EP1185674A1

EP1185674A1 - Cationic lipids for gene transfer and preparation method thereof

Info

Publication number: EP1185674A1
Application number: EP00935672A
Authority: EP
Inventors: Jong Sang Park; Joon Sig Choi; Eun Jung Lee; Hyung Suk Jang
Original assignee: Choi Joon Sig; Lee Eun Jung; Park Jong Sang
Current assignee: Choi Joon Sig; Lee Eun Jung; Park Jong Sang
Priority date: 1999-05-28
Filing date: 2000-05-29
Publication date: 2002-03-13
Also published as: JP2003501363A; WO2000073471A1; KR20020013528A; AU5110200A; KR100454881B1; KR20010069179A

Abstract

Disclosed are cationic lipids for gene transfer, which are capable of transferring nucleic acid materials into cells at very high efficiency with low cell toxicity. 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. Further, disclosed are liposomes made of the cationic lipids. These liposomes are 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

TECHNICAL FIELD

The present invention relates to cationic lipids for gene transfer and, more particularly, to gene-transferring vectors consisting of a cholesterol compound dangling with an amino acid moiety such as lysine or ornithine. Also, the present invention is concerned with a method for preparing such cationic lipids.

BACKGROUND ART

Until recently, viral vector systems taking advantage of adenovirus or retrovirus have been extensively used as vehicles for use in gene therapy. Highly efficient as they are in gene transfer, the viral vector systems find difficulty in application for gene therapy because they cannot carry macromolecules of large molecular weight and are apt to cause serious side effects, including virus reinfection, immune response induction and carcinogenic gene activation.

At present, accordingly, active research is directed to non-viral gene transfer, which is expected to prevail in the bioengineering field. Chemically synthesized polymers are commonly adopted as non-viral vectors for use in gene transfer, exemplified by poly-L-lysine, poly-L-ornithine, polyethylenimine (PEI), polyspermine, and dentrimer. More prevalently utilized are cationic lipids. As reported (Nature, vol. 337, pp 387-388, 1989), methods of utilizing cationic lipids in gene transfer have already been disclosed. Accordingly, with attraction of great attention, a variety of cationic lipids have been developed as next generation gene transfer materials and commercialized, such as DC-chol (Biochem. Biophys. Res. Commun. Vol. 179, pp280-285, 1991), spermidine-cholesterol (Biochem. Biophys. Res. Commun. Vol. 179, pp 82-88, 1996), B.G.T.C. (Proc, Natl. Acad. Sci. USA, vol. 93, pp 9682-9686), Lipofectin (Gibco-BRL), Lipofectamine (Gibco-BRL), Transfectam (Promega), Escourt (Sigma), Effectene (Qiagen), Geneporter (Genetherapy Systems).

Cationic polymers or lipids are advantageous in that they are easy to chemically synthesize, able to transfer DNA molecules of large molecular weights, and safe for the body. However, cationic polymers and lipids suffer from disadvantages of being poor in transfection efficiency and high in production cost.

DISCLOSURE OF THE INVENTION

Leading to the present invention, the intensive and thorough research on cationic lipids suitable as vehicles for use in gene therapy or gene transfer, repeated by the present inventors aiming to overcome the above problems encountered in prior arts, resulted in the finding that lysinamide- or ornithinamide-cholesterol derivatives are of high efficiency in transferring nucleic acid materials into cells with a low cell toxicity and that the cationic lipids are formed into liposomes which can deliver pharmaceutically active ingredients into cells. The present inventors also developed a solid-phase method for synthesizing such cationic lipids, which can be applied for mass production.

Accordingly, it is an object of the present invention to provide cationic lipids which are highly efficient for gene transfer with low cell toxicity.

It is another object of the present invention to provide liposomes consisting mainly of cationic lipids, which can be used as carriers for pharmaceutical active materials.

It is a further object of the present invention to provide a method for preparing such cationic lipids using a solid support.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which: Fig. la is a synthesis scheme for lysinamide-cholesterols Kl-Chol and K2- Chol;

Fig. lb is a synthesis scheme for omithinamide-cholesterols 01 -Choi and O2-Chol; Fig. 2a is a graph elucidating the toxicity of gene-transfer vehicles to

HepG2 cells;

Fig. 2b is a graph elucidating the toxicity of gene-transfer vehicles to NIH3T3 cells;

Fig. 2c is a graph elucidating the toxicity of gene-transfer vehicles to 293 cells;

Fig. 3 is a photograph showing the measured size of the K-Chol/DNA composite;

Fig. 4 is a histogram elucidating transfection results of the cationic lipid of the present invention into 293T and HepG2 cells; Fig. 5 is a histogram elucidating transfection results of the cationic liposome of the present invention into 293T, NIH3T3 and HepG2 cells;

Fig. 6 is a photograph showing the identification of transfected HepG2 cells by X-gal staining; and

Fig. 7 is a graph elucidating the serum-dependent transfection efficiency for 293 cells with regard to transfection time period.

BEST MODES FOR CARRYING OUT THE INVENTION

In accordance with an embodiment, the present invention contemplates cationic lipids, represented by the following general formula I:

wherein, 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. As recognized from the general formula I, the cationic lipid of the present invention consists of an amino acid moiety having a positively charged residue and a cholesterol moiety featured by four hydrophobic rings.

In accordance with the present invention, lysinamide (K) or ornithinamide (O) is suitable for use in the amino acid moiety for the cationic lipid. Under a normal physiological condition, almost neutral in pH, the side amino groups of lysine and ornithine exist as ε-NH₃ ⁺ and δ-NH₃ ⁺, respectively, so that the molecule of the general formula I is positively charged in total. The positive charges enable the cationic lipids of the general formula I to combine with various oligonucleotides, which are negatively charged in a neutral pH range in addition to being helpful in adhering the cationic lipids to target membranes which are relatively negative in charge.

As for the hydrophobic moiety of the general formula I, it is preferably a cholesterol residue. In vivo, cholesterol, which is hydrophobic in its entirety except for the hydroxy radical on the carbon in position 3, is a membrane constituent serving as a support for other membrane lipids. When being utilized in vehicles for transfection, cholesterol enables the vehicles to fuse to target cells.

As illustrated above, the cationic lipid of the present invention is an amphiphathic compound consisting of a hydrophilic amino acid moiety and a hydrophobic lipid moiety. It is preferably synthesized through the carbamate-ester formation between the amine group (-NH₂) of the amino acid moiety and the hydroxy group (OH) on the carbon of position 3 in cholesterol. For the synthesis, the hydroxy group is activated by chloroformate.

In addition, either of homo-oligopeptides consisting of lysine or ornithine only or hetero-oligopeptides consisting of a combination of lysine and ornithine may be used as the amino acid moiety. The number of the amino acid residues is preferably on the order of 1 to 10, more preferably 1 to 5, and most preferably 1 to 2 when account is taken of transfection efficiency and economical favor. Where n is 1 or 2, examples of the cationic lipids include 3β[L-lysinamide- carbamoyl] cholesterol, 3 β [di-L-lysinamide-carbamoyl]cholesterol, 3 β [L- ornithinamide-carbamoyljcholesterol, and 3β[di-L-ornithinamide-carbamoyl] cholesterol.

Along with macromolecules, the cationic lipids of the present invention form complexes which are suitable to be transfected into cells. As the macromolecules to be transferred into cells, linear or circular polynucleotides may be used. Examples of the polynucleotides include DNAs, plasmids, and RNAs.

Available DNAs may be of single or double strands. As for RNAs, they include not only mRNA, tRNA, and rRNA, but also antisense RNA sequences complementary to target DNA or RNA sequences. Besides, rybozymes can be transfected by means of the cationic lipids of the present invention. As a rule, polynucleotides to be transfected into cells comprise structural genes or expression control factors. Typically, the structural genes encode polypeptides which are related to the treatment and/or diagnosis of diseases. For example, peptide hormones, histocompatible antigens, cell-adhesive proteins, cytokines, antibodies, cellular receptors, intracellular or extracellular enzymes and fragments thereof may be newly expressed after their corresponding genes are transfected with the aid of the cationic lipids of the present invention. The expression control factors which can be targets of the cationic lipids of the present invention may be exemplified by transcription promoters, enhancers, silencers, operators, terminators, attenuators, and so on. In accordance with another embodiment of the present invention, there is provided a liposome which can transfect a polynucleotide of interest into cells. The liposome is composed essentially of the cationic lipids of the present invention. Hereinafter, this liposome composed of the cationic lipids will be referred to as "cationic liposome". Liposomes are vesicles which have membranous envelopes consisting of lipids with an inclusion of water-soluble materials. At present, liposomes are used to carry matters, e.g., pharmaceutically useful materials, into target cells. In the pharmaceutical industry, liposomes can be utilized as sustained-release preparations because of the slow resolution of their lipid membranes. Such liposomes can be administered orally or by injection. A surprising feature of the liposomes is to localize their contents into desired targets or their environs.

In accordance with the present invention, the cationic liposomes may comprise only the cationic lipids of the general formula I. Preferably, the cationic liposomes of the present invention consist, by mol, of 10-90 % the cationic lipids of the general formula I and 90-10 % neutral lipids. When applied for the construction of the liposomes, the cationic lipid of the general formula I preferably has an amino acid moiety consisting of two to five amino acid residues when taking transfection efficiency and economical favor into account. More preferably, the number of the amino acid residues is on the order of 1 to 2. Where n is 1 or 2, examples of the cationic lipids include 3 β[L-lysinamide-carbamoyl] cholesterol, 3β[di-L-lysinamide-carbamoyl]cholesterol, 3β[L-ornithinamide- carbamoyljcholesterol, and 3β[di-L-ornithinamide-carbamoyl]cholesterol.

Illustrative, but non-limitative examples of available neutral lipids are dioleoyl phosphatidylethanolamine (DOPE) and cholesterol.

The construction of the cationic liposomes can be achieved by common techniques. For instance, after the lipid molecules are suspended in an aqueous medium, the suspension is subjected to ultrasonication to give globular vesicles which are very uniform in size. Alternatively, a solution of lipids in ethanol is rapidly mixed with water to afford globular vesicles.

In accordance with a further embodiment of the present invention, there is provided a pharmaceutical composition comprising the cationic lipid or liposome as a vector of carrying pharmaceutically active ingredients into cells. In this regard, the pharmaceutically active ingredients may be oligo- or polynucleotides suitable for use in gene therapy. When the cationic lipid or liposome is applied to the pharmaceutical composition, its amount is determined depending on the dose of the pharmaceutically active ingredients, patient conditions, disease severity, and other circumstances. In accordance with still another embodiment of the present invention, there is provided a method for synthesizing the cationic lipids of the general formula I. As mentioned earlier, the cationic lipid of the present invention is synthesized through the carbamate ester linkage formation between a lysinamide or an ornithinamide residue or an oligopeptide and a cholesteryl chloroformate. Herein, the present invention should not be construed to be limited by this method, but comprise modified techniques which are obvious to those who are skilled in the art.

A detailed description will be given of the synthesis method in conjunction with drawings, below. As shown in Fig. 1, the cationic lipids of the general formula I can be synthesized by conducting the following steps:

1) a Rink amide resin is swelled in an appropriate organic solvent and treated for a period of time in a solution of piperidine in an organic solvent to remove a protecting Fmoc (9-fluoromethoxycarbonyl) group.

2) an Nα-Fmoc-Nε-tBoc(tertiary butoxycarbonyl)-lysine or an Nα-Fmoc- Nε-tBoc-ornithine is activated at the carboxy group and linked to the deprotected resin, followed by the removal of the protecting Fmoc group.

This step, when it is desired, may be repeated to give a homo- or hetero- oligonucleotide consisting of lysine and/or ornithine residues. In this case, the preferable repeating number is within the range of 1-10 and more preferably within the range of 1 -5, as mentioned previously.

3) cholesteryl chloroformate is dissolved in methylene chloride and reacted with DIPEA (diisopropylethylamine) to link the free amine group of the amino acids to the activated hydroxy group of the cholesterol.

4) the resultant of the step 3) is added with TFA (trifluoroacetic acid)/methylene chloride to leave lysine-cholesterol or ornithine cholesterol and the side-chain protecting group from the solid support.

At its surface, the cationic liposome may be linked with hydrophilic polymeric chains which allow the cationic liposome to circulate in blood for a longer period of time. Examples of the hydrophilic polymers suitable for use in this purpose include polyethylene glycol (PEG), polylacetic acid, polyglycolic acid, polyvinyl pyrrolidone, polymethyl oxazoline, and polyethyl oxazoline with preference to polyethylene glycol. This polymer is an amphiphatic compound comprising a hydrophilic moiety and a hydrophobic moiety therein. When being chemically bonded to the cationic lipid composite or the cationic liposome composite of the present invention, the hydrophilic polymer has an effect of greatly attenuating the antigenicity of the composite. This hydrophilic polymer may be bonded to the cationic lipids via a linkage to the ε-amino group of lysine or the δ- amino group of ornithine or via a linkage to the amide group on the terminal carboxyl group, as shown in the following general formula II. The latter is preferred.

The polyethylene glycol which is attached to the cationic lipid preferably ranges, in molecular weight, from 0.5 to 20 kD.

To bind specifically to target cells, the cationic lipids of the present invention may be tailed by a marker or a ligand specific for the cationic lipids.

Preferable as the marker or ligand are antigens, transferrin, biotin, folic acid, low- density lipoprotein (LDL), monosaccharides. such as mannose, glucose and galactose, and disaccharides such as lactose. Selection of markers or ligands are determined depending on cell types because a certain type of cells possess characteristic receptors which can specifically bind to a type of ligands or markers.

Markers or ligands corresponding to certain cells are well known to those skilled in the art and no further descriptions are given in this regard.

The mechanism for the uptake or transfer of a material carried by the cationic lipid or liposome into cells is explained by endocytosis or fusion. When the material binds to a receptor at an area of cell membrane, the area of membrane engulfs the material so as for the material to go inside the cell. As for the cationic liposome composite, it fuses with cell membrane. When endocytosis occurs, endosomes which are directly responsible for transfection are formed within cells. In the case of the fusion, the liposomal membrane is incorporated into the membrane of a target cell while the contents of the liposome composite enter the cell, implementing transfection. No conclusive transfection mechanisms are known. However, it can be deduced that the endosomes comprising the cationic lipid composites fuse with large endoplasmic reticulums connected to the nuclear envelope to move the lipid composite into the perinulcear cistenae and then, the oligo- or polynucleotide enters the nucleus to participate in transcription and protein synthesis. An alternative deducible mechanism is that, while the cationic lipid composite electrostatically interchanges with anionic lipids of the endosome, the oligo or polynucleotide is dissociated from the cationic lipid, released into the cytoplasm and transferred into the nucleus to participate in transcription and protein synthesis.

The cationic lipids and the cationic liposomes of the present invention can be enzymatically degraded within cells. In addition, they are found to be remarkably low in toxicity to cells compared with conventional vehicles for gene transfer, as elucidated in Fig. 2.

A better understanding of the present invention may be obtained in light of the following examples which are set forth to illustrate, but are not to be construed to limit the present invention.

EXAMPLE I: Synthesis of Lysine-Cholesterol and Ornithine-Cholesterol

50-100 mg of a rink amide resin (Anaspec, USA) was immersed for more than one hour in DMF (N,N-dimethylformamide) until being sufficiently swelled. Thereafter, the resin was treated for more than 30 min in a solution of piperidine in

DMF to remove a protecting Fmoc (9-fluoromethoxycarbonyl) group. Using a solution of one equivalent of HBTU (2-(lH-nenzotriazole-l-yl)-l,l,3,3-tetramethyl- uronium hexafluorophosphate), one equivalent of HOBt (N-hydroxybenzotriazole- H₂O), and two equivalents of DIPEA (Diisopropylethylamine) in DMF, the carboxyl group of the Nα-Fmoc-N_ε-tBoc(tertiary butoxycarbonyl)-lysine or Nα-Fmoc-N_ε- tBoc-ornithine is activated and linked to the deprotected resin which was then washed many times with DMF. Subsequently, the protecting Fmoc group was removed from the amino acid by use of piperidine, followed by washing with DMF. This amino acid linking process, if necessary, was repeated once to attach, in series, two residues of lysine or ornithine to the resin. Afterwards, cholesteryl chloroformate was dissolved in methylene chloride and reacted with the amino acid bound to the resin in the presence of DIPEA to form a bond between the hydroxy group of the cholesterol and the amine group of the amino acid. The resulting resin was sufficiently washed with DMF, then with methylene chloride and finally with methanol and allowed to stand overnight under vacuum for drying.

From the solid support, the lysinamide-cholesterol (hereinafter referred to as "K-Chol") or the ornithinamide-cholesterol (hereinafter referred to as "O-Chol") was released, followed by the removal of the side-chain protecting group from the amino acid. This procedure was conduced by use of TFA (trifluoroacetic acid)/methylene chloride. The cationic lipid thus synthesized was allowed to precipitate in ethylether, washed several times, and dissolved in water before freeze- drying. A measurement was made of its molecular weight by FAB MS (fast atom bombardment mass spectroscopy) or MALDI-TOF MS (matrix-assisted laser desorption ionization-time of-flight mass spectroscopy).

Kl-Chol: IH NMR (300MHz, d6-OMSO) 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)

M.w. (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, -NH₂ of Lys) 7.7 (br, -CO-NH- of Lys).

M.w. (FAB-MS) m/z 686 [M+HJ+

Note: Kl : one lysine residue, K2: two lysine residues Ol-Chol: IH NMR (300MHz, 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) 7.2 (br, -NH₂ of Orn) 7.7 (br, -CO-NH- of Orn)

M.w. (MALDI-TOF MS) m/z 560.2 [M+Na]+

02-Chol: IH NMR (300MHz, 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)

M.w. (MALDI-TOF MS) m/z 673.2 [M+Na]+

Note: Ol : one ornithine residue, O2: two ornithine residues

EXAMPLE II: Preparation of Liposome

An appropriate amount of the K-chol synthesized in Example I was dissolved in water and stored in a refrigerator. An appropriate amount of DOPE, a commonly used, neutral lipid, was dissolved in chloroform, and dried with a blow of nitrogen gas and then, thoroughly under vacuum. Next, the cationic lipid in water was added with the dried DOPE at the same mass ratio and mixed by shaking the aqueous solution which was then allowed to stand overnight in a refrigerator. This chilled suspension was subjected to ultrasonication for several minutes in a bath to form liposomes. They were found to be about 146±8.2 in mean size as measured five times by a dynamic light scattering technique with the aid of Malvern 4700 system (Malvern Instrument Ltd., UK). The measurement records are given in Table 1 , below.

TABLE 1

Rounds Size (nanometer)

1 138.3

2 143.8

3 151.3

4 158.8

5 142.3 EXAMPLE III Toxicity Assay to 293, NIH3T3, HepG2 Cells

293. NIH3T3 and HepG2 cells are respectively cultured at 37 °C on 96-well plates containing a MEM (minimum essential media) supplemented with 10% FBS (fetal bovine serum) in 5% CO₂ atmosphere. Appropriate amounts of polyethylenimine and various cationic liposomes were added to the media. After two days of incubation, a solution of MTT (3-4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide) in PBS (phosphate buffered saline was added to the cells which were then further cultured for 4 hours in the same 5% CO₂ incubator. The cultured cells were added with DMSO (dimethylsulfoxide) before the measurement of absorbance at 570 nm. For comparison, cells treated with no polyethylenimine and cationic lipids were used as controls. The measurements of absorbance at 570 were visualized in plots of relative cell viability against the dose of the lipids, as shown in Fig. 2. The IC₅₀ values of the vehicles tested against the cells were summarized in Table 2, below.

As apparent from Fig. 2 and Table 2, the K-Chol/DOPE liposome and the O-Chol/DOPE liposome both are far lower in cell toxicity than are conventional gene-transfer vehicles, including Lipofectin, DC-Chol/DOPE liposome and PEI (polyethylenimine) .

TABLE 2 Cell Toxicity of Vehicles for Gene Transfer

Transfection IC₅o -g/m«)

Reagent 293 NIH3T3 HepG2

PEI 9.45 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

ND (not determined): The toxicity did not reach IC₅₀ even after the concentration of the reagent was over 60 μg/ml. EXAMPLE IV: Volumetric Measurement of K-Chol/DNA Composite

A beta-galactosidase gene carrying plasmid, such as that manufactured by Promega, identified as "pSV-β-gal", and K-Chol were mixed in HEPES (25 mM, pH7.4, 10 mM MgCl₂) to form a K-Chol/DNA composite which was subsequently allowed to stand for 30 min at room temperature. 1 μl of aliquots of the composite solution was placed on and allowed to be absorbed onto a fresh split mica disc surface for 1 -2 min. Filtration was conducted by means of a filter, followed by washing with pure water. The mica was dried at room temperature with a blow of N₂ gas. The K-Chol/plasmid DNA composite thus obtained was found to range, in size, from 150 to 200 nm as measured by an atomic force microscopic method using NanoScope Ilia System (Digital Instruments, Inc., Santa Barbara CA., USA). The results are shown in Fig. 3.

EXAMPLE V: Transfection into 293T and HepG2 Cells Using Cationic Lipids

Each of stocks of 293 T cells (embryonic human kidney cells) and HepG2 cells (human liver carcinoma cell line) were allotted on 24-well plates containing a DMEM (Dulbecco's modified eagle medium) or a MEM (minimum essential medium) and cultured. Separately, a plasmid carrying a beta-galactosidase gene was mixed with K-Chol to form a composite. This composite was transfected into the cultured cells. For comparison, Lipofectin and polyethylenimine were used as controls. A measurement was made of the quantity of the beta-galactosidase expressed in the cells and the results are shown in Fig. 4 and summarized in Table 3, below. As recognized from the histogram of Fig. 4, K-Chol is higher in gene transfer efficiency than are the controls. Particularly when applied for 48 hours to cells cultured in the presence of 10% FBS (fetal bovine serum), the vehicle of the present invention exhibited far superior gene transfer and expression efficiency to the other vehicles. TABLE 3 Gene Transfer Efficiencies of Vehicles

Expression of β-Galactosidase (mUits / well)

Gene Transfer HepG2 293T 293T Vehicle (-)FBS 4hrs (-)FBS 4hrs (+)FBS 48hrs

Lipofectin 2.25+ 0.12 12.7+ 0.50 3.53± 0.83

PEI 20.9± 1.8 14.1+ 0.93 0+ 0

K-Chol 11.4+ 2.4 19.8+ 1.6 12.3+ 0.62

Note: numerical values are a mean±standard deviation obtained after three rounds of measurement.

EXAMPLE VI: Transfection into 293T, NIH3T3 and HepG2 Cells Using Cationic Liposomes

A transfection experiment was made on 293 T (human embryonic kidney cell), NIH3T3 (mouse embryonic fibroblast cell), and HepG2 (human liver carcinoma cell line). For this, 293T and NIH3T3 cells were respectively cultured in DMEM (Dulbecco's modified eagle medium) on 24-well plates while HepG2 was done in MEM (minimum essential medium). The K-Chol/DOPE liposome and the O-Chol/DOPE liposome of the present invention were used as transfection vehicles.

Loading of a beta-galactosidase carrying plasmid into the vehicles was achieved by mixing at room temperature. These composites thus obtained were transfected into the cells for four hours in the absence of FBS. For comparison, Lipofectin, a DC- Chol/DOPE liposome and polyethylenimine were used as controls. In order to evaluate the gene transfer performance of the vehicles, quantities of the beta- galactosidase expressed in the cells were measured and the results are shown in Fig. 5. A summary of the measured results are given in Table 4, below. As apparent from the histogram of Fig. 5 and Table 4, the K-Chol/DOPE liposome and the O- Chol/DOPE exhibit high DNA transfection efficiencies over various cells. Particularly, the O-Chol/DOPE liposome is far superior in DNA transfer and expression performance. TABLE 4 DNA Transfer and Expression Yields

Expression of β-Galactosidase (mUnits/mg protein)

Gene Transfer (-) FBS, 4hr transfection

Reagent NIH3T3 HepG2 293

PEI 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+ 283.32 830.63± 159.87 1059.75+ 156.82 O-Chol/DOPE 978.84+ 284.1 1364.641 261.34 2064.61+ 87.46

EXAMPLE VII Identification of Transfection into HepG2 Cells Using X-Gal

With the aid of the lysine-cholesterol (K-Chol) prepared in Example I, a plasmid carrying a beta-galactosidase gene was transfected into HepG2 cells.

These cells were grown on agar plates containing X-gal to determine whether cells were transformed or not. Fig. 6 shows dyed cells which had beta-galactosidase expressed therein.

EXAMPLE VIII Transfection Efficiency For 293 Cells According to Time Period and FBS Influence

Into 293 cells, the K-Chol/DNA composite was transfected for various time periods. In order to examine the influence of serum on transfection efficiency, 293 cells were incubated in a 10% FBS-containing transfection medium and a serum-free transfection medium. After various predetermined time periods of incubation, the cells were transferred into fresh culture media supplemented with 10% FBS and cultured therein. After 48 hours of culture in fresh media, the cells discriminated according to the transfection period of time and FBS provision were measured for the expression of beta-galactosidase in order to determine the transfection efficiency. Fig. 7 shows the quantity of beta-galactosidase expressed in the cells with regard to transfection time period according to whether FBS is present or absent. As shown in these curves, the greatest transfection efficiency was obtained when the cells were incubated for 2-4 hours in the transfection media and the transfection performance of the composite is not dependent on the presence of serum.

Only a slight change of transfection efficiency was detected even after the cells were cultured for 48 hours, indicating that the cationic lipid of the present invention is almost free of cell toxicity. On the other hand, when the transfection medium was not supplemented with serum, the transfection efficiency was sharply decreased. This is believed to result from the cell death due to starvation.

INDUSTRIAL APPLICABILITY

As described hereinbefore, the cationic lipids of the present invention are capable of transferring nucleic acid materials into cells at very high efficiency with low cell toxicity. Also, the cationic lipids of the present invention perform good transfection over various cells, taking advantages over conventional vehicles, such as Lipofectin, DC-Choi and polyethylenimine in terms of transfection efficiency and cell toxicity. In addition, various cationic lipids could be synthesized fast and efficiently by the solid-phase method according to the present invention. Thus, the solid-phase synthesis method allows the automatic mass production of the vehicles for gene transfer. Consequently, the amino acid-cholesterol derivatives themselves and their liposomal forms according to the present invention are very useful in transfecting nucleic materials into cells as well as in delivering pharmaceutically active materials into target cells.

The present invention has been described in an illustrative manner, and it is to be understood that the terminology used is intended to be in the nature of description rather than of limitation. Many modifications and variations of the present invention are possible in light of the above teachings. Therefore, it is to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

Claims

1. An cationic lipid for transferring a nucleic material into cells, represented by the following general formula I:

wherein, 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.

2. The cationic lipid as set forth in claim 1, wherein n is an integer of 1-5.

3. The cationic lipid as set forth in claim 1, wherein said nucleic material is selected from the group consisting of DNAs, plasmids, RNAs, mRNAs, tRNAs, rRNAs, Rybozymes, and fragments thereof.

4. The cationic lipid as set forth in claim 3, wherein said DNA encodes a polypeptide suitable for use in medical treatment and diagnosis of diseases.

5. The cationic lipid as set forth in claim 4, wherein said polypeptide is selected from the group consisting of hormones, histocompatible antigens, cell- adhesion proteins, cytokines, antibodies, cell receptors, intracellular or extracellular enzymes, and fragments thereof.

6. The cationic lipid as set forth in claim 1, wherein said cationic lipid is 3β[L-lysinamide-carbamoly]cholesterol which is biodegradable.

7. The cationic lipid as set forth in claim 1, wherein said cationic lipid is 3 β[di-L-lysinamide-carbamoly] cholesterol which is biodegradable.

8. The cationic lipid as set forth in claim 1, wherein said cationic lipid is 3 β[L-ornithinamide-carbamoly] cholesterol which is biodegradable.

9. The cationic lipid as set forth in claim 1, wherein said cationic lipid is 3β[di-L-ornithinamide-carbamoly]cholesterol which is biodegradable.

10. The cationic lipid as set forth in claim 1, wherein a polyethylene glycol chain with a molecular weight of 0.5-20 kD is linked to the ε-amino group of the lysine residue, the δ-amino group of the ornithine residue, and/or the amide group on the terminal carboxyl group of the polypeptide.

11. The cationic lipid as set forth in claim 1 , wherein said cationic lipid is tailed by a marker or ligand selected from the group consisting of antigens, transferrins, biotin, folic acid, low-density proteins, mannose, glucose, galactose and lactose, said marker or ligand binding specifically to a target cell.

12. A pharmaceutical composition, comprising the cationic lipid of claim 1, and a nucleic acid material carrying a gene suitable for use in gene therapy.

13. The pharmaceutical composition as set forth in claim 12, wherein the gene encodes a polypepetide selected from the group consisting of hormones, histocompatible antigens, cell adhesion proteins, cytokines, antibodies, cell receptors, intracellular or extracellular enzymes, and fragments thereof.

14. A liposome for transferring a nucleic acid material into cells, consisting, by mole, of 10-90 % the cationic lipid of claim 1 and 90-10 % a neutral lipid.

15. The cationic liposome as set forth in claim 14. wherein said neutral lipid is selected from the group consisting of dioleoyl phosphatidylethanolamine, cholesterol and mixtures thereof.

16. The cationic liposome as set forth in claim 14, wherein said nucleic material is selected from the group consisting of DNAs, plasmids, RNAs, mRNAs, tRNAs, rRNAs, Rybozymes, and fragments thereof.

17. The cationic liposome as set forth in claim 14, wherein said DNA encodes a polypeptide suitable for use in medical treatment and diagnosis of diseases.

18. The cationic liposome as set forth in claim 18, wherein said polypeptide is selected from the group consisting of hormones, histocompatible antigens, cell- adhesion proteins, cytokines, antibodies, cell receptors, intracellular or extracellular enzymes, and fragments thereof.

19. The cationic liposome as set forth in claim 14, wherein said cationic lipid is 3 β[L-lysinamide-carbamoly] cholesterol which is biodegradable.

20. The cationic liposome as set forth in claim 14. wherein said cationic lipid is 3β[di-L-lysinamide-carbamoly]cholesterol which is biodegradable.

21. The cationic liposome as set forth in claim 14, wherein said cationic lipid is 3 β[L-ornithinamide-carbamoly] cholesterol which is biodegradable.

22. The cationic liposome as set forth in claim 14, wherein said cationic lipid is 3 β[di-L-ornithinamide-carbamoly] cholesterol which is biodegradable.

23. A pharmaceutical composition, comprising the cationic lipid of claim 1, and a nucleic acid material carrying a gene suitable for use in gene therapy.

24. The pharmaceutical composition as set forth in claim 23, wherein the gene encodes a polypepetide selected from the group consisting of hormones, histocompatible antigens, cell adhesion proteins, cytokines, antibodies, cell receptors, intracellular or extracellular enzymes, and fragments thereof.

25. A method for preparing a cationic lipid, represented by the following general formula I, comprising the formation of a carbamate ester linkage between lysine or ornithine residue and a cholesteryl chloroformate:

wherein, 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.