Bioconjugate Chem. 2007, 18, 1349−1354
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Alkyl Chain Moieties of Polyamidoamine Dendron-Bearing Lipids Influence Their Function as a Nonviral Gene Vector Toshinari Takahashi, Chie Kojima, Atsushi Harada, and Kenji Kono* Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan. Received October 6, 2006; Revised Manuscript Received February 21, 2007
We recently developed a novel family of cationic lipids consisting of a polyamidoamine (PAMAM) dendron and two dodecyl chains. Their transfection activity increases with increasing generation of the dendron moiety [Takahashi et al. (2003) Bioconjugate Chem. 14, 764-773]. In the present study, to elucidate the effect of hydrophobic tail moieties of the dendron-bearing lipids, two kinds of PAMAM G3 dendron-bearing lipids were synthesized with different alkyl lengths, DL-G3-2C18 and DL-G3-2C12. Their functions as gene vectors were compared. Irrespective of their different alkyl chain lengths, these dendron-bearing lipids formed complexes with plasmid DNA with similar efficiency. However, their complex sizes differed markedly: DL-G3-2C18 lipoplexes exhibited much smaller diameters than DL-G3-2C12 lipoplexes. Interaction of the lipoplexes with heparin revealed that the DL-G3-2C18 lipoplexes required more heparin than DL-G3-2C12 lipoplexes to cause dissociation of plasmid DNA from the lipoplexes. Although the DL-G3-2C12 lipoplexes and DL-G3-2C18 lipoplexes transfected CV1 cells with similar efficiency in the absence of serum, only the latter retained high transfection activity in the presence of serum. These results indicate that hydrophobic interaction of alkyl chain moieties plays an important role in the increment of stability and the serum-resistant transfection activity for dendron-bearing lipid lipoplexes.
INTRODUCTION Preparation of nonviral vectors that deliver therapeutic genes to target cells has been demanded for efficient and safe gene therapy. Among these nonviral vectors, various types of cationic lipids have been studied intensively, but their activity requires improvement. These vectors can associate with plasmid DNA and form complexes, which are termed lipoplexes (1, 2). Lipoplexes bind to the cell surface through electrostatic interactions and are taken up by cells mainly via endocytosis. Subsequently, some parts of plasmid DNA contained in the complexes reach the nucleus, where gene transcription occurs. Nevertheless, most complexes are likely to be trapped in the endosome and be eventually degraded in the lysosome. Therefore, avoidance of plasmid DNA degradation in the lysosome and its transfer into cytosol is considered to be a key process for efficient transfection (3). Cationic polymers are another type of nonviral vector that has been studied intensively (2). Polyamidoamine (PAMAM) dendrimers have been shown to achieve efficient gene transfection of cells among cationic polymers: PAMAM dendrimers possess many tertiary amine groups, which become protonated under weakly acidic conditions (4-6). Those amine groups suppress the lowering of pH in endosomes and lysosomes by adsorbing protons and prohibiting degradation of DNA in the lysosome. In addition, the endosome buffering ability of PAMAM dendrimers is thought to induce osmotic swelling of the endosome interior, engendering rupture of the endosome and the subsequent release of DNA into the cytoplasm (7). Such a function of these tertiary amine groups of PAMAM dendrimers, the so-called proton sponge effect, is considered to contribute to their high transfection activity (8). In a previous study (9), we developed a novel type of cationic lipid that consists of two dodecyl groups and a PAMAM * Corresponding author. Kenji Kono, Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan. E-mail:
[email protected]: +81-722549330.Fax: +81-722549330.
dendron as a head group and showed that these dendron-bearing lipids are promising as a potent nonviral vector that exhibits higher transfection activity than the PAMAM dendrimer of the same generation. Their molecular structure, e.g., that of the head group of the dendron moiety and the tail moieties of alkyl chains, will influence their transfection activity. We showed in a previous study that their transfection activity increases concomitant with increasing generation of the head moiety (9). Nevertheless, it remains unclear how alkyl chains affect their transfection activity. In this study, we synthesized the PAMAM dendron-bearing lipid with two octadecyl chains DL-G3-2C18 and compared its capability as a gene vector to that of DL-G3-2C12 (Figure 1). The importance of the alkyl chain moiety for stability and transfection activity of the dendron-bearing lipid lipoplexes is described.
EXPERIMENTAL PROCEDURES General Methods. Dioctadecylamine was purchased from Fluka (Tokyo, Japan). Methyl acrylate, ethylenediamine, and tris(hydroxymethyl)aminomethane (Tris) were obtained from Kishida Chemical (Osaka, Japan). Sodium cyanide and ethidium bromide were supplied from Wako Pure Chemical Industries (Osaka, Japan). Merck Kieselgel 60 (230-400 mesh) was used for silica gel chromatography. Fetal calf serum (FCS) was obtained from PAA Laboratories GmbH (Pasching, Austria). Dulbecco’s modified Eagle’s medium (DMEM) was from Nissui Pharmaceutical (Tokyo, Japan). Heparin sodium salt was purchased from Sigma (St. Louis, MO). Agarose was purchased from nacalai tesque (Kyoto, Japan). DL-G3-2C12 was synthesized by the method previously reported (9). Plasmid DNA pCMV-Luc, which contains the cDNA of firefly luciferase driven by a human cytomegalovirus immediate-early promoter, was a gift from Dr. Kazuo Maruyama, Teikyo University, and amplified in E. coli, isolated, and purified using a QIAGEN plasmid Maxi Kit. Synthesis of Dendron-Bearing Lipids. DL-G3-2C18 was synthesized by repetition of exhaustive Michael addition with
10.1021/bc060311k CCC: $37.00 © 2007 American Chemical Society Published on Web 05/09/2007
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Figure 1. Structures of DL-G3-2C12 and DL-G3-2C18.
methyl acrylate using dioctadecylamine as the core material and subsequent exhaustive amidation with ethylenediamine. DL-G-0.5-2C18. Dioctadecylamine (2.0 g, 3.8 mmol) was dissolved in methyl acrylate (35 mL, 0.39 mol), and the resultant solution was refluxed under nitrogen. After 18 h, unreacted methyl acrylate was removed under vacuum and the residue was chromatographed on silica gel using petroleum etherdiethyl ether (2/1, v/v) as eluent. Yield: 87.9%. 1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)15-), δ 1.26 (s, CH3(CH2)15-), δ 1.41 (br, -CH2CH2N-), δ 2.35-2.46 (m, -CH2COOCH3, -CH2N), δ 2.77 (t, -CH2CH2COOCH3), δ 3.66 (s, -OCH3). DL-G0-2C18. DL-G-0.5-2C18 (2.0 g, 3.4 mmol) was dissolved in methanol (40 mL). The resultant solution was added dropwise to distilled ethylenediamine (70 mL, 1.05 mol) containing sodium cyanide (33 mg, 0.67 mmol) and stirred at 50 °C under nitrogen. After 1 week, methanol and unreacted ethylenediamine were removed from the reaction mixture under vacuum. The obtained DL-G0-2C18 was chromatographed on silica gel using chloroform-methanol-water (60/35/5, v/v/v) as eluent. Yield: 53.2%. 1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)15-), δ 1.26 (s, CH3(CH2)15-), δ 1.44 (br, -CH2CH2N-), δ 2.33-2.45 (m, -CH2CONH-, -CH2N-), δ 2.65 (t, -CH2CH2CONH-), δ 2.79 (t, -CH2NH2), δ 3.28 (q, -CH2CH2NH2), δ 8.65 (m, -CONH-) CONH). MALDI-TOF-MS m/z 636.5 ([M + H]+). DL-G0.5-2C18. DL-G0-2C18 (1.2 g, 1.9 mmol) was dissolved in methanol (20 mL) and added to methyl acrylate (34 mL, 0.38 mol). The mixed solution was stirred at 35 °C for 50 h under nitrogen. The obtained material was chromatographed on silica gel using petroleum ether-diethyl ether (2/1, v/v) and then chloroform-methanol (9/1, v/v) as eluent. Yield: 97.7%. 1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)15-), δ 1.26 (s, CH3(CH2)15-), δ 1.44 (br, -CH2CH2N-), δ 2.35 (m, -CH2CONH-), δ 2.43 (m, -CH2COOCH3, -CH2N-), δ 2.54 (t, -CONHCH2CH2), δ 2.69-2.80 (m, -CH2CH2CONH-, -CH2CH2COOCH3), δ 3.29 (q, -CONHCH2-), δ 3.67 (s, -OCH3), δ 7.76 (br, -CONH). DL-G1-2C18. DL-G0.5-2C18 (1.5 g, 1.8 mmol) was dissolved in methanol (50 mL) and added to ethylenediamine (65 mL, 0.92 mol) containing sodium cyanide (18 mg, 0.37 mmol). The mixed solution was stirred at 45 °C for 60 h under nitrogen. The obtained DL-G1-2C18 was purified with a Sephadex LH20 column using chloroform as eluent. Yield: 94.3%. 1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)15-), δ 1.26 (s, CH3(CH2)15-), δ 1.42 (br, -CH2CH2N-), δ 2.31 (m, -CH2CONH-), δ 2.35-2.43 (m,-CH2CONHCH2CH2NH2,-CH2N-),δ2.51(t,-CONHCH2CH2), δ 2.67 (t, -CH2CH2CONH-), δ 2.75 (t, -CH2CH2CONHCH2CH2NH2), δ 2.83 (t, -CH2NH2), δ 3.24-3.31 (m, -CONHCH2), δ 7.37 and 8.65 (br, -CONH-). MALDI-TOF-MS m/z 862.9 ([M + H]+).
Takahashi et al.
DL-G1.5-2C18. DL-G1-2C18 (1.3 g, 1.5 mmol) was dissolved in methanol (60 mL) and added to methyl acrylate (110 mL, 1.23 mol). The mixed solution was stirred at 35 °C for 52 h under nitrogen. The obtained material was chromatographed on silica gel using chloroform and then chloroform-methanol (9/ 1, v/v) as eluent. Yield: 71.9%. 1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)15-), δ 1.25 (s, CH3(CH2)15-), δ 1.43 (br, -CH2CH2N), δ 2.37 (t, -CH2CONH-), δ 2.43 (t, -CH2COOCH3, -CH2N-), δ 2.52-2.58 (m, -CONHCH2CH2-), δ 2.75 (t, -CH2CH2CONH-, -CH2CH2COOCH3), δ 2.82 (t, -CH2CH2CONH-), δ 3.30 (m, -CONHCH2-), δ 3.68 (s, -OCH3), δ 7.00 and 8.07 (br, -CONH). DL-G2-2C18. DL-G1.5-2C18 (1.3 g, 1.1 mmol) was dissolved in methanol (30 mL) and added to ethylenediamine (95 mL, 1.42 mol) containing sodium cyanide (21 mg, 0.43 mmol). The mixed solution was stirred at 45 °C for 50 h under nitrogen. The obtained DL-G2-2C18 was purified with a Sephadex LH20 column using methanol as eluent. Yield: 92.2%. 1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)15-), δ 1.25 (s, CH3(CH2)15-), δ 1.42 (br, -CH2CH2N-), δ 2.32-2.42 (m, -CH2CONHCH2CH2NH2, -CH2CONH-, -CH2N-), δ 2.52 (m, -CONHCH2CH2-), δ 2.73 (m, -CH2CH2CONH-, -CH2CH2CONHCH2CH2NH2), δ 2.83 (t, -CH2NH2), δ 3.25-3.30 (m, -CONHCH2-), δ 7.73, 7.93, and 8.58 (br, -CONH-). MALDI-TOF-MS m/z 1320.6 ([M + H]+). DL-G2.5-2C18. DL-G2-2C18 (0.94 g, 0.71 mmol) dissolved in methanol (60 mL) was added to methyl acrylate (55 mL, 0.61 mol). The mixed solution was stirred at 30 °C for 50 h under nitrogen. The obtained material was chromatographed on silica gel using chloroform-methanol (95/5 and then 8/2, v/v) as eluent. Yield: 67.4%. 1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)15-), δ 1.25 (s, CH3(CH2)15-), δ 1.47 (br, -CH2CH2N-), δ 2.37 (m, -CH2CONH-), δ 2.44 (m, -CH2COOCH3, -CH2N-), δ 2.55 (m, -CONHCH2CH2-), δ 2.74-2.81 (m, -CH2CH2CONH-, -CH2CH2COOCH3), δ 3.29 (m, -CONHCH2-), δ 3.67 (s, -OCH3), δ 7.10, 7.65, and 8.09 (br, -CONH-). DL-G3-2C18. DL-G2.5-2C18 (0.92 g, 0.46 mmol) was dissolved in methanol (40 mL) and added to ethylenediamine (60 mL, 0.9 mol) containing sodium cyanide (8.9 mg, 0.18 mmol). The mixed solution was stirred at 45 °C for 55 h under nitrogen. The obtained DL-G3-2C18 was purified with a Sephadex LH20 column using methanol as eluent. Yield: 90.7%. 1H NMR (CDCl3): δ 0.88 (m, CH3(CH2)15-), δ 1.25 (s, CH3(CH2)15-), δ 1.42 (br, -CH2CH2N-), δ 2.32-2.37 (m, -CH2CONHCH2CH2NH2, -CH2CONH-, -CH2N-), δ 2.53 (m, -CONHCH2CH2-), δ 2.73 (br, -CH2CH2CONH-, -CH2CH2CONHCH2CH2NH2), δ 2.83 (t, -CH2NH2), δ 3.24-3.30 (m, -CONHCH2-), δ 7.82, 8.02, and 8.52 (br, -CONH-). MALDI-TOF-MS m/z 2234.3 ([M + H]+). Preparation of Lipoplexes. To a dry thin membrane of the dendron-bearing lipid, phosphate-buffered saline (PBS) was added and sonicated for 2 min using a bath-type sonicator to afford a lipid suspension. Plasmid DNA (1 µg) was dissolved in 20 mM Tris-HCl buffer (pH 7.4, 50 µL), mixed with the lipid suspension (50 µL), and incubated for 30 min at room temperature to afford a lipoplex with a given ratio of primary amine of DL-G3 to DNA phosphate (N/P ratio). Agarose Gel Electrophoresis. The dendron-bearing lipidDNA complexes with varying N/P ratios were prepared by mixing plasmid DNA (1 µg) dissolved in 20 mM Tris-HCl buffer (5 µL) and lipid suspension (5 µL). After 30 min incubation at room temperature, the samples (10 µL) were electrophoresed on 0.6 wt % agarose gel in 40 mM Tris, 20 mM sodium acetate, and 2 mM EDTA buffer (pH 8.0) containing 1 µg/mL ethidium bromide at 100 V for 30 min. The ethidium bromide stained bands were visualized using a
Technical Notes
LAS-1000UVmini (Fujifilm, Tokyo, Japan) and analyzed with Science Lab 2003 Multi Gauge software (Fujifilm, Tokyo, Japan). Dynamic Light Scattering (DLS) Measurement. The diameters of lipoplexes were estimated by DLS using an electrophoretic light scattering spectrometer ELS-8000F (Otuka Electronics Co, Ltd., Osaka, Japan). Lipoplexes with various N/P ratios were prepared by mixing plasmid DNA (20 µg) and a given amount of the dendron-bearing lipid and incubated for 30 min as described above. The data were obtained at a detection angle of 90° at 25 °C and were analyzed by the cumulant method. Atomic Force Microscopy (AFM). AFM measurements were performed by SPI3800 probe station and SPA400 unit system of the scanning probe microscopy system (Seiko Instruments Inc., Chiba, Japan). The cantilever was made of silicon (SI-DF20; Seiko Instruments Inc., Chiba, Japan), and its spring constant was 14 N/m. Lipoplexes were formed at a total of 100 µg/mL of plasmid DNA concentration with N/P ratio of 4. Lipoplexes containing 0.5 µg of plasmid DNA were applied to freshly cleaved mica and incubated on it for 5 min. After incubation, excess fluid was dried under air flow. The measurements were performed in a dynamic force mode (noncontact mode). Raw AFM images have been processed only for background removal (flattening) using the microscope manufacturer’s image-processing software. Interaction of Lipoplexes with Heparin. The dendronbearing lipid-DNA lipoplexes were prepared by mixing plasmid DNA (1 µg) dissolved in 20 mM Tris-HCl buffer (2.5 µL) and lipid suspension (2.5 µL). After 30 min incubation at room temperature, the lipoplexes were added to a given amount (0-2 µg) of heparin dissolved in PBS (5 µL) and incubated for 30 min at room temperature. The samples (10 µL) were electrophoresed on 0.6 wt % agarose gel in 40 mM Tris, 20 mM sodium acetate, and 2 mM EDTA buffer (pH 8.0) containing 1 µg/mL ethidium bromide at 100 V for 30 min. The ethidium bromide stained bands were visualized using a LAS-1000UVmini (Fujifilm, Tokyo, Japan) and analyzed with Science Lab 2003 Multi Gauge software (Fujifilm, Tokyo, Japan). Transfection. Transfection of CV1 cells was done according to the following procedures unless otherwise noted in the text. The cells were seeded in 0.5 mL of DMEM supplemented with 10% FCS in 24-well culture plates at 5.0 × 104 cells per well the day before transfection. The cells were washed with PBS containing 0.36 mM CaCl2 and 0.42 mM MgCl2 [PBS(+)] and then covered with DMEM in the presence or absence of 10% FCS (1 mL). The lipoplexes containing plasmid DNA were added gently to the cells and incubated for 4 h at 37 °C. Then, the cells were rinsed with PBS(+), covered with DMEM containing 10% FCS, and incubated at 37 °C. After 40 h, the cells were lysed by adding 50 µL of Luc-PGC-50 detergent (Toyo Ink, Tokyo, Japan). A 20 µL aliquot was taken from each dish and used for one luciferase assay using a kit (Toyo Ink) and a Lumat LB9507 luminometer (Berthold, Bad Wildbad, Germany). The protein content of the lysate was measured by Coomassie protein assay reagent (Pierce, IL) using bovine serum albumin as the standard.
RESULTS AND DISCUSSION Synthesis of Dendron-Bearing Lipid with Two Octadecyl Groups. In a previous study, we developed a new type of cationic lipids consisting of two dodecyl groups and PAMAM dendron of varying generations (9). Transfection activity of these dendron-bearing lipids was shown to increase with increasing dendron generation, demonstrating the importance of the head moiety of the dendron-bearing lipids. To elucidate the effect of the hydrophobic tail moiety of the lipids, a PAMAM G3
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Figure 2. Agarose gel electrophoretic analysis for complexation of DL-G3-2C18 (A) and DL-G3-2C12 (B) with plasmid DNA at varying N/P ratios. Percent of free plasmid DNA was evaluated and plotted against N/P ratio (C) for complexation of DL-G3-2C18 (9) and DLG3-2C12 (O) with plasmid DNA. Each point represents the mean ( SD (n ) 3).
dendron-bearing lipid with two octadecyl chains DL-G3-2C18 was designed in this study (Figure 1). This lipid was synthesized through repetition of exhaustive Michael addition with methyl acrylate using dioctadecylamine as the core material and subsequent exhaustive amidation with ethylenediamine, as reported by Tomalia et al. (10-12). Characterization using 1H NMR showed good correspondence between the theoretically expected and experimentally obtained values for the synthesized products, as shown in the Experimental Section. Lipoplex Formation with Plasmid DNA. The ability of the dendron-bearing lipid to form a lipoplex with plasmid DNA was investigated using gel retardation assay. The plasmid DNA (1 µg) was incubated with varying amounts of DL-G3-2C18 or DL-G3-2C12 for 30 min and electrophoresed on an agarose gel. Figure 2 shows that the amount of free plasmid DNA decreased with increasing N/P ratio, indicating that these lipids can form a lipoplex with plasmid DNA. In the case of DL-G3-2C18, migration of plasmid DNA band was not observed for N/P ratios greater than 1.6 (Figure 2A), whereas migration of plasmid DNA was inhibited at N/P ratios greater than 1.0 for DL-G3-2C12 (Figure 2B). This result indicates that DL-G3-2C12 can form a lipoplex with plasmid DNA more efficiently than DL-G3-2C18, even though their abilities of lipoplex formation are not markedly different. Their complex formation is driven by electrostatic interaction between the dendron moiety and DNA. Therefore, the difference of alkyl chains of these dendronbearing lipids might only slightly affect their lipoplex formation.
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Figure 4. Release of plasmid DNA from DL-G3-2C18 (9) and DLG3-2C12 (O) lipoplexes induced by the addition of heparin. Percent release of DNA was determined by agarose gel electrophoresis of DLG3 lipoplexes with N/P ratio of 2 in the presence of varying amounts of heparin, and plotted against the charge ratio of phosphate (P) groups of DNA and sulfonate (S) and carboxylate (C) groups of heparin to primary amine groups (N) of dendron-bearing lipid. Each point represents the mean ( SD (n ) 3).
Figure 3. (A) Mean diameters of DL-G3-2C18 (9) and DL-G3-2C12 lipoplexes (O) as a function of N/P ratio. Mean diameters were estimated using DLS. Each point represents the mean ( SD (n ) 3). AFM images of DL-G3-2C12 lipoplexes with N/P ratios of 2 (B) and 4 (C), and DL-G3-2C18 lipoplexes with N/P ratios of 2 (D) and 4 (E).
Particle Size and Morphology of Lipoplexes. Influence of the alkyl chain length of the dendron-bearing lipids on their lipoplex size was investigated using DLS. Figure 3A shows that the DL-G3-2C12 lipoplexes showed roughly the same diameter of about 2 µm, irrespective of the N/P ratio. In contrast, particle sizes of the DL-G3-2C18 lipoplexes depended greatly on the N/P ratio. Large particles of about 2 µm were observed at the N/P ratio of 1.0, but the lipoplex size decreased sharply with an increasing N/P ratio from 1 to 4 and became constant around 250 nm. The lipoplexes particles were further characterized using AFM. Figure 3B,C shows that large particles with a diameter greater than 2 µm were observed for DL-G3-2C12 lipoplexes with N/P ratios of 2 and 4. These particles have irregular shapes and rough surfaces. Therefore, it seems that they might be formed by aggregation of smaller particles of the lipid-DNA complexes. Particles with similar morphology and diameter of ca. 1 µm were visible in the AFM image of the DL-G3-2C18 lipoplexes with the N/P ratio of 2 (Figure 3D). However, in the image of the DL-G3-2C18 lipoplexes with the N/P ratio of 4, spherical particles with 200-300 nm diameter were observed (Figure 3E). These sizes of lipoplexes, which were observed by AFM, were consistent with the DLS results (Figure 3A).
Charge-neutralized lipoplexes with an N/P ratio around 1 are well-known to tend to form large aggregates because of their hydrophobic interaction (13). Therefore, both dendron-bearing lipids might form large lipoplexes at N/P ratio of 2, at which ratio their charge density was low. In contrast, at the N/P ratio of 4, the lipoplexes might possess sufficient net charge to suppress aggregation of small lipoplexes. In fact, we observed small particles with a diameter of 200-300 nm for the DLG3-2C18 lipoplex. However, much larger particles appeared for the DL-G3-2C12 lipoplex with the N/P ratio of 4, although both lipoplexes possess the same charge density. Because of strong hydrophobic interaction of the long alkyl chains, the DL-G32C18 lipoplex particles might have a tightly packed interior and a positively charged surface, prohibiting aggregation and fusion of lipoplexes. However, because the DL-G3-2C12 has shorter alkyl chains, its lipoplex particles might be less stabilized than the DL-G3-2C18 lipoplex. Such low stability of the lipoplex particles might induce their aggregation and fusion, thereby generating larger particles. Estimation of Lipoplex Stability. Some polyanions, such as heparin, are known to induce dissociation of DNA from lipoplexes by binding to cationic lipids of lipoplexes through electrostatic interaction (14, 15). These polyanions have been used to estimate the lipoplexes’ stability (16). Therefore, we examined the effect of addition of heparin on dissociation of plasmid DNA from the DL-G3-2C18 and DL-G3-2C12 lipoplexes to evaluate their stability. Lipoplexes with an N/P ratio of 2 were incubated for 30 min with various amounts of heparin; the amount of the liberated plasmid DNA was estimated using agarose gel electrophoresis. Figure 4 shows the percentage of plasmid DNA released from the lipoplexes as a function of the charge balance of the lipoplex and added heparin, which is expressed as the ratio of negative charges of phosphate of DNA and sulfate and carboxylate of heparin and positive charges of primary amines of the dendronbearing lipid [(P + S + C)/N]. The negative charges of heparin were calculated by assuming the structure of heparin as repeats of (1 f 4)-O-(R-L-idopyranosyluronic acid 2-sulfate)-(1f 4)(2-deoxyl-2-sulfamino-R-D-glucopyranosyl 6-sulfate) (17, 18). Figure 4 shows that release of plasmid DNA from the DL-G32C12 lipoplex occurred at -/+ charge ratios greater than 1.15,
Technical Notes
Figure 5. Luciferase activities of CV1 cells treated with DL-G3-2C12 lipoplexes (open bars) and DL-G3-2C18 lipoplexes (closed bars) in the absence (A) and presence (B) of 10% FCS. Each bar represents the mean ( SD (n ) 3).
whereas the release of plasmid DNA was observed at -/+ charge ratios greater than 1.58 for the DL-G3-2C18 lipoplex, which indicates that 1.7 times more heparin was necessary to dissociate plasmid DNA from the DL-G3-2C18 lipoplexes than in the case of the DL-G3-2C12 lipoplex with the same N/P ratio. This result indicates clearly that hydrophobic interaction between the longer alkyl chains of DL-G3-2C18 increases the stability of the complex formed with plasmid DNA. Transfection Activity of Dendron-Bearing Lipid-DNA Lipoplexes. Finally, we investigated the influence of the alkyl chain length of dendron-bearing lipids on their transfection activity. The transfection activity of lipoplexes varies depending on their N/P ratio (19). For that reason, we prepared lipoplexes consisting of the dendron-bearing lipid, DL-G3-2C12 or DLG3-2C18, and plasmid DNA containing luciferase gene at various N/P ratios and examined transfection of CV1 cells using these lipoplexes. Figure 5A represents the expression of luciferase in the cells treated with these lipoplexes in the absence of serum. The transfection activity of these lipoplexes increased with increasing N/P ratio from 1 to 2 and reached an approximately constant level at N/P ratios greater than 2. Although the DL-G3-2C18 lipoplexes with the low N/P ratios seem to have a slightly higher activity than DL-G3-2C12 lipoplexes with the same N/P ratios, both lipoplexes exhibited similarly high transfection activities at higher N/P ratios. However, marked differences were apparent when the transfection was performed in the presence of serum. Figure 5B shows that the DL-G3-2C12 lipoplexes with N/P ratios of 1-4 did not induce expression of luciferase in CV1 cells in the presence of FCS. In contrast, the DL-G3-2C18 lipoplexes caused transfection of CV1 cells efficiently. Especially, DL-G3-2C18 lipoplexes with N/P ratios of 1.5 and 2 induced a high level of luciferase expression, which is comparable to the level of transfection in the absence of serum (Figure 5A). In addition, we observed that viability of cells treated with the DL-G3-2C18 lipoplexes was about 100% under the experimental conditions, indicating that these lipoplexes were able to achieve efficient transfection without cellular damage (see Supporting Information). Many studies have indicated that the transfection activity of lipoplexes is reduced dramatically in the presence of serum (2023). As shown in Figure 5B, the DL-G3-2C12 lipoplexes also abolished their transfection activity in the presence of serum. It is likely that the DL-G3-2C12 lipoplexes are decomposed through strong interaction with serum proteins because these lipoplexes might be stabilized weakly by short alkyl chains. In contrast, the DL-G3-2C18 lipoplexes retained high transfection activity even in the presence of serum. Hydrophobic interaction
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of the long alkyl chains of DL-G3-2C18 might greatly improve the stability of the lipoplexes and impart a high serum-resistant property to them. In fact, we observed better stability of the DL-G3-2C18 lipoplex against interaction with heparin than that of the DL-G3-2C12 lipoplex (Figure 4). These results indicate the importance of hydrophobic tail moieties of the dendronbearing lipid for retention of their high transfection activity in the presence of serum. In conclusion, we demonstrated the importance of the hydrophobic tail moieties of dendron-bearing lipids for their lipoplex formation with plasmid DNA and their transfection activity. The newly synthesized dendron-bearing lipid DL-G32C18 produced highly potent, small lipoplexes with high transfection activity in the presence of serum. In a previous study, we showed that addition of dioleoylphosphatidylethanolamine, which increases fusion ability of lipoplexes, enhanced the transfection activity of the DL-G3-2C12 lipoplexes (9, 16). Therefore, inclusion of this fusogenic lipid might further elevate transfection activity of the DL-G3-2C18 lipoplexes. For that reason, the DL-G3-2C18 might be a promising gene vector. We are currently examining the influence of addition of dioleoylphosphatidylethanolamine on transfection activity of the DL-G3-2C18 lipoplexes.
ACKNOWLEDGMENT This work was partly supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture in Japan. T. Takahashi thanks the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (JSPS). Supporting Information Available: Viability of CV1 cells treated with DL-G3-2C18 or DL-G3-2C12 lipoplexes. This material is available free of charge via the Internet at http:// pubs.acs.org.
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