Bioconjugate Chem. 2004, 15, 1095−1101
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Gene-Transferring Efficiencies of Novel Diamino Cationic Lipids with Varied Hydrocarbon Chains Hong Sung Kim,† Jaeho Moon,† Keun Sik Kim,† Myung Min Choi,† Ji Eun Lee,† Yeon Heo,‡ Dae Hyan Cho,‡ Doo Ok Jang,‡ and Yong Serk Park*,† Department of Biomedical Laboratory Science and Department of Chemistry, Yonsei University, Wonju 220-710, Korea. Received March 13, 2004
Utilizing three biocompatible components, a series of novel cationic lipids has been chemically synthesized and tested for their gene-transferring capabilities in 293 transformed kidney cells and B16BL6 mouse melanoma cells. The synthesized cationic lipids consisting of a core of lysine and aspartic acid with hydrocarbon chains of varied length were assigned the acronyms DLKD (O,O′dilauryl N-lysylaspartate), DMKD (O,O′-dimyristyl N-lysylaspartate), DPKD (O,O′-dipalmityl Nlysylaspartate), and DSKD (O,O′-distearyl N-lysylaspartate). The gene-transferring capabilities of these cationic lipids were found to be dependent on the hydrocarbon chain length. Under similar experimental conditions, the order of gene transfection efficiency was DMKD > DLKD > DPKD > DSKD. Addition of cholesterol or dioleoyl phosphatidylethanolamine (DOPE) as a colipid did not change this order. Colipid addition affected the transfection efficiency positively or negatively depending on the length of the cationic lipid acyl chain. On the whole, the length of the hydrophobic carbon chain was a major factor governing the gene-transferring capabilities of this series of cationic lipids. The observed differences in transfection efficiency may be due to differing binding affinities to DNA molecules as well as differences in the surface charge potential of the liposome-DNA complexes (lipoplexes) in the aqueous environment.
INTRODUCTION
Cationic liposomes have become established as one of the leading nonviral gene transfer systems for both clinical and nonclinical purposes. The ease and rapidity of preparation of liposome-DNA complexes (lipoplexes) coupled with their relative safety, demonstrated in a number of preclinical and clinical tests, has expanded their applications despite relatively low transfection efficiency as compared to that of viral vector systems (1). The low transfection efficiency is related to the biophysical characteristics of cationic liposomes as well as the biological activity of plasmid DNA inside mammalian cells (2). Compared with viral infection, internalization of DNA vectors mediated by cationic lipids is a less specific and less active process. In addition, plasmid DNA transferred into the cytoplasm is incapable of selfreplication and is vulnerable to intracellular degradation. However, starting from the initial interactions of lipoplexes with cells and ending with gene expression, the many obstacles encountered along the way suggest that, if they can be resolved, additional possibilities for clinical application of this delivery system may lie in the future. Potential clinical applications of cationic liposomemediated gene delivery have been demonstrated in disease models of various cancers (3,4) and some genetic diseases such as cystic fibrosis (5,6). However, at present, the low transfection efficiency of cationic lipidic vectors has hindered their application in those areas of gene therapy such as genetic diseases that require persistent
gene expression at a therapeutic level. Hence, a great deal of effort, including synthesis of new cationic lipids (7) and providing lipoplexes with new characteristics (8), has been expended in the search for more effective cationic lipid vectors. A number of preclinical and clinical studies have claimed that lipoplexes are safe when delivered locally in relatively low doses (9). However, it has been reported that, in a mouse model, intravenous administration of high doses of lipolexes induces acute toxicity (10). Several groups have been working on establishment of a structure-function relationship to define the requirements for the safe and efficient gene transfection of cationic lipids (11). To begin to address transfection efficiency and safety concerns, we synthesized a series of novel cationic lipids incorporating the biocompatible and biodegradable components, lysine, aspartate, and fatty acyl chains. In this series of cationic lipids based on lysylaspartate (KD), diacyl derivatives with hydrocarbon chains of differing length (C12, C14, C16, and C18) exhibited different DNAbinding affinities which could be correlated with their different in vitro gene-transferring capabilities. The KD derivatives with the short acyl chains (C12 and C14) exhibited stronger DNA-binding affinity and more efficient transfection than ones with long acyl chains. Biocompatible cationic liposomes of the type described here may be more suitable for preclinical and clinical applications. EXPERIMENTAL PROCEDURES
* To whom correspondence should be addressed. Phone: +82-33-760-2448. Fax: +82-33-763-5224. E-mail: yspark@ dragon.yonsei.ac.kr. † Department of Biomedical Laboratory Science. ‡ Department of Chemistry.
Reagents. Dioleoyl phosphatidylethanolamine (DOPE) and N-[1-(2,3-dioleoyloxy)]-N,N,N-trimethylammonium propane methyl sulfate (DOTAP) were purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol was
10.1021/bc049934t CCC: $27.50 © 2004 American Chemical Society Published on Web 08/14/2004
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Figure 1. Synthetic outline for the KD series of cationic lipids
purchased from Sigma (St. Louis, MO). All organic reagents employed in the synthesis of cationic lipids were obtained from Aldrich (Milwaukee, WI) and used as received. Synthesis of Dialkyl N-Carbobenzyloxy-L-aspartate (3a-d). Chemical synthesis of the KD series of cationic lipids is outlined in Figure 1. A solution of N-carbobenzyloxy-L-aspartic acid (1) (500 mg, 1.87 mmol), 1-dodecanol (2a) (1.06 mL, 4.68 mmol), and p-toluenesulfonic acid monohydrate (71 mg, 0.37 mmol) in toluene (30 mL) was heated at reflux with a Dean-Stark trap. After 16 h, the solution was washed with water and dried over anhydrous MgSO4. After filtration, the solvent was removed in vacuo and the residue purified by column chromatography over silica gel (eluent: hexane-EtOAc ) 9.5:0.5) to afford 960 mg of 3a (85%). 1H NMR (CDCl3) δ 0.90-1.90 (m, 50H), 2.99 (m, 2H), 4.17 (m, 4H), 4.94 (m, 1H), 5.40 (m, 2H), 5.65 (m, 1H), 7.30-7.45 (m, 5H). Compounds 3b, 3c, and 3d were synthesized using the same method as described above for compound 3a in 66, 62, and 65% yields, respectively. 3b: 1H NMR (CDCl3) δ 0.93-2.00 (m, 54H), 3.11 (m, 2H), 4.19 (m, 4H), 4.94 (m, 1H), 5.43 (m, 2H), 5.60 (m, 1H), 7.26-7.38 (m, 5H). 3c: 1 H NMR (CDCl3) δ 0.95-1.60 (m, 62H), 2.99 (m, 2H), 4.26 (m, 4H), 4.60 (m, 1H), 5.53 (s, 2H), 5.80 (m, 1H), 7.3-7.5 (m, 5H). 3d: 1H NMR (CDCl3) δ 0.82-1.84 (m, 70H), 2.99 (m, 2H), 4.18 (m, 4H), 4.60 (m, 1H), 5.21 (s, 2H), 5.80 (m, 1H), 7.3-7.5 (m, 5H). Synthesis of Dialkyl L-Aspartate (4a-d). A solution of compound 3a (523 mg, 0.87 mmol) and 10% Pd/C (523 mg, 0.49 mmol) in THF (20 mL) was stirred at room temperature under a hydrogen atmosphere until the reaction was complete. The catalyst was filtered off through a Celite pad and the filtrate concentrated and purified by flash column chromatography on silical gel (eluent: hexane-EtOAc ) 8:2) to afford 387 mg of 4a
(95%). 1H NMR (CDCl3) δ 0.78-1.71 (m, 50H), 2.72 (m, 2H), 3.80 (m, 1H), 4.12 (m, 4H). Compounds 4b, 4c, and 4d were synthesized using the same method as described above for compound 4a in 94, 91, and 92% yields, respectively. 4b: 1H NMR (CDCl3) δ 0.82-1.72 (m, 54H), 2.74 (m, 2H), 3.81 (m, 1H), 4.12 (m, 4H). 4c: 1H NMR (CDCl3) δ 0.67-1.63 (m, 62H), 2.39 (m, 2H), 3.39 (m, 1H), 4.11 (m, 4H). 4d: 1H NMR (CDCl3) δ 0.82-1.62 (m, 70H), 2.74 (m, 2H), 3.80 (m, 1H), 4.12 (m, 4H). Synthesis of Bis(N,N′-tert-butoxycarbonyl)aspartic Acid (6). L-Lysine monohydrochloride (200 mg, 1.01 mmol) was dissolved in aqueous NaOH solution (131.5 mg of NaOH in 3 mL of H2O) followed by addition of a THF (3 mL) solution of di-tert-butyl dicarbonate (477.9 mg, 2.19 mmol). The mixture was stirred at room temperature overnight and then was neutralized with 1 M HCl aqueous solution and extracted with CH2Cl2. The organic layer was dried over anhydrous MgSO4 and concentrated to give 311 mg of 6 (82%). 1H NMR (CDCl3) δ 1.21-1.91 (m, 24H), 3.12 (m, 2H), 3.76 (m, 1H), 4.28 (br, 1H), 6.03 (br, 1H). Synthesis of 2-(2,6-Bis-tert-butoxycarbonylaminohexanoylamino) Succinic Acid Dialkyl Ester (7ad). A solution of compound 6 (260 mg, 0.75 mmol), N-hydroxysuccinimide (87 mg, 0.75 mmol), and 1,3dicyclohexylcarbodiimide (388 mg, 1.88 mmol) in THF (30 mL) was stirred for 1 h at room temperature. Compound 4a (294 mg, 0.63 mmol) was added to the above solution, and the resulting mixture was stirred for an additional 6 h. The reaction mixture was washed with water, dried, and concentrated. The residue was purified by column chromatography on silica gel (eluent: hexane-EtOAc ) 8:2) to furnish 377 mg of 7a (75%). 1H NMR (CDCl3) δ 0.80-1.58 (m, 74H), 2.73-3.12 (m, 4H), 4.15 (m, 4H), 4.64 (br, 1H), 4.82 (m, 2H), 5.15 (br, 1H), 6.91 (br, 1H). Compounds 7b, 7c, and 7d were synthesized using the
Cationic Liposome-Mediated Gene Transfer
same method as described above for compound 7a in 62, 61, and 62% yields, respectively. 7b: 1H NMR (CDCl3) δ 0.82-1.58 (m, 78H), 2.76-3.14 (m, 4H), 4.14 (m, 4H), 4.65 (br, 1H), 4.86 (m, 2H), 5.15 (br, 1H), 6.91 (br, 1H). 7c: 1 H NMR (CDCl3) δ 0.96-1.62 (m, 82H), 2.74-3.11 (m, 4H), 4.07 (m, 4H), 4.65 (br, 1H), 4.68 (m, 2H), 5.13 (br, 1H), 6.90 (br, 1H). 7d: 1H NMR (CDCl3) δ 0.82-1.83 (m, 86H), 2.77-3.16 (m, 4H), 4.17 (m, 4H), 4.69 (br, 1H), 4.91 (m, 2H), 5.14 (br, 1H), 6.91 (br, 1H). Synthesis of 2-(2,6-Bis-tert-butoxycarbonylaminohexanoylamino) Succinic Acid Dialkyl Ester Dihydrochloride (8a-d). Compound 7a (494 mg, 0.62 mmol) was dissolved in THF (10 mL) and treated with 4 M HCl in 1,4-dioxane (10 mL) at 0 °C. After 6 h, the reaction mixture was concentrated and the residue was washed with Et2O, affording 344 mg of 8a (93%). Similarly, 8b, 8c, and 8d were prepared in 93, 94, and 92% yields, respectively. Cells and Cell Culture. Transformed kidney cells (293 cells) were purchased from the American Type Culture Collection (ATCC, Manassa, VA). The 293 cells were grown at 37 °C under a 5% CO2 atmosphere in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Tulsa, OK), supplemented with 10% fetal bovine serum, 1 mM sodium bicarbonate, 50 µg/mL streptomycin, and 500 U/mL penicillin. Highly metastatic B16BL6 mouse melanoma cells were provided by Dr. Fidler (M. D. Anderson Cancer Center, Houston, TX). The melanoma cells were grown in Minimum Essential Medium Eagle (MEM) supplemented with 5% FBS, 100 µM vitamin solution, 100 µM sodium pyruvate, 10 µM nonessential amino acid, and penicillin/streptomycin antibiotics. Preparation of Plasmid DNA-Lipid Complexes. The plasmid used in this study was pAAVCMV (prepared by Dr. M. J. During, Jefferson Medical College, Philadelphia, PA) containing a luciferase gene under the control of adeno-associated virus inverted terminal repeats and the cytomegalovirus promoter. The plasmid was prepared with a plasmid maxiprep kit (QIAGEN, Germany). Appropriate amounts of cationic lipids (DLKD, DSKD, DPKD, DMKD, or DOTAP) alone or with helper lipids (DOPE or cholesterol) were mixed in chloroform. The organic solvent was evaporated under a stream of N2 gas. Vacuum desiccation for about 2 h ensured removal of the residual organic solvent. The dried lipid films were hydrated in an appropriate amount of saline and then vigorously mixed with a vortex mixer for 5 min. Varied amounts of plasmid DNA in saline were gently mixed with appropriate amounts of the prepared liposomal solution. To provide stable lipoplex formation, the lipidDNA mixtures were incubated at room temperature for 30 min before they were added to cultured cells. Gel Retardation Analysis of Lipoplexes. Various amounts of cationic liposomes (0.3-12 nmol in 50 µL of saline) were added to 1 µg of DNA in 50 µL of saline, which were then incubated at room temperature for 30 min. A small aliquot of ethidium bromide (EtBr) solution was added to 20 µL of the lipoplex solution, which was run on 0.7% agarose gel and visualized by UV illumination (Fluor-S MultiImage, Bio-Rad, Herculus, CA). ζ-Potential Measurement. The ζ-potentials of the lipoplexes were measured using a zetasizer (Zetasizer4, Malvern, U.K.). The lipoplex suspensions in Hepes buffer (pH 7.5) were loaded in the capillary cell mounted on the zetasizer, and their ζ-potentials were measured five times per sample at 25 °C. In Vitro Gene Transfection. The 293 cells (1 × 105 cells/well) were plated onto 24-well plates and incubated
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at 37 °C under 5% CO2 for 24 h. The various lipoplex formulations were added onto the 293 or B16BL6 cells in serum-free media, which were then incubated further for 4 h. After transfection, the cell culture medium containing the lipoplexes was replaced with a fresh serum-containing medium and the transfected cells were incubated for an additional 48 h. After removal of the culture medium by aspiration, the transfected cells in each well were carefully washed twice with 3 mL of PBS. The cells were lysed with 100 µL of lysis buffer (0.1 M Tris-HCl, 2 mM EDTA, and 0.1% Triton X-100, pH 7.8). The luciferase activity in a 10 µL aliquot of the cell lysate was measured with a luminometer (Minilumat LB9506, EG&G, Germany) and a luciferase assay kit (Promega, Madison, WI). The protein concentration of each cell lysate was determined by a standard protein assay (BioRad Protein Assay, Bio-Rad, Hercules, CA). The luciferase activity in each sample was normalized to the relative light unit (RLU) per microgram of protein. Cytotoxicity of Cationic Liposomes. The cytotoxicity of cationic liposomes was determined by MTT assay (12). The B16BL6 cells were plated into 96-well plates (5 × 103 cells per well) and cultured for 24 h. The cells were treated with various concentrations of cationic liposomes and then cultured for 24 h. Fifty microliters of MTT solution (1 mg/mL) was added to the cells, which were further cultured for 4 h. After the media containing MTT were removed, 100 µL of DMSO was added to solubilize the MTT-formazan product. The absorbance at 540 nm was measured with a microplate reader (Molecular Devices, Sunnyvale, CA). RESULTS
Synthesis of Diacyl Lysylaspartate Cationic Lipids. We have synthesized a series of biocompatible cationic lipids that exhibit potent gene-transferring capability. These KD cationic lipids, composed of three biocompatible molecular moieties, lysine (the cationic headgroup), aspartic acid (the anchor), and two acyl chains (the hydrophobic tails), were synthesized as outlined in Figure 1. Esterification of N-carbobenzyloxy-L-aspartic acid (1) with alcohols 2a-d in the presence of p-toluenesulfonic acid monohydrate was carried out to obtain the corresponding diesters 3a-d in 62-85% yields. Deprotection of the N-carbobenzyloxy group of the diesters 3a-d with 10% Pd/C under hydrogen atmosphere gave the dialkyl aspartates 4a-d. L-Lysine monohydrochloride (5) was transformed into 6 with di-tert-butyl dicarbonate, which was coupled with the compounds 4a-d in the presence of NHS (N-hydroxysuccinimide) and DCC at room temperature, affording compounds 7a-d in 61-75% yields. The desired cationic lipids 8a-d were prepared by treatment of 7a-d with 4.0 M HCl in dioxane at 0 °C. Formation of DLKD-, DSKD-, DPKD-, and DMKDBased Lipoplexes and Their Surface Charges. (DLKD ) O,O′-dilauryl N-lysylaspartate, DMKD ) O,O′-dimyristyl N-lysylaspartate, DPKD ) O,O′-dipalmityl Nlysylaspartate, and DSKD ) O,O′-distearyl N-lysylaspartate.) It has been well documented that electronic interactions between the headgroups of the cationic lipids and DNA molecules cause the formation of liposomeDNA complexes, called “lipoplexes”. However, the strength of these interactions is dependent on the biophysical characteristics of the cationic lipids and the polymorphism of the liposomal membranes. The gel retardation assay is a simple method that can be used to show the relative strength of electric interac-
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Figure 2. Gel retardation of pDNA complexed to KD liposomes. An aliquot of EtBr solution was added to plasmid DNA complexed to DLKD, DMKD, DPKD, DSKD, and DOTAP liposomes at various charge ratios. The lipoplexes were run on agarose gel and visualized by UV illumination.
Figure 3. ζ-potentials of KD lipoplexes. The DLKD, DMKD, DPKD, DSKD, and DOTAP lipoplexes were prepared at various charge ratios of lipid to pDNA and loaded into the capillary cell mounted on a zetasizer. The numbers given were calculated from five measurements at 25 °C.
tions between cationic liposomes and DNA molecules. In this assay, a cationic liposome suspension and a DNA solution are mixed in various charge ratios from 0.4:1 to 12:1 (lipid/DNA) and run on an agarose gel. Stable lipoplexes do not penetrate into the agarose gel, but free pDNA, not complexed to cationic liposomes, localizes in the same pattern as the control of pDNA in the absence of cationic liposomes (Figure 2). The DMKD liposomes and DLKD liposomes were able to complete complex formation at approximately 1:1 and 2:1 charge ratios of lipid to DNA, respectively. Meanwhile, the DPKD liposomes and DSKD liposomes completed complex formation at charge ratios higher than 6:1. The DNA-binding affinity of DOTAP liposomes, one of the well-established cationic liposomes, was similar to that of the DPKD liposomes. It is possible that the configuration of cationic charges on the DMKD liposomes and DLDK liposomes confers an advantage in binding pDNA and lipoplex formation over that of the other liposomes. ζ-potential measurement of cationic liposome-DNA complexes also indicates completion of lipoplex formation, as well as the resulting surface charge, another major parameter governing gene transfection activity. The DMKD lipoplexes exhibited a positively charged surface at a 1:1 (lipid/DNA) charge ratio (Figure 3). The DLKD and DOTAP lipoplexes became positively charged at charge ratios higher than 1:1, and their patterns of surface charge change were similar to each other. However, The DPKD and DSKD lipoplexes exhibited their positive surface charge at a 6:1 charge ratio. These results also indicate that DMKD and DLKD liposomes form stable lipoplexes more effectively than the other liposomes, as was suggested previously (Figure 2). In Vitro Transfection Activities of KD Cationic Liposomes. To evaluate the transfection efficiencies of
Kim et al.
KD liposomes, various lipoplex formulations were prepared and tested in 293 transformed human kidney cells and B16BL6 mouse melanoma cells. After a 4 h transfection and an additional 24 h incubation, luciferase expression in the cells was measured by a luciferase activity assay. As is shown in Figure 4, the DMKD liposomes clearly have the most potent transfection efficiency among the series of KD liposomes, and the DLKD liposomes are next. When various amounts of pDNA complexed to the DMKD or DLKD liposomes (1: 6, DNA wt/lipid wt) were added to 293 and B16BL6 cells in 24 well plates (Figure 4A and C), luciferase expression was nearly maximal at 0.5 µg of pDNA and saturated at higher DNA concentrations. Transfection with 5 µg of pDNA greatly diminished the luciferase expression in 293 cells. The gene expression pattern by the DPKD and DSKD was different from that by the DMKD and DLKD liposomes. The overall level of gene expression mediated by the DPKD and DSKD liposomes in the two cell lines was significantly lower than that by the DMKD liposomes and was not saturated up to 5 µg of pDNA. Meanwhile, 293 cells and B16BL6 cells were transfected with 1 µg of pDNA complexed to various amounts of the KD series of cationic lipids to find the optimal ratio for in vitro transfection. In both cell lines, luciferase expression mediated by the DMKD liposomes was maximal at a 1:3-1:6 weight ratio of DNA and lipid and then decreased slightly on addition of more cationic lipids (Figure 4B and D). At all ratios tested, the DMKD liposomes exhibited a transfection efficiency superior to that of the other cationic liposomes including DOTAP liposomes. Under the same transfection conditions, the DLKD liposomes exhibited a similar transfection pattern to that of the DMKD liposomes but a lower transfection activity. In both cell lines, transfection by the DPKD or DSKD liposomes was not saturated up to the ratio 1:9, but their transfection efficiencies were far lower than those of any of the DMKD or DLKD liposomes. Under the best transfection conditions for each liposomal formulation, the order of transfection activities was DMKD > DLKD > DOTAP > DPKD > DSKD. These results implied that the optimal transfection conditions were dependent on the type of cationic liposomes as well as the characteristics of the cell lines. It has been well documented that helper lipids, such as cholesterol and DOPE, generally enhance the transfection activity of cationic lipids (13). We prepared a series of the KD cationic liposomes containing cholesterol or DOPE (50 mol %) and compared their transfection activities in 293 and B16BL6 cells with those of liposomes consisting of only KD lipids. The transfection patterns of the helper lipid-containing liposomes were similar in both cell lines (Figure 5). Addition of cholesterol enhanced the transfection activities of the DSKD and DPKD liposomes 2.6- and 4.0-fold in 293 cells and 10.4- and 6.3fold in B16BL6 cells, respectively. However, the DMKD and DLKD liposomes containing cholesterol exhibited 5.8- and 2.2-fold lower transfection activity in 293 cells and 4.5- and 2.1-fold lower activity in B16Bl6 cells, respectively. Meanwhile, addition of DOPE further enhanced the transfection activities of the DSKD liposomes (19.8-fold in 293 cells and 33.5-fold in B16BL6 cells) and DPKD liposomes (20.0-fold in 293 cells and 19.1-fold in B16BL6 cells). However, DOPE addition reduced the transfection activities of the DMKD liposomes (28.4-fold in 293 cells and 2.1-fold in B16BL6 cells) and the DLKD liposomes (10.9-fold in 293 cells and 1.3-fold in B16BL6 cells). These results indicated that addition of the helper lipids cholesterol or DOPE could affect the transfection
Cationic Liposome-Mediated Gene Transfer
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Figure 4. Transfection activities of KD lipoplexes in 293 and B16BL6 cells. Various amounts of pDNA complexed to KD liposomes and DOTAP liposomes (1:6 wt ratio of DNA and lipid) were added to 293 cells (A) and B16BL6 cells (C). Also 1 µg of pDNA complexed to KD liposomes and DOTAP liposomes at various weight ratios was added to 293 cells (B) or B16BL6 cells (D). The cells were transfected for 4 h and incubated for an additional 24 h. The luciferase activity of the cell lysate was measured with a luminometer and a luciferase assay kit. The mean luciferase activity was calculated from three different measurements.
activities of cationic liposomes positively or negatively, depending on the type and characteristics of the cationic lipids. Cell Toxicity of KD Cationic Liposomes. The B16BL6 cells were incubated in the presence of various concentrations of KD liposomes and DOTAP liposomes to measure their cell toxicities and compare them to each other. In the ranges of lipid concentration for in vitro transfection dipalmytyl > distearyl (15). These results and those previously reported strongly indicate that cationic lipid polymorphism and liposomal membrane integrity affect the DNA-binding affinity of cationic liposomes. Even though the mechanism is not fully understood, there is a certain optimal membrane fluidity of liposomal bilayers for more suitable binding and accommodation of pDNA molecules, which may be more effective in interaction with cellular membranes due to proper lipid mixing. It has been well documented that addition of cholesterol (16) or DOPE affects lipid polymorphism in liposomal membranes (17,18). Liposomal membrane fluidity can be controlled by the amount of cholesterol added (19). The addition of DOPE, a typical phospholipid that prefers a non-bilayer hexagonal phase, increases the fusogenic properties of the liposomes (18). Generally, incorporation of these two ingredients enhances the transfection activity of cationic liposomes (20,21). However, their effects on the transfection activities of the KD liposomes were varied depending on the structure of the individual cationic lipids. As is shown in Figure 5, addition of cholesterol or DOPE can interfere with the transfection activity of the DMKD and DLKD liposomes. However, their addition affected the transfection activities of the DSKD and DPKD liposomes both positively. At this moment it is not clear how these two molecules affect the membrane integrity and polymorphism of KD cationic liposomes. However, it can be stated that better gene transfection by cationic liposomes is derived from optimization of the membrane integrity and maximization of their surface charge. Also, the conditions for transfection by cationic liposomes can be changed depending on the characteristics of the target cells. The KD series of cationic liposomes was expected to exhibit less cytotoxicity than other commercially available cationic liposomes, such as DOTAP-based liposomes, because they consist of lysine, aspartic acid, and two acyl hydrocarbon chains. Unexpectedly, the KD cationic liposomes were relatively more cytotoxic than the DOTAP liposomes. We speculated that the cytotoxicity of the KD liposomes may be due to physical membrane damage induced by detergent-like cationic lipid molecules and not to biochemical damage because their components are all biocompatible and their degradation does not produce any toxic end products in the cell. In summary, among the KD series of cationic liposomes, the DMKD liposomes were the most effective in gene transfection into 293 and B16BL6 cells, and the DLKD liposomes were next. Since the DMKD liposomes needed much smaller amounts of pDNA and lipid to reach maximal gene transfection than the DOTAP liposomes, it can be stated that the DMKD liposomes are superior to the DOTAP liposomes. The potent transfection activity observed may be attributed to the strong DNA-binding affinity and effective distribution of positive surface charge on the lipoplex surface, which may mediate efficient interactions between cells and the lipoplexes. We have now undertaken in vivo transfection studies using the KD series of cationic lipids. KD liposome transfection systems optimized in vivo will be utilized for preclinical or clinical applications.
Cationic Liposome-Mediated Gene Transfer ACKNOWLEDGMENT
The authors wish to acknowledge the financial support of the Korea Ministry of Commerce, Industry, and Energy given in the program year of 2002. LITERATURE CITED (1) Huang, L., and Viroonchatanpan, E. (1999) Nonviral vectors for gene therapy (Huang, L., et al., Eds.) pp 3-22, Academic Press, San Diego, CA. (2) Lasic, D. D. (1999) Nonviral vectors for gene therapy (Huang, L., et al., Eds.) pp 69-89, Academic Press, San Diego, CA. (3) Hui, K. M., Ang, P. T., Huang, L., and Tay, S. K. (1997) Phase I study of immunotherapy of cutaneous metastases of human carcinoma using allogeneic and xenogeneic MHC DNA-liposome complexes. Gene Ther. 8 783-790. (4) Rini, B. I., Selk, L. M., and Vogelzang, N. J. (1999) Phase I study of direct intralesional gene transfer of HLA-B7 into metastatic renal carcinoma lesions. Clin. Cancer Res. 5, 2766-2772. (5) Hyde, S. C., Southern, K. W., Gileadi, U., Fitzjohn, E. M., Mofford, K. A., Waddell, B. E., Gooi, H. C., Goddard, C. A., Hannavy, K., Smyth, S. E., Egan, J. J., Sorgi, F. L., Huang, L., Cuthbert, A. W., Evans, M. J., Colledge, W. H., Higgins, C. F., Webb, A. K., and Gill, D. R. (2000) Repeat administration of DNA/liposomes to the nasal epithelium of patients with cystic fibrosis. Gene Ther. 7, 1156-1165. (6) Noone, P. G., Hohneker, K. W., Zhou, Z., Johnson, L. G., Foy, C., Gipson, C., Jones, K., Noah, T. L., Leigh, M. W., Schwartzbach, C., Efthimiou, J., Pearlman, R., Boucher, R. C., and Knowles, M. R. (2000) Safety and biological efficacy of a lipid-CFTR complex for gene transfer in the nasal epithelium of adult patients with cystic fibrosis. Mol. Ther. 1, 105-114. (7) Liu, D., Ren, T., and Gao, X. (2003) Cationic transfection lipids. Curr. Med. Chem. 10, 1005-1013. (8) Hofland, H. E., Masson, C., Iginla, S., Osetinsky, I., Reddy, J. A., Leamon, C. P., Scherman, D., Bessodes, M., and Wils, P. (2002) Folate-targeted gene transfer in vivo. Mol. Ther. 5, 739-744. (9) Stewart, M. J., Plautz, G. E., Del Buono, L., Yang, Z. Y., Xu, L., Gao, X., Huang, L., Nabel, E. G., and Nabel, G. J. (1992) Gene transfer in vivo with DA-liposome complexes: Safety and acuty toxicity in mice. Hum. Gene Ther. 3, 267275. (10) Song, Y. K., Liu, F., Chu, S., and Liu, D. (1997) Characterization of cationic liposome-mediated gene transfer in vivo by intravenous administration. Hum. Gene Ther. 8, 15851594.
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