventors found that the transfection activity of liposomes having a lipid membrane modified with an agent as a single membrane or as the outermost membrane of a plurality of membranes was remarkably improved, and completed the following inventions.

(1) A lipid membrane structure having a lipid membrane modified with a cationic surfactant as a single membrane or as an outermost membrane of a plurality of membranes.

［式中：Ｒ_１はアルキル基であり、Ｒ_２は疎水基である。］の陽イオン性界面活性剤である、（１）に記載の脂質膜構造体。(2) The cationic surfactant is represented by the following formula (Formula 1)

[Wherein, R ₁ is an alkyl group, and R ₂ is a hydrophobic group. The lipid membrane structure according to (1), which is a cationic surfactant.

または次式（化３）

の基である、（２）に記載の脂質膜構造体。(3) R ₁ is an alkyl group having 12 or more carbon atoms, and R ₂ is represented by the following formula (Formula 2):

Or the following formula (Formula 3)

The lipid membrane structure according to (2), which is a group of

の基である、（２）に記載の脂質膜構造体。(4) R ₁ is an alkyl group having 16 carbon atoms, and R ₂ is represented by the following formula (Formula 4)

The lipid membrane structure according to (2), which is a group of

(5) The lipid membrane structure according to any one of (1) to (4), which is a negatively charged lipid membrane structure.

(6) The lipid membrane structure according to any one of (1) to (5), which is a liposome.

(7) A method for producing a lipid membrane structure having a lipid membrane modified with a cationic surfactant as a single membrane or as an outermost membrane of a plurality of membranes, comprising the following (i), (ii) Or (iii) the method having the step; (i) a step of preparing a lipid film in which a cationic surfactant is modified by mixing a lipid and a cationic surfactant; A step of preparing a lipid film in which the cationic surfactant is modified by adding a cationic surfactant; (iii) a single membrane or a plurality of lipid membrane structures having a single membrane or a plurality of membranes A step of modifying the outermost membrane of the membrane with a cationic surfactant.

(8) (i) a step of preparing a lipid film in which a lipid and a cationic surfactant are mixed to modify the cationic surfactant; or (ii) adding a cationic surfactant to the lipid film. And (iv) a method for preparing a lipid film in which a cationic surfactant is modified, and (iv) hydrating the prepared lipid film. A step of preparing a lipid membrane structure.

(9) A method of encapsulating one target substance with a single lipid membrane, the method comprising the following step (i) or (ii): (i) mixing a lipid and a cationic surfactant A step of preparing a lipid film modified with a cationic surfactant, and (ii) a step of preparing a lipid film modified with a cationic surfactant by adding a cationic surfactant to the lipid film. .

(10) The method according to (9), comprising the following step (iv); (iv) a step of preparing a lipid membrane structure by hydrating the prepared lipid film.

［式中：Ｒ_１はアルキル基であり、Ｒ_２は疎水基である。］の陽イオン性界面活性剤である、（７）から（１０）のいずれかに記載の方法。(11) The cationic surfactant is represented by the following formula (Formula 5):

[Wherein, R ₁ is an alkyl group, and R ₂ is a hydrophobic group. ] The method in any one of (7) to (10) which is a cationic surfactant.

または次式（化７）

の基である、（１１）に記載の方法。(12) R ₁ is an alkyl group having 12 or more carbon atoms, and R ₂ is represented by the following formula (Formula 6):

Or the following formula (Formula 7)

The method according to (11), wherein

の基である、（１１）に記載の方法。(13) R ₁ is an alkyl group having 16 carbon atoms, and R ₂ is represented by the following formula (Formula 8):

The method according to (11), wherein

  According to the lipid membrane structure and the method for producing the same according to the present invention, a fine and uniform lipid membrane structure, and a lipid membrane structure in which the number of substances to be encapsulated (target substance) and the number of lipid membranes are controlled are obtained. be able to. Moreover, according to the minute and uniform lipid membrane structure, it can be efficiently delivered to a disease site such as a tumor or liver parenchyma, and according to the lipid membrane structure in which the number of lipid membranes is controlled, the target site Can be delivered efficiently. For example, when the number of the lipid membranes of the lipid membrane structure according to the present invention is 3, the membrane or mitochondria is obtained by membrane fusion three times, and when the number of lipid membranes is one, the membrane is one time. By fusion, the target substance can be efficiently delivered to the cytoplasm. Furthermore, the lipid membrane structure according to the present invention can deliver a target substance to a target site without damaging the target substance, and can exert the function of the target substance at the target site. Can be applied. On the other hand, according to the method for producing a lipid membrane structure according to the present invention, a lipid membrane structure having the above-described characteristics can be produced easily and inexpensively under mild preparation conditions. Furthermore, according to the method of encapsulating one target substance according to the present invention with one lipid membrane, a lipid membrane structure corresponding to the size of the target substance can be obtained, and this method is performed one or more times. Thus, a lipid membrane structure having a desired number of lipid membranes can be obtained. The method for producing a lipid membrane structure according to the present invention and the method for encapsulating one target substance with a single lipid membrane can be performed regardless of the type of lipid. The lipid composition of the lipid membrane can be appropriately selected according to the above.

The figure which shows the outline | summary of the manufacturing method in the lipid membrane structure which has the lipid membrane modified with the conventional liposome and the cationic surfactant based on this invention, the number of the enclosed substance (target substance), and the number of lipid membranes It is. It is a figure which shows the one aspect | mode of the lipid membrane by which a cationic surfactant based on this invention is modified. Benzyldodecyldimethylammonium bromide (BB-1), benzyltetradecyldimethylammonium bromide (BB-2), benzylhexadecyldimethylammonium bromide (BB-3), benzyloctadecyldimethylammonium bromide (BB-4), tonzonium bromide It is a figure which shows the structure of (TB) and n-Octyl (beta) -D-glucopyranoside (OG). Nucleic acid nanoparticles, liposomes having lipid membranes not modified with surfactant (a; control), liposomes having lipid membranes modified with BB-1 (b, c), lipid membranes modified with BB-2 (D, e) having liposomes, liposomes (f, g) having lipid membranes modified with BB-3, liposomes (h, i) having lipid membranes modified with BB-4, and OG were modified It is a figure (A and B) which shows the result of having measured the particle diameter about the liposome (j, k) which has a lipid membrane, and the figure (C and D) which shows the result of measuring a zeta potential. For nucleic acid nanoparticles, TB unmodified liposomes (a), and TB modified liposomes (b, c, d, e and f) with modified concentrations of 11, 16.5, 22, 27.5 and 55 mol%, A diagram showing the result of measuring the particle size (A), a diagram showing the result of measuring the zeta potential (B), and a schematic diagram for explaining the particle size of a liposome in which one nucleic acid nanoparticle is encapsulated with one lipid membrane (C). Particle size for TB unmodified empty liposomes (g) and TB modified empty liposomes (h, i, j, k and l) with TB modified concentrations of 11, 16.5, 27.5, 55 and 110 mol% It is a figure (B) which shows the result of having measured zeta potential, and figure (B) which shows the result of having measured zeta potential. Lipofectamine PLUS (LFN PLUS; A, B), TB unmodified liposome (C, D), 11 mol% TB modified liposome (E, F), 16.5 mol% TB modified liposome (G, H) and 22 mol% TB modified liposome The figure (right figure) which shows the result of having measured the luciferase activity in the HeLa cell which transfected plasmid DNA (pDNA) by which Enhanced Green luminescence Protein (EGFP) -luciferase fusion protein gene was encoded by (I, J), and It is a figure (left figure) which shows the result of having calculated the cell viability. TB-modified liposomes (a), TB-unmodified liposomes (b), multilamellar liposomes (MLV; c) and unilamellar liposomes (SUVs) labeled with Nitro-2-1,3-Benzoxadiazol-4-yl (NBD) The figure which shows the result of having continuously measured the fluorescence intensity of NBD about (d) (upper figure), and the schematic diagram explaining the quenching effect of NBD at the time of adding hydrosulfite sodium to the liposome labeled with NBD ( (Figure below). The TB modified positively charged lipid film (a), the TB unmodified positively charged lipid film (b), the TB modified negatively charged lipid film (c) and the TB unmodified negatively charged lipid film (d) are negatively charged. It is a figure which shows the result of having measured the fluorescence intensity of NBD continuously about each sample which sealed the membrane liposome. It is a figure which shows the outline | summary of the process of encapsulating 1 bilamellar liposome with 1 lipid membrane and preparing a trilamellar liposome. It is a figure which shows the result of having measured the luciferase activity in the JAWSII cell which transfected the pDNA by which the EGFP-luciferase fusion protein gene was encoded by LFN PLUS, 4 membrane liposome, and TB modification 3 membrane liposome. It is a figure which shows the outline | summary of the process of preparing TB modification | reformation trilamellar liposome which each encapsulated PLL stabilization bilayer liposome (a) and BSA stabilization bilayer liposome (b). The fluorescence intensity of NBD was continuously measured for the TB-modified trilamellar liposome encapsulating the PLL-stabilized bilayer liposome (a) and the BSA-stabilized bilayer liposome (b) and the negatively charged bilayer liposome, respectively. It is a figure which shows the measurement result. LGFP PLUS and negatively charged bilayer liposome (a), PLL-stabilized bilayer liposome (b) and BSA-stabilized bilayer liposome (c) were encapsulated in EGFP- It is a figure which shows the result of having measured the luciferase activity in the JAWSII cell which transfected pDNA by which the luciferase fusion protein gene was encoded. The photograph when the liposome of this invention is observed with a transmission electron microscope is shown. In the figure, the scale bar indicates 50 nm.

  Hereinafter, a lipid membrane structure according to the present invention, a method for producing the lipid membrane structure, and a method for encapsulating one target substance with a single lipid membrane will be described in detail. The lipid membrane structure according to the present invention has a lipid membrane modified with a cationic surfactant as a single membrane or as an outermost membrane of a plurality of membranes.

  In the present invention, the “lipid membrane” refers to a membrane having lipid as a main constituent, which the lipid membrane structure has. The lipid membrane in the present invention may be composed of a lipid bilayer in which lipid molecules are associated with each other and formed in two layers, and the hydrophobic portion is formed with the hydrophobic portion facing inward or outward. It may consist of a single layer.

  As a preferable form of the lipid membrane structure in the present invention, a closed vesicle having a lipid membrane composed of a lipid bilayer can be mentioned, and as such a lipid membrane structure, for example, a liposome can be mentioned.

  In the present invention, the number of lipid membranes included in the lipid membrane structure may be one, or two or more. When the lipid membrane structure according to the present invention has a plurality of membranes, at least the outermost membrane has a lipid membrane in which a cationic surfactant is modified, but not only the outermost membrane but also one other than the outermost membrane. Alternatively, a plurality of membranes may be lipid membranes obtained by modifying a cationic surfactant.

［式中：Ｒ_１はアルキル基であり、Ｒ_２は疎水基である。］で示される陽イオン性界面活性剤が好ましく、上記式（化１）において、Ｒ_１が炭素数が１２以上のアルキル基であり、かつＲ_２が次式（化２）

またはＲ_２が次式（化３）

で示される陽イオン性界面活性剤であることがさらに好ましい。In the present invention, the “cationic surfactant” refers to a surfactant having a cationic hydrophilic group, and the ionic value is not particularly limited, but is preferably monovalent. Specific examples of the cationic surfactant include alkyltrimethylammonium salts such as trimethyltetradecylammonium chloride and cetyltrimethylammonium bromide, and dialkyldimethylammonium such as dodecylethyldimethylammonium bromide and ethylhexadecyldimethylammonium bromide. Salt, alkyldimethylbenzylammonium salt such as benzyldodecyldimethylammonium bromide, benzyltetradecyldimethylammonium bromide, benzylhexadecyldimethylammonium bromide, benzyloctadecyldimethylammonium bromide, benzalkonium chloride, tonzonium bromide, benzethonium chloride, cetyl chloride Pyridinium, decalinium chloride, second Grade amine salts, secondary amine salts, there may be mentioned cations such as from tertiary amine salts such as N-methyl-bis-hydroxyethyl amine fatty acid ester hydrochloride salt, of which the following formula (Formula 1)

[Wherein, R ₁ is an alkyl group, and R ₂ is a hydrophobic group. In the above formula (Formula 1), R ₁ is an alkyl group having 12 or more carbon atoms, and R ₂ is represented by the following formula (Formula 2).

Or R ₂ represents the following formula (Formula 3)

It is further preferable that the surfactant be a cationic surfactant.

  The “hydrophobic group” in the present invention includes, for example, groups represented by the above formulas (Chemical Formula 2) and (Chemical Formula 3), alkyl groups such as a methyl group, an ethyl group, a propyl group, and an isopropyl group, and an alkenyl group. In addition, a hydrocarbon group such as vinyl group, aryl group such as phenyl group or naphthyl group, a heterocyclic ring, or a combination of a hydrophilic group and a hydrophobic group can be cited as a group which is hydrophobic as a whole.

  In the present invention, as an aspect of the lipid membrane in which the cationic surfactant is modified, for example, as shown in FIG. 2, the cationic surfactant is located in the gap between the lipid molecules constituting the lipid membrane. In addition to the embodiment, there may be mentioned an embodiment in which a cationic surfactant is bound to a lipid molecule constituting the lipid membrane by a covalent bond, a hydrogen bond, an ionic bond, a hydrophobic bond, or a van der Waals bond.

  In the present invention, the lipid membrane structure may be any of positive chargeability, nonchargeability, both (positive / negative) chargeability, and negative chargeability, but is preferably negatively chargeable. The “negatively charged lipid membrane structure” in the present invention refers to a lipid membrane structure that is negatively charged as a whole. For example, positively charged lipids or both (positive and negatively) chargeable lipids as lipids constituting the lipid membrane. Because it contains lipids, non-charged lipids, or because the cationic surfactant is modified, even if it is locally positively charged, both (positive and negative) charged, or uncharged, If it is negatively charged as a whole, it is included in the “negatively charged lipid membrane structure”.

  The lipid constituting the lipid membrane structure in the present invention may be any of positively charged lipids, neutral (including both positive and negative) charged lipids and uncharged lipids, and negatively charged lipids. Examples thereof include phospholipids, glycolipids, sterols, long-chain aliphatic alcohols and glycerin fatty acid esters, and one or more of these can be used.

  Examples of the phospholipid include phosphatidylcholine (for example, dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine), phosphatidylglycerol (for example, dioleoylphosphatidylglycerol, dilauroylphosphatidylglycerol, Dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, etc., phosphatidylethanolamine (eg dioleoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipal Toylphosphatidylethanolamine, distearoylphosphatidylethanolamine (DSPE), dioleoylglycerophosphoethanolamine (DOPE), etc.), phosphatidylserine, phosphatidylinositol, phosphatidic acid (PA), cardiolipin, or hydrogenated products thereof, egg yolk , Natural lipids derived from soybeans and other animals and plants (for example, egg yolk lecithin, soybean lecithin, etc.) and the like, and one or more of these can be used.

  Examples of glycolipids include glyceroglycolipids such as sphingomyelin, sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride and glycosyl diglyceride, and sphingoglycolipids such as galactosyl cerebroside, lactosyl cerebroside and ganglioside. 1 type, or 2 or more types of these can be used.

  Examples of sterols include sterols derived from animals such as cholesterol, cholesterol succinic acid, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol, sterols derived from plants such as stigmasterol, sitosterol, campesterol, and brassicasterol (tytosterol). And sterols derived from microorganisms such as ergosterol, and one or more of these can be used. In addition, these sterols can generally be used to physically or chemically stabilize the lipid bilayer or to adjust the fluidity of the membrane.

  As the long-chain fatty acid or long-chain aliphatic alcohol, a fatty acid having 10 to 20 carbon atoms or an alcohol thereof can be used. Examples of such long-chain fatty acids or long-chain aliphatic alcohols include palmitic acid, stearic acid, lauric acid, myristic acid, pentadecylic acid, arachidic acid, margaric acid, tuberculostearic acid and other saturated fatty acids, palmitoleic acid, Mention of unsaturated fatty acids such as oleic acid, arachidonic acid, vaccenic acid, linoleic acid, linolenic acid, arachidonic acid, eleostearic acid, oleyl alcohol, stearyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, linolyl alcohol 1 type, or 2 or more types of these can be used.

  Examples of the glycerin fatty acid ester include monoacyl glycerides, diacyl glycerides, and triacyl glycerides, and one or more of these can be used.

  Examples of the positively charged lipid include dioctadecyldimethylammonium chloride (DODAC), N- (2,3-oleyloxy) propyl-N, N, N-trimethylammonium (N-) in addition to the above-described lipids. (2,3-dioleoyloxy) propyl-N, N, N-trimethylammonium (DOTMA), didodecylammonium bromide (DDAB), 1,2-dioleoyloxy-3-trimethylammonium propane (1,2-dioleoyloxy) -3-trimethylaminopropane, DOTAP), 3β-N- (N ′, N′-dimethylaminoethane) ) Carbamol cholesterol (3β-N- (N ′, N ′,-dimethyl-aminoethane) -carbamol cholesterol, DC-Chol), 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium (1,2- dimyristoyoxypropyl-3-dimethylhydroxyl ammonium (DMRIE), 2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propaneammonium trifluoroacetate (2,3-dioleoyloxy-N) -[2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanamine trifluo oacetate, DOSPA) and the like can be illustrated, may be used alone or two or more thereof.

  Examples of neutral lipids including both (positive and negative) charged lipids and non-charged lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide and the like in addition to the above-described lipids. Or 2 or more types can be used. Examples of the negatively charged lipid include diacylphosphatidylserine, diacylphosphatidic acid, N-succinylphosphatidylethanolamine (N-succinyl PE), phosphatidylethylene glycol, cholesteryl hemisuccinate (CHEMS) and the like in addition to the lipids described above. 1 type, or 2 or more types of these can be used.

  In addition to the lipids described above, the lipid membrane of the lipid membrane structure according to the present invention has positive charges such as tocopherol, propyl gallate, ascorbyl palmitate, butylated hydroxytoluene, stearylamine, oleylamine and the like. Charged substances to be added, charged substances to give negative charges such as dicetyl phosphate, membrane proteins such as membrane surface proteins and integral membrane proteins, and peptides that impart cell permeability and nuclear translocation ability to lipid membrane structures It can be combined or contained, and the amount and content of the bond can be adjusted as appropriate.

  The lipid membrane structure having a lipid membrane modified with the cationic surfactant according to the present invention as a single membrane or as an outermost membrane of a plurality of membranes is a hydration method, an ultrasonic treatment method, an ethanol injection method, Although it can be produced using a known method such as an ether injection method, a reverse phase evaporation method, a surfactant method, or a freezing / thawing method, in particular, as shown in the lower part of FIG. By preparing a modified lipid film and carrying out a hydration method, it can be easily produced. In the present invention, the “lipid film” is mainly composed of lipid molecules formed so as to stick to the bottom surface or side surface of the container by dissolving the lipid in the organic solvent and then evaporating and removing the organic solvent. This refers to a membrane as a constituent component.

Next, the present invention provides a method for producing a lipid membrane structure. The method for producing a lipid membrane structure according to the present invention is a method for producing a lipid membrane structure having a lipid membrane modified with a cationic surfactant as a single membrane or as an outermost membrane of a plurality of membranes. There,
(I) a step of preparing a lipid film in which a cationic surfactant is modified by mixing a lipid and a cationic surfactant;
(Ii) adding a cationic surfactant to the lipid film to prepare a lipid film in which the cationic surfactant is modified;
(Iii) modifying the cationic surfactant on the outermost membrane of one or more membranes of a lipid membrane structure having one or more membranes;
The process (i), (ii), or (iii) is included. In the method for producing a lipid membrane structure according to the present invention, the description of the same or equivalent configuration as the configuration of the above-described lipid membrane structure according to the present invention is omitted.

  In the step (i), a method for preparing a lipid film in which a cationic surfactant is modified by mixing a lipid and a cationic surfactant is obtained by, for example, dissolving a lipid in an organic solvent. After mixing the solution (lipid solution) and the solution obtained by dissolving the cationic surfactant in an organic solvent (cationic surfactant solution), the cationic surfactant is added to the lipid solution. After mixing or dissolving, after adding lipid to the cationic surfactant solution and mixing or dissolving, or after dissolving lipid and cationic surfactant in the organic solvent at the same time, The method of evaporating and removing can be mentioned.

  In the present invention, examples of the organic solvent for dissolving the lipid and the cationic surfactant include hydrocarbons such as pentane, hexane, heptane and cyclohexane, halogenated hydrocarbons such as methylene chloride and chloroform, and benzene. Aromatic hydrocarbons such as toluene, lower alcohols such as methanol and ethanol, esters such as methyl acetate and ethyl acetate, ketones such as acetone, etc. may be used alone or in combination of two or more. it can.

  In the step (ii), as a method for preparing a lipid film in which a cationic surfactant is modified by adding a cationic surfactant to the lipid film, for example, a cationic surfactant solution is added to the lipid film. A method of removing the solvent by evaporation after the addition can be mentioned.

  In the step (iii), as a method of modifying the cationic surfactant on the outermost membrane of the single membrane or the plural membranes of the lipid membrane structure having a single membrane or a plurality of lipid membranes, for example, Examples thereof include a method of adding a cationic surfactant to a lipid membrane structure having a membrane or a plurality of membranes.

Moreover, when the method for producing a lipid membrane structure according to the present invention includes the step (i) or the step (ii),
(Iv) hydrating the prepared lipid film to prepare a lipid membrane structure;
The step (iv) can also be included.

  In step (iv), the lipid film structure is prepared by hydrating the lipid film. For example, a method of adding a liquid such as water to the lipid film for hydration, followed by stirring or ultrasonic treatment That is, the hydration method can be mentioned.

Next, the present invention provides a method of encapsulating one target substance with one lipid membrane. The method of encapsulating one target substance according to the present invention with one lipid membrane is as follows:
(I) a step of preparing a lipid film in which a cationic surfactant is modified by mixing a lipid and a cationic surfactant;
(Ii) adding a cationic surfactant to the lipid film to prepare a lipid film in which the cationic surfactant is modified;
The step (i) or (ii) is included. In addition, in the method of encapsulating one target substance according to the present invention with a single lipid membrane, the configuration equivalent to or corresponding to the configuration of the lipid membrane structure according to the present invention or the method for producing the lipid membrane structure described above is described. The description will not be repeated.

Moreover, the method of encapsulating one target substance according to the present invention with one lipid membrane is as follows:
(Iv) hydrating the prepared lipid film to prepare a lipid membrane structure;
The step (iv) can also be included.

  In the present invention, the “target substance” refers to a substance to be encapsulated in a lipid membrane structure or an encapsulated substance. In the present invention, the target substance may be any substance, and examples thereof include various physiologically active substances such as drugs, nucleic acids, peptides, proteins, sugars or complexes thereof, and lipid membrane structures such as liposomes and micelles, It can select suitably according to the objectives, such as a diagnosis and treatment. The target substance can be encapsulated in the aqueous phase inside the lipid membrane structure, for example, by adding it to an aqueous solvent used when the lipid membrane is hydrated in the production of the lipid membrane structure.

  For example, in a method of encapsulating one target substance according to the present invention with one lipid membrane, if a liposome having one lipid membrane is the target substance, a liposome having two lipid films can be obtained. If liposomes having two lipid membranes are used as the target substance, liposomes having three lipid membranes can be obtained. The lipid membrane structure of the target substance may be modified with various functional molecules. Examples of the modifying substance include peptides that improve migration to a target site and cell permeability, and lipid membrane structures. Examples include PLL, BSA, and charged substances that improve body stability.

  The lipid membrane structure according to the present invention can be used by being dispersed in an appropriate aqueous solvent such as physiological saline, phosphate buffer, citrate buffer, and acetate buffer. Additives such as saccharides, polyhydric alcohols, water-soluble polymers, nonionic surfactants, antioxidants, pH adjusters, and hydration accelerators may be appropriately added to the dispersion. Moreover, the lipid membrane structure according to the present invention can be stored in a state in which the dispersion is dried. Furthermore, the lipid membrane structure according to the present invention can be administered orally, and can also be administered parenterally to veins, intraperitoneally, subcutaneously, nasally and the like.

  In addition, in the lipid membrane structure of the present invention described so far, the method for producing a lipid membrane structure, and the method of encapsulating one target substance in one lipid membrane, a cationic surfactant to be added to the lipid film The particle diameter of the lipid membrane structure produced or encapsulated with the target substance can be controlled in the range of approximately 50 nm to 200 nm by making the amount of the range of 10 to 30 mol% with respect to the total lipid. Is possible. In addition, when encapsulating particles such as nucleic acids, it is preferable to adjust the amount of the cationic surfactant within the above range according to the size of the encapsulated particles.

  Hereinafter, a lipid membrane structure according to the present invention, a method for producing the lipid membrane structure, and a method for encapsulating one target substance with a single lipid membrane will be described based on examples. Note that the technical scope of the present invention is not limited to the features shown by these examples.

<Example 1> Examination of particle diameter of liposome having lipid membrane modified with cationic surfactant (1) Preparation of nucleic acid nanoparticles [1-1] Preparation of plasmid DNA (pDNA) 10 mg / mL kanamycin Escherichia coli (BD Bioscience Clontech) having a plasmid DNA encoding an Enhanced Green luminescence protein (EGFP) -luciferase fusion protein gene downstream of the CMV promoter was inoculated into 10 mL of LB medium supplemented with 10 μL of an aqueous solution. The first E. coli suspension was prepared by shaking culture for 8-9 hours.

  Subsequently, 5 mL of the first Escherichia coli suspension was added to 1 L of LB medium supplemented with 1 mL of 10 mg / mL kanamycin aqueous solution, followed by shaking culture at 37 ° C. for about 16 hours, thereby suspending the second Escherichia coli suspension. A liquid was prepared.

  Next, the second E. coli suspension was centrifuged at 4 ° C. and 4000 × g for 15 minutes, and then the supernatant was removed to prepare an E. coli pellet. Subsequently, DNA was extracted and purified using Endo Free Plasmid Mega Kit in accordance with the attached specifications to prepare a crude purified pDNA aqueous solution.

  Subsequently, 17.5 mL of isopropanol was added to 17.5 mL of the crude purified pDNA aqueous solution, and then immediately centrifuged at 4 ° C. and 15000 × g for 30 minutes. After carefully removing the supernatant, 7.5 mL of 70% (v / v) ethanol aqueous solution prepared with endotoxin-free water was added, and centrifuged again at 4 ° C. and 15000 × g for 30 minutes. . The supernatant was carefully removed and the resulting pellet was dried appropriately, and then an appropriate amount of sterilized water was added and dissolved to obtain a purified pDNA aqueous solution. Subsequently, an appropriate amount was taken from the purified pDNA aqueous solution and diluted 200-fold with sterilized water, and then the absorbance was measured at 260 nm to measure the concentration of the purified pDNA aqueous solution. Based on the measurement result of the concentration, the concentration of the purified pDNA aqueous solution was adjusted to 1.0 mg / mL by diluting with sterilized water. Thereafter, 100 μL each was dispensed into an Eppendorf tube and stored at −20 ° C.

[1-2] Preparation of nucleic acid nanoparticles Poly-L-Lysine (PLL; molecular weight 27400; Sigma-Aldrich), which is a cationic polymer, is dissolved in sterile water to prepare a 1.0 mg / mL PLL aqueous solution. did. Subsequently, the PLL aqueous solution and the purified pDNA aqueous solution of this Example (1) [1-1] were diluted with 10 mmol / L HEPES buffer (pH 7.35-7.40) to obtain 0.1 mg / mL. PLL solution and 0.1 mg / mL pDNA solution were prepared.

  A solution (nucleic acid nanoparticle solution) containing particles (nucleic acid nanoparticles) in which PLL and pDNA are condensed by electrostatic interaction by gently dropping the same amount of pDNA solution into the PLL solution while vortexing. A final concentration of 0.05 mg / mL) was prepared.

[1-3] Measurement of Particle Diameter and Zeta Potential of Nucleic Acid Nanoparticles For the nucleic acid nanoparticles of Example (1) [1-2], dynamic light scattering method using Zetasizer Nano ZS (Malvern Instruments). Were used to measure the particle size, and the zeta potential was measured by electrophoresis.

(2) Preparation of lipid film [2-1] Preparation of surfactant solution Benzyldodecyldimethylammonium bromide (BB-1; Sigma-Aldrich) and benzyltetradecyldimethylammonium bromide (BB) which are cationic surfactants -2; Sigma-Aldrich), benzylhexadecyldimethylammonium bromide (BB-3; Sigma-Aldrich), benzylhexadecyldimethylammonium bromide (BB-3; Sigma-Aldrich) and benzyloctadecyldimethylammonium bromide (BB) -4; Sigma-Aldrich), and non-ionic surfactant n-Octyl β-D-glucopyranoside (OG; Tokyo Chemical Industry Co., Ltd.) BB-1 solution, BB-2 solution, BB-3 solution, BB-4 solution, and OG solution were prepared by dissolving in ethanol so that each would be 3.03 mmol / L. The structures of BB-1, BB-2, BB-3, and BB-4 are shown at the top of FIG. 3, and the structure of OG is shown at the bottom of FIG.

[2-2] Preparation of mixed lipid solution Dioleoylglycerophosphoethanolamine (DOPE; Avanti Polar Lipids), which is both (positive and negative) charged lipids, becomes ethanol: chloroform = 1: 1 (v / v). A DOPE solution was prepared by dissolving in an ethanol / chloroform solution mixed so as to be 15 mmol / L. Moreover, the CHEMS solution was prepared by melt | dissolving the cholesteryl hemisuccinate (CHEMS; Sigma-Aldrich) which is a negatively charged lipid in ethanol so that it might become 1 mmol / L.

  The mixed lipid solution (final concentration 0.55 mmol / L) having a molar ratio of DOPE: CHEMS = 9: 2 was prepared by mixing 7.5 μL of the DOPE solution and 25 μL of the CHEMS solution, and a, b, c, d, e, f, g, h, i, j and k.

[2-3] Preparation of lipid film In this example (2) [2-2], a, b, c, d, e, f, g, h, i, j and k were used in this example (2). The BB-1 solution, BB-2 solution, BB-3 solution, BB-4 solution and OG solution of [2-1] are added as follows, and the mol% (modification concentration) of surfactant to lipid is 0. 11 / 16.5 surfactant / mixed lipid solutions were prepared.

Modification concentration (mol%)
a: not added (control) 0
b: BB-1 solution 5 μL 11
c; BB-1 solution 7.5 μL 16.5
d; BB-2 solution 5 μL 11
e; BB-2 solution 7.5 μL 16.5
f; BB-3 solution 5 μL 11
g; BB-3 solution 7.5 μL 16.5
h; BB-4 solution 5 μL 11
i; BB-4 solution 7.5 μL 16.5
j: OG solution 5 μL 11
k: OG solution 7.5 μL 16.5

  Subsequently, after drying by reduced-pressure drying using a vacuum pump, 250 μL of chloroform was added and dissolved again, and a reduced-pressure drying treatment was performed again, whereby a lipid film with an unmodified surfactant ( a), BB-1 modified lipid film (b and c), BB-2 modified lipid film (d and e), BB-3 modified lipid film (f and g), BB- Lipid films modified with 4 (h and i) and lipid films modified with OG (j and k) were prepared.

(3) Preparation of liposome encapsulating nucleic acid nanoparticles In this example (2), a, b, c, d, e, f, g, h, i, j, and k were used. -2] was added in an amount of 250 μL each and allowed to stand at room temperature for 15 minutes to hydrate each lipid film. Subsequently, the surfactant is modified as a liposome encapsulating nucleic acid nanoparticles (liposome encapsulating nucleic acid nanoparticles) by performing ultrasonic treatment using a bath type sonicator (AU-25C; Aiwa Medical Industry Co., Ltd.). Liposome (a) having a lipid membrane not modified, liposome (b, c) having a lipid membrane modified with BB-1, liposome (d, e) having a lipid membrane modified with BB-2, BB- Liposomes (f, g) having a lipid membrane 3 modified, liposomes (h, i) having a lipid membrane modified BB-4, and liposomes (j, k) having a lipid membrane modified OG-4 Prepared.

(4) Measurement of liposome particle diameter and zeta potential For a, b, c, d, e, f, g, h, i, j and k in Example (3), Example (1) [1 The particle size and zeta potential were measured by the method described in [3].

  The experiment of Example (1) [1-2] to (4) was repeated three times or more, and the average value and standard deviation (SD) of the results obtained for each sample were calculated. The results are shown in Table 1 below. 4A and B show graphs of the measurement results of the particle diameter, and FIGS. 4C and D show graphs of the measurement results of the zeta potential.

  As shown in Table 1, FIGS. 4A and B, the particle size of the liposomes is small for b, c, d, e, f, g, h and i compared to a, while j and k are a and It was almost the same in comparison. In comparison with liposomes having lipid membranes modified with the same type of surfactant, b> c, d> e, f> g and h> i, whereas j≈k.

  From these results, the liposome having a lipid membrane modified with a cationic surfactant has a smaller particle size than the liposome having a lipid membrane not modified with a surfactant, and a cationic interface. It was found that the particle size decreases as the modified concentration of the active agent increases. On the other hand, liposomes having lipid membranes modified with nonionic surfactants, compared with liposomes having lipid membranes not modified with surfactants, regardless of the modified concentration of nonionic surfactants, It became clear that the particle size was almost the same.

  That is, it was revealed that liposomes having a minute particle diameter can be obtained by performing a hydration method using a lipid film modified with a cationic surfactant.

  Moreover, as shown in Table 1 and FIG. 4A, when comparing liposomes having the same surfactant modification concentration, the particle diameters were b> d> f≈h and c> e> g≈i. From these results, when the carbon number of the alkyl group of the cationic surfactant modified on the lipid membrane is smaller than 16, the larger the carbon number, the smaller the liposome particle size, whereas the carbon number When larger than 16, it became clear that the particle size of the liposome did not decrease any more and became almost constant.

  Also, as shown in Table 1, FIGS. 4C and D, the nucleic acid nanoparticles are positive for zeta potential, whereas a, b, c, d, e, f, g, h, i, j and k. Were both negative. From these results, the liposome having a lipid membrane modified with a cationic surfactant has a lipid membrane with no surfactant modified when the modification concentration is at least 11 mol% and 16.5 mol%. It was also revealed that negatively charged liposomes that maintain the negative chargeability derived from the constituent lipids were obtained in the same manner as liposomes having lipid membranes modified with nonionic surfactants.

<Example 2> Examination of particle diameter of liposome having lipid membrane modified with tonzonium bromide (TB) (1) Preparation of nucleic acid nanoparticles Example 1 (1) [1-2] and [1-3] ], Nucleic acid nanoparticles were prepared, and the particle diameter and zeta potential were measured.

(2) Preparation of lipid film [2-1] Preparation of surfactant solution Tonzonium bromide (TB; Sigma-Aldrich), which is a cationic surfactant, was added to ethanol so as to have a concentration of 3.03 mmol / L. A TB solution was prepared by dissolving. The structure of TB is shown in the middle part of FIG.

[2-2] Preparation of Mixed Lipid Solution Six samples of mixed lipid solutions were prepared by the method described in Example 1 (2) [2-2], which were designated as a, b, c, d, e, and f, respectively. .

[2-3] Preparation of lipid film About a, b, c, d, e and f of Example (2) [2-2], the method described in Example 1 (2) [2-3] A lipid film was prepared. However, instead of the BB-1 solution, the BB-2 solution, the BB-3 solution, the BB-4 solution, and the OG solution, the TB solution of Example (2) [2-1] was used. The amount of TB solution added and the modification concentration of TB were as follows.

Modification concentration (mol%)
a: not added (control) 0
b: TB solution 5 μL 11
c: TB solution 7.5 μL 16.5
d: TB solution 10 μL 22
e; TB solution 12.5 μL 27.5
f; TB solution 25 μL 55

(3) Preparation of liposome encapsulating nucleic acid nanoparticles, and measurement of particle diameter and zeta potential Example 1 (2) About a, b, c, d, e and f of [2-3] According to the method described in 3), as a liposome encapsulating a nucleic acid nanoparticle, a liposome having a lipid membrane not modified with TB (TB unmodified liposome; a) and a liposome having a lipid membrane modified with TB (TB modified liposome; b, c, d, e and f) were prepared. Thereafter, the particle diameter and zeta potential were measured by the method described in Example 1 (1) [1-3].

  The experiment of this Example (1)-(3) was repeated 3 times or more, and the average value and standard deviation (SD) of the result obtained about each sample were computed. The results are shown in Table 2 below. Further, a graph of the measurement result of the particle diameter is shown in FIG. 5A, and a graph of the measurement result of the zeta potential is shown in FIG. 5B.

  As shown in Table 2 and FIG. 5A, the particle size of the liposome was a> b> c> d≈e≈f.

  From these results, it was clarified that the TB modified liposome has a smaller particle size than the TB unmodified liposome. When the modification concentration of TB is at least 22 mol%, the liposome particle size decreases as the modification concentration increases. On the other hand, when the modification concentration is at least 22 mol%, the liposome particle size does not decrease any more. It became clear that it was almost constant.

  Furthermore, as shown in Table 2 and FIG. 5A, the particle diameters of d, e, and f were about 130 nm, which was a value that was very close to about 100 nm, which is the particle diameter of nucleic acid nanoparticles. Here, as shown in FIG. 5C, the thickness of the lipid membrane is about 4 nm, and the total thickness of the inner aqueous phase is about 15 nm. Therefore, one nucleic acid nanoparticle is added to one lipid membrane. The particle diameter of the liposome encapsulated in is considered to be about 100 nm + about 15 nm × 2 = about 130 nm. From this, it became clear that d, e, and f are liposomes in which one nucleic acid nanoparticle is encapsulated with one lipid membrane. That is, it is clear that the liposome obtained by performing the hydration method using a lipid film in which TB is modified by at least about 22 mol% is a liposome in which one target substance is encapsulated by one lipid membrane. became.

  From the above results, it has been clarified that one target substance can be encapsulated with one lipid membrane by performing a hydration method using a lipid film modified with a cationic surfactant.

  In addition, as shown in Table 2 and FIG. 5B, the zeta potential was positive for nucleic acid nanoparticles, whereas a, b, c, d, e, and f were all negative. From these results, it becomes clear that TB-modified liposomes having a modified TB concentration of less than 55 mol% become negatively charged liposomes that maintain the negative chargeability derived from the constituent lipids, as in the case of TB-unmodified liposomes. It was.

(4) Preparation of liposome without encapsulating nucleic acid nanoparticles (empty liposome) and measurement of particle diameter and zeta potential [4-1] Preparation of mixed lipid solution As described in Example 1 (2) [2-2] According to the above method, 6 samples of mixed lipid solutions were prepared, which were designated as g, h, i, j, k, and l, respectively.

[4-2] Preparation of lipid film For g, h, i, j, k, and l of Example (4) [4-1], the method described in Example (2) [2-3] was used. A lipid film was prepared. However, the amount of TB solution added and the modified concentration of TB were as follows.

Modification concentration (mol%)
g; not added (control) 0
h; TB solution 5 μL 11
i: TB solution 7.5 μL 16.5
j: TB solution 10 μL 27.5
k: TB solution 12.5 μL 55
l; TB solution 25 μL 110

[4-3] Preparation of empty liposomes and measurement of particle diameter and zeta potential In this example (4), g, h, i, j, k, and l of [4-1] are described in Example 1 (3). In the described method, a liposome having a lipid film not modified with TB (TB unmodified empty liposome; g) as a liposome not encapsulating nucleic acid nanoparticles (empty liposome), replacing the nucleic acid nanoparticle solution with sterile water; ) And liposomes with TB-modified lipid membranes (TB-modified empty liposomes; h, i, j, k and l). Thereafter, the particle diameter and zeta potential were measured by the method described in Example 1 (1) [1-3].

  The experiment of this example (4) [4-1] to [4-3] was repeated three times or more, and the average value and standard deviation (SD) of the results obtained for each sample were calculated. The results are shown in Table 3. In addition, FIG. 6A shows a graph of the measurement result of the particle diameter, and FIG. 6B shows a graph of the measurement result of the zeta potential.

  As shown in Table 3 and FIG. 6A, the particle size of empty liposomes was smaller than that of g in all of h, i, j, k and l. Further, h> i> k> j> l. From these results, it was revealed that TB-modified empty liposomes have a smaller particle size than TB unmodified empty liposomes.

  That is, by performing a hydration method using a lipid film modified with a cationic surfactant, a liposome having a minute particle size can be obtained regardless of whether the target substance is encapsulated or not. It was revealed.

  Further, as shown in FIG. 6B, the zeta potential was negative for g, h, i, and j, but positive for k and l. From these results, TB-modified empty liposomes having a modified TB concentration of at least 55 mol% or more are positively charged liposomes, whereas TB-modified empty liposomes having a modified TB concentration of less than 55 mol% are not modified with TB. It became clear that it becomes a negatively charged liposome in which the negatively charged property derived from the constituent lipid is maintained, like the empty liposome.

<Example 3> Examination of transfection activity of TB modified liposome (1) Preparation of R8 / TB modified liposome [1-1] Preparation of TB modified liposome and TB unmodified liposome Example 2 (1), (2) [ 2-1], [2-2], [2-3] and (3), as a nucleic acid nanoparticle-encapsulated liposome, TB unmodified liposome, modified concentrations of 11 mol%, 16.5 mol% and 22 mol% TB modified liposomes (11 mol% TB modified liposome, 16.5 mol% TB modified liposome and 22 mol% TB modified liposome) were prepared.

[1-2] Modification of R8 R8 (RRRRRRRR: SEQ ID NO: 6), a peptide known to confer a certain cell permeability to liposomes (Kentaro Kogure, Pharmaceutical Journal, Vol. 127, No. 10, Peptide 1685-1691, 2007) Peptide-modified fatty acid (STR-R8) with stearic acid (STR) added to the C-terminal is purchased from KUROBO and dissolved in sterile water to 2.0 mg / mL. Thus, an STR-R8 solution was prepared.

  In this example (1) [1-1], 100 mL of the liposome-containing solution was mixed with 2.33 mL of the STR-R8 solution, and then incubated at room temperature for 30 minutes, whereby the total lipids of the liposomes were obtained. R8 was modified to 5 mol%.

(2) Examination of Transfection Activity [2-1] Preparation of Medium After preparing Dulbecco's modified Eagle medium (DMEM) containing no antibiotics according to a conventional method, filter sterilization is performed in a clean bench, and this is DMEM. (-) Medium was used. Moreover, Fetal bovine serum (FBS) was added to DMEM so that it might become 20% (v / v), and this was made into the DMEM (+) culture medium. DMEM (−) medium and DMEM (+) medium were stored at 4 ° C. until use.

[2-2] Preparation of Liposome-Containing Medium For each liposome of Example (1) [1-2], 0.04 μg pDNA equivalent and 0.1 μg pDNA equivalent of Example (2) [2-1] were used. 0.25 mL of liposome-containing medium is prepared by adding to DMEM (−) medium, and a-1, a-2, b-1, b-2, c-1, c-2, d-1 and d -2. The types of liposomes and the amount of pDNA contained in each liposome-containing medium are as follows.

Liposome type pDNA amount a-1; TB unmodified liposome 0.04 μg
a-2; TB unmodified liposome 0.1 μg
b-1; 0.04 μg of 11 mol% TB-modified liposome
b-2; 11 mol% TB-modified liposome 0.1 μg
c-1; 16.5 mol% TB-modified liposome 0.04 μg
c-2; 16.5 mol% TB-modified liposome 0.1 μg
d-1; 22 mol% TB modified liposome 0.04 μg
d-2; 22 mol% TB-modified liposome 0.1 μg

[2-3] Pre-culture of HeLa cells Twelve plates prepared by seeding 0.5 mL (equivalent to 4 × 10 ⁴ ) of HeLa cell suspension derived from human cervical cancer in a 24-well plate (Corning), A, B, C, D, E, F, G, H, I, J, K, and L. To these, the DMEM (+) medium of Example (2) [2-1] was added and pre-cultured in an environment of 37 ° C. and 5% CO ₂ for 24 hours.

[2-4] Transfection into HeLa cells The media of A, B, C, D, E, F, G, H, I, J, K, and L in Example (2) [2-3] are removed. Then, 0.5 mL of PBS was added and washed. Next, for A and B, using commercially available transfection reagent Lipofectamine PLUS (LFN PLUS), according to the attached specifications, 0.04 μg of pDNA of the purified pDNA aqueous solution of Example 1 (1) [1-1]. Equivalent amounts and pDNA equivalents of 0.1 μg were each transfected. C, D, E, F, G, H, I and J include a-1, a-2, b-1, b-2, c-1, c-2, d-1, d-. 2 was added, and K and L were respectively added with an amount corresponding to 0.04 μg pDNA and an amount corresponding to 0.1 μg pDNA of the purified pDNA aqueous solution of Example 1 (1) [1-1]. Transfection using liposomes was performed by incubating in the environment of ₂ for 3 hours.

  Thereafter, the medium of A, B, C, D, E, F, G, H, I, J, K, and L was replaced with the DMEM (+) medium of Example (2) [2-1]. Culturing was further carried out after 21 hours in the environment.

(3) Measurement of luciferase activity and calculation of cell viability Medium of A, B, C, D, E, F, G, H, I, J, K and L of Example (2) [2-4] Then, 0.5 mL of PBS was added to wash the cells, and reporter lysis buffer (Promega) was added in an amount of 75 μL. After sealing a 24-well plate using parafilm, the plate was incubated at −80 ° C. for 30 minutes or more. Thereafter, centrifugation was performed at 4 ° C. and 15000 × g for 5 minutes, and 50 μL of the supernatant was collected and used as a cell lysate for each sample.

  After adding 50 μL of luciferase assay reagent (Promega) to 20 μL of the collected cell lysate, the amount of luminescence was measured using a luminometer (Luminescence-PSN; ATTO) according to the attached specifications. In addition, the protein concentration of the cell lysate was measured using a BCA protein assay kit (PIERCE) according to the attached specifications. Based on these results, the value of luciferase activity was determined using the following formula (1).

  Formula (1); Luciferase activity (RLU / mg) = Luminescence (RLU / 20 μL) × 50 / Protein concentration (mg / mL)

  Further, the cell viability is calculated using the following formula (2), assuming that K is set to 100% for A, C, E, G and I, and L is set to 100% for B, D, F, H and J, respectively. Calculated.

  Formula (2); cell viability (%) = {A, B, C, D, E, F, G, H, I or J protein concentration (mg / mL) / K or L protein concentration (mg / mL)} × 100

  The experiments of Examples (1) and (2) [2-2] to (3) were repeated three times or more, and the average value and standard deviation (SD) of the results obtained for each sample were calculated. The measurement results of luciferase activity are shown in Table 4 below, and the calculation results of cell viability are shown in Table 5, respectively. In addition, a graph of the luciferase activity is shown in the left diagram of FIG. 7, and a graph of the calculation result of the cell viability is shown in the right diagram of FIG.

  As shown in Table 4 and the left figure of FIG. 7, the luciferase activity when 0.04 μg of DNA was transfected was C <A <E <G <I. Further, the luciferase activity when 0.1 μg of DNA was transfected was D <F <H <B <J.

  From these results, when 0.04 μg of DNA is transfected, the TB modified liposome has an activity to introduce a gene into the nucleus of the cell (transfection activity) as compared with the TB unmodified liposome and a commercially available transfection reagent. Became clear. In addition, when 0.1 μg of DNA was transfected, TB modified liposomes were found to have higher transfection activity than TB unmodified liposomes. Furthermore, when 0.1 μg of DNA was transfected, it was revealed that 22 mol% TB-modified liposomes had higher transfection activity than commercially available transfection reagents.

  That is, it has been clarified that the transfection activity of liposomes having a lipid membrane modified with a cationic surfactant as a single membrane or multiple membranes is remarkably improved.

  Moreover, as shown in Table 5 and the right figure of FIG. 7, the cell viability was larger than 80% in all of A, B, C, D, E, F, G, H, I, J, K, and L. From these results, it was revealed that TB modified liposomes having modified concentrations of at least 11 mol%, 16.5 mol% and 22 mol% are not cytotoxic.

<Example 4> Examination of the number of lipid membranes of liposomes (1) Preparation of lipid film DOPE (NBD-labeled DOPE) to which a fluorescent dye, Nitro-2-1,3-Benzoxadiazol-4-yl (NBD) is bound; NBD-labeled DOPE solution was prepared by dissolving (Avanti Polar Lipids) in ethanol so as to be 0.1 mmol / L.

  Subsequently, an NBD-labeled DOPE solution was added to the mixed lipid solution prepared by the method described in Example 1 (2) [2-2], and 1 mol% of the total lipid was NBD-labeled DOPE. Were prepared as a, b, c and d.

  Next, with respect to a, b, c and d, in the method described in Example 2 (2) [2-3], the amount of TB solution added and the modification concentration of TB were changed as follows, and TB was modified. Lipid films (a) and lipid films (b, c and d) not modified with TB were prepared.

Modification concentration (mol%)
a: TB solution 10 μL 22
b; not added 0
c; not added 0
d; not added 0

(2) Preparation of TB modified liposome and TB unmodified liposome About a and b of this Example (1), TB modification was carried out as a nucleic acid nanoparticle enclosure liposome labeled with NBD by the method as described in Example 1 (3). Liposomes (a) and TB unmodified liposomes (b) were prepared.

(3) Preparation of multilamellar liposomes (MLV) By adding 250 μL of 10 mmol / L HEPES buffer (pH 7.4) to c of Example (1), and allowing to stand at room temperature for 15 minutes, The lipid film was hydrated. Subsequently, NBD-labeled multilamellar liposomes (MLV) were prepared by vortexing for 1 minute.

(4) Preparation of single membrane liposome (SUV) By adding 2 mL of 10 mmol / L HEPES buffer solution (pH 7.4) to d of Example (1), and allowing to stand at room temperature for 15 minutes. The lipid film was hydrated. Subsequently, ultrasonic treatment was performed for 1 minute using a bus type sonicator (AU-25C; Aiwa Medical Industry Co., Ltd.). Furthermore, using a probe type sonicator, ultrasonic treatment was performed for 10 minutes under cooling with ice water. Next, the whole amount was transferred to a 2.0 mL capacity Eppendorf tube and centrifuged at 4 ° C. and 15000 rpm for 5 minutes to precipitate titanium nanoparticles generated by ultrasonication. The supernatant was collected leaving 100 μL of the lower layer and transferred to another Eppendorf tube. NBD-labeled single membrane liposomes (SUV) were prepared by performing this centrifugation and supernatant recovery operation three times in total.

(5) Fluorescence intensity measurement Hydrosulfite sodium (Wako Pure Chemical Industries, Ltd.) was dissolved in 1 mmol / L Tris buffer solution (pH 10) to a concentration of 0.5 mol / L to prepare a hydrosulfite sodium solution.

  With respect to a and b in this Example (2), c in this Example (3), and d in this Example (4), the fluorescence intensity of NBD was continuously measured for 200 seconds using a luminometer. During 10 seconds from the start of the measurement, 2.75 μL of the hydrosulfite sodium solution was added to each sample and mixed for 5 seconds. Furthermore, 12.5 μL of a 10% (v / v) Triton X-100 solution was added to each sample at the elapse of 105 seconds from the start of measurement and mixed for 5 seconds. The results are shown in Table 6 below. Moreover, the measurement result of fluorescence intensity is shown in the upper diagram of FIG.

  Hydrosulfite sodium is a quencher that reduces the NBD chromophore by a reducing action and loses its fluorescence. As shown in the lower figure of FIG. 8, since hydrosulfite sodium does not have lipid membrane permeability, in this example, hydrosulfite sodium added to the liposome solution is the outermost lipid bilayer of the liposome. Only the fluorescence of NBD present in the layer in contact with the aqueous phase disappears. Therefore, it is considered that the greater the number of lipid membranes possessed by the liposome, the greater the fluorescence intensity after addition of hydrosulfite sodium.

  As shown in Table 6 and the upper diagram of FIG. 8, the fluorescence intensity between the addition of sodium hydrosulfite and the addition of Triton X-100 was c> b> d> a. From these results, it became clear that TB modified liposomes have fewer lipid membranes than TB unmodified liposomes and MLV which is a liposome prepared by a conventional method.

  That is, it has been clarified that the number of lipid membranes is suppressed in liposomes obtained by performing hydration using a lipid film modified with a cationic surfactant.

  Note that the fluorescence intensity of TB-modified liposomes is smaller than that of SUV, which is a single-membrane liposome, because, in TB-modified liposomes, lipid-molecule flip-flop occurs due to the modification of TB to the lipid membrane, This is probably because the NBD existing on the side in contact with the inner aqueous phase was exposed to the liposome surface and quenched.

<Example 5> Encapsulation of liposomes with TB-modified lipid membrane (1) Preparation of negatively charged bilayer liposomes Negatively charged bilayer liposomes were prepared according to a previous report (International Publication WO2007 / 037444). Specifically, first, phosphatidic acid (PA) which is a negatively charged lipid is added to an ethanol / chloroform solution mixed so that ethanol: chloroform = 1: 1 (v / v) to 1 mmol / L. A PA solution was prepared by dissolving. In addition, a DOPE solution and a CHEMS solution were prepared by the method described in Example 1 (2) [2-2], and an NBD-labeled DOPE solution was prepared by the method described in Example 4 (1).

  A negatively charged mixed lipid solution (total 152.5 nmol) having a molar ratio of DOPE: CHEMS: PA = 9: 2: 1.2 was prepared by mixing 60 μL of the DOPE solution, 200 μL of the CHEMS solution, and 120 μL of the PA solution. An NBD-labeled DOPE solution was added thereto to prepare an NBD negatively charged mixed lipid solution (total 152.5 nmol) in which 1 mol% of the total lipid was NBD-labeled DOPE.

  For the NBD negatively charged mixed lipid solution, an SUV was prepared by the method described in Example 4 (4). After gently adding a half amount (v / v) of the nucleic acid nanoparticle solution prepared by the method described in Example 1 (1) [1-1] and [1-2] to the SUV-containing solution, By vortexing for 5 seconds, negatively charged bilayer liposomes encapsulating nucleic acid nanoparticles and labeled with NBD were prepared.

(2) Encapsulation of liposomes with lipid membrane [2-1] Preparation of positively charged mixed lipid solution Cholesterol, which is an uncharged lipid, and 1,2-dioleyloxy-3, which is a positively charged lipid -Ethanol / chloroform solution in which trimethylammonium propane (1,2-dioleoyl-3-trimethylammiopropane; DOTAP; Avanti Polar Lipids) was mixed in ethanol and ethanol / chloroform = 1: 1 (v / v), respectively. A Chol solution and a DOTAP solution were prepared by dissolving the solution to 15 mmol / L. Moreover, the DOPE solution was prepared by the method as described in Example 1 (2) [2-2].

  29.4 μL of DOPE solution, 22 μL of DOTAP solution and 22 μL of Chol solution were mixed to prepare 2 samples of positively charged mixed lipid solution (total 137.5 nmol) having a molar ratio of DOPE: CHEMS: Chol = 3: 4: 3 And a and b, respectively.

[2-2] Preparation of negatively charged mixed lipid solution A negatively charged mixed lipid solution having a molar ratio of DOPE: CHEMS: PA = 9: 2: 1.2 was prepared by the method described in this Example (1). Two samples were prepared, designated c and d, respectively.

[2-3] Preparation of Negatively Charged Lipid Film and Positively Charged Lipid Film a and b of Example (2) [2-1] and c and d of Example (2) [2-2] In the method described in Example 2 (2) [2-3], the amount of added TB solution and the modified concentration of TB are as follows, and the positively charged lipid film (a; TB modified with TB) Modified positively charged lipid film), positively charged lipid film not modified with TB (b; TB unmodified positively charged lipid film), negatively charged lipid film with modified TB (c; TB modified negatively charged) Lipid film) and negatively charged lipid film (d; TB unmodified negatively charged lipid film) not modified with TB were prepared.

Modification concentration (mol%)
a: TB solution 10 μL 22
b; not added 0
c: TB solution 10 μL 22
d; not added 0

[2-4] Encapsulation of liposomes TB modified positively charged lipid film (a), TB unmodified positively charged lipid film (b), TB modified negatively charged lipid film of Example (2) [2-3] Add 250 μL each of the solution containing the negatively charged bilayer liposome of Example (1) to (c) and the TB unmodified negatively charged lipid film (d), and incubate at room temperature for 15 minutes. Thus, negatively charged bilayer liposomes were encapsulated.

(3) Fluorescence Intensity Measurement This example (2) a, b, c, d of [2-4] and the negatively chargeable bilayer liposome of this example (1) are described in Example 4 (5). The fluorescence intensity was measured by this method. The results are shown in Table 7 below. In addition, FIG. 9A shows a graph of results for a, b and negatively charged bilayer liposomes, and FIG. 9B shows a graph of results for c, d and negatively charged bilayer liposomes. Show.

  In addition, a, b, c, and d are liposomes prepared with the intention of encapsulating liposomes labeled with NBD (labeled liposomes) with lipid membranes not labeled with NBD (unlabeled lipid membranes). Therefore, when the fluorescence of NBD is reduced by the addition of sodium hydrosulfite, the labeled liposome is not encapsulated with the unlabeled lipid membrane, or the membrane fusion between the lipid membrane of the labeled liposome and the unlabeled lipid membrane occurs. Conceivable. On the other hand, when the degree of decrease in NBD fluorescence due to the addition of sodium hydrosulfite is smaller than that of the labeled liposome, it is considered that the labeled liposome was encapsulated with an unlabeled lipid membrane.

  As shown in Table 7 and FIG. 9A, the negatively charged bilayer liposome has a constant fluorescence intensity during the period from the addition of sodium hydrosulfite to the addition of Triton X-100, whereas a and b Shortly after the addition of sodium hydrosulfite, the fluorescence intensity decreased significantly, and almost no fluorescence was detected immediately before adding Triton X-100. From these results, when negatively charged bilayer liposomes are encapsulated with a positively charged lipid membrane, it becomes clear that membrane fusion occurs regardless of the presence or absence of TB modification in the positively charged lipid membrane. It was.

  On the other hand, as shown in Table 7 and FIG. 9B, between the addition of sodium hydrosulfite and the addition of Triton X-100, the fluorescence intensity of c is larger than that of the negatively charged bilayer liposome, The fluorescence intensity was almost the same as that of the negatively charged bilayer liposome. From these results, when negatively charged bilayer liposomes are encapsulated with a negatively charged lipid membrane, they can be encapsulated if TB is modified in the negatively charged lipid membrane, whereas TB is modified. It became clear that it could not be sealed without it.

  From the above results, it is clear that negatively charged liposomes can be encapsulated with a negatively charged lipid membrane by performing a hydration method using a negatively charged lipid film modified with a cationic surfactant. became.

  That is, as shown in FIG. 10, after obtaining a bilayer liposome according to the previous report, a hydration method is performed using a lipid film in which a cationic surfactant is modified, whereby one bilayer liposome is obtained. It was revealed that a triple membrane liposome can be obtained by encapsulating with a single lipid membrane.

Example 6 Examination of Transfection Activity of TB Modified Trilamellar Liposomes (1) Preparation of TB Modified Trilamellar Liposomes Negatively charged 2 encapsulating nucleic acid nanoparticles by the method described in Example 5 (1) Sheet membrane liposomes were prepared. However, NBD-labeled DOPE solution was not added. Subsequently, negatively charged bilayer liposomes were encapsulated in a TB-modified negatively charged lipid film by the method described in Example 5 (2) [2-2], [2-3] and [2-4]. This was designated as a TB-modified trilamellar liposome. Subsequently, R8 was modified on the TB-modified trilamellar liposome by the method described in Example 3 (1) [1-2].

(2) Preparation of quadrilamellar liposomes In the method described in a previous report (Akita H. et al., Biomaterials, No. 15, Vol. 30, Nos. 2940-2949, 2009), the lipid membrane composition was changed to DOPE: CHEMS: Four membrane liposomes modified with R8 were prepared with PA = 9: 2: 1.2.

(3) Examination of transfection activity [3-1] Preparation of medium The following reagent is added to α-modified minimal essential medium (α-MEM) so as to have the following final concentration, and this is added to α-MEM (−). The medium was used.

L-glutamine 4mmol / L
Sodium pyruvate 1mmol / L
Granulocyte macrophage colony-stimulating factor
5 ng / mL

  Moreover, the following reagent was added to α-MEM so as to have the following final concentration, and this was used as α-MEM (+) medium.

Deactivated FBS 20% (v / v)
L-glutamine 4mmol / L
Sodium pyruvate 1mmol / L
Penicillin 100 U / mL
Streptomycin 100 μg / mL

  α-MEM (−) medium and α-MEM (+) medium were stored at 4 ° C. until use.

[3-2] Preparation of liposome-containing medium About the TB-modified trilamellar liposome of Example (1) and the tetralamellar liposome of Example (2), an equivalent amount of 0.04 μg of pDNA was used in this Example (3). [3-1] α-MEM (−) medium was added to prepare 0.25 mL of trilamellar liposome-containing medium and tetralamellar liposome-containing medium.

[3-3] Pre-culture of JAWSII cells Three non-dividing JAWSII cells (American Type Culture Collection) seeded in a 24-well plate (Corning) at 1.6 × 10 ⁴ cells / well Prepared as A, B and C. The α-MEM (+) medium of Example (3) [3-1] was added thereto, and precultured in an environment of 37 ° C. and 5% CO ₂ for 48 hours.

[3-4] Transfection into JAWSII cells The medium of A, B and C in Example (3) [3-2] was removed, and 0.5 mL of PBS was added and washed. Next, with respect to A, an amount corresponding to 0.04 μg of pDNA in the purified pDNA aqueous solution of Example 1 (1) [1-1] was transfected using LFN PLUS according to the attached specifications. Further, to B and C, the three-membrane liposome-containing medium and the four-membrane liposome-containing medium of Example (3) [3-2] were added, respectively, in an environment of 37 ° C. and 5% CO _2. Transfection was performed by incubating for 3 hours.

  Thereafter, the medium of A, B and C was replaced with the α-MEM (+) medium of Example (3) [3-1], and further cultured for 21 hours in the same environment.

(4) Measurement of luciferase activity About A, B, and C of this Example (3) [3-3], the luciferase activity was measured by the method as described in Example 4 (3).

  The experiments of Examples (1), (2) and (3) [3-2] to (4) were repeated three times or more, and the average value and standard deviation (SD) of the results obtained for each sample were calculated. . The result is shown in FIG.

As shown in FIG. 11, the luciferase activity was 3.3 × 10 ⁶ (3258833) for A, 1.2 × 10 ^{5 for} B, and 7.5 × 10 ⁵ for C. From these results, it was revealed that the transfection activity of TB-modified trilamellar liposomes is lower than that of commercially available transfection reagents, but is about 6 times higher than that of tetralamellar liposomes.

  That is, not only in dividing cells such as HeLa cells, but also in non-dividing cells, the transfection activity of liposomes having a lipid membrane modified with a cationic surfactant as the outermost membrane of multiple membranes is significantly improved. It became clear.

<Example 7> Examination of optimization of TB-modified trilamellar liposome (1) Preparation of PLL-stabilized bilayer liposome and BSA-stabilized bilamellar liposome Negative chargeability by the method described in Example 5 (1) A solution containing bilamellar liposomes was prepared. Two samples of 100 μL each of the solution containing the negatively charged bilayer liposome were prepared, and one of them was prepared as 1.0 mg / mL PLL solution 4 prepared by the method described in Example 1 (1) [1-2]. 0.0 μL was added and 0.5 mg / mL bovine serum albumin (BSA) solution was added to the other half. Then, the solution containing negatively charged bilayer liposomes (PLL-stabilized bilayer liposomes) stabilized by electrostatically binding PLL and non-specifically adsorbing BSA are incubated at room temperature for 15 minutes. A solution containing negatively charged bilayer liposomes (BSA-stabilized bilayer liposomes) that had been stabilized was prepared.

(2) Preparation of TB-modified trilamellar liposomes By the method described in Example 5 (2) [2-3], two samples of negatively charged lipid films modified with TB were prepared as a and b. Subsequently, in the method described in Example 5 (2) [2-4], instead of the solution containing the negatively charged bilayer liposome of Example 5 (1), a is used in this Example (2). A solution containing a PLL-stabilized bilayer liposome was added, and for b, a solution containing a BSA-stabilized bilayer liposome was added to prepare a TB-modified trilamellar liposome. The outline of the preparation process of these TB modified trilamellar liposomes is shown in FIG.

(3) Fluorescence intensity measurement For a and b of this Example (2) and the negatively chargeable bilayer liposome of this Example (1), the fluorescence intensity was measured by the method described in Example 4 (5). It was. The results are shown in Table 8 below. Moreover, the measurement result of fluorescence intensity is shown in FIG.

  As shown in Table 8 and FIG. 13, between the addition of sodium hydrosulfite and the addition of Triton X-100, the fluorescence intensity of a is smaller than that of negatively charged bilayer liposomes, whereas b The fluorescence intensity of was higher than that of negatively charged bilayer liposomes.

  From these results, when encapsulated with a negatively charged lipid film modified with TB, PLL-stabilized bilayer liposomes cause membrane fusion, whereas BSA-stabilized bilayer liposomes membrane-fuse. It became clear that it was possible to encapsulate with almost no generation.

<Example 8> Examination of transfection activity of optimized TB-modified trilamellar liposomes (1) Preparation of TB-modified trilamellar liposomes By the method described in Example 7 (1), negatively charged bilayer liposomes Then, a solution containing each of the PLL-stabilized bilayer liposome and the BSA-stabilized bilayer liposome was prepared. However, NBD-labeled DOPE solution was not added.

  Next, three samples of negatively charged lipid films modified with TB were prepared by the method described in Example 5 (2) [2-3], and designated as a, b, and c. Subsequently, in the method described in Example 5 (2) [2-4], instead of the solution containing the negatively charged bilayer liposome of Example 5 (1), a is a negatively charged bilayer membrane. A solution containing liposomes, a solution containing PLL-stabilized bilayer liposomes for b, and a solution containing BSA-stabilized bilayer liposomes for c were added to prepare TB-modified trilamellar liposomes.

  Subsequently, R8 was modified for a, b and c by the method described in Example 3 (1) [1-2].

(2) Examination of transfection activity Using a, b and c of this Example (1), it is described in Example 6 (3) [3-1], [3-2] and [3-3]. The method was used to transfect JAWSII cells. Thereafter, the luciferase activity was measured by the method described in Example 4 (3).

  The experiments of Examples (1) and (2) were repeated three times or more, and the average value and standard deviation (SD) of the results obtained for each sample were calculated. The result is shown in FIG.

  As shown in FIG. 14, the luciferase activity when transfection is performed with a and b is lower than that of a commercially available transfection reagent, whereas the luciferase activity when transfection is performed with c is commercially available. Compared to the transfection reagent.

  From these results, the transfection activity of TB-modified trilamellar liposomes encapsulating BSA-stabilized bilayer liposomes is as follows: TB-encapsulated PLL-stabilized bilayer liposomes and non-stabilized negatively charged bilayer liposomes It was found to be higher than the modified trilamellar liposome and equivalent to a commercially available transfection reagent.

  That is, by stabilizing the bilamellar liposomes with BSA, the transfection activity of the trilamellar liposomes encapsulating the bilamellar liposomes using a lipid film modified with a cationic surfactant is remarkably increased. It became clear that it improved.

<Example 9> Photomicrograph of single-membrane liposome 10 μL of the TB modified liposome d / HEPES suspension of the present invention prepared in Example 2 (3) was rapidly frozen using liquid nitrogen and fixed. The observation was made with a transmission electron microscope. A part of the observation photograph is shown in FIG.

  The average particle size of the particles (the liposome of the present invention) observed in a microscopic field was in the range of 50 to 100 nm. This value is consistent with the particle diameter measurement data measured in Example 2 (3), considering that the liquid encapsulated in the TB modified liposome is removed by rapid freezing. In addition, all the observed particles had an inner black outline and an outer white outline. The black outline is considered to correspond to the encapsulated core particle. The white outline had a thickness of about 3.5 nm, and this value approximates the theoretical thickness of one lipid membrane of 4.0 nm.

  The above-mentioned microscopic observation results show that the liposome of the present invention has a single lipid membrane.

[0001]
Technical field [0001]
TECHNICAL FIELD The present invention relates to a lipid membrane structure, a method for producing a lipid membrane structure, and a method for encapsulating one target substance with a single lipid membrane, and more particularly, a lipid membrane modified with a cationic surfactant. The present invention relates to a lipid membrane structure having a single membrane or a plurality of outermost membranes, a method for producing the lipid membrane structure, and a method for encapsulating one target substance in a single lipid membrane.
Background art [0002]
A vector for reliably delivering a substance such as a protein, a drug, or a nucleic acid to a target site of a living individual has been actively developed. Vectors developed so far are roughly classified into viral vectors and non-viral vectors. Viral vectors are generally excellent in gene transfer ability to introduce the target gene into the nucleus of the target cell, but there are problems such as difficulty in mass production, antigenicity, and toxicity. As a few non-viral vectors, lipid membrane structures represented by liposomes have attracted attention.
[0003]
Liposomes are lipid membrane structures having a lipid membrane composed of artificially prepared lipid bilayers as a basic component, and have the ability to encapsulate various substances and send them into target cells. In addition to the advantage that liposomes can improve the directivity to the target site by introducing functional molecules such as antibodies, proteins, sugar chains, etc. on the surface, liposomes can be decomposed in vivo. It has the advantage of being protected from action and metabolic action, and the advantage of preventing the substance from acting at a site other than the target (side effects). So far, for example, a liposome having a peptide containing a plurality of continuous arginine residues on its surface and having a nuclear translocation ability has been developed (Patent Document 1).

［式中：Ｒ_１はアルキル基であり、Ｒ_２は疎水基である。］の陽イオン性界面活性剤である、（１）に記載の脂質膜構造体。
［００１３］
（３）Ｒ_１が炭素数が１２以上のアルキル基であり、かつＲ_２が次式（化２）[0003]
Therefore, it is difficult to put it to practical use (Jeffs LB, Pharm Res., Vol. 22, No. 3, 362-72, 2005).
[0009]
The present invention has been made in order to solve such problems, and can be easily produced and includes a lipid membrane structure having a minute and uniform particle diameter and a substance to be encapsulated (target substance). It is an object to provide a lipid membrane structure in which the number and the number of lipid membranes are controlled, a method for producing the same, and a method for encapsulating one target substance with one lipid membrane.
Means for Solving the Problems [0010]
As a result of earnest research, the present inventors have a fine and uniform particle size by performing a hydration method using a lipid film modified with a cationic surfactant as shown in the lower part of FIG. The ability to obtain liposomes in which the number of liposomes and substances to be encapsulated (target substances) and the number of lipid membranes are controlled, the ability to encapsulate one target substance with a single lipid membrane, and cationic surface activity The inventors found that the transfection activity of liposomes having a lipid membrane modified with an agent as a single membrane or as the outermost membrane of a plurality of membranes was remarkably improved, and completed the following inventions.
[0011]
(1) A lipid membrane structure having a lipid membrane modified with a cationic surfactant as a single membrane or as an outermost membrane of a plurality of membranes.
[0012]
(2) The cationic surfactant is represented by the following formula (Formula 1)
[Chemical 1]

[Wherein, R ₁ is an alkyl group, and R ₂ is a hydrophobic group. The lipid membrane structure according to (1), which is a cationic surfactant.
[0013]
(3) R ₁ is an alkyl group having 12 or more carbon atoms, and R ₂ is represented by the following formula (Formula 2):

または次式（化３）
［化３］

の基である、（２）に記載の脂質膜構造体。
［００１４］
（４）Ｒ_１が炭素数が１６のアルキル基であり、かつＲ_２が次式（化４）
［化４］

の基である、（２）に記載の脂質膜構造体。
［００１５］
（５）負帯電性脂質膜構造体である、（１）から（４）のいずれかに記載の脂質膜構造体。
［００１６］
（６）リポソームである、（１）から（５）のいずれかに記載の脂質膜構造体。
［００１７］
（７）陽イオン性界面活性剤で修飾された脂質膜を１枚膜としてまたは[0004]
[Chemical formula 2]

Or the following formula (Formula 3)
[Chemical formula 3]

The lipid membrane structure according to (2), which is a group of
[0014]
(4) R ₁ is an alkyl group having 16 carbon atoms, and R ₂ is represented by the following formula (Formula 4)
[Chemical formula 4]

The lipid membrane structure according to (2), which is a group of
[0015]
(5) The lipid membrane structure according to any one of (1) to (4), which is a negatively charged lipid membrane structure.
[0016]
(6) The lipid membrane structure according to any one of (1) to (5), which is a liposome.
[0017]
(7) A lipid membrane modified with a cationic surfactant as a single membrane or

［式中：Ｒ_１はアルキル基であり、Ｒ_２は疎水基である。］の陽イオン性界面[0005]
A method for producing a lipid membrane structure having an outermost membrane of a plurality of membranes, the method comprising the following steps (i), (ii) or (iii): (i) a lipid and a cationic surfactant To prepare a lipid film modified with a cationic surfactant, and (ii) adding a cationic surfactant to the lipid film to form a lipid film modified with a cationic surfactant A step of preparing, (iii) a step of modifying a cationic surfactant on the outermost membrane of a single membrane or a plurality of membranes of a lipid membrane structure having a single membrane or a plurality of membranes.
[0018]
(8) (i) a step of preparing a lipid film modified with a cationic surfactant by mixing a lipid and a cationic surfactant; or (ii) adding a cationic surfactant to the lipid film. And (iv) a method of preparing a lipid film modified with a cationic surfactant, and (iv) hydrating the prepared lipid film. A step of preparing a lipid membrane structure.
[0019]
(9) A method of encapsulating one target substance with a single lipid membrane, the method comprising the following step (i) or (ii): (i) mixing a lipid and a cationic surfactant A step of preparing a lipid film modified with a cationic surfactant, and (ii) a step of preparing a lipid film modified with a cationic surfactant by adding a cationic surfactant to the lipid film .
[0020]
(10) The method according to (9), comprising the following step (iv); (iv) a step of preparing a lipid membrane structure by hydrating the prepared lipid film.
[0021]
(11) The cationic surfactant is represented by the following formula (Formula 5):
[Chemical formula 5]

[Wherein, R ₁ is an alkyl group, and R ₂ is a hydrophobic group. ] Cationic interface

[0007]
A membrane structure, a lipid membrane structure in which the number of substances to be encapsulated (target substance) and the number of lipid membranes are controlled can be obtained. Moreover, according to the minute and uniform lipid membrane structure, it can be efficiently delivered to a disease site such as a tumor or liver parenchyma, and according to the lipid membrane structure in which the number of lipid membranes is controlled, the target site Can be delivered efficiently. For example, when the number of the lipid membranes of the lipid membrane structure according to the present invention is 3, the membrane or mitochondria is obtained by membrane fusion three times, and when the number of lipid membranes is one, the membrane is one time. By fusion, the target substance can be efficiently delivered to the cytoplasm. Furthermore, the lipid membrane structure according to the present invention can deliver a target substance to a target site without damaging the target substance, and can exert the function of the target substance at the target site. Can be applied. On the other hand, according to the method for producing a lipid membrane structure according to the present invention, a lipid membrane structure having the above-described characteristics can be produced easily and inexpensively under mild preparation conditions. Furthermore, according to the method of encapsulating one target substance according to the present invention with one lipid membrane, a lipid membrane structure corresponding to the size of the target substance can be obtained, and this method is performed one or more times. Thus, a lipid membrane structure having a desired number of lipid membranes can be obtained. The method for producing a lipid membrane structure according to the present invention and the method for encapsulating one target substance with a single lipid membrane can be performed regardless of the type of lipid. The lipid composition of the lipid membrane can be appropriately selected according to the above.
BRIEF DESCRIPTION OF THE DRAWINGS [0025]
[FIG. 1] Production method in conventional liposome and lipid membrane structure having lipid membrane modified with cationic surfactant according to the present invention, number of encapsulated substance (target substance) and number of lipid membranes It is a figure which shows an outline.
FIG. 2 is a view showing one embodiment of a lipid membrane modified with a cationic surfactant according to the present invention.
[FIG. 3] Benzyldodecyldimethylammonium bromide (BB-1), benzyltetradecyldimethylammonium bromide (BB-2), benzylhexadecyldimethylammonium bromide (BB-3), benzyloctane

[0010]
It is a figure which shows the result of having measured the luciferase activity in the JAWSII cell which carried out.
FIG. 15 shows a photograph of the liposome of the present invention observed with a transmission electron microscope. In the figure, the scale bar indicates 50 nm.
MODE FOR CARRYING OUT THE INVENTION [0026]
Hereinafter, a lipid membrane structure according to the present invention, a method for producing the lipid membrane structure, and a method for encapsulating one target substance with a single lipid membrane will be described in detail. The lipid membrane structure according to the present invention has a lipid membrane modified with a cationic surfactant as a single membrane or as an outermost membrane of a plurality of membranes.
[0027]
In the present invention, the “lipid membrane” refers to a membrane having lipid as a main constituent, which the lipid membrane structure has. The lipid membrane in the present invention may be composed of a lipid bilayer in which lipid molecules are associated with each other and formed in two layers, and the hydrophobic portion is formed with the hydrophobic portion facing inward or outward. It may consist of a single layer.
[0028]
As a preferable form of the lipid membrane structure in the present invention, a closed vesicle having a lipid membrane composed of a lipid bilayer can be mentioned, and as such a lipid membrane structure, for example, a liposome can be mentioned.
[0029]
In the present invention, the number of lipid membranes included in the lipid membrane structure may be one, or two or more. When the lipid membrane structure according to the present invention has a plurality of membranes, at least the outermost membrane has a lipid membrane modified with a cationic surfactant, but not only the outermost membrane but also 1 or other than the outermost membrane A plurality of membranes may be lipid membranes modified with a cationic surfactant.
[0030]
In the present invention, the “cationic surfactant” refers to a surfactant having a cationic hydrophilic group, and the ionic value is not particularly limited, but is preferably monovalent. Specific examples of the cationic surfactant include alkyltrimethylammonium salts such as trimethyltetradecylammonium chloride and cetyltrimethylammonium bromide, dodecylethyldimethylammonium bromide, and ethylhexadecyldimethylan.

で示される陽イオン性界面活性剤であることがさらに好ましい。
［００３１］
なお、本発明における「疎水基」としては、例えば、上記式（化２）および（化３）で示される基のほか、メチル基、エチル基、プロピル基、イソプロピル基などのアルキル基、アルケニル基、ビニール基、フェニル基やナフチル基などのアリール基といった炭化水素基や複素環、または親水基と疎水基が組み合わされて、全体として疎水性である基を挙げることができる。
［００３２］
本発明において、陽イオン性界面活性剤で修飾された脂質膜の態様としては、例えば、図２に示すように、脂質膜を構成する脂質分子の間隙に陽イオン性界面活性剤が位置する態様の他、脂質膜を構成する脂質分子に、共有結合、水素結合、イオン結合、疎水結合、あるいはファンデルワールス結合により陽イオン性界面活性剤が結合する態様などを挙げることができる。
［００３３］
また、本発明において、脂質膜構造体は、正帯電性、非帯電性、両（正負）帯電性および負帯電性のいずれでもよいが、負帯電性であることが好ましい。なお、本発明における「負帯電性脂質膜構造体」とは、全体として負帯電性である脂質膜構造体をいい、例えば、脂質膜の構成脂質として正帯電性脂質や両（正負）帯電性脂質、非帯電性脂質を含むため、または陽イオン性界面活性剤が修飾されているために、局所的に正帯電性や両（正負）帯電性、非帯電性であるものであっても、全体として負帯電性であれば「負帯電性脂質膜構造体」に包含される。
［００３４］
本発明における脂質膜構造体を構成する脂質は、正帯電性脂質、両（正負）帯電性脂質や非帯電性脂質を含む中性脂質、負帯電性脂質のいずれでもよ[0012]
[Chemical formula 3]

It is further preferable that the surfactant be a cationic surfactant.
[0031]
The “hydrophobic group” in the present invention includes, for example, groups represented by the above formulas (Chemical Formula 2) and (Chemical Formula 3), alkyl groups such as a methyl group, an ethyl group, a propyl group, and an isopropyl group, and an alkenyl group. In addition, a hydrocarbon group such as vinyl group, aryl group such as phenyl group or naphthyl group, a heterocyclic ring, or a combination of a hydrophilic group and a hydrophobic group can be cited as a group which is hydrophobic as a whole.
[0032]
In the present invention, as an embodiment of the lipid membrane modified with a cationic surfactant, for example, as shown in FIG. 2, an embodiment in which a cationic surfactant is located in the gap between lipid molecules constituting the lipid membrane. In addition, examples include a mode in which a cationic surfactant is bound to a lipid molecule constituting the lipid membrane by a covalent bond, a hydrogen bond, an ionic bond, a hydrophobic bond, or a van der Waals bond.
[0033]
In the present invention, the lipid membrane structure may be any of positive chargeability, nonchargeability, both (positive / negative) chargeability, and negative chargeability, but is preferably negatively chargeable. The “negatively charged lipid membrane structure” in the present invention refers to a lipid membrane structure that is negatively charged as a whole. For example, positively charged lipids or both (positive and negatively) chargeable lipids as lipids constituting the lipid membrane. Because it contains lipids, non-charged lipids, or because the cationic surfactant is modified, even if it is locally positively charged, both (positive and negative) charged, or uncharged, If it is negatively charged as a whole, it is included in the “negatively charged lipid membrane structure”.
[0034]
The lipid constituting the lipid membrane structure in the present invention may be a positively charged lipid, a neutral lipid containing both (positive and negative) charged lipids and an uncharged lipid, or a negatively charged lipid.

[0016]
A peptide that imparts cell permeability and nuclear translocation ability can be bound or contained in the membrane structure, and the binding amount and content can be appropriately adjusted.
[0043]
A lipid membrane structure having a lipid membrane modified with a cationic surfactant according to the present invention as a single membrane or as an outermost membrane of a plurality of membranes comprises a hydration method, an ultrasonic treatment method, an ethanol injection method, an ether Although it can be produced using a known method such as injection method, reverse phase evaporation method, surfactant method, freezing / thawing method, etc., it is particularly modified with a cationic surfactant as shown in the lower part of FIG. The prepared lipid film is prepared and subjected to a hydration method, whereby it can be easily produced. In the present invention, the “lipid film” is mainly composed of lipid molecules formed so as to stick to the bottom surface or side surface of the container by dissolving the lipid in the organic solvent and then evaporating and removing the organic solvent. This refers to a membrane as a constituent component.
[0044]
Next, the present invention provides a method for producing a lipid membrane structure. The method for producing a lipid membrane structure according to the present invention is a method for producing a lipid membrane structure having a lipid membrane modified with a cationic surfactant as a single membrane or as an outermost membrane of a plurality of membranes. And
(I) a step of preparing a lipid film modified with a cationic surfactant by mixing a lipid and a cationic surfactant;
(Ii) adding a cationic surfactant to the lipid film to prepare a lipid film modified with the cationic surfactant;
(Iii) modifying the cationic surfactant on the outermost membrane of one or more membranes of a lipid membrane structure having one or more membranes;
The process (i), (ii), or (iii) is included. In the method for producing a lipid membrane structure according to the present invention, the description of the same or equivalent configuration as the configuration of the above-described lipid membrane structure according to the present invention is omitted.
[0045]
In the step (i), as a method for preparing a lipid film modified with a cationic surfactant by mixing a lipid and a cationic surfactant, for example, it was obtained by dissolving a lipid in an organic solvent. A solution (lipid solution) and a solution obtained by dissolving a cationic surfactant in an organic solvent (cationic surfactant solution) are mixed.

[0017]
After adding a cationic surfactant to the lipid solution and mixing or dissolving it, after adding the lipid to the cationic surfactant solution and mixing or dissolving it, or after adding the lipid to the cationic interface An example is a method in which an activator is dissolved in an organic solvent at the same time and then the organic solvent is removed by evaporation.
[0046]
In the present invention, examples of the organic solvent for dissolving the lipid and the cationic surfactant include hydrocarbons such as pentane, hexane, heptane and cyclohexane, halogenated hydrocarbons such as methylene chloride and chloroform, and benzene. Aromatic hydrocarbons such as toluene, lower alcohols such as methanol and ethanol, esters such as methyl acetate and ethyl acetate, ketones such as acetone, etc. may be used alone or in combination of two or more. it can.
[0047]
As a method for preparing a lipid film modified with a cationic surfactant by adding a cationic surfactant to the lipid film in step (ii), for example, a cationic surfactant solution is added to the lipid film. A method of removing the solvent by evaporation after the addition can be mentioned.
[0048]
In the step (iii), as a method of modifying the cationic surfactant on the outermost membrane of the single membrane or the plural membranes of the lipid membrane structure having a single membrane or a plurality of lipid membranes, for example, Examples thereof include a method of adding a cationic surfactant to a lipid membrane structure having a membrane or a plurality of membranes.
[0049]
Moreover, when the method for producing a lipid membrane structure according to the present invention includes the step (i) or the step (ii),
(Iv) hydrating the prepared lipid film to prepare a lipid membrane structure;
The step (iv) can also be included.
[0050]
In step (iv), the lipid film structure is prepared by hydrating the lipid film. For example, a method of adding a liquid such as water to the lipid film for hydration, followed by stirring or ultrasonic treatment That is, the hydration method can be mentioned.

[0018]
[0051]
Next, the present invention provides a method of encapsulating one target substance with one lipid membrane. The method of encapsulating one target substance according to the present invention with one lipid membrane is as follows:
(I) a step of preparing a lipid film modified with a cationic surfactant by mixing a lipid and a cationic surfactant;
(Ii) adding a cationic surfactant to the lipid film to prepare a lipid film modified with the cationic surfactant;
The step (i) or (ii) is included. In addition, in the method of encapsulating one target substance according to the present invention with a single lipid membrane, the configuration equivalent to or corresponding to the configuration of the lipid membrane structure according to the present invention or the method for producing the lipid membrane structure described above is described. The description will not be repeated.
[0052]
Moreover, the method of encapsulating one target substance according to the present invention with one lipid membrane is as follows:
(Iv) hydrating the prepared lipid film to prepare a lipid membrane structure;
The step (iv) can also be included.
[0053]
In the present invention, the “target substance” refers to a substance to be encapsulated in a lipid membrane structure or an encapsulated substance. In the present invention, the target substance may be any substance, and examples thereof include various physiologically active substances such as drugs, nucleic acids, peptides, proteins, sugars or complexes thereof, and lipid membrane structures such as liposomes and micelles, It can select suitably according to the objectives, such as a diagnosis and treatment. The target substance can be encapsulated in the aqueous phase inside the lipid membrane structure, for example, by adding it to an aqueous solvent used when the lipid membrane is hydrated in the production of the lipid membrane structure.
[0054]
For example, in a method of encapsulating one target substance according to the present invention with one lipid membrane, if a liposome having one lipid membrane is the target substance, a liposome having two lipid films can be obtained. If liposomes having two lipid membranes are used as the target substance, liposomes having three lipid membranes can be obtained. The lipid membrane structure of the target substance may be modified with various functional molecules. Examples of the modified substance include improved migration to the target site and cell permeability.

[0019]
Peptide, lipid membrane structure, PLL, BSA, and charged substance that improve the stability of the lipid membrane structure.
[0055]
The lipid membrane structure according to the present invention can be used by being dispersed in an appropriate aqueous solvent such as physiological saline, phosphate buffer, citrate buffer, and acetate buffer. Additives such as saccharides, polyhydric alcohols, water-soluble polymers, nonionic surfactants, antioxidants, pH adjusters, and hydration accelerators may be appropriately added to the dispersion. Moreover, the lipid membrane structure according to the present invention can be stored in a state in which the dispersion is dried. Furthermore, the lipid membrane structure according to the present invention can be administered orally, and can also be administered parenterally to veins, intraperitoneally, subcutaneously, nasally and the like.
[0056]
In addition, in the lipid membrane structure of the present invention described so far, the method for producing a lipid membrane structure, and the method of encapsulating one target substance in one lipid membrane, a cationic surfactant to be added to the lipid film The particle diameter of the lipid membrane structure produced or encapsulated with the target substance can be controlled in the range of approximately 50 nm to 200 nm by making the amount of the range of 10 to 30 mol% with respect to the total lipid. Is possible. In addition, when encapsulating particles such as nucleic acids, it is preferable to adjust the amount of the cationic surfactant within the above range according to the size of the encapsulated particles.
[0057]
Hereinafter, a lipid membrane structure according to the present invention, a method for producing the lipid membrane structure, and a method for encapsulating one target substance with a single lipid membrane will be described based on examples. Note that the technical scope of the present invention is not limited to the features shown by these examples.
Example [0058]
<Example 1> Examination of particle diameter of liposome having lipid membrane modified with cationic surfactant (1) Preparation of nucleic acid nanoparticles [1-1] Preparation of plasmid DNA (pDNA) 10 mg / mL kanamycin To 10 mL of LB medium supplemented with 10 μL of aqueous solution, Enhanced Green luor downstream of the CMV promoter

［００７５］
表１、図４ＡおよびＢに示すように、リポソームの粒子径は、ｂ、ｃ、ｄ、ｅ、ｆ、ｇ、ｈおよびｉはａと比較して小さいのに対し、ｊおよびｋはａと比較してほとんど同じであった。また、同種の界面活性剤が修飾された脂質膜を有するリポソームにおいて比較すると、ｂ＞ｃ、ｄ＞ｅ、ｆ＞ｇおよびｈ＞ｉであるのに対し、ｊ≒ｋであった。
［００７６］
これらの結果から、陽イオン性界面活性剤で修飾された脂質膜を有するリポソームは、界面活性剤が修飾されていない脂質膜を有するリポソームと比較して粒子径が小さいこと、および陽イオン性界面活性剤の修飾濃度が大きいほど粒子径が小さくなることが明らかになった。一方、非イオン性界面活性剤が修飾された脂質膜を有するリポソームは、界面活性剤が修飾されていない脂質膜を有するリポソームと比較して、非イオン性界面活性剤の修飾濃度にかかわらず、粒子径がほとんど同じであることが明らかになった。
［００７７］
すなわち、陽イオン性界面活性剤で修飾された脂質フィルムを用いて水和法を行うことにより、微小な粒子径を有するリポソームが得られることが明らかになった。
［００７８］
また、表１および図４Ａに示すように、界面活性剤の修飾濃度が同じであるリポソーム同士を比較すると、粒子径はｂ＞ｄ＞ｆ≒ｈおよびｃ＞ｅ＞ｇ≒ｉであった。これらの結果から、脂質膜に修飾された陽イオン性界面活性剤が有するアルキル基の炭素数が１６より小さい場合は、炭素数が大きいほ[0024]
Are shown in FIGS. 4C and D, respectively.
[0074]
[Table 1]

[0075]
As shown in Table 1, FIGS. 4A and B, the particle size of the liposomes is small for b, c, d, e, f, g, h and i compared to a, while j and k are a and It was almost the same in comparison. In comparison with liposomes having lipid membranes modified with the same type of surfactant, b> c, d> e, f> g and h> i, whereas j≈k.
[0076]
These results indicate that liposomes with lipid membranes modified with a cationic surfactant have a smaller particle size than liposomes with lipid membranes that are not modified with a surfactant, and cationic interfaces. It was found that the particle size decreases as the modified concentration of the active agent increases. On the other hand, liposomes having lipid membranes modified with nonionic surfactants, compared with liposomes having lipid membranes not modified with surfactants, regardless of the modified concentration of nonionic surfactants, It became clear that the particle size was almost the same.
[0077]
That is, it was revealed that liposomes having a minute particle diameter can be obtained by performing a hydration method using a lipid film modified with a cationic surfactant.
[0078]
Moreover, as shown in Table 1 and FIG. 4A, when comparing liposomes having the same surfactant modification concentration, the particle diameters were b>d> f≈h and c>e> g≈i. From these results, when the carbon number of the alkyl group of the cationic surfactant modified on the lipid membrane is smaller than 16, the carbon number is larger.

[0025]
It was revealed that the particle size of the liposome is small, whereas when the carbon number is larger than 16, the particle size of the liposome does not decrease any more and becomes almost constant.
[0079]
Also, as shown in Table 1, FIGS. 4C and D, the nucleic acid nanoparticles are positive for zeta potential, whereas a, b, c, d, e, f, g, h, i, j and k. Were both negative. From these results, the liposome having a lipid membrane modified with a cationic surfactant has a lipid membrane whose surfactant is not modified when the modification concentration is at least 11 mol% and 16.5 mol%. It was also revealed that negatively charged liposomes that maintain the negative chargeability derived from the constituent lipids were obtained in the same manner as liposomes having lipid membranes modified with nonionic surfactants.
[0080]
<Example 2> Examination of particle diameter of liposome having lipid membrane modified with tonzonium bromide (TB) (1) Preparation of nucleic acid nanoparticles Example 1 (1) [1-2] and [1-3] ], Nucleic acid nanoparticles were prepared, and the particle diameter and zeta potential were measured.
[0081]
(2) Preparation of lipid film [2-1] Preparation of surfactant solution Tonzonium bromide (TB; Sigma-Aldrich), which is a cationic surfactant, was added to ethanol so as to have a concentration of 3.03 mmol / L. A TB solution was prepared by dissolving. The structure of TB is shown in the middle part of FIG.
[0082]
[2-2] Preparation of Mixed Lipid Solution Six samples of mixed lipid solutions were prepared by the method described in Example 1 (2) [2-2], which were designated as a, b, c, d, e, and f, respectively. .
[0083]
[2-3] Preparation of lipid film About a, b, c, d, e and f of Example (2) [2-2], the method described in Example 1 (2) [2-3] A lipid film was prepared.

［００８８］
表２および図５Ａに示すように、リポソームの粒子径は、ａ＞ｂ＞ｃ＞ｄ≒ｅ≒ｆであった。
［００８９］
これらの結果から、ＴＢ修飾リポソームは、ＴＢ未修飾リポソームと比較して、粒子径が小さくなることが明らかになった。また、ＴＢの修飾濃度が少なくとも２２ｍｏｌ％より小さい場合は、修飾濃度が大きいほど、リポソームの粒子径が小さくなるのに対し、少なくとも２２ｍｏｌ％より大きい場合は、リポソームの粒子径はそれ以上減少せず、ほぼ一定となることが明らかになった。
［００９０］
さらに、表２および図５Ａに示すように、ｄ、ｅおよびｆの粒子径は、約１３０ｎｍであり、核酸ナノ粒子の粒子径である約１００ｎｍにかなり近い値であった。ここで、図５Ｃに示すように、脂質膜の厚さは約４ｎｍであり、これに内水相の厚さを合わせると約１５ｎｍであることから、１の核酸ナノ粒子を１枚の脂質膜で封入したリポソームの粒子径は、約１００ｎｍ＋約１５ｎｍ×２＝約１３０ｎｍとなると考えられる。このことから、ｄ、ｅおよびｆは、１の核酸ナノ粒子を１枚の脂質膜で封入したリポソームであることが明らかになった。すなわち、ＴＢが少なくとも２２ｍｏｌ％程度修飾された脂質フィルムを用いて水和法を行うことにより得られたリポソームは、１の目的物質が１枚の脂質膜で封入されたリポソームであることが明らかになった。
［００９１］
以上の結果から、陽イオン性界面活性剤で修飾された脂質フィルムを用いて水和法を行うことにより、１の目的物質を１枚の脂質膜で封入することができることが明らかになった。[0027]
[Table 2]

[0088]
As shown in Table 2 and FIG. 5A, the particle size of the liposome was a>b>c> d≈e≈f.
[0089]
From these results, it was clarified that the TB modified liposome has a smaller particle size than the TB unmodified liposome. When the modification concentration of TB is at least 22 mol%, the liposome particle size decreases as the modification concentration increases. On the other hand, when the modification concentration is at least 22 mol%, the liposome particle size does not decrease any more. It became clear that it was almost constant.
[0090]
Furthermore, as shown in Table 2 and FIG. 5A, the particle diameters of d, e, and f were about 130 nm, which was a value that was very close to about 100 nm, which is the particle diameter of nucleic acid nanoparticles. Here, as shown in FIG. 5C, the thickness of the lipid membrane is about 4 nm, and the total thickness of the inner aqueous phase is about 15 nm. Therefore, one nucleic acid nanoparticle is added to one lipid membrane. The particle diameter of the liposome encapsulated in is considered to be about 100 nm + about 15 nm × 2 = about 130 nm. From this, it became clear that d, e, and f are liposomes in which one nucleic acid nanoparticle is encapsulated with one lipid membrane. That is, it is clear that the liposome obtained by performing the hydration method using a lipid film in which TB is modified by at least about 22 mol% is a liposome in which one target substance is encapsulated by one lipid membrane. became.
[0091]
From the above results, it has been clarified that one target substance can be encapsulated with one lipid membrane by performing a hydration method using a lipid film modified with a cationic surfactant.

［００９９］
表３および図６Ａに示すように、空リポソームの粒子径は、ｈ、ｉ、ｊ、ｋおよびｌのいずれも、ｇの粒子径と比較して小さかった。また、ｈ＞ｉ＞ｋ＞ｊ＞ｌであった。これらの結果から、ＴＢ修飾空リポソームは、ＴＢ未修飾空リポソームと比較して、粒子径が小さくなることが明らかになった。
［０１００］
すなわち、陽イオン性界面活性剤で修飾された脂質フィルムを用いて水和法を行うことにより、目的物質が封入されているか否かにかかわらず、微小な粒子径を有するリポソームが得られることが明らかになった。
［０１０１］
また、図６Ｂに示すように、ゼータ電位は、ｇ、ｈ、ｉおよびｊはいずれも負であるのに対し、ｋおよびｌは正であった。これらの結果から、ＴＢの修飾濃度が少なくとも５５ｍｏｌ％以上であるＴＢ修飾空リポソームは、正帯電性リポソームとなるのに対し、ＴＢの修飾濃度が５５ｍｏｌ％より小さいＴＢ修飾空リポソームは、ＴＢ未修飾空リポソームと同様に、構成脂質に由来する負帯電性が維持された負帯電性リポソームとなることが明らかになった。
［０１０２］
＜実施例３＞ＴＢ修飾リポソームのトランスフェクション活性の検討[0029]
H; i, j, k and l) were prepared. Thereafter, the particle diameter and zeta potential were measured by the method described in Example 1 (1) [1-3].
[0097]
The experiment of this example (4) [4-1] to [4-3] was repeated three times or more, and the average value and standard deviation (SD) of the results obtained for each sample were calculated. The results are shown in Table 3. In addition, FIG. 6A shows a graph of the measurement result of the particle diameter, and FIG. 6B shows a graph of the measurement result of the zeta potential.
[0098]
[Table 3]

[0099]
As shown in Table 3 and FIG. 6A, the particle size of empty liposomes was smaller than that of g in all of h, i, j, k and l. Further, h>i>k>j> l. From these results, it was revealed that TB-modified empty liposomes have a smaller particle size than TB unmodified empty liposomes.
[0100]
That is, by performing a hydration method using a lipid film modified with a cationic surfactant, liposomes having a minute particle size can be obtained regardless of whether the target substance is encapsulated or not. It was revealed.
[0101]
Further, as shown in FIG. 6B, the zeta potential was negative for g, h, i, and j, but positive for k and l. From these results, TB-modified empty liposomes having a modified TB concentration of at least 55 mol% or more are positively charged liposomes, whereas TB-modified empty liposomes having a modified TB concentration of less than 55 mol% are not modified with TB. It became clear that it becomes a negatively charged liposome in which the negatively charged property derived from the constituent lipid is maintained, like the empty liposome.
[0102]
<Example 3> Examination of transfection activity of TB modified liposome

Claims (13)

  A lipid membrane structure having a lipid membrane modified with a cationic surfactant as a single membrane or as an outermost membrane of a plurality of membranes. The cationic surfactant is represented by the following formula (Formula 1)

[Where:
R ₁ is an alkyl group;
R ₂ is a hydrophobic group. ]
The lipid membrane structure according to claim 1, which is a cationic surfactant. R ₁ is an alkyl group having 12 or more carbon atoms, and R ₂ is represented by the following formula (Formula 2)

Or the following formula (Formula 3)

The lipid membrane structure according to claim 2, which is a group of R ₁ is an alkyl group having 16 carbon atoms, and R ₂ is represented by the following formula (Formula 4)

The lipid membrane structure according to claim 2, which is a group of   The lipid membrane structure according to any one of claims 1 to 4, which is a negatively charged lipid membrane structure.   The lipid membrane structure according to any one of claims 1 to 5, which is a liposome. A method for producing a lipid membrane structure having a lipid membrane modified with a cationic surfactant as a single membrane or as an outermost membrane of a plurality of membranes, comprising the following (i), (ii) or (iii) The method comprising the steps of
(I) a step of preparing a lipid film in which a cationic surfactant is modified by mixing a lipid and a cationic surfactant;
(Ii) adding a cationic surfactant to the lipid film to prepare a lipid film in which the cationic surfactant is modified;
(Iii) A step of modifying a cationic surfactant on the outermost membrane of a single membrane or a plurality of membranes of a lipid membrane structure having a single membrane or a plurality of membranes. (I) preparing a lipid film in which a lipid and a cationic surfactant are mixed to modify the cationic surfactant; or (ii) adding a cationic surfactant to the lipid film to form a cation. The method according to claim 7, which comprises the following step (iv) when the functional surfactant has a step of preparing a modified lipid film;
(Iv) A step of preparing a lipid membrane structure by hydrating the prepared lipid film. A method of encapsulating one target substance with one lipid membrane, the method comprising the following step (i) or (ii):
(I) a step of preparing a lipid film in which a cationic surfactant is modified by mixing a lipid and a cationic surfactant;
(Ii) A step of preparing a lipid film in which a cationic surfactant is modified by adding a cationic surfactant to the lipid film. The method according to claim 9, which comprises the following step (iv):
(Iv) A step of preparing a lipid membrane structure by hydrating the prepared lipid film. The cationic surfactant is represented by the following formula (Formula 5):

[Where:
R ₁ is an alkyl group;
R ₂ is a hydrophobic group. ]
The method according to any one of claims 7 to 10, which is a cationic surfactant. R ₁ is an alkyl group having 12 or more carbon atoms, and R ₂ is represented by the following formula (Formula 6):

Or the following formula (Formula 7)

The method according to claim 11, wherein R ₁ is an alkyl group having 16 carbon atoms, and R ₂ is represented by the following formula (Formula 8):

The method according to claim 11, wherein

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