KR100373844B1 - Cationic lipids and method for preparing the same - Google Patents

Abstract

The present invention relates to a cationic lipid represented by Chemical Formula 1, a method for preparing the same, and a method for effectively delivering an anionic molecular material into a cell using the cationic lipid. The lipids are suitable for gene therapy because of their high stability and delivery efficiency.

Formula 1

Wherein y and z are natural numbers between 1 and 20, the same as or different from each other; R ₁ to R ₅ are the same or different hydrogen, an alkyl or hydroxy alkyl group having 1 to 10 carbon atoms, or an aryl or alalkyl group having 7 to 11 carbon atoms; R ₆ is a cholesterol radical; X is a pharmaceutically acceptable halogen anion.

Description

Cationic lipids for gene and biologically active drug delivery and preparation method thereof

The present invention relates to a cationic lipid capable of delivering a gene or a biologically active drug into a cell and a method of preparing the same. More specifically, the present invention relates to anionic molecular material, ie, DNA, RNA, The present invention relates to a method for effectively delivering a protein or a biologically active drug.

As a means of delivering genes developed so far, methods using viral vectors using retroviruses or adenoviruses, methods using liposome vectors via cationic amphiphilic compounds, DEAE dextran method or synthetic polymers or gene injection Or a gene gun.

Viral vectors have high gene expression efficiency, but it is difficult to maintain constant expression efficiency, and upon repeated administration, the viral vector may stimulate the body's immune system and worsen lesions, and there is no selectivity for target cells or target tissues. In addition, there is a disadvantage that it is difficult to prepare a large amount in a quick time because it takes a long time to culture and does not grow as easily as desired. Furthermore, DNA integration in the body can induce oncogenesis, and can be rather lethal by genetic recombination with potential allogeneic viruses.

In this regard, it is preferable to use non-viral vectors using cationic lipids, synthetic and natural polymers, or small particulates, rather than using viral vectors for which safety has not yet been established. There is a drawback in terms of efficiency of transfection. However, given the recent remarkable achievements in the field of synthetic vector development, the potential is expected to be quite significant.

On the other hand, literatures describing techniques using liposomes which are cationic lipids include the following; Hazinski, et al, Science, 249, 1285-1288, 1991; Wang and Huang, Proc. Natl. Acad. Sci. (USA), 84, 7851-7855, 1987; Felgner, et al., Proc. Natl. Acad. Sci. USA, 84, 7413-7416, 1987. Compared to viral vectors, liposomes have the advantage of being able to deliver genes through various infusion routes. For example, it can be injected directly into tissue, absorbed into the skin, digestive tract, pulmonary, or can be administered systemically via intravenous injection. This includes conventional methods using cationic polymers such as calcium phosphate, DEAE dextran, polylysine, polybrene, and gene guns that directly insert DNA into tissues or cells. Unlike electroporation methods, synthetic vectors designed theoretically at the molecular level are expected to have a large market as a new type of expensive drug for gene therapy.

Currently, the most common gene therapy methods include the delivery of genes in vitro and the transplantation of cells expressing the genes into the body (ex vivo). However, this method is not only complicated, but also difficult to be clinically universal from an economic point of view. Thus, gene transfer technology is directed toward direct in vivo, and is furthering toward the goal of systemic gene therapy that can deliver and express genes specifically to target cells. Of particular interest as non-viral vectors for use are the synthesis of cationic lipid-based lipid carriers, molecular conjugates using the receptor-mediated endocytosis principle, and synthesis. Polymer vectors and the like.

Cationic lipids can be transported into cells by cell membrane fusion or natural endocytosis by forming complex particles with stable ionic bonds with anionic DNA. In 1987, Dr. Felgner and his colleagues used cationic quaternary amino lipids N- [1- (2,3-dioleyloxy) propyl] -N, N, N-triethylammonium chloride (N- [1- (2,3) -dioleyloxy) Cationic liposomes consisting of dioleoylphosphatidylethanolamine (DOPE), known to have cell membrane fusion activity with propyl] -N, N, N-triethylammonium chloride (DOTMA), are useful for gene transfer Since the first publication of the results, various cationic lipids have been synthesized and used for gene transfer. DOTMA is a highly efficient cationic lipid whose hydrophobic group is an aliphatic group of 18 carbon atoms having a double bond. It also has a quaternary ammonium salt bound to the opposite side of the hydrophobic group bonded via a 3-carbon spacer arm and two ether linker bonds. Although it has high delivery efficiency, it is toxic, requires many synthetic processes, and Lipofectin, which is commercially available by mixing DOTMA and DOPE, is sold at a high price.

Cationic lipids used in gene therapy are amphipathic compounds, commonly having a molecular structure consisting of three parts: a cationic head portion, a spacer, and a hydrophobic terminal portion. The hydrophobic terminal portion is mainly used for fatty acid derivatives such as oleic acid or myristic acid, and serves as an anchor for the cation of ammonium groups to contact the surface of cells or liposomes. . Glycerol or the like as a spacer is bonded thereto and is usually a hydrophilic group having 2 to 15 carbon atoms, and a primary, secondary, tertiary or quaternary ammonium group, which is positively charged at neutral pH, is bonded to the cation head portion. have.

In order to further increase the efficiency of intracellular gene transfer, another form of derivative of DOTMA, 1,2-dimylyloxypropyl-3-dimethylhydroxyethyl ammonium bromide (DMRIE), 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 trifluoroacetate (2,3-dioleyloxy- N- [2- (sperminecarboxyamide) ethyl] -N, N-dimethyl-1-propane ammonium trifluoroacetate (DOSPA) has been developed.

In addition, a cholesterol derivative, 3β- [N- (N ', N'-dimethylaminoethane) carbamoyl] cholesterol (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), dioctadecylamidoglycylspermine (DOGS), and the like have been synthesized and used for gene transfer purposes. These cationic lipids are of the type spermine or polylysine, with quaternary amines, tertiary amines, or hydroxyethylated quaternary amines connected to the head of the lipid to provide a single positive charge. This may be divided into types that provide several positive charges in connection.

Another cationic lipid used for gene transfer is detergent, the quaternary ammonium salt. These are single chains such as cetrimethylammonium bromide or double chains such as dimethyldioctadecyl ammonium bromide detergents, all of which are gene transferable to animal cells. The amino groups belonging to these amphiphiles are quaternary, and single chains are attached to primary amine groups without spacer arms or linker bonds. These amphiphiles are also toxic to cells.

Other amphiphiles include 1,2-dioleoyl-3- (4'-trimethylammonio) butanoyl-cin-glycerol similar in structure to DOTMA (1,2-dioleoyl-3- (4'-trimethylammonio) butanoyl-sn-glycerol; DOBT), cholesteryl (4'-trimethylammonino) butanoate (choTB), 1,2-dioleoyl-3-ricinyl-cin 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC) and the like. The transfection activities of these amphiphiles are generally low.

However, there are two other amphiphiles, L-5-carboxyspermine, containing primary and secondary ammonium groups. Also called lipopolyamines are dioctadecyl-amidologlycylspermine (DOGS) and dipalmitoyl phosphatidylethanolamido-spermine (dipalmitoyl phosphatidylethanolamido-spermine; DPPES). . They are particularly efficient at transfecting primary endocrine cells without cellular toxicity.

On the other hand, liphopolylysine has been reported, which contains polylysine as an ammonium group in phospholipid (N-glutaryl-phosphatidyl ethanolamine). The spacer arm is the side chain of lysine and the head group of the phospholipid The hydrophobic group is a double chain linked to the spacer arm via two ester bonds, although it is efficient for transfection and not cytotoxic. The cells must be scraped for activity to be active, so this is clearly not convenient, and no in vivo experiments can be performed.

Liposomes are microscopic vesicles made up of concentrated lipid bilayers that vary in size and shape, from structurally long tubes to spheres, and range in size from several hundred Angstroms to several millimeters. (millimeter). Bilayer structure is generally composed of concentrated lamellae and an aqueous layer separating each lamellae from each other. The diameter of the vesicles is usually between about 20 to 30,000 nm and the liquid film between the lamellae is approximately 3 to 10 nm. Liposomes are largely divided into three groups depending on their overall size and the nature of the lamellae structure. That is, multi-lamellar vesicles (MLVs), small uni-lamellar vesicles (SUVs), large uni-lamellar vesicles (LUVs).

SUVs are approximately 20-50 nm in diameter and consist of a single lipid bilayer surrounding the aqueous solution portion. Uni-lamellar vesicles can also be produced in sizes of about 50-600 nm in diameter. Whereas uni-lamellae vesicles are single compartmental vesicles of uniform size, MLVs that vary greatly in size to 10,000 nm are multi-compartmental in structure and have one or more bilayers. ) Is included. LUV liposomes with large diameters range from about 600 to 30,000 nm and may include one or more bistructures.

Liposomes can be prepared in a variety of ways, but three methods are often used.

Firstly, metal probes are directly immersed in suspensions of MLVs by means of ultrasonic dispersion, and are often used in the manufacture of SUVs. Secondly, as a method for preparing MLV liposomes, the lipid is dissolved in a suitable organic solvent and then the solvent is removed by gas or air. Thus, when the aqueous solution is put into a thin film of dried lipid remaining on the surface of the container, the lipid is dispersed in the form of a lipid aggregate or liposome. Thirdly, a method of preparing LUV liposomes is a method of slowly hydrating a thin membrane of lipids with distilled water or some aqueous solution. Another method is lyophilization to a film. The film is again dissolved in volatile solvent, frozen and again removed from the freeze drying apparatus. To prepare a phamarceutical formulation containing a drug, a drug solution is added to the lyophilized lipids, whereby liposomes are formed. Various methods for the preparation of liposomes have been published in the literature and patents (Felgner, PL and Ringold, GM, Nature, 337,387-388, (1989); WO 93/05162, US Pat. No. 5,994,317; US Pat. No. 6,056,938) ).

Liposomes in this broad range are made from one or more lipids. Although it is believed that lipids such as cationic, neutral, and anionic can be used for the preparation of liposomes, positively charged liposomes have some problems, although they are not completely known to date. The amines used to prepare cationic liposomes to date are chemically unstable, resulting in poor shelf shelf life of vesicles. Another amine, dimethyl dioctadecyl ammonium bromide, lacked the appropriate molecular bond structure for the optimal formation of the duplex structure constituting the liposome structure.

Encapsulation by liposomes has been carried out by allowing a variety of biological substances to contact liposomes with lipids after contact with the substance. A common problem with this method is that the liposome encapsulated material is generally less than 50%, usually less than 20%, thus requiring another step to remove the unencapsulated material. Moreover, after removing the unencapsulated material, the liposomes of the encapsulated material may be lacking. Therefore, the stability problem of liposomes is very important in this field.

Meanwhile, liposomes have been used to introduce DNA into cells, microinjection, protoplast fusion, liposome fusion, calcium phosphate precipitation, and electroporation. Many DNA delivery methods, such as electroporation and retrovirus, have been used. These methods all have some problems. That is, it is too inefficient, toxic, and complex to use conveniently and efficiently with large scale biological or therapeutic protocols. For example, calcium phosphorus precipitation method is too low efficiency because it can deliver only 1/10 ⁴ ~ 1/10 ⁷ cells. Although microinjection is efficient, it is practically impossible to apply to numerous cells or many patients. Protoplast fusion is more efficient than calcium phosphorus precipitation, but requires propylene glycol, which is toxic to cells. The electroporation method is efficient but requires special equipment. Retroviruses are efficient enough, but the infection or cancer is concerned because the virus must be introduced into the patient. Liposomes have also been used but have not been as efficient as calcium phosphorus precipitation. The most desirable delivery method should have a very high efficiency without the involvement of toxic or infectious substances and should be simple to perform without sophisticated instruments.

Therefore, in the present invention, as a result of extensive research to solve the above problems, it was found that not only has a very high efficiency without the intervention of toxic or infectious substances, but also a cationic lipid that can be performed without sophisticated instruments. Completed on this basis.

Accordingly, it is an object of the present invention to provide cationic lipids capable of effectively delivering anionic molecules, ie, DNA, RNA, proteins, peptides or biologically active drugs, etc. into cells.

Another object of the present invention is to provide a method for preparing the cationic lipid.

Still another object of the present invention is to provide a method for effectively delivering DNA, RNA, protein, peptide or biologically active drug into cells using the cationic lipids.

Cationic lipids of the present invention for achieving the above object is represented by the following formula (1).

Wherein y and z are natural numbers between 1 and 20, the same as or different from each other; R ₁ to R ₅ are the same or different hydrogen, an alkyl or hydroxy alkyl group having 1 to 10 carbon atoms, or an aryl or alalkyl group having 7 to 11 carbon atoms; R ₆ is a cholesterol radical; X is a pharmaceutically acceptable halogen anion.

According to another aspect of the present invention, there is provided a method for preparing a cationic lipid in which a oxyalkyl halide represented by the following formula (2) is added dropwise to an organic base containing a nitrogen atom and a hydroxy alkyl halide in the presence of a solvent. After obtaining a halide, it is reacted with hydrogen or diamine substituted with one or more alkyl groups or hydroxy alkyl groups in the presence of a solvent, followed by reaction with hydrogen or hydroxy alkyl or alkyl halides to produce a diammonium salt.

X- (CH ₂ ) zO-R ₆

Wherein X, z and R ₆ are as described above.

According to another aspect of the present invention, there is provided a method for delivering a cationic lipid, which comprises (a) contacting an anionic molecular material with a composition comprising an effective amount of the cationic lipid to form a complex with another lipid to form a lipid complex. Making a step; And (b) contacting the lipid complex with a cell.

FIG. 1 is an electrophoretic photograph of naked pCMV-p53 plasmid DNA showing no contamination by RNA or DNA of E coli . Most DNAs consist of cyclic supercoil plasmid DNA. Serial number of the DNA batch, M is the lambda Hind III marker.

FIG. 2 is a DNA electrophoresis (0.8% agarose gel) photograph of the binding of pCMV-p53 plasmid DNA.

Lane 1 is a size marker (1kb ladder),

Lane 2 is bare pCMV-p53 plasmid DNA showing electromigration without contamination by RNA, naked DNA, and chromosomal DNA.

Lane 3 is a complex of pCMV-p53 plasmid DNA and a lipid: DOPE liposome (1: 1 molar ratio) according to the present invention,

Lane 4 is a complex of pCMV-p53 plasmid DNA and lipid: DOPE liposome (1: 1 molar ratio) according to the present invention,

Lane 5 is a complex of pCMV-p53 plasmid DNA without lipid electrophoresis and showing complete binding of DNA and liposomes with a lipid: DOPE liposome (1: 4 molar ratio) according to the present invention,

Lane 6 is a complex of pCMV-p53 plasmid DNA showing a complete binding of DNA and liposomes with no electrophoresis of DNA and a lipid: DOPE liposome (1: 6 molar ratio) according to the present invention,

Lane 7 is a complex of pCMV-p53 and lipid: DOPE: PLL (1: 1: 0.375 by weight) according to the present invention.

Figure 3 according to the invention CEHA (N- [2- (N'-Cholesteryloxyethyl-N ', N'-dimethylamino)] ethyl-N, N-dimethyl-N-hydroxyethyl ammonium chloride) and DOPE (Phosphatidylethanolamine dioleyl) A scanning electron microscopy (SEM) photograph of liposomes prepared at a ratio of 1: 1 (mol / mol).

Looking at the present invention in more detail as follows.

As described above, the cationic lipid according to the present invention is represented by the following formula (1).

Formula 1

Wherein y and z are natural numbers between 1 and 20, the same as or different from each other; R ₁ to R ₅ are the same or different hydrogen, an alkyl or hydroxy alkyl group having 1 to 10 carbon atoms, or an aryl or alalkyl group having 7 to 11 carbon atoms; R ₆ is a cholesterol radical; X is a pharmaceutically acceptable halogen anion. More preferably y and z are natural numbers between 2 and 12 equal to or different from each other; R ₁ to R ₅ are one or more hydrogen atoms same as or different from each other, or an alkyl or hydroxy alkyl group having 1 to 7 carbon atoms; X is Br, Cl or I.

Cationic lipids, such as Formula 1, according to the present invention are non-viral delivery agents that are highly efficient for animal cells in the laboratory. Such cationic lipids can be transported into cells by cell membrane fusion or natural endocytosis by forming complex particles by anionic molecular material, for example, stable ionic bonds with anionic DNA (PL Felgner, Adv.Drug Delivery Review, 5, 163 (1990)). At this time, when the anion portion of the DNA is bonded to the cationic portion of the cationic lipid and coated on the DNA molecular chain, the lipids that are located outside are bound together by hydrophobic bonds to make DNA molecules into dense particles. The particles are cationic as a whole and have a diameter of several hundred nanometers. These cationic DNA / lipid particles act nonspecifically with anionic cell membranes and destabilize the membrane structure and enter the cell. In the cell, the gene is expressed by the endosome, which is entrapped in the cell and transported to the lysosome, by the escape to the cytoplasm followed by the transfer to the nuclear chromosome.

Cationic lipid according to the present invention represented by the formula (1) is added to the oxy alkyl halide and the organic base containing a nitrogen atom to the cholesterol oxy derivative in the presence of a solvent to obtain a cholesterol oxy alkylene halide represented by the following formula (2) Next, it is prepared by reacting hydrogen, or diamine substituted with one or more alkyl groups or hydroxy alkyl groups in the presence of a solvent, and then reacting with hydrogen or hydroxy alkyl or alkyl halides to produce a diammonium salt.

Formula 2

X- (CH ₂ ) zO-R ₆

Wherein X, z and R ₆ are as described above.

Typical cholesterol derivatives used in the present invention include cholesteryl mesylate and the like, and organic bases including the nitrogen atom include pyridine and triethylamine, and the hydroxy alkyl halide is 2-bromine. Moethanol and the like, and diamine in which the hydrogen or at least one alkyl group or hydroxy group is substituted includes tetramethylethylenediamine and the like. In addition, most organic solvents such as chloroform and acetone can be used as the solvent.

The cationic lipids according to the present invention may form complexes with one or more other lipids and may carry anionic molecular materials by contacting the anionic molecular materials with the composition comprising the lipids of the present invention. The lipid complex thus obtained can be contacted with a cell to deliver anionic molecular material to the cell.

Other lipids that may be used in the present invention may be selected from one or more from the group consisting of DOPE, Dioleoyl Phosphatidyl Choline (DOPC), Phosphhatidyl Choline (PC), and cholesterol. For example, when liposomes are prepared by mixing an appropriate amount of mPEG-cholesterol synthesized as in Example 3 of the present invention, they are less lost by proteins or RES (Reticular endotherial system) in vivo and stay in the body for a long time. Can maintain the efficacy. In addition, the cells to which the present invention is applicable include, but are not limited to, HeLaS 3, HepG 2, Hep 3B, NIH 3T3, CHRC 2, or COS 7.

Cationic positive lipids of the present invention are made into liposomes as in Example 3 below, which are mixed with DNA or drug as in Example 7 to form a complex, and the complex is ex vivo in cells as in Example 8 When applied or in vivo, injection or systemic administration through a vein close to a specific target site may result in an excellent delivery effect.

As described above, the cationic lipids of the present invention are inexpensive to manufacture, can be obtained in high yield with high yield, and are anionic molecular materials, that is, DNA, RNA, protein, peptide, and drug. And the like may be usefully used for delivery to cells.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples.

Example 1

Preparation of N-[(3-Cholesteryloxyethyl-N, N-dimethylamino) ethyl] -N ', N'-dimethylammonium chloride hydrobromide salt (N-[(3-Cholesteryloxyethyl-N, N-dimethylamino) ethyl] -N ', N'-dimethylammonium chloride hydrobromide)

As shown in Scheme 1, 2-bromoethyl cholesteryl ether (5 g, 10.13 mmol) and tetramethylethylenediamine (10 g, 146 mmol) were simultaneously added to 50 mL of dried acetone, followed by stirring at 50 ° C. for 48 hours. It was. The resulting crystals were filtered using a glass filter, dried and separated using a silica gel column with a chloroform: methanol (10: 1) solution, and then recrystallized with ethanol to obtain white crystals (6.5 g, 73%). The compound thus obtained was dissolved in 30 mL of anhydrous ethanol, and 0.1N HCl solution was added dropwise until pH 4.0. The obtained crystals were recrystallized twice with anhydrous ethanol after filtration. The product thus obtained was vacuum dried for 12 hours. Yield (3 g, 46%).

¹ H NMR (CDCl ₃ , ppm): 5.3 (t, 1H, C = C H of cholesterol)

4.0 (m, 8H, NC H ₂ )

3.5 (s, 15H, NC H ₃ )

Example 2

Preparation of N- [3- (Cholesteryl) -oxyethyl] -N'-hydroxylethyl-N, N, N ', N'-tetramethylethylenediammonium bromide (N- [3- (Cholesteryl)- oxyethyl] -N'-hydroxyethyl-N, N, N ', N'-tetramethylethylene diammonium bromide)

As shown in Scheme 2, 2-bromoethyl cholesteryl ether (5 g, 10.13 mmol) and tetramethylethylenediamine (10 g, 146 mmol) were simultaneously added to 50 mL of dried acetone, followed by stirring at 50 ° C. for 48 hours. . The resulting crystals were filtered and dried, and then separated by chloroform: methanol (10: 1), and then recrystallized with ethanol to obtain white crystals (6.5 g, 73%). 100 mL of dry acetonitrile was added to the obtained compound, and 20 mL of 2-bromoethanol was added thereto, followed by stirring at 50 ° C. for 48 hours. Acetonitrile, a reaction solvent, was distilled under reduced pressure at about 50 mL, and the resulting crystals were vacuum dried for 12 hours after filtration.

¹ H NMR (CDCl ₃ , ppm): 5.3 (t, 1H, C = C H of cholesterol)

4.2 (t, 2H, HOCH ₂ CH ₂ N-)

3.5 to 4.0 (m, 20H, HOCH ₂ C H ₂ (C H ₃ ) NC H ₂ C H ₂ N (C H ₃ ) C H ₂ CH ₂ O)

Example 3

Preparation of Cationic Liposomes

Cationic lipid of Example 1: DOPE: mPEG-Cholesterol (1: 1: 0.2, molar ratio) and Cationic lipid of Example 2: DOPE (1: 1, molar ratio) take the lipid 2mL Wheaton Lipids were placed in glass septum vials, dissolved in excess chloroform to mix lipids, and then rotated at low speed until almost all chloroform evaporated in a nitrogen environment to form a lipid film. The lipid film was vacuum dried for another 12 hours to completely remove the solvent. To make a large multi-lamellar vesicle (MLV), 1 mL of endotoxin-free tertiary purified water was added thereto, and the vial was sealed at 37 ° C. and vortexed for 1 minute after sealing. Small uni-lamellae vesicles (SUVs) were prepared by 30 passes of 0.1 μm polycarbonate membranes using a mini-extruder (Avanti Polar Lipids, Inc). Meanwhile, small ultrasonically treated uni-lamellae vesicles (SUV) were sonicated for 60 minutes using an inverted cup sonicator (Heat systems) in a nitrogen environment. The liposomes thus prepared were filtered using a 0.22 μm filter and stored at 4 ° C. until use. The obtained liposome was about 100-150 nm in average particle size as a result of light scattering measurement.

Example 4

Cell culture

HeLaS3, HepG2, Hep3B, NIH3T3 and COS-7 (SV-40 virus transformed monkey kidney cell line) cell lines were purchased from the American Type Culture Collection (ATCC), CRL 1651, and used with 10% w / v fetal calf serum. Gibco BRL, Gaitherburg, Md.) And DMEM (Gibco BRL, Gaitherburg, Md.) Containing 100 units / mL penicillin and 100 μg / mL streptomycin. For transfection, the cells were grown to a uniform distribution of about 60-80% in 6-well plates (Falcon Ware, Benton Dickinson, Franklin Lakes, NJ), and then the DNA: liposomal complexes were introduced. 1 μg of pCMV-beta-Gal per well was introduced into each well after mixing with lipids and media was replaced after 4 hours. 48 hours for X-gal staining in a 37 ℃ / CO ₂ incubator (incubator), 24 hours for luciferase assay (luciferase assay) and then used for measuring the expression efficiency.

Example 5

Plasmid DNA Preparation

As strains for plasmid DNA isolation, DCM5 E. coli transformed with pCMV-beta-gal and E. coli transformed with pCIS-CAT were used, and QUIAGEN plasmid purification kit was used for plasmid DNA separation. DNA was isolated as follows.

First, inoculation bacteria were obtained from colonies cultured in LB-ampicillin solid medium and inoculated into LB liquid medium containing 20 µg / mL ampicillin, followed by shaking culture at 37 ° C for 16 hours. Cells were then centrifuged at 5,000 × g for 10 minutes at 4 ° C. to separate cells and resuspended with 10 mL of P1 buffer. Then 10mL of P2 buffer solution was added and left at room temperature for 5 minutes, 10mL of P3 buffer solution was added and left for 20 minutes on ice. After centrifugation at 4 ° C. and 30,000 × g for 30 minutes, the supernatant was placed in a column equilibrated with QBT buffer, washed twice with 30 mL QC buffer, eluted with 5 mL QF buffer, and then 0.7. A volume of isopropanol was added and the DNA was precipitated by centrifugation at 4 ° C./20,000×g for 30 minutes. DNA was washed with 70% ethanol, dried in air, dissolved in distilled water, and quantified by UV spectrophotometer. In addition, the endotoxin (LPS) level of the obtained DNA was 10 EU / mg or less as a result of a limulus amebocyte lysate (LAL) test, and was run again on 1% agarose gel. The shape of the cyclic supercoiled plasmid DNA was confirmed. The results are shown in FIG.

Example 6

DNA / cationic liposome binding ratio test

In order to determine the ratio of cationic liposomes to DNA and whether they bind completely, lipids were mixed and electrophoresed. The cationic liposomes prepared with DOPE and the lipids of Examples 1 and 2 at a ratio of 1: 1 (mol / mol), respectively, were λHind III Marker DNA and 0.5, 1, 2, 3, 4, 5, 6, After slowly mixing at a weight ratio of 7, 8: 1, it was allowed to stand at room temperature for 15 minutes. After careful loading of the DNA / liposomal complex on 0.8% -agarose gel, electrophoresis was performed at 50V. As a result, the lipids of Examples 1 and 2 showed excellent binding ability with DNA, and it was confirmed that the DNA was completely bound by all liposomes at a ratio of about 1: 3 of DNA: cationic liposomes. It means that the charge is the point with the cationic tendency. Similar results were also obtained with liposomes composed of the respective lipids: DOPE: mPEG-cholesterol (1: 1: 1 0.2 molar ratio) of Examples 1 and 2. The results are shown in FIG.

Example 7

Preparation of DNA / Cationic Liposome Complex for In Vitro Gene Transfer Efficiency Test

Plasmid DNA and liposome complexes were prepared by slowly mixing 0.5 mL (mg / mL) of DNA and 0.5-4 mL (mg / mL) of liposomes. The thus prepared complex was used for testing after incubation at room temperature for 15 minutes before transfection.

Example 8

In Vitro Gene Transfections

Each cell was seeded 1 × 10 ⁵ / well dogs into 6-well plates the day before transfection. Transfection was performed when the cells of each plate grew uniformly about 60 to 80%. DNA and each liposome were mixed at a ratio of 1: 4, 1: 5, 1: 6 and left at room temperature for 15 minutes. Plates were washed with media without serum and DNA: liposomal complexes were introduced into each well. After 4 hours in a 37 ° C./CO ₂ incubator, add 1 mL of media containing serum to each well of the plate and incubate for 24 or 48 hours. Intracellular Transfection Protocol is as follows.

1.Put 100 μl of serum-free medium into two ependorp tubes (× plate number of wells) and add DNA and liposomes at each ratio.

2. Add DNA to liposomes slowly, pipet slowly 2 to 3 times, and leave at room temperature for 15 minutes.

3. After 10 minutes, remove the 6-well plate from the incubator, remove the medium, and place 2 mL of medium-free media in each well.

4. Add 800 μl of serum-free medium to an open drop tube containing 200 μl of DNA / liposomal complex solution.

5. Tilt the plate left and right several times to allow the cells to wash well, then remove the medium in the plate.

6. Add 1 mL of serum-free media to the control wells.

7. Add solution (1 mL) in an open drop tube to each well, tilt it left and right to spread evenly across the wells, and observe the cell condition under a microscope.

8. Place in a 37 ° C./5%-CO ₂ incubator and leave for 4 hours.

9. Add 1 mL of media containing serum to each well, and allow it to stand for 24 or 48 hours depending on the desired experimental conditions.

Example 9

Expression efficiency measurement through X-Gal staining

The cells obtained in Example 8 were first washed twice with PBS for X-gal staining, then the cells were fixed in 0.5% glutaaldehyde PBS solution for 15 minutes at room temperature. The fixed solution was removed and washed twice with PBS, and then stained with X-gal (Stratagene, USA) solution at 37 ° C. for 3-5 hours. 0.45㎛ filter after mixing X-gal 1mL / mL DMSO solution with X-gal buffer solution (0.01 Sodium deoxycholate, 0.02% NP-40, 2mM MgCl ₂ , 5mM ferrocyanide, 5mM ferricyanide, 0.01M PBS) It was used by filtration. As a result, it showed high expression efficiency in the majority of cells, the highest expression efficiency in COS 7, Hep 3B, NIH 3T3 cells, and X-gal staining in more than 25% of the total cell number.

Example 10

CAT analysis

Place the cell in 100 μl of 200 mM Tris-HCl (pH 7.4). After several freezing / thawings, 50 μl of supernatant was taken and 40 μl of Tris-HCl containing ¹⁴ C-labeled chloramphenicol (0.1 μCi; specific activity, 47 μCi / mmol; 1 Ci = 37 GBq) Was added. After 5 minutes at 37 ° C., 20 μl of 4 mM acetyl CoA was added. After 1 hour at 37 ° C., chloramphenicol and acetylated derivatives were extracted with ethyl acetate and separated by TLC to make an radioradiograph. Radiographs were analyzed with an LKB UltroScan densitometer. As a result, the CAT treatment with the cationic liposomes and DNA showed a higher CAT activity than the control.

Example 10

Electron microscopy

The sample is dispersed in water using an ultrasonic disperser to form a vesicle, which is colored by mixing 1: 1 with an aqueous solution of the sample and 2% uranyl acetate. Add 0.02 mL of sample solution and 0.02 mL of uranil acetate (2% solution) onto the slide glass and mix well. A small sample is taken here, the sample solution is dropped onto a grid coated with a formvar film, allowed to air dry for 3 hours, and electron micrographs are taken. At this time, if the drying time is shortened, the form bar film is ruptured when scanning the electron beam during electron microscopy. Therefore, the longer the drying is better, it is appropriate to dry at room temperature for about 24 hours. Scanning electron micrographs as shown in FIG. 3 show that the cationic lipids developed in the present invention form a spherical multilayer structure having an average diameter of 100 to 150 nm in an aqueous solution.

As described above, the cationic amphoteric lipid of the present invention contains a polyvalent cation, which enables the excellent delivery of biologically active substances such as genes into cells, and does not have severe toxicity or instability, and thus is suitable for drug delivery in clinical practice. It can be a system. In addition, it is relatively easy to manufacture and purify, and thus commercially excellent.

Claims (10)

Cationic lipid represented by the following formula (1). Formula 1 Wherein y and z are natural numbers between 1 and 20, the same as or different from each other; R ₁ to R ₅ are the same or different hydrogen, an alkyl or hydroxy alkyl group having 1 to 10 carbon atoms, or an aryl or alalkyl group having 7 to 11 carbon atoms; R ₆ is a cholesterol radical; X is a pharmaceutically acceptable halogen anion. The cationic lipid according to claim 1, wherein X is Br, Cl or I. In the presence of a solvent, an organic base containing a nitrogen atom and a hydroxy alkyl halide are added dropwise to the cholesterol oxy derivative to obtain a cholesterol oxy alkylene halide represented by the following formula (2), and then hydrogen or one or more alkyl groups in the presence of a solvent or A method of producing a cationic lipid represented by the following Chemical Formula 1, wherein the hydroxy alkyl group is reacted with a substituted diamine and then reacted with hydrogen or hydroxy alkyl or an alkyl halide to produce a diammonium salt. Formula 2 X- (CH ₂ ) zO-R ₆ Formula 1 Wherein y and z are natural numbers between 1 and 20, the same as or different from each other; R ₁ to R ₅ are the same or different hydrogen, an alkyl or hydroxy alkyl group having 1 to 10 carbon atoms, or an aryl or alalkyl group having 7 to 11 carbon atoms; R ₆ is a cholesterol radical; X is a pharmaceutically acceptable halogen anion. The method of claim 3, wherein the cholesterol oxy derivative is cholesterol mesylate. The method of claim 3, wherein the organic base is pyridine, the hydroxy alkyl halide is 2-bromoethanol, and the diamine is tetramethylethylenediamine. (a) contacting anionic molecular material with a composition comprising an effective amount of the cationic lipid according to claim 1 to form a complex with the other lipid to form a lipid complex; And (b) contacting the lipid complex with the cell. The method of claim 6, wherein the composition comprises one or more additional lipids. 7. The method of claim 6, wherein said additional lipid is selected from the group consisting of DOPE, DOPC, PC and Cholesterol. The method of claim 6, wherein the anionic molecular material is DNA, RNA, protein, peptide or biologically active drug. The method of claim 6, wherein the cell is HeLaS 3, HepG 2, Hep 3B, NIH 3T3, CHRC 2 or COS 7.