JP2013245190A - Agent for imparting ph dependant cationic property to lipid membrane structure, the lipid membrane structure given the ph dependant cationic property thereby, and production method for lipid membrane structure - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an agent which has YSK05 as an effective component and imparts a pH dependant cationic property to a lipid membrane structure, to provide the lipid membrane structure given the pH dependent cationic property thereby, and to provide a production method for the lipid membrane structure.SOLUTION: An effective component of an agent is 1-methyl-4,4-bis[(9Z, 12Z)-octadeca-9,12-dien-1-yloxy]piperidine. A lipid structure impart a pH dependant cationic property to a lipid structure without giving an influence to a property such as a particle diameter and PDI of the lipid structure, improves membrane fusing performance with a biomembrane as the pH decreases, effectively involves a substance such as a siRNA and a drug, and also protects the involved substance from decomposing, etc. in a serum to stably hold it.

Description

  The present invention relates to an agent for imparting pH-dependent cationic property to a lipid membrane structure, a lipid membrane structure imparted with pH-dependent cationic property thereby, and a method for producing the lipid membrane structure. More specifically, lipids containing 1-methyl-4,4-bis [(9Z, 12Z) -octadeca-9,12-dien-1-yloxy] piperidine (hereinafter referred to as “YSK05”) as an active ingredient. An agent that imparts pH-dependent cationicity to a membrane structure, a lipid membrane structure imparted with pH-dependent cationicity by YSK05, and a base sequence that is the same or complementary to all or part of the messenger RNA of the target gene The present invention relates to a target gene expression inhibitor comprising the lipid membrane structure containing RNA as an active ingredient, and a method for producing a lipid membrane structure having a lipid membrane containing YSK05 as a constituent lipid.

  Cationic lipid membrane structures have been used as carriers to deliver nucleic acids and drugs into cells. Since cells and nucleic acids are generally negatively charged, cationic lipid membrane structures are useful in that they can stably hold nucleic acids and can be efficiently introduced into cells. is there. Therefore, various cationic lipids that can be used as constituent lipids of the cationic lipid membrane structure have been researched and developed. Patent Document 1 discloses a cationic lipid that has low toxicity to hepatocytes. Yes.

  On the other hand, when a cationic lipid membrane structure is administered to a living body by a method such as intravenous injection, it has a non-specific interaction with a negatively charged blood cell or serum protein. Has the problem of being difficult to selectively deliver. Therefore, a pH-dependent cationic lipid membrane structure that is neutral in a neutral environment such as blood and can be made cationic in an acidic environment such as endosome and transfer from the endosome to the cytoplasm. Is required. Examples of the lipid that can be used as a constituent lipid of the pH-dependent cationic lipid membrane structure include 1,2-dioleoyl-3-dimethylamine probe (DODAP).

International Publication Pamphlet WO2011 / 090965

  However, the cationic lipid described in Patent Document 1 has a property that the lipid itself is easily decomposed in an acidic environment (Patent Document 1; FIG. 1, FIG. 2), and is therefore rapidly degraded in the liver. It is a cationic lipid having low toxicity to cells (Patent Document 1; page 1, lines 22 to 27, FIG. 4), and does not impart pH-dependent cationicity to the lipid membrane structure. Further, DODAP is another substance having a different structure from YSK05, as shown in Examples described later.

  The present invention has been made in order to solve such problems, and is an agent that imparts pH-dependent cationic property to a lipid membrane structure, which has YSK05 as an active ingredient, and thereby has pH-dependent cationic property. It is an object of the present invention to provide a given lipid membrane structure and a method for producing the lipid membrane structure.

  As a result of intensive studies, the present inventors have shown that YSK05 imparts pH-dependent cationicity to a lipid membrane structure, and by preparing a lipid membrane containing YSK05 as a constituent lipid, pH-dependent cationicity is imparted. Found that a lipid membrane structure containing a siRNA against a target gene and having a pH-dependent cationic property imparted by YSK05 suppresses the expression of the target gene. Each invention was completed.

(1) Giving pH-dependent cationic property to a lipid membrane structure containing 1-methyl-4,4-bis [(9Z, 12Z) -octadeca-9,12-dien-1-yloxy] piperidine as an active ingredient Agent to do.

(2) 1-methyl-4,4-bis [(9Z, 12Z) -octadeca-9,12-dien-1-yloxy] piperidine having a lipid membrane containing as a constituent lipid A lipid membrane structure to which pH-dependent cationicity is imparted by bis [(9Z, 12Z) -octadeca-9,12-dien-1-yloxy] piperidine.

(3) The lipid membrane structure according to (2) having the lipid membrane of (A) or (B) below; (A) (a) 1-methyl-4, 4-bis [(9Z, 12Z)- octadeca-9,12-dien-1-yloxy] piperidine, (b) cholesterol and (c) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoeethanolamine (POPE) in molar ratio (a) / (b ) + (C) ≧ 2/3 and 3/7 ≦ (b) / (c) ≦ 7/3 as a constituent lipid, (B) (a) 1-methyl-4,4-bis [(9Z, 12Z) -octadeca-9,12-dien-1-yloxy] piperidine and (b) cholesterol in a molar ratio of 13/7 ≦ (a) / ( ) In a proportion of ≦ 3 lipid membrane comprising as a component lipid.

(4) The lipid membrane structure according to (2) or (3), which has a lipid membrane containing a lipid bonded to polyethylene glycol as a constituent lipid.

(5) 1,2-dimyristol-sn-glycerol (DMG) and / or 1,2-disteayl-sn-glycerol (DSG) bound to polyethylene glycol, wherein the lipid bound to polyethylene glycol is bound to polyethylene glycol. The lipid membrane structure according to (4).

(6) The target gene expression inhibitor, which contains RNA having the same or complementary base sequence as the whole or part of the messenger RNA of the target gene (2) to (5) The said agent which uses the lipid membrane structure of this as an active ingredient.

(7) A method for producing a lipid membrane structure, comprising the step of preparing a lipid membrane of the following (I) or (II): (I) (a) 1-methyl-4,4-bis [(9Z, 12Z) -Octadeca-9,12-dien-1-yloxy] piperidine, (b) cholesterol, (c) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoeethanolamine (POPE) and (d) polyethylene glycol conjugated 1,2-dimyristol-sn-glycerol (DMG) has a molar ratio of (a) / (b) + (c) ≧ 2/3, 3/7 ≦ (b) / (c) ≦ 7/3 and 0 < (D) / (a) + (b) + (c) ≦ 1/10 lipid membrane containing as constituent lipid at a ratio of (II) (a) 1-methyl- , 4-bis [(9Z, 12Z) -octadeca-9,12-dien-1-yloxy] piperidine, (b) cholesterol and (d) polyethylene glycol-conjugated 1,2-dimethylristol-sn-glycerol (DMG) Membrane as a constituent lipid at a molar ratio of 13/7 ≦ (a) / (b) ≦ 3 and 0 <(d) / (a) + (b) ≦ 1/10.

(8) The method for producing a lipid membrane structure according to (7), comprising a step of adding 1,2-distearoy-sn-glycerol (DSG) bonded to polyethylene glycol to the prepared lipid membrane.

  According to the agent for imparting pH-dependent cationic property to the lipid membrane structure according to the present invention, the pH-dependent cation can be added to the lipid membrane structure without affecting the physical properties of the lipid membrane structure, such as particle diameter and PDI. Sex can be imparted. Next, according to the lipid membrane structure to which pH-dependent cationic property is imparted by YSK05 according to the present invention and the production method thereof, the lipid membrane structure whose membrane fusion ability with the biological membrane is improved as the pH is lowered. Can be obtained. In addition, a lipid membrane structure can be obtained in which a substance such as siRNA or a drug can be efficiently encapsulated, and the encapsulated substance can be stably retained while being protected from degradation in serum. Further, since the inclusion can be rapidly released into the cytoplasm after being taken into the cell and before being degraded by lysosome, a lipid membrane structure capable of efficiently delivering the inclusion to the cytoplasm can be obtained. Furthermore, a lipid membrane structure capable of delivering inclusions to any tissue can be obtained not only by direct administration to a target tissue but also by blood administration.

  On the other hand, according to the target gene expression inhibitor according to the present invention, since the expression of the target gene can be efficiently suppressed, the dose can be reduced, and the burden on the administration subject can be reduced. Moreover, expression of a target gene in an arbitrary tissue or cell can be suppressed not only by direct administration to a target tissue but also by blood administration. In particular, the target gene expression inhibitor according to the present invention has high retention in the blood, and therefore can be delivered to a tumor tissue using the enhanced permeability and retention (EPR) effect. In addition to suppressing expression, tumors can be treated. Here, the EPR effect means that the neovascularization of the tumor tissue is more fragile than the blood vessels of the normal tissue, and the permeability is increased, so that fine particles such as a minute lipid membrane structure are present in the blood for a long time. Circulates in the blood and passively leaks from the blood vessels of the cancer tissue and accumulates in the cancer tissue (Matsumura Y et al., Cancer Res., 46, 6387-6392, 1986).

1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-3-dimethylammonium propane (DODAP) and 1-methyl-4,4-bis [(9Z, 12Z) -octadeca-9,12- It is a figure which shows the structure of dien-1-yloxy] piperidine (YSK05). It is a figure which shows the relative fluorescence intensity of the buffer solution of various pH which added the lipid membrane structure which has a lipid membrane which contains DOTAP, DODAP, or YSK05 as a constituent lipid, and the TNS reagent. It is a figure which shows the membrane fusion rate of the empty lipid membrane structure which has a lipid membrane which contains DOTAP, DODAP, or YSK05 as a constituent lipid, and erythrocytes in various pH. The figure which shows the expression suppression rate (Luc / KD rate) of the luciferase gene of the lipid membrane structure which contains the siRNA (anti-luc) with respect to a firefly luciferase gene messenger RNA, and has a lipid membrane containing DOTAP, DODAP, or YSK05 as a constituent lipid It is. The result of measuring the Luc / KD ratio (right figure) of a lipid membrane structure containing an anti-luc and having a lipid membrane of composition 3-7, and after being incubated in serum for 0 to 48 hours, then encapsulated It is a figure which shows each result (left figure) which detected anti-luc by 20% polyacrylamide gel electrophoresis. It is a figure which shows Luc / KD rate of the lipid membrane structure which encapsulates anti-luc and has a lipid membrane of composition 8-13 (left figure) and composition 14-16 (right figure). It is a figure which shows the membrane fusion rate of the empty lipid membrane structure (Empty YSK lipid membrane structure and empty YSK / A lipid membrane structure) which has the lipid membrane of the composition 3 and the composition 10, and erythrocytes in various pH. . Lipid membrane structure containing anti-luc and having lipid membranes of composition 3 and composition 10 (luc-encapsulated YSK lipid membrane structure and luc-encapsulated YSK / A lipid membrane structure), and commercially available transfection reagent (LF2k) It is a figure which shows the Luc / KD rate. Add lipid membrane structures (including Cy5-luc-encapsulated DODAP lipid membrane structure and Cy5-luc-encapsulated YSK / A lipid membrane structure) that encapsulate siRNA labeled with Cy5 and have a lipid membrane containing DODAP and YSK05 as constituent lipids It is a figure which shows the fluorescence of Cy5 and Hoechst33342 in the cell which dye | stained the cell nucleus by Hoechst33342 after this. In the figure, arrows indicate main locations where Cy5 fluorescence is detected in the form of dots. Presence of chloroquine or ammonium chloride in a lipid membrane structure containing anti-luc and having a lipid membrane containing DOTAP and YSK05 as constituent lipids (luc-encapsulated DOTAP lipid membrane structure and luc-encapsulated YSK / A lipid membrane structure) It is a figure which shows the Luc / KD ratio in the bottom. 0-24 hours in serum for anti-luc, a mixture of siRNA / protamine complex and empty YSK lipid membrane structure, luc-encapsulated YSK / A lipid membrane structure, and PEG / luc-encapsulated YSK / A lipid membrane structure It is a figure which shows the result of having detected anti-luc by 20% polyacrylamide gel electrophoresis, after incubating. It is a figure which shows the relative expression level of PLK1 in the tumor tissue of the tumor model mouse which administered various lipid membrane structures, and the tumor model mouse which does not administer anything (control). It is a figure which shows the PCR amplification product of 5'RACE PCR using a PLK1 gene specific primer. In the figure, an arrow indicates the position of a band indicating DNA obtained by specifically amplifying an RNA fragment resulting from cleavage of PLK1 gene mRNA. It is a figure which shows the relative SRBI expression level in the liver of the mouse | mouth which included siRNA (anti-sr) with respect to SRBI gene messenger RNA, and administered the lipid membrane structure which has a lipid membrane of the composition 17-19, and PBS. It is a figure which shows the relative SRBI expression level in the liver of the mouse | mouth which encapsulated anti-sr and administered the lipid membrane structure which has a lipid membrane of a composition 20-25, and PBS. It is a figure which shows the relative SRBI expression level in the liver of the mouse | mouth which encapsulated anti-sr and which administered the lipid membrane structure which has a lipid membrane of the composition 17, 26, and 27, and PBS. The figure which shows the membrane fusion rate of the empty lipid membrane structure (Empty YSK / 28 lipid membrane structure and empty YSK / B lipid membrane structure) which has the lipid membrane of the composition 28 and the composition 23, and erythrocytes in various pH It is. Relative in the liver of mice administered anti-sr and administered with lipid membrane structures having composition 28 and composition 23 lipid membranes (sr-encapsulated YSK / 28 lipid membrane structure and sr-encapsulated YSK / B lipid membrane structure) It is a figure which shows SRBI expression level. Lipid membrane structures containing siRNA (anti-f7) against the factor 7 gene messenger RNA and having lipid membranes of composition 28 and composition 23 (f7-encapsulated YSK / 28 lipid membrane structure and f7-encapsulated YSK / B lipid membrane structure) FIG. 7 is a graph showing the ratio of factor 7 in the serum of mice administered with the body. It is a figure which shows the change accompanying the elapsed days after administration about the 7th factor ratio in the serum of the mouse | mouth which administered f7 inclusion YSK / B lipid membrane structure and PBS. Administration of 1,2-distearoyl-sn-glycerol (DSG) labeled with a radioisotope and conjugated with polyethylene glycol (PEG), that is, a lipid membrane structure having a lipid membrane added with 0 to 3 mol% of PEG-DSG It is a figure which shows the radiation density | concentration in the plasma of the mouse | mouth which performed. It is a figure which shows the relative expression level of PLK1 in the tumor tissue of the tumor model mouse which administered various lipid membrane structures and PBS. In the figure, the number at the tip of the bar indicates the dose of siRNA (mg / kg).

  Hereinafter, an agent for imparting pH-dependent cationic property to the lipid membrane structure according to the present invention, a lipid membrane structure imparted with pH-dependent cationic property thereby, a target gene expression inhibitor, and production of the lipid membrane structure The method will be described in detail.

  The agent that imparts pH-dependent cationicity to the lipid membrane structure according to the present invention is 1-methyl-4,4-bis [(9Z, 12Z) -octadeca-9,12-dien-1-yloxy] piperidine ( Hereinafter, it is referred to as “YSK05”) as an active ingredient.

  The “pH-dependent cationic” in the present invention refers to the property that the amount of charge increases as the pH decreases. Accordingly, the lipid membrane structure to which pH-dependent cationic property is imparted in the present invention includes a lipid membrane structure having a positive charge (cationic lipid membrane structure, positively charged lipid membrane structure), Negatively charged (anionic lipid membrane structure, negatively charged lipid membrane structure) or non-charged (uncharged lipid membrane structure), both positively and negatively charged (Both (positive and negative) charged lipid membrane structures).

  YSK05 can be synthesized using a method that can be appropriately selected by those skilled in the art. As such a method, for example, according to the method described in the previous report (Sodeoka M, et al., J. Med. Chem., 44, 3216-3222, 2001), first, (9Z, 12Z) -Octadecadien-1-OL is synthesized, dissolved in anisole, 1-methyl-4-piperidone4 and p-toluenesulfonic acid are added and heated to reflux. Thereafter, the organic phase is recovered, diluted and washed, and then the solvent is removed to obtain a crude product, which can be purified by silica gel column chromatography, as well as the method described in Patent Document 1. it can.

YSK05 can be used after being modified as necessary by a method such as binding a modifying substance. Examples of such modifiers include PEG chains, amino acids and peptides, monosaccharides, polysaccharides, proteins, nucleic acids, radioisotopes (RI) such as ³² P, ³ H, ¹⁴ C, ¹³ C, and ¹⁵ N, and fluorescence. Mention may be made of labeling substances such as dyes.

  Next, the present invention provides a lipid membrane structure to which pH-dependent cationicity is imparted by YSK05. In addition, in the lipid membrane structure to which pH-dependent cationic property is imparted by YSK05, the configuration equivalent to or equivalent to the configuration of the agent that imparts pH-dependent cationic property to the lipid membrane structure according to the present invention described above is again described. The description of is omitted.

  Whether or not the lipid membrane structure to which pH-dependent cationic property is imparted by YSK05 according to the present invention has pH-dependent cationic property can be confirmed according to a conventional method, for example, using a negatively charged TNS reagent Can be confirmed easily. The TNS reagent is a reagent that enters a cationic lipid membrane and emits fluorescence. In this method, the lipid membrane structure and the TNS reagent are mixed in a buffer solution prepared to have various pHs, and then the buffer solution is mixed. Measure fluorescence intensity. The correlation between the measured fluorescence intensity and the pH of the buffer solution is confirmed, and when the fluorescence intensity increases as the pH decreases, it can be determined that the lipid membrane structure has pH-dependent cationic properties.

  A preferred form of the lipid membrane structure to which pH-dependent cationic property is imparted by YSK05 according to the present invention is a closed vesicle having a lipid membrane composed of a lipid bilayer, and such a lipid membrane structure. Examples thereof include liposomes. Here, the number of lipid membranes included in the lipid membrane structure to which pH-dependent cationic property is imparted by YSK05 according to the present invention may be one, or may be two or more.

  The lipid membrane structure to which pH-dependent cationic property is imparted by YSK05 according to the present invention has a lipid membrane containing YSK05 as a constituent lipid. A method for preparing a lipid membrane containing YSK05 as a constituent lipid can be performed according to a conventional method. As such a method, for example, YSK05 is dissolved in a solvent, and using this, simple hydration method, ultrasonic treatment method, ethanol injection method, ether injection method, reverse phase evaporation method, surfactant method, freezing method -The method of preparing a lipid membrane structure by well-known methods, such as a melting method, can be mentioned. Alternatively, after preparing a lipid membrane structure by these methods without using YSK05, YSK05 may be added to the lipid membrane of the lipid membrane structure.

  The lipid membrane of the lipid membrane structure to which pH-dependent cationic property is imparted by YSK05 according to the present invention may contain only YSK05 as a constituent lipid, and may contain lipids other than YSK05 in addition to YSK05. Lipids other than YSK05 may be cationic lipids, neutral (including both positive and negative) charged lipids and non-charged lipids, and anionic lipids. Examples of lipids include phospholipids, glycolipids, A sterol, a long chain aliphatic alcohol, a glycerol fatty acid ester, etc. can be mentioned, These 1 type (s) or 2 or more types can be used.

  Examples of the phospholipid include phosphatidylcholine (for example, 1,2-Diolyl-sn-3-phosphatidylCholine (DOPC), dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyyl phosphatidylcholine (DPPC), distearoylphosphatidylphosphatidylcholine -Oleyl-sn-glycero-3-phosphatidylCholine (SOPC), etc., phosphatidylglycerol (eg dioleoylphosphatidylglycerol, dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphine Thiodylglycerol), phosphatidylethanolamine (eg dioleoylphosphatidylethanolamine, dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine (DSPE), dioleoylglycero Phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), phosphatidylserine, phosphatidylinositol, phosphatidic acid (PA), cardiolipin, or hydrogenated products thereof, Natural lipids derived from egg yolk, soybeans and other animals and plants (eg egg yolk 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.

  Sterols derived from animals such as cholesterol (Chol), cholesterol succinic acid, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol, plant-derived sterols (phytosterols) such as stigmasterol, sitosterol, campesterol and brassicasterol And sterols derived from microorganisms such as timosterol and 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 Specific examples thereof include 1,2-dimyristol-sn-glycerol (DMG) and 1,2-distearoyl-sn-glycerol (DSG).

  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 cationic lipid include dioctadecyldimethylammonium chloride (DODAC), N- (2,3-oleyloxy) propyl-N, N, N-trimethylammonium (N- (). 2,3-dioyloxy) propyl-N, N, N-trimethylammonium, DOTMA), didodecylammonium bromide (DDAB), 1,2-dioleoyl-3-trimethylammonium 1,2 -dipropylepropylene, Tropropylene 3-dimethylammonium probe (DODAP), 3β-N- (N , N′-dimethylaminoethane) carbamol cholesterol (3β-N- (N ′, N ′,-dimethyl-aminoethane) -carbamol cholesterol, DC-Chol), 1,2-dimyristoyloxypropyl-3-dimethylhydroxy Ethylammonium (1,2-dimethyloxypropyl-3-dimethylhydroxyl ammonium, DMRIE), 2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propaneammonium trifluoroacetate ( 2,3-dioyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propa aminum trifluoroacetate, 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 anionic 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.

  The lipid membrane of the lipid membrane structure to which pH-dependent cationic property is imparted by YSK05 according to the present invention can contain the above-described lipids in an arbitrary ratio, but (a) YSK05, (b) Chol and ( c) POPE is contained in a molar ratio of (a) / (b) + (c) ≧ 2/3 and 3/7 ≦ (b) / (c) ≦ 7/3, or (a) It is preferable that YSK05 and (b) Chol are included in a molar ratio of 13/7 ≦ (a) / (b) ≦ 3.

  Moreover, it is preferable that the lipid membrane of the lipid membrane structure to which pH-dependent cationic property is imparted by YSK05 according to the present invention contains a lipid bonded to PEG. Examples of the lipid conjugated with PEG in this case include those in which the above-described lipid is conjugated with PEG. Among them, 1,2-dimyristol-sn-glycerol (DMG) conjugated with PEG, that is, PEG -DMG or 1,2-distearoy-sn-glycerol (DSG) conjugated with PEG, that is, PEG-DSG is more preferable.

  The lipid membrane of the lipid membrane structure to which pH-dependent cationic property is imparted by YSK05 according to the present invention includes antioxidants such as tocopherol, propyl gallate, ascorbyl palmitate, butylated hydroxytoluene in addition to the above-described lipids. Agents, charged substances that impart positive charges such as stearylamine and oleylamine, charged substances that impart negative charges such as dicetyl phosphate, membrane proteins such as membrane surface proteins and integral membrane proteins, antibodies, lipid membrane structures Can bind or contain functional peptides such as peptides that confer cell permeability and nuclear translocation ability, targeting ligands and other functional elements for drug delivery, and the binding amount and content can be adjusted appropriately. it can.

  Moreover, the lipid membrane structure to which pH-dependent cationic property is imparted by YSK05 according to the present invention can be used by encapsulating various substances inside the lipid membrane. The substance to be encapsulated in this case may be any substance, for example, various physiologically active substances such as drugs, nucleic acids, peptides, proteins, sugars or complexes thereof, labeling substances, and lipid membrane structures such as liposomes and micelles. And can be appropriately selected depending on the purpose of diagnosis, treatment, and the like. A method for encapsulating these substances in a lipid membrane structure to which pH-dependent cationic property is imparted by YSK05 can be performed according to a standard method. Examples of such a method include standard methods for polar solvents and nonpolar solvents. And a method for preparing a lipid membrane containing YSK05 described above as a constituent lipid.

  The lipid membrane structure to which pH-dependent cationic property is imparted by YSK05 according to the present invention is used by dispersing in a suitable aqueous solvent such as physiological saline, phosphate buffer, citrate buffer, and acetate buffer. Can do. 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.

  Next, the present invention provides a target gene expression inhibitor. The target gene expression inhibitor according to the present invention is imparted with pH-dependent cationicity by YSK05, which contains RNA having the same or complementary base sequence as the whole or part of the messenger RNA (mRNA) of the target gene. The lipid membrane structure is an active ingredient. It should be noted that, in the target gene expression inhibitor, it is equivalent to the above-described agent that imparts pH-dependent cationicity to the lipid membrane structure according to the present invention or the lipid membrane structure that is imparted with pH-dependent cationic property by YSK05. Or a repetitive description of the corresponding configuration is omitted.

  In the target gene expression inhibitor according to the present invention, the target gene is not particularly limited. Therefore, its expression can be suppressed.

  The RNA having a base sequence complementary to all or part of the messenger RNA of the target gene may be single-stranded or double-stranded complementary. In addition, examples of the structure include a cyclic structure, a linear structure, a complementary double-stranded structure, and a structure in which these are mixed. The base length is not particularly limited, and may be, for example, 5 mer, 10 mer, 15 mer, 20 mer, 21 mer, 25 mer, 30 mer, 40 mer, etc., and may be longer or shorter.

  Specific examples of RNA having a base sequence complementary to all or part of the messenger RNA of the target gene include, for example, small interfering RNA (short interfering RNA; siRNA), micro RNA (miRNA), and small temporal RNA (stRNA). ), Primary miRNA (pri-mRNA), pre-miRNA, morpholino antisense oligo, and the like. Specific examples of RNA having the same base sequence as all or part of the messenger RNA of the target gene include: And anti-miRNA oligonucleotide (AMO) that suppresses miRNA.

  Commercially available siRNA can be used for RNA having the same or complementary base sequence as all or part of the messenger RNA of the target gene, and can be synthesized using methods that can be appropriately selected by those skilled in the art. Such methods include, for example, a method of chemically synthesizing using a synthesizer, a polymerase chain using RNA polymerase using a base sequence identical or complementary to all or part of the messenger RNA of the target gene as a template. A method of synthesis by reaction (PCR), a recombinant vector prepared by inserting a base sequence identical or complementary to all or part of the messenger RNA of the target gene into a vector such as a plasmid vector or a viral vector. Examples thereof include a method of culturing and proliferating a transformant obtained by introducing A into a suitable host cell, and collecting from the proliferated transformant. In addition, any synthesis method other than a general method widely known by those skilled in the art can be used for any synthesis method.

  The method of encapsulating RNA having the same or complementary base sequence as all or part of the messenger RNA (mRNA) of the target gene in a lipid membrane structure to which pH-dependent cationic property has been imparted by YSK05 is performed according to a conventional method. be able to. As such a method, for example, RNA having the same or complementary base sequence as that of all or part of the messenger RNA of the target gene is dissolved in an aqueous solvent, and YSK05 described above is contained as a constituent lipid using this. Mention may be made of a method for preparing a lipid membrane. In this case, RNA having the same or complementary base sequence as all or part of the messenger RNA (mRNA) of the target gene may be pretreated by forming a complex with protamine in advance to form nanoparticles. Good.

  Methods known to those skilled in the art can be used to formulate an agent that imparts pH-dependent cationicity to the lipid membrane structure according to the present invention and a target gene expression inhibitor. The dosage form may also be a dosage form that can be appropriately selected by those skilled in the art. Examples of such a dosage form include tablets, granules, powders, capsules, and coating agents in the case of preparation as an oral dosage formulation. In the case of preparations for parenteral administration, forms such as inhalants, injections, drops, suppositories, coating agents, sprays, patches etc. it can. In addition, the dosage can be appropriately set depending on the formulation form of the pharmaceutical composition, the administration method, the purpose of use, and the age, weight and symptom of the administration subject applied thereto.

Finally, 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 comprises:
(I) (a) YSK05, (b) Chol, (c) POPE, and (d) PEG-DMG at a molar ratio of (a) / (b) + (c) ≧ 2/3, 3/7 ≦ (b ) / (C) ≦ 7/3 and 0 <(d) / (a) + (b) + (c) ≦ 1/10 as a constituent lipid,
(II) (a) YSK05, (b) Chol, and (d) PEG-DMG have a molar ratio of 13/7 ≦ (a) / (b) ≦ 3 and 0 <(d) / (a) + (b) Lipid membrane containing as constituent lipid at a ratio of ≦ 1/10,
It has the process of preparing the lipid membrane of the above (I) or (II). In the method for producing a lipid membrane structure, the agent for imparting pH-dependent cationicity to the above-described lipid membrane structure according to the present invention, the lipid membrane structure to which pH-dependent cationicity is imparted by YSK05, or a target gene The description of the same or equivalent configuration of the expression suppressor is omitted.

  Examples of the method for preparing the lipid membrane of (I) or (II) include a method of preparing a lipid membrane containing YSK05 described above as a constituent lipid. In addition, the method for producing a lipid membrane structure according to the present invention preferably includes a step of adding PEG-DSG to the prepared lipid membrane after the step (I) or (II). The ratio of adding PEG-DSG in this case is not particularly limited, and can be appropriately set according to the purpose of use of the lipid membrane structure, the type and ratio of the constituent lipid, the type of inclusion, and the like. It can be 1 mol%, 2 mol%, 3 mol%, 5 mol%, 7 mol%, 10 mol%, 15 mol%, 20 mol%, etc. with respect to the total lipid other than DSG.

  The method for producing a lipid membrane structure according to the present invention can include other steps as long as the characteristics of the present invention are not impaired. Examples of such steps include a purification step, a labeling step, a staining step, Examples include a filtration step, a sizing step, a concentration step, a dilution step, a modification step, and the like.

  Hereinafter, an agent for imparting pH-dependent cationic property to the lipid membrane structure according to the present invention, a lipid membrane structure imparted with pH-dependent cationic property thereby, a target gene expression inhibitor, and production of the lipid membrane structure A method is demonstrated based on an Example. Note that the technical scope of the present invention is not limited to the features shown by these examples.

<Example 1> Synthesis of 1-methyl-4,4-bis [(9Z, 12Z) -octadeca-9,12-dien-1-yloxy] piperidine (1) (9Z, 12Z) -Octadecadien-1-OL
With reference to the structure of a commercially available cationic lipid, 1,2-dioleoyl-3-trimethylammonium probe (DOTAP) and a commercially available pH-dependent cationic lipid, 1,2-dioleoyl-3-dimethylaminopropane (DODAP) 1-methyl-4,4-bis [(9Z, 12Z) -octadeca-9,12-dien-1-yloxy] piperidine (hereinafter referred to as “YSK05”) was designed. The structures of YSK05, DOTAP and DODAP are shown in FIG.

  In order to synthesize YSK05, first, a method described in the previous report (Sodeoka M, et al., J. Med. Chem., 44, 3216-3222, 2001) was modified by a method (9Z, 12Z). -Octadecadien-1-OL was synthesized. Specifically, 375 mg (20 nmol) of lithium aluminum hydride was suspended in 50 mL of tetrahydrofuran (THF) and cooled to 4 ° C. Thereto was added dropwise 1.65 mL (10 mmol) of linolenic acid, and the mixture was stirred for 30 minutes. Then, it refluxed for 2 hours, heating with an oil bath. After cooling this, 5 mL of water was added, and then 50 mL of a 1 mol / L aqueous sodium hydroxide solution was added to stop the reaction. Next, 200 mL of ethyl acetate was added for dilution, followed by filtration, and the filtrate collected using saturated saline was washed twice. Subsequently, the organic phase was recovered, and anhydrous sodium sulfate was added thereto for dehydration. After filtering this, the solvent was distilled off using a rotary evaporator to obtain a crude product. The crude product was purified by subjecting it to silica gel column chromatography {elution solvent; hexane: ethyl acetate (continuous gradient)} to obtain 2.4 g (9 mmol) of (9Z, 12Z) -Octacadecaden-1-OL. The yield was about 90%.

Proton Nuclear Magnetic Resonance ( ¹ H NMR; 500 MHz) data δ0.88 (t, 3H, J = 7.2 Hz), 1.25 to 1.36 (m, 16H), 1.53 to 1.58 of (9Z, 12Z) -Octadecadien-1-OL (m, 2H), 2.02 to 2.06 (m, 4H), 2.76 (t, 2H, J = 6.9 Hz), 3.62 (t, 2H, J = 6.9 Hz), 5.29 to 5.40 (m, 4H).

(2) YSK05
After dissolving (9Z, 12Z) -Octadecadien-1-OL of Example 1 (1) in 50 mL of anisole, 455 μL (3.7 mmol) of 1-methyl-4-piperidone was added, followed by p-toluenesulfonic acid. 780 mg (4.1 mmol) was added. A Dean-Stark device and a Dimroth were attached and heated to reflux for 16 hours. After cooling, the organic phase was collected and diluted by adding 200 mL of ethyl acetate. This was washed with a saturated aqueous sodium hydrogen carbonate solution and further washed with a saturated saline solution. Subsequently, anhydrous sodium sulfate was added for dehydration, and the solvent was distilled off using a rotary evaporator to obtain a crude product. The crude product was purified by subjecting it to silica gel column chromatography {elution solvent; dichloromethane: methanol (continuous gradient)} to obtain 1.55 g (2.4 mmol) of YSK05 as an oily substance. The yield was 62%.

YSK05 proton nuclear magnetic resonance ( ¹ H NMR; 400 MHz) data δ0.89 (t, 6H, J = 7.1 Hz), 1.23-1.38 (m, 36H), 1.50? 1.55 (m, 4H), 1.77? 1.82 ( m, 4H), 2.02? 2.07 (m, 8H), 2.28 (s, 3H), 2.40 (br, 3H), 2.77 (t, 2H, J = 6.6 Hz), 3.36 (t, 2H, J = 6.9 Hz ), 5.29? 5.39 (m, 8H) .MS (FD): (M) + Calcd for C42H77NO2: 627, found 627.

<Example 2> Production of lipid membrane structure (1) Lipid membrane structure encapsulating siRNA DOTAP, DODAP, YSK05, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol) and 1,2-Dimyristol-sn-glycerol (DMG) (PEG-DMG) combined with polyethylene glycol (PEG) is adjusted so that the lipid composition of the following compositions 1 to 3 and the total lipid concentration are 1.5 mmol / L. Diluted in tert-butyl alcohol to obtain a lipid solution. 68.6 μg of siRNA (anti-luc; SEQ ID NO: 1, 2) against firefly luciferase gene messenger RNA was dissolved in 1 mmol / L citrate buffer (pH 4.5), and the nitrogen / phosphate ratio (N Protamine was added dropwise such that the / P ratio was 1.1 to form a complex of siRNA and protamine (siRNA / protamine complex). A solution containing siRNA / protamine complex (siRNA complex solution) was dropped into a lipid solution and vigorously stirred, and then tert-butyl was added by adding 1 mmol / L citrate buffer (pH 4.5) while stirring. The alcohol concentration was 20% (v / v) or less. Thereafter, ultrafiltration is performed using Amicon Ultra filter units (Millipore) and phosphate buffered saline (pH 7.4; PBS) to replace the external solution with PBS, and a lipid membrane structure containing siRNA. Got. The lipid membrane structures obtained from the lipid solutions of compositions 1, 2 and 3 were respectively luc-encapsulated DOTAP lipid membrane structure, luc-encapsulated DODAP lipid membrane structure and luc-encapsulated YSK lipid membrane structure.

Lipid composition of lipid solution (molar ratio)
Composition 1: DOTAP: DOPE: Chol: PEG-DMG = 30: 40: 30: 3
Composition 2; DODAP: DOPE: Chol: PEG-DMG = 30: 40: 30: 3
Composition 3: YSK05: DOPE: Chol: PEG-DMG = 30: 40: 30: 3

(2) Lipid membrane structure not encapsulating siRNA By performing the method described in Example 2 (1) using 1 mmol / L citrate buffer (pH 4.5) instead of the siRNA complex solution , A lipid membrane structure not containing siRNA was obtained. The lipid membrane structures obtained from the lipid solutions of compositions 1, 2 and 3 were designated as empty DOTAP lipid membrane structure, empty DODAP lipid membrane structure and empty YSK lipid membrane structure, respectively.

(3) Physical Properties of Lipid Membrane Structure The average particle diameter, polydispersity (PDI) and zeta potential of the lipid membrane structure of Example 2 (1) were measured using Zetasizer Nano ZS ZE3600 (MALVERN Instrument). . The results are shown in Table 1. As shown in Table 1, the average particle diameter and PDI were values that approximated each lipid membrane structure. On the other hand, the zeta potential is a value at which the luc-encapsulated DOTAP lipid membrane structure is cationic, and the luc-encapsulated DODAP lipid membrane structure and the luc-encapsulated YSK lipid membrane structure are values that are almost neutral. It was. From this result, YSK05 does not affect the physical properties of lipid membrane structures such as the average particle diameter and PDI, and measurement environment (neutral environment) using Zetasizer Nano ZS ZEN3600 (MALVERN Instrument) It became clear that it showed neutrality.

(4) siRNA encapsulation rate The siRNA encapsulation rate was measured using a Ribogreen reagent that emits fluorescence by intercalating into a double-stranded nucleic acid. Specifically, the lipid membrane structure of Example 2 (1) was diluted in 10 mmol / L Hepes buffer (pH 7.4) containing 20 μg / mL dextran sulfate and Ribogreen reagent, and an excitation wavelength of 500 nm and The fluorescence intensity was measured at a fluorescence wavelength of 525 nm, and was defined as the fluorescence intensity before decomposition. Then, the lipid membrane structure was decomposed by adding Triton X-100 so as to be 0.1% (w / v), and the fluorescence intensity was measured again at the same wavelength to obtain the fluorescence intensity after decomposition. Subsequently, the siRNA encapsulation rate was calculated by Formula 1; siRNA encapsulation rate (%) = {(fluorescence intensity after degradation−fluorescence intensity before degradation) / fluorescence intensity after degradation} × 100. The results are shown in Table 1. As shown in Table 1, the siRNA encapsulation rate of the luc-encapsulated YSK lipid membrane structure was approximately 90%. This value was high compared to the luc-encapsulated DOTAP lipid membrane structure, and was comparable to the luc-encapsulated DODAP lipid membrane structure. From this result, it was revealed that siRNA can be efficiently encapsulated in a lipid membrane structure having a lipid membrane containing YSK05 as a constituent lipid.

<Example 3> Function of lipid membrane structure (1) pH-dependent cationic property The cationic property of the lipid membrane structure was evaluated using a negatively charged TNS reagent. Since the TNS reagent enters the cationic lipid membrane and emits fluorescence, the fluorescence intensity is a cationic indicator of the lipid membrane. Specifically, 20 mmol / L citrate buffer, sodium phosphate buffer or Tris HCl containing NaCl having a final concentration of 130 mmol / L prepared to have various pH values in the range of pH 2.5 to 9.5. A buffer solution was prepared. To these buffers, the lipid membrane structure of Example 2 (1) was added to a lipid concentration of 30 μmol / L, followed by the addition of TNS reagent to 6 μmol / L to a final volume of 200 μL. . Thereafter, the fluorescence intensity was measured at an excitation wavelength of 321 nm and a fluorescence wavelength of 447 nm at 37 ° C. The relative fluorescence intensity was calculated as a percentage with the maximum value of the fluorescence intensity in each lipid membrane structure as 100%. The pH at which the relative fluorescence intensity was 50% was defined as pKa. The result is shown in FIG.

  As shown in FIG. 2, the relative fluorescence intensity of the buffer containing the luc-encapsulated DOTAP lipid membrane structure was constant regardless of the pH. In contrast, in the buffer solution containing the luc-encapsulated DODAP lipid membrane structure and the luc-encapsulated YSK lipid membrane structure, the lower the pH, the greater the relative fluorescence intensity, and the greater the pH, the smaller the relative fluorescence intensity. The pKa of the buffer containing the luc-encapsulated DODAP lipid membrane structure and the luc-encapsulated YSK lipid membrane structure was 5.7 and 6.6, respectively. From this result, the lipid membrane structure having a lipid membrane containing YSK05 as a constituent lipid has the property that the amount of charge increases with decreasing pH (pH-dependent cationic property), and YSK05 has a lipid membrane structure. It has been shown to impart pH-dependent cationic properties to the body.

  Further, when the relative fluorescence intensities of the buffer containing the luc-encapsulated YSK lipid membrane structure and the luc-encapsulated DODAP lipid membrane structure around pH 6-7 were compared, the luc-encapsulated YSK lipid membrane structure was more luc-encapsulated DODAP lipid membrane structure. The relative fluorescence intensity was larger than that of the body. That is, it has been clarified that the amount of charge in the luc-encapsulated YSK05 lipid membrane structure increases more rapidly than the luc-encapsulated DODAP lipid membrane structure in accordance with the change from the neutral environment to the acidic environment. From this result, the lipid membrane structure having a lipid membrane containing YSK05 as a constituent lipid is transferred from the extracellular (neutral environment) into the cell as compared with the lipid membrane structure having a lipid membrane containing DODAP as a constituent lipid. After being taken in, it was suggested that it is rapidly released from the endosome (acidic environment) into the cytoplasm.

(2) Membrane fusion ability The membrane fusion ability of a lipid membrane structure with a biological membrane was evaluated using erythrocytes. When the lipid membrane structure fuses with the red blood cell membrane, hemoglobin leaks into the solution, and the amount of hemoglobin leaked is an indicator of membrane fusion ability. Specifically, blood was collected from male ICR mice, and red blood cells were collected and suspended in physiological saline. After adjusting PBS to pH 7.5, 6.5 and 5.5, a physiological saline containing a certain amount of red blood cells was added. Subsequently, 3.3 μL, 10 μL, and 30 μL of PBS containing the lipid membrane structure of Example 2 (2) were added. Moreover, what added the same amount PBS without a lipid membrane structure was made into negative control (NC), and after adding the same amount PBS without a lipid membrane structure, 0.5% (w / v) Triton X-100 was added so that erythrocytes were lysed so as to be used as a positive control (PC). These were incubated at 37 ° C. for 30 minutes, centrifuged at 4 ° C. under 400 × g for 5 minutes, the supernatant was collected, and the amount of hemoglobin was measured by measuring the absorbance at 545 nm. did. Subsequently, each measurement value was expressed as a percentage with the measurement value of the positive control (PC) as 100%, and this was defined as the membrane fusion rate (%). The result is shown in FIG.

  As shown in FIG. 3, the membrane fusion rate was constant regardless of the pH in the empty DOTAP lipid membrane structure and empty DODAP lipid membrane structure, whereas in the empty YSK lipid membrane structure, the pH was small. It was so big. That is, it became clear that the empty YSK lipid membrane structure is more likely to cause membrane fusion with erythrocytes as the pH is lower. From this result, it was shown that the lipid membrane structure having a lipid membrane containing YSK05 as a constituent lipid becomes easier to fuse with a biological membrane, that is, the membrane fusion ability is improved as the pH is lowered.

(3) Suppression of Luciferase Gene Expression HeLa cells stably expressing two types of firefly luciferase and Renilla luciferase (dluc-HeLa cells) are seeded in a 96-well plate at a concentration of 5 × 10 ³ cells / well, 37 ° C., for 24 hours at an atmosphere of 5% CO _2. The medium is a DMEM medium containing 10% (v / v) calf serum, 100 U / mL penicillin, 100 mg / mL streptomycin and 0.4 mg / mL G418 at a final concentration (hereinafter referred to as “reagent-containing DMEM medium”). .) Was used. Thereafter, the cells were washed with PBS, and a reagent-containing DMEM medium containing the lipid membrane structure of Example 2 (1) at a final concentration of 1 to 243 nmol / L (siRNA concentration) was added and cultured for 24 hours. Further, a reagent-containing DMEM medium not containing a lipid membrane structure was added and cultured in the same manner as a control. Subsequently, after washing with PBS, the cells were collected, and the luminescence intensity of luciferase was measured using Dual Luciferase Reporter Assay System (Promega). Next, the relative luminescence intensity was calculated by dividing the Renilla luciferase luminescence intensity by the firefly luciferase luminescence intensity. Subsequently, the relative luminescence intensity was expressed as a percentage (%) with the value of the relative activity of the control as 100%, and this was defined as the expression suppression rate (Luc / KD rate) of the luciferase gene. The result is shown in FIG.

  As shown in FIG. 4, the Luc / KD ratio was higher in the luc-encapsulated YSK lipid membrane structure than in the luc-encapsulated DOTAP lipid membrane structure and the luc-encapsulated DODAP lipid membrane structure at any siRNA concentration. The Luc / KD ratio of the luc-encapsulated YSK lipid membrane structure was a high value of about 50% or more when the siRNA concentration was 30 nmol / L or more. From these results, the lipid membrane structure having a lipid membrane containing YSK05 as a constituent lipid and encapsulating RNA having the same or complementary base sequence as all or part of the messenger RNA of the target gene is expressed in the target gene. It was shown that the expression can be suppressed, and that the effect of suppressing the expression is greater than that of YSK05 not included as a constituent lipid.

<Example 4> Production of lipid membrane structure of preferred lipid composition A (1) Examination of lipid types [1-1] Inhibition of luciferase gene expression In addition to DOPE, 1-palmitoyl-2-oleoyl-sn-glycero -3-phosphoethanolamine (POPE), 1,2-Dioleyl-sn-3-phosphophatylcholine (DOPC), 1-Stheroyl-2-oleyl-sn-glycero-3-phosphophatylylline (SOPC) Using 3-phosphatidylCholine (DSPC), the composition of the lipid solution was changed to the following compositions 3 to 7, and a lipid membrane structure was obtained by performing the method described in Example 2 (1).

Composition 3: YSK05: DOPE: Chol: PEG-DMG = 30: 40: 30: 3
Composition 4: YSK05: POPE: Chol: PEG-DMG = 30: 40: 30: 3
Composition 5; YSK05: DOPC: Chol: PEG-DMG = 30: 40: 30: 3
Composition 6; YSK05: SOPC: Chol: PEG-DMG = 30: 40: 30: 3
Composition 7; YSK05: DSPC: Chol: PEG-DMG = 30: 40: 30: 3

  Subsequently, the Luc / KD ratio was measured for these lipid membrane structures by the method described in Example 3 (3). However, siRNA concentrations in the reagent-containing DMEM medium were 9 nmol / L and 27 nmol / L. The result is shown in the left figure of FIG. As shown in the left diagram of FIG. 5, the lipid membrane structure having the lipid membrane of composition 4 is the lipid having the lipid membranes of compositions 3 and 5-7 when the siRNA concentration is 9 nmol / L and 27 nmol / L. The Luc / KD ratio was the highest as compared with the membrane structure. That is, anti-luc encapsulated in a lipid membrane structure having a lipid membrane containing POPE as a constituent lipid is an siRNA encapsulated in a lipid membrane structure having a lipid membrane containing DOPE, DOPC, SOPC and DSPC as a constituent lipid. It was revealed that the effect of the expression efficiency of the luciferase gene is greater than that of.

[1-2] Serum tolerance Serum was collected from male ICR mice, and the lipid membrane structure of Example 4 (1) [1-1] was added. Immediately after adding the lipid membrane structure (after 0 hours) and after incubation at 37 ° C for 6 hours, 24 hours and 48 hours, freezing at -80 ° C stopped the degradation of siRNA by serum. Then, after thawing at room temperature, phenol / chloroform / isoamyl alcohol extraction was quickly performed to collect siRNA encapsulated in the lipid membrane structure. The collected siRNA was subjected to 20% polyacrylamide gel electrophoresis (150 V, 2 hours), and then the gel was immersed in 1 μg / mL ethidium bromide aqueous solution and stained. The result is shown in the right figure of FIG. As shown in the right figure of FIG. 5, the fluorescence intensity of the band showing siRNA was the same between the lipid membrane structures having lipid membranes of compositions 3 to 7 after 0 hours and after 6 hours, After 24 hours and 48 hours, the lipid membrane structure having the lipid membrane of composition 4 was the largest compared to the lipid membrane structure having the lipid membranes of compositions 3 and 5-7. From this result, siRNA encapsulated in a lipid membrane structure having a lipid membrane containing POPE as constituent lipid is siRNA encapsulated in a lipid membrane structure having a lipid membrane containing DOPE, DOPC, SOPC and DSPC as constituent lipids. As compared with, it was revealed that the remaining time without being decomposed in the serum was long, that is, the serum resistance was high.

(2) Examination of the ratio of lipid The lipid membrane structure was obtained by making the composition of a lipid solution into the following composition 8-16 and performing the method as described in Example 2 (1). Subsequently, the Luc / KD ratio was measured for the lipid membrane structures having compositions 8 to 16 by the method described in Example 3 (3). However, the siRNA concentration in the reagent-containing DMEM medium was varied in the range of 1 to 27 nmol / L. The results of the lipid membrane structures having compositions 8 to 13 are shown in the left diagram of FIG. 6, and the results of the lipid membrane structures having compositions 14 to 16 are shown in the right diagram of FIG. 6.

Composition 8; YSK05: POPE: Chol: PEG-DMG = 50: 15: 35: 3
Composition 9; YSK05: POPE: Chol: PEG-DMG = 50: 20: 30: 3
Composition 10; YSK05: POPE: Chol: PEG-DMG = 50: 25: 25: 3
Composition 11; YSK05: POPE: Chol: PEG-DMG = 50: 30: 20: 3
Composition 12; YSK05: POPE: Chol: PEG-DMG = 50: 35: 15: 3
Composition 13; YSK05: POPE: Chol: PEG-DMG = 50: 40: 10: 3
Composition 14; YSK05: POPE: Chol: PEG-DMG = 40: 20: 40: 3
Composition 15; YSK05: POPE: Chol: PEG-DMG = 50: 20: 30: 3
Composition 16; YSK05: POPE: Chol: PEG-DMG = 60: 20: 20: 3

  As shown in the left diagram of FIG. 6, when the Luc / KD ratio is compared between lipid membrane structures having lipid membranes of composition 8 to 13, the lipid membrane having lipid membrane of composition 10 at any siRNA concentration Lipid membrane structures with the largest structures and lipid membranes of composition 8, 9, 11 and 12 were relatively large. Further, as shown in the right diagram of FIG. 6, when the Luc / KD ratio was compared between lipid membrane structures having lipid membranes having compositions 14 to 16, the composition 14 was larger in the order of <15 <16. From these results, the molar ratio of YSK05, POPE and Chol in the lipid membrane of the lipid membrane structure including anti-luc is 15/35 ≦ POPE / Chol ≦ 35/15, that is, 7/3 ≦ POPE / Chol ≦ 3. When YSK05 / (POPE + Chol) ≧ 40 / (20 + 40), that is, YSK05 / POPE + Chol ≧ 2/3, the effect of suppressing the expression of the luciferase gene was found to be large.

  From the results of the above Example 4 (1) [1-1] and [1-2] and (2), RNA having the same or complementary base sequence as all or part of the messenger RNA of the target gene is obtained. The preferred composition of the constituent lipid in the lipid membrane of the lipid membrane structure having a lipid membrane containing YSK05 as a constituent lipid to be included is YSK05 / Chol + POPE ≧ 2/3 and 3/7 ≦ Chol / POPE ≦ 7/3 ( Molar ratio; hereinafter referred to as “preferred lipid composition A”).

<Example 5> Function of lipid membrane structure of preferred lipid composition A (1) Production and physical properties of lipid membrane structure, siRNA inclusion rate and pKa
The lipid composition in the lipid solution is the composition 10 of Example 4 (2), and the lipid membrane structure is obtained by carrying out the method described in Example 2 (1), and this is used as the luc-encapsulated YSK / A lipid membrane structure. It was. In addition, the average particle diameter, PDI and zeta potential of the luc-encapsulated YSK / A lipid membrane structure were determined by the method described in Example 2 (3), and the siRNA encapsulation rate was determined by the method described in Example 2 (4). pKa was measured by the method described in Example 3 (1). As a result, the average particle size of the luc-encapsulated YSK / A lipid membrane structure was 116 ± 5 nm, PDI was 0.09 ± 0.02, zeta potential was + 4.5 ± 2.1 mV, and siRNA encapsulation was 93 ± 4. % And pKa were 6.4. These values approximated the values in the luc-encapsulated YSK lipid membrane structure of Example 2 (1) (Table 1, FIG. 2).

(2) Membrane fusion ability The lipid composition in the lipid solution was set to composition 10 of Example 4 (2), and a lipid membrane structure was obtained by carrying out the method described in Example 2 (2). A lipid membrane structure was obtained. For the empty YSK / A lipid membrane structure, the membrane fusion rate was determined by the method described in Example 3 (2). For comparison, it is shown in FIG. 7 together with the membrane fusion rate of the empty YSK lipid membrane structure obtained in Example 3 (2). As shown in FIG. 7, the membrane fusion rate was similar between the lipid membrane structures at pH 7.4, but the empty YSK / A lipid membrane structure was more empty YSK at pH 6.5 and pH 5.5. It was significantly larger than the lipid membrane structure. From this result, it became clear that the membrane fusion ability is improved by setting the lipid composition in the lipid membrane of the lipid membrane structure having a lipid membrane containing YSK05 as a constituent lipid to the preferred lipid composition A.

(3) Suppression of Luciferase Gene Expression The Luc / KD ratio of the luc-encapsulated YSK / A lipid membrane structure of Example 5 (1) was measured by the method described in Example 3 (3). Further, using Lipofectamine 2000 (LF2k; Carlsbad), which is a commercially available transfection reagent, anti-luc was introduced into dluc-HeLa cells according to the attached instruction, and the Luc / HeLa cell was prepared by the method described in Example 3 (3). The KD rate was measured. The results are shown in FIG. 8 together with the Luc / KD ratio of the luc-encapsulated YSK lipid membrane structure obtained in Example 3 (3). As shown in FIG. 8, the Luc / KD ratio was greater in the luc-encapsulated YSK / A lipid membrane structure than in the luc-encapsulated YSK lipid membrane structure and LF2k regardless of the siRNA concentration. From these results, the lipid in the lipid membrane of the lipid membrane structure having a lipid membrane containing YSK05 as a constituent lipid, which contains RNA having the same or complementary base sequence as all or part of the messenger RNA of the target gene It was revealed that the effect of suppressing the expression of the target gene is increased by setting the composition to the preferred lipid composition A.

<Example 6> Examination of gene expression suppression mechanism of lipid membrane structure (1) Observation of transition state of siRNA to cytoplasm anti-luc (Cy5-anti-luc; SEQ ID NO :) instead of anti-luc 3 and 4), the lipid composition in the lipid solution was set as the composition 2 of Example 2 (1) and the composition 10 of Example 4 (2), and the method described in Example 2 (1) was performed to obtain the lipid. A membrane structure was obtained. The lipid membrane structures obtained from the lipid solutions of compositions 2 and 10 were designated as Cy5-luc-encapsulated DODAP lipid membrane structure and Cy5-luc-encapsulated YSK / A lipid membrane structure, respectively.

Dluc-HeLa cells were seeded on a glass bottom dish at a concentration of 1 × 10 ⁵ cells / well to give a sample for 1 hour and a sample for 6 hours. After culturing the sample for 1 hour and the sample for 6 hours in a reagent-containing DMEM medium at 37 ° C. in an atmosphere of 5% CO ₂ for 24 hours, Cy5-luc inclusion YSK / A lipid membrane structure and Cy5-luc inclusion The DODAP lipid membrane structure was added to the medium so as to be 100 nmol / L (siRNA concentration), and incubated at 37 ° C. for 1 hour. Thereafter, the cells were washed three times with chilled PBS. Subsequently, for 1 hour samples, 4% (v / v) paraformaldehyde / PBS was added and incubated for 10 minutes to fix the cells. On the other hand, for the 6-hour sample, a reagent-containing DMEM medium was added, incubated at 37 ° C. for 6 hours, washed 3 times with cooled PBS, and cells were similarly fixed. After 1 hour sample and 6 hour sample after fixation were washed with PBS, Hoechst 33342 was added, and cell nuclei were stained for 10 minutes, and then using a confocal laser microscope Nikon A1 (Nikon) and a 60 × objective lens. The fluorescence of Cy5 and Hoechst 33342 was observed. The result is shown in FIG.

  As shown in FIG. 9, in the sample for 1 hour, the Cy5 fluorescence was observed in a dot-like manner when either the Cy5-luc-encapsulated DODAP lipid membrane structure or the Cy5-luc-encapsulated YSK / A lipid membrane structure was added. It was. That is, it is clear that both siRNA encapsulated in Cy5-luc-encapsulated DODAP lipid membrane structure and Cy5-luc-encapsulated YSK / A lipid membrane structure are taken up into cells by endocytosis and localized in endosomes. Became. On the other hand, in the sample for 6 hours, when Cy5-luc-encapsulated DODAP lipid membrane structure was added, Cy5 fluorescence was seen in the form of dots, whereas Cy5-luc-encapsulated YSK / A lipid membrane structure was added. In some cases, Cy5 fluorescence was diffused into the cells. That is, the siRNA encapsulated in the Cy5-luc-encapsulated DODAP lipid membrane structure is still localized in the endosome after a certain period of time after being taken into the cell, whereas the Cy5-luc-encapsulated YSK / A It became clear that siRNA encapsulated in the lipid membrane structure migrated to the cytoplasm and diffused. From these results, a lipid membrane structure having a lipid membrane containing YSK05 as a constituent lipid and containing RNA having the same or complementary base sequence as all or part of the messenger RNA of the target gene is taken into the cell. After that, it was shown that the encapsulated RNA was rapidly released into the cytoplasm.

(2) Gene Expression Suppression in the Presence of Acidification Inhibiting Substance Example 3 Regarding the luc-encapsulated YSK / A lipid membrane structure of Example 5 (1) and the luc-encapsulated DOTAP lipid membrane structure of Example 2 (1) The Luc / KD ratio was measured by the method described in (3). However, chloroquine or ammonium chloride, which is a substance that inhibits acidification, was added to the reagent-containing DMEM medium containing the lipid membrane structure at various concentrations. The result is shown in FIG.

  As shown in FIG. 10, in the luc-encapsulated DOTAP lipid membrane structure, the Luc / KD ratio tended to increase as the concentration of chloroquine or ammonium chloride increased, whereas the luc-encapsulated YSK / A lipid membrane structure. Then, the Luc / KD ratio decreased as the concentration of chloroquine or ammonium chloride increased. That is, in the luc-encapsulated YSK / A lipid membrane structure, the effect of suppressing gene expression decreased due to inhibition of acidification. As a result, based on the results of Example 5 (4) and Examples 3 (1), (2) and (3), it has the same or complementary base sequence as all or part of the messenger RNA of the target gene. A lipid membrane structure containing a lipid membrane containing YSK05 as a constituent lipid, which encapsulates RNA, is incorporated into the cell, then becomes cationic by the acidification of the endosome, membrane-fused with the endosome, and the encapsulated RNA is cytoplasmic It was shown to suppress the expression of the target gene.

<Example 7> Function of a lipid membrane structure containing PEG-DSG as a constituent lipid and having a lipid membrane having a preferred lipid composition A (1) Addition of PEG-DSG Luc-encapsulated YSK / A lipid of Example 5 (1) Ethanol was added to PBS containing the membrane structure and the luc-encapsulated DOTAP lipid membrane structure and luc-encapsulated DODAP lipid membrane structure of Example 2 (1) so that the concentration was 10% (v / v), and the PEG-conjugated 1 , 2-distearoyl-sn-glycerol (DSG) (PEG-DSG) was added at 3 mol% with respect to the total lipid other than PEG-DSG, followed by incubation at 45 ° C. for 45 minutes. Here, since DSG has 18 carbon atoms and has a relatively long carbon chain, it enters the lipid membrane of the lipid membrane structure and is stable. That is, according to the above operation, PEG-DSG is added to the lipid membrane of the lipid membrane structure to obtain a lipid membrane structure having a lipid membrane containing PEG-DSG as a constituent lipid, and this is converted into PEG / luc-encapsulated YSK / A lipid membrane structure, PEG / luc-encapsulated DOTAP lipid membrane structure, and PEG / luc-encapsulated DODAP lipid membrane structure were used.

(2) Serum resistance anti-luc, siRNA / protamine complex of Example 2 (1) and empty YSK lipid membrane structure of Example 2 (2), luc-encapsulated YSK / A lipid membrane of Example 5 (1) For the structure and the PEG / luc-encapsulated YSK / A lipid membrane structure of Example 7 (1), the method described in Example 4 (1) [1-2] was performed. However, siRNA / protamine complex and empty YSK lipid membrane structure were mixed and added to serum. Incubation times were 0, 3, 6 and 24 hours, and before and after incubation, Triton-X100 was added to 0.05% (w / v) and not added. The result is shown in FIG.

  As shown in FIG. 11, when Triton-X100 is not added, the anti-luc and siRNA / protamine complex and the mixture of empty YSK lipid membrane structure have a lower fluorescence intensity of the band showing siRNA as the incubation time elapses. It was confirmed that siRNA was decomposed because the width became wider and the migration speed increased. In contrast, in the luc-encapsulated YSK / A lipid membrane structure, after 3 and 6 hours, the fluorescence intensity, width, and migration speed of the band are equivalent to those after 0 hour, and after 24 hours, the fluorescence intensity of the band and Although the width was slightly reduced, the electrophoresis speed did not decrease. In addition, in the PEG / luc-encapsulated YSK / A lipid membrane structure, the band fluorescence intensity, width, and migration rate hardly changed after 0 to 24 hours. That is, it was confirmed that siRNA was hardly degraded in the luc-encapsulated YSK lipid membrane structure and the luc-encapsulated YSK / A lipid membrane structure. From these results, it was shown that siRNA encapsulated in a lipid membrane structure having a lipid membrane containing YSK05 as a constituent lipid has higher serum resistance than non-encapsulated siRNA. In addition, it was revealed that the serum resistance of the encapsulated siRNA is increased by adding PEG-DSG to the lipid membrane.

(3) PLK1 gene expression suppression [3-1] Production of lipid membrane structure In place of anti-luc, siRNA against Polo-like kinase 1 (PLK1) gene messenger RNA (anti-plk1; SEQ ID NOs: 5 and 6) is used. Thus, a lipid membrane structure was obtained by the method described in Example 2 (1). The lipid membrane structures obtained from the lipid solutions having compositions 1 to 3 were designated as a pl-encapsulated DOTAP lipid membrane structure, a pl-encapsulated DODAP lipid membrane structure, and a pl-encapsulated YSK lipid membrane structure, respectively. In addition, the lipid composition in the lipid solution was set to composition 10 of Example 4 (2), and in addition to anti-luc, anti-plk1 was used to obtain a lipid membrane structure by the method described in Example 2 (1). Luc-encapsulated YSK / A lipid membrane structure and pl-encapsulated YSK / A lipid membrane structure, respectively. Subsequently, by performing the method described in Example 7 (1) for the lipid membranes of these lipid membrane structures, a lipid membrane structure having a lipid membrane containing PEG-DSG as a constituent lipid is obtained. PEG / pl-encapsulated DOTAP lipid membrane structure, PEG / pl-encapsulated DODAP lipid membrane structure, PEG / pl-encapsulated YSK lipid membrane structure, PEG / luc-encapsulated YSK / A lipid membrane structure, and PEG / pl-encapsulated YSK / A lipid membrane structure was obtained.

[3-2] Administration to tumor model mouse Human renal cell carcinoma-derived OSRC2 cells were treated with 10% (v / v) FBS, 100 U / mL penicillin and 100 mg / mL streptomycin in a RPMI 1640 medium (reagent-containing RPMI medium). Was cultured at 37 ° C. in a 5% CO ₂ atmosphere and suspended in PBS. Subsequently, 1000000 OSRC2 cells per mouse were transplanted subcutaneously into the back of male BALB / c nude mice to form tumor model mice. Breeding while measuring the volume of the tumor formed by the transplanted OSRC2 cells over time, and when the tumor volume reached about 100 mm ³ , the lipid membrane structure of Example 7 (3) [3-1] was obtained. It was administered directly to the tumor tissue and reared for 24 hours. The tumor volume was calculated by the formula 2; tumor volume = 1/2 × (tumor major axis) × (tumor minor axis) ² . The dose was 10 μg (siRNA amount) / one animal. Tumor model mice to which nothing was administered were similarly raised and used as controls.

[3-3] Quantitative real-time PCR
A tumor tissue was collected from the mouse of Example 6 (3) [3-2], and about 30 mg of the tumor mass was homogenized and centrifuged at 4 ° C. and 15000 rpm for 10 minutes. The supernatant was recovered and total RNA was recovered using RNeasy kit (Qiagen). Using 1 μg of total RNA as a template, reverse transcription reaction was performed using High Capacity RNA-to-cDNA kit (Applied Biosystems) according to the attached instruction manual to obtain cDNA. For 2 ng cDNA, quantitative real-time PCR was performed according to a conventional method using Fast SYBR Green Master Mix (Applied Biosystems) and Lightcycler 480 (Roche) to measure the messenger RNA expression level of the human PLK1 gene. The reaction solution scale of quantitative real-time PCR was 15 μL, and the following sequences were used as PCR primers for the human PLK1 gene and the human GAPDH gene. In addition, the messenger RNA expression level of human (Glyceraldehyde-3-phosphate dehydrogenase) GAPDH gene was corrected as an internal standard. The corrected messenger RNA expression level of the human PLK1 gene was expressed as a relative value with the messenger RNA expression level of the human PLK1 gene in the control being 1.0, and this was defined as the relative PLK1 expression level. The result is shown in FIG.

Primer forward of human PLK1 gene; 5′-AATAAAGGCTTGGAGAACCC-3 ′ (SEQ ID NO: 7)
Reverse; 5′-ACCTCACCCTGTTCTCGAGA-3 ′ (SEQ ID NO: 8)
Primer forward of the human GAPDH gene; 5′-CCTCTGACTTCAACAGCGAC-3 ′ (SEQ ID NO: 9)
Reverse; 5'-CGTTGTCATACCGGAAATGAG-3 '(SEQ ID NO: 10)

  As shown in FIG. 12, the relative expression level of PLK1 is controlled when PEG / luc-encapsulated YSK / A lipid membrane structure, PEG / pl-encapsulated DOTAP lipid membrane structure and PEG / pl-encapsulated DODAP lipid membrane structure are administered. It was almost equivalent. In contrast, when the PEG / pl-encapsulated YSK lipid membrane structure and the PEG / pl-encapsulated YSK / A lipid membrane structure were administered, they were significantly smaller than the control. From this result, the expression of the PLK1 gene in the tumor tissue is suppressed by directly administering to the tumor tissue a lipid membrane structure having a lipid membrane containing PEG-DSG as a constituent lipid in addition to YSK05 encapsulating anti-plk1. It was shown that

  In addition, the relative expression level of PLK1 was smaller when the PEG / pl-encapsulated YSK / A lipid membrane structure was administered than when the PEG / pl-encapsulated YSK lipid membrane structure was administered. From this result, it was clarified that the effect of suppressing the expression of the PLK1 gene was increased by setting the lipid composition in the lipid membrane to a suitable lipid composition A. Inhibition of PLK1 gene expression by setting the lipid composition in the lipid membrane of the lipid membrane structure having a lipid membrane containing PEG-DSG as a constituent lipid in addition to YSK05, which contains anti-plk1, to a suitable lipid composition A Increased.

[3-4] 5'RACE
5′RACE PCR was performed to specifically amplify the RNA fragment resulting from the cleavage of the PLK1 gene mRNA, and confirm whether anti-plk1 caused RNA interference.

Specifically, first, tumor tissue was collected from the mouse of Example 7 (3) [3-2], and about 30 mg of the tumor mass was homodulized in TRIzol (Invitrogen) to obtain total RNA. Extracted. In addition, OSRC2 cells cultured for 24 hours at 37 ° C. in a 5% CO ₂ atmosphere using a reagent-containing PRMI medium are prepared, and the PEG / pl-encapsulated YSK / A lipid membrane structure is 100 nmol / L (siRNA concentration). The cells were added to the medium and incubated at 37 ° C. for 24 hours as positive control cells (PC cells), and the cells incubated in the same manner without adding the lipid membrane structure were used as negative control cells (NC cells). Total RNA was also extracted from PC cells and NC cells using TRIzol (Invitrogen). 5 μg of total RNA was heated at 65 ° C. for 5 minutes and then placed at 4 ° C. Subsequently, T4 RNA ligase (TaKaRa) and 1.2 μg of RNA adapter (5′-NH2-CGACUGGAGCACGAGAGACACUGACAUGGACUGAAGGAGAGAAAAA-3 ′; SEQ ID NO: 11) were added, and a ligation reaction was performed by incubating at 37 ° C. for 1 hour. It was. Next, RNA was extracted according to a conventional method using phenol / chloroform / isoamyl alcohol, ethanol-precipitated, and then dissolved in 10 μL of water to obtain an RNA aqueous solution. Using this RNA aqueous solution as a template, cDNA was obtained by performing a reverse transcription reaction using SuperScript III (Invitrogen) and a PLK1 gene-specific primer (5′-GGACAAGGCTGTAGAACCCCACAC-3 ′; SEQ ID NO: 12). The conditions for the reverse transcription reaction were 4 ° C after heat denaturation at 65 ° C for 5 minutes, 50 ° C for 1 hour and 70 ° C for 15 minutes. Subsequently, 5 ′ RACE PCR (touch-down PCR and nested PCR) was performed using cDNA as a template under the following conditions to obtain a 5 ′ RACE PCR product. The 5′RACE PCR product was electrophoresed on a 2% TBE agarose gel, stained with 1 ug / mL ethidium bromide and observed. The result is shown in FIG.

Condition template for touch-down PCR; cDNA
Forward primer 1 (f1); (corresponding to the 5 ′ region of the RNA adapter) 5′-CGACTGGAGCACGAGGACACTGA-3 ′ (SEQ ID NO: 13)
Reverse primer 1 (r1); (corresponding to the 3 ′ end region of PLK1 gene messenger RNA) 5′-GCTTGTCCACCATATGGCGG-3 ′ (SEQ ID NO: 14)
PCR device; 2720 Thermal Cycler (Applied Biosystems)
Reaction conditions: 94 ° C. for 2 minutes, followed by 5 cycles of 94 ° C. for 30 seconds and 72 ° C. for 40 seconds as one cycle, followed by 5 cycles of 94 ° C. for 30 seconds and 70 ° C. for 40 seconds as one cycle, Subsequently, 25 cycles of 94 ° C. for 30 seconds, 66 ° C. for 30 seconds and 72 ° C. for 40 seconds, followed by 72 ° C. for 10 minutes.

Nested PCR condition template; touch-down PCR PCR product forward primer; (corresponds to 3 ′ region of RNA adapter (3 ′ region of f1)) 5′-GGACACTGACATGGACTGAAGGAGTA-3 ′ (SEQ ID NO: 15)
Reverse primer; (corresponding to 5 ′ region of PLK1 gene messenger RNA extended by r1) 5′-TCCTTGCAGCAGCCGTACTC-3 ′ (SEQ ID NO: 16)
PCR device; 2720 Thermal Cycler (Applied Biosystems)
Reaction conditions: 94 ° C. for 2 minutes, 94 ° C. for 30 seconds, 64 ° C. for 30 seconds and 72 ° C. for 40 seconds for 20 cycles, then 72 ° C. for 10 minutes.

  As shown in FIG. 13, in the PC cell, a band indicating DNA amplified specifically for the RNA fragment resulting from cleavage of the PLK1 gene mRNA was clearly confirmed. And a band corresponding to this was not seen in the mice administered with the control and PEG / luc-encapsulated YSK / A lipid membrane structure, whereas the mice administered with PEG / pl-encapsulated YSK / A lipid membrane structure It was clearly seen. That is, it was revealed that PLK1 gene messenger RNA was cleaved in the tumor tissue of mice administered with PEG / pl-encapsulated YSK / A lipid membrane structure. From this result, it is possible to cause RNA interference to the PLK1 gene messenger RNA by administration of a lipid membrane structure including a lipid membrane containing anti-plk1 and containing PEG-DSG as a constituent lipid in addition to YSK05. confirmed.

<Example 8> Production of lipid membrane structure of preferred lipid composition B (1) Examination of lipid types [1-1] Production of lipid membrane structure siRNA against SRBI gene messenger RNA instead of anti-luc (anti- sr; SEQ ID NOS: 17 and 18) were replaced with 1 mmol / L citrate buffer (pH 4.5) and 20 mmol / L citrate buffer (pH 4.0). By performing the method described in Example 2 (1) with compositions 17 to 19, lipid membrane structures having a lipid membrane containing YSK05 as a constituent lipid and containing anti-sr were obtained. Composition 17 was set with reference to a previous report (Sample SC, Nat. Biotechnol., Vol. 28, No. 2, 2010).

Composition 17; YSK05: DSPC: Chol: PEG-DMG = 57.1: 7.1: 34.3: 7.5
Composition 18; YSK05: dipalmitoyyl phosphatidyl Choline (DPPC): Chol: PEG-DMG = 57.1: 7.1: 34.3: 7.5
Composition 19; YSK05: Chol: PEG-DMG = 60: 40: 7.5

[1-2] Suppression of SRBI gene expression The lipid membrane structure of Example 8 (1) was administered to the tail vein of 5-6 week old male ICR mice anesthetized with diethyl ether or Nembutal. The dose was 10 to 12.5 mL / kg (1.5 mg / kg; siRNA amount). A mouse administered with the same amount of PBS was used as a control. After 24 hours from the administration, the mice were anesthetized with diethyl ether or Nembutal, then laparotomized and the liver was collected. The collected liver was subjected to quantitative real-time PCR by the method described in Example 6 (3) [3-3] to determine the relative SRBI expression level. However, the PCR primers for quantitative real-time PCR used the following sequences, and the messenger RNA expression level of the mouse GAPDH gene was used as an internal standard. The result is shown in FIG.

Primer forward of mouse SRBI gene; 5′-AATAAAGGCTTGGAGAACCC-3 ′ (SEQ ID NO: 19)
Reverse; 5′-ACCTCACCCTGTCTTCGAGA-3 ′ (SEQ ID NO: 20)
Primer forward of mouse GAPDH gene; 5′-AGCAAGGACACTGAGCAAG-3 ′ (SEQ ID NO: 21)
Reverse; 5'-TAGGCCCCTCCCTGTATTTATG-3 '(SEQ ID NO: 22)

  As shown in FIG. 14, the relative SRBI expression level was remarkably small as compared with the control when the lipid membrane structure having any lipid membrane of composition 17 to 19 was administered. That is, it was revealed that the expression of the SRBI gene in the liver was suppressed by administration in blood of a lipid membrane structure having a lipid membrane containing YSK05 as a constituent lipid and containing anti-sr. Moreover, the relative SRBI expression level when a lipid membrane structure having a lipid membrane of composition 19 is administered is smaller than that when a lipid membrane structure having a lipid membrane of composition 17 is administered, and the lipid membrane structure of composition 18 is provided. Compared with the case where the lipid membrane structure was administered. That is, even if DSPC or DPPC is not included as a constituent lipid in the lipid membrane, it has been clarified that the expression of SRBI gene can be suppressed to the same level or higher than those including those lipids.

(2) Examination of ratio of lipid The composition of the lipid solution was set to the following composition 20 to 25, and a lipid membrane structure was obtained by performing the method described in Example 8 (1) [1-1]. Subsequently, the relative SRBI expression level of the lipid membrane structure having a lipid membrane having a composition of 20 to 25 was determined by the method described in Example 8 (1) [1-2]. However, the dose was 0.2, 0.2, 1.0, or 2.0 mg / Kg (siRNA amount). The result is shown in FIG. As shown in FIG. 15, a lipid membrane structure having a lipid membrane of composition 23 at doses of 0.2 and 0.4 mg / kg, compositions 23 and 24 at doses of 1.0 mg / kg, and composition 22 at doses of 2.0 mg / kg. When the body was administered, the relative SRBI expression level was remarkably small. From these results, the molar ratio of YSK05 to Chol in the lipid membrane of the lipid membrane structure having a lipid membrane containing YSK05 as a constituent lipid encapsulating anti-sr is 75/35 ≦ YSK05 / Chol ≦ 75 / 25, that is, 13/7 ≦ YSK05 / Chol ≦ 3, it was revealed that the SRBI gene expression suppression effect is large.

Composition 20; YSK05: Chol: PEG-DMG = 55: 45: 3
Composition 21; YSK05: Chol: PEG-DMG = 60: 40: 3
Composition 22; YSK05: Chol: PEG-DMG = 65: 35: 3
Composition 23; YSK05: Chol: PEG-DMG = 70: 30: 3
Composition 24; YSK05: Chol: PEG-DMG = 75: 25: 3
Composition 25; YSK05: Chol: PEG-DMG = 80: 20: 3

  From the results of the above Example 8 (1) and (2), the preferred composition of the constituent lipid in the lipid membrane of the lipid membrane structure having a lipid membrane containing YSK05 as the constituent lipid is 13/7 ≦ YSK05 / It was revealed that Chol ≦ 3 (molar ratio; hereinafter referred to as “preferred lipid composition B”).

(3) Examination of ratio of PEG-DMG As the composition of the lipid solution, the composition 17 of this Example 8 (1) [1-1] and the following compositions 26 and 27 were used, and this Example 8 (1) [1-1] ] Was performed to obtain a lipid membrane structure. Subsequently, the relative SRBI expression level of the lipid membrane structure having lipid membranes of compositions 17, 26 and 27 was determined by the method described in Example 8 (1) [1-2]. However, the dose was 1.5 mg / Kg (siRNA amount). The result is shown in FIG. As shown in FIG. 16, the relative SRBI expression level was small when a lipid membrane structure having any lipid membrane of composition 17, 26 and 27 was administered. That is, the molar ratio of PEG-DMG in the lipid membrane of the lipid membrane structure having a lipid membrane containing YSK05 as a constituent lipid encapsulating anti-sr is 10 mol% or less with respect to the total lipid other than PEG-DMG. , It was found that the SRBI gene expression suppression effect is large. From these results, the ratio of PEG-DMG in the lipid membrane of the lipid membrane structure having a lipid membrane containing YSK05 as a constituent lipid is preferably 10 mol% or less with respect to the total lipid other than PEG-DMG. Became clear.

Composition 17; YSK05: DSPC: Chol: PEG-DMG = 57.1: 7.1: 34.3: 7.5
Composition 26; YSK05: DSPC: Chol: PEG-DMG = 57.1: 7.1: 34.3: 3
Composition 27; YSK05: DSPC: Chol: PEG-DMG = 57.1: 7.1: 34.3: 10

<Example 9> Function of lipid membrane structure of preferred lipid composition B (1) Production of lipid membrane structure and confirmation of physical properties, siRNA inclusion rate and pKa Composition of lipid solution is composition 28; YSK05: DSPC: Chol: PEG -DMG = 50: 10: 40: 3 and composition 23 of Example 8 (2), a lipid membrane structure was obtained by carrying out the method described in Example 8 (1) [1-1]. The lipid membrane structures obtained from the lipid solutions having compositions 28 and 23 were used as the sr-encapsulated YSK / 28 lipid membrane structure and the sr-encapsulated YSK / B lipid membrane structure, respectively. For the sr-encapsulated YSK / B lipid membrane structure, the mean particle size, PDI and zeta potential were determined by the method described in Example 2 (3), and the siRNA encapsulation rate was determined by the method described in Example 2 (4). When measured by the method described in Example 3 (1), each value approximated the value in the luc-encapsulated YSK lipid membrane structure of Example 2 (1) (Table 1, FIG. 2) ( Results are not shown).

(2) Membrane fusion ability The lipid composition in the lipid solution is the composition 28 of Example 9 (1) and the composition 23 of Example 8 (2), and instead of 1 mmol / L citrate buffer (pH 4.5). A lipid membrane structure was obtained by performing the method described in Example 2 (2) using 20 mmol / L citrate buffer (pH 4.0). Membrane fusion was carried out by the method described in Example 3 (2), using lipid membrane structures obtained from lipid solutions of compositions 28 and 23 as empty YSK / 28 lipid membrane structures and empty YSK / B lipid membrane structures, respectively. The rate was determined. The result is shown in FIG. As shown in FIG. 17, the empty YSK / B lipid membrane structure is better than the empty YSK / 28 lipid membrane structure when the pH is 7.4, 6.5, 6.0, or 5.5. The membrane fusion rate was also large. From this result, it became clear that the membrane fusion ability is improved by setting the lipid composition in the lipid membrane of the lipid membrane structure having a lipid membrane containing YSK05 as a constituent lipid to the preferred lipid composition B.

(3) Suppression of SRBI gene expression The sr-encapsulated YSK / B lipid membrane structure and the sr-encapsulated YSK / 28 lipid membrane structure of Example 9 (1) are described in Example 8 (1) [1-2]. The relative SRBI expression level was determined by the above method. However, various doses were used in the range of 0.05 to 4 mg / kg (siRNA amount). The result is shown in FIG. As shown in FIG. 18, the siRNA dosage (ED50 / SRBI) at which the relative SRBI expression level is 0.5 was approximately 1-2 mg / kg in the sr-encapsulated YSK / 28 lipid membrane structure. The sr-encapsulated YSK / B lipid membrane structure was approximately 0.1 mg / kg. That is, it was revealed that the sr-encapsulated YSK / B lipid membrane structure can efficiently suppress the expression of the SRBI gene with a small amount of siRNA compared to the sr-encapsulated YSK / 28 lipid membrane structure.

(4) Inhibition of expression of factor 7 gene The composition of the lipid solution was changed to that of Example 9 (1) using siRNA (anti-f7; SEQ ID NOs: 23 and 24) against factor 7 gene messenger RNA instead of anti-sr. ) And composition 23 of Example 8 (2), and the method described in Example 8 (1) [1-1] was performed to obtain a lipid membrane structure. The lipid membrane structures obtained from the lipid solutions having compositions 28 and 23 were designated as f7-encapsulated YSK / 28 lipid membrane structure and f7-encapsulated YSK / B lipid membrane structure, respectively. Subsequently, each lipid membrane structure was administered to the tail vein of 5-6 week old male ICR mice anesthetized with diethyl ether or Nembutal. The dose was 20 to 25 mL / kg (0.03 to 1 mg / kg; siRNA amount). In addition, a mouse administered with the same amount of PBS containing no lipid membrane structure was used as a control. 48 hours after the administration, blood was collected from mice according to a conventional method to obtain plasma. The amount of factor 7 in the plasma was measured using an olorimetric Biophen VII assay kit (Aniara) according to the attached instructions. For the measurement results, a calibration curve was created with the measured values of the control, and expressed as a percentage with the measured value of the control being 100%, and this was defined as the seventh factor ratio (%). The result is shown in FIG.

  As shown in FIG. 19, the ratio of the factor 7 decreased depending on the dose when either the f7-encapsulated YSK / 28 lipid membrane structure or the f7-encapsulated YSK / B lipid membrane structure was administered. That is, it was revealed that the expression of the factor 7 gene in plasma was suppressed by administration in the blood of a lipid membrane structure having a lipid membrane containing YSK05 as a constituent lipid encapsulating anti-f7.

  In addition, at any dose of 0.1, 0.2 and 1.0 mg / kg, the case where the f7-encapsulated YSK / B lipid membrane structure is administered is more than the f7-encapsulated YSK / 28 lipid membrane structure. The siRNA dose (ED50 / F7) at which the factor 7 ratio was significantly smaller and the factor 7 ratio was 50% was 0.8 mg / kg for the f7-encapsulated YSK / 28 lipid membrane structure. In the f7-encapsulated YSK / B lipid membrane structure, the amount was 0.06 mg / kg. That is, it was revealed that the f7-encapsulated YSK / B lipid membrane structure can efficiently suppress the expression of the factor 7 gene with a small amount of siRNA as compared with the f7-encapsulated YSK / 28 lipid membrane structure.

  From the results of the above Examples 9 (3) and (4), a lipid membrane containing YSK05 as a constituent lipid encapsulating RNA having the same or complementary base sequence as all or part of the messenger RNA of the target gene It has been clarified that the effect of suppressing the expression of the target gene is increased by setting the lipid composition of the lipid membrane structure having a suitable lipid composition B.

<Example 10> Duration of gene expression inhibitory effect For the f7-encapsulated YSK / B lipid membrane structure of Example 9 (4), the method described in Example 9 (4) was performed to determine the factor 7 ratio. . However, the dose was 1.0 mg / kg (siRNA amount), and serum was collected immediately after administration and 1, 3, 5, 7, 10 and 14 days after administration. The result is shown in FIG. As shown in FIG. 20, the factor 7 ratio was 50% or less in any of 1 to 14 days after administration. From this result, a lipid membrane structure having a lipid membrane containing YSK05 as a constituent lipid and containing RNA having the same or complementary base sequence as all or part of the messenger RNA of the target gene is 14 days after administration. It was shown that the expression of the target gene can be suppressed over time.

<Example 11> Function of a lipid membrane structure containing PEG-DSG as a constituent lipid and having a lipid membrane having a preferred lipid composition B (1) Addition of PEG-DSG The composition of a lipid solution is the composition of Example 8 (2) 23, and ³ H-cholesteryl hexadecylethyl (CHE), which is a radioisotope, was added to the lipid solution so as to be approximately 4 ng / mL, and the method described in Example 8 (1) [1-1] was performed. As a result, a lipid membrane structure in which the lipid membrane was labeled with a radioisotope was obtained. Subsequently, the method described in Example 7 (1) was performed with the addition amount of PEG-DSG being 0, 1, 2, and 3 mol% based on the total lipid other than PEG-DSG, and PEG-DSG was used as the constituent lipid. In addition, a lipid membrane structure having a lipid membrane labeled with a radioisotope was obtained.

(2) Retention in blood The lipid membrane structure of Example 11 (1) was administered from the tail vein of male ICR mice (5-6 weeks old) anesthetized with diethyl ether or Nembutal. After 10 minutes and 2 hours from the administration, blood was collected according to a conventional method to obtain plasma. The radiation dose in plasma was measured using a liquid scintillation counter, and the radiation concentration per mL of plasma was expressed as a percentage (% ID) relative to the total dose administered (injected dose) (% ID / mL). The result is shown in FIG.

  As shown in FIG. 21, when the radiation concentrations at 10 minutes and 2 hours after administration were compared, the larger the amount of PEG-DSG added, the smaller the difference in radiation concentration between 10 minutes and 2 hours later. . That is, it has been clarified that as the amount of PEG-DSG added to the lipid membrane increases, the amount of the lipid membrane structure in plasma that decreases with time is small. From these results, it was shown that the retention in blood is improved by adding PEG-DSG to the lipid membrane of a lipid membrane structure having a lipid membrane containing YSK05 as a constituent lipid.

(3) Inhibition of PLK1 gene expression Using anti-plk1 and anti-luc instead of anti-sr, the composition of the lipid solution was set to composition 23 of Example 8 (2), and Example 8 (1) [1- 1] to obtain a lipid membrane structure encapsulating anti-plk1 and anti-luc, respectively, and pl-encapsulating YSK / B lipid membrane structure and luc-encapsulating YSK / B lipid membrane structure, did. Subsequently, the method described in Example 7 (1) was performed, and the lipid membrane structure obtained by adding PEG-DSG to the lipid membrane was converted into a PEG / pl-encapsulated YSK / B lipid membrane structure and PEG / luc. An encapsulated YSK / B lipid membrane structure was obtained. Next, this was administered to tumor model mice by the method described in Example 7 (3) [3-2]. However, in addition to OSRC2 cells, human liver cancer-derived Huh7 cells and human liver cancer-derived HepG2 cells are used as tumor cell types used to create tumor model mice, and the administration method is intravenous administration instead of direct administration to tumor tissue, The dosage of the lipid membrane structure containing anti-plk1 is 0.3, 0.75, 1.5, 3, 4 mg / kg (siRNA amount), and the dosage of the lipid membrane structure containing anti-luc is The concentration was 3 mg / kg (siRNA amount). Then, relative PLK1 expression level was calculated | required by the method as described in Example 7 (3) [3-3]. The result is shown in FIG.

  As shown in FIG. 22, the relative PLK1 expression level in the tumor tissue of Huh7 cells was almost equivalent to the control when PEG / luc-encapsulated YSK / B lipid membrane structure was administered. In contrast, when the PEG / pl-encapsulating YSK / B lipid membrane structure was administered, the relative PLK1 expression level in any tumor tissue of OSRC2 cells, Huh7 cells, and HepG2 cells was the same as that of the control. It was remarkably small compared. From this result, the expression of the PLK1 gene in tumor tissue was suppressed by administering a lipid membrane structure containing a lipid membrane containing PEG-DSG as a constituent lipid in addition to YSK05 encapsulating anti-plk1 in blood. It was shown that.

Claims (8)

  1-methyl-4,4-bis [(9Z, 12Z) -octadeca-9,12-dien-1-yloxy] An agent that imparts pH-dependent cationicity to a lipid membrane structure containing piperidine as an active ingredient.   1-methyl-4,4-bis [(9Z, 12Z) -octadeca-9,12-dien-1-yloxy] piperidine, a lipid membrane containing lipidine as a constituent lipid, 1-methyl-4,4-bis [( [9Z, 12Z) -lipid membrane structure to which pH-dependent cationic property is imparted by octadeca-9,12-dien-1-yloxy] piperidine. The lipid membrane structure according to claim 2, which has the following lipid membrane (A) or (B);
(A) (a) 1-methyl-4,4-bis [(9Z, 12Z) -octadeca-9,12-dien-1-yloxy] piperidine, (b) cholesterol and (c) 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphoethanolamine (POPE) at a molar ratio of (a) / (b) + (c) ≧ 2/3 and 3/7 ≦ (b) / (c) ≦ 7/3 A lipid membrane containing as a constituent lipid,
(B) (a) 1-methyl-4,4-bis [(9Z, 12Z) -octadeca-9,12-dien-1-yloxy] piperidine and (b) cholesterol in a molar ratio of 13/7 ≦ (a ) / (B) A lipid membrane containing as constituent lipids at a ratio of ≦ 3.   The lipid membrane structure according to claim 2 or 3, which has a lipid membrane containing a lipid bonded to polyethylene glycol as a constituent lipid.   5. The lipid conjugated with polyethylene glycol is 1,2-dimethyl-sn-glycerol (DMG) conjugated with polyethylene glycol and / or 1,2-disteayl-sn-glycerol (DSG) conjugated with polyethylene glycol. The lipid membrane structure described in 1.   6. The lipid membrane according to any one of claims 2 to 5, which is a target gene expression inhibitor and contains RNA having the same or complementary base sequence as all or part of the messenger RNA of the target gene. The said agent which uses a structure as an active ingredient. A method for producing a lipid membrane structure, comprising a step of preparing a lipid membrane of the following (I) or (II):
(I) (a) 1-methyl-4,4-bis [(9Z, 12Z) -octadeca-9,12-dien-1-yloxy] piperidine, (b) cholesterol, (c) 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and (d) 1,2-dimyristol-sn-glycerol (DMG) conjugated with polyethylene glycol in a molar ratio of (a) / (b) + (c) ≧ 2 / 3, 3/7 ≦ (b) / (c) ≦ 7/3 and 0 <(d) / (a) + (b) + (c) ≦ 1/10 as a constituent lipid lipid membrane,
(II) (a) 1-methyl-4,4-bis [(9Z, 12Z) -octadeca-9,12-dien-1-yloxy] piperidine, (b) cholesterol and (d) polyethyleneglycol bound 1 , 2-dimyristol-sn-glycerol (DMG) in a molar ratio of 13/7 ≦ (a) / (b) ≦ 3 and 0 <(d) / (a) + (b) ≦ 1/10 Lipid membrane containing as lipid.   The method for producing a lipid membrane structure according to claim 7, further comprising a step of adding 1,2-distearoyl-sn-glycerol (DSG) bound to polyethylene glycol to the prepared lipid membrane.