Cationic Nucleoside Lipids for Gene Delivery - American Chemical

Feb 8, 2006 - 33076 Bordeaux Cedex, France, Laboratoire de Chimie Biologique UMR A408 Faculté des sciences d'Avignon 33, rue Louis. Pasteur, 84000 ...
6 downloads 0 Views 290KB Size
Download PDF
Bioconjugate Chem. 2006, 17, 466−472

466

Cationic Nucleoside Lipids for Gene Delivery Pauline Chabaud,† Michel Camplo,† Dominique Payet,⊥ Guillaume Serin,| Louis Moreau,‡ Philippe Barthe´le´my,*,‡ and Mark W. Grinstaff§ Groupe de Chimie Organique et des Mate´riaux Mole´culaires (UMR-CNRS 6114), Faculte´ des Sciences de Luminy, case 901, 13288 Marseille Cedex 09, France, INSERM U386, Universite´ Victor Segalen Bordeaux 2, 146 rue Le´o Saignat 33076 Bordeaux Cedex, France, Laboratoire de Chimie Biologique UMR A408 Faculte´ des sciences d’Avignon 33, rue Louis Pasteur, 84000 Avignon, France, Centre d’Immunologie de Marseille Luminy, Parc scientifique de Luminy, 13288 Marseille, Cedex 9, France, Oncodesign, Parc de la Toison d’Or, 28 rue Louis de Broglie, 21000 Dijon, France, and Departments of Biomedical Engineering and Chemistry, Boston University, Boston, Massachusetts 02215. Received June 7, 2005; Revised Manuscript Received January 4, 2006

A novel uridine-based nucleo-lipid, DOTAU (N-[5′-(2′,3′-dioleoyl)uridine]-N′,N′,N′-trimethylammonium tosylate) was prepared by using a convenient four-step synthetic pathway. From the preliminary physicochemical studies (quasielastic light scattering and light microscopy), this amphiphilic structure forms supramolecular organizations in aqueous solution. In addition, in the presence of nucleic acids, transmission electronic microscopy experiments (TEM) and small angle X-ray scattering (SAXS) reveal the formation of multilamellar structures similar to lipoplexes (cationic liposome-DNA complexes) with cationic lipids. The formation of a complex was confirmed by fluorescence spectroscopic assays involving ethidium bromide. Transfection assays of mammalian cell lines (HeLa and MCF-7) indicate that DOTAU can transfect efficiently an expression vector (pEGFP) encoding GFP. Proliferation assays realized on these cell lines show that DOTAU does not inhibit cell proliferation and is less toxic than the commercial Lipofectamine 2000.

INTRODUCTION The transport of nucleic acids through a cell membrane for subsequent transcription is fundamental for the delivery of therapeutic genes (1-3). Viruses are efficient vectors for transportation of DNA, but there are risks associated with their clinical application (4). Consequently, there is significant basic and clinical interest in nonviral vectors, and a large variety of synthetic gene-delivery systems have been investigated including cationic lipids, dendritic macromolecules, and linear polymers (5-12). New cationic lipids have been synthesized and evaluated for plasmid DNA delivery (13) since the discovery of N-[1(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)1 as a cationic transfecting agent (5, 14). In general, cationic lipids are attractive materials for this application because (1) of their reduced cytotoxicity compared to viral vectors, (2) * To whom correspondence should be addressed. E-mail: [email protected]. † Groupe de Chimie Organique et des Mate ´ riaux Mole´culaires (UMRCNRS 6114). ‡ Laboratoire de chimie biologique UMR A408 Faculte´ des sciences d’Avignon 33 and Universite´ Victor Segalen Bordeaux 2. ⊥ Centre d’Immunologie de Marseille Luminy. | Oncodesign. § Boston University. 1 Abbreviation: TEM (transmission electron microscopy); SAXS (small-angle X-ray scattering); CT DNA (calf thymus DNA); GFP (green fluorescent protein); FACS (fluorescence activated cell sorter); DOTAP, (N-[1-(2,3-dioleoyloxy)propyl]-N′,N′,N′-trimethylammonium chloride) or DOTAP (16); DOTAU, N-[5′-(2′,3′-dioleoyl)uridine]N′,N′,N′-trimethylammonium tosylate; DCC, dicyclohexylcarbodiimide; DCU, dicyclohexylurea; DMAP, (dimethylamino)pyridine; DCM, methylene dichloride; DMF, dimethylformamide; TEA, triethylamine; THF, tetrahydrofuran; SUV, small unilamellar vesicles; BrdU, 5-bromo-2′deoxyuridine; LIPO, Lipofectamine 2000; EB, ethidium bromide; NBA, 3-nitrobenzyl alcohol.

of their well-defined structure (compared to linear polymers), (3) tuneable chemical structure, and (4) ease of synthesis. Four primary motivations for this research are to improve the transfection activity or modulate the level of gene expression, reduce the cytotoxicity, broaden the scope of the nucleic acid payload, and expand the repertoire of chemical structures under investigation. Consequently, numerous structural features have been altered on the basic cationic lipid structure, leading to compositions such as DOTAP (N-[1-(2,3-dioleoyloxy)propyl]N′,N′,N′-trimethylammonium chloride) which are now commercially available and under clinical evaluation (Figure 1) (15). Among the chemical variants/alterations of the lipid structure explored, the incorporation of natural moieties such as cholesterol, oleic acids, and amino acids or multicationic charges have been investigated. Currently, all the synthetic vectors used relay on either electrostatic interactions or electrostatic and hydrophobic interactions for DNA binding and supramolecular assembly formation required for gene delivery. We are interested in molecular interactions such as hydrogen bonding and π-π stacking to further modulate the interactions between DNA and the synthetic vector. Within this framework, hybrid molecules bearing both nucleic acid units and amphiphilic moieties would be the simplest prototype. To the best of our knowledge, there are no previous examples of synthetic gene carriers derived from cationic nucleoside-based lipids. Herein, we describe a promising nucleic acid vector based on a cationic amphiphilic uridine. We report the synthesis, physicochemical studies, cytotoxicity, and in vitro transfection results of this new nucleoside-based lipid (DOTAU, Figure 1).

MATERIALS AND METHODS General Experiments and Analytical Conditions. Unless noted otherwise, all starting materials were obtained from Aldrich and were used without further purification, and the solvents were redistilled over calcium chloride, calcium hydride, potassium hydroxide, or sodium according to the solvent used.

10.1021/bc050162q CCC: $33.50 © 2006 American Chemical Society Published on Web 02/08/2006

Cationic Nucleoside Lipids for Gene Delivery

Figure 1. Chemical structures of DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N′,N′,N′-trimethylammonium chloride) and DOTAU (N-[5′(2′,3′-dioleoyl)uridine]-N′,N′,N′-trimethylammonium tosylate).

All compounds were characterized using standard analytical and spectroscopic techniques such as 1H, 13C, and 31P NMR spectroscopy (apparatus Bruker Avance DPX-300, 1H at 300.13 MHz, 13C at 75.46 MHz, and 31P at 121.49 MHz) and mass spectrometry (Instrument JEOL SX 102, NBA matrix). The NMR chemical shifts are reported in ppm relative to tetramethylsilane for 1H and 13C. The 1H NMR coupling constants, J, are reported in Hz. TEM microscopy experiments were performed on a Philips CM 10 (negative staining with ammonium molybdate 1% in water, Cu/Pd carbon coated grids). A Wyatt QELS system was used to obtain the Rh. Silica gel 60 (particle size: 40-60 µm) was employed for flash chromatography. Thin layer chromatograms were performed with aluminum plates coated with silica gel 60 F254 (Merck). Varian silica gel reverse phase C18 Mega BE-C18, 2GM 12 mL, and Sephadex LH-20 (25-100 µm) were used for quantitative chromatography. 13C chemical shifts were determined by HMQC. 2′,3′-O-Isopropylideneuridine, 1. To a stirring solution of uridine (1.5 g, 6.14 mmol) in dry acetone (135 mL) was added a catalytic amount of H2SO4 dropwise, at room temperature. After being stirred for 2 h, the solution became clear and was quenched with BaCO3 until neutralization. The heterogeneous mixture was filtered on a Celite pad, and the filtrate was evaporated under reduced pressure. The crude product was immediately purified by flash column chromatography (CH2Cl2/MeOH: 90/10) to give 1 (1.24 g, 71%) as a white solid. mp ) 161 °C. 1H NMR (D3CCOCD3): δ 7.84 (1H, d, J ) 8.06 Hz), 5.92 (1H, d, J ) 2.69 Hz), 5.62 (1H, d, J ) 8.06 Hz), 4.92 (2H, 2 × dd, J ) 2.76 Hz, 6.3 Hz and J ) 3.24 Hz, 6.3 Hz), 4.19 (q, 1H, J ) 3.47 Hz), 3.78 (2H, t, J ) 3.70 Hz), 1.54 (3H, s), 1.34 (1H, s). 13C (D3CCOCD3): δ 163.64, 152.00, 142.71, 114.28, 102.67, 93.04, 87.63, 85.14, 81.59, 62.74, 29.55 FAB MS m/z: 285 (M + 1), 307(M + 23). 2′,3′-O-Isopropylidene-5′-O-tosyluridine, 2. 2′,3′-O-Isopropylideneuridine 1 (1.2 g, 4.22 mmol) was dissolved in dry pyridine (15 mL), and the solution was cooled to 0 °C. Tosyl chloride (2.4 g, 12.67 mmol) was added in small portions. After the addition, the solution which was allowed to warm to room temperature, was stirred overnight, and was quenched with methanol. The resulting mixture was stirred for 30 min, diluted with CH2Cl2, and successively washed with brine, aqueous NaHCO3 5% w/v, and brine. The organic layer was dried on Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (cyclohexane/EtOAc: 50/50) to give 2 (1.32 g, 71%) as a white solid. mp ) 67 °C. 1H NMR (CDCl3): δ 9.48 (1H, s), 7.75 (2H, d, J ) 8.29 Hz), 7.32 (2H, d, J ) 7.98 Hz), 7.23 (1H, d, J ) 8.13 Hz), 5.71 (1H, d, J ) 8.06 Hz), 5.63 (1H, d, J ) 2.05 Hz), 4.93 (1H, dd, J ) 2.05 Hz, 6.40 Hz), 4.78 (1H, dd, J ) 3.63 Hz, 6.40 Hz), 4.32 (1H, m), 4.27 (2H, m), 2.43 (3H, s), 1.53 (3H, s), 1.32 (3H, s). 13C (CDCl3): δ 163.41, 150.10,

Bioconjugate Chem., Vol. 17, No. 2, 2006 467

145.42, 142.41, 132.59, 130.03, 128.09, 114.79, 102.83, 94.96, 85.16, 84.45, 80.94, 69.45, 27.15, 23.31, 21.81 FAB-MS m/z 439 (M + 1). 2′,3′-Dioleyl-5′-O-tosyluridine, 3. At 0 °C, 2′,3′-O-isopropylidene-5′-O-tosyluridine 2 (200 mg, 0.46 mmol) was dissolved in 3.5 mL of an aqueous trifluoroacetic acid/H2O (6/1) solution and stirred for 1 h at 0°C. The solvents were coevaporated with methanol under reduced pressure, and the residue was dried in vacuo. The crude product was dissolved in 10 mL of CH2Cl2, and the solution was cooled to 0 °C. Dicyclohexylcarbodiimide (388 mg, 1.88 mmol), (dimethylamino)pyridine (230 mg, 1.88 mmol), and oleic acid (531 mg, 1.88 mmol) were added, and the mixture was stirred at room temperature overnight. DCU was then filtered through a Celite pad, and the filtrate was successively washed with aqueous 2 N HCl and aqueous saturated NaHCO3 solutions. The organic layer was dried on Na2SO4, filtered, and evaporated under reduced pressure. The crude product was purified by flash column chromatography (cyclohexane/EtOAc: 90/10 to 70/30) to give 3 (349 mg, 82%) as an oil which solidify upon standing. 1H NMR (DMSO-d6): δ 7.80 (2H, d, J ) 8.37 Hz), 7.62 (1H, d, J ) 8.21 Hz), 7.48 (2H, d, J ) 8.06 Hz), 5.83 (1H, d, J ) 5.05 Hz), 5.70 (1H, d, J ) 8.06 Hz), 5.21-5.49 (6H, m), 4.17-4.40 (3H, m), 2.24-2.30 (4H, m), 1.88-2.00 (8H, m), 1.17-1.39 (44H, m), 0.80-0.87 (6H, m) 13C NMR (DMSO-d6): 171.48, 162.84, 150.098, 145.06, 141.45, 131.83, 130.00, 129.38, 129.37, 127.62, 102.26, 88.16, 78.63, 71.42, 69.03, 33.626, 31.22, 29.02, 28.80, 28.64, 28.58, 28.44, 28.39, 28.3, 26.5, 24.16, 22.02, 21.01, 13.79 FAB MS m/z: 927 (M + 1), 949 (M + 23). Tosylate Salt of 2′,3′-Dioleyl-5′-(trimethylammonio)uridine, 4. Anhydrous trimethylamine (1 mL) was transferred to a pressure tube cooled at -50 °C via a syringe. Next, anhydrous acetonitrile (2 mL) and a solution of 3 (200 mg, 0.46 mmol) in dry THF (2 mL) were added. The tube was sealed and heated in an oil bath at 50 °C during 48 h and then cooled to -20 °C and opened. The solvents were evaporated under reduced pressure to give 4 (229 mg, quantitative) as a white solid. 1H NMR (DMSO-d6): δ 8.06 (1H,d, J ) 8.13 Hz), 7.69 (2H, d, J ) 8.06 Hz), 7.32 (2H, d, J ) 7.90 Hz), 6.09 (1H, d, J ) 4.27 Hz), 5.92 (1H, d, J ) 9.80 Hz), 5.60-5.67 (1H, m), 5.52 (6H, m), 4.00 (2H, broad s), 3.34 (9H, s), 2.29-2.34 (4H, m), 1.912.02 (8H, m), 1.13-1.35 (44H, m), 0.82-0.86 (6H, m). 13C NMR (DMSO-d6): δ 172.00, 162.93, 150.10, 145.71, 142.35, 137.44, 129.38, 127.92, 125.40, 102.30, 89.13, 74.73, 70.89, 70.41, 66.53, 52.99, 33.25, 31.21, 28.20-29.20, 26.49, 24.20, 22.01, 20.01, 14.00 FAB MS m/z: 815 (M cation). (4+H2O) Anal. Cal. C, 65.77; H, 9.33; N, 4.18. Found Cal. C, 65.22; H, 9.51; N, 4.61. Materials. pEGFP-N3 (Clontech) plasmid was amplified in E. coli and purified with endotoxin free plasmid preparation kit (Qiagen). Calf thymus DNA (CAS regist. no. 91080-16-9) and ethidium bromide (EB) were obtained from Sigma. EB was weighed, and a stock solution (0.6 mg/mL) was prepared in distilled water and filtered (0.22 µm) water. The buffer solutions were also prepared in distilled and filtered (0.22 µm) water, and the buffers were pH adjusted to 7.4 with 0.1 N NaOH. A stock solution (1 mL) of calf thymus DNA (1 mg/mL) for the fluorescence exclusion assay and for TEM and SAXS studies was dissolved in Tris buffer: 20 mM Tris, 100 mM NaCl, 10 µM EDTA, pH 7.4. Exclusion Assay. Five micrograms (5 µL of 1 mg/mL solution) of DNA and a varying amount of lipid (dependent on the lipid/DNA ratio required) were diluted to 1000 µL with buffer (Tris buffer: 20 mM Tris, 100 mM NaCl, 10 µM EDTA, pH 7.4). The solutions were mixed on a benchtop vortex and incubated for 60 min at room temperature. Each solution was then diluted to 3 mL with buffer. Immediately prior to the

468 Bioconjugate Chem., Vol. 17, No. 2, 2006

analysis, 3 µL of an ethidium bromide (EB) solution (0.6 mg/mL, 1.3 mM) was added, the sample was mixed on a benchtop vortex, and the fluorescence was measured (λexc ) 546 nm, λem ) 600 nm; 1 cm path length glass cuvette, slit width 3 nm). The fluorescence was expressed as the percentage of the maximum fluorescence signal when EB was bound to the DNA in the absence of lipid. All assays were run in triplicate. Transmission Electronic Microscopy. Samples containing lipoplexes were obtained from the mixture of 3 mg/mL of extruded (50 nm) liposome and 1.25 mg/mL DNA solutions (Tris buffer: 20 mM Tris, 100 mM NaCl, 10 µM EDTA, pH ) 7.4). Before TEM imaging, the mixture was incubated for 5 h at 4 °C. TEM microscopy experiments were realized on a Philips CM 10 (negative staining with ammonium molybdate 1% in water, Cu/Pd carbon coated grids). X-ray Diffraction. A typical procedure for lipoplex formation with DOTAU and CT-DNA in a Tris buffer (20 mM Tris, 100 mM NaCl, 10 µM EDTA, pH ) 7.4) is described. The samples were prepared from 10 mg/mL of extruded (50 nm) liposome solutions (33 µL) and 10 mg/mL DNA solution (20 µL), in 53 µL of buffer. The mixture was incubated for 5 h at 4 °C and then centrifuged in an eppendorf tube. The supernatant was removed, and the pellet was suspended in 50 µL of buffer. Lipoplexes were studied by placing a small amount of this suspension in a sealed quartz capillary (diameter 1 mm) and then placing the capillary in a sample holder. SAXS measurements were conducted at the University of Montpellier II, Laboratoire GDPC, on a diffraction system equipped with a twodimensional position-sensitive area detector. The sample-todetector distance was 30 cm, source: “Rigaku” copper rotating anode working at 40 kV, 100 mA, monochromator confocal Max Flux Osmic optic. Diffraction patterns were obtained using an image plate 2D detector. The low angle reflections were in accordance with Bragg’s law 2d sin θ ) hλ, where λ is the wavelength (1.54 Å), d is the repeat period, h is the number of the diffraction order, and θ is the Bragg angle. Cell Culture and Transfection. The human epitheloid carcinoma Hela cells and the human breast adenocarcinoma MCF-7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were grown on plastic tissue dishes at 37 °C in a 5% CO2 containing atmosphere. One day before transfection, cells were seeded in 24-well plates (1 × 105 cells/well), to reach 70-90% confluency at the time of the transfection. Just prior to transfection, the complete medium was replaced by serumfree DMEM (350 µL/well). DOTAU and DOTAP solutions at 1 mg/mL in water or Lipofectamine 2000 (Invitrogen) were mixed with DNA (2.5 µg/well) in serum-free medium. Various liposome/DNA ratios were tested. The lipoplexes were formed at room temperature during 20 min and then loaded on cells which were incubated at 37 °C. At 4 h posttransfection, 350 µL of DMEM supplemented with 20% FBS was added to the cells. Cells were analyzed 48 h after transfection. Flow Cytometry. The transfection efficiency was defined by the expression of the green fluorescent protein (GFP). After transfection, cells were detached from the plastic dish with trypsin/EDTA and then resuspended in PBS. Then single-cell suspensions were analyzed by flow cytometry using a fluorescence-activated cell sorter (FACS) Becton-Dickinson model BD-LSR II device and the FlowJo software. Proliferation Assay. Proliferation in cell population was defined using the 5-bromo-2′-deoxyuridine (BrdU) labeling and detection kit III (Roche) accordingly to the manufacturer recommendations. Briefly, after 48 h of incubation with the liposomes, the cells cultured in 96-well plates were incubated during 3 h with BrdU, fixed with ethanol, and incubated with an anti-BrdU monoclonal antibody labeled with peroxidase.

Chabaud et al. Scheme 1. Synthetic Route to DOTAUa

a Conditions: (a) TsCl, pyridine, 14 h, rt, 71%; (b) TFA/H2O, 1 h, 0 °C then oleic acid, DCC, DMAP, CH2Cl2, 14 h, rt, 82%; (c) NMe3.HCl, THF, CH3CN, 48 h, quant.

Upon addition of its substrate, the peroxidase catalyzes a colored reaction which was quantified by the measure of the absorbance at 405 nm.

RESULTS AND DISCUSSION Lipid-DNA interactions control many of the steps in the path to efficient gene transfection. There are two primary mechanisms for a lipid to interact with DNA. The lipid can possess one or more cationic charges that interact with the negatively charged phosphate backbone of DNA, as is typical. Alternatively, the lipid possesses a functional group for nonionic interactions with DNA (e.g., Watson-Crick/Hoogsteen hydrogen-bonding and base π-π stacking) to form a lipid/DNA supramolecular assembly (17). Both of these interaction features can be combined in one molecular structure to affect the binding between DNA and the lipid. Thus, we prepared a new lipid possessing a DNA unit (e.g., nucleobase), a cationic ammonium group, and hydrophobic oleyl chains connected by an ester linkage to the ribose hydroxyls. The amphiphilic polar head bears a cationic charge to ensure formation of a stable DNA complex. The cationic charge is required, since our previous studies on a noncationic derivative of 4, which possesses a phosphocholine group, does not strongly bind DNA (18). This is not entirely unexpected, given that the binding constant for a single U-U base-base interaction in a non-hydrogen bonding organic solvent (e.g., CHCl3), is estimated to be less than 102 M-1 and orders of magnitude weaker than known cationic lipids such as DOTAP binding to DNA in water (19). Lipid 4 also possesses a hydrophobic section, which entails two oleyl chains. These chains were selected since previous studies have shown that this chain length and unsaturation was the most efficient for gene transfer in the cationic lipid family (20). Herein, we report the synthesis of 2′,3′-dioleyl-5′-(trimethylammonio)uridine (DOTAU), the lipoplex formed between DNA and DOTAU, cytotoxicity, and successful in vitro gene delivery. Synthesis. A nucleoside-based cationic lipid derived from uridine was synthesized as shown in Scheme 1. After preparation of the uridine acetonide 1 in acidic conditions, this intermediate was treated with tosyl chloride in pyridine to afford the 5′-tosylate nucleoside derivative, 2. Hydrolysis of the acetonide protecting group followed by a coupling reaction with oleic acid using the standard reagent DCC to the 2′ and 3′ secondary alcohols of 2 afforded the intermediate 3. Next, 3 was transferred to a pressure tube and treated for 2 days with trimethylamine in acetonitrile to give the final lipid product, 4. Importantly,

Cationic Nucleoside Lipids for Gene Delivery

Bioconjugate Chem., Vol. 17, No. 2, 2006 469

Figure 2. (A) Light micrograph of DOTAU “worm-like” structures upon hydration (internal circle is 14 µm, method A). (B) TEM images of SUV obtained after extrusion of DOTAU through a 50 nm filter. (C) TEM micrograph of DOTAU lipoplexes resulting from the incubation of CT-DNA.

Figure 3. SAXS profiles of (a) CT-DNA/DOTAU, (b) poly A/DOTAU, (c) CT-DNA/DOTAP, and (d) poly A/DOTAP complexes showing Bragg peaks at Q (0.1041 Å-1), Q (0.1241 Å-1), Q (0.1061 Å-1), and Q (0.1131 Å-1), and corresponding lamellar repeat distances d of 6.0 nm, 5.0, 5.9, and 5.6 nm, respectively. SAXS measurements realized on samples containing only DOTAU in the absence of nucleic acids (data not shown) give a repeat period of 4.6 ( 0.2 nm. (e) Schematic drawing of a CL-DNA complex in the LRC phase, displaying a condensed multilamellar structure with DNA rods intercalated between lipid bilayers. The key length scales are the bilayer spacing d and the interaxial DNA spacing dDNA.

the synthetic strategy developed allows easy access in four steps to only one stereoisomer of the lipid. Supramolecular Assembly Studies. The supramolecular structures formed by 4 in aqueous solution with and without DNA were studied by light and transmission electronic microscopy (TEM). Lipid 4 was observed to self-assemble at room temperature into liposome-like structures in aqueous solutions. Under these conditions, at room temperature, the oleyl chains are in a “fluid” state. This result is consistent with that reported previously for phosphocholine uridine derivatives (18). Interestingly, the presence of both a nucleobase and a cationic charge on the molecular structure does not prohibit the formation of lamellar phases. Depending on the procedure used, different sizes of liposomes ranging from 60 nm to 15 µm can be prepared. Light microscopy and TEM experiments were initially performed on samples without DNA. Solid lipid material can be hydrated directly on a glass slide (method A). Under this experimental setup, hydration was followed visually under a microscope and vesicular organization and/or multilamellar systems were observed. A light micrograph of supramolecular organizations forming on a hydrated thin film of DOTAU is shown in Figure 2. Another typical procedure (method B) to form self-assembled structures involves extrusion. In this procedure, 3 mg of the lipid was first dissolved in 1 mL of buffer (Tris buffer: 20 mM Tris, 100 mM NaCl, 10 µM EDTA, pH 7.4). After 20 min of agitation at room temperature, the liposomes were extruded through a polycarbonate filter (50 nm) 10 times at 21 °C to obtain small unilamellar vesicles (SUV). Particle sizing using a QELS showed supramolecular organiza-

tions of approximately 60 nm. The presence of this SUV population was confirmed by TEM experiments (see Figure 2b). TEM experiments were also performed on samples prepared with DNA. For this experiment, a SUV population of the lipid was prepared as described above, and then these vesicles were incubated for 5 h in the presence of calf thymus DNA (CTDNA). TEM micrographs of the DNA-lipid supramolecular assemblies exhibited a typical multilamellar structure called a lipoplex as shown in Figure 2c. The apparent size of the bilayer was estimated to be roughly 6-7 nm, suggesting that the DNA macromolecules are entrapped within the organized structures. SAXS Studies. To further characterize the molecular organization within the DNA supramolecular assemblies, small-angle X-ray scattering (SAXS) experiments were performed with lipid 4 in the presence of either calf thymus double stranded or polyadenylic acid single strand DNA. For lamellar lipoplexes, the repeat distance of the lipid-DNA multilayer d is related to the membrane thickness d1 and solvent thickness containing DNA in the intermembrane space dw via the expression: d ) dw + d1 (Figure 3e). Data collected from several SAXS experiments at room temperature show that CT-DNA/lipid complexes arrange into LRC lamellar phases. For reference, supramolecular assemblies of DOTAP/CT-DNA and DOTAU/ CT-DNA at 20 °C possess lamellar repeat periods d of 5.9 ( 0.2 nm and 6.0 ( 0.2 nm, respectively (Figure 3c and 3a). A priori, since DOTAU possesses a larger polar head volume than DOTAP, such a result indicates that the uracil moiety of DOTAU does not increase the dw distance. It can be postulated that the uracil is closely associated to the DNA double helix.

470 Bioconjugate Chem., Vol. 17, No. 2, 2006

Chabaud et al.

Figure 4. EB exclusion assays with DOTAP and DOTAU in the presence of CT-DNA (fluorescence intensity versus charge ratio).

Figure 5. Effects of nucleoside lipids on cell proliferation. MCF-7 (top) and HeLa cells (bottom) were incubated with various quantities of lipids then BrdU was added to the cells.

As expected, the distance d decreases when a poly A single strand is added to the liposome solutions, indicating more compact structures, which are likely due to smaller dw distances. Interestingly, the DOTAU/poly A lipoplexes exhibit a repeat period of d ) 5.0 ( 0.2 nm (Figure 3b), which is 0.6 nm smaller than DOTAU/poly A lipoplexes (d ) 5.6 ( 0.2 nm) (Figure 3d). In that case, the smaller d value observed may be attributed to tighter association involving the uracil base of DOTAU and adenine of the poly A single strand. The infrared spectrum of the DOTAU/poly A lipoplex in the KBr pellet shows a carbonyl stretch at 1712 cm-1 for the C2dO uracil-associated stretching vibration and both CdN and CdC ring vibrations characteristic of adenine upon duplexation in RNA (21), confirming U-A hydrogen bonding in the DOTAU/poly A complex. Ethidium Bromide Exclusion Assays. The formation of the DNA-lipid complexes was also studied using an ethidium bromide (EB) exclusion assay (22). The binding affinity of the nucleoside-based lipid to CT-DNA was investigated by the fluorescence titration of EB with a premixed solution of DNA and various concentrations of lipid 4. In the presence of DNA, the fluorescence emission of EB is enhanced relative to that in water as a result of EB intercalation between the DNA base pairs (23). The subsequent addition of lipids to the DNA-EB solution results in lipid binding to DNA and displacement of EB from the DNA helix to water, which yields a decrease in fluorescence intensity. As expected, the fluorescence intensity decreases rapidly for DOTAU and DOTAP (Figure 4). The binding curves for DOTAP and DOTAU indicate that 1:1 and 1:1.5 charge complexes are formed, respectively, between DNA and the lipids. The residual fluorescence intensity of 45% measured for DOTAU above a charge ratio of 1.5

Figure 6. Transfection efficiency of nucleoside lipids. The pEGFP vector (2.5 µg) was transiently transfected into HeLa (top histograms) or MCF-7 cells (bottom histograms) using various quantities of lipotransfectant. After transfection, cells were analyzed by FACS. In part A, the number of GFP positive cells is reported. The MFI of GFP positive cells is reported in part B.

indicates that certain portions of DNA are still exposed to EB (24). In the same conditions, DOTAP induces a residual fluorescence intensity of 20%. These results would suggest that DOTAU exhibits a lesser ability to condense DNA compare to DOTAP. Cell Proliferation. The effect on mammalian cell lines proliferation of the nucleoside lipids was compared to a commercially available transfectant, Lipofectamine 2000 (LIPO). As shown in Figure 5, the incorporation of BrdU into HeLa or MCF-7 DNA is not affected by DOTAU or DOTAP, as the absorbance does not decrease in the presence of these cationic lipids. In contrast, in both cell types, the incorporation of BrdU decreases in the presence of Lipofectamine 2000. The effect is much stronger in HeLa cells than in MCF-7 cells as the absorbance is respectively reduced by 50% and 15% in the presence of Lipofectamine 2000. These proliferation assays show that the nucleoside lipids do not inhibit cell proliferation and appear to be less toxic than the commercial Lipofectamine 2000. Transfection. To investigate the efficiency of DOTAU to transfer DNA into cells, we performed transient transfection assay of mammalian cell lines (HeLa and MCF-7). The transfected DNA is an expression vector (pEGFP) encoding GFP which fluorescence can be detected by FACS. Various quantities of cationic lipids were mixed with 2.5 µg of pEGFP, and then lipoplexes were incubated with HeLa or MCF-7 cells. Singlecell suspensions were analyzed by FACS. The GFP negative and positive gates were defined using the control experiment

Cationic Nucleoside Lipids for Gene Delivery

carried out in absence of lipoplex. In each experiment 5000 cells were sorted, the results are shown in Figure 6 and in Supporting Information Figure 1. DOTAU is efficient for transfection at a concentration of 6 µg/well. At a concentration of 36 µg/well, approximately 400 cells are GFP positive. With DOTAP, 2 µg/well is the best concentration to transfect MCF-7 cells. Higher quantities are necessary to transfect 20% of Hela cells; however, an equivalent transfection efficiency is obtained with concentrations of 6, 18, and 36 µg/well. In comparison, the commercial Lipofectamine 2000 is efficient only at 2 µg/well, while higher quantities induce a strong decrease of transfection efficiency. Therefore, it appears that the nucleoside lipid is efficient in a wider range of concentration compared to Lipofectamine 2000; likely this feature might be linked to the low cell toxicity of these products. It must be underlined that ongoing transfection studies on nucleolipid analogues have shown similar results with high transfection efficiency only at high charge ratios. The mean fluorescence intensities (MFI) defined for GFP positive cells reveal that the level of GFP expression with a higher MFI value correlated with a high GFP expression. Surprisingly, transfection carried out with DOTAU is characterized by low MFI values (around 1000 for Hela cells and 400 for MCF-7). DOTAP and Lipofectamine 2000 lead respectively to medium MFI values (around 1900) and high MFI values (around 3000) (Figure 6). This study reveals that cells transfected using DOTAU express GFP at a low level. For the moment we can only speculate about the basis of this observation. One possibility is that DOTAU/ DNA lipoplex might contain less DNA molecules than the lipoplex formed with DOTAP or Lipofectamine 2000. Another possibility is that the vectors do not reach the nuclei where DNA is transcribed because DOTAU/DNA lipoplex might dissociate in the cytoplasm instead of the nuclei. Only at higher nucleolipid concentrations compared to Lipofectamine 2000 can we observe increased transfection efficiencies. Further studies are in progress to confirm these hypotheses.

CONCLUSION The results obtained by SAXS, TEM studies, ethidium bromide exclusion assays, and FTIR clearly indicate that the cationic nucleolipid DOTAU interacts with both DNA double helix and polyadenylic acid single strand. Transfection assays of mammalian cell lines (HeLa and MCF-7) reveal that DOTAU can efficiently transfect a high number of cells with a low level of gene expression by cells (compare to DOTAP and Lipofectamine). This aspect could be of interest in the case of gene overexpression; the possibility of this novel nucleolipid to tune gene expression could be exploited when transfection of toxic proteins is needed, for example. The proliferation assays realized on the same cell lines show that the nucleoside lipid DOTAU does not inhibit cell proliferation and is less toxic than the commercial Lipofectamine 2000. In summary, the data reported here suggest that the presence of a nucleoside moiety on the cationic lipid has an impact on the lipoplex efficiency and cell toxicity. The results collected for this biocompatible lipid suggest its potential use as a transfection reagent for mammalian cells and in vitro applications for gene therapy. Further cell biology experiments, such as cellular uptake studies and intracellular trafficking experiments aimed at understanding subcellular localizations of the nucleolipid/DNA complexes, are planned to gain better mechanistic insights into the origin of the contrasting transfection profiles of these novel nucleolipids.

ACKNOWLEDGMENT This research was supported by the Army Research Office (Grant DAAD 19-02-1-0386) and the “Association pour la

Bioconjugate Chem., Vol. 17, No. 2, 2006 471

Recherche sur le Cancer” (ARC). The authors would like to thank Dr. Philippe Genne and Dr. Nicolas Guilbaud of the Oncodesign company (Dijon, France), Dr. Stephen J. Lee (ARO, US Army), Dr. Carla A. H. Prata (Boston University), Dr. Philippe Dieudonne´ (University of Montpellier, France), and Prof. Mohamed El Maataoui (University of Avignon, France). Supporting Information Available: FACS details. This material is available free of charge via the Internet at http://pubs.acs.org.

LITERATURE CITED (1) Morgan, R. A., and Anderson, W. F. (1993) Human gene therapy. Annu. ReV. Biochem. 62, 191-217. (2) Bennett, C. F., Chiang, M. Y., Chan, H., Shoemaker, J. E., and Mirabelli, C. K. (1992) Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides. Mol. Pharm. 41, 1023-1033. (3) Debs, R. J., Freedman, L. P., Edmunds, S., Gaensler, K. L., Duzgunes, N., and Yamamoto, K. R. (1990) Regulation of gene expression in vivo by liposome-mediated delivery of a purified transcription factor. J. Biol. Chem. 265, 10189-10192. (4) Marshall E. (2000) FDA Halts All Gene Therapy Trials at Penn. Science 287, 565-567. (5) Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M., and Danielsen, M. (1987) Lipofection: a highly efficient, lipid mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. U.S.A. 84, 7413-7417. (6) Leventis, R., and Silvius, J. R. (1990) Interactions of mammalian cells with lipid dispersions containing novel metabolizable cationic lipids. Biochim. Biophys. Acta 1023, 124-132. (7) Gao, X., and Huang, L. (1991) A novel cationic liposome reagent for efficient transfection of mammalian cells. Biochem. Biophys. Res. Commun. 179, 280-285. (8) Rose, J. K., Buonocore, L., and Whitt, M. A. (1991) A new cationic liposome reagent mediating nearly quantitative transfection of animal cells. Biotechniques 10, 520-525. (9) Barthel, F., Remy, J. S., Loeffler, J. P., and Behr, J. P. (1993) Gene transfer optimization with lipospermine-coated DNA. DNA Cell Biol. 12, 553-560. (10) Solodin, I., Brown, C. S., Bruno, M. S., Chow, C. Y., Jang, E. H., Debs, R. J., and Heath, T. D. (1995) A novel series of amphiphilic imidazolinium compounds for in vitro and in vivo gene delivery. Biochemistry 34, 13537-13544. (11) Akao, T., Nakayama, T., Takeshia, K., and Ito, A. (1994) Design of a new cationic lipid with efficient DNA transfection ability. Biochem. Mol. Biol. Int. 34, 915-920. (12) Bichko, V., Netter, H. J., and Taylor, J. (1994) Introduction of hepatitis delta virus into animal cell lines via cationic liposomes. J. Virol. 68, 5247-5252. (13) Gao, X., and Huang, L. (1995) Cationic liposome-mediated gene transfer. Gene Ther. 2, 710-22. (14) Rolland, A. P. (1998) From genes to gene medicines: recent advances in nonviral gene delivery. Crit. ReV. Ther. Drug Carrier Syst. 15, 143-98. (15) Choi, J. S., Lee, E. J., Jang, H. S., and Park, J. S. (2001) New cationic liposomes for gene transfer into mammalian cells with high efficiency and low toxicity. Bioconjugate Chem. 12, 108-113 and references herein. (16) Godbey, W. T., and Mikos, A. G. (2001) Recent progress in gene delivery using non-viral transfer complexes J. Controlled Release. 72, 115-125. (17) Kim, Y. H., Gihm, S. H., Park, C. R., Lee, K. H., Kim, T. W., Kwon, I. C., Chung, H., and Jeong, S. Y. (2001) Structural characteristics of size-controlled self-aggregates of deoxycholic acidmodified chitosan and their application as a DNA delivery carrier. Bioconjugate Chem. 12, 932-938. (18) Moreau, L., Barthe´le´my, P., El Maataoui, M., and Grinstaff, M. W. (2004)Supramolecular Assemblies of Nucleoside-based Lipids. J. Am. Chem. Soc. 126, 7533-7539.

472 Bioconjugate Chem., Vol. 17, No. 2, 2006 (19) Pozharski, E., and MacDonald, R. C. (2002) Thermodynamics of Cationic Lipid-DNA Complex Formation as Studied by Isothermal Titration Calorimetry. Biophys J. 83, No. 1, 556-565. (20) Regelin, A. E., Fankhaenel, S., Gu¨rtesch, L, Prinz, C., von Kiedrowski, G., Massing, U. (2000) Biophysical and lipofection studies of DOTAP analogs. Biochim. Biophys. Acta 1464, 151-164. (21) Banyay, M., Sarkar, M.; Gra¨slund, A. (2003) A library of IR bands of nucleic acids in solution Biophys. Chem. 104, Issue 2, 477488. (22) Geall, A. J., and Blagbrough, I. S. (2000) Rapid and sensitive ethidium bromide fluorescence quenching assay of poly-

Chabaud et al. amine conjugate-DNA interactions for the analysis of lipoplex formation in gene therapy. J. Pharm. Biomed. Anal. 22, 849859. (23) LePecq, J. B., and Paoletti, C. (1967) A fluorescent complex between ethidium bromide and nucleic acids. Physical-chemical characterization J. Mol. Biol. 27, 87-106. (24) Faneca, H., Simo˜es S., and Pedroso de Lima, M. C. (2002) Evaluation of lipid-based reagents to mediate intracellular gene delivery. Biochim. Biophys. Acta 1567, 23-33. BC050162Q