KR20020013528A - Cationic lipids for gene transfer and preparation method thereof - Google Patents

Abstract

PURPOSE: Provided are cationic lipids for gene transfer and, more particularly, gene-transferring vectors consisting of a cholesterol compound dangling with an amino acid moiety such as lysine or ornithine. Also, provided is a method for preparing such cationic lipids. CONSTITUTION: Cationic lipids for gene transfer, which are capable of transferring nucleic acid materials into cells at very high efficiency with low cell toxicity, are represented by the following general formula. They are synthesized by forming a carbamate ester linkage between lysine or ornithine residue and a cholesteryl chloroformate in a solid-phase manner. This solid-phase synthesis method allows the automatic mass production of the vehicles suitable for use in gene therapy and transfer. In the formula, R is-CH2-CH2-CH2-CH2-NH3+ or-CH2-CH2-CH2-NH3+ and n is an integer of 1-10 with a proviso that the amino acid moiety is a homo-oligopeptide consisting of lysine or ornithine alone or a hetero-oligopeptide consisting of lysine and ornithine. Further, liposomes are made of the cationic lipids and very useful in transfecting nucleic materials into cells as well as in delivering pharmaceutically active materials into target cells.

Description

Cationic lipids for gene transfer and preparation method thereof {Cationic lipids for gene transfer and Preparation Method etc}

Recently, many developed countries have been actively researching this field, and related gene transfer materials have been commercialized to form a very large market. In this regard, however, domestic research and new material development are very poor, and the demand for pure domestic proprietary technologies is urgent in view of the enormous biotechnology market to be formed.

In general, as a vehicle used for gene therapy, a viral vector system using mainly adenovirus or retrovirus has been used recently. Such viral vector systems have high efficiency but are difficult to deliver high molecular weight DNA, and can cause serious side effects such as virus re-infection, immune response, or oncogenic gene activation. have.

Accordingly, many gene delivery methods that do not use viruses are currently being studied, and it is expected that such methods will dominate in the biotechnology field in the future. As a material used in a gene transfer method that does not use a virus, chemically synthesized vectors are used first of all, cationic polymers are widely used, and poly-L-lysine and polyornithine L-ornithine, polyethylenimine (PEI), polyspermine, dendrimer, and the like. In addition, there are various cationic lipids that are used more than this, and a method using cationic lipids (Nature, vol. 337, pp 387-388, 1989) is as follows. That is, BC-Col (Biochem. Biophys. Res. Commun. Vol. 179, pp280-285, 1991), supermidine-cholesterol (Biochem. Biophys. Res. Commun. Vol. 221, pp82-88, 1996), b. Proc. Natl. Acad. Sci. USA vol. 93, pp9682-9686, Lipofectin (Promega), Transfectam (Promega), Escort (Sigma), Effecten ( Lipids such as Qiagen) and Genotherapy Systems (Genetherapy Systems) are in the spotlight as next generation gene transfer materials, and as such, they are already commercialized and sold by famous foreign molecular biology or chemical companies.

The method of using the cationic polymer or cationic lipid for gene transfer has the advantage that it is easy to synthesize raw materials, deliver DNA having high molecular weight, and can be safely used when applied to a living body. Compared to the method of using, the efficiency is lower and the unit price has a problem.

The present invention relates to a cationic lipid for gene delivery and a method for manufacturing the same, and more particularly, to a gene delivery vector required for gene therapy or transformation by introducing an amino acid group such as lysine or ornithine into cholesterol and a method for synthesizing the same. will be.

Figure 1a is a synthetic diagram of lysine-cholesterol (K1-Chol, K2-Chol),

Figure 1b is a synthetic diagram of ornithine-cholesterol (O1-Chol, O2-Chol).

Figure 2a is a graph showing the results of toxicity experiments on HepG2 cells.

Figure 2b is a graph showing the results of toxicity experiments on NIH3T3 cells.

Figure 2c is a graph showing the results of toxicity experiments on 293 cells.

Figure 3 is a photograph showing the size measurement results of the K-Chol / DNA complex.

Figure 4 is a graph showing the transfection results for 293T cells and HepG2 cells using the cationic lipid of the present invention.

5 is a graph showing the results of transfection of 293T cells, NIH3T3 cells, and HepG2 cells using cationic liposomes.

Figure 6 is a photograph stained using X-gal transfected HepG2 cells.

Figure 7 shows the time and serum-dependent transfection test results measured by varying the transfection time for 293 cells

The present inventors are generally used in gene therapy or gene delivery methods that do not use viruses, and are cationic lipids that have many future applications, and have high gene transfer efficiency, low toxicity to cells, and biometabolism, and thus are applicable to the human body. The present invention was completed by finding out that lysine-cholesterol or ornithine-cholesterol can meet these requirements while studying new harmless cationic lipids.

Accordingly, it is an object of the present invention to provide a new type of cationic lipid with high gene transfer efficiency and low toxicity to cells,

Another object of the present invention to provide a liposome prepared by using cationic lipids,

It is still another object of the present invention to provide a solid phase synthesis method for easily synthesizing these cationic lipids using a solid support.

The present invention includes a cationic lipid represented by the following general formula.

In the above, R represents one kind selected from -CH ₂ -CH ₂ -CH ₂ -CH ₂ -NH ₃ ⁺ or -CH ₂ -CH ₂ -CH ₂ -NH ₃ ⁺ , and the polymer is lysine or ornithine alone. Or n = 1 to 10 in the mixed homo or hetero.

The cationic lipid of the present invention represented by the above general formula is a group of amino acids having a positively charged amino residue and hydrophobic cholesterol characterized by four ring structures are involved in binding.

The amino acid group constituting the cationic lipid of the present invention is lysine (Lysine, K) or ornithine (Ornithine, O), and the two amino acids are ε in the neutral region where pH is normal in the cellular environment. Since -NH ₃ ⁺ and ornithine are present in the form of δ-NH ₃ ⁺ , the compound of general formula (I) has a positively charged state in the cell as a whole. The cationic property of the compound (I) enables the cationic lipids of the present invention to form complexes with various oligonucleotides that are negatively charged in the neutral region, and also effectively adheres to relatively negatively charged target cell membranes. To help you transition.

In addition, lipids, which are hydrophobic sites in the general formula (I), preferably use cholesterol. Cholesterol is a compound that acts as a supporter between lipids that make up the cell membrane with hydrophobicity except the hydroxyl group (-OH) of carbon 3, as well as fusion with target cells when used as a vehicle for transfection. Make it possible.

The cationic lipid of the present invention is an amphiphilic compound composed of an amino acid group which is a hydrophilic region and a lipid region which is a hydrophobic region, and is activated with an amine group (-NH ₂ ) and chloroformate of an alpha group of an amino acid group having a carboxyl group in an amide form. It is preferable to form by carbamate ester formation reaction between hydroxyl group (-O-CO-Cl) of carbon 3 of the lipid which is the lipid which is the lipid.

Lysine or ornithine in the amino acid moiety is also suitable for the practice of the present invention in either homo or hetero form upon polymerization. In the polymerization, the value of n is preferably set to 1 to 10, and more preferably, n is set to 1 to 5 in consideration of transfection efficiency and economic efficiency in the cell. More preferably, the cationic lipids contain 3β [L-lysineamide-carbamoyl] cholesterol, 3β [di-L-lysineamide-carbamoyl] cholesterol, 3β [L-ornithine, in which n ranges from 1 to 2. Amide-carbamoyl] cholesterol, 3β [di-L-lysineamide-carbamoyl] cholesterol.

The cationic lipid of the present invention can be transfected into a target cell by forming a complex with a macromolecule. Macromolecules forming the complex include oligonucleotides and oligomers, and the like, wherein the oligonucleotides include single- or double-chain DNA or RNA and plasmids.

The oligonucleotides may be plasmids, RNAs, including DNA or genes encoding any protein, and RNAs are mRNAs, tRNAs, rRNAs as well as antisense RNAs and ribozymes complementary to the target DNA, RNA sequence of any cell. (Rybozyme).

Genes encoding such proteins include polypeptides associated with the treatment or diagnosis of a disease. The polypeptide may include various hormones, tissue-adhesive antigens, cell adhesion proteins, cytokines, various antibodies, cell receptors, intracellular or extracellular enzymes, fragments thereof, and the like. In addition, the oligonucleotides preferably include gene expression regulators, such as transcriptional promoters, enhancers, silencers, operators, terminators, attenuators, and other expression regulators.

The cationic lipid of the present invention also includes the form of liposomes (hereinafter, liposomes composed of cationic lipids will be referred to as 'cationic liposomes').

Liposomes are vesicles made of synthetic lipid membranes and refer to structures containing water-soluble substances therein. Liposomes function to carry various pharmaceutically useful substances to target cells. When the liposome is used in pharmaceutical form, the lipid membrane slowly dissolves, so it can be a sustained-release drug, and can be absorbed in the body by oral administration or injection, and the concentration of the drug only in the vicinity of the target organ or tissue. There is an advantage that can be increased.

The cationic liposomes of the present invention may be constituted using only the cationic lipids of the present invention described above, and preferably 10 to 90 mol% of cationic lipids and neutral auxiliary lipids 10 to 90 mol%. It consists of 90 mol%. The cationic lipid used for the liposome is preferably 1 to 5 in consideration of the transfection efficiency and economic efficiency in the cell, and more preferably the cationic lipid is in the range of 1 to 2. 3β [L-lysineamide-carbamoyl] cholesterol, 3β [di-L-lysineamide-carbamoyl] cholesterol, 3β [L-ornithineamide-carbamoyl] cholesterol, 3β [di-L-lysineamide- Carbamoyl] cholesterol.

The neutral auxiliary lipids include, but are not limited to, dioleoyl phosphatidylethanolamine (DOPE) and cholesterol.

The cationic liposomes may form spherical vesicles of very uniform size by floating the lipid molecules in an aqueous solution and then ultrasonically treating the cationic liposomes. Also, the cationic liposomes may be formed by rapid mixing of the lipid solution in ethanol with water. It is preferable in the practice of the invention.

The present invention also includes a pharmaceutical composition comprising the aforementioned cationic lipid or liposome as a vector for gene delivery. The pharmaceutical composition is preferably a cationic lipid complex consisting of a cationic lipid (or cationic liposome) and an oligonucleotide containing a gene useful for the treatment of disease. When used as a pharmaceutical composition, the addition amount does not require any particular limitation because the allowable amount varies depending on the relationship with other compositions to be injected, the condition of the patient, and the type of disease.

The present invention also includes methods of synthesizing cationic lipids.

The cationic lipid of the present invention is made by lysine or ornithine by cholesteryl chloroformate and carbamate ester coupling reaction as shown in FIG. However, the cationic lipids of the present invention should not be construed as being limited by the above method, and it is also possible for those skilled in the art to prepare the cationic lipids of the present invention by a procedure different from the method of the present invention.

Cationic lipids according to the present invention as shown in Figure 1

(1) Rinkamide resin (Sinkamideresin) in a suitable organic solvent to swell, and then treated with an organic solvent containing piperidine for a period of time to remove the Fmoc (9-fluoromethoxycarbonyl) protecting group.

(2) activating and linking a carboxyl group of Nα-Fmoc-Nε-tBoc (tertiarybutoxycarbonyl) -lysine or Nα-Fmoc-Nε-tBoc-ornithine to the resin treated by the above procedure Removing Fmoc protector,

In step (2), lysine and ornithine may be polymerized into a homo or heteropolymer through the same process as necessary. In this case, the preferable value of n is 1 to 10, more preferably 1 to 5, as described above.

(3) dissolving cholesteryl chloroformate in methylene chloride and adding DIPEA thereto to link amine groups of amino acids.

(4) adding TFA (trifluoroacetic acid) / methylene chloride to the resultant obtained in step (3) to desorb lysine-cholesterol or ornithine cholesterol and side chain protecting groups from the solid support to finally obtain the desired cationic lipid. Will be synthesized.

In addition, the cationic liposome of the present invention may have a hydrophilic polymer chain effective on prolonging the blood circulation time of the liposome complex. The hydrophilic polymer may include polyethylene glycol (PEG), polylactic acid, polyglycolic acid, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline and the like. Preferably, the hydrophilic polymer is polyethylene glycol, and the compound is a polymer obtained by polymerizing ethylene glycol. The polyethylene glycol is an amphiphilic substance having a hydrophobic portion and a hydrophilic portion in a molecule thereof, and is used in the cationic lipid complex / liposomal complex of the present invention. Chemical binding has the effect of reducing antigenicity. The hydrophilic polymer is capable of binding to the ε-amino group or the δ-amino group of the lysine or ornithine of the cationic lipid, and preferably to the amide group of the polypeptide terminal carboxy group as represented by the following general formula (II). It involves doing.

When polyethyleneglycol is bonded as a hydrophilic polymer as mentioned above, it is preferable to use the thing of the molecular weight 0.5-20K dalton in the practice of this invention.

In addition, the cationic lipids of the present invention may add 'markers' or ligands specific for the cationic lipids so that they can be specifically bound to the target cells. As the marker or ligand, various antibodies, transferrin, biotin, folic acid, low-density lipoprotein (LDL), and monosaccharides mannose, glucose, galactose, and disaccharide lactose as carbohydrates are preferable. The identifiers differ from cell to cell, and each cell has a corresponding receptor to react specifically. The identifier for a particular cell is optional to those skilled in the art from the description of the present invention and will not be described in further detail.

The mechanism of transition of the cationic lipid complex or liposome complex into the target cell is achieved by endocytosis or by fusion of a cell membrane with a liposome or complex. In the case of endocytosis, transfection occurs through the process of forming endosomes in the cell, and in the case of fusion, the membrane of the liposome is integrated with the target cell membrane and the contents of the liposome are transduced into the cytoplasm. Will be specified. The exact mechanism of transfection is not yet known, but after the transfer of the lipid complex around the nucleus by fusion of the endosome containing the complex into the large vesicles connected to the nuclear membrane around the cell nucleus, the oligo nutotide Is involved in transcription and protein synthesis, or the cationic lipid complex is electrostatically exchanged with the anionic lipids of the endosomes. You will be involved in the process.

The cationic lipids and cationic liposomes of the present invention are capable of intracellular degradation, and as shown in FIG. 2, the intracellular toxicity is significantly lower than that of conventional gene carriers, and thus is very excellent as a gene carrier.

Hereinafter, the content of the present invention will be described in detail through examples. However, the following examples are merely illustrative for the purpose of describing the present invention and they should not be used in any sense to limit the scope of the present invention.

Example 1 Synthesis of Lysine-Cholesterol (K-Chol) and Ornithine-Cholesterol (O-kol)

Rin Amide resin (Anaspec, USA) of about 50 to 100 mg was added to DMF (N, N-dimethylformamide) for at least 1 hour, followed by swelling, and then a DMF solution containing piperidine was added. Treatment for more than a minute removes the Fmoc (9-Fluoromethoxycarbonyl) protecting group. Then, 1 equivalent of HBTU (2- (1H-Benzotriazole-1-yl) -1,1,3,3-tetramethyl-uronium hexafluoro phosphate), 1 equivalent of HOBt (N-hydroxybenzotriazole-H ₂ O), 2 The carboxyl group of N _α -Fmoc-N _ε -Boc (tertiarybutoxycarbonyl) -lysine or N _α -Fmoc-N _ε -Boc-ornithine was activated using a DMF solution containing an equivalent amount of DIPEA (diisopropylethylamine). After connection the resin was washed several times with DMF. The Fmoc protecting group of amino acids was then removed with piperidine and then also washed with DMF. If necessary, lysine or ornithine was repeated one more time so that up to two amino acids were added to the resin.

Then, cholesteryl chloroformate was dissolved in methylene chloride and added DIPEA to it, and then linked to the amine group of the amino acid. The resin was then sufficiently washed in order using DMF, methylene chloride and methanol, and dried overnight in vacuo.

Desorption of lysine-cholesterol (hereinafter referred to as K-Chol) or ornithine-cholesterol (hereinafter referred to as O-Chol) from the solid support and desorption of the side chain protecting group were carried out by addition of trifluoroacetic acid (TFA) / methylene chloride. . Cationic lipids synthesized by the above procedure were precipitated on ethyl ether, washed three or more times, and then dissolved in water and lyophilized. Its molecular weight was confirmed by FAB MS (fast atom bombardment mass spectroscopy) method or MALDI-TOF MS (matrix-assisted laser desorption ionization- time-of-flight mass spectroscopy) method.

K1-Chol: 1 H NMR (300 MHz, d6 -DMSO) δ in ppm 0.65-2.26 (m, skeleton of cholesterol,-(CH2) 3- of Lys), 2.75 (br, s, -CH2 of Lys), 4.3 ( br, s, -CH of Lys) 7.2 (br, -NH2 of Lys) 7.7 (br, -CO-NH- of Lys).

Molecular weight (FAB-MS) m / z 558 [M + H] < + >, (MALDI-TOF MS) m / z 576 [M + Na] < + >.

K2-Chol: 0.65-2.26 (m, skeleton of cholesterol,-(CH2) 3- of Lys), 2.75 (br, s, -CH2 of Lys), 4.3 (br, d, -CH of Lys), 7.2 ( br, -NH 2 of Lys), 7.7 (br, -CO-NH- of Lys). Molecular weight (FAB-MS) m / z 686 [M + H] < + >.

* K1: 1 lysine, K2: 2 lysine

O1-Chol: 1 H NMR (300 MHz, d6 -DMSO) δ in ppm 0.65-2.26 (m, skeleton of cholesterol,-(CH2) 2- of Orn), 2.75 (br, s, -CH2 of Orn), 4.3 ( br, s, -CH of Orn) 7.2 (br, -NH2 of Orn) 7.7 (br, -CO-NH- of Orn)

Molecular weight (MALDI-TOF MS) m / z 560.2 [M + Na] < + >.

O2-Chol: 1 H NMR (300 MHz, d6 -DMSO) δ in ppm 0.65-2.26 (m, skeleton of cholesterol,-(CH2) 2- of Orn), 2.75 (br, s, -CH2 of Orn), 4.3 ( br, s, -CH of Orn) 7.2 (br, -NH2 of Orn) 7.7 (br, -CO-NH- of Orn)

Molecular weight (MALDI-TOF MS) m / z 673.2 [M + Na] < + >.

* O1: 1 ornithine, O2: 2 ornithine

Example 2 Preparation of Liposomes.

K-Chol synthesized in Example 1 was dissolved in an appropriate amount of water and stored in a refrigerator. In addition, a moderate amount of DOPE, a commonly used neutral auxiliary lipid, was taken in an appropriate amount and dissolved in chloroform, dried by blowing with nitrogen gas, and dried completely in a vacuum. Next, the cationic lipids dissolved in water were added to DOPE and the mass ratio was about 50:50, and the aqueous solution was stirred for a while, and then put in a refrigerator to stand overnight. Then, liposomes were formed by treatment for about several minutes using a tank-shaped sonicator. The size of the liposome was measured by the dynamic light scattering method using a Malvern 4700 system (Malvern Instrument Ltd., UK). As shown in Table 1, the average of five measurements was 146.9 ± 8.2.

TABLE 1

Count Size (nanometer) One 138.3 2 143.8 3 151.3 4 158.8 5 142.3

Example 3 Toxicity Experiments on 293, NIH3T3, HepG2 Cells.

293, NIH3T3, and HepG2 cells were cultured in a 96-well plate using medium essential medium (MEM) containing 10% FBS (fetal bovine serum) in a medium at 37 ° C and 5% carbon dioxide. And various cationic liposomes were added. After two more cells, MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl tetrazolium bromide) was dissolved in PBS (phosphate buffered saline) and added to the cells for 4 hours. Grew even more. The reaction was then dissolved by adding DMSO (dimethylsulfoxide), and the absorbance at 570 nm was measured and compared with the control without treatment of cells. As shown in Table 2 and FIGS. 2 (a), (b) and (c), K-Chol / DOPE liposomes and O-Chol / DOPE liposomes are other gene transfer agents, Lipofectin and DC-Chol. Compared with / DOPE liposomes and polyethyleneimine (PEI), the cytotoxicity was found to be significantly lower in the same concentration range.

TABLE 2 Cytotoxicity Comparison

Transfection Reagent IC ₅₀ (μg / ml) 293 NIH3T3 HepG2 Polyethyleneimine 9.45 Less than 4.54 14.7 Lipofectin 35.5 17.9 21.2 DC-Chol / DOPE ND * 17.2 15.5 K-Chol / DOPE 57.5 21.9 39.2 O-Chol / DOPE ND 40.9 ND

* Not determined (ND): It means that the concentration of each reagent exceeds 60 µg / ml, but the IC ₅₀ has not yet been reached.

Example 4 Size Measurement of K-Chol / DNA Complexes.

K-Chol / DNA complex was formed by mixing plasmid DNA (brand name: pSV-β-gal, Promega) and K-Chol with HEPES buffer (25 mM, pH 7.4, 10 mM MgCl ₂ ) into which beta galactose degrading enzyme gene was introduced. , And allowed to stand at room temperature for 30 minutes. 1 μl of the complex solution was dispensed on a fresh split Mica disk surface and adsorbed for 1 to 2 minutes, and then the filtrate was removed by filtration with filter paper and washed with pure water. N ₂ gas was blown onto the mica surface and dried at room temperature. The shape and particle size of the K-Chol and plasmid DNA complexes obtained by the above procedure were measured using the Atomic force microscopy method (NanoScope IIIa system, Digital Instruments, Inc., Santa Barbara, CA). It confirmed that it is -200nm.

Example 5 Transfection of 293T Cells and HepG2 Cells Using Cationic Lipids.

293T cells (embryonic human kidney cells) and HepG2 cells (human liver carcinoma cell line) were grown in Dulbecco's modified eagle medium or DME (Minimum essential medium) medium, respectively. After dividing the appropriate amount of cells in a 24well plate, plasmid DNA expressing a gene synthesizing beta galactose degrading enzyme and K-Chol were mixed to form a complex and subjected to transfection. Lipofectin and polyethyleneimine were used as controls, respectively. As can be seen in Figure 4, it was confirmed that K-Chol generally has a high gene transfer efficiency. In particular, as shown in Table 3, when treated for about 48 hours in the presence of 10% FBS (fetal bovine serum), it was confirmed that the gene transfer and expression effect is very superior to other substances.

<Table 3> Comparison table of gene transfer efficiency

Expression level of beta galactose degrading enzyme (mUnits / well) Gene carrier HepG2, (-) FBS, 4 hours transfection 293T, (-) FBS, 4 hours transfection 293T, (+) FBS, 48 hours transfection Lipofectin 2.25 ± 0.12 12.7 ± 0.50 3.53 ± 0.83 Polyethyleneimine 20.9 ± 1.8 14.1 ± 0.93 0 ± 0 almost not measured. K-Chol 11.4 ± 2.4 19.8 ± 1.6 12.3 ± 0.62

* The figures in the table are the average ± standard error of three experiments.

Example 6 Transfection of 293T Cells, NIH3T3 Cells, and HepG2 Cells Using Cationic Liposomes.

293T cells (human embryonic kidney cells), NIH3T3 cells (mouse embryonicfobroblast cells) and HepG2 cells (human liver carcinoma cell line) were transfected. 293T and NIH3T3 were grown in Dulbecco's modified eagle medium (DMEM) and HepG2 cells were grown in Minimum essential medium (MEM) medium. Divide the appropriate amount of cells in a 24well plate, mix the plasmid DNA expressing the gene synthesizing beta galactose degrading enzyme with K-Chol / DOPE liposomes and O-Chol / DOPE liposomes to form a complex at room temperature to transfect the cells. Was carried out. Transfection was performed for 4 hours in the absence of FBS. Lipofectin, DC-Chol / DOPE liposomes and polyethyleneimine were tested as controls. As shown in Table 4 and FIG. 5 below, it was confirmed that K-Chol / DOPE liposomes and O-Chol / DOPE liposomes have high gene transfer and expression efficiency for various cells. In particular, it was found that O-Chol / DOPE liposomes have very good gene transfer and expression efficiency.

<Table 4> Comparison table of gene delivery and expression efficiency

Gene carrier Expression level of beta galactose degrading enzyme (mUnits / mg protein) (-) FBS, 4 hours transfection result NIH3T3 cells HepG2 cells 293 cells Polyethyleneimine 1267.60 ± 23.05 ^* 1210.82 ± 217.89 2601.91 ± 286.69 Lipofectin 51.00 ± 9.93 137.40 ± 68.95 358.81 ± 158.64 DC-Chol / DOPE 35.41 ± 1.10 107.46 ± 14.90 784.87 ± 97.20 K-Chol / DOPE 831.92 ± 288.32 830.63 ± 159.87 1059.75 ± 156.82 O-Chol / DOPE 978.84 ± 284.1 1364.64 ± 261.34 2604.61 ± 97.46

* The figures in the table are the average ± standard error of three experiments.

Example 7 X-gal Staining of Transfected HepG2 Cells

Plasmid DNA was transfected into HepG2 cells using lysine-cholesterol (K-Chol) prepared in Example 1. Next, X-gal was added to confirm the beta-galactose degrading enzyme-expressing cells. 6 is a photograph of the stained cells.

Example 8 Measurement of Transfection Efficiency and FBS Effect over Time for 293 Cells.

Figure 7 shows the time and serum-dependent transfection test results measured by varying the transfection time for 293 cells. 293 cells were treated with the K-Chol / DNA complex under optimal transfection conditions. To test serum dependence on transfection efficiency, cells were cultured in transfection medium with and without 10% FBS and transfected with the complex after a predetermined time period for each culture time. Sean medium was removed and the cells were transferred to fresh medium containing FBS and cultured. As a result of measuring the beta galactose degrading enzyme activity of expressing cells separated by incubation time in a fresh medium for 48 hours is shown in FIG. 7. According to the result The maximum transfection efficiency was shown in the cell group with the incubation time of 2 to 4 hours, and the transfection capacity was not dependent on the presence of serum in the medium. In addition, the transfection efficiency was not changed even if the cells were grown for 48 hours in the presence of the complex, and it was confirmed that the cationic lipids of the present invention almost alleviated cytotoxicity. On the other hand, in the absence of serum in the transfection medium, the efficiency drops sharply, which is believed to be due to cell death due to starvation.

According to the present invention, it is possible not only to provide a new type of cationic lipids having low cytotoxicity and high gene transfer efficiency, but also to provide a method for rapidly and efficiently synthesizing various cationic lipids through a solid phase synthesis method. On the other hand, such a solid phase synthesis method allows for automated synthesis and mass synthesis. In addition, lysine-cholesterol (K-Chol) and ornithine-cholesterol (O-Chol) according to the present invention was very low toxicity compared to lipofectin (Lipofectin), DC-Chol and polyethyleneimine (PEI) In addition, it was confirmed that the gene transfer and expression effects were also excellent for various cells. Therefore, the amino acid-cholesterol derivative of the present invention is a new type of cationic lipid for gene delivery, and can be said to have great utility value in itself or in liposome form.

Claims (25)

In cationic lipids for gene delivery, Cationic lipid represented by the following general formula. In the above, R represents one kind selected from -CH ₂ -CH ₂ -CH ₂ -CH ₂ -NH ₃ ⁺ or -CH ₂ -CH ₂ -CH ₂ -NH ₃ ⁺ , and the polymer is lysine or ornithine alone. Or n = 1 to 10 in the mixed homo or hetero. The cationic lipid according to claim 1, wherein n is 1 to 5 in the general formula. The method of claim 1, wherein the gene delivered by the cationic lipid may be DNA, or plasmid or RNA including a gene encoding any protein, and the RNA may be a target of any cell as well as mRNA, tRNA, rRNA. Cationic lipids, characterized in that they are complementary DNA and RNA sequences and antisense RNA and ribozyme. 4. The cationic lipid of claim 3, wherein the gene encoding the protein is a polypeptide associated with the treatment or diagnosis of a disease. The method according to claim 4, wherein the polypeptide is cationic, characterized in that it comprises a variety of hormones, histocompatibility antigens, cell adhesion proteins, cytokines, various antibodies, cell receptors, intracellular or extracellular enzymes and fragments thereof. Lipid. The cationic lipid of claim 1, wherein the cationic lipid is biodegradable 3β [L-lysineamide-carbamoyl] cholesterol. The cationic lipid according to claim 1, wherein the cationic lipid is biodegradable 3β [di-L-lysineamide-carbamoyl] cholesterol. The cationic lipid of claim 1, wherein the cationic lipid is biodegradable 3β [L-ornithineamide-carbamoyl] cholesterol. The cationic lipid of claim 1, wherein the cationic lipid is biodegradable 3β [di-L-ornithineamide-carbamoyl] cholesterol. 2. The molecular weight of 0.5 to 20K daltons according to claim 1, wherein the amide group of the polypeptide terminal carboxy group as represented by the ε-amino group or δ-amino group of the lysine or ornithine of the cationic lipid or general formula (II). Biodegradable cationic lipids combined with phosphorus polyethylene glycol. [Claim 2] The cationic lipid according to claim 1, wherein the cationic lipid has a marker or ligand attached to the cationic lipid to specifically bind to the cell; Various antibodies, transferrin, biotin, folic acid, low specific lipoprotein, carbohydrate, monosaccharides mannose, glucose, galactose and disaccharides lactose A pharmaceutical composition comprising the cationic lipid complex comprising the cationic lipid of claim 1 and an oligonucleotide comprising a gene useful for treating a disease. The method of claim 12, wherein the gene comprises a gene encoding various hormones, histocompatibility antigens, cell adhesion proteins, cytokines, various antibodies, cell receptors, intracellular or extracellular enzymes and fragments thereof. A pharmaceutical composition comprising the cationic lipid complex as an active ingredient. In the cationic liposome for gene delivery, the cationic liposome for gene delivery, characterized in that the cationic lipid of claim 1 alone or mixed 10 to 90 mol% of the cationic lipid and 10 to 90 mol% of the neutral lipid. 15. The cationic liposome for gene delivery according to claim 14, wherein the neutral lipid is dioleoyl phosphatidylethanolamine or cholesterol. 15. The method of claim 14, wherein the gene delivered by the cationic liposomes may be DNA, or a plasmid, RNA, including a gene encoding any protein, and the RNA is a target of any cell as well as mRNA, tRNA, rRNA. Cationic liposomes for gene delivery, characterized in that the DNA, RNA sequence complementary antisense RNA and ribozyme (Rybozyme). 17. The cationic liposome for gene delivery according to claim 16, wherein the gene encoding the protein is a polypeptide associated with the treatment or diagnosis of a disease. 18. The gene transfer method according to claim 17, wherein the polypeptide includes various hormones, tissue-adhesive antigens, cell adhesion proteins, cytokines, various antibodies, cellular receptors, intracellular or extracellular enzymes and fragments thereof. Cationic liposomes. 15. The cationic liposome of claim 14, wherein the cationic lipid is biodegradable 3β [L-lysineamide-carbamoyl] cholesterol. 15. The cationic liposome of claim 14, wherein the cationic lipid is biodegradable 3β [di-L-lysineamide-carbamoyl] cholesterol. 15. The cationic liposome according to claim 14, wherein the cationic lipid is biodegradable 3β [L-ornithineamide-carbamoyl] cholesterol. The cationic liposome according to claim 14, wherein the cationic lipid is biodegradable 3β [di-L-ornithineamide-carbamoyl] cholesterol. A pharmaceutical composition comprising, as an active ingredient, a cationic lipid complex composed of an oligonucleotide comprising a cationic lipid of claim 14 and a gene useful for treating a disease. The method of claim 23, wherein The gene is effective for cationic lipid complexes comprising genes encoding various hormones, tissue-adhesive antigens, cell adhesion proteins, cytokines, various antibodies, cell receptors, intracellular or extracellular enzymes and fragments thereof. Pharmaceutical compositions comprising the ingredients. In the method of producing a cationic lipid for gene delivery, to prepare a cationic lipid for gene delivery represented by the following general formula by the lysine or ornithine by cholesteryl chloroformate and carbamate ester coupling reaction Way. In the above, R represents one kind selected from -CH ₂ -CH ₂ -CH ₂ -CH ₂ -NH ₃ ⁺ or -CH ₂ -CH ₂ -CH ₂ -NH ₃ ⁺ , and the polymer is lysine or ornithine alone. Or n = 1 to 10 in the mixed homo or hetero.