KR100909786B1 - Novel cationic lipids modified with oligopeptides, preparation methods thereof, and carriers comprising the same - Google Patents

Abstract

The present invention provides novel cationic lipids modified with oligopeptides, methods for their preparation and nucleic acid carriers comprising the same. Cationic lipids of the present invention are used for the preparation of nucleic acid carriers. The cationic lipids of the present invention have high economical efficiency in mass production due to the simple manufacturing and purification process. In addition, the nucleic acid carrier including the cationic lipid of the present invention significantly enhances the efficiency of transporting oligonucleotide medicines such as ribonucleic acid, small interfering ribonucleic acid, antisense oligonucleic acid, nucleic acid aptamer, etc. to be delivered into the cell. In addition to reducing the cytotoxicity, it is useful for the purpose of enhancing the therapeutic efficacy of nucleic acid medicine.

Description

Oligo peptide-derived novel cationic lipid, a preparation method of the same and a delivery system comprising the same

The present invention relates to novel cationic lipids modified with oligopeptides, methods for their preparation, and carriers comprising the same.

In the mid-1960s, attempts have been made to treat various genetic disorders caused by gene therapy since a technique has been proposed that can be used to treat hereditary diseases by inserting normal gene sequences into cells of hereditary patients with incomplete gene sequences. Recently, as medicinal uses of various nucleic acid materials such as plasmid deoxyribonucleic acid (small DNA), small interfering double-stranded ribonucleic acid (siRNA), microribonucleic acid (micro RNA), antisense oligonucleotide, etc. have been identified, The importance of nucleic acid delivery materials for efficiently delivering these nucleic acid materials into cells has also increased.

Various techniques and carriers, particularly vectors, have been proposed to transfer genes into various cells, but techniques used for delivery or expression of nucleic acids can be largely divided into methods using viral and non-viral vectors. In particular, cationic liposomes or cationic polymers in the non-viral vector have been studied as gene carriers because they have a characteristic of forming a complex by binding to the anionic charge of a gene by positive charges due to structural features.

Gene delivery methods using such synthetic materials are simpler to manufacture than viral gene transporters using lentiviruses or adenoviruses, and are not limited in size to the genes to be delivered, and are repeatedly administered by viral capsid proteins. There is little immune side effect, no safety problems caused by the genes owned by the virus itself, and there is a commercially advantageous advantage in terms of manufacturing cost or time.

Cationic polymer transporter in the gene transfer method using a cationic material, DEAE dextran, polylysine of repeating lysine amino acid, polyethyleneimine of repeating ethyleneimine, polyamidoamine (polyamidoamine) ( US Patent No. 6,020,457) has been studied as a transporter of genes. However, it has been pointed out that in the case of transporters using these cationic polymers, transport efficiency into cells in the body is lower than that in viral transporters that are effectively transported through receptors on the cell surface. On the other hand, in the case of cationic phospholipid transporters, nanocharged particles such as cationic liposomes are prepared by mixing positively charged lipids in a phospholipid composition and mixing these particles with genes to complex positively charged phospholipid particles with genes. Has been used to enhance the expression of genes by treating the cell lines (US Pat. No. 5,858,784).

Conventionally developed cationic lipids have amines such as primary amine, secondary amine, tertiary amine, or quarternary ammonium salt in neutral fatty acid chains. The method of combining compounds to impart cationicity was used. Early cationic lipids for nucleic acid delivery include N- [1- (2,3-dioleyloxy) propyl] -N, N, N-triethyl, a cationic quaternary amino lipid synthesized by Dr. Felgner's team in 1987. Ammonium chloride (N- [1- (2,3-dioleyloxy) propyl] -N, N, Ntriethylammonium chloride; DOTMA). DOTMA is used for gene delivery by forming cationic liposomes with dioleoylphosphatidylethanolamine (DOPE), which is known to have cell membrane fusion activity. The hydrophobic group of DOTMA is an aliphatic group of 18 carbon atoms having a double bond, and a quaternary ammonium salt is bonded to the opposite side of the hydrophobic group chemically linked through a spacer arm and an ether linker bond. . In the case of DOTMA, gene transfer efficiency is relatively high, but it is cytotoxic and has the disadvantage of having to go through the synthesis process of various processes.

In order to solve the high toxicity of DOTMA and increase the efficiency of intracellular nucleic acid delivery, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide (1,2-dimyristyloxypropyl-3-), another form of DOTMA derivative dimethylhydroxyethyl ammonium bromide; DM RIE), N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium methyl sulfate (N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium methyl sulfate; DOTAP), 2,3-dioleyloxy-N- [2- (sperminecarboxyamide) ethyl] -N, N-dimethyl-1-propane ammonium trifluoro Acetate (2,3-dioleyloxy-N- [2-sperminecarboxyamide) ethyl] -N, N-dimethyl-1-propane ammonium trifluoroacetate; DOSPA).

In addition, 3β- [N- (N ', N'-dimethylaminoethane) carbamoyl] cholesterol (cholesterol derivative) (3β- [N- (N', N'-dimethylaminoethane) carbamoyl] cholesterol; DC-Chol ), Dimethyl-dioctadecyl ammonium bromide (DDAB), N- (α-trimethylammonioacetyl) -didodecyl-D-glutamate chloride (N- (α-trimethylammonioacetyl)-didodecyl-D- Glutamate chloride (TMAC) and dioctadecyl amidoglysylspermine (DOGS) have been synthesized and used for the delivery of nucleic acid substances such as deoxyribonucleic acid. These cationic lipids are categorized by quaternary amines, tertiary amines, or hydroxyethylated quaternary amines (such as DC-Chol, DDAB, TMAC, etc.) and spermine (such as DC-Chol, DDAB, TMAC, etc.) connected to the head portion of the lipid. Polyamines, such as spermine, can be classified into different types (DOGS), which provide several positive charges.

Another example of a cationic lipid used for nucleic acid delivery is a surfactant, which is a quarternary ammonium salt. These are surfactants such as single chains such as cetrimethylammonium bromide or double chains such as dimethyldioctadecyl ammonium bromide, all of which are capable of delivering nucleic acids to animal cells. Do. For these quaternary ammonium salt cationic lipids, the amine groups belonging to the amphiphile are quaternary, with a single chain of lipids attached to the primary amine groups without spacer arms or linker bonds. have. However, formulations using these amphipathic surfactants also generally have serious problems with cytotoxicity. Other amphiphiles include 1,2-dioleoyl-3- (4'-trimethylammonio) butanoyl-glycerol similar in structure to DOTMA (1,2-dioleoyl-3- (4'-trimethylammonio) butanoyl- sn-glycerol), cholesteryl (4'-trimethylammonino) butanoate, 1,2-dioleoyl-3-succinyl-glycerol choline ester (1,2 -dioleoyl-3-succinylsn-glycerol choline esters. These amphiphiles generally have low intracellular nucleic acid delivery efficiency.

Cationic lipids in non-viral vectors are simpler to manufacture than viral nucleic acid transporters such as lentiviruses and adenoviruses, have less immune side effects upon repeated doses by viral capsid proteins, and the genes owned by the virus itself. It does not pose a safety problem due to the body, there is an industrially advantageous advantage in the manufacturing cost or manufacturing process. However, many of the previously disclosed cationic lipids for nucleic acid delivery still need to be complemented in terms of synthetic methods, cytotoxicity and intracellular nucleic acid delivery efficiency. Therefore, there is a need for the development of a technology that delivers nucleic acid materials efficiently into cells while having a short synthetic process and low cytotoxicity.

Therefore, the present inventors synthesized a new cationic lipid using an oligopeptide having a positive charge to increase the intracellular delivery efficiency of nucleic acid and the like. In the case of a nucleic acid carrier including the cationic lipid, the delivery of nucleic acid in human cancer cell lines The present invention was completed by confirming that not only significantly increased the efficiency but also significantly reduced the cytotoxicity.

It is an object of the present invention to provide novel cationic lipids modified with oligopeptides, methods for their preparation and transporters comprising the same. Cationic lipids of the present invention can be formulated into various forms of nucleic acid carriers and used to enhance delivery of the desired material into cells.

The present invention provides cationic lipids of the general formula (I).

Wherein n is an integer of 3 to 6,

R is a saturated or unsaturated hydrocarbon radical having 12 to 20 carbon atoms, preferably oleyl, myristyl, palmityl, stearyl, lauryl, linoleyl ( linoleyl), and arachidyl.

The cationic lipid of the present invention represented by general formula (I) is formed by combining a cationic amino acid group, preferably an oligolysine group, with a saturated or unsaturated fatty acid amine derivative having 12 to 20 carbon atoms. The cationic lipid of the present invention is an amphiphilic compound composed of a hydrophilic amino acid group and a hydrophobic fatty acid moiety, and is an amide bond between a carboxyl group (-COOH) of an amino acid and an amine group (-NH ₂ ) of a fatty acid amine derivative. Is done. As the amino acid group constituting the cationic lipid of the present invention, oligolysine having 3 to 6 lysine (Lysine, K) among amino acids having a positive charge is preferable.

The present invention also relates to a carboxyl group (-COOH) of oligolysine represented by the following general formula (II)

The amine group (-NH2) of the fatty acid amine derivative represented by the following general formula (III)

H ₂ NR (III)

It provides a method for producing a cationic lipid of formula (I) comprising the step of connecting by an amide bond, wherein m is an integer of 1 to 4, R is a saturated or unsaturated hydrocarbon having 12 to 20 carbon atoms It is a radical.

The fatty acid amine derivative represented by the formula (III) is oleylamine, oleylamine, myristylamine, palmitylamine, stearylamine, laurylamine, linoleylamine linoleylamine, and arachidylamine.

The present invention also provides cationic lipids prepared by the above method.

Cationic lipids of the present invention can be synthesized in high yield by a simple process using amino acids that are components of biological proteins. In the case of the cationic lipid of the present invention, since the amine group of the oligo lysine is present in the form of a positive charge in the neutral region pH of a normal in vivo environment, the cationic lipid of the general formula (I) generally has a positively charged state in the cell. Will have. The cationic charge of the cationic lipids makes it possible to form complexes with various nucleic acid materials that are negatively charged at neutral pH and also increase contact with target cell membranes having relatively negative charges in vivo. .

The present invention therefore also provides a nucleic acid delivery system, preferably an oligonucleic acid delivery system, into a cell comprising said cationic lipid of formula (I). In the present invention, a nucleic acid carrier refers to a nucleic acid carrier medium capable of forming a complex that can normally enter a cell by binding to the nucleic acid by interaction with a negatively charged nucleic acid sequence.

In the present invention, nucleic acids include ribonucleic acid (RNA), small interfering RNA (small interfering RNA), antisense oligonucleotide, nucleic acid aptamer and the like.

Cationic lipids of the present invention can be used in various forms of nucleic acid delivery formulations such as liposomes, micelles, emulsions, nanoparticles, etc. for nucleic acid delivery into cells.

In embodiments of the invention, nucleic acid carriers comprising cationic lipids of the invention are ribonucleic acid (RNA), small interfering RNA, antisense oligonucleotides, nucleic acid aptamers, and the like. Mediates delivery of oligonucleotides comprising cells into cells.

In addition to the cationic lipid component of the present invention, the nucleic acid carrier may further include one or more selected from the group consisting of galactose lipid derivatives, mannose lipid derivatives, folate lipid derivatives, polystyrene glycol lipid derivatives, and biotin lipid derivatives.

In one embodiment of the invention, the nucleic acid carrier may be made of a liposome formulation containing the cationic lipids and cell fusion phospholipids. Such cell fusion phospholipids can be, for example, dioleoylphosphatidylethanolamine (DOPE), or 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (1,2-diphytanoyl-sn- glycero-3-phospho ethanolamine).

In another embodiment of the present invention, the nucleic acid carrier may be composed of a micelle formulation additionally containing a surfactant in the cationic lipid. Surfactants that can be used in the micelle formulation are, for example, Tween 20, polyethylene glycol monooleyl ether, ethylene glycol monododecyl ether, diethylene glycol mono Hexyl ether (diethylene glycol monohexyl ether), trimethylhexadecyl ammonium chloride, dodecyltrimethyl ammonium bromide, cyclohexylmethyl β-D-maltoside, pentaerythryl palmitate (pentaerythrityl palmitate), lauryldimethylamine-oxide, or laurosalcosine sodium salt (N-lauroylsarcosine sodium salt) and the like.

In another embodiment of the present invention, the nucleic acid carrier may be composed of an emulsion formulation additionally containing a surfactant in the cationic lipid. Surfactants that can be used in emulsion formulations are classified into cationic, zwitterionic, nonionic, and the like. As the cationic surfactant, for example, cetyl trimethylammonium bromide, hexadecyl trimethyl ammonium bromide, etc. may be used, and as amphoteric surfactant, for example, dodecyl betaine betaine), dodecyl dimethylamine oxide, dimethylpalmitoylammonoprovane sulfonate (3- (N, N-dimethylpalmitylammonio) propane sulfonate) and the like can be used. Tween 20, Tween 80, Triton-X-100, polyethylene glycol monooleyl ether, triethylene glycol monododecyl ether ( triethylene glycol monododecyl ether), octyl glucoside, and nonanoylmethylglucamine (N-nonanoyl-N-methyl glucamine) can be used.

Nucleic acid carriers consisting of formulations of cationic liposomes, micelles, emulsions, nanoparticles and the like according to the invention can significantly enhance the transport efficiency of the desired nucleic acid in animal cells and also reduce the toxicity to the cells.

Nucleic acid carriers containing cationic lipids of the present invention can be delivered to any animal cell, depending on the intended use of the nucleic acid to be delivered. In the following examples, the tumor cells of the nucleic acid carrier (HeLa cell line, which is a human cervical cancer epithelial cell, A549 cell line, which is a lung cancer cell, Huh-7, a hepatic cancer cell line, WM266.4, a melanoma, and a U87 cell line, which are glioma) Nucleic acid delivery efficiency).

Block IT (Invitrogen, USA) double-stranded ribonucleic acid with fluorescent labeling forms complexes with various formulations containing cationic lipids and delivers them into cells, and when observed under a fluorescence microscope, delivers the cationic lipids into cells. Specifically, the nucleic acid transport ability can be measured. In addition, the cytotoxicity of the nucleic acid carrier of the present invention can be evaluated using the color development method (MTT) using tetrazolium 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide.

Nucleic acid carriers such as liposomes, micelles, emulsions, nanoparticles, and the like comprising the cationic lipids described in the present invention not only increase the intracellular transport of nucleic acids in various cells but also significantly reduce cytotoxicity. In addition, the present invention can be effectively used for treatment with oligonucleotide medicines such as small interfering ribonucleic acid, antisense oligonucleotide, and nucleic acid aptamer.

The present invention also provides a complex of the nucleic acid carrier and the nucleic acid, preferably a complex of the oligonucleotide carrier and the oligo nucleic acid. Nucleic acid carriers of the present invention having a formulation of liposomes, micelles, emulsions, etc. are positively charged due to cationic lipids, and thus, nucleic acid through electrostatic bonding by a simple mix by positive charge of nucleic acid carriers and anionic charge of nucleic acid. Complexes between the carrier and the nucleic acid can be efficiently formed.

The complex of nucleic acid carrier and nucleic acid may be introduced into cells for the treatment of various diseases caused by overexpression of pathogenic proteins including tumors, arthritis, cardiovascular, endocrine diseases and the like. The nucleic acid carrier of the present invention is excellent in nucleic acid delivery efficiency and low in cytotoxicity, thereby inhibiting overexpression of pathogenic proteins in cells, thereby achieving an excellent disease treatment effect.

Accordingly, the present invention also provides a nucleic acid medicament therapeutic agent comprising the complex of the nucleic acid carrier and the nucleic acid as an active ingredient, the use of the nucleic acid carrier and the complex of the nucleic acid for the manufacture of a nucleic acid medicament therapeutic agent and a therapeutically effective amount of the complex of the nucleic acid carrier and the nucleic acid. It provides a method for the treatment of various diseases caused by overexpression of pathogenic proteins, including tumors, arthritis, cardiovascular, endocrine diseases, and the like, comprising introducing into a cell of a subject.

The introduction of a desired nucleic acid into a cell in vivo or ex vivo through the nucleic acid medicament treatment of the present invention serves to selectively reduce the expression of the target protein or to correct for mutations in the target gene. It is possible to treat diseases caused by expression or diseases caused by target genes. In the present invention, the therapeutically effective amount of the complex of the nucleic acid carrier and the nucleic acid means an amount required for administration in order to expect a therapeutic effect of the disease. Therefore, the type of disease, the severity of the disease, the type of nucleic acid to be administered, the type of formulation, the age, weight, general health, sex and diet of the patient, the time of administration, the route of administration and the duration of treatment, the chemotherapy drug used simultaneously, etc. It can be adjusted according to various factors including a drug. It is preferable to administer the nucleic acid medicament therapeutic agent to an adult at a dose of 0.001 mg / kg to 100 mg / kg once daily.

The cationic lipids of the present invention can also be used as components of diagnostic kits using nucleic acid aptamers in vitro. For example, coating the surface of a diagnostic plate with cationic lipids and binding aptamers on these coating surfaces can be used to diagnose the presence of substances that selectively react with aptamers in a sample. The present invention therefore provides a diagnostic kit comprising a plate coated with the cationic lipid of the present invention. An aptamer may be bound to the cationic lipid coating side of the diagnostic kit.

As described above, the cationic lipid of the present invention is easy to manufacture and purify, and thus economical in mass production. In addition, the nucleic acid carrier comprising the cationic lipid of the present invention not only significantly enhances the efficiency of transporting oligonucleotide medicines such as desired ribonucleic acid, small interfering ribonucleic acid, antisense oligonucleic acid, nucleic acid aptamer, etc. into cells. In addition, it is useful for the purpose of reducing the cytotoxicity to enhance the therapeutic efficacy of the drug based nucleic acid or protein.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the technical field to which the present invention pertains. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

Synthesis of New Cationic Lipids

Example 1 Synthesis of Oleyl Trilysinamide

1-1). Trilysine (1g, 2.48mmole, PEPTRON, Inc., Korea) was dissolved in ice water in a ratio of H ₂ O: p -Dioxane (Sigma-Aldrich, USA) = 1: 10, followed by DMAP (4- (Dimethylamino) pyridine ) (0.91g, 7.45mmole, FLUKA, USA), (Boc) ₂ O (Di-tert-butyl pyrocarbonate) (1.62g, 7.45mmole, FLUKA, USA) is added and stirred for 12 hours. The product obtained after the reaction was concentrated under reduced pressure, dissolved in ethyl acetate (ethylacetate, Sigma-Aldrich, USA), washed three times each with a supersaturated sodium bicarbonate solution and a supersaturated sodium chloride solution to remove unreacted substances. Sigma-Aldrich, USA) and dried to obtain a white solid product. The reaction was confirmed by thin layer chromatography (TLC, thin layer chromatograpy, Merck & Co., Inc. USA).

1-2). The reaction product obtained in Example 1-1 was dissolved in dichloromethane, and then 3 equivalents of DCC (N, N′-Dicyclohexylcarbodiimide, FLUKA, USA) and DMAP (4- (Dimethylamino) pyridine), respectively, were used in an ice water bath for about 1 hour. Stir. 1.5 equivalents of oleylamine (oleukamine, FLUKA, USA) are dissolved in anhydrous dichloromethane (Sigma-Aldrich, USA), slowly added dropwise and reacted at room temperature for 12 hours. After the reaction, dichloromethane was concentrated under reduced pressure to remove the obtained product, and the obtained product was dissolved in ethylacetate, and washed three times with supersaturated sodium bicarbonate solution and supersaturated sodium chloride solution to remove unreacted substances. After removal, the mixture was dried to obtain a colorless liquid product. The reaction was confirmed by TLC.

1-3). Trifluoroacetic acid (TFA, Trifluoreacetic acid, Sigma-Aldrich, USA): dichloromethane = 1: 1 was slowly added dropwise to the reaction product obtained in Example 1-2 under ice water, followed by stirring for about 1 hour. After the reaction, the TFA was removed with ethylacetate, and dried to obtain a slightly yellow liquid product. The reaction was confirmed by TLC.

[Scheme 1]

R in Scheme 1 is oleyl as an unsaturated hydrocarbon radical having 18 carbon atoms.

Example 2 Synthesis of palmityl tetralysinamide

2-1) Tetralysine (1 g, 1.88 mmol, PEPTRON, Inc., Korea) and (Boc) ₂ O (3.08 g, 14.13 mmol) were reacted in the same manner as in Example 1-1 to obtain a lysine derivative product.

2-2) The reaction product obtained in Example 2-1 was dissolved in anhydrous dichloromethane, and then reacted in the same manner as in Example 1-2 using 1.5 equivalents of palmitylamine to obtain a colorless liquid product. . The reaction was confirmed by TLC.

2-3) After the reaction product obtained in Example 2-2 was reacted in the same manner as in Example 1-3, a slightly yellow liquid product was obtained. The reaction was confirmed by TLC.

Scheme 2

R in Scheme 2 is palmityl as a saturated hydrocarbon radical having 16 carbon atoms.

Example 3 linoleyl pentalysinamide

3-1) Pentalysine (1 g, 1.5 mmol, Sigma-Aldrich, USA) and (Boc) ₂ O (2.98 g, 13.66 mmol) were reacted in the same manner as in Example 1-1 to obtain a lysine derivative product.

3-2) The reaction product obtained in Example 3-1 was dissolved in anhydrous dichloromethane, and then reacted in the same manner as in Example 1-2 using 1.5 equivalents of linoleylamine, whereby a colorless liquid product was obtained. . The reaction was confirmed by TLC.

3-3) After the reaction product obtained in Example 3-2 was reacted in the same manner as in Example 1-3, a slightly yellow liquid product was obtained. The reaction was confirmed by TLC.

Scheme 3

R in Scheme 3 is linoleyl as a C18 double unsaturated hydrocarbon radical.

Example 4. Lauryl hexalysinamide

4-1) Reaction with hexalysine (1 g, 1.27 mmol, PEPTRON, Inc., Korea) and (Boc) ₂ O (2.77 g, 12.7 mmol) in the same manner as in Example 1-1 to obtain a lysine derivative product.

4-2) The reaction product obtained in Example 4-1 was dissolved in anhydrous dichloromethane, and then reacted in the same manner as in Example 1-2 using 1.5 equivalents of laurylamine, to thereby give a colorless liquid product. . The reaction was confirmed by TLC.

4-3) After reacting the reaction product obtained in Example 4-2 in the same manner as in Example 1-3, a slightly yellow liquid product was obtained. The reaction was confirmed by TLC.

Scheme 4

R in Scheme 4 is lauryl as a saturated hydrocarbon radical having 12 carbon atoms.

Comparative Example 1. Lauryl lysinamide

1-1) Lysine (1 g, 6.84 mmol, Sigma-Aldrich, USA) and (Boc) ₂ O (4.48 g, 20.5 mmol) were reacted in the same manner as in Example 1-1 to obtain a lysine derivative product.

1-2) The reaction product obtained in Comparative Example 1-1 was dissolved in anhydrous dichloromethane, and then reacted in the same manner as in Example 1-2 using 1.5 equivalents of laurylamine, to thereby give a colorless liquid product. . The reaction was confirmed by TLC.

1-3) The reaction product obtained in Comparative Example 1-2 was reacted in the same manner as in Example 1-3, to give a slightly yellow liquid product. The reaction was confirmed by TLC.

Comparative Example 2. Oleyl Nonalysinamide

2-1) Nonlysine (1 g, 0.85 mmol, PEPTRON, Inc., Korea) and (Boc) ₂ O (2.79 g, 12.8 mmol) were reacted in the same manner as in Example 1-1 to obtain a lysine derivative product.

2-2) The reaction product obtained in Comparative Example 2-1 was dissolved in anhydrous dichloromethane, and then reacted in the same manner as in Example 1-2 using 1.5 equivalents of oleylamine to obtain a colorless liquid product. . The reaction was confirmed by TLC.

2-3) After reacting the reaction product obtained in Comparative Example 2-2 in the same manner as in Example 1-3, a slightly yellow liquid product was obtained. The reaction was confirmed by TLC.

Comparative Example 3 stearyl decalysinamide

3-1) Decalysin (1 g, 0.77 mmol, PEPTRON, Inc., Korea) and (Boc) ₂ O (3.56 g, 15.3 mmol) were reacted in the same manner as in Example 1-1 to obtain a lysine derivative product.

3-2) The reaction product obtained in Comparative Example 3-1 was dissolved in dichloromethane, and then reacted in the same manner as in Example 1-2 using 1.5 equivalents of stearylamine (stearylamine, Sigma-Aldrich, USA), and then colorless. The liquid product of was obtained. The reaction was confirmed by TLC.

3-3) After reacting the reaction product obtained in Comparative Example 3-2 in the same manner as in Example 1-3, a slightly yellow liquid product was obtained. The reaction was confirmed by TLC.

Comparative Example 4. linoleyl argininamide

4-1) After dissolving Boc-Arg (Pbf) -OH (1 g, 1.89 mmol, PEPTRON, Inc., Korea) in anhydrous dichloromethane, Example 1-2 and 1.5 equivalents of linoleylamine were used. After the reaction in the same manner, a colorless liquid product was obtained. The reaction was confirmed by TLC.

4-2) After adding TFA: H ₂ O: TIS (triisopropyl silane, Sigma-Aldrich, USA) = 90: 5: 5 to the reaction product obtained in Comparative Example 4-1, the mixture was stirred for 2 hours. After the reaction, it was dried to give a yellow liquid product. The reaction was confirmed by TLC.

Scheme 5

R in Scheme 5 is linoleyl as a C18 double unsaturated hydrocarbon radical.

Comparative Example 5. myristyl pentaargininamide

5-1) A penta arginine (1 g, 0.46 mmol, PEPTRON, Inc., Korea) having the same protecting group as in Comparative Example 4-1 was dissolved in anhydrous dichloromethane, followed by using 1.5 equivalents of myristylamine. After the reaction in the same manner as in 1-2, a colorless liquid product was obtained. The reaction was confirmed by TLC.

5-2) TFA: H ₂ O: TIS (triisopropyl silane) = 90: 5: 5 was added to the reaction product obtained in Comparative Example 5-1, followed by stirring for 2 hours. After the reaction, it was dried to give a yellow liquid product. The reaction was confirmed by TLC.

Comparative Example 6. stearyl octaargininamide

6-1) Example 1-1, dissolving octaarginine (1 g, 0.29 mmol, PEPTRON, Inc., Korea) having the same protecting group in anhydrous dichloromethane, and then using 1.5 equivalents of stearylamine. After the reaction in the same manner as in 1-2, a colorless liquid product was obtained. The reaction was confirmed by TLC.

6-2) TFA: H ₂ O: TIS (triisopropyl silane) = 90: 5: 5 was added to the reaction product obtained in Comparative Example 6-1, followed by stirring for 2 hours. After the reaction, it was dried to give a yellow liquid product. The reaction was confirmed by TLC.

Comparative Example 7. arachidyl nonaargininamide

7-1) Non-arginine (1 g, 0.26 mmol, PEPTRON, Inc., Korea) having the same protective group as Comparative Example 4-1 was dissolved in anhydrous dichloromethane, followed by using 1.5 equivalents of arachidylamine. After the reaction in the same manner as in 1-2, a colorless liquid product was obtained. The reaction was confirmed by TLC.

7-2) TFA: H ₂ O: TIS (triisopropyl silane) = 90: 5: 5 was added to the reaction product obtained in Comparative Example 7-1, followed by stirring for 2 hours. After the reaction, it was dried to give a yellow liquid product. The reaction was confirmed by TLC.

Comparative Example 8. oleyl dacaargininamide

8-1) A decaarginine (1 g, 0.24 mmol, PEPTRON, Inc., Korea) having the same protecting group as in Comparative Example 4-1 was dissolved in anhydrous dichloromethane, followed by using 1.5 equivalents of oleylamine. After the reaction in the same manner as in 1-2, a colorless liquid product was obtained. The reaction was confirmed by TLC.

8-2) TFA: H ₂ O: TIS (triisopropyl silane) = 90: 5: 5 was added to the reaction product obtained in Comparative Example 8-1, followed by stirring for 2 hours. After the reaction, it was dried to give a yellow liquid product. The reaction was confirmed by TLC.

<Production of Nucleic Acid Carrier Containing Cationic Lipids>

Example 5 Preparation of Cationic Liposomes Containing Oleyl Trilysinamide of Example 1

Oleyl trilysinamide, a cationic lipid prepared in Example 1, and DOPE (Avanti Polar Lipid Inc., USA), a cell-compatible phospholipid, were dissolved in 1 ml of chloroform, respectively, at a molar ratio of 1: 1. The mixture was taken into a Pyrex 10 ml glass diaphragm vial and mixed, followed by rotary evaporation at low speed until all chloroform evaporated in a nitrogen environment to prepare a lipid thin film. In order to prepare lipid multilamellar vesicles, 1 ml of a phosphate buffer solution was added to the thin film, and the vial was sealed at 37 ° C., and then stirred (vortexing) for 3 minutes. To make a uniform size, it was prepared by three passes of a 0.2 μm polycarbonate membrane using a particle homogenizer (extruder, Northern Lipid Inc., Canada). The cationic liposomes obtained were stored at 4 ° C. until use.

Example 6 Preparation of Cationic Liposomes Containing Linoleyl Pentalysinamide of Example 3

A molar ratio of 1: 1 after dissolving linoleyl pentalysinamide, a cationic lipid prepared in Example 3, and DPhPE (Avanti Polar Lipid Inc., USA), a cell phospholipid, were dissolved in 1 ml of chloroform, respectively. After taking into a Pyrex 10ml glass septum vial and mixing, a cationic liposome was prepared in the same manner as in Example 5.

Example 7 Preparation of Cationic Micelles Containing Linoloeyl Pentalysinamide of Example 3

Linoleyl pentalysinamide, a cationic lipid prepared in Example 3, and polyethyleneglycol monooleyl ether, a surfactant, were mixed in a molar ratio of 1: 1, and then the mixed solution and the phosphate buffer solution were mixed. Cationic micelles were prepared by mixing in a volume ratio of 1:10, vortexing several times and using an ultrasonic generator for about 1 minute.

Example 8 Preparation of Cationic Micelles Containing Palmityl Tetralysinamide of Example 2

A cationic lipid prepared in Example 2, palmithyl tetralysinamide and a surfactant, Tween 20, were mixed at a molar ratio of 1: 1, to prepare a cationic micelle in the same manner as in Example 7. It was.

Example 9 Preparation of Cationic Emulsion Containing Lauryl hexalysinamide of Example 4

The cationic lipid lauryl hexalysinamide prepared in Example 4 and Tween 80 were mixed in a molar ratio of 1: 0.1, and the mixture was added to a phosphate buffer solution at a volume ratio of 1:10, and then homogenized. (homogenizer) was used to homogenize for about 2 minutes to prepare an oil-in-water (o / w type) cationic emulsion in which the oil phase was dispersed in the water phase.

Example 10 Preparation of Cationic Emulsions Containing Palmityl Tetralysinamide of Example 2

Palmityl tetralysinamide, a cationic lipid prepared in Example 2, and Tween 20 were mixed at a molar ratio of 1: 0.1, and the mixture was added to a phosphate buffer solution at a volume ratio of 1:10. Cationic emulsion was prepared in the same manner as in 9.

Example 11 Preparation of Cationic Liposomes Containing Palmityl Tetralysinamide and Folic Acid Lipid Derivatives of Example 2

Palmityl tetralysinamide, a cationic lipid prepared in Example 2, DOPE, a cell-compatible phospholipid, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [folate ( After dissolving polyethylene glycol) -2000 (Avanti Polar Lipid Inc., USA) in 1 ml of chloroform, the molar ratio of 1: 1: 0.05 was taken and the folate moiety was present on the surface in the same manner as in Example 5. Cationic liposomes were prepared.

Example 12 Preparation of Cationic Liposomes Containing Lauryl Hexalysinamide and Polyethylene Glycol Lipid Derivatives of Example 4

Lauryl hexalysinamide, a cationic lipid prepared in Example 4, DPhPE, a cell-fusion phospholipid, and 1,2-diacyl-sn-glycero-3, phosphoethanolamine-N- [methoxy (polyethylene glycol) -3000 (Avanti Polar Lipid Inc., USA) was dissolved in 1 ml of chloroform, and then taken at a molar ratio of 1: 1: 0.05, and cationic cationic glycol groups exist on the surface in the same manner as in Example 5. Liposomes were prepared.

Example 13 Preparation of Cationic Micelles Containing Oleyl Trilysinamide and Galactose Lipid Derivatives of Example 1

Oleyl trilysinamide, a cationic lipid prepared in Example 1, cerebroside, a galactose lipid derivative (Avanti Polar Lipid Inc., USA), and a surfactant, Triton X-100, were respectively prepared. Cationic micelles containing galactose moiety on the surface were prepared in the same manner as in Example 7, after mixing in a molar ratio of: 0.05: 1.

Example 14 Preparation of Cationic Emulsion Containing Linoleyl Pentalysinamide of Example 3

Linoleyl pentalysinamide, a cationic lipid prepared in Example 3, and Tween 20 were mixed at a molar ratio of 1: 0.1, and the mixture was added to a phosphate buffer solution at a volume ratio of 1:10. Cationic emulsion was prepared in the same manner as in 9.

Example 15 Preparation of Cationic Liposomes Containing Oleyl Trilysinamide and Polyethylene Glycol Lipid Derivatives of Example 1

Oleyl trilysinamide, a cationic lipid prepared in Example 1, DPhPE, a cell soluble phospholipid, and 1,2-diacyl-sn-glycero-3, phosphoethanolamine-N- [methoxy (polyethylene glycol) -3000 (Avanti Polar Lipid Inc., USA) was dissolved in 1 ml of chloroform, and then taken at a molar ratio of 1: 1: 0.05, and cationic cationic glycol groups exist on the surface in the same manner as in Example 5. Liposomes were prepared.

Comparative Example 9. Existing Cationic Liposomes

Lipofectamine (2000) (Invitrogen, USA), a cationic liposome that is commercially available for conventional nucleic acid delivery experiments, was purchased and used.

Comparative Example 10 Preparation of Cationic Liposomes Containing Lauryl Lysamide In Comparative Example 1

Lauryl lysinamide, a cationic lipid prepared in Comparative Example 1, and DOPE (Avanti Polar Lipid Inc., USA), a cell-compatible phospholipid, were dissolved in 1 ml of chloroform, and then taken in a ratio of 1: 1. Cationic liposomes were prepared in the same manner as in Example 5 after mixing into a Pyrex 10 ml glass septum vial.

Comparative Example 11. Cationic liposome preparation containing myristyl pentaargininamide of Comparative Example 5

Myristyl pentaargininamide, a cationic lipid prepared in Comparative Example 5, and DPhPE (Avanti Polar Lipid Inc., USA), a cell-compatible phospholipid, were dissolved in 1 ml of chloroform, respectively, in a ratio of 1: 1. Cationic liposomes were prepared in the same manner as in Example 5 after mixing into a Pyrex 10ml glass septum vial and mixing.

Comparative Example 12. Preparation of Cationic Liposomes Containing Stearyl octaargininamide of Comparative Example 6

Stearyl octaargininamide, a cationic lipid prepared in Comparative Example 6, and DPhPE (Avanti Polar Lipid Inc., USA), a cell-fusion phospholipid, were dissolved in 1 ml of chloroform, respectively, at a molar ratio of 1: 1. Cationic liposomes were prepared in the same manner as in Example 5 after mixing into a Pyrex 10ml glass septum vial and mixing.

Comparative Example 13 Preparation of Cationic Liposomes Containing Oleyl Nonalysinamide of Comparative Example 2

Oleyl nonalysinamide, a cationic lipid prepared in Comparative Example 2, and DPhPE (Avanti Polar Lipid Inc., USA), a cell-compatible phospholipid, were dissolved in 1 ml of chloroform, respectively, in a ratio of 1: 1. Cationic liposomes were prepared in the same manner as in Example 5 after mixing into a Pyrex 10ml glass septum vial and mixing.

Comparative Example 14. Cationic liposome preparation containing linoleyl argininamide of Comparative Example 4

Linoleyl argininamide, a cationic lipid prepared in Comparative Example 4, and DOPE (Avanti Polar Lipid Inc., USA), a cell-compatible phospholipid, were dissolved in 1 ml of chloroform, respectively, and were taken at a molar ratio of 1: 1. Cationic liposomes were prepared in the same manner as in Example 5 after mixing into a Pyrex 10 ml glass septum vial.

Comparative Example 15 Preparation of Cationic Micelle Containing Stearyl Decalysinamide of Comparative Example 3

Stearyl decalysinamide, a cationic lipid prepared in Comparative Example 3, and tween 20, a surfactant, were taken at a molar ratio of 1: 1, and then mixed, and a cationic micelle was prepared in the same manner as in Example 7. It was.

Comparative Example 16 Preparation of Cationic Emulsion Containing Arachidyl Nonaargininamide of Comparative Example 7

Arachidyl nonaargininamide, a cationic lipid prepared in Comparative Example 7, and Tween 80 were mixed in a molar ratio of 1: 0.1, and the mixture was added to a phosphate buffer solution in a volume ratio of 1:10, and then carried out. A cationic emulsion was prepared in the same manner as in Example 9.

Comparative Example 17 Preparation of Cationic Emulsion Containing Oleyl Dacaargininamide of Comparative Example 8

Oleyl dacaargininamide, a cationic lipid prepared in Comparative Example 8, and Tween 20 were mixed at a molar ratio of 1: 0.1, and the mixture was added to a phosphate buffer solution at a volume ratio of 1:10. Cationic emulsion was prepared in the same manner as in 9.

Comparative Example 18 Preparation of Cationic Liposomes Containing Linoleyl Argininamide and Folic Acid Lipid Derivatives of Comparative Example 4

Linoleoyl argininamide, a cationic lipid prepared in Comparative Example 4, DOPE, a cell-fusion phospholipid, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [folate (polyethylene Cation dissolving glycol) -2000 (Avanti Polar Lipid Inc., USA) in 1 ml of chloroform and taking a molar ratio of 1: 1: 0.05, and a folate moiety is present on the surface in the same manner as in Example 5. Sex liposomes were prepared.

Comparative Example 19 Preparation of Cationic Micelle Containing Lauryl lysinamide and Galactose Lipid Derivative of Comparative Example 1

Lauryl lysinamide, a cationic lipid prepared in Comparative Example 1, cerebroside, a galactose lipid derivative (Avanti Polar Lipid Inc., USA) and Triton X-100, a surfactant, were each 1: After mixing in a molar ratio of 0.05: 1, cationic micelles having a galactose moiety on their surface were prepared in the same manner as in Example 7.

Experimental Example

Cell culture

Human cervical carcinoma epithelial cell HeLa cell line, lung cancer cell A549 cell line, liver cancer cell line Huh-7, melanoma WM266.4 and A375 cell line, glioma U87 cell line are purchased from ATCC (American Type Culture Collection, USA) It was. HeLa, A549 cell lines RPMI (Gibco, USA), Huh-7, A375, U87 cell lines DMEM (Dulbecco's modified eagles medium, Gibco, USA), WM266.4 cell lines to MEM (Minimum Essential Medium, Gibco, USA) Incubated with% fetal calf serum w / v (HyClone laboratories Inc, USA) and 100 unit / ml penicillin or 100 μg / ml streptomycin.

Experimental Example I Evaluation of Nucleic Acid Delivery Efficiency Using Small Interfering Ribonucleic Acid Labeled with Fluorescent Markers

 I-1. Evaluation of Delivery Efficiency of Small Interfering Ribonucleic Acids in WM266.4 Cell Line

50 μl of serum-free medium was added to the Eppendorf tubes when the WM266.4 cell line was seeded in 48 well plates at the day before the experiment and seeded at 3 × 10 μL per well and cells of each plate were grown uniformly by 60-70%. 5 μl of BlockiT (Invitrogen, USA), a small interfering ribonucleic acid substance labeled with a fluorescent marker, and 2 μl of cationic liposomes prepared in Comparative Examples 10, 14 and 5 were added. These were slowly pipetted and mixed and left at room temperature for 20 minutes. The complex thus prepared was added to a well plate and incubated in a CO 2 incubator at 37 ° C. for 24 hours. The medium of the cultured cells was replaced with 200 μl of fresh media per well, and then the ribonucleic acid transfer efficiency was observed under a fluorescence microscope.

Figure 1 shows the nucleic acid transfer efficiency of the cationic liposomes prepared from Comparative Examples 10, 14 and Example 5 with a phase difference and a fluorescence microscope (DM IL, Leica, Germany), Figures 1A and C treated Comparative Examples 10, 14 It is a phase contrast microscope photograph at the time, and FIG. 1E is a phase difference microscope photograph at the time of the composition process of Example 5. FIG. 1B and D are fluorescence micrographs showing intracellular delivery of small interfering ribonucleic acid labeled with fluorescent markers in Comparative Examples 10 and 14, and FIG. 1F is fluorescence micrographs in the composition treatment of Example 5. FIG. From this figure, the cationic liposomes prepared by containing the cationic lipids of the present invention in Example 5 increased the delivery efficiency of small interfering ribonucleic acid in WM266.4 cells than the liposomes of the known composition prepared in Comparative Examples 10 and 14. It can be seen that.

 I-2. Evaluation of Delivery Efficiency of Small Interfering Ribonucleic Acids in HeLa Cell Lines

HeLa cell lines were seeded in 3 wells per 48 well plates on the day before the experiment, and when the cells of each plate were evenly grown by 60-70%, 50 μl of serum-free medium was added to the Eppendorf tube. BlockiT 5 pmole, a small interfering ribonucleic acid substance labeled with a fluorescent marker, and 2 μl of cationic liposomes prepared in Comparative Examples 11, 13 and 6 were added, respectively. These were slowly pipetted and mixed and left at room temperature for 20 minutes. The complex thus prepared was added to a well plate and incubated in a CO 2 incubator at 37 ° C. for 24 hours. The media of the cultured cells were replaced with 200 µl of fresh media per well, and then the delivery efficiency of ribonucleic acid was observed under a fluorescence microscope.

FIG. 2 shows the nucleic acid transfer efficiency of the cationic liposomes prepared from Comparative Examples 11 and 13 and Example 6 using a phase contrast microscope and a fluorescence microscope. FIGS. 2A and C are phase contrast micrographs when the Comparative Examples 11 and 13 were treated. 2E is a phase contrast micrograph at the time of the composition treatment of Example 6. FIG. 2B and D are fluorescence micrographs showing intracellular delivery of small interfering ribonucleic acid labeled with fluorescent markers in Comparative Examples 11 and 13, and FIG. 2F is fluorescence micrographs in the composition treatment of Example 6. FIG. The fact that cationic liposomes prepared with the cationic lipids of the present invention in Example 6 from this figure increases the delivery efficiency of small interfering ribonucleic acid in HeLa cells than liposomes of known composition prepared in Comparative Examples 11 and 13 It can be seen.

I-3. Evaluation of Delivery Efficiency of Small Interfering Ribonucleic Acids in U87 Cell Line

When the U87 cell line was seeded at 3 × 10 mm / well in a 48 well plate on the day before the experiment, and the cells of each plate were uniformly grown by 60-70%, 50 μl of serum-free medium was added to the Eppendorf tube. BlockiT 5 pmole, a small interfering ribonucleic acid substance labeled with a fluorescent marker, and 2 μl of cationic liposomes and micelles prepared in Comparative Examples 12, 19 and 13 were added, respectively. These were slowly pipetted and mixed and left at room temperature for 20 minutes. The complex thus prepared was added to a well plate and incubated in a CO 2 incubator at 37 ° C. for 24 hours. The medium of the cultured cells was replaced with 200 μl of fresh media per well, and then the ribonucleic acid transfer efficiency was observed under a fluorescence microscope.

3 is a phase contrast microscope and a fluorescence microscope of the nucleic acid transfer efficiency of the cationic liposomes and micelles prepared from Comparative Examples 12, 19 and 13, and FIGS. 3A and C are phase contrast micrographs of Comparative Examples 12 and 19. 3E is a phase contrast micrograph at the time of the composition treatment of Example 13. FIG. 3B and D are fluorescence micrographs showing intracellular delivery of small interfering ribonucleic acid labeled with fluorescent markers in Comparative Examples 12 and 19, and FIG. 3F is fluorescence micrographs in the composition treatment of Example 13. FIG. From this figure, the cationic micelle prepared with the cationic lipid of the present invention in Example 13 increased the delivery efficiency of the interfering ribonucleic acid in U87 cells smaller than the liposomes and micelles of known composition prepared in Comparative Examples 12 and 19. It can be seen that.

Experimental Example II Measurement of Delivery Efficiency of Small Interfering Ribonucleic Acid Using Fluorescence Activated Cell Sorting (FACS)

II-1. Measurement of Delivery Efficiency of Small Interfering Ribonucleic Acid Using Fluorescence Activated Cell Sorting (FACS) in A549 Cell Line

A549 cell lines were seeded 3 × 10 ⁵ per well in 6 well plates the day before the experiment. When the cells of each plate grew uniformly by 60-70%, 100 μl of serum-free medium was added to the Eppendorf tube, and 20 pmole of BlockiT, a fluorescently labeled double-stranded ribonucleic acid, and Comparative Examples 12 and 15, respectively. 6 μl of cationic micelles or liposomes prepared in 8 were added, respectively. These were slowly pipetted and mixed and left at room temperature for 20 minutes.

The complex thus prepared was added to a well plate and incubated for 24 hours in a CO 2 incubator at 37 ℃. Cultured cells were collected and washed twice with PBS. Cells containing fluorescently labeled double-stranded ribonucleic acid were analyzed for intracellular delivery efficiency by shifting the fluorescence intensity peak using a fluorescence flow cytometer, BD FACS CALIBUR (BD Bioscience, USA). This is shown in FIG. The untreated group, which was not treated to the cells of FIG. 4A, had no transfer of ribonucleic acid into the cells as a control, and almost no peak shifted. FIG. 4B shows liposomes prepared in Comparative Example 12, and FIG. In the cell group treated with the micelles of the composition, ribonucleic acid was 44.79% in FIG. 4B and 58.93% in FIG. 4C. On the other hand, in the case of the treatment group of Example 8 of the present invention, 84.78% was shown, and the intracellular delivery efficiency was increased compared to the comparative examples 12 and 15 treatment groups.

Therefore, it was found that the cationic micelles prepared in Example 8 of the present invention had increased ribonucleic acid transfer efficiency in A549 cells than liposomes or micelles prepared in Comparative Examples 12 and 15.

II-2. Measurement of Delivery Efficiency of Small Interfering Ribonucleic Acid Using Fluorescence Activated Cell Sorting (FACS) in A375 Cell Line

A375 cell lines were seeded 3 × 10 ⁵ per well in 6 well plates the day before the experiment. When the cells of each plate grew uniformly about 60-70%, 100 μl of medium without serum was added to the Eppendorf tube and 20 pmole of BlockiT, a fluorescently labeled double-stranded ribonucleic acid, and Comparative Examples 14 and 16, respectively. 6 μl of cationic liposomes or emulsion prepared in 9 were added respectively. These were slowly pipetted and mixed and left at room temperature for 20 minutes.

The complex thus prepared was added to a well plate and incubated for 24 hours in a CO 2 incubator at 37 ℃. Cultured cells were collected and washed twice with PBS. Cells containing fluorescently labeled double-stranded ribonucleic acid were analyzed for intracellular delivery efficiency by shift of fluorescence intensity peak using a fluorescence flow cytometer, BD FACS CALIBUR. This is shown in FIG. The untreated group, which was not treated to the cells of FIG. 5A, had no transfer of ribonucleic acid into the cells as a control, and almost no peak shifted. FIG. 5B is a cell group treated with a known composition of liposome prepared in Comparative Example 14, and FIG. 5C. The cell group treated with the emulsion of the known composition prepared in Comparative Example 16, 55.59% of genes, 51.89% of Figure 5C was delivered. On the other hand, in the case of the treatment group of Example 9 of the present invention showed 89.69%, the intracellular delivery efficiency was increased compared to the comparative examples 14, 16 treatment group.

Therefore, it was found that the cationic emulsion prepared in Example 9 of the present invention had increased ribonucleic acid transfer efficiency in A375 cells than the liposomes and emulsion prepared in Comparative Examples 14 and 16.

II-3. Measurement of Delivery Efficiency of Small Interfering Ribonucleic Acid Using Fluorescence Activated Cell Sorting (FACS) in Huh-7 Cell Line

Huh-7 cell lines were seeded 3 × 10 ⁵ per well in 6 well plates the day before the experiment. When the cells of each plate were grown uniformly by 60-70%, 100 μl of the medium containing no serum was added to the Eppendorf tube and the fluorescently labeled nucleic acid aptamer and the cations prepared in Comparative Examples 10, 18 and Example 15. 6 μl of sex liposomes were added respectively. These were slowly pipetted and mixed and left at room temperature for 20 minutes.

The complex thus prepared was added to a well plate and incubated for 24 hours in a CO 2 incubator at 37 ℃. Cultured cells were collected and washed twice with PBS. Cells containing fluorescently labeled double-stranded ribonucleic acid were analyzed for intracellular delivery efficiency by shift of fluorescence intensity peak using a fluorescence flow cytometer, BD FACS CALIBUR. This is shown in FIG. 6. In the untreated group, which was not treated to the cells of FIG. 6A, the peak was hardly shifted because the gene was not transferred into the cells as a control, and FIG. 6B was treated with liposomes of the known composition prepared in Comparative Example 10 and FIG. 6C. 6B shows 42.08% ribonucleic acid and 6C in one cell population. 38.16% were delivered. On the other hand, in the case of the treatment group of Example 15 of the present invention showed 85.53%, the intracellular delivery efficiency was increased compared to the comparative examples 10, 18 treatment group.

Therefore, it was found that the cationic liposomes prepared in Example 15 of the present invention had increased ribonucleic acid delivery efficiency in Huh-7 cells than the liposomes prepared in Comparative Examples 10 and 18.

Experimental Example III Evaluation of Small Interfering Ribonucleic Acid Delivery Efficacy Using Reverse Transcriptase Polymerase Chain Reaction

III-1. Evaluation of Small Interfering Ribonucleic Acid Delivery Efficacy by Reverse Transcription Polymerase Chain Reaction in A375 Cell Line

A375 cell lines were seeded 8 × 10 μs of cells per well into 24 well plates the day before the experiment. When the cells of each plate were grown uniformly by about 60-70%, 25 μl of medium without serum was added to the eppendorf tube, and the siRNA and the cationic liposomes prepared in Comparative Examples 9, 13, 16 and Example 6 Or 4 μl of emulsion each. SiRNA for inducing expression of survivin gene (Gene bank accession number: NM_001168) was purchased from Samchully Pharmaceuticals, Seoul, Korea. The final concentration of siRNA included in the media was adjusted to 100 nM. After slowly pipetting and mixing, the mixture was allowed to stand at room temperature for 20 minutes, and the complex thus prepared was added to a well plate and incubated in a CO 2 cell incubator at 37 ° C. for 24 hours. After 24 hours, total ribonucleic acid (RNA) in cells was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and the RNA was reverse transcribed into cDNA using AccuPowerRT PreMix (Bioneer, Daejeon, Korea). . The primers specific for survivin are 5'-GGACCACCGCATCTCTACAT-3 '(forward) and 5'-CTTTCTCCGCAGTTTCCTCA-3' (reverse), and the polymerase chain reaction was performed at 95 ° C for 5 minutes, and then 95 ° C. 1 minute, 59 minutes at 1 ℃, 30 seconds at 72 ℃ was repeated 30 times and further reacted at 72 ℃ 5 minutes. The size of the product was 347 base pairs. The level of survivin gene expression was measured by adjusting the band density of survivin-specific chain reaction product to the band density of amplified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. .

Figure 7 compares the expression of transcripts of survivin, a target gene, in A375 cells when each composition was treated. In the control group, the gene was not delivered into the cell, and there was no change in the expression of the survivin gene. The cationic liposome prepared in Comparative Example 13, the cationic emulsion prepared in Comparative Example 16, and Comparative Example 9, which are commercially available, were used in Example 6. It was observed that the expression reduction effect of survivin gene was lower than the prepared cationic liposomes. As can be seen from these results, the cationic liposome prepared in Example 6 was found to deliver a small interfering ribonucleate into the melanoma cells to selectively inhibit the expression of the target gene.

III-2. Evaluation of Antisense Oligonucleotide Delivery Efficacy by Reverse Transcription Polymerase Chain Reaction in HeLa Cell Line

HeLa cell lines were seeded 8 × 10 μs of cells per well in 24 well plates the day before the experiment. When the cells of each plate were grown uniformly by 60-70%, 25 μl of the medium containing no serum was added to the Eppendorf tube, and the antisense oligonucleic acid and the cationic liposomes or emulsions prepared in Comparative Examples 9, 11 and 16 were used. And 4 µl of the cationic liposomes prepared in Example 12 were added. Antisense oligonucleotides for inducing the expression inhibition of Bcl2 gene (Gcl bank accession number: NM_000633) were synthesized and ordered from Bioneer (Bioneer, Deajeon, Korea) (5'-TCT CCC AGC GTG CGC CAT- 3 ') was used. The final concentration of antisense oligonucleotide included in the media was set to 100 nM. After slowly pipetting and mixing, the mixture was allowed to stand at room temperature for 20 minutes, and the complex thus prepared was added to a well plate and incubated in a CO 2 cell incubator at 37 ° C. for 24 hours. After 24 hours, total ribonucleic acid (RNA) in cells was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and the RNA was reverse transcribed into cDNA using AccuPowerRT PreMix (Bioneer, Daejeon, Korea). . The sequences of primers specific for BC2 (Bcl2) are 5'-ATG GCG CAC GCT GGG AGA AC-3 '(left), 5'-GCG GTA GCG GCG GGA GAA GT-3' (right) and polymerase chains. After 5 minutes of reaction at 95 ° C, 1 minute at 95 ° C, 1 minute at 53 ° C, and 50 seconds at 72 ° C were repeated 32 times, followed by further 5 minutes at 72 ° C. The size of the product was 327 base pairs. The degree of Bcl2 transcript expression was measured by adjusting the band density of the Bcl2 specific chain reaction product to the band density of amplified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. It was.

Figure 8 compares the expression of transcripts of BC2, a target gene, in HeLa cells after treatment with each composition. In the control group, there was no change in the expression of the BC2 gene because the gene was not transferred into the cell, and the cationic liposome prepared in Comparative Example 11, the cationic emulsion prepared in Comparative Example 16 and the commercially available Comparative Example 9 were used in Example 12. It was observed that the expression inhibitory effect of BCL2 gene was significantly lower than the prepared cationic liposomes. As can be seen from these results, the cationic liposome prepared in Example 12 was found to deliver the antisense oligonucleotide material into the cervical cancer cell line to selectively inhibit the expression of the target gene.

III-3. Evaluation of Small Interfering Ribonucleic Acid Delivery Efficacy by Reverse Transcription Polymerase Chain Reaction in A549 Cell Line

The A549 cell line was seeded 8 × 10 μs of cells per well in a 24-well plate the day before the experiment. When the cells of each plate were grown uniformly by 60-70%, 25 μl of serum-free medium was added to the Eppendorf tube, and the survivin siRNA prepared in Comparative Examples 9, 15, 19 and Example 11 were prepared. 4 μl of cationic liposomes or micelles were added respectively. The final concentration of siRNA included in the media was set to 100 nM. After slowly pipetting and mixing, the mixture was allowed to stand at room temperature for 20 minutes, and the complex thus prepared was added to a well plate and incubated in a CO 2 cell incubator at 37 ° C. for 24 hours. After 24 hours, total ribonucleic acid (RNA) in cells was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and the RNA was reverse transcribed into cDNA using AccuPowerRT PreMix (Bioneer, Daejeon, Korea). . The level of survivin gene expression was measured by adjusting the band density of survivin-specific chain reaction product to the band density of amplified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. .

9 compares the expression of transcripts of survivin, a target gene, in A549 cells after treatment with each composition. The control group had no change in the expression of survivin gene because the gene was not delivered into cells, and Comparative Example 9, which is a cationic micelle prepared in Comparative Examples 15 and 19 and a commercially available cationic liposome, was used as a cationic liposome prepared in Example 11. It was shown that the suppression effect of survivin gene was significantly lower. As can be seen from the results, it was found that the cationic liposome prepared in Example 11 delivers a small interfering ribonucleate into the lung cancer cell line to more effectively suppress the expression of the target gene.

Experimental Example IV Cytotoxicity Evaluation Using 3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyl tetrazolium bromide (MTT)

IV-1. Cytotoxicity Assessment in WM266.4 Cell Line

In order to evaluate the cytotoxicity of the nucleic acid carrier containing a novel cationic lipid of the present invention, the experiment was carried out as follows. WM266.4 cells were treated with the complex composition of the cationic micelle of Example 7, the cationic liposomes or emulsions prepared in Comparative Examples 9, 12, and 16 and small interfering ribonucleic acids, and the cytotoxicity was evaluated. In order to clearly assess the cytotoxicity of the nucleic acid carrier only, siRNA used an inert scrambled sequence in the cell. Cytotoxicity was assessed by the method with 3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) reagent.

The cells were seeded in 48 wells for 2 × 10 ⁴ cells per well and incubated for 12 hours, followed by cationic micelles of Example 7 and cationic liposomes or emulsions prepared in Comparative Examples 9, 12, and 16. The complex composition of and siRNA was treated. After 24 hours, MTT solution was added to 10% of the medium, incubated for 4 hours more, the supernatant was removed, and 0.04 N isopropanol solution was added, followed by ELISA reader, Sunrise-Basic TECAN, M, Its absorbance was measured at 570 nm. As a control, no cells were used. 10 is a result of performing a cytotoxicity test of the cationic micelles and siRNA complexes prepared in Example 7 compared to the cationic formulations of Comparative Examples 9, 12 and 16 as a result of the synthesis of the cationic micelles and siRNA of Example 7 It was found that the cationic micelles of the present invention prepared in Example 7 did not show significant toxicity to human melanoma cell lines. It can be seen that the toxicity is increased in Comparative Example 12 and Comparative Example 16 made of a lipid having a large number of modified cationic amino acids.

IV-2. Cytotoxicity Assessment in Huh-7 Cell Line

In order to evaluate the cytotoxicity of the nucleic acid carrier containing a novel cationic lipid of the present invention, the experiment was carried out as follows. Huh-7 cells were treated with a complex composition of cationic emulsions or liposomes prepared in Example 10, Comparative Examples 9, 13, and 17 and small interfering ribonucleic acids, and cytotoxicity was evaluated. In order to clearly assess the cytotoxicity of the nucleic acid carrier only, siRNA used an inert scrambled sequence in the cell. Cytotoxicity was assessed by the method with 3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) reagent.

The cells were seeded in 48 wells for 2 × 10 ⁴ cells per well and incubated for 12 hours, followed by cationic formulations prepared in Examples 10, Comparative Examples 9, 13, and 17 and siRNA. The composite composition of was processed. After 24 hours, each MTT solution was added to 10% of the medium, incubated for another 4 hours, the supernatant was removed, and 0.04 N isopropanol solution was added, and then the resultant was removed at 570 nm using an ELISA reader. Absorbance was measured. As a control, no cells were used.

11 is a result of performing the cytotoxicity test of the cationic emulsion and siRNA complex prepared in Example 10, the complex of the cationic emulsion and siRNA of Example 10 was larger than the carriers of Comparative Examples 9, 13, 17 It was seen that the cationic emulsions of the present invention prepared in Example 10 did not show cytotoxicity and therefore do not show severe toxicity to human melanoma cell lines. In addition, it can be seen from the toxicity results of Comparative Examples 13 and 17 that the toxicity increases when the number of cationic amino acids modified more than the cationic lipid of the present invention increases.

IV-3. Cytotoxicity Assessment in U87 Cell Line

In order to evaluate the cytotoxicity of the nucleic acid carrier containing a novel cationic lipid of the present invention, the experiment was carried out as follows. U87 cells were treated with a complex composition of cationic emulsions, liposomes, or micelles and small interfering ribonucleic acids prepared in Example 14 and Comparative Examples 9, 13, and 15 and evaluated for cytotoxicity. In order to clearly assess the cytotoxicity of the nucleic acid carrier only, siRNA used an inert scrambled sequence in the cell. Cytotoxicity was assessed by the method with 3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) reagent.

The cells were seeded in 48 wells for 2 × 10 ⁴ cells per well and incubated for 12 hours, and then cationic emulsions, micelles, or liposomes and siRNA prepared in Example 14 and Comparative Examples 9, 13, and 15 were used. The composite composition of was processed. After 24 hours, each MTT solution was added to 10% of the medium, incubated for another 4 hours, the supernatant was removed, and 0.04 N isopropanol solution was added, and then the resultant was removed at 570 nm using an ELISA reader. Absorbance was measured. As a control, no cells were used.

12 shows the cytotoxicity test of the cationic emulsion prepared in Example 14 and the siRNA complex. As a result, the complex of the cationic emulsion and siRNA of Example 14 was added to the complex of the carrier and siRNA of Comparative Examples 9, 13, and 15. There was no significant cytotoxicity in comparison. Therefore, it can be seen that the cationic emulsion prepared in Example 14 from FIG. 12 does not show serious toxicity to human glioma cell lines, and also has a higher number of cationic amino acids modified than the cationic lipid of the present invention. It can be seen that in Comparative Examples 13 and 15 prepared with increased cytotoxicity.

FIG. 1 is a photograph showing intracellular nucleic acid delivery efficiency of a nucleic acid carrier containing a cationic lipid of the present invention in human melanoma cells WM266.4 using double-stranded ribonucleic acid labeled with fluorescent labels.

Figure 2 is a photograph showing the intracellular nucleic acid delivery efficiency of the nucleic acid carrier containing a cationic lipid of the present invention in HeLa, a human cervical cancer cell using a double-stranded ribonucleic acid with a fluorescent label.

Figure 3 is a photograph showing the intracellular nucleic acid delivery efficiency of the nucleic acid carrier containing a cationic lipid of the present invention in human glioma cells U87 using a double-stranded ribonucleic acid with a fluorescent label.

Figure 4 is analyzed using a fluorescence flow cytometer (FACS) the degree of intracellular nucleic acid delivery efficiency of the nucleic acid carrier containing a cationic lipid of the present invention in human lung cancer cells A549 using a double-stranded ribonucleic acid with a fluorescent label One result.

FIG. 5 shows the degree of intracellular nucleic acid delivery efficiency of a nucleic acid carrier containing a cationic lipid of the present invention in A375, a human melanoma cell, using a double-stranded ribonucleic acid labeled with a fluorescent flow cytometer (FACS). The result of the analysis.

FIG. 6 shows a fluorescence flow cytometer (FACS) of intracellular nucleic acid delivery efficiency of a nucleic acid carrier containing a cationic lipid of the present invention in Huh-7, a human liver cancer cell, using a double-stranded ribonucleic acid labeled with a fluorescent label. The result of the analysis.

Figure 7 shows the effect of inhibiting the expression of survivin transcript mediated by survivin transcript siRNA using a nucleic acid carrier comprising a cationic lipid of the present invention in reverse transcriptase polymerase chain reaction in human melanoma cell line A375. Show the result.

Figure 8 is a reverse transcriptase polymerase chain in HeLa, a cervical cancer cell line of human cervical cancer cell line using the nucleic acid carrier containing a cationic lipid of the present invention to inhibit the BCL2 transcript expression mediated by BCL2 expression inhibition antisense oligonucleotide Show the results confirmed by the reaction.

Figure 9 shows the effect of inhibiting the expression of survivin transcript mediated by survivin expression siRNA using a nucleic acid carrier containing a cationic lipid of the present invention by reverse transcriptase polymerase chain reaction in human lung cancer cell line A549 Show results.

Figure 10 shows the cytotoxicity of the nucleic acid carriers comprising the cationic lipids of the present invention using tetrazolium 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) staining. The results confirmed in the species cell line WM266.4.

Figure 11 shows the cytotoxicity of nucleic acid carriers comprising cationic lipids of the present invention using MTT (tetrazolium 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) staining. The results confirmed in the cell line Huh-7 are shown.

Figure 12 shows the cytotoxicity of nucleic acid carriers comprising cationic lipids of the present invention using tetrazolium 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) staining. The results confirmed in glioma cell line U87 are shown.

Claims (29)

Cationic lipids represented by formula (I)

Wherein n is an integer of 3 to 6 and R is an unsaturated hydrocarbon radical having 12 to 20 carbon atoms. The cationic lipid of claim 1, wherein R is oleyl or linoleyl. An oligonucleic acid delivery system into a cell comprising the cationic lipid of claim 1. The oligonucleotide carrier of claim 3, wherein the oligonucleotide is selected from the group consisting of ribonucleic acid, small interfering ribonucleic acid (siRNA), antisense oligonucleotide, and nucleic acid aptamers. The oligonucleotide carrier of claim 4, wherein the formulation of the oligonucleotide carrier is selected from the group consisting of liposomes, micelles, emulsions, and nanoparticles. The oligonucleic acid carrier according to claim 4, further comprising at least one selected from the group consisting of galactose lipid derivatives, mannose lipid derivatives, folic acid lipid derivatives, polystyrene glycol lipid derivatives, and biotin lipid derivatives. The oligonucleic acid carrier of claim 5, further comprising a cell fusion phospholipid. 8. The method of claim 7, wherein the cell fusion phospholipid is dioleoylphosphatidylethanolamine (DOPE) or 1,2-dipitanoyl-sn-glycero-3-phosphoethanolamine (1,2-diphytanoyl- sn-glycero-3-phosphoethanolamine). The oligonucleic acid carrier of claim 5, further comprising a surfactant. The method of claim 9, wherein the surfactant is Tween 20, polyethylene glycol monooleyl ether, ethylene glycol monododecyl ether, diethylene glycol monohexyl ether ( diethylene glycol monohexyl ether), trimethylhexadecyl ammonium chloride, dodecyltrimethyl ammonium bromide, cyclohexylmethyl β-D-maltoside, pentaerythyl palmitate palmitate palmitate ), Lauryldimethylamine-oxide, and laurosalcosine sodium salt (N-lauroylsarcosine sodium salt). The oligonucleic acid carrier of claim 5, further comprising a surfactant. The method of claim 11, wherein the surfactant is cetyl trimethylammonium bromide, hexadecyl trimethyl ammonium bromide, dodecyl betaine, dodecyl dimethylamine oxide , 3- (N, Ndimethylpalmitylammonio) propane sulfonate, Tween-20, Tween 80, Triton X-100, Polyethylene With glycol monooleyl ether, triethylene glycol monododecyl ether, octyl glucoside, and nonanoylmethylglucamine (N-nonanoyl-N-methylglucamine) Oligonucleic acid carriers selected from the group consisting of. The oligonucleic acid carrier of claim 3 and a complex of oligonucleic acid. The complex of oligonucleic acid carrier and oligonucleic acid according to claim 13, wherein the oligonucleic acid is selected from the group consisting of ribonucleic acid, small interfering ribonucleic acid, antisense oligonucleic acid, nucleic acid aptamer. delete delete delete Oligonucleic acid delivery system into a cell comprising a cationic lipid represented by formula (I):

Wherein n is an integer of 3 to 6 and R is a saturated hydrocarbon radical having 12 to 20 carbon atoms. The oligonucleotide carrier of claim 18, wherein the oligonucleotide is selected from the group consisting of ribonucleic acid, small interfering ribonucleic acid (siRNA), antisense oligonucleotide, and nucleic acid aptamers. The oligonucleotide carrier of claim 19, wherein the formulation of the oligonucleotide carrier is selected from the group consisting of liposomes, micelles, emulsions, and nanoparticles. 20. The oligonucleotide carrier according to claim 19, further comprising at least one selected from the group consisting of galactose lipid derivatives, mannose lipid derivatives, folate lipid derivatives, polystyrene glycol lipid derivatives, and biotin lipid derivatives. 21. The oligonucleic acid carrier of claim 20, further comprising a cell fusion phospholipid. The method of claim 22, wherein the cell fusion phospholipid is dioleoylphosphatidylethanolamine (DOPE) or 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (1,2-diphytanoyl- sn-glycero-3-phosphoethanolamine). The oligonucleic acid carrier of claim 20, further comprising a surfactant. The method of claim 24, wherein the surfactant is Tween 20, polyethylene glycol monooleyl ether, ethylene glycol monododecyl ether, diethylene glycol monohexyl ether ( diethylene glycol monohexyl ether), trimethylhexadecyl ammonium chloride, dodecyltrimethyl ammonium bromide, cyclohexylmethyl β-D-maltoside, pentaerythyl palmitate palmitate palmitate ), Lauryldimethylamine-oxide, and laurosalcosine sodium salt (N-lauroylsarcosine sodium salt). The oligonucleic acid carrier of claim 20, further comprising a surfactant. 27. The method of claim 26, wherein the surfactant is cetyl trimethylammonium bromide, hexadecyl trimethyl ammonium bromide, dodecyl betaine, dodecyl dimethylamine oxide , 3- (N, Ndimethylpalmitylammonio) propane sulfonate, Tween-20, Tween 80, Triton X-100, Polyethylene With glycol monooleyl ether, triethylene glycol monododecyl ether, octyl glucoside, and nonanoylmethylglucamine (N-nonanoyl-N-methylglucamine) Oligonucleic acid carriers selected from the group consisting of. A complex of the oligonucleic acid carrier and oligonucleic acid of claim 18. 29. The complex of oligonucleic acid carrier and oligonucleic acid according to claim 28, wherein the oligonucleic acid is selected from the group consisting of ribonucleic acid, small interfering ribonucleic acid, antisense oligonucleic acid, nucleic acid aptamer.

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