Transfection with Fluorinated Lipoplexes Based on New Fluorinated

Bioconjugate Chem. 2004, 15, 901−908

901

Transfection with Fluorinated Lipoplexes Based on New Fluorinated Cationic Lipids and in the Presence of a Bile Salt Surfactant Caroline Boulanger, Christophe Di Giorgio, Je´roˆme Gaucheron, and Pierre Vierling* Laboratoire de Chimie Bioorganique, UMR 6001 CNRS, Universite´ de Nice Sophia-Antipolis, Faculte´ des Sciences, 06108 Nice Ce´dex 2, France. Received March 8, 2004; Revised Manuscript Received April 21, 2004

The synthesis of two fluorinated cationic lipids, which are analogues of frequently used synthetic gene carrier agents (including the cationic 2,3-dioleoyloxy-N-[2-(spermine-carboxamido)ethyl]-N,Ndimethyl-1-propanaminium (DOSPA) component of the commercially available liposomal Lipofectamine), and the disintegration and DNA accessibility (evaluated by the ethidium bromide (BET) intercalation assay) as well as the in vitro transfection efficacy of cationic lipoplexes formulated with these new lipids in conjunction with conventional or fluorinated helper lipids, in the absence or presence of sodium taurocholate (STC), a powerful anionic bile salt detergent, is reported. A higher stability, with respect to the STC lytic activity and DNA accessibility, of the fluorinated cationic lipoplexes as compared with their respective lipofectamine-based ones was demonstrated. Indeed, while the Lipofectamine lipoplexes were fully disintegrated at a [STC]/[lipid] molar ratio of 2000, only 40-60% of the DNA intercalation sites of the lipoplexes based on the fluorinated analogue of DOSPA were accessible to ethidium bromide. A higher transfection potential in the presence of STC was further found for the lipoplexes formulated with the fluorinated analogue of DOSPA as compared with the Lipofectamine preparation. For a STC concentration of 7.5 mM, lipofection mediated with these fluorinated lipoplexes was significantly higher (nearly 30- to 50-fold, p < 0.05) than with the Lipofectamine ones. These results confirm the remarkable transfection potential of fluorinated lipoplexes.

INTRODUCTION

Synthetic gene transfer systems based on (poly)cationic lipids, liposomes, or polymers (e.g. lipoplexes or polyplexes, respectively) have become very promising novel forms of molecular medicine (for recent reviews, see refs 1-8 and references therein). Although the gene expression levels obtained with these systems are transient and lower than with viral vectors, they present several advantages including low-cost and large-scale production, safety, and capacity to deliver large gene fragments. However, there is still a need for the development of gene transfer systems with improved and original properties. Progress in this field also requires systems which allow efficient gene expression in various biological fluids. In the field of gene transfer and gene therapy of cystic fibrosis and of cystic fibrosis-associated diseases for example, systems which enable efficient gene expression in the presence of various surfactants (pulmonary ones for the gene transfer to the respiratory epithelium, bile salts for the biliar epithelium) are required. Indeed, pulmonary surfactants constitute a barrier to transfection of the endobronchial airway (9). Such surfactants were also found to inhibit lipofection of various cell lines with cationic lipids (9-12). In our laboratory, fluorinated cationic lipoplexes, i.e., lipoplexes resulting from the formulation of DNA with highly fluorinated cationic lipids (analogues of DOTMA, DMRIE, DPPES, and DOGS)1 (13, 14) and/or highly fluorinated helper lipids (analogues of DOPE) (15, 17) * Corresponding author. Phone: 33 4 92 07 61 43. Fax: 33 4 92 07 61 51. E-mail: [email protected].

(see structures in Figure 1), were shown to constitute very promising transfection systems. Such fluorinated cationic lipoplexes exhibited in vitro (13-17) and in vivo (15) a higher transfection potential than their conventional lipoplex counterparts and more particularly in the presence of a powerful anionic surfactant (16). Their remarkable transfection potency was attributed to the unique lipophobic and hydrophobic character of the fluorinated lipids used for their formulation, thus preventing DNA from interactions with lipophilic and hydrophilic biocompounds, and from degradation (16). We report herein on the synthesis of F-DMAEA and F-DOSPA which are fluorinated analogues of frequently used synthetic gene carrier agents, i.e., GAP-DLRIE (18) and DOSPA (which is the cationic component of commercially available liposomal Lipofectamine) (19) (see structure in Figure 1), and on their ability to compact, transfer, and express DNA into cells in the absence or presence of a powerful anionic detergent (e.g. sodium taurocholate, STC, which is a model of bile salts). The 1 Abbreviation: BET, ethidium bromide; CL, cationic lipid; DMRIE, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide; DPPES, 1,2-dipalmitoylphosphatidylethanolamidospermine-4 trifluoroacetate; DOGS, N,N-dioctadecylamidoglycylspermine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOSPA, 2,3-dioleoyloxy-N-[2-(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate; DOTMA, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride; F-PE: fluorinated analogue of DOPE.; GAPDLRIE: N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)1-propanammonium bromide; HL: helper lipid; N/P, N ) number of amine equivalents of the cationic lipid; P ) number of DNA phosphate equivalents; STC, sodium taurocholate.

10.1021/bc049942+ CCC: $27.50 © 2004 American Chemical Society Published on Web 05/25/2004

902 Bioconjugate Chem., Vol. 15, No. 4, 2004

Boulanger et al.

Figure 1. Chemical structure and code name of the fluorinated and conventional cationic and helper) lipids used and/or mentioned in this study. F-DOSPA, F-DMAEA, F-DOGS (14), F-DOTMA, F-DMRIE, F-DPPES (13), and F-PE (15, 17) are fluorinated analogues of conventional DOSPA (19), GAP-DLRIE (18), DOGS (25), DOTMA (26), DMRIE (27), DPPES (28), and DOPE, respectively.

goals of this study were to extend the library of fluorinated cationic lipids that enable gene transfer and expression and to confirm or infirm the transfection potential of fluorinated cationic lipoplexes in the presence of surfactants (16). As the use of helper lipids (HL) enhances the transfection activity of most (poly)cationic lipids (including the fluorinated analogues of DOGS) (14, 17), we report on the transfection efficacy of these new fluorinated cationic lipids (CL) used in conjunction with DOPE or F-PE, a fluorinated analogue of DOPE (see structure in Figure 1) which was found in vitro and in vivo to be a more efficient helper lipid than DOPE (15, 17). In this paper, we show that, in the presence of STC, the cationic fluorinated F-DOSPA/F-PE lipoplexes display a greater stability with respect to detergent-triggered disintegration and DNA accessibility, and a significantly larger in vitro transfection potential than conventional lipoplexes formulated with Lipofectamine. MATERIALS AND METHODS

General Experimental and Analytical Conditions. Most of the reactions were performed in anhydrous solvents under dry and oxygen-free nitrogen. Anhydrous solvents were prepared by standard methods. Purifications by column chromatography were carried out using silica gel 60 (Merck, 70-230 mesh) and chloroform (CHCl3), dichloromethane (CH2Cl2), methanol (MeOH), or mixtures thereof as indicated. Unless noted otherwise, the ratios describing the composition of solvent mixtures represent relative volume. Advancement of the reaction was followed by thin-layer chromatography (TLC) on silica plates F254 (Merck). The following developing systems were used: UV light, KMnO4, H2SO4/EtOH, Dragendorff reagent, ninhydrin reagent (Sigma). TetraN,N′,N′′,N′′′-(tert-butyloxycarbonyl)-spermine-5-carboxylic acid [(Boc)4SperCOOH] and N-[1-(2,3-di-11-(F-butyl)undecyloxy)-propyl]-N-[2-hydroxyethyl]-N,N-dimethylammonium bromide [F-DMHEA] were synthesized according to published procedures (13, 14). rac-3-[11-(FOctyl)undec-10-enyl]-2-(hexadecyl)glycero-1-phosphoethanolamine, F-PE, was from our laboratory (15, 17). 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was purchased from Sigma. All other organic chemicals were purchased from Sigma, Aldrich, or Fluka. Lipofectamine was purchased from Invitrogen (Cergy Pon-

toise, France). 1H, 13C, and 19F NMR spectra were recorded at 200, 50.3, and 188.3 MHz, respectively, on a Bruker AC-200. Chemical shifts were measured relative to CHCl3 (δ 7.26 ppm) or CH3OD (δ 3.31 ppm) for 1H, relative to CDCl3 (δ 77.1 ppm) for 13C and expressed indirectly in relation to TMS, and relative to CCl3F as internal reference for 19F. The following abbreviations are used to describe the signal multiplicities: s (singlet), d (doublet), t (triplet), q (quadruplet), and m (multiplet). Chemical shifts are expressed in parts per million and listed as follows: shift in parts per million (multiplicity, coupling, integration, and attribution). Electrospray ionization mass spectrometry (ESI-MS) in positive mode was performed on a Finnigan MAT LCQ equipped with an atmospheric pressure ionization (API) source. Synthesis of the Fluorinated Cationic Lipids. Synthesis of N-[1-(2,3-di-11-(F-butyl)undecyloxy)-propyl]N,N-dimethyl-N-(2-aminoethyl)ammonium hydroxide [FDMAEA]. To a solution of F-DMHEA (13) (600 mg, 0.6 mmol) in THF (20 mL) were added 0.7 mL of a freshly prepared 1.6 M solution of hydrazoic acid (HN3, 1.1 mmol) in chloroform followed by 0.22 mL of a THF solution of diisopropyl azodicarboxylate (DIAC, 1.1 mmol) and then 10 mL of a THF solution of triphenylphosphine (0.52 g, 2 mmol) (20). The resulting mixture was stirred at 50 °C overnight. Water (1 mL) was added, and the temperature was maintained at 50 °C for 3 h. After evaporation in vacuo of the solvents, the residue was dissolved in dichloromethane. The organic phase was washed three times with water and then dried over Na2SO4. Flash chromatography of the residue obtained after evaporation over a silica gel column (60 g, from CHCl3/MeOH 9/1 to CHCl3/MeOH 8/2 and then CHCl3/MeOH/NH4OH 8/2/0.2) led to 0.27 g (0.27 mmol, 45%) of a white powder consisting into F-DMAEA as its hydroxide. TLC (CHCl3/ MeOH/NH4OH, 8/2/0.2 v/v; H2SO4, Dragendorff, ninhydrin): Rf ) 0.35. 1H NMR (CDCl3/CD3OD): δ 1.02-1.31 (s large, 28H, CF2(CH2)2(CH2)7); 1.31-1.55 (m, 8H, CF2CH2CH2, CH2CH2O); 1.90 (tt, 3J ) 8.3 Hz, 3JHF ) 18.8 Hz, 4H, CF2CH2); 2.93-3.09 (m, 2H,CH2NH2); 3.09-3.19 (s large, 6H, CH3); 3.23-3.61 (m, 10H, CH2O, CH2NCH2); 3.80-3.95 (m, 1H, CH). 13C NMR (CDCl3/CD3OD): δ 19.9 (t, 3JCF ) 3.7 Hz, CF2CH2CH2); 25.9, 26.0 (CH2(CH2)2O); 28.9, 29.0, 29.2, 29.3, 29.4 (CF2(CH2)2(CH2)6); 29.9 (CH2-

Transfection with Fluorinated Lipoplexes

CH2O); 30.6 (t, 2JCF ) 22.3 Hz, CF2CH2); 35.4 (CH2NH2); 52.3, 52.9 (CH3); 66.4 (CH2NCH2); 68.6, 69.3, 71.9 (CH2O); 73.0 (CH).19F NMR (CDCl3/CD3OD): δ -78.2 (3F, CF3); -111.1 (2F, CF2CH2); -129.4 (2F, CF2CF2CH2); -127.5 (2F, CF3CF2). MS (ESI+): m/z ) 907.6 [M]+, in agreement with the mass calculated for [M]+ ) C37H61F18N2O2. Synthesis of N-[1-(2,3-Di-11-(F-butyl)undecyloxy)propyl]-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethylammonium as a Trifluoroacetate Salt, [FDOSPA]. To a solution of (Boc)4SperCOOH (14) (212 mg, 0.33 mmol) in 15 mL of CH2Cl2 were added N-hydroxysuccinimide (HOSu) (49 mg, 0.43 mmol) and dicyclohexylcarbodiimide (DCC) (88 mg, 0.43 mmol) with a few drops of THF. The mixture was stirred at room temperature during 12 h and was then cooled to 0 °C and filtered. Then, 270 mg (0.27 mmol) of F-DMAEA and 75 µL (0.54 mmol) of triethylamine were added, and the resulting mixture was stirred at room temperature during 12 h. The organic phase was washed with 5% sodium carbonate, 5% citric acid, and water until neutrality and then dried over Na2SO4. Flash chromatography over a silica gel column of the residue obtained after evaporation (20 g, CH2Cl2/MeOH: from 99/1 to 90/10 then CH2Cl2/MeOH/ NH4OH, 8/2/0.2 v/v) led to 200 mg (0.12 mmol, 46%) of a colorless oil consisting of N-[1-(2,3-di-11-(F-butyl)undecyloxy)propyl]-N-[2-(tetrabutoxycarbonyl-sperminecarboxamido)ethyl]-N,N-dimethylammonium hydroxide [F-DOSPA(Boc)]. TLC (CHCl3/MeOH/ NH4OH: 8/2/0.2; Dragendorff, ninhydrin): Rf ) 0.51. 1H NMR (CDCl3/CD3OD): δ 0.74-1.83 (m, 80H, CF2CH2(CH2)9, CH3(Boc), CH2CH2N(H)Boc, CH2CH2NBoc, CH2CHC(O)); 1.97 (tt, 3 JHH ) 7.5 Hz and 3JHF ) 19.1 Hz, 4H, CF2CH2); 2.924.26 (m, 30H, N(CH3)2, CH2O, CHO, CH2N, CHC(O)NH). 13 C NMR (CDCl3/CD3OD): δ 20.1 (t, 3JCF ) 3.7 Hz, CF2CH2CH2); 26.1, 26.2 (CH2(CH2)2O); 28.5 (CH3(Boc); 29.1, 29.3, 29.4, 29.6, 29.7, 30.0 (CF2(CH2)2(CH2)6, CH2CH2O, CH2CH2NBoc, CH2CHNBoc); 30.8 (t, 2JCF ) 22.3 Hz, CF2CH2); 38.3, 44.0, 46.4, 46.7 (CH2NBoc); 52.6 (CH2NHC(O)); 53.3, 53.5 (N(CH3)2); 60.6 (CHC(O)NH); 68.6, 69.5, 72.1 (CH2O, CHCH2NCH2); 73.3 (CHO); 78.9, 79.6, 80.7 (C(CH3)3); 155.6, 155.7, 156.2 (C(O)). 19F NMR (CDCl3/CD3OD): δ -81.6 (3F, CF3); -115.1 (2F, CF2CH2); -125.0 (2F, CF2CF2CH2); -126.6 (2F, CF3CF2). MS (ESI+): m/z ) 1536 [M]+, in agreement with the calculated mass for [M]+ ) C68H117F18N6O11. The Boc-deprotection of F-DOSPA(Boc) was quantitatively achieved in a large excess of a TFA/CH2Cl2 mixture during 1 h at room temperature. The excess TFA was removed by coevaporation with cyclohexane yielding F-DOSPA as a TFA salt (2.6 TFA according to 19F NMR). 1 H NMR (CD3OD): δ 1.22-2.29 (m, 48H, CF2(CH2)10, CH2CH2NH, CH2CHC(O)); 2.84-3.81 (m, 29H, N(CH3)2, CH2O, CHO, CH2N); 4.01-4.17 (m, 1H, CHC(O)NH). 13C NMR (CD3OD): δ 21.2 (t, 3JCF ) 3.8 Hz, CF2CH2CH2); 23.1 (CH2CHC(O)); 25.3 (CH2(CH2)2OCH2); 26.3 (CH2(CH2)2OCH); 27.2, 27.3, 28.6, 29.4, 30.1, 30.4, 30.5, 30.6, 30.7, 31.1 (CF2(CH2)2(CH2)6, CH2CH2O); 31.6 (t, 2JCF ) 22.4 Hz, CF2CH2); 34.5 (CH2CH2NH2); 37.8 (CH2NHC(O)); 38.5, 45.3, 45.9, 48.4 (CH2NH, CH2NH2); 52.8, 53.1 (N(CH3)2); 61.6 (CHC(O)NH); 64.4, 67.4, 70.1, 70.2 (CH2O, CHCH2NCH2); 72.8 (CHO); 163.1 (q, 2JCF ) 34.5 Hz, CF3COO-); 171.9 (C(O)NH).19F NMR (CD3OD): δ -77.2 (8F, CF3COO-); -83.0 (3F, CF3); -116.1 (2F, CF2CH2); -125.9 (2F, CF2CF2CH2); -127.7 (2F, CF3CF2). MS (ESI+): m/z ) 1135.7 and m/2z ) 568.6 in agreement with the calculated mass for [M]+ ) C48H85F18N6O3 and [M + H]2+/2, respectively. Preparation of the DNA Complexes. The plasmid pTG11236 (pCMV-SV40-luciferase-SV40pA) used for the

Bioconjugate Chem., Vol. 15, No. 4, 2004 903

preparation of the DNA complexes and for transfection assays was produced by Transge`ne (Strasbourg, France). It is a plasmid of 5739 bp. The endotoxin content of the plasmid preparation was checked using a Limulus Amebocyte Lysat kit (Biogenic, France). This value was below 1 endotoxin unit/mg of plasmid, hence below the 5 e.u./ mg of DNA recommended for in vivo protocols.The quantities of lipids used for the N/P 5 lipoplexes were calculated according to the DNA concentration of 0.1 mg/ mL in the lipoplex preparation, the molar weight, and the number of amino groups in the selected cationic lipid (CL is F-DMAEA, F-DOSPA or DOSPA). The N/P ratio of 5 corresponds to the molar amount of CL necessary to have a ratio of five amino group nitrogens (for 1 mol CL) per one phosphate in the DNA (330 Da mean MW), as described elsewhere (13-17). Liposomal dispersions containing CL and DOPE (or F-PE) as helper lipid (HL) at a [CL]/[HL] 60/40 molar ratio and at a CL concentration of 1.0 mg/mL in a 5% glucose solution were first prepared. Thus, for the preparation of N/P 5 F-DOSPA/HL/DNA complexes, 100 µL of a F-DOSPA solution (10 mg/mL in CHCl3/MeOH 9/1) and 34.8 µL of DOPE or 54.7 µL of F-PE solutions (10 mg/mL in CHCl3 or CHCl3/MeOH 9/1, respectively) were transferred to a borosilicate glass tube. The solvents were evaporated in a Rotavap evaporation system (45 °C, 0.2 bar, 40 rotations/min). Then, 1.0 mL of a 5% glucose solution was added to the film obtained. The preparation was vortexed for 12 h and then sonicated for 5 min to yield a liposomal preparation. The F-DOSPA/HL/DNA lipoplexes were then formulated by adding 64.8 µL of the F-DOSPA/HL liposomal preparation to 85.2 µL of a DNA solution in glucose 5% (0.176 mg/mL) to reach a final DNA concentration of 0.1 mg/mL. This preparation was vortexed for 10 s and was used within 1 h for particle size measurements, agarose gel electrophoresis, ethidium bromide (BET) exclusion, and/or in vitro transfection experiments. Measurement of the Size of the Lipoplexes. The average sizes were measured by photon correlation spectroscopy using a Coulter N4Plus particle size analyzer, as described elsewhere (14). Agarose Gel Electrophoresis. Each sample of the above-described lipoplex preparation (0.5 µg of plasmid) was electrophoresed for about 30 min under 150 V, through a 0.8% agarose gel in TAE 1 x (Tris-acetateEDTA) buffer and stained by spreading a 1 µL/mL BET solution in TAE buffer. The absence of BET-stained DNA bands after photographing on a UV transilluminator confirmed that all the DNA was complexed. Its integrity was confirmed by electrophoresis after disrupting the lipoplexes with a large excess of sodium dodecyl sulfate, following procedures described elsewhere (14). Lipoplex Stability in the Presence of Sodium Taurocholate Dye-Exclusion Assays. The BET intercalation into the N/P 5 lipid-complexed DNA at a concentration of 15.0 µM for F-DOSPA or DOSPA (respectively 37.5 µM for F-DMAEA) and 15 µM DNA phosphate in the presence of various concentrations of STC (from 0 to 60 mM) was assessed by fluorescence monitoring at 2.5 µM BET which correspond to a BET: nucleotide ratio of 1:6. Under these conditions, the fluorescence of BET intercalated into DNA was found to be directly proportional to the percentage of accessible DNA (21). The above-described lipoplex preparation (0.1 mg DNA/mL) (75 µL) in a 5% glucose solution was added to 750 µL of BET (5 µM in a 5% glucose solution) and to 675 µL of STC in glucose 5% to reach a final concentration of STC in the 0-60 mM range. The resulting


preparations were incubated at 37.0 ( 0.5 °C in a thermoregulated cell with magnetic stirring for 10 min. Then, the fluorescence F was measured using a PerkinElmer Spectrofluorometer LS 50B (excitation at 306 nm, emission at 622 nm, and a 530 nm emission cutoff filter). The fluorescence signal, Fmax, which corresponds to 100% BET intercalation, was determined for each detergent concentration tested on a 2.5 µM BET and naked DNA 0.1 mg/mL glucose solution. The fluorescence of each sample was corrected for the background fluorescence F0 of BET in the absence of DNA and in the presence of STC at each concentration tested. The percentage of accessible DNA to BET for each sample and for each detergent concentration was calculated from the equation (F - F0)/ (Fmax - F0). This percentage can exceed 100% owing to diffusion background fluorescence resulting from the presence of lipospermine-based aggregates (in the present experiments F could not be corrected for this effect). The given means were calculated from at least three independent experiments. In Vitro Transfection of A549 Cells. For in vitro transfection, A549 cells (epithelial cells derived from human pulmonary carcinoma) were grown 24 h before the experiments in Dulbeco-modified Eagle culture medium (DMEM) (Gibco-BRL), containing 10% foetal calf serum, FCS (Sigma), in 96-well plates (2 × 104 cells per well), in a wet (37 °C) and 5% CO2/95% air atmosphere. Five microliters of CL/HL/DNA lipoplex preparation was diluted to 50 µL in DMEM. Ten minutes before addition to the cells, this dispersion was diluted with 50 µL of STC in DMEM to obtain the desired detergent concentration (from 0 to 17.5 mM). The cell culture medium was removed and replaced with this 100 µL of lipoplex/ detergent/DMEM solution which corresponds to a DNA concentration of 0.5 µg and a lipid concentration of 15.1 µM for F-DOSPA and 37.9 µM for F-DMAEA in each well. After 4 and 24 h, 50 and 100 µL of DMEM supplemented with 30% and 10% FCS, respectively, were added. Fortyeight hours after transfection, the culture medium was discarded and the cells were washed twice with 100 µL of phosphate-buffered saline (PBS) and then lysed with 50 µL of lysis buffer (Promega, Charbonnie`res, France). The lysates were frozen at -40 °C awaiting for luciferase activity analysis. This measurement was done for 10 s on 10 µL of lysis mixture in a LB96P luminometer (Berthold, Evry, France) in dynamic mode, using the ‘Luciferase’ determination system (Promega) in 96-well plates. The total protein concentration per well was determined by the BCA test (Pierce, Montluc¸on, France). For cells grown in the absence of lipoplexes or STC, a well contained approximately 30-50 µg of proteins. Luciferase activity was calculated as femtograms (fg) of luciferase per milligram (mg) of proteins. The percentage cell viability of the lipoplexes (without or with STC) and of STC was calculated as the ratio of the total protein per well of the transfected cells (without or with STC) relative to that measured for untreated cells or for cells incubated with the detergent × 100%, respectively. The given means ( SEM were calculated from three independent experiments. Statistical Analysis. Statistical tests were performed with STATGRAPHICS Plus5.1 software. Analysis of variance (Anova) was run on the logarithmic transformation of transfection levels (log10(fg luciferase/mg protein)) and on the cell viability to fit normal distributions of the data. One factor, i.e., the nature of the formulation, for a given amount of STC added (in the 0-60 mM range) was analyzed as source of the variation of cell viability percentages and of logarithmic transformation of the

Boulanger et al. Scheme 1. F-DOSPAa

Synthetic

Route

to

F-DMAEA

and

a Reagents: (i) HN , PPh , DIAC; (ii) DCC, HOSu, CH Cl / 3 3 2 2 THF; (iii) excess TFA/CH2Cl2. R represents F(CF2)4(CH2)11.

transfection levels using a one-way comparison procedure. The Tukey’s honestly significant difference (HSD) method was used to discriminate among the means of cell viability percentages and the logarithmic transformations of luciferase expression levels. RESULTS AND DISCUSSION

Our main objectives were to extend the library of fluorinated cationic lipids and lipoplex formulations that enable gene transfer and expression (13, 14) and to confirm the most promising transfection potential of fluorinated cationic lipoplexes in the presence of a powerful anionic surfactant (e.g. sodium taurocholate, STC) (16). Therefore, we (i) performed the synthesis of F-DMAEA and F-DOSPA, (ii) evaluated, as compared with control lipoplexes formulated with the commercially available DOSPA/DOPE Lipofectamine mixture, their ability to maintain, in the presence of STC, the integrity (in terms of DNA accessibility) of fluorinated lipoplexes they form, and (iii) explored their potential as gene transfer vectors and the impact of their hydrophobic and lipophobic character on detergent-triggered transfection inhibition. The fluorinated F-DMAEA and F-DOSPA lipids were designed according to previous studies conducted in our laboratory on fluorinated analogues of DOGS (14, 16). Their framework consists of a hydrophobic part which includes two F(CF2)4(CH2)11 chains. These chains were shown to lead to one of the most efficient gene transfer agent in the fluorinated DOGS series (14, 16). Synthesis. The synthesis of the fluorinated polycationic lipid F-DOSPA relies on F-DMAEA which himselfrelies on F-DMHEA precursor previously reported by our laboratory (Scheme 1) (13). The transformation of FDMHEA into F-DMAEA consisted of a Mitsunobu reaction which converts the alcohol function into an amine (20). This was achieved by the use of hydrazoic acid (HN3) in the presence of diisopropyl azodicarboxylate (DIAC) and triphenylphosphine. The conversion of F-DMAEA into F-DOSPA (isolated as its 2.6TFA salt as attested by 19 F NMR) was then performed using a conventional twostep coupling/deprotection procedure consisting into DCC/ HOSu activation of tetra-tert-butoxycarbonylspermine5-carboxylic acid (14) and condensation with F-DMAEA, followed by the quantitative removal of the Boc-protect-


ing groups with TFA. The chemical structure of the fluorinated F-DMAEA and F-DOSPA lipids was clearly attested by ESI/MS as well as by 1H, 13C, and 19F NMR. Lipoplex Formation and Characterization. The fluorinated lipoplexes were formulated at a N/P ratio of 5 with the fluorinated cationic lipid F-DOSPA or FDMAEA in conjunction with a helper lipid (HL ) DOPE or F-PE). These lipids were used at a molar ratio of 60/ 40 as for the control ones formulated with commercial Lipofectamine (i.e. DOSPA/DOPE 60/40). These studies were performed with pTG11236 plasmid also used for the detergent-triggered disintegration and the in vitro transfection assays (see below). The procedure applied for the lipoplex preparation relies on the dilution from liposomal CL/HL 60/40 dispersion in 5% glucose, using a N/P ratio of 5. All the lipoplex preparations consisted into a single population of stable DNA particles of mean diameters in the 120 (50) to 300 (150) nm range, as shown by light scattering measurements (data not shown). These formulations were also analyzed by gel electrophoresis (results not shown) which indicated the presence of fully complexed plasmid (not accessible to ethidium bromide intercalation). It should be mentioned that only N/P 5 lipoplexes were formulated and analyzed. We selected this N/P ratio as we found that in our transfection protocol and among the control N/P 0.8 to 10 Lipofectamine lipoplexes tested, the N/P 5 ones led to the highest level of luciferase expression (see Transfection section below). Lipoplex Stability in the Presence of the Detergent. To evaluate the ability of the fluorinated lipids, as compared to Lipofectamine, to prevent lipoplex disintegration in the presence of surfactants, we examined the effects of STC on BET intercalation into the complexed plasmid. We used the fluorescence monitoring of BET intercalation into DNA which has been reported for characterizing DNA complexes (21) and applied for the first generation of fluorinated lipoplexes (16). When DNA is not accessible to BET, as in the N/P 5 lipoplexes, a fluorescence signal of very weak intensity (F0) corresponding to the free dye in solution is measured upon mixing BET with the lipoplexes. When surfactant-triggered dissociation of the lipoplexes occurs and all the BET intercalation sites of DNA become accessible, BET fluorescence is enhanced. For naked DNA, an approximately 40-fold fluorescence enhancement is obtained upon intercalation into DNA, the maximal fluorescence intensity (Fmax) being measured for a BET:nucleotide ratio of 1:6. The intensity varies further linearly between F0 and Fmax for accessible DNA between zero and six nucleotides per BET. This allows the degree of DNA accessibility to be observed fluorometrically. Figure 2 shows the evolution of the percentage of DNA accessibility to BET from the CL/HL (CL ) F-DOSPA, or F-DMAEA; HL ) DOPE or F-PE) and control Lipofectamine N/P 5 lipoplexes with the detergent/lipid [STC]/ ([CL]+[HL]) molar ratio. The percentage of DNA accessibility was seen to increase for all formulations when the STC concentration and, consequently, the detergent/ lipid ratio were raised. However, lipoplexes formulated with the fluorinated or conventional pentaamino DOSPA were substantially more resistant to the lytic activity of STC than those formulated with the diamino F-DMAEA lipid. Indeed, for the F-DMAEA-based lipoplexes, a STC concentration of only 5 to 10 mM (which corresponds to a detergent/lipid molar ratio of 80-160) was sufficient to promote 80 to 100% of DNA accessibility (hence almost complete disintegration), whereas for the Lipofectamine and F-DOSPA-based lipoplexes and these STC concen-


Figure 2. Evolution of the percentage of DNA accessibility to ethidium bromide (BET) from various lipoplexes with the [STC]/ ([CL]+[HL])] molar ratio (STC ) sodium taurocholate; CL ) cationic lipid ) DOSPA, F-DOSPA, or F-DMAEA; HL ) helper lipid DOPE or F-PE). The lipoplexes prepared in 5% glucose solution were made of the cationic lipids and helper lipids (structure shown in Figure 1) and plasmid pTG11236 (DNA) for a N/P ratio of 5 and for a [CL]/[HL] molar ratio of 60/40. The BET intercalation into lipid-complexed DNA at a concentration of 15.0 µM for F-DOSPA or DOSPA (respectively 37.5 µM for F-DMAEA) and 15 µM DNA phosphate in the presence of various concentrations of STC (from 0 to 60 mM) was assessed by fluorescence monitoring at 2.5 µM BET and at a BET: nucleotide ratio of 1:6. The given means ( SEM were calculated from at least three independent experiments. For more details, see Materials and Methods.

trations (which correspond to detergent/lipid molar ratios of 200-400), only 40-45% of the DNA intercalation sites became accessible to BET. Furthermore, much higher STC concentrations (50 and 60 mM, respectively), which correspond to detergent/lipid molar ratios of 2000-2400, were necessary to disintegrate actively these latter lipoplexes. Interestingly, our data show further that the stability of the lipoplexes, with respect to the lytic activity of STC, is increasing along the sequence Lipofectamine < F-DOSPA/DOPE < F-DOSPA/F-PE, indicating that the higher the fluorination degree of the lipoplexes, the higher their stability. These results are in line with the pioneering study from this laboratory which highlighted a greater resistance to STC-triggered disintegration of lipoplexes formulated with fluorinated analogues of Transfectam (or DOGS) rather than with Transfectam itself (16). They confirm that the enhanced lipophobic and hydrophobic character of the fluorinated cationic or helper lipids, and consequently of the fluorinated lipoplexes they form, prevents these lipoplexes from disintegration by the detergent and, consequently, prevents DNA from degradation and from interactions with lipophilic and hydrophilic biocompounds. The main impact of the fluorinated lipids likely stems from the reduction of the detergent solubility into the film they form which surrounds DNA, owing to the very low miscibility between hydrocarbon (e.g. STC) and fluorinated lipids (22-24). Transfection. The transfection potential of F-DOSPA and F-DMAEA with DOPE or F-PEhelper lipid was assayed in vitro on A549 cells, a commonly used cell model for in vitro gene delivery. These assays were performed by incubating the N/P 5 lipoplexes (at a DNA dose of 0.5 µg/well) with the cells in the presence of 10% fetal calf serum for 24 h and in the presence of increasing concentrations of STC (from 0 to 17.5 mM) and, consequently, of various detergent/lipid molar ratios (from 0


Boulanger et al.

Figure 3. Means and 95.0% Tukey HSD intervals of the logarithmic transformation of transfection levels (log10(fg luciferase/mg protein); panels A-C) and of the percentage of cell viability (panels D-F) for the fluorinated F-DMAEA- or F-DOSPA-based N/P 5 lipoplexes coformulated with DOPE or F-PE, and for control N/P 5 formulations made with lipofectamine and incubated without STC (panels A and D), with 5 mM STC (panels B and E) or with 7.5 mM STC (panels C and F). Luciferase expression and percentage of cell viability were determined in A549 cells for a DNA dose of 0.5 µg per well. For the F-DMAEA-based lipoplexes, a STC concentration of 5 and 7.5 mM corresponds to a [STC]/[lipid] molar ratio of 80 and 120, respectively. For the (F-)DOSPA-based lipoplexes, a STC concentration of 5 and 7.5 mM corresponds to a [STC]/[lipid] molar ratio of 200 and 300, respectively. For more details, see Materials and Methods.

to 693 for (F-)DOSPA and 0 to 277 for F-DMAEA). Their transfection efficiency (expressed in femtograms (fg) of luciferase/mg of protein) was further evaluated as compared to naked DNA and to reference N/P 5 lipoplexes based on Lipofectamine. These conditions (N/P 5 and DNA dose of 0.5 µg/well) were shown to give the highest luciferase expression level and percentage of cell viability for the transfection of A549 cells with the control lipoplexes formulated with Lipofectamine among the N/P 0.8 to 10 ratio and DNA dose of 0.5 and 1 µg/well tested (data not shown). Cells treated with naked DNA under equivalent conditions showed expression levels of about 102-3 fg of luciferase/mg of protein. The cell viability of the lipoplexes without and with STC at the different concentrations tested was also checked by determining the total protein amount per well of the transfected cells relative to that measured for untreated cells (for which the total protein amount per well is in a 30-60 µg/well range). Figure 3 represents a variance analysis of (i) the logarithmic transformation of the transfection levels (panels A-C) and (ii) the percentage cell viability (panels D-F) obtained with the various N/P 5 lipoplexes without or with increasing amounts of STC. Although analysis was performed throughout the 0-17.5 mM STC concentration range, only data for a 0-7.5 mM STC concentration range are presented. For a STC concentration higher than 7.5 mM, cell toxicity of the lipoplexes and of STC was indeed too important to allow an unambiguous interpretation of the transfection results. However, it should be noted that in the presence of a STC concentration of 5 and 7.5 mM, cell viability of the lipoplexes was improved as compared with that of the lipoplexes alone (see Figure 3, panel E and F vs panel D). This indicates likely that, in this STC concentration range, STC neutralizes the deleterious effects on cells of the cationic lipids present in excess in the N/P 5 lipoplexes. Concerning the transfection experiments performed in the absence of surfactant (Figure 3, panel A), our data show that the N/P 5 lipoplexes formulated with the

fluorinated lipospermine F-DOSPA (whether used with DOPE or the fluorinated F-PE helper lipid) and the control Lipofectamine-based lipoplexes display comparable transfection efficiencies. This indicates that there is no benefit from using F-DOSPA/DOPE (or F-PE) instead of DOSPA/DOPE for transfecting A549 cells and is in line with the data obtained in our laboratory for fluorinated analogues of DOGS/DOPE (14). It can be further observed that the lipoplexes coformulated with the dicationic fluorinated F-DMAEA lipid and DOPE are less efficient for transfecting A549 cells than those formulated with the pentacationic lipospermine (F-)DOSPA and DOPE. Moreover, coformulation of the F-DMAEA lipoplexes with the fluorinated F-PE analogue of DOPE resulted in a substantial enhancement of transfection efficiency, confirming the remarkable transfection helper properties of F-PE (15, 17). This transfection increase is however accompanied by a substantial decrease of cell viability (Figure 3, panel D). It should further be noticed that the N/P 5 F-DMAEA-based lipoplexes exhibit a lower cell viability than their respective lipospermine-based ones. This is likely related to the fact that, for a given N/P ratio, the concentration of the lipids in the transfection wells is 2.5-fold higher for a dicationic (e.g. F-DMAEA) than for a pentacationic lipid (e.g. (F-)DOSPA). More importantly, concerning the transfection experiments performed in the presence of STC (Figure 3, panels B and C), and based on the stability study described in the precedent section, lipofection was expected to be inhibited by the detergent for all the lipoplexes but to a lesser extent (i) for any of the (F-)DOSPA lipoplexes as compared with the F-DMAEA-based ones, and (ii) for the fluorinated F-DOSPA lipoplexes as compared with the Lipofectamine controls and particularly with increasing the fluorination degree of the formulation. These were the observed trends as can be seen in Figure 3 (panels B and C vs panel A). Indeed, lipofection when mediated by the N/P 5 F-DMAEA-based lipoplexes was fully inhibited for a STC concentration of 5 mM which corresponds to a


[STC]/[lipid] molar ratio of 80. By contrast, only a 6-, 15-, and 50-fold inhibition of transfection (p < 0.05) was observed for the N/P 5 DOSPA/DOPE, F-DOSPA/F-PE, and F-DOSPA/DOPE formulations, respectively, although for these latter formulations a STC concentration of 5 mM corresponds to a higher [STC]/[lipid] molar ratio (i.e. 200). For a STC concentration of 7.5 mM (panel C), lipofection mediated with complexes formulated with both the fluorinated F-DOSPA and helper lipid F-PE was significantly higher than with Lipofectamine (nearly 50fold, p < 0.05) or even with F-DOSPA/DOPE (nearly 30fold, p < 0.05). It is thus the formulation displaying the highest fluorination degree of the formulations investigated here which possesses the largest lipofection potential in the presence of STC. That F-DOSPA/F-PE lipoplexes are more efficient for transfecting A549 cells than the F-DOSPA/DOPE ones illustrates again the remarkable helper properties of F-PE, a fluorinated analogue of DOPE. The present study which demonstrates the improved lipofection capability of fluorinated lipoplexes in the presence of a powerful surfactant is in line with a previous study from this laboratory which was performed with N/P 5 lipoplexes made from fluorinated DOGS analogues (16). However, for these fluorinated DOGS formulations higher detergent concentrations (up to 17.5 mM vs 10 mM for F-DOSPA/F-PE) and higher [STC]/ [lipid] molar ratios (up to 462 vs 400 for F-DOSPA/FPE, data not shown) were necessary to inhibit lipofection of A549 cells. The lower cell transfection ability of F-DOSPA/F-PE (or DOPE) lipoplexes in the presence of STC as compared with that of the fluorinated DOGS analogues is likely related to the presence of the helper lipid (DOPE or F-PE) for the former formulations. STC displays a greater miscibility with DOPE and even with the mixed-chain F-PE (which contains a fluorinated and hydrocarbon chain) than with the fluorinated DOGS which contain two fluorocarbon chains (22-24). One expects therefore a lower stability (with respect to disintegration) in the presence of STC for the F-DOSPA/ F-PE (or DOPE) lipoplexes than for the lipoplexes formulated only with fluorinated double-chain DOGS analogues. Within the [STC]/[lipid] molar ratio range of 0-300, only subtle differences in disintegration of the (F-)DOPSA-based lipoplexes were observed by the dyeexclusion assay (Figure 2), while more drastic differences were observed in transfection. This is due to the different experimental conditions employed for each assay type, as discussed elsewhere (16). CONCLUSION

The extent of gene therapy using synthetic vectors depends on the development of new approaches to improve the features of lipoplexes aimed at facilitating their use in vivo. This study, which shows improved stability of fluorinated lipoplexes and higher levels of lipofection with fluorinated lipoplexes in the presence of surfactants, definitely confirms that fluorinated lipoplexes display a most promising potential as gene transfer vectors, and that they constitute a very attractive alternative to their more conventional homologues. The data reported here, together with the higher in vivo lung transfection efficiency found for fluorinated lipoplexes (15), suggest that their enhanced lipophobic and hydrophobic character prevents them from disintegration and, consequently, DNA from degradation and from interac-


tions with lipophilic and hydrophilic biocompounds responsible for lipofection inhibition. ACKNOWLEDGMENT

We wish to thank TRANSGENE, SA (Strasbourg, France) for the generous gift of pcTG11236. LITERATURE CITED (1) Tranchant I., Thompson, B., Nicolazzi, N., Mignet, N., and Scherman, D. (2004) Physicochemical optimisation of plasmid delivery with cationic lipids. J. Gene Med. 6, S24-S35. (2) Liu, D., Ren, T., and Gao, X. (2003) Cationic transfection lipids. Curr. Med. Chem. 10, 1307-1315. (3) Miller, A. D. (2003) The problem with cationic liposome/ micelle-based nonviral vector systems for gene therapy. Curr. Med. Chem. 10, 1195-1211. (4) Niculescu-Duvaz, D., Heyes, J., and Springer, C. J. (2003) Structure-activity relationship in cationic lipid mediated gene transfection. Curr. Med. Chem. 10, 1233-1261. (5) Ilies, M. A., Seitz, W. A., and Balaban, A. T. (2002) Cationic lipids in gene delivery: principles, vector design and therapeutical applications. Curr. Pharm. Des 8, 2441-2473. (6) Kichler, A. (2004) Gene transfer with modified polyethylenimines. J. Gene Med. 6, S3-S10. (7) Merdan, T., Kopecek, J., and Kissel, T. (2002) Prospects for cationic polymers in gene and oligonucleotide therapy against cancer. Adv. Drug Delivery Rev. 54, 715-758. (8) Merlin, J. L., N’Doye, A., Bouriez, T., and Dolivet, G. (2002) Polyethylenimine Derivatives as Potent Nonviral Vectors for Gene Transfer. Drug News Perspect. 15, 445-451. (9) Raczka, E., Kukowska-Latallo, J. F., Rymaszewski, M., Chen, C., and Baker, J. R., Jr. (1998) The effect of synthetic surfactant Exosurf on gene transfer in mouse lung in vivo. Gene Ther. 5, 1333-1339. (10) Duncan, J. E., Whitsett, J. A., and Horowitz, A. D. (1997) Pulmonary surfactant inhibits cationic liposome-mediated gene delivery to respiratory epithelial cells in vitro. Hum Gene Ther. 8, 431-438. (11) Tsan, M. F., Tsan, G. L., and White, J. E. (1997) Surfactant inhibits cationic liposome-mediated gene transfer. Hum Gene Ther. 8, 817-825. (12) Ernst, N., Ulrichskotter, S., Schmalix, W. A., Radler, J., Galneder, R., Mayer, E., Gersting, S., Plank, C., Reinhardt, D., and Rosenecker, J. (1999) Interaction of liposomal and polycationic transfection complexes with pulmonary surfactant. J. Gene Med. 1, 331-340. (13) Gaucheron, J., Santaella, C., and Vierling, P. (2002) Transfection with fluorinated lipoplexes based on fluorinated analogues of DOTMA, DMRIE and DPPES. Biochim. Biophys. Acta 1564, 349-358. (14) Gaucheron, J., Santaella, C., and Vierling, P. (2001) Highly fluorinated lipospermines for gene transfer: synthesis and evaluation of their in vitro transfection efficiency. Bioconjugate Chem. 12, 114-128. (15) Boussif, O., Gaucheron, J., Boulanger, C., Santaella, C., Kolbe, H. V. J., and Vierling, P. (2001) Enhanced in vitro and in vivo cationic lipid-mediated gene delivery with a fluorinated glycerophospho-ethanolamine helper lipid. J. Gene Med. 3, 109-114. (16) Gaucheron, J., Santaella, C., and Vierling, P. (2001) Improved in vitro gene transfer mediated by fluorinated lipoplexes in the presence of a bile salt surfactant. J. Gene Med. 3, 338-344. (17) Gaucheron, J., Boulanger, C., Santaella, C., Sbirrazzuoli, N., Boussif, O., and Vierling, P. (2001) In vitro cationic lipidmediated gene delivery with fluorinated glycerophosphoethanolamine helper lipids. Bioconjugate Chem. 12, 949-963. (18) Wheeler, C. J., Felgner, P. L., Tsai, Y. J., Marshall, J., Sukhu, L., Doh, S. G., Hartikka, J., Nietupski, J., Manthorpe, M., Nichols, M., Plewe, M., Liang, X., Norman, J., Smith, A., and Cheng, S. H. (1996) A novel cationic lipid greatly enhances plasmid DNA delivery and expression in mouse lung. Proc. Natl. Acad. Sci. U.S.A. 93, 11454-11459.

908 Bioconjugate Chem., Vol. 15, No. 4, 2004 (19) Hawley-Nelson, P., Ciccarone, V., Gebeyehu, G., Jessee, J., and Felgner, P. L. (1993) Lipofectamine reagent: a new, higher efficiency polycationic liposome transfection reagent. Focus 73-79. (20) Fabiano, E., Golding, B. T., and Sadeghi, M. M. (1987) A simple conversion of alcohols into amines. Synthesis 190-192. (21) Eastman, S. J.; Siegel, C.; Tousignant, J.; Smith, A. E.; Cheng, S. H.; and Scheule, R. K. (1997) Biophysical characterization of cationic lipid: DNA complexes. Biochim. Biophys. Acta 1325, 41-62. (22) Gadras, C., Santaella, C., and Vierling, P. (1999) Improved stability of highly fluorinated phospholipid-based vesicles in the presence of bile salts. J. Controlled Release 57, 29-34. (23) Vierling, P., Santaella, C., Riess J. G. (1995) Fluorinated Liposomes. Liposomes: New Systems and New Trends in their Applications (Puisieux, F., Couvreur, P., Delattre J., and Devissaguet J. P., Eds.) Editions de Sante´, Paris, pp 295-313. (24) Vierling, P., Santaella, C., and Greiner, J. (2001) Highly fluorinated amphiphiles as drug and gene carrier and delivery systems. J. Fluorine Chem. 107, 337-354.

Boulanger et al. (25) Remy, J. S., Abdallah, B., Zanta, M. A., Boussif, O., Behr, J. P., and Demeneix, B. A. (1998) Gene transfer with lipospermines and polyethylenimines. Adv. Drug Delivery Rev. 30, 85-95. (26) 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, lipidmediated DNA-transfection procedure. Proc. Natl. Acad. Sci. U.S.A. 84 7413-7417. (27) Felgner, J. H., Kumar, R., Sridhar, C. N., Wheeler, C. J., Tsai, Y. J., Border, R., Ramsey, P., Martin, M., and Felgner, P. L. (1994) Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J. Biol. Chem. 269, 2550-2561. (28) Behr, J.-P., Demeneix, B., Loeffler, J. P., and Perez-Mutul, J. (1989) Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine-coated DNA. Proc. Natl. Acad. Sci. U.S.A. 86, 6982-6986.

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Transfection with Fluorinated Lipoplexes Based on New Fluorinated

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