KR20110112305A - Vesicle preparation - Google Patents

Abstract

The serum component transports vitamin E to hepatic parenchymal cells, so that vitamin E is adsorbed or chemically bonded to the surface of the endoplasmic reticulum, thereby efficiently and specifically delivering the bioactive substances contained in the endoplasmic reticulum to the parenchymal cells. Made it clear.

Description

Vesicle preparation

The present invention relates to a endoplasmic reticulum and its use for efficiently introducing a bioactive substance into hepatic parenchymal cells and to effectively function the bioactive substance in hepatic parenchymal cells.

The present invention is directed to antiviral agents, chemotherapeutic agents, peptides, nucleotides, oligonucleotides, antisense nucleic acids, aptamers, decoys and their flexibles, micronucleic acid molecules, for example siRNAs. The present invention relates to a endoplasmic reticulum for efficiently introducing bioactive substances such as, miRNA, dsRNA, or shRNA into hepatic parenchymal cells, and to effectively effect the bioactive substances in hepatic parenchymal cells.

Examples are known in which hepatic parenchymal cells are introduced with nucleic acids such as genes and bioactive substances such as antitumor agents to attempt gene expression, gene control, and tumor reduction.

In the case of the introduction of nucleic acid-related, a paper such as Scherr et al. (Non-Patent Document 1) and an open patent (Patent Document 1), an example of a hydrodynamic injection introduced into a cell by applying water pressure as an example of using a viral vector, such as Song et al. (Non-Patent Document 2) and Open Patent (Patent Document 2). However, introduction methods using viral vectors are problematic for safety in humans and are not suitable for widely general therapeutic purposes. In addition, although the introduction method by applying water pressure can be introduced into cells in vitro, it is not suitable for use in humans for therapeutic purposes.

In addition, a method of using receptor-mediated endocytosis that targets a sugar receptor on the surface of the liver is known as a method of transport to hepatic parenchymal cells. The asialo glycoprotein receptor (Non Patent Literature 3) specifically binds to a glycoprotein having a galactose end, such as an asialo-orthomucoid, specifically for hepatocytes. The liposome composition (patent document 3) containing the galactose modified cationic cholesterol using this principle is introduced. However, in the example of carrying siRNA, the effect is slightly improved compared to the case of not modifying galactose, but the gene expression is also seen in the lung, kidney, spleen and heart. It turns out that it is not made and is not a valid technique.

A composition in which lactose is bound to a phosphatidylethanolamine of phospholipid and blended into a cationic liposome (Non Patent Literature 4), or a transporter targeting a sugar receptor in liver in the same way as liposomes using a sugar-modified peptide (Patent Literature 4) Proposed.

However, in the case of transporting liposomes targeting the sugar receptor in the liver, there has been a problem that after insertion into endocytosis, the nucleic acid is degraded in the endosomes so that the effect of gene expression and gene control is hardly exerted.

On the other hand, a technique for avoiding the reticulo-endothelial system (RES), which is a defense system of the living body, improves blood retention, and as a result, a technique for improving the transport efficiency to hepatic parenchymal cells has been introduced (Patent Document 5). . Doxoilcin is a commercially available liposome formulation of the anticancer drug Doxorubicin, which is called a technique of chemically bonding a water-soluble polymer (methoxypolyethylene glycol: mPEG) to the surface of a liposome and avoiding foreign material recognition by RES, or passive targeting. This is the same technology that is being used. In addition to the features that tend to accumulate in tumor tissues and neovascularized areas due to vascular permeability, they are also partially inserted into RES cells, and thus are not an efficient transport technology to hepatic parenchymal cells.

Vitamin E (tocopherol), a fat-soluble vitamin, is used in a form in which a small amount (generally about 0.2%) is added to the endoplasmic reticulum for the purpose of preventing oxidation of the unsaturated fatty acid in the lipid endoplasmic reticulum due to its antioxidant function (Patent Document 6). , 7).

In addition, vitamin E derived from a meal is absorbed from the small intestine and transported to the liver by the serum component chiromicron to the liver, and vitamin E other than the meal derived from HDL (high specific lipoprotein) or LDL (low specific gravity) in the blood. Nishina et al. Have reported an example of using α-tocopherol, which is a main component of vitamin E, with a siRNA to carry it to the liver by chemically bonding α-tocopherol, which is easy to be transported to lipoproteins) (Non-Patent Document 5). However, due to the difficulty in production on an industrial scale for combining siRNA and α-tocopherol 1: 1 and high purity as a prodrug, removal of unreacted raw materials and by-products is required. The low yields from these manufacturing and purification processes, as well as the release of siRNA by hydrolysis after soy sauce is introduced, and the economical problem that a large amount of expensive siRNA must be used to obtain a therapeutic effect. There was.

In addition, commercially available cationic liposomes for introduction of cells into cells are mixed with nucleic acids such as plasmids and siRNAs to form an emulsion, and a method of transporting them to hepatic satellite cells is known. (Patent Document 8) . Hepatic cells, like reticuloendothelial cells, are nonparenchymal cells that have high levels of vitamin A in their fat droplets. In the delivery method to non-parenchymal cells using vitamin A, when a large amount of vitamin A is present in the blood, it is easy to be transported to hepatic cells (non-parenchymal cells) by vitamin A-binding proteins in the blood to control particle size. There is no need to adjust the surface charge. However, in the case of delivering a physiologically active substance specifically in hepatic parenchymal cells, it is necessary to appropriately adjust the particle size and surface charge of the whole particle so that the particles are not trapped in the nonparenchymal cells. No optimized carrier has been developed.

Japanese Patent Publication No. 2007-110995 Japanese Patent Publication No. 2008-054681 Japanese Patent Publication No. 2007-112768 Japanese Patent Publication No. 1999-290073 Japanese Patent Publication No. 2004-196675 Japanese Patent Publication No. 2007-112768 Japanese Patent Publication No. 2008-530215 Japanese Patent Publication No. 2008-50375

 Scherr, M. et al., Oligonucleotides, 2003; 13: 353-363  Song, E. et al., Nature Medicine, 2003; 9: 347-351  Wu, GY and Wu, CH, J. Biol. Chem., 1987; 262: 4429-4432  Watanabe, T. et al., J. Hepatol., 2007; 47: 744-750  Nishina, K. et al., Molecular Therapy, 2008; 16 (4): 734-740

SUMMARY OF THE INVENTION The present invention has been made in view of the above situation, and an object thereof is to efficiently and specifically transport and introduce a bioactive substance into hepatic parenchymal cells, and to effectively operate the bioactive substance in hepatic parenchymal cells. It relates to a composition for.

The present inventors have diligently studied the transport technology to hepatic parenchymal cells. As a result, the serum components are adsorbed or chemically bound to the surface of the endoplasmic reticulum containing the bioactive substances, so that the serum components are bioactive. The present invention has been accomplished by finding that the endoplasmic reticulum containing the substance can be transported to the liver parenchymal cells very efficiently, and that the bioactive substance can exert the effect quickly.

It has been found that a very safe and efficient method of using vitamin E as a transporting factor of a bioactive substance, without the property of being easily transported to hepatic parenchymal cells in the blood, through the binding of the bioactive substance.

That is, this invention relates to the invention of the following [1]-[24].

[1] A endoplasmic reticulum comprising (i) and (ii), wherein vitamin E is adsorbed on the surface of the endoplasmic reticulum, or vitamin E is chemically bonded to an amphiphilic substance constituting the endoplasmic reticulum.

(i) amphiphilic substances

(ii) an organic material comprising a material having a cationicity and / or a material having anionicity

[2] The endoplasmic reticulum of [1], wherein the cationic substance is a cationic lipid.

[3] The endoplasmic reticulum of [2], wherein the cationic lipid is represented by the following general formula (I).

[Formula I]

(In formula, R <^1> and R <^2> means the same or different C12-C22 saturated or unsaturated hydrocarbon group, R <^3> means a C1-C6 alkyl group or a C1-C6 hydroxyalkyl group, m means an integer of 1 to 10, X means a halogen atom.)

[4] The cationic lipids are saturated or unsaturated aliphatic primary amines (stearylamine, oleylamine), saturated or unsaturated secondary, tertiary amines (distearoyltrimethylammonium propane, dioleoyltrimethylammonium propane ) And quaternary amine salts (DODAC (dioctadecyldimethylammonium chloride), DTDAB (ditetradecyldimethylammonium bromide), DOTMA (N- (2,3-dioleyloxy) propyl-N , N, N-trimethylammonium: N- (2,3-dioleyloxy) propyl-N, N, N-trimethylammonium), DDAB (didodecylammonium bromide), didodecylammonium bromide), DTAB (ditetradecylammonium bromide) , DOTAP (1,2-dioleoyloxy-3-trimethylammonio propane: 1,2-dioleoyloxy-3-trimethylammonio propane), O, O'-ditetradecaneoil-N- (α-trimethylammonio- Acetyl) -diethanolamine chloride: O, O'-ditetradecanoyl-N- ( -trimethylammonio-acetyl) -diethanolamine chloride, O, O'-dihexadecanyl-N- (α-trimethylammonio-acetyl) -diethanolamine chloride: O, O'-dihexadecanoyl-N- (α-trimethylammonio- acetyl) -diethanolamine chloride, O, O'-dioctadecanyl-N- (α-trimethylammonio-acetyl) -diethanolamine chloride: O, O'-dioctadecanoyl-N- (α-trimethylammonio-acetyl)- diethanolamine chloride, DC-Chol (3β-N- (N ', N'-dimethylaminoethane) carbamol cholesterol hydrochloride: 3β-N- (N', N'-dimethyl-aminoethane) -carbamol cholesterol hydrochloride), DMRIE (1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium: 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethyl ammonium), DOSPA (2,3-dioleyloxy-N- [2 (sper) Uncarboxamido) ethyl] -N, N-dimethyl-1-propanaminum trifluoroacetate: 2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminum trifluoroacetate)) The endoplasmic reticulum of [2], characterized in that it contains at least one or more types selected from these.

[5] The amphipathic substances include phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, methoxypolyethylene glycol (mPEG) addition phosphatidylethanolamine, phosphatidic acid, sphingomyelin, and cardiolipin. And at least one or more phospholipids selected from the group consisting of plasmaogens.

[6] The phosphatidylcholine, yolk phosphatidylcholine, hydrogenated yolk phosphatidylcholine, soybean phosphatidylcholine, hydrogenated soybean phosphatidylcholine, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dispalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine , Palmitoyl-oleoyl phosphatidylcholine, dilinoleyl phosphatidylcholine, dilinolenoyl phosphatidylcholine, didecanoyl phosphatidylcholine, diethylcoyl phosphatidylcholine, dieticosanoyl phosphatidylcholine, dieticosatrienoyl phosphatidylcholine, dietoisyl phosphatidylcholine Cosaenoyl phosphatidylcholine and didocasahexaenoyl phosphatidylcholine,

The phosphatidyl ethanolamine is egg yolk phosphatidyl ethanolamine, hydrogenated egg yolk phosphatidyl ethanolamine, soybean phosphatidyl ethanol amine, hydrogenated soybean phosphatidyl ethanol amine, dilauroyl phosphatidyl ethanol amine, dimyristoyl phosphatidyl ethanol amine, dipalmitoyl phosphatidyl ethanol amine Ethanolamine, distearoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, palmitoyl-oleoyl phosphatidylethanolamine, dilinoleoyl phosphatidylethanolamine, dilinolenoyl phosphatidylethanolamine, didecanoyl phosphatidylethanolamine, Diethylcoyl phosphatidylethanolamine, diethasanoyl phosphatidylethanolamine, diethasatrienoyl phosphatidylethanolamine, dieticosapentaenoyl phosphatidylethanolamine, dietocosaenoyl phosphatidylethanolamine and dietocosahexaenoyl phosphine Selected from the group consisting of patylethanolamine,

The phosphatidylglycerol, egg yolk phosphatidylglycerol, hydrogenated egg yolk phosphatidylglycerol, soybean phosphatidylglycerol, hydrogenated soybean phosphatidylglycerol, dilauroyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidyl Glycerol, Dioleoyl Phosphatidylglycerol, Palmitoyl-Oleoyl Phosphatidylglycerol, Dilinoleoyl Phosphatidylglycerol, Dilinolenoyl Phosphatidylglycerol, Didecanoyl Phosphatidylglycerol, Dieticoyl Phosphatidylglycerol, Dieticosanyl Phosphatidylglycerol Selected from the group consisting of trienoyl phosphatidylglycerol, diecosapentaenoyl phosphatidylglycerol, didicosaenoyl phosphatidylglycerol and didocasahexaenoyl phosphatidylglycerol Selected,

The phosphatidylserine, egg yolk phosphatidylserine, hydrogenated egg yolk phosphatidylserine, soybean phosphatidylserine, hydrogenated soybean phosphatidylserine, dilauroyl phosphatidylserine, dimyristoyl phosphatidylserine, dispalmitoyl phosphatidylserine, distearoyl phosphatidyl Serine, dioleoyl phosphatidylserine, palmitoyl-oleoyl phosphatidylserine, dirinoleoyl phosphatidylserine, dilinolenoyl phosphatidylserine, diedecanoyl phosphatidylserine, dieticoyl phosphatidylserine, dieticosanyl phosphatidylserine Trienoyl phosphatidylserine, diecosapentaenoyl phosphatidylserine, didicosaenoyl phosphatidylserine and didicosahexaenoyl phosphatidylserine,

The phosphatidyl inositol, egg yolk phosphatidyl inositol, hydrogenated egg yolk phosphatidyl inositol, soybean phosphatidyl inositol, hydrogenated soybean phosphatidyl inositol, dilauroyl phosphatidyl inositol, dimyristoyl phosphatidyl inositol, dipalmitoyl phosphatidyl inositol, disteamiloyl phosphatidylinositol Inositol, dioleoyl phosphatidyl inositol, palmitoyl-oleoyl phosphatidyl inositol, diolinoleyl phosphatidyl inositol, dilinolenoyl phosphatidyl inositol, diedecanoyl phosphatidyl inositol, diethylcoyl phosphatidyl inositol, dietycosinoyl Selected from the group consisting of trienoyl phosphatidyl inositol, diecosapentaenoyl phosphatidyl inositol, didicosaenoyl phosphatidyl inositol and didicosahexaenoyl phosphatidyl inositol Selected,

The said methoxy polyethylene glycol (mPEG) addition phosphatidyl ethanolamine is mPEG2000- egg yolk phosphatidyl ethanolamine, mPEG3400- egg yolk phosphatidyl ethanolamine, mPEG5000- egg yolk phosphatidyl ethanolamine, mPEG2000- hydrogenated egg yolk phosphatidyl ethanolamine, mPEG3400- hydrogenated egg yolk Ethanolamine, mPEG5000-hydrogenated egg yolk phosphatidylethanolamine, mPEG2000- soybean phosphatidylethanolamine, mPEG3400- soybean phosphatidylethanolamine, mPEG5000- soybean phosphatidylethanolamine, mPEG2000- hydrogenated soybean phosphatidylethanolamine, mPEG3400- hydrogenated soybeans , mPEG5000-hydrogenated soybean phosphatidylethanolamine, mPEG2000-dilauroyl phosphatidylethanolamine, mPEG3400-dilauroyl phosphatidylethanolamine, mPEG5000-dilauroyl phosphatidylethanolamine, mPEG2000-dimyristoyl phosphatidylethanolamine, mPEG3400-Dimitristo Phosphatidylethanolamine, mPEG5000-dimyristoyl phosphatidylethanolamine, mPEG2000-dipalmitoyl phosphatidylethanolamine, mPEG3400-dipalmitoyl phosphatidylethanolamine, mPEG5000-dipalmitoyl phosphatidylethanolamine, mPEG2000-distearoyl phosphatidylethanamine , mPEG3400-distearoyl phosphatidylethanolamine, mPEG5000-distearoyl phosphatidylethanolamine, mPEG2000-dioleoyl phosphatidylethanolamine, mPEG3400-dioleoyl phosphatidylethanolamine and mPEG5000-dioleoyl phosphatidylethanolamine Is selected from

The phosphatidic acid, egg yolk phosphatidic acid, hydrogenated yolk phosphatidic acid, soybean phosphatidic acid, hydrogenated soybean phosphatidic acid, dilauroyl phosphatidic acid, dimyristoyl phosphatidic acid, dipalmitoyl force Fatty Acid, Distearoyl Phosphate Acid, Dioleoyl Phosphate Acid, Dilinoleyl Oil Phosphate Acid, Palmitoyl-Oleoyl Phosphate Acid, Dilinolenoyl Phosphate Acid, Didecanoyl Phosphate Acid, Consisting of dietycoyl phosphatidic acid, diecosanoyl phosphatidic acid, dieticosatrienoyl phosphatidic acid, diecosapentaenoyl phosphatidic acid, didicosaenoyl phosphatidic acid and didicosahexaenoyl phosphatidic acid Selected from the group,

The sphingomyelin is egg yolk-derived sphingomyelin or milk-derived sphingomyelin,

The cardiolipin, egg yolk cardiolipin, hydrogenated egg yolk cardiolipin, soy cardiolipin, hydrogenated soy cardiolipin, dilauroyl cardiolipin, dimyristoyl cardiolipin, dipalmitoyl car Diolipine, Distearoyl Cardiolipin, Dioleoyl Cardiolipin, Palmitoyl-Oleoyl Cardiolipin, Dilinoleyl Oil Cardiolipin, Dilinolenoyl Cardiolipin, Didecanoyl Cardiolipin, Consisting of dietycoyl cardiolipin, diecosanoyl cardiolipin, diecosartenoyl cardiolipin, diecosapentaenoyl cardiolipin, didicosaenoyl cardiolipin, and didicosahexaenoyl cardiolipin The endoplasmic reticulum of [5], characterized by being selected from the group.

[7] The vesicle according to [1], wherein the substance having cationicity is a cationic polymer.

[8] The vesicle according to [7], wherein the cationic polymer is polyethyleneimine or polylysine.

[9] The vesicle according to [1], wherein the anionic substance is an acidic phospholipid.

[10] The acidic phospholipid comprises at least one or more selected from the group consisting of phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, methoxy polyethylene glycol addition phosphatidylethanolamine and phosphatidic acid. Endoplasmic reticulum as described in.

[11] The phosphatidylglycerol, egg yolk phosphatidylglycerol, hydrogenated egg yolk phosphatidylglycerol, soybean phosphatidylglycerol, hydrogenated soybean phosphatidylglycerol, dilauroyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distea Royl phosphatidylglycerol, dioleoyl phosphatidylglycerol, palmitoyl-oleoyl phosphatidylglycerol, dirinoleoyl phosphatidylglycerol, dilinolenoyl phosphatidylglycerol, diedecanoyl phosphatidylglycerol, diethyl yl phosphatidylglycerol , Diecosatrienoyl phosphatidylglycerol, diecosapentaenoyl phosphatidylglycerol, didicosaenoyl phosphatidylglycerol and didocasahexaenoyl phosphatidylglycerol Selected and,

The phosphatidylserine, egg yolk phosphatidylserine, hydrogenated egg yolk phosphatidylserine, soybean phosphatidylserine, hydrogenated soybean phosphatidylserine, dilauroyl phosphatidylserine, dimyristoyl phosphatidylserine, dispalmitoyl phosphatidylserine, distearoyl phosphatidyl Serine, dioleoyl phosphatidylserine, palmitoyl-oleoyl phosphatidylserine, dirinoleoyl phosphatidylserine, dilinolenoyl phosphatidylserine, diedecanoyl phosphatidylserine, dieticoyl phosphatidylserine, dieticosanyl phosphatidylserine Trienoyl phosphatidylserine, diecosapentaenoyl phosphatidylserine, didicosaenoyl phosphatidylserine, didicosahexaenoyl phosphatidylserine,

The phosphatidyl inositol, egg yolk phosphatidyl inositol, hydrogenated egg yolk phosphatidyl inositol, soybean phosphatidyl inositol, hydrogenated soybean phosphatidyl inositol, dilauroyl phosphatidyl inositol, dimyristoyl phosphatidyl inositol, dipalmitoyl phosphatidyl inositol, disteamiloyl phosphatidylinositol Inositol, dioleoyl phosphatidyl inositol, palmitoyl-oleoyl phosphatidyl inositol, diolinoleyl phosphatidyl inositol, dilinolenoyl phosphatidyl inositol, diedecanoyl phosphatidyl inositol, diethylcoyl phosphatidyl inositol, dietycosinoyl Selected from the group consisting of trienoyl phosphatidyl inositol, diecosapentaenoyl phosphatidyl inositol, didicosaenoyl phosphatidyl inositol and didicosahexaenoyl phosphatidyl inositol Selected,

The said methoxy polyethylene glycol (mPEG) addition phosphatidyl ethanolamine is mPEG2000- egg yolk phosphatidyl ethanolamine, mPEG3400- egg yolk phosphatidyl ethanolamine, mPEG5000- egg yolk phosphatidyl ethanolamine, mPEG2000- hydrogenated egg yolk phosphatidyl ethanolamine, mPEG3400- hydrogenated egg yolk Ethanolamine, mPEG5000-hydrogenated egg yolk phosphatidylethanolamine, mPEG2000- soybean phosphatidylethanolamine, mPEG3400- soybean phosphatidylethanolamine, mPEG5000- soybean phosphatidylethanolamine, mPEG2000- hydrogenated soybean phosphatidylethanolamine, mPEG3400- hydrogenated soybeans , mPEG5000-hydrogenated soybean phosphatidylethanolamine, mPEG2000-dilauroyl phosphatidylethanolamine, mPEG3400-dilauroyl phosphatidylethanolamine, mPEG5000-dilauroyl phosphatidylethanolamine, mPEG2000-dimyristoyl phosphatidylethanolamine, mPEG3400-Dimitristo Phosphatidylethanolamine, mPEG5000-dimyristoyl phosphatidylethanolamine, mPEG2000-dipalmitoyl phosphatidylethanolamine, mPEG3400-dipalmitoyl phosphatidylethanolamine, mPEG5000-dipalmitoyl phosphatidylethanolamine, mPEG2000-distearoyl phosphatidylethanamine , mPEG3400-distearoyl phosphatidylethanolamine, mPEG5000-distearoyl phosphatidylethanolamine, mPEG2000-dioleoyl phosphatidylethanolamine, mPEG3400-dioleoyl phosphatidylethanolamine and mPEG5000-dioleoyl phosphatidylethanolamine Is selected from

The phosphatidic acid, egg yolk phosphatidic acid, hydrogenated yolk phosphatidic acid, soybean phosphatidic acid, hydrogenated soybean phosphatidic acid, dilauroyl phosphatidic acid, dimyristoyl phosphatidic acid, dipalmitoyl force Fatty Acid, Distearoyl Phosphate Acid, Dioleoyl Phosphate Acid, Dilinoleyl Oil Phosphate Acid, Palmitoyl-Oleoyl Phosphate Acid, Dilinolenoyl Phosphate Acid, Didecanoyl Phosphate Acid, Consisting of dietycoyl phosphatidic acid, diecosanoyl phosphatidic acid, dieticosatrienoyl phosphatidic acid, diecosapentaenoyl phosphatidic acid, didicosaenoyl phosphatidic acid and didicosahexaenoyl phosphatidic acid The endoplasmic reticulum according to [10], which is selected from the group.

[12] The vesicle according to [1], wherein the vitamin E includes at least one or more selected from the group consisting of α-tocopherol, β-tocopherol, γ-tocopherol and δ-tocopherol.

[13] The endoplasmic reticulum of [1], wherein the vitamin E is dl-α-tocopherol.

[14] The vesicle according to any one of [1] to [13], wherein the substance having a cationic property is contained in a range of 10 to 50% of the number of moles of the entire substance constituting the endoplasmic reticulum.

[15] The vesicle according to any one of [1] to [14], wherein the vitamin E is adsorbed or chemically bonded in the range of 3 to 30% of the total number of moles of the substance constituting the endoplasmic reticulum.

[16] The vesicle according to any one of [1] to [14], wherein the vitamin E is adsorbed or chemically bonded in a range of 5 to 20% of the total number of moles of the substance constituting the endoplasmic reticulum.

[17] The vesicle according to any one of [1] to [16], wherein the vesicle has a particle size in the range of 50 to 150 nm.

[18] The endoplasmic reticulum contains at least one bioactive substance selected from the group consisting of plasmid DNA, siRNA, miRNA, antisense oligonucleotides, antiviral agents and cytotoxic substances, and / or its biological activity The vesicle according to any one of [1] to [17], wherein the substance binds hydrophobicly or electrostatically to a component of the vesicle.

[19] The endoplasmic reticulum of [18], wherein the zeta potential on the endoplasmic reticulum is anionic.

[20] The vesicle according to [18], wherein the physiologically active substance is an antiviral agent.

[21] The cytotoxic substance includes at least one or more selected from the group consisting of metabolic antagonists, platinuming agents, alkylating agents, plant alkaloids, molecular target drugs, biological response modifiers and anticancer antibiotics. The vesicle of [18] characterized by the above-mentioned.

[22] The metabolic antagonists include enositabine, capecitabine, carmopur, cladribine, gemcitabine, cytarabine, cytarabine oxphosphate, tegapur, tegapur uracil, tegapur and kigi. Meracyl-Otherasil Potassium, Doxyfluidine, Nellarabine, Hydroxycarbamide, Fluorouracil, Fludarabine, Pemetrexed, Pentostatin, Mercaptopurine and Methotrexate,

The platinum agent is selected from the group consisting of oxaliplatin, carboplatin, cisplatin and nedaplatin,

The alkylating agent is selected from the group consisting of ifosfamide, cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, melphalan and rannimustine,

The plant alkaloid is selected from irinotecan, etoposide, sorbic acid, docetaxel, nogitecan, paclitaxel, vinorelbine, vincristine, vindesine, vinblastine,

The molecular targeting agent is selected from the group consisting of imatinib, erlotinib, sunitinib, cetuximab, sorafenib, panitumumab, bevacizumab, bortezomib,

The biological response modulator is selected from the group consisting of interferon-α, interferon-β, interferon-γ, interleukin,

The anti-cancer antibiotic is selected from the group consisting of aclarubicin, epirubicin, daunorubicin, doxorubicin, pyrarubicin, bleomycin, mitomycin C and mitoxantrone, [21] Endoplasmic reticulum as described in].

[23] A kit used for delivering a physiologically active substance to hepatic parenchymal cells in the presence of serum, comprising the vesicle according to any one of [18] to [22] and a pharmaceutically acceptable carrier.

[24] A method for delivering a physiologically active substance to hepatic parenchymal cells in a subject, comprising the step of administering the vesicles according to any one of [18] to [22] to the subject.

1 is a diagram showing the results of the introduction of siRNA by liposomes in vitro. SiRNA using the liposome dispersion described in [Example 1] and [Example 2] prepared by adding a liposome dispersion or a tocopherol of [Comparative Example 1] to HCV replicon cells R6 FLR41-14 cells containing a luciferase gene Was introduced and its HCV replication inhibitory activity was examined. The results were shown at high concentrations.
Fig. 2 shows the results of introduction of siRNA by liposomes in vitro. [Example 1] and [Example 2], which were prepared by adding liposome dispersions or tocopherols of [Comparative Example 1] and [Comparative Example 2] to HCV replicon cells R6 FLR41-14 cells containing a luciferase gene. SiRNA was introduced using a liposome dispersion to examine its HCV replication inhibitory activity. The results were shown at low concentrations.
Figure 3 is a diagram showing the gene expression principle of transgenic (Cre / loxP / HCV-MxCre Tg) mouse switching expression of the HCV gene.
FIG. 4 is a diagram showing changes in HCV core gene expression in transgenic (Cre / loxP / HCV-MxCre Tg) mice expressing switching HCV genes.
FIG. 5 shows that Cre / loxP / HCV-MxCre Tg mice 43 days after gene expression were administered with liposome dispersion prepared in [Example 1] and si197-1 complex (0.3 mg siRNA / kg body weight) to inhibit HCV gene expression. It is a figure which shows the evaluation result of activity.

The present invention relates to a endoplasmic reticulum comprising the following (i) and (ii), wherein vitamin E is adsorbed on the surface of the vesicle, or vitamin E is chemically bonded to an amphiphilic substance constituting the endoplasmic reticulum. It is about.

(i) amphiphilic substances

(ii) an organic material comprising a material having a cationicity and / or a material having anionicity

Amphiphilic materials used in the present invention are preferably associated with each other in water to form submicron vesicles in nano. In addition, as an amphiphilic substance of the present invention, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, polyethylene glycol (PEG) addition phosphatidylethanolamine, phosphatidic acid, sphingomyelin, cardiolipin, plasmamarogen At least one phospholipid and / or at least one polymer axis selected from the group consisting of polypolysaccharides (flurane, mannan, etc.) condensates of phospholipids and cholesterol, and polyamino acids (polyaspartic acid, etc.) condensates of polyethylene glycol Coalescence is mentioned. In this invention, the said phospholipid and / or the said polymer condensate and cholesterol may be used together as an amphiphilic substance.

In the present invention, as phosphatidylcholine, yolk phosphatidylcholine, hydrogenated yolk phosphatidylcholine, soybean phosphatidylcholine, hydrogenated soybean phosphatidylcholine, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, diol Phosphatidylcholine, Palmitoyl-Oleoyl Phosphatidylcholine, Dilinoleyl Oil Phosphatidylcholine, Dilinolenoyl Phosphatidylcholine, Didecanoyl Phosphatidylcholine, Dieticoyl Phosphatidylcholine, Diecosanoyl Phosphatidylcholine, Diecosatrienoyl Phosphatidyl Paphosyldicholine Didicosaenoyl phosphatidylcholine, didicosahexaenoyl phosphatidylcholine, etc. can be illustrated.

Examples of phosphatidylethanolamine include egg yolk phosphatidylethanolamine, soybean phosphatidylethanolamine, hydrogenated soybean phosphatidylethanolamine, dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidyl ethanolamine, and distearoyl phosphatidyl ethanolamine. Ethanolamine, dioleoyl phosphatidylethanolamine, palmitoyl-oleoyl phosphatidylethanolamine, dilinoleyl phosphatidylethanolamine, dilinolenoyl phosphatidylethanolamine, diedecanoyl phosphatidylethanolamine, diethycoyl phosphatidylethanolamine Cosanoyl phosphatidyl ethanolamine, diecosatrienoyl phosphatidyl ethanolamine, diecosapentaenoyl phosphatidyl ethanolamine, didicosaenoyl phosphatidyl ethanolamine, didocosahexaenoyl phosphatidyl ethanolamine, etc. can be illustrated.

Examples of phosphatidylglycerols include egg yolk phosphatidylglycerol, hydrogenated yolk phosphatidylglycerol, soybean phosphatidylglycerol, hydrogenated soybean phosphatidylglycerol, dilauroyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol and distearoyl phosphatidylglycerol , Dioleoyl phosphatidylglycerol, palmitoyl-oleoyl phosphatidylglycerol, dilinoleyl phosphatidylglycerol, dilinolenoyl phosphatidylglycerol, didecanoyl phosphatidylglycerol, diethylcoyl phosphatidylglycerol, diecosanoyl phosphatidylglycerol Enoyl phosphatidylglycerol, diecosapentaenoyl phosphatidylglycerol, didicosaenoyl phosphatidylglycerol, didicosahexaenoyl phosphatidylglycerol, etc. can be illustrated.

As phosphatidylserine, yolk phosphatidylserine, hydrogenated egg yolk phosphatidylserine, soybean phosphatidylserine, hydrogenated soybean phosphatidylserine, dilauroyl phosphatidylserine, dimyristoyl phosphatidylserine, dispalmitoyl phosphatidylserine, distearoyl phosphatidylserine , Dioleoyl phosphatidylserine, palmitoyl-oleoyl phosphatidylserine, diolinoleyl phosphatidylserine, dilinolenoyl phosphatidylserine, didecanoyl phosphatidylserine, dietycoyl phosphatidylserine, diecosanoyl phosphatidylserine Enoyl phosphatidylserine, diecosapentaenoyl phosphatidylserine, didicosaenoyl phosphatidylserine, didicosahexaenoyl phosphatidylserine and the like can be exemplified.

Examples of the phosphatidylinositol include egg yolk phosphatidyl inositol, hydrogenated egg yolk phosphatidyl inositol, soybean phosphatidyl inositol, hydrogenated soybean phosphatidyl inositol, dilauroyl phosphatidyl inositol, dimyristoyl phosphatidyl inositol, dipalmitoyl phosphatidyl inositol, and distearyl inositol. , Dioleoyl phosphatidyl inositol, palmitoyl-oleoyl phosphatidyl inositol, dirinoleoyl phosphatidyl inositol, dilinolenoyl phosphatidyl inositol, diedecanoyl phosphatidyl inositol, dietyrisoyl phosphatidyl inositol, dieicosinosyl trisinoyl trisinoyl phosphatidyl inositol Enoyl phosphatidyl inositol, dieicosapentaenoyl phosphatidyl inositol, didicosaenoyl phosphatidyl inositol, didicosahexaenoyl phosphatidyl inositol and the like can be exemplified.

As methoxy polyethyleneglycol (mPEG) addition phosphatidyl ethanolamine, mPEG2000- egg yolk phosphatidyl ethanolamine, mPEG3400- egg yolk phosphatidyl ethanolamine, mPEG5000- egg yolk phosphatidyl ethanolamine, mPEG2000- hydrogenated egg yolk phosphatidyl ethanolamine, mPEG3400- hydrogen ethane ethanol sulphate Amine, mPEG5000-hydrogenated egg yolk phosphatidylethanolamine, mPEG2000- soybean phosphatidylethanolamine, mPEG3400- soybean phosphatidylethanolamine, mPEG5000- soybean phosphatidylethanolamine, mPEG2000- hydrogenated soybean phosphatidylethanolamine, mPEG3400- hydrogenated soybean diethylethane mPEG5000-hydrogenated soybean phosphatidylethanolamine, mPEG2000-dilauroyl phosphatidylethanolamine, mPEG3400-dilauroyl phosphatidylethanolamine, mPEG5000-dilauroyl phosphatidylethanolamine, mPEG2000-dimiristoyl phosphatidylethanolamine, mPEG3400 Dimiristo Il phosphatidylethanolamine, mPEG5000-dimyristoyl phosphatidylethanolamine, mPEG2000-dipalmitoyl phosphatidylethanolamine, mPEG3400-dipalmitoyl phosphatidylethanolamine, mPEG5000-dipalmitoyl phosphatidylethanolamine, PEG2000-distearoyl phosphatidylethanol Amine, mPEG3400-distearoyl phosphatidylethanolamine, mPEG5000-distearoyl phosphatidylethanolamine, mPEG2000-dioleoyl phosphatidylethanolamine, mPEG3400-dioleoyl phosphatidylethanolamine, mPEG5000-dioleoyl phosphatidylethanolamine and the like You can choose.

Examples of phosphatides include egg yolk phosphatides, hydrogenated yolk phosphatides, soybean phosphatides, hydrogenated soy phosphatides, dilauroyl phosphatides, dimyristoyl phosphatides, dipalmitoyl phosphatides Deic acid, distearoyl phosphatidic acid, dioleoyl phosphatidic acid, dilinoleyl phosphatidic acid, palmitoyl-oleoyl phosphatidic acid, dilinolenoyl phosphatidic acid, didecanoyl phosphatidic acid, die Examples include lucicoyl phosphatidic acid, diecosanoyl phosphatidic acid, diecosattrienoyl phosphatidic acid, diecosapentaenoyl phosphatidic acid, didicosaenoyl phosphatidic acid, diedocosahexaenoyl phosphatidic acid, and the like. can do.

As sphingomyelin, egg yolk-derived sphingomyelin or milk-derived sphingomyelin can be illustrated,

Cardiolipins include egg yolk cardiolipin, hydrogenated egg yolk cardiolipin, soy cardiolipin, hydrogenated soy cardiolipin, dilauroyl cardiolipin, dimyristoyl cardiolipin, dipalmitoyl cardiolipin , Distearoyl cardiolipin, dioleoyl cardiolipin, palmitoyl-oleoyl cardiolipin, diolinoleyl cardiolipin, diolinolenoyl cardiolipin, didecanoyl cardiolipin, dietierus Illustrated coiled cardiolipin, diecosanoyl cardiolipin, diecosartrienoyl cardiolipin, diecosapentaenoyl cardiolipin, didicosaenoyl cardiolipin, didicosahexaenoyl cardiolipin, etc. Can be.

In addition, what was described here is an example, If it has a structure of the same kind, it can use without being limited to the compound described here.

In the present invention, in the case of using a polyanionic material such as a nucleic acid as a physiologically active substance, a substance having cationicity for electrical bonding can be used as a component of the endoplasmic reticulum. As a substance which has cationicity, Preferably cationic lipid can be illustrated, More preferably, the compound represented by following formula (I) can be illustrated.

[Formula I]

(In formula, R <^1> and R <^2> is the same or different, and means a C12-C22 saturated or unsaturated hydrocarbon group, R <^3> means a C1-C6 alkyl group or a C1-C6 hydroxyalkyl group. M means an integer of 1 to 10, and X means a halogen atom.)

Examples of the compound include N- (α-trimethylammonoacetyl) -didodecyl-D-glutamate chloride, N- (α-trimethylammonioacetyl) -didodecyl-L-glutamate chloride and N- (α-trimethylammonio Acetyl) -Dioctadecyl-D-glutamate chloride, N- (α-trimethylammonioacetyl) -dioleyl-D-glutamate chloride, N-ω-trimethylammoniodecanyl dehexadecyl-D-glutamate bromide, N -ω-trimethylammoniodecane oil dehexadecyl-L-glutamate bromide etc. can be illustrated, These can use what is marketed (Sogo Pharmaceutical Co., Ltd.). Other cationic substances include saturated or unsaturated aliphatic primary amines such as stearylamine and oleylamine; saturated or unsaturated secondary and tertiary amines such as distearoyltrimethylammonium propane and dioleoyltrimethylammonium propane; Or DODAC (dioctadecyldimethylammonium chloride), DOTMA (N- (2,3-dioleyloxy) propyl-N, N, N-trimethylammonium: N- (2,3-dioleyloxy) propyl-N , N, N-trimethylammonium), DDAB (didodecylammonium bromide), DOTAP (1,2-dioleoyloxy-3-trimethylammonopropane: 1,2-dioleoyloxy-3-trimethylammonio propane),

DC-Chol (3β-N- (N ', N'-dimethylaminoethane) carbamol cholesterol hydrochloride: 3β-N- (N', N'-dimethyl-aminoethane) -carbamol cholesterol hydrochloride), DMRIE (1 , 2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium: 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethyl ammonium), DOSPA (2,3-dioleyloxy-N- [2 (sperminecarboxa) Mido) ethyl] -N, N-dimethyl-1-propanaminum trifluoroacetate: with 2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminum trifluoroacetate You may use at least 1 sort (s) or more types of mixtures chosen from the group which consisted of.

It is also possible to use polyethyleneimine or polylysine which is a polycation.

In the present invention, in the case of using a non-polar substance such as cytotoxic substance, a low molecular weight antiviral agent or a substance which does not have many polar groups as the physiologically active substance, (1) it is contained in the endoplasmic reticulum or (2) the membrane component and the minority Either method of combining and placing in the membrane is used. In these cases, a substance having an anion can be used for the purpose of preventing aggregation of the endoplasmic reticulum by using electrostatic repulsion. As a substance which has anionicity, Preferably acidic phospholipid can be illustrated.

Examples of the acidic phospholipids include phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, methoxy polyethylene glycol addition phosphatidylethanolamine and phosphatidic acid.

Examples of phosphatidylglycerols include egg yolk phosphatidylglycerol, hydrogenated yolk phosphatidylglycerol, soybean phosphatidylglycerol, hydrogenated soybean phosphatidylglycerol, dilauroyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol and distearoyl phosphatidylglycerol , Dioleoyl phosphatidylglycerol, palmitoyl-oleoyl phosphatidylglycerol, dilinoleyl phosphatidylglycerol, dilinolenoyl phosphatidylglycerol, didecanoyl phosphatidylglycerol, diethylcoyl phosphatidylglycerol, diecosanoyl phosphatidylglycerol Enoyl phosphatidylglycerol, diecosapentaenoyl phosphatidylglycerol, didicosaenoyl phosphatidylglycerol and didocasahexaenoyl phosphatidylglycerol can be exemplified.

As phosphatidylserine, yolk phosphatidylserine, hydrogenated egg yolk phosphatidylserine, soybean phosphatidylserine, hydrogenated soybean phosphatidylserine, dilauroyl phosphatidylserine, dimyristoyl phosphatidylserine, dispalmitoyl phosphatidylserine, distearoyl phosphatidylserine , Dioleoyl phosphatidylserine, palmitoyl-oleoyl phosphatidylserine, diolinoleyl phosphatidylserine, dilinolenoyl phosphatidylserine, didecanoyl phosphatidylserine, dietycoyl phosphatidylserine, diecosanoyl phosphatidylserine Enoyl phosphatidylserine, diecosapentaenoyl phosphatidylserine, didicosaenoyl phosphatidylserine, didicosahexaenoyl phosphatidylserine can be exemplified.

Examples of the phosphatidylinositol include egg yolk phosphatidyl inositol, hydrogenated egg yolk phosphatidyl inositol, soybean phosphatidyl inositol, hydrogenated soybean phosphatidyl inositol, dilauroyl phosphatidyl inositol, dimyristoyl phosphatidyl inositol, dipalmitoyl phosphatidyl inositol, and distearyl inositol. , Dioleoyl phosphatidyl inositol, palmitoyl-oleoyl phosphatidyl inositol, dirinoleoyl phosphatidyl inositol, dilinolenoyl phosphatidyl inositol, diedecanoyl phosphatidyl inositol, dietyrisoyl phosphatidyl inositol, dieicosinosyl trisinoyl trisinoyl phosphatidyl inositol Enoyl phosphatidyl inositol, diecosapentaenoyl phosphatidyl inositol, didicosaenoyl phosphatidyl inositol and didicosahexaenoyl phosphatidyl inositol can be exemplified.

As methoxy polyethyleneglycol (mPEG) addition phosphatidyl ethanolamine, mPEG2000- egg yolk phosphatidyl ethanolamine, mPEG3400- egg yolk phosphatidyl ethanolamine, mPEG5000- egg yolk phosphatidyl ethanolamine, mPEG2000- hydrogenated egg yolk phosphatidyl ethanolamine, mPEG3400- hydrogen ethane ethanol sulphate Amine, mPEG5000-hydrogenated egg yolk phosphatidylethanolamine, mPEG2000- soybean phosphatidylethanolamine, mPEG3400- soybean phosphatidylethanolamine, mPEG5000- soybean phosphatidylethanolamine, mPEG2000- hydrogenated soybean phosphatidylethanolamine, mPEG3400- hydrogenated soybean diethylethane mPEG5000-hydrogenated soybean phosphatidylethanolamine, mPEG2000-dilauroyl phosphatidylethanolamine, mPEG3400-dilauroyl phosphatidylethanolamine, mPEG5000-dilauroyl phosphatidylethanolamine, mPEG2000-dimiristoyl phosphatidylethanolamine, mPEG3400 Dimiristo Il phosphatidylethanolamine, mPEG5000-dimyristoyl phosphatidylethanolamine, mPEG2000-dipalmitoyl phosphatidylethanolamine, mPEG3400-dipalmitoyl phosphatidylethanolamine, mPEG5000-dipalmitoyl phosphatidylethanolamine, mPEG2000-distearoyl phosphatidylethanol Examples of amines, mPEG3400-distearoyl phosphatidylethanolamine, mPEG5000-distearoyl phosphatidylethanolamine, mPEG2000-dioleoyl phosphatidylethanolamine, mPEG3400-dioleoyl phosphatidylethanolamine and mPEG5000-dioleoyl phosphatidylethanolamine can do.

Examples of phosphatides include egg yolk phosphatides, hydrogenated yolk phosphatides, soybean phosphatides, hydrogenated soy phosphatides, dilauroyl phosphatides, dimyristoyl phosphatides, dipalmitoyl phosphatides Deic acid, distearoyl phosphatidic acid, dioleoyl phosphatidic acid, dilinoleyl phosphatidic acid, palmitoyl-oleoyl phosphatidic acid, dilinolenoyl phosphatidic acid, didecanoyl phosphatidic acid, die Illustrates lucicoyl phosphatidic acid, diecosanoyl phosphatidic acid, diecosartrinoyl phosphatidic acid, diecosapentaenoyl phosphatidic acid, didicosaenoyl phosphatidic acid and didicosahexaenoyl phosphatidic acid Can be.

In the present invention, the vitamin E adsorbed or chemically bonded to the surface of the endoplasmic reticulum is not particularly limited, but preferably at least one kind selected from the group consisting of α-tocopherol, β-tocopherol, γ-tocopherol and δ-tocopherol. The above vitamin E can be illustrated. Moreover, dl- (alpha) -tocopherol is mentioned as a more preferable example of vitamin E.

The amount of vitamin E adsorbed or chemically bonded to the surface of the endoplasmic reticulum is a sufficient amount recognized by the particles in the serum. It is necessary to be present, and more preferably, it is required to be adsorbed or chemically bonded in the range of 5 to 20%. If the ratio of these bonds is small, the serum E perception ability of the serum particles is weak, resulting in poor transport efficiency to hepatic parenchymal cells. In too many cases, the adsorbed vitamin E tends to associate with the endoplasmic reticulum by hydrophobic bonding, so the average particle diameter increases. As the average particle diameter increases, it is more likely to be trapped by the RES in the blood, and as a result, the transport efficiency to the hepatic parenchymal cells also decreases. In the case of the coupling | bonding, the ester etc. which couple | bonded dibasic acids, such as glutaric acid, to the front-end | tip of the amino group of phosphatidylethanolamine which are an amphiphilic substance, and condensed the hydroxyl group of one carboxylic acid and vitamin E are used. Since the serum component can be recognized, polyethyleneglycol whose terminal is substituted with an active group may be used instead of dibasic acid. The method of incorporating vitamin E is not limited to these, and any of generally known methods may be used.

By adding vitamin E within the above range, the recognition power by the serum particles is optimized, and the association of the endoplasmic reticulum can also be prevented, so that efficient delivery to the parenchymal cells can be realized.

The particle size of the endoplasmic reticulum may be 20 to 500 nm in the case of delivery to hepatic parenchymal cells in vitro, but is controlled by 50 to 150 nm in the case of intravenous intravenous administration, so that it is not trapped in the RES and is represented by LDL in serum. It is carried by the particles to the liver parenchymal cells.

If the particle size of the endoplasmic reticulum is too small, the efficiency of transporting the physiologically active substance is poor. If the endoplasmic reticulum is too large, it is trapped in the RES in vivo and difficult to transport to the liver parenchymal cells. Therefore, in order to fully exhibit the transport effect in vivo, it is more preferable that the endoplasmic reticulum having a particle size of 50 to 150 nm is 60% or more of the whole. The present invention also includes a endoplasmic reticulum, wherein the endoplasmic reticulum having a particle size of 50 nm to 150 nm is 60% or more of the entire endoplasmic reticulum population.

The present invention relates to an endoplasmic reticulum containing at least one bioactive substance selected from the group consisting of plasmid DNA, siRNA, miRNA, antisense oligonucleotide, antiviral agent and cytotoxic substance, and / or It relates to a vesicle that hydrophobicly or electrostatically binds to the components of the endoplasmic reticulum.

The present invention relates to an endoplasmic reticulum in which the zeta potential of the endoplasmic reticulum surface is anionic. The physiologically active substance is preferably anionic by electrostatically binding to the components of the endoplasmic reticulum, but the form thereof is not particularly limited.

Although siRNA used as a bioactive substance in this invention is not specifically limited, Preferably, the siRNA containing the oligoribonucleotide of SEQ ID NO: 1 and the oligonucleotide of SEQ ID NO: 2 can be illustrated.

The antiviral agent used as a physiologically active substance in the present invention is not particularly limited as long as it is a drug for the treatment of viral liver disease such as an antiHCV agent or an anti-HBV agent, but is preferably an antiHCV agent. The antiviral agent of the present invention includes not only nucleic acid-based drugs such as plasmid DNA, siRNA, miRNA, antisense oligonucleotide to HCV and HBV, but also low molecular antiviral agents. The low molecular weight antiviral agent is not particularly limited, but may be an inhibitor such as a polymerase or protease encoded by a viral gene, or a low molecular weight compound having other antiviral activity. Specific examples of the compound include ribavirin (1-β-D-ribofuranosyl-1H-1, 2,4-tri-azole-3-carboxamide) etc. can be illustrated.

In the present invention, the cytotoxic substance may be a cytotoxic agent comprising at least one or more selected from the group consisting of metabolic antagonists, platinum agents, alkylating agents, plant alkaloids, molecular target drugs, biological response modifiers, and anticancer antibiotics. Sex materials can be exemplified.

Examples of metabolic antagonists include enositabine, capecitabine, carmopur, cladribine, gemcitabine, cytarabine, cytarabine oxphosphate, tegapur, tegapur uracil, tegapur gimerasil and o Teracyl potassium, doxyfluidine, ellarabine, hydroxycarbamide, fluorouracil, fludarabine, pemetrexed, pentostatin, mercaptopurine and methotrexate.

Examples of the platinum agent include oxaliplatin, carboplatin, cisplatin, and nedaplatin.

Examples of the alkylating agent include phosphamide, cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, melphalan, and ranimustine.

As the plant alkaloid, it is selected from irinotecan, etoposide, sorbic acid, docetaxel, nogitecan, paclitaxel, vinorelbine, vincristine, vindesine, vinblastine,

Molecular targeting agents can exemplify imatinib, erlotinib, sunitinib, cetuximab, sorafenib, panitumumab, bevacizumab, bortezomib.

Examples of biological response modifiers include interferon-α, interferon-β, interferon-γ, and interleukin.

Examples of the anticancer antibiotics include aclarubicin, epirubicin, daunorubicin, doxorubicin, pyrarubicin, bleomycin, mitomycin C, and mitoxantrone.

The present invention relates to a kit used for delivering a bioactive substance to hepatic parenchymal cells in the presence of serum, including the endoplasmic reticulum and a pharmaceutically acceptable carrier.

Pharmaceutical compositions and kits of the present invention may contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, sterile water, saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphate, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.). , Chelating agents (EDTA, etc.), binders and the like. Other low molecular weight polypeptides, proteins such as serum albumin, gelatin or immunoglobulin, amino acids such as glycine, glutamine, asparagine, arginine and lysine, sugars such as polysaccharides and monosaccharides, sugar alcohols such as carbohydrates, mannitol and sorbitol You may do it. In the case of the aqueous solution for injection, for example, isotonic solution containing saline solution, glucose or other auxiliary agent, for example, D-sorbitol, D-mannose, D-mannitol, sodium chloride and the like can be mentioned. For example, you may use together alcohol (ethanol etc.), polyalcohols (propylene glycol, PEG, etc.), nonionic surfactant (polysorbate 80, HCO-50).

Also, if necessary, it is enclosed in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin, poly [methylmethacrylic acid]), or colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles and nanoparticles). Capsules).

In addition, the kit of the present invention may be accompanied by an instruction manual describing a method for using the kit.

The present invention relates to a method of delivering a physiologically active substance to hepatic parenchymal cells in a subject, comprising the step of administering the endoplasmic reticulum to the subject. After the vesicles of the present invention are administered to a subject, they are selectively delivered to hepatic parenchymal cells by serum components in the subject.

In the present invention, administration to the subject may be either oral or parenteral, but is preferably parenteral. There is no restriction | limiting in particular as a form (formula) of the composition which becomes a vesicle of this invention, An injection form, a non-administrative form, a transpulmonary form, a transdermal form, a lyophilized form, a solution form, etc. are mentioned.

As an example of an injection formulation, it can be administered systemically or locally, for example by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection or the like. Moreover, the administration method can be suitably selected according to the state of the subject (when the subject is a human patient, the age and symptoms of the patient).

The endoplasmic reticulum containing the physiologically active substance carried by the serum particle exerts a physiologically active action in hepato parenchymal cells immediately. For example, when the physiologically active substance is siRNA, while the physiologically active substance carried through the glycoprotein receptor on the surface of the liver cells of the prior art is metabolized in the lysosome, the present invention shows a knockdown effect within 48 hours.

In addition, cationic liposomes in which siRNAs are electrostatically bonded to the surface of the endoplasmic reticulum are generally known, but for the interaction with the cell surface, anionic siRNA is added in a lower amount than the cationic lipid or cationic polymer, and zeta on the surface of the endoplasmic reticulum is It is used with the potential being positive (cationic). However, when the blood is administered with the remaining cationicity, it is easy to trap or bind to albumin in RES or serum present in the lung, and thus the transport efficiency to hepatic parenchymal cells is lowered. In order to avoid this, it is very important that the zeta potential on the surface of the endoplasmic reticulum is negative, so that the amount of addition of the nucleic acid physiologically active substance such as siRNA to the endoplasmic reticulum should be used more than the isoelectric point of the cationic lipid or cationic polymer.

In addition, all the prior art documents referred in this specification are contained in this specification as a reference.

Example

1. Delivery of siRNA to hepatic parenchymal cells in vitro

Example 1

N- (α-trimethylammonioacetyl) -dioleyl-D-glutamate chloride (SOGO Pharmaceutical Co., Ltd., Japan; product name: DC-3-18: 1), dioleoyl phosphatidylethanolamine (Nichiyu Corporation, Japan) And cholesterol (Wako Pure Chemical Co., Ltd.) were dissolved in an appropriate amount of chloroform at a compounding ratio of 40:30:30 (molar ratio), and the resultant was dried with distilling off the solvent under reduced pressure to obtain a lipid mixture.

After drying in a desiccator until the solvent can be reliably removed, a 9% sucrose solution is added to the lipid mixture, and the whole is indirectly ultrasonically irradiated with a sonicator at 65 ° C warming. A liposome crude dispersion having a lipid concentration of 2.5 mM was obtained. Next, in order to make the particle size of a liposome even, two filters of 0.2 micrometers of pore diameters were piled up and set in the extruder, and the extrusion filtration was carried out under about 65 degreeC heating and pressurization. Furthermore, extrusion filtration was carried out similarly using two filters of 0.1 micrometers of pore diameters, and this was made into the empty liposome dispersion liquid. The average particle diameter was 136.1 nm.

To 5 μmol of DL-α-tocopherol (Tokyo Kasei Co., Ltd.) was dissolved at a ratio of 400 μL of ethyl alcohol, a liquid (tocopherol solution) filtered through a 0.2 μm filter was prepared.

In a 50 mL vial, 10 mL of the liposome dispersion was added and 400 μL of the tocopherol solution was further added (20% relative to the total number of moles of lipids), followed by mixing and stirring for 20 seconds with a stirrer. Furthermore, it incubated at room temperature for 1 hour, and obtained vitamin E adsorption liposome dispersion. The average particle diameter was 124.2 nm.

[Example 2]

To liquid 2.5-mol of DL- (alpha) -tocopherol (Tokyo Chemical Co., Ltd., Japan) was melt | dissolved in the ratio of 400 microliters of ethyl alcohol, the liquid (tocopherol liquid) filtered with the 0.2 micrometer filter was prepared.

In a 50 mL vial, 10 mL of the liposome dispersion prepared in [Example 1] was added, and 400 μL of the tocopherol solution was further added (10.0% based on the total number of moles of lipids), followed by mixing and stirring for 20 seconds with a stirrer. Furthermore, it incubated at room temperature for 1 hour, and obtained vitamin E adsorption liposome dispersion. The average particle diameter was 136.7 nm.

Example 3

To 3.75 μmol of DL-α-tocopherol (Tokyo Kasei Co., Ltd.) was dissolved in a ratio of 400 μL of ethyl alcohol, and a liquid (tocopherol solution) filtered through a 0.2 μm filter was prepared.

In a 50 mL vial, 10 mL of the liposome dispersion prepared in [Example 1] was added, and 400 μL of the tocopherol solution was further added (15.0% based on the total number of moles of lipids), followed by mixing and stirring for 20 seconds with a stirrer. Furthermore, it incubated at room temperature for 1 hour, and obtained vitamin E adsorption liposome dispersion. The average particle diameter was 133.5 nm.

Example 4

N- (α-trimethylammonioacetyl) -dioleyl-D-glutamate chloride (SOGO Pharmaceutical Co., Ltd., Japan; product name: DC-3-18: 1D), dioleoyl phosphatidylethanolamine (Nichiyu Corporation, Japan) And cholesterol (Wako Pure Chemical Co., Ltd., Japan) were dissolved in an appropriate amount of chloroform at a compounding ratio of 40:30:30 (molar ratio), which was dried while distilling off the solvent under reduced pressure to obtain a lipid mixture. After drying in a desiccator until the solvent can be reliably removed, a 9% sucrose solution was added to the lipid mixture, and heated to 65 ° C. and indirectly sonicated with a sonicator to obtain a total lipid concentration of 2.5 mM. A liposome crude dispersion was obtained. Next, in order to make the particle size of a liposome even, two filters of 0.2 micrometers of pore diameters were piled up, set in the extruder, and the extrusion filtration was carried out under about 65 degreeC heating and pressurization. Furthermore, extrusion filtration was carried out similarly using two filters of 0.1 micrometers of pore diameters, and this was made into the empty liposome dispersion liquid. The average particle diameter was 138.7 nm.

Subsequently, the nano-Mizer mark II NM2-L200 (Yoshida Machinery Co., Ltd., Japan) was further subjected to three micronization treatment under 100 MPa pressurization, and the average particle size was 55.7 nm.

To 5 μmol of DL-α-tocopherol (Tokyo Kasei Co., Ltd.) was dissolved at a ratio of 400 μL of ethyl alcohol, a liquid (tocopherol solution) filtered through a 0.2 μm filter was prepared.

In a 50 mL vial, 10 mL of liposome dispersion was added and 320 μL of the tocopherol solution (16% relative to the total number of moles of lipids) was mixed and stirred for 20 seconds with a stirrer. Furthermore, it incubated at room temperature for 1 hour, and obtained vitamin E adsorption liposome dispersion. The average particle diameter was 58.0 nm.

Comparative Example 1

The liposome dispersion liquid with an average particle diameter of 136.1 nm produced in [Example 1] was used as it was (tocopherol no addition).

Comparative Example 2

20 µmol of DL-α-tocopherol (Tokyo Chemical Co., Ltd.) was dissolved in 400 µL of ethyl alcohol, and a liquid (tocopherol liquid) filtered through a 0.2 µm filter was prepared.

In a 50 mL vial, 5 mL of the liposome dispersion prepared in [Example 1] was added, and 200 μL of the prepared tocopherol solution was further added (80.0% based on the total number of moles of lipids), followed by mixing and stirring for 20 seconds with a stirrer. Furthermore, it incubated at room temperature for 1 hour, and obtained vitamin E adsorption liposome dispersion. The average particle diameter was 190.1 nm.

In vitro testing into hepatic parenchymal cells (in vitro) and mice using various liposomes prepared as described above (Examples 1 to 3 and Comparative Examples 1 to 2). A furnace introduction test (in vivo test) was performed to compare the examples with the comparative examples, and the effects of the present invention are shown below.

In addition, an Example is not limited only to this, It specifies that this Example is only an example.

[Cell Test Example 1] Study of Hepatitis C Virus (HCV) Replicon Replication Inhibitory Activity of Designed siRNA

Hepatitis C is one of the most serious infections in Japan, with more than 2 million people in Japan and more than 200 million people worldwide. The great feature is that when hepatitis C virus (HCV) is infected with humans, it is persistently infected at a high rate of 80 to 90%, causing chronic hepatitis and even hepatocellular carcinoma. Although treatment results have been improved by current peginterferon-ribavirin combination therapy, the therapeutic effect is not sufficient, and the development of a more effective and less effective side effect is urgently needed. Thus, attempts were made to control HCV by short double stranded RNA (siRNA).

Short double-stranded RNA (siRNA) has been widely used as a gene knockdown method from the discovery that it avoids the interferon (IFN) response of mammalian cells and causes RNAi. Recently, however, siRNA has also been reported to induce IFN responses by the Off Target effect, and not only shorten the length of double stranded RNA (dsRNA), but also improvements in sequence, nucleic acid modification, and the like have been investigated. The inventors have progressed HCV replication inhibition by RNA interference (RNAi), which is expected as a novel antiviral therapy, and HCV targeting by siRNA for therapeutic applications. However, it is difficult to efficiently introduce siRNA into the liver, which is a target organ, and the development of a drug delivery system (DDS) is urgent. In order to solve this problem, the following examination was performed.

<Transfection of siRNA into HCV Replicon Cells>

In this example, siRNA assays were performed using R6 FLR41-14 cells (Watanabe, T. et al., Gene Theraphy 13: 883-892 (2006)) as HCV replicon cells containing the luciferase gene. It was. Count the number of cells in each cell and suspend in DMEM + GlutaMax-I containing 10% immobilizer FCS, and R6 FLR41-14 cells are seeded in 96 wells at 4500 cells / 100 μL / well, the next day in the trans of siRNA Specification was performed. siRNA used si197-1 (sense strand 5'-AUCAUGAGCACAAAUCCUAAA-3 ', SEQ ID NO: 1, antisense strand (3'-CGUAGUACUCGUGUUUAGGAU-5', SEQ ID NO: 2). The final concentration of siRNA was 0.01, 0.04. , 0.11, 0.33, 1, 1.1, 3, 3.3, 10, 30, 90 nM.

Each siRNA sample was prepared with opti-MEM containing 0.23% NaHCO _3, and a dilution sequence of 10 steps was prepared in three-fold dilutions from 90 nM. To 50 μL of siRNA solution, 0.5 μL of the liposome dispersion of [Comparative Example 1] or the liposome dispersion according to [Example 1] and [Example 2] prepared by adding tocopherol were added, respectively. After sufficient mixing, the mixture was incubated at room temperature for 20 minutes to prepare an siRNA-Liposome complex. Samples of each dilution series were placed in multiplate 96FII (white) for cell culture for luciferase assay (Sumitomo Bakelite, Japan cat. # MS-8096W) and 96 well plate for measuring cytotoxicity (BD, cat. # 353072), respectively. 10 μL / 96-well was added with n = 3. The wells (Lipo CTRL) in which only the liposome dispersions described in [Example 1] and [Example 2] prepared by adding the liposome dispersion or the tocopherol of [Comparative Example 1] were added as controls. Incubated at 37 ° C., 5% CO ₂ , followed by a cytotoxicity test by luciferase assay and WST-8 after 72 hours.

The particle diameter and zeta potential before and after each conjugate are shown in Table 1.

From Table 1, it can be seen that before and after conjugation of the siRNA, the zeta potential is converted from cationic to anionic.

<Method of measuring luciferase activity>

In measuring luciferase activity, Bright-Glo Luciferase Assay System (Promega, cat. # E2620) was used. The method was according to the article (Watanabe, T. et al., Gene Therapy 13: 883-892 (2006)).

The medium of the entire well of a 96 well plate for luciferase assay was discarded, and medium exchange was performed by adding 75 μL / well of DMEM + GlutaMax-I with 5% immobilizer FCS. Bright-Glo Luciferase Assay System was added at 75 μL / well, and shaken with Mithras LB 940 (Berthold) for 1 minute, followed by luciferase activity.

Cytotoxicity Measurement

For cytotoxicity measurement, Cell Counting Kit-8 (Dojindo, cat. # 343-07623) was used.

The Cell Counting Kit-8 solution was diluted to 7% with DMEM + GlutaMax-I with 5% immobilizer FCS to prepare a solution for assay. The medium of the whole well of the 96 well plate for cytotoxicity test was discarded, and the above-mentioned assay solution was added at 100 µL / well and incubated for 1 hour at 37 ° C., 5% CO ₂ . Using a microplate reader (Bio-Rad model 550), wavelength 450 nm (reference wavelength 655 nm) was measured.

As a result, the liposome dispersions prepared in [Example 1] and [Example 2] to which tocopherol was added showed strong HCV replicon replication inhibitory activity, and it was clear that the cytotoxicity was mild. Compared to the liposome dispersion of [Comparative Example 1], the liposome prepared in [Example 2] showed about 100-fold, and the liposome dispersion prepared in [Example 1] showed HCV replicon replication inhibitory activity (about 1, 2).

2. Delivery of siRNA to hepatic parenchymal cells in vivo

[Animal test example 1] Inhibition of designed siRNA in HCV transgenic mice

<HCV transgenic mouse>

In order to elucidate the mechanism of sustained infection of HCV and the mechanism of transition to chronic hepatitis, cirrhosis, and liver cancer, transgenic (Tg) mice have been produced in a condition model. As a model closer to HCV infection, HCV structural protein region expressing Tg mice (CN2 mice) using a Cre / loxP recombinant system capable of switching expression of HCV genes at any time were established. Until now, intravenous administration of recombinant adenovirus (AxCANCre) expressing Cre DNA recombinase has been performed. However, in the case of the AxCANCre method, an immune response to adenovirus in mice is also caused. Accordingly, the present inventors crossed a transgenic mouse incorporating the HCV gene into the Cre / loxP system and a Mx-Cre transgenic mouse inducing Cre, thereby transgenic (Cre / loxP / HCV) switching expression of the HCV gene at any time. MxCre Tg) mice were constructed. In MxCre Tg mice, since Cre DNA recombinase is expressed by the interferon (IFN) reactive Mx1 promoter, switching occurs by induction of IFN by administration of poly-IpC (hereinafter referred to as pIpC, double stranded RNA). . The mice were intraperitoneally administered pIpC three times daily. Expression of HCV core protein was recognized from 0.5 days after the first administration, and peaked at 915 pg / mg (total liver protein amount) on day 7. Thereafter, there was a tendency to decrease, but expression continued at 435 pg / mg even on day 21. Moreover, this expression was maintained at the same level even after 6 months (FIGS. 3 and 4).

Administration of siRNA to HCV Transgenic Mice

Cre / loxP / HCV-MxCre Tg mice 43 days after gene expression were administered with the liposome dispersion prepared in [Example 1] and si197-1 complex (0.3 mg siRNA / kg body weight) to evaluate its RNAi activity. Two days after administration, autopsy was performed and the liver tissue was sampled. The amount of HCV core protein in this liver was quantified by ELISA method (ortho HCV core protein ELISA kit), and RNAi activity was evaluated. In the fasting and non-fasting groups, a significant decrease in the amount of HCV core protein was observed as compared to the siCTRL (Damacon Corporation; siCTRL3) administration group (FIG. 5). It is evident that this shows a 50-fold stronger inhibitory activity than that reported so far (Watanabe, T. et al., J. Hepatology 47: 744-750 (2007)).

When the composition of the present invention is used, a bioactive substance can be introduced into the liver parenchymal cells not only in vitro but also in vivo very efficiently in the presence of serum, and thus is very useful not only as a reagent but also as a medicine.

In addition, the present invention, by adsorbing or chemically binding the vitamin E to the particles, LDL (low specific gravity lipoprotein) and the like in the serum recognizes the vitamin E and delivers the particles in the liver parenchymal cells through the receptor of the liver parenchymal cells. It is characterized by. Since the particle size and surface charge of the particles are optimized, these particles are not inserted into the nonparenchymal cells, and the vesicle particles can be specifically delivered into the parenchymal cells.

                         SEQUENCE LISTING <110> CHUGAI SEIYAKU KABUSHIKI KAISHA        TOKYO METROPOLITAN ORGANIZATION FOR MEDICAL RESEARCH        HOKKAIDO SYSTEM SCIENCE CO., LTD.   <120> vesicle formulation <130> C1-A0807P <150> JP 2008-301763 <151> 2008-11-26 <160> 2 <170> PatentIn version 3.4 <210> 1 <211> 21 <212> RNA <213> Artificial <220> <223> An artificially synthesized sequence for siRNA <400> 1 aucaugagca caaauccuaa a 21 <210> 2 <211> 21 <212> RNA <213> Artificial <220> <223> An artificially synthesized sequence for siRNA <400> 2 uaggauuugu gcucaugaug c 21

Claims (24)

A vesicle comprising (i) and (ii), wherein the vesicle is adsorbed on the surface of the vesicle, or vitamin E is chemically bonded to an amphiphilic substance constituting the vesicle.
(i) amphiphilic substances
(ii) an organic material comprising a material having a cationicity and / or a material having anionicity The method of claim 1,
The vesicles, characterized in that the cationic substance is a cationic lipid. The method of claim 2,
Vesicles, characterized in that the cationic lipid is represented by the following formula (I).
(I)

(In formula, R <^1> and R <^2> means the same or different C12-C22 saturated or unsaturated hydrocarbon group, R <^3> means a C1-C6 alkyl group or a C1-C6 hydroxyalkyl group, m means an integer of 1 to 10, X means a halogen atom.) The method of claim 2,
The cationic lipids are saturated or unsaturated aliphatic primary amines (stearylamine, oleylamine), saturated or unsaturated secondary, tertiary amines (distearoyltrimethylammonium propane, dioleoyltrimethylammonium propane) and agent Quaternary amine salts (DODAC (dioctadecyldimethylammonium chloride), DTDAB (ditetradecyldimethylammonium bromide), DOTMA (N- (2,3-dioleyloxy) propyl-N, N, N-trimethylammonium: N- (2,3-dioleyloxy) propyl-N, N, N-trimethylammonium), DDAB (didodecylammonium bromide), didodecylammonium bromide), DTAB (ditetradecylammonium bromide), DOTAP (DOTAP) 1,2-dioleoyloxy-3-trimethylammonio propane: 1,2-dioleoyloxy-3-trimethylammonio propane), O, O'- ditetradecane oil-N- (α-trimethylammonio-acetyl)- Diethanolamine chloride: O, O'-ditetradecanoyl-N- (α-tr imethylammonio-acetyl) -diethanolamine chloride, O, O'-dihexadecanyl-N- (α-trimethylammonio-acetyl) -diethanolamine chloride: O, O'-dihexadecanoyl-N- (α-trimethylammonio-acetyl ) -diethanolamine chloride, O, O'-dioctadecanyl-N- (α-trimethylammonio-acetyl) -diethanolamine chloride: O, O'-dioctadecanoyl-N- (α-trimethylammonio-acetyl) -diethanolamine chloride, DC-Chol (3β-N- (N ', N'-dimethylaminoethane) carbamol cholesterol hydrochloride: 3β-N- (N', N'-dimethyl-aminoethane) -carbamol cholesterol hydrochloride), DMRIE (1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium: 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethyl ammonium), DOSPA (2,3-dioleyloxy-N- [2 (spermine Carboxamido) ethyl] -N, N-dimethyl-1-propanaminum trifluoroacetate: 2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminum trifluoroacetate From the group consisting of An endoplasmic reticulum comprising at least one or more types selected. The method of claim 1,
The amphiphilic substance consists of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, methoxypolyethylene glycol (mPEG) addition phosphatidylethanolamine, phosphatidic acid, sphingomyelin, cardiolipin, and plasmamarogen. The endoplasmic reticulum is at least one or more phospholipids selected from the group. The method of claim 5,
The phosphatidylcholine, egg yolk phosphatidylcholine, hydrogenated egg yolk phosphatidylcholine, soybean phosphatidylcholine, hydrogenated soybean phosphatidylcholine, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dispalmitoyl phosphatidylcholine, disoalloyl phosphatidylcholine -Oleoyl phosphatidylcholine, dirinoleoyl phosphatidylcholine, dilinolenoyl phosphatidylcholine, didecanoyl phosphatidylcholine, diethylcoyl phosphatidylcholine, dieticosanoyl phosphatidylcholine, diecosatrienoyl phosphatidylcholine, dieicosapentaenoyl phosphatienoyl phosphatienoyl Phosphatidylcholine and didicosahexaenoyl phosphatidylcholine,
The phosphatidyl ethanolamine is egg yolk phosphatidyl ethanolamine, hydrogenated egg yolk phosphatidyl ethanolamine, soybean phosphatidyl ethanol amine, hydrogenated soybean phosphatidyl ethanol amine, dilauroyl phosphatidyl ethanol amine, dimyristoyl phosphatidyl ethanol amine, dipalmitoyl phosphatidyl ethanol amine Ethanolamine, distearoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, palmitoyl-oleoyl phosphatidylethanolamine, dilinoleoyl phosphatidylethanolamine, dilinolenoyl phosphatidylethanolamine, didecanoyl phosphatidylethanolamine, Diethylcoyl phosphatidylethanolamine, diethasanoyl phosphatidylethanolamine, diethasatrienoyl phosphatidylethanolamine, dieticosapentaenoyl phosphatidylethanolamine, dietocosaenoyl phosphatidylethanolamine and dietocosahexaenoyl phosphine Selected from the group consisting of patylethanolamine,
The phosphatidylglycerol, egg yolk phosphatidylglycerol, hydrogenated egg yolk phosphatidylglycerol, soybean phosphatidylglycerol, hydrogenated soybean phosphatidylglycerol, dilauroyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidyl Glycerol, Dioleoyl Phosphatidylglycerol, Palmitoyl-Oleoyl Phosphatidylglycerol, Dilinoleoyl Phosphatidylglycerol, Dilinolenoyl Phosphatidylglycerol, Didecanoyl Phosphatidylglycerol, Dieticoyl Phosphatidylglycerol, Dieticosanyl Phosphatidylglycerol Selected from the group consisting of trienoyl phosphatidylglycerol, diecosapentaenoyl phosphatidylglycerol, didicosaenoyl phosphatidylglycerol and didocasahexaenoyl phosphatidylglycerol Selected,
The phosphatidylserine, egg yolk phosphatidylserine, hydrogenated egg yolk phosphatidylserine, soybean phosphatidylserine, hydrogenated soybean phosphatidylserine, dilauroyl phosphatidylserine, dimyristoyl phosphatidylserine, dispalmitoyl phosphatidylserine, distearoyl phosphatidyl Serine, dioleoyl phosphatidylserine, palmitoyl-oleoyl phosphatidylserine, dirinoleoyl phosphatidylserine, dilinolenoyl phosphatidylserine, diedecanoyl phosphatidylserine, dieticoyl phosphatidylserine, dieticosanyl phosphatidylserine Trienoyl phosphatidylserine, diecosapentaenoyl phosphatidylserine, didicosaenoyl phosphatidylserine and didicosahexaenoyl phosphatidylserine,
The phosphatidyl inositol, egg yolk phosphatidyl inositol, hydrogenated egg yolk phosphatidyl inositol, soybean phosphatidyl inositol, hydrogenated soybean phosphatidyl inositol, dilauroyl phosphatidyl inositol, dimyristoyl phosphatidyl inositol, dipalmitoyl phosphatidyl inositol, disteamiloyl phosphatidylinositol Inositol, dioleoyl phosphatidyl inositol, palmitoyl-oleoyl phosphatidyl inositol, diolinoleyl phosphatidyl inositol, dilinolenoyl phosphatidyl inositol, diedecanoyl phosphatidyl inositol, diethylcoyl phosphatidyl inositol, dietycosinoyl Selected from the group consisting of trienoyl phosphatidyl inositol, diecosapentaenoyl phosphatidyl inositol, didicosaenoyl phosphatidyl inositol and didicosahexaenoyl phosphatidyl inositol Selected,
The said methoxy polyethylene glycol (mPEG) addition phosphatidyl ethanolamine is mPEG2000- egg yolk phosphatidyl ethanolamine, mPEG3400- egg yolk phosphatidyl ethanolamine, mPEG5000- egg yolk phosphatidyl ethanolamine, mPEG2000- hydrogenated egg yolk phosphatidyl ethanolamine, mPEG3400- hydrogenated egg yolk Ethanolamine, mPEG5000-hydrogenated egg yolk phosphatidylethanolamine, mPEG2000- soybean phosphatidylethanolamine, mPEG3400- soybean phosphatidylethanolamine, mPEG5000- soybean phosphatidylethanolamine, mPEG2000- hydrogenated soybean phosphatidylethanolamine, mPEG3400- hydrogenated soybeans , mPEG5000-hydrogenated soybean phosphatidylethanolamine, mPEG2000-dilauroyl phosphatidylethanolamine, mPEG3400-dilauroyl phosphatidylethanolamine, mPEG5000-dilauroyl phosphatidylethanolamine, mPEG2000-dimyristoyl phosphatidylethanolamine, mPEG3400-Dimitristo Phosphatidylethanolamine, mPEG5000-dimyristoyl phosphatidylethanolamine, mPEG2000-dipalmitoyl phosphatidylethanolamine, mPEG3400-dipalmitoyl phosphatidylethanolamine, mPEG5000-dipalmitoyl phosphatidylethanolamine, mPEG2000-distearoyl phosphatidylethanamine , mPEG3400-distearoyl phosphatidylethanolamine, mPEG5000-distearoyl phosphatidylethanolamine, mPEG2000-dioleoyl phosphatidylethanolamine, mPEG3400-dioleoyl phosphatidylethanolamine and mPEG5000-dioleoyl phosphatidylethanolamine Is selected from
The phosphatidic acid, egg yolk phosphatidic acid, hydrogenated yolk phosphatidic acid, soybean phosphatidic acid, hydrogenated soybean phosphatidic acid, dilauroyl phosphatidic acid, dimyristoyl phosphatidic acid, dipalmitoyl force Fatty Acid, Distearoyl Phosphate Acid, Dioleoyl Phosphate Acid, Dilinoleyl Oil Phosphate Acid, Palmitoyl-Oleoyl Phosphate Acid, Dilinolenoyl Phosphate Acid, Didecanoyl Phosphate Acid, Consisting of dietycoyl phosphatidic acid, diecosanoyl phosphatidic acid, dieticosatrienoyl phosphatidic acid, diecosapentaenoyl phosphatidic acid, didicosaenoyl phosphatidic acid and didicosahexaenoyl phosphatidic acid Selected from the group,
The sphingomyelin is egg yolk-derived sphingomyelin or milk-derived sphingomyelin,
The cardiolipin, egg yolk cardiolipin, hydrogenated egg yolk cardiolipin, soy cardiolipin, hydrogenated soy cardiolipin, dilauroyl cardiolipin, dimyristoyl cardiolipin, dipalmitoyl car Diolipine, Distearoyl Cardiolipin, Dioleoyl Cardiolipin, Palmitoyl-Oleoyl Cardiolipin, Dilinoleyl Oil Cardiolipin, Dilinolenoyl Cardiolipin, Didecanoyl Cardiolipin, Consisting of dietycoyl cardiolipin, diecosanoyl cardiolipin, diecosartenoyl cardiolipin, diecosapentaenoyl cardiolipin, didicosaenoyl cardiolipin, and didicosahexaenoyl cardiolipin Endoplasmic reticulum characterized by being selected from the group. The method of claim 1,
The vesicles characterized in that the cationic material is a cationic polymer. The method of claim 7, wherein
The vesicles, characterized in that the cationic polymer is polyethyleneimine or polylysine. The method of claim 1,
The vesicles characterized in that the anionic substance is an acidic phospholipid. 10. The method of claim 9,
The said phospholipid contains at least 1 sort (s) chosen from the group which consists of phosphatidylglycerol, phosphatidylserine, phosphatidyl inositol, methoxy polyethyleneglycol addition phosphatidylethanolamine, and phosphatidic acid. The method of claim 10,
The phosphatidylglycerol, egg yolk phosphatidylglycerol, hydrogenated egg yolk phosphatidylglycerol, soybean phosphatidylglycerol, hydrogenated soybean phosphatidylglycerol, dilauroyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidyl Glycerol, Dioleoyl Phosphatidylglycerol, Palmitoyl-Oleoyl Phosphatidylglycerol, Dilinoleoyl Phosphatidylglycerol, Dilinolenoyl Phosphatidylglycerol, Didecanoyl Phosphatidylglycerol, Dieticoyl Phosphatidylglycerol, Dieticosanyl Phosphatidylglycerol Selected from the group consisting of trienoyl phosphatidylglycerol, diecosapentaenoyl phosphatidylglycerol, didicosaenoyl phosphatidylglycerol and didocasahexaenoyl phosphatidylglycerol Selected,
The phosphatidylserine, egg yolk phosphatidylserine, hydrogenated egg yolk phosphatidylserine, soybean phosphatidylserine, hydrogenated soybean phosphatidylserine, dilauroyl phosphatidylserine, dimyristoyl phosphatidylserine, dispalmitoyl phosphatidylserine, distearoyl phosphatidyl Serine, dioleoyl phosphatidylserine, palmitoyl-oleoyl phosphatidylserine, dirinoleoyl phosphatidylserine, dilinolenoyl phosphatidylserine, diedecanoyl phosphatidylserine, dieticoyl phosphatidylserine, dieticosanyl phosphatidylserine Trienoyl phosphatidylserine, diecosapentaenoyl phosphatidylserine, didicosaenoyl phosphatidylserine, didicosahexaenoyl phosphatidylserine,
The phosphatidyl inositol, egg yolk phosphatidyl inositol, hydrogenated egg yolk phosphatidyl inositol, soybean phosphatidyl inositol, hydrogenated soybean phosphatidyl inositol, dilauroyl phosphatidyl inositol, dimyristoyl phosphatidyl inositol, dipalmitoyl phosphatidyl inositol, disteamiloyl phosphatidylinositol Inositol, dioleoyl phosphatidyl inositol, palmitoyl-oleoyl phosphatidyl inositol, diolinoleyl phosphatidyl inositol, dilinolenoyl phosphatidyl inositol, diedecanoyl phosphatidyl inositol, diethylcoyl phosphatidyl inositol, dietycosinoyl Selected from the group consisting of trienoyl phosphatidyl inositol, diecosapentaenoyl phosphatidyl inositol, didicosaenoyl phosphatidyl inositol and didicosahexaenoyl phosphatidyl inositol Selected,
The said methoxy polyethylene glycol (mPEG) addition phosphatidyl ethanolamine is mPEG2000- egg yolk phosphatidyl ethanolamine, mPEG3400- egg yolk phosphatidyl ethanolamine, mPEG5000- egg yolk phosphatidyl ethanolamine, mPEG2000- hydrogenated egg yolk phosphatidyl ethanolamine, mPEG3400- hydrogenated egg yolk Ethanolamine, mPEG5000-hydrogenated egg yolk phosphatidylethanolamine, mPEG2000- soybean phosphatidylethanolamine, mPEG3400- soybean phosphatidylethanolamine, mPEG5000- soybean phosphatidylethanolamine, mPEG2000- hydrogenated soybean phosphatidylethanolamine, mPEG3400- hydrogenated soybeans , mPEG5000-hydrogenated soybean phosphatidylethanolamine, mPEG2000-dilauroyl phosphatidylethanolamine, mPEG3400-dilauroyl phosphatidylethanolamine, mPEG5000-dilauroyl phosphatidylethanolamine, mPEG2000-dimyristoyl phosphatidylethanolamine, mPEG3400-Dimitristo Phosphatidylethanolamine, mPEG5000-dimyristoyl phosphatidylethanolamine, mPEG2000-dipalmitoyl phosphatidylethanolamine, mPEG3400-dipalmitoyl phosphatidylethanolamine, mPEG5000-dipalmitoyl phosphatidylethanolamine, mPEG2000-distearoyl phosphatidylethanamine , mPEG3400-distearoyl phosphatidylethanolamine, mPEG5000-distearoyl phosphatidylethanolamine, mPEG2000-dioleoyl phosphatidylethanolamine, mPEG3400-dioleoyl phosphatidylethanolamine and mPEG5000-dioleoyl phosphatidylethanolamine Is selected from
The phosphatidic acid, egg yolk phosphatidic acid, hydrogenated yolk phosphatidic acid, soybean phosphatidic acid, hydrogenated soybean phosphatidic acid, dilauroyl phosphatidic acid, dimyristoyl phosphatidic acid, dipalmitoyl force Fatty Acid, Distearoyl Phosphate Acid, Dioleoyl Phosphate Acid, Dilinoleyl Oil Phosphate Acid, Palmitoyl-Oleoyl Phosphate Acid, Dilinolenoyl Phosphate Acid, Didecanoyl Phosphate Acid, Consisting of dietycoyl phosphatidic acid, diecosanoyl phosphatidic acid, dieticosatrienoyl phosphatidic acid, diecosapentaenoyl phosphatidic acid, didicosaenoyl phosphatidic acid and didicosahexaenoyl phosphatidic acid Endoplasmic reticulum, characterized in that selected from the group. The method of claim 1,
The vesicles characterized in that the vitamin E includes at least one or more selected from the group consisting of α-tocopherol, β-tocopherol, γ-tocopherol and δ-tocopherol. The method of claim 1,
The vesicles of the vitamin E is dl-α-tocopherol. The method according to any one of claims 1 to 13,
The vesicles of which the said cationic substance is contained in 10 to 50% of range of the number-of-moles of the whole substance which comprises a ER. The method according to any one of claims 1 to 14,
The vesicles are characterized in that the vitamin E is adsorbed or chemically bonded in the range of 3 to 30% of the total number of moles of the material constituting the endoplasmic reticulum. The method according to any one of claims 1 to 14,
The vesicles are characterized in that the vitamin E is adsorbed or chemically bonded in the range of 5 to 20% of the total number of moles of the material constituting the endoplasmic reticulum. The method according to any one of claims 1 to 16,
A vesicle, characterized in that the particle size of the endoplasmic reticulum is 50 ~ 150 nm range. The method according to any one of claims 1 to 17,
The endoplasmic reticulum encapsulates at least one or more bioactive substances selected from the group consisting of plasmid DNA, siRNA, miRNA, antisense oligonucleotides, antiviral agents and cytotoxic substances, and / or the bioactive substances are incorporated into the components of the endoplasmic reticulum. Vesicles characterized in that they bind hydrophobicly or electrostatically. The method of claim 18,
An endoplasmic reticulum, characterized in that the zeta potential of the endoplasmic reticulum is anionic. The method of claim 18,
The vesicles, characterized in that the bioactive substance is an antiviral agent. The method of claim 18,
The endoplasmic reticulum comprises at least one or more selected from the group consisting of metabolic antagonists, platinum-based agents, alkylating agents, plant alkaloids, molecular target drugs, biological response modifiers and anti-cancer antibiotics. The method of claim 21,
The metabolic antagonists include enositabine, capecitabine, carmopur, cladribine, gemcitabine, cytarabine, cytarabine oxphosphate, tegapur, tegapur and uracil, tegapur and kimerasil. Is selected from the group consisting of utereracil potassium, doxyfluidine, ellarabine, hydroxycarbamide, fluorouracil, fludarabine, pemetrexed, pentostatin, mercaptopurine and methotrexate,
The platinum agent is selected from the group consisting of oxaliplatin, carboplatin, cisplatin and nedaplatin,
The alkylating agent is selected from the group consisting of ifosfamide, cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, melphalan and rannimustine,
The plant alkaloid is selected from irinotecan, etoposide, sorbic acid, docetaxel, nogitecan, paclitaxel, vinorelbine, vincristine, vindesine, vinblastine, and the molecular target drug is imatinib, erlotinib, Snitinib, cetuximab, sorafenib, panitumumab, bevacizumab, voltezomib,
The biological response modulator is selected from the group consisting of interferon-α, interferon-β, interferon-γ, interleukin,
The anticancer antibiotic is characterized in that the vesicles are selected from the group consisting of aclarubicin, epirubicin, daunorubicin, doxorubicin, pyrarubicin, bleomycin, mitomycin C and mitoxantrone. 23. A kit used for delivering a physiologically active substance to hepatic parenchymal cells in the presence of serum, comprising the endoplasmic reticulum according to any one of claims 18 to 22 and a pharmaceutically acceptable carrier. A method of delivering a physiologically active substance to hepatic parenchymal cells in a subject, comprising the step of administering the vesicles according to any one of claims 18 to 22 to the subject.

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