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Bioconjugate Chem. 2001, 12, 108−113
New Cationic Liposomes for Gene Transfer into Mammalian Cells with High Efficiency and Low Toxicity Joon Sig Choi, Eun Jung Lee, Hyung Suk Jang, and Jong Sang Park* School of Chemistry & Molecular Engineering, Seoul National University, San 56-1, Shillim-dong, Kwanak-ku, Seoul, 151-742, Korea. July 10, 2000
Novel cationic amphiphiles, based on hydrophobic cholesterol linked to L-lysinamide or L-ornithinamide, were designed and tested as nonviral gene transfer vectors. Each amide form of amino acid was conjugated to cholesterol by a carbamate ester bond to facilitate efficient degradation in animal cells. Cytotoxicity tests were performed for some cell lines. The transfection efficiency of the amphiphiles on different cell lines was evaluated as a liposomal solution in the presence of the fusogenic helper lipid, dioleoyl phosphatidylethanolamine (DOPE). The efficiency was also compared with other generally used gene carriers, such as lipofectin, 3β[N-(N′N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) liposome, and polyethylenimine (PEI).
INTRODUCTION
Gene therapy is generally considered as a promising approach not only in the treatment of diseases with genetic defects but also in the development of strategies for treatment and prevention of chronic diseases such as cancer, cardiovascular diseases, and rheumatoid arthritis. Intensive investigation has been conducted during the past decade into methods for transferring genes to cells (1-4). The methods used so far make use of either viral systems (retroviruses or adenoviruses) or nonviral systems (polymers, electroporation, calcium-phosphate precipitation, peptides, and liposomes). Even though some viral systems showed remarkable levels of expression, they are limited for general use in vivo due to their low capacity for large-sized therapeutic genes as well as short and long-term risks such as generation of host immune responses and the possibility of inserted genes combining with endogenous viruses or activation of oncogenes (2, 4). Consequently, there has been increasing attention focused on the development of safe and efficient nonviral gene transfer vectors (5), which are either polycationic polymers or cationic lipids. Polycationic polymers, e.g., poly-L-lysine, poly-L-ornithine, and polyethylenimine (6, 7), that could interact with DNA forming polyionic complexes, have been introduced for gene delivery. Various cationic lipids have also been proven to form lipoplexes with DNA and induce efficient transfection of a large number of eukaryotic cells (5, 8, 9). Among such kinds of synthetic vectors, cationic lipids are attractive and widely used because it is possible to design and synthesize various kinds of derivatives that are outstanding in the aspects of transfection efficiency, biodegradability, and low toxicity (10). Some cationic lipids are commercially available, and several lipids have already been used in the clinical setting. Among them, cationic cholesterol derivatives are known to be very useful for their high transfection efficiency and low toxicity (1117). However, there are still many issues to be tackled * To whom correspondence should be addressed. Phone: (82) 2 880 6660. Fax: (82) 2 889 1568. E-mail: pfjspark@ plaza.snu.ac.kr.
such as the gene transfer potency, the half-life in the blood stream, and the biocompatibility deserving less cytotoxicity. In light of the above considerations, we have prepared and described in this paper new promising DNA delivery agents composed of L-lysinamide, L-ornithinamide and cholesterol, which are designed to be readily susceptible to metabolic degradation after incorporation into animal cells. 3β[L-lysinamide-carbamoyl] cholesterol (K-Chol)1 and 3β[L-ornithinamide-carbamoyl] cholesterol (O-Chol) were synthesized by the solid-phase synthesis method which has several advantages such as simplification of reaction procedures, easy separation of the supported products, and possibility of application to automatic systems. It was demonstrated that the newly designed cationic cholesterol derivatives were less toxic and more efficient in transfection than lipofectin and DC-Chol. The results for the biocompatible lipids suggest their potential use as general reagents for transfection of mammalian cells and in vivo applications for gene therapy. MATERIALS AND METHODS
Materials. Ν-R-Fmoc-Ν--tBOC-L-lysine, Ν-R-Fmoc-Ν-tBOC-L-ornithine, N-hydroxybenzotriazole (HOBt), and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were purchased from AnaSpec, Inc. (San Jose, CA), and cholesteryl chloroformate, N,Ndiisopropylethylamine (DIPEA), N,N-dimethylformamide (DMF), piperidine, dichloromethane, and polyethylenimine (PEI, 25 kDa) were from Aldrich (St. Louis, MO). N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES), ethidium bromide (EtBr), o-nitrophenyl-β-Dgalactopyranoside (ONPG), 1,2-dioleoyl-sn-glycero-3phosphatidylethanolamine (DOPE), and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO). Rink amide 1 Abbreviations: K-Chol, 3β[L-lysinamide-carbamoyl] cholesterol; O-Chol, 3β[L-ornithinamide-carbamoyl] cholesterol; DCChol, 3β[N-(N′N′-dimethylaminoethane)-carbamoyl] cholesterol; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine; PEI, polyethylenimine; ONPG, o-nitrophenyl-β-D-galactopyranoside.
10.1021/bc000081o CCC: $20.00 © 2001 American Chemical Society Published on Web 01/17/2001
Cationic Liposomes for Gene Transfer Scheme 1. Outline Synthesis of K-Chola
of
Bioconjugate Chem., Vol. 12, No. 1, 2001 109 Step-wise
Solid-Phase
a (i) Fmoc-Lys(Boc)-OH, HOBt, HBTU, DIPEA, DMF. (ii) 30 % piperidine. (iii) Cholesteryl chloroformate, DIPEA, dichloromethane. (iv) TFA/DCM (50:50).
resin was from Novabiochem (San Diego, CA). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), and Dulbecco’s phosphate-buffered saline (DPBS) were purchased from GIBCO (Gaithersburg, MD). Minimal essential medium (MEM) was from Hyclone (Logan, UT). 5× Reporter lysis buffer, lipofectin, and pSV-β-gal plasmid (EMBL accession no. X65335) were all purchased from Promega (Madison, WI). DCChol was synthesized according to Gao and Huang (12). Synthesis of K-Chol and O-Chol. As shown in Scheme 1, L-lysine was attached to the rink amide resin by using fluoren-9-ylmethoxycarbonyl (Fmoc) chemistry (18, 19). Coupling of amino acid to the resin was performed with 6 equiv of HOBt, HBTU, N-R-Fmoc-N-tBOC-L-lysine (AnaSpec, Inc., San Jose, CA), and DIPEA, respectively, in anhydrous DMF. Piperidine (30%) was used for deprotection of the Fmoc group of lysine. Cholesteryl chloroformate was added to the resinbound lysine (6 equiv, DCM, 25 °C, 4 h). The progress of each reaction was monitored by ninhydrin test until completion. After treating TFA (50:50 TFA-DCM v/v, 4 °C, 1.5 h), the final product was precipitated in ethyl ether and washed with excess ether. The product was solubilized in water and collected by freeze-drying yielding colorless powder (85% yield based on the initial loading level of rink amide resin). The compound gave a single spot on a TLC plate (ethyl acetate:n-butanol:acetic acid:H2O ) 2:1:1:1 v/v/v/v, Rf ) 0.7). 1H NMR, (300 MHz, d6-DMSO) δ (ppm) 0.65-2.26 (m, skeleton of cholesterol, -(CH2)3- of Lys), 2.75 (br, s, -CH2 of Lys), 4.3 (br, s, R-CH of Lys) 7.2 (br, -NH2 of Lys) 7.7 (br, -CO-NH- of Lys). Matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectra, m/z 576 [M + Na]+. The product was resuspended in water and stored in refrigerator before use. The compound was stable in aqueous solution over 6 months at 4 °C. The overall synthesis scheme for O-Chol was the same as described above. Ν-R-Fmoc-Ν--tBOC-L-ornithine was used for the preparation of O-Chol. The compound also gave a single spot on a TLC plate (Rf ) 0.63). 1H NMR (300 MHz, d6-DMSO) δ (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, -CONH- of Orn). MS (MALDI) m/z 560.2 [M + Na]+. Liposome Preparation. DOPE in chloroform was evaporated under a stream of N2 gas and further dried under vacuum. Each cationic lipid solubilized in water was added to the dried DOPE and vortex mixed (weight ratio of cationic lipid: DOPE ) 1:1). After overnight incubation at 4 °C for hydration, the mixture was sonicated using a bath-type sonicator. The shape and size distribution of the liposome was determined by atomic force microscopy as described below. Plasmid Preparation. Plasmid pSV-β-gal, where the β-galactosidase reporter gene is expressed was amplified in Escherichia coli and prepared as described previously (20). Agarose Gel Electrophoresis. Lipoplexes were formed at different charge ratios between K-Chol and pSV-β-gal plasmid by incubation in serum-free DMEM at room temperature for 30 min. Each sample was then analyzed by electrophoresis on a 0.7% agarose gel containing ethidium bromide (0.5 µg/mL of gel). Atomic Force Microscopy. Atomic force microscopy (Nanoscope IIIa system, Digital Instruments, Inc., Santa Barbara. CA) was used for imaging the shape and particle size of the liposomes and liposome-DNA complexes, as previously reported (21). Briefly, 2 µL aliquots of either liposome solution or aqueous liposome-DNA complex solutions were loaded onto the center of a freshly split mica disk. The charge ratio of liposome/DNA was set to 1.7, at which the transfection efficiency was optimal. After adsorption for 1-2 min at room temperature, excess complex solution was removed by absorption onto filter paper and the mica surface was further dried at room temperature before imaging. The image mode was set to tapping mode and the average scanning speed was 5 Hz. Cell Culture. Human embryonic kidney 293 and mouse embryonic fibroblast NIH3T3 cells (ATCC, Rockville, MD) were grown in DMEM with 10% FBS. Human liver carcinoma HepG2 cells (Korean Cell Line Bank) were propagated in MEM supplemented with 10% FBS. All cells were routinely maintained on plastic tissue culture dishes (Falcon) at 37 °C in a humidified 5% CO2/ 95% air containing atmosphere. Determination of Cell Toxicity. Each of the cell lines was seeded in 96 well plates (1-2 × 104 cells/well) and incubated for 1 day prior to experiment. Lipofectin, PEI, and cationic liposomes were introduced to the cells and incubated for 48 h. The cytotoxicity was determined by comparing the amount of MTT reduced by the cells treated with carriers to that reduced by control cells (22). Transfection Procedures. For transfection, (1-1.5) × 105 cells/well were seeded in 24 well plates 1 day prior to transfection experiments, and grown in the appropriate medium with 10% FBS. The cell lines were 60-70% confluent at the time of transfection. Complexes were prepared by mixing each reagent with plasmid DNA (2.0 µg/well) in FBS-free medium. Each complex solution was further incubated for 30 min at room temperature and added to the cells. Transfection was performed in serumfree medium for 4 h. Each medium was replaced with a fresh complete medium and gene expression was assayed 48 h post-transfection. Control transfections were performed by using DC-Chol liposome and commercially available transfection reagents, such as lipofectin and PEI. Transfection Assay. The expressed β-galactosidase activity was measured by a standard method as recommended by the manufacturer (23). Briefly, each cell in
110 Bioconjugate Chem., Vol. 12, No. 1, 2001
Figure 1. The structures of the cationic lipids. (A) DOTMA (lipofectin). (B) DC-Chol. (C) K-Chol. (D) O-Chol.
the 24 well plates was washed with DPBS and lysed with Reporter lysis buffer. The cell lysates were analyzed using the colorimetric ONPG assay in a 96-well plate format. The protein concentration was determined by the Pierce Micro BCA Protein Assay Reagent Kit, using BSA as standard. β-Galactosidase activity is expressed in total milliunits per mg of protein. One milliunit is defined as the amount of β-galactosidase that hydrolyzes 1 nmol of ONPG/min at pH 7.5 at 37 °C. RESULTS AND DISCUSSION
Syntheses and Features of K-Chol and O-Chol. The cationic lipids, K-Chol and O-Chol, were prepared in good yields by the solid-phase synthesis method and their structures are presented in Figure 1. The amphiphiles are composed of naturally occurring lipid and amino acid that could be metabolized after administration into animal cells. The amphiphiles are structurally related to the various cationic cholesterol derivatives previously reported, such as ChoSC or ChoTB (11), DCChol (12), spermine-cholesterol (13), cholesteryl-spermidine (14), BGSC or BGTC (15), and some DC-Chol derivatives (16, 17). As the primary amino group is abundant in poly-L-lysine and PEI, which are generally used in polymer-mediated gene transfer, that of lysinamide or ornithinamide is considered to be capable of interacting electrostatically with polyphosphate anions of plasmid DNA. The amphiphiles contain a carbamate ester bond that could be efficiently degraded in the cells (12). Charge Ratio-Dependent Complex Formation and Transfection Activity. To assess the complex formation of K-Chol with DNA, agarose gel electrophoresis of cationic lipid:DNA complexes was performed at various charge ratios. As shown in Figure 2A, plasmid DNA became completely saturated at a charge ratio of around 2 ((). In addition, some completely retarded
Choi et al.
Figure 2. (A) Agarose gel retardation of K-Chol/DNA complexes as a function of cationic lipid to plasmid DNA charge ratio. pSV-β-gal plasmid DNA (1.0 µg) only (lane 1), charge ratio of K-Chol/DNA ) 0.2, 0.3, 0.6, 1.7, and 2.8 (lanes 2, 3, 4, 5, and 6, respectively). (B) β-Galactosidase expression as a function of the mean ionic charge ratio of the lipid/DNA complexes (-NH3+ group of the cationic lipid to DNA phosphate anion). HepG2 cells were transfected at a fixed amount of DNA (2.0 µg/well) with various amounts of cationic liposome. Data are shown as mean ( SD (n ) 3).
complexes were observed at charge ratios even below 1, which indicates that the mode of K-Chol binding to DNA is cooperative (24). The results shown in Figure 2B demonstrate the dose dependent transfection activity of K-Chol and O-Chol liposomes for HepG2 cells, with an optimal charge ratio of 1.7 ((). It was apparent that the transfection efficiency showed a bell-shape dependence on the liposome concentration. Low transfection efficiency at charge ratios below 1 is generally presumed to be due to reduced cell membrane binding, and decreased efficiency at high charge ratios seems to be due to the toxicity of the cationic lipid (15). AFM Images of the Liposomes and the Liposome-Plasmid DNA Complexes. To examine the shape and size distribution of liposomes and liposomeDNA complexes, atomic force microscopy was introduced. As shown in Figure 3, panels A and B, K-Chol/DOPE liposome gave small vesicles with a diameter of 40-110 nm and O-Chol/DOPE liposome was somewhat larger in size, ranging from ca. 60 to 150 nm. When these liposomes were complexed with plasmid DNA, larger heterogeneous particles were observed and their shapes varied from spherical vesicles to amorphous aggregates (Figure 3, panels C and E). Uncomplexed free liposomes were also observed. As for K-Chol/DOPE-DNA complexes, the particles were 0.3-0.6 µm in size and O-Chol/DOPEDNA complexes gave larger particles in the range 0.41.2 µm. As presented in Figure 3, panels C and E, fiberlike structures were also observed surrounding spherical vesicle-like structures. Some sections of Figure 3, panels C and E, are enlarged and presented in panels
Cationic Liposomes for Gene Transfer
Bioconjugate Chem., Vol. 12, No. 1, 2001 111
Figure 3. AFM images of liposomes and liposome-DNA complexes. (A) K-Chol/DOPE liposome; (B) O-Chol/DOPE liposome; (C), (D) K-Chol/DOPE liposome-DNA complex; (E), (F) O-Chol/DOPE liposome-DNA complex. The white color indicates a height in excess of the designated nm above the mica surface. The x and y dimensions are scaled as shown.
D and F, respectively. These complexes showed aggregates connected in rows by fibrous structures, and this result conforms to previous electron microscopy studies that have reported large aggregates surrounded by thin fibers (25). Even though the structure and location of complexed DNA could not be definitely distinguished in the presented images, the fibrous structures are presumed to result from the interaction of DNA with the cationic liposomes. Effects of the Liposomes on the Viability of Cells. The cytotoxicity of our cationic liposomes was compared with that of lipofectin, DC-Chol liposome, and PEI, all are widely used as gene transfer reagents. As shown in Figure 4, the viability of the cells decreased abruptly accordingly as the amount of PEI or lipofectin increased. Cationic cholesterol liposomes showed a lower toxicity level for 293 cells compared to lipofectin and PEI (Figure 4A). In the case of NIH3T3 cells, toxicity was also observed for all the reagents at concentrations much higher than those required for optimal transfection, even though K-Chol and O-Chol liposomes showed much
reduced toxicity toward the cells at the same concentration ranges (Figure 4B). For HepG2 cells, K-Chol and O-Chol liposomes were noticeably less toxic to the cells than other reagents (Figure 4C). The IC50 values of the transfection reagents are shown in Table 1. An important feature of our cationic liposomes is their relatively low toxicity toward the cells at concentrations required for optimal transfection, for cytotoxicity is one of the major hurdles in the application of many cationic amphiphiles. The toxicity of some of the commercially available cationic lipids and synthetic polycationic polymers, such as lipofectin and PEI, has been attributed to their nonnatural, nonbiodegradable nature. Transfection Efficiency on Different Cell Lines. To investigate the effectiveness of K-Chol and O-Chol for in vitro transfection of mammalian cells, we performed in vitro transfection tests with some cell lines routinely used in laboratories. It already has been shown that some cell types, especially NIH3T3 cells, are not susceptible to transfection with lipofectin (8, 26, 27) or DC-Chol (27). It was therefore of interest to examine the versatility in
112 Bioconjugate Chem., Vol. 12, No. 1, 2001
Choi et al. Table 1. Comparison of Cytotoxicity for Different Transfection Reagentsa IC50 (µg/mL) transfection reagents
293
NIH3T3
HepG2
polyethylenimine lipofectin DC-Chol/DOPE K-Chol/DOPE O-Chol/DOPE
9.45 35.5 ND 57.5 ND
