In Vitro Cationic Lipid-Mediated Gene Delivery with Fluorinated

Bioconjugate Chem. 2001, 12, 949−963

949

In Vitro Cationic Lipid-Mediated Gene Delivery with Fluorinated Glycerophosphoethanolamine Helper Lipids Je´roˆme Gaucheron,† Caroline Boulanger,† Catherine Santaella,†,| Nicolas Sbirrazzuoli,‡ Otmane Boussif,§,⊥ and Pierre Vierling*,† Laboratoire de Chimie Bioorganique, UMR 6001 CNRS, Laboratoire de Thermodynamique Expe´rimentale, Universite´ de Nice-Sophia Antipolis, 06108 Nice Ce´dex 2, France, and Transge`ne, SA, 11, rue de Molsheim 67082 Strasbourg Ce´dex, France. Received March 19, 2001; Revised Manuscript Received July 3, 2001

There is a need for the development of nonviral gene transfer systems with improved and original properties. “Fluorinated” lipoplexes are such candidates, as supported by the remarkably higher in vitro and in vivo transfection potency found for such fluorinated lipoplexes as compared with conventional ones or even with PEI-based polyplexes (Boussif, O., Gaucheron, J., Boulanger, C., Santaella, C., Kolbe, H. V. J., Vierling, P. (2001) Enhanced in vitro and in vivo cationic lipid-mediated gene delivery with a fluorinated glycerophosphoethanolamine helper lipid. J. Gene Med. 3, 109-114). Here, we describe the synthesis of fluorinated glycerophosphoethanolamines (F-PEs), close analogues of dioleoylphosphatidylethanolamine (DOPE), and report on their lipid helper properties vs that of DOPE, as in vitro gene transfer components of fluorinated lipoplexes based on pcTG90, DOGS (Transfectam), or DOTAP. To evaluate the contribution of the F-PEs to in vitro lipoplex-mediated gene transfer, we examined the effect of including the F-PEs in lipoplexes formulated with these cationic lipids (CL) for various CL:DOPE:F-PE molar ratios [1:(1 - x):x with x ) 0, 0.5 and 1; 1:(2 y):y with y ) 0, 1, 1.5, and 2], and various N/P ratios (from 10 to 0.8, N ) number of CL amines, P ) number of DNA phosphates). Irrespective of the F-PE chemical structure, of the colipid F-PE:DOPE composition, and of the N/P ratio, comparable transfection levels to those of their respective control DOPE lipoplexes were most frequently obtained when using one of the F-PEs as colipid of DOGS, pcTG90, or DOTAP in place of part of or of all DOPE. However, a large proportion of DOGS-based lipoplexes were found to display a higher transfection efficiency when formulated with the F-PEs rather than with DOPE alone while the opposite tendency was evidenced for the DOTAP-based lipoplexes. The present work indicates that “fluorinated” lipoplexes formulated with fluorinated helper lipids and conventional cationic lipids are very attractive candidates for gene delivery. It confirms further that lipophobicity and restricted miscibility of the lipoplex lipids with the endogenous lipids does not preclude efficient gene transfer and expression. Their transfection potency is rather attributable to their unique lipophobic and hydrophobic character (resulting from the formulation of DNA with fluorinated lipids), thus preventing to some extent DNA from interactions with lipophilic and hydrophilic biocompounds, and from degradation.

INTRODUCTION

Gene (or DNA) transfer systems into cells have become powerful tools for gene and cellular therapy that are very promising novel forms of molecular medicine. Successful gene therapy depends on the efficient delivery of genetic material to cells and its effective expression within these cells. Although at present, the in vivo expression levels of synthetic nonviral gene transfer vectors based on (poly)cationic lipids, liposomes, and polymers (e.g., lipoplexes and polyplexes, respectively (1)) are lower than for viral vectors and gene expression is transient, these * To whom correspondence should be addressed. E-mail: [email protected]. Phone: 33 (0)4 92 07 61 43. Fax: 33 (0)4 92 07 61 51. † Laboratoire de Chimie Bioorganique. ‡ Laboratoire de Thermodynamique Expe ´ rimentale. § Transge ` ne. | Present address: CEA Cadarache, DSV:DEVM, Laboratoire d′Ecologie Microbienne de la Rhizosphe`re, UMR CNRS-CEA 163, 13108 Saint Paul lez Durance, France. ⊥ Present address: Gencell, a division of Aventis Pharma, Process Development, Non Viral Formulation, 13, Quai Jules Guesde, 94400 Vitry/Seine, France.

systems are likely to present several advantages including low-cost and large-scale production, safety, lower immunogenicity, and capacity to deliver large gene fragments. Such systems have therefore gained wide acceptance over the past decade for in vitro and/or in vivo gene delivery (2-5). However, there is still a need for the development of gene transfer systems with improved and original properties. Our approach with the development of “fluorinated” lipoplexes based either on highly fluorinated lipospermines (6, 7) or on a fluorinated glycerophosphoethanolamine helper lipid (e.g., [F8E11][C16]OPE in Scheme 1 which is an analogue of dioleoylphosphatidylethanolamine, DOPE) (8) proved to some extent successful as these systems showed a higher in vitro and in vivo transfection potential than conventional lipoplexes (68) or PEI polyplexes (8). More particularly, the use of [F8E11][C16]OPE (which on its own is inactive in promoting transfection) as colipid of the cationic lipopolyamine pcTG90 (see structure in Scheme 1) increased the in vitro and in vivo gene transfer capability of pcTG90 to a larger extent than DOPE (8). Although the exact mechanism which governs lipoplex-mediated gene delivery and ex-

10.1021/bc010033j CCC: $20.00 © 2001 American Chemical Society Published on Web 10/27/2001

950 Bioconjugate Chem., Vol. 12, No. 6, 2001 Scheme 1

Gaucheron et al.

potential) relationships. We report here on their synthesis and on their helper lipid properties as in vitro gene transfer components of pcTG90, DOGS (Transfectam), and DOTAP-based lipoplexes (see structures in Scheme 1), as compared with DOPE. These (poly)cationic lipids were selected for their well-documented high transfection efficiency which is significantly improved with DOPE (for pcTG90, see (8, 14, 15), for DOGS, see (16) and for DOTAP, see (17, 18)). They were also chosen for their specific structural and geometrical features (linear or branched polyamino or single quaternary ammonium polar head) which might have quite different effects on the phases these cationic lipids associated to the F-PEs are susceptible to form. The thermotropic phase behavior of the F-PEs in water was also investigated by differential scanning calorimetry (DSC) in order to highlight its role on lipoplex formulation and lipofection (10, 12). In our initial study (8), the optimized conditions for the in vitro and in vivo transfection tests, which led us to show a significantly higher transfection helper potential of [F8E11][C16]OPE as compared with that of DOPE, were corresponding to pcTG90-based lipoplexes formulated in HEPES and in 5% glucose, respectively. However, in vitro, when the lipoplexes were formulated in 5% glucose, both colipids displayed a comparable transfection helper potential. In anticipation of in vivo testing, lipofection with the F-PEs as colipids was investigated here only for lipoplexes formulated in 5% glucose. EXPERIMENTAL PROCEDURES

pression is unclear, several reasons may account for the gene expression improvement resulting from the use of [F8E11][C16]OPE in place of DOPE. This improvement could be due to the larger ability of the fluorinated colipid to preserve the integrity of complexed DNA in a biological environment. Indeed, owing to its increased hydrophobic and lipophobic character, this colipid is expected to prevent the fluorinated lipoplexes it forms with cationic lipids from interactions with lipophilic and hydrophilic biocompounds, and from degradation to a larger extent than conventional helper lipids, as shown for fluorinated lipoplexes formed from fluorinated lipospermines as compared with conventional ones (7). This improvement could also be related to the larger propensity of the fluorinated colipid to promote fusion with and destabilization of the endosome membrane allowing more efficient DNA release in the cytosol, thus preventing internalized DNA from endosome and lysosome degradation to a larger extent than when DOPE is used as helper lipid (9-12). This fluorinated PE displays also a more pronounced cone-shape geometry than DOPE as a consequence of the larger size of its fluorinated chain when compared with hydrocarbon ones (13). One therefore expects a greater tendency for [F8E11][C16]OPE to promote a lamellar to inverted hexagonal HII phase transition (13) and, thus, a greater effectiveness in disrupting membranes than DOPE (the role in membrane fusion of DOPE was indeed attributed to its capability to initiate such a phase transition (11, 12)). The goals of the present study was aimed at extending the library of fluorinated glycerophosphoethanolamines (F-PEs), and at examining (i) their lipid helper effect on lipofection and (ii) the cationic lipid specificity of their transfection enhancement potential, as compared with that of DOPE. The molecular structure of this second generation of F-PEs (listed in Scheme 1) follows a modular design (one or two fluorinated chains, saturated or unsaturated chains, ester or ether bond) aimed at the establishment of structure/properties (transfection helper

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. The 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. Advancing 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 (Sigma), ninhydrin reagent (Sigma). The perfluoroalkyl iodides were purified over neutral alumina before use. DF4C11OPE, DOGS (or Transfectam), and DOTAP were prepared as described in refs 19, 6, and 17, respectively. The lipopolyamine pcTG90 was from Transge`ne (Strasbourg, France) (20). DOPE was purchased from Sigma. All other organic chemicals were from Aldrich, Fluka, or Novabiochem. 1H, 13C, 31P{1H}, and 19F NMR spectra were recorded on a Bruker AC-200 at 200, 50.3, 81, and 188.3 MHz, respectively. Chemical shifts were measured relative to CHCl3 (δ 7.27 ppm) or CH3OD (δ 3.35 ppm) for 1H, to CDCl3 (δ 76.9 ppm) for 13C and expressed indirectly in relation to TMS, to external ref 75% H3PO4 for 31P, and 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 ppm and listed as follow: shift in ppm (multiplicity, coupling, integration and attribution). Elemental analyses were carried out by the Service Central de Microanalyses du CNRS. Synthesis of the Glycerol Derivatives 2a-c. rac2,3-Di-[11-(F-butyl)undecanoyl)]glycerol, 2a. The synthesis of 2a from rac-1-O-benzylglycerol was performed according to published procedures (21).

In Vitro Cationic Lipid-Mediated Gene Delivery

rac-3-[11-(F-Octyl)undec-10-enyloxy]-2-(hexadecyloxy)propan-1-ol, 2b. The syntheses of derivatives 4 to 6 from 1,3-O-benzylideneglycerol, 3, were performed according to published procedures (19). rac-3-(Benzyloxy)-2-(hexadecyloxy)propan-1-ol, 4. The alkylation of 3 with 1-bromohexadecane under phase-transfer catalysis conditions gave 5-hexadecyloxy2-phenyl-1,3-dioxane [white powder, 73%; TLC (hexane/ ethyl acetate 8:2, UV and H2SO4): Rf ) 0.72. 1H NMR (CDCl3): δ 0.84 (t, 3J ) 6.1 Hz, 3H, CH3); 1.20-1.50 (broad s, 26H, CH3(CH2)13); 1.51-1.80 (m, 2H, CH2CH2O); 3.10-3.20 (m, part X of a ABX system, 1H, CH2CHCH2); 3.68 (t, 3J ) 6.6 Hz, 2H, CH2CH2OCH); 4.05, 4.38 (part AB of a ABX system, 2JAB ) 12.4 Hz, 3JAX ) 1.6 Hz, 3JBX ) 1.3 Hz, 4H, CH2CHO); 5.60 (s, 1H, CHPh); 7.31-7.49, 7.50-7.65 (m, m, 3H, 2H, Ph). 13C NMR (CDCl3): δ 14.3 (CH3); 22.8 (CH3CH2); 26.3 (CH2CH2CH2O); 29.5, 29.6, 29.7, 29.8, 29.9 (CH3(CH2)2(CH2)9, CH2CH2O); 32.1 (CH3CH2CH2); 69.2, 69.3 (CH2O); 70.7 (CH2OCH); 101.4 (CHPh); 126.2, 128.2, 128.8, 138.3 (Ph)]. The reductive cleavage of the 1,3-O-benzylidene group in 5-hexadecyloxy-2-phenyl-1,3-dioxane with BH3‚THF gave 4 (colorless oil, 85%). TLC (CHCl3; UV and H2SO4): Rf ) 0.25. 1H NMR (CDCl3): δ 0.90 (t, 3J ) 6.1 Hz, 3H, CH3); 1.101.40 (broad s, 26H, CH3(CH2)13); 1.50-1.70 (m, 2H, CH2CH2O); 2.22 (broad s, 1H, OH); 3.35-3.78 (m, 7H, CH2O, CHO); 4.56 (s, 2H, CH2Ph); 7.20-7.35 (m, 5H, Ph). 13C NMR (CDCl ): δ 14.3 (CH ); 22.8 (CH CH ); 26.3 3 3 3 2 (CH2CH2CH2O); 29.5, 29.6, 29.7, 29.8, 29.9 (CH3(CH2)2(CH2)9, CH2CH2O); 32.1 (CH3CH2CH2); 63.0 (CH2OH); 70.2, 70.6 (CH2O); 73.7 (CH2Ph); 78.7 (OCH); 127.6, 127.7, 128.4, 138.1 (Ph). rac-1-[3-(Undec-10-enyloxy)-2-(hexadecyloxy)propyloxy]benzene, 5. The alkylation of 4 via its anion with 11-bromoundecene gave 5 (colorless oil, 84%). TLC (CHCl3; UV and H2SO4): Rf ) 0.47. 1H NMR (CDCl3): δ 0.90 (t, 3J ) 6.1 Hz, 3H, CH3); 1.10-1.40 (broad s, 38H, CH3(CH2)13, (CH2)6(CH2)2O); 1.41-1.70 (m, 4H, CH2CH2O); 2.00-2.20 (m, 2H, CH2dCHCH2); 3.35-3.78 (m, 9H, CH2O, CHO); 4.56 (s, 2H, CH2Ph); 4.80-5.10 (m, 2H, CH2dCH); 5.65-5.95 (m, 1H, CH2dCH); 7.20-7.35 (m, 5H, Ph). rac-1-[3-(11-(F-Octyl)undec-10-enyloxy]-2-(hexadecyloxy)propyloxy]benzene, 6. The addition of CF3(CF2)7I on the terminal CH2dCH bond of 5 gave 6 (colorless oil, 73%). TLC (CH2Cl2; UV and H2SO4): Rf ) 0.40. 1H NMR (CDCl3): δ 0.92 (t, 3J ) 6.1 Hz, 3H, CH3); 1.10-1.40 (broad s, 38H, CH3(CH2)13, (CH2)6(CH2)2O); 1.41-1.70 (m, 4H, CH2CH2O); 2.10-2.30 (m, 2H, CHd CHCH2); 3.35-3.78 (m, 9H, CH2O, CHO); 4.56 (s, 2H, CH2Ph); 5.30-5.80 (m, 1H, CF2CHdCH); 6.30-6.55 (m, 1H, CF2CHdCH); 7.20-7.35 (m, 5H, Ph). 13C NMR (CDCl3): δ 14.2 (CH3); 22.8 (CH3CH2); 26.2, 26.3 (CH2CH2CH2O); 28.1, 29.1, 29.2, 29.4, 29.5, 29.6, 29.7, 29.8, 29.9, 30.3 (CH2CH2O, CH3(CH2)2(CH2)10, CHdCHCH2(CH2)5); 32.1 (CH3CH2CH2, dCHCH2); 70.5, 70.8, 70.9 (CH2O); 73.5 (CH2Ph); 78.6 (CHO); 116.9 (t, 2JCF ) 23 Hz, CF2CHdCH); 126.2, 128.2, 128.8, 138.3 (Ph); 143.4 (t, 3JCF ) 9 Hz, CF2CHdCH trans); 144.0 (t, 3JCF ) 5 Hz, CF2CHdCH, cis). 19F NMR (CDCl3): δ -81.3 (3F, CF3); -107.2 (0.2F, CF2CH cis); -112.7 (1.8F, CF2CH trans); -122.0, -122.5, -123.2, -124.0 (2F, 4F, 2F, 2F, CF3CF2(CF2)5); -126.6 (2F, CF3CF2). rac-3-[11-(F-Octyl)undec-10-enyloxy]-2-(hexadecyloxy)propan-1-ol, 2b. A suspension of FeCl3 and 4.4 g of 6 in 15 mL CH2Cl2 was stirred for 90 min at room temperature. The organic phase was then washed, dried over Na2SO4 and evaporated, and the residue was purified by silica gel chromatography (CH2Cl2:MeOH 100:0

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to 99:1) to afford 2.9 g of 2b (white powder, 73%). TLC (CHCl3 ; H2SO4): Rf ) 0.20. 1H NMR (CDCl3): δ 0.85 (t, 3H, 3J ) 6.5 Hz, CH3); 1.05-1.45 (broad s, 38H, (CH2)6CH2CH2O, CH3(CH2)13); 1.45-1.70 (m, 4H, CH2CH2O); 2.07-2.28 (m, 2H, CHdCHCH2); 2.48 (broad s, 2H, CH2OH); 3.30-3.80 (m, 9H, CH2O, CHO); 5.40-5.74 (m, 1H, CF2CHdCH); 6.35-6.50 (m, 1H, CF2CHdCH). 13C NMR (CDCl3): δ 13.8 (CH3) 22.6 (CH3CH2); 26.0 (CH2CH2CH2O); 27.9, 28.9, 29.2, 29.3, 29.6, 30.0 (CH2CH2O, CH3(CH2)2(CH2)9, dCHCH2(CH2)5); 31.8 (CH3CH2CH2, dCHCH2); 62.8 (CH2OH); 70.3, 70.7, 71.6 (CH2O); 78.3 (CHO); 116.7 (t, 2JCF ) 23 Hz, CF2CHdCH); 143.0 (t, 3JCF ) 9 Hz, CF2CHdCH trans); 145.0 (CF2CHdCH cis). 19F NMR (CDCl3) identical to that of 6. rac-3-[11-(F-Octyl)undecyloxy]-2-(hexadecyloxy)propan-1-ol, 2c. The simultaneous benzyl deprotection and CdC hydrogenation of 4 (1.8 g;1.8 mmol) in 15 mL of EtOH and 0.2 mL of AcOH was performed at room temperature under 40 atm H2 pressure with Pd/C (10%) as catalyst. Usual workup and silica gel chromatography (CH2Cl2) afforded 1.0 g (60%) of 2c as a white powder (60%). TLC (CH2Cl2; H2SO4): Rf ) 0.55. 1H NMR (CDCl3): δ 0.88 (t, 3H, 3J ) 6.5 Hz, CH3); 1.15-1.48 (broad s, 40H, (CH2)7CH2CH2O, CH3(CH2)13); 1.48-1.72 (m, 4H, CH2CH2O, CF2CH2CH2); 2.06 (t, 3J ) 7.5 Hz, 3JHF ) 19.0 Hz, 2H, CF2CH2); 2.22-2.34 (broad s, 1H, CH2OH); 3.383.83 (m, 9H, CH2O, CHO). 13C NMR (CDCl3): δ 14.0 (CH3); 20.6 (t, 3JCF ) 4 Hz, CF2CH2CH2); 22.6 (CH3CH2); 26.1 (CH2CH2CH2O); 29.2, 29.3, 29.6, 30.0 (CH2CH2O, CH3(CH2)2(CH2)10, CF2(CH2)2(CH2)6); 30.9 (t, 2JCF ) 22 Hz, CF2CH2); 31.8 (CH3CH2CH2); 63.1 (CH2OH); 70.4, 70.9, 71.8 (CH2O); 78.3 (CHO). 19F NMR (CDCl3): δ -79.6 (3F, CF3); -113.3 (2F, CF2CH2); -120.7 (6F, (CF2)3CF2CH2); -121.6 (2F, CF3(CF2)2CF2); -122.4 (2F, CF3CF2CF2); -125.0 (2F, CF3CF2). Synthesis of the F-PE Derivatives. The phosphorylation of the glycerol derivatives 2a-c and the deprotection step of the BOC-protected intermediates 7a-c with TFA were performed according to the procedures described for the synthesis of DF4C11OPE (19). The F-PE derivatives were isolated as TFA salts. rac-2,3-Di[11-(F-butyl)undecanoyl)glycero-1-phosphoethanolamine, DF4C11PE. rac-1,2-Di[10-(Fbutyl)undecanoyl]glycero-3-phospho(tert-butoxycarbonyl)ethanolamine, 7a (30% yield): 1H NMR (CDCl3:CD3OD): δ 1.02-1.37 (m, 37H, (CH2)7(CH2)2C(O), CH3 (BOC); 1.37-1.57 (m, 4H, CH2CH2C(O)); 1.92 (tt, 3J ) 7.5 Hz, 3JHF ) 19.0 Hz, 4H, CF2CH2); 2.09-2.26 (m, 4H, CH2C(O)); 3.07-3.26 (m, 2H, CH2N); 3.66-3.90 (m, 4H, CH2OPOCH2); 3.95-4.32 (m, 2H, C(O)OCH2); 5.035.16 (m, 1H, CH). 13C NMR (CDCl3/CD3OD): δ 20.3 (t, 3J CF ) 4 Hz, CF2CH2CH2); 25.0, 25.1 (CH2CH2C(O)); 28.4 (C(CH3)3); 29.5, 29.6 ((CH2)6(CH2)2C(O)); 30.9 (t, 2JCF ) 22 Hz, CF2CH2); 34.2, 34.4 (CH2C(O)); 41.1 (d, 3JCP ) 6 Hz, CH2N); 62.9 (OCH2); 64.1 (d, 2JCP ) 5 Hz, POCH2CH2N); 65.1 (d, 2JCP ) 5 Hz, CHCH2OP); 71.1 (d, 3JCP ) 8 Hz, CH); 80.1 (C(CH3)3); 157.1 (NC(O)); 173.6, 174.6 (CH2C(O)). 19F NMR (CDCl3/CD3OD): δ -80.0 (3F, CF3); -113.5 (2F, CF2CH2); -123.3, -124.9 (2F, 2F, CF3(CF2)2). 31 P{1H} NMR (CDCl3/CD3OD): δ -8.1 (s). DF4C11PE (15% yield from 7a): TLC (CH2Cl2/MeOH 7:3; ninhydrine): Rf ) 0.70. 1H NMR (CDCl3/CD3OD): δ 0.80-1.18 (m, 28H, (CH2)7(CH2)2C(O)); 1.18-1.50 (m, 4H, CH2CH2C(O)); 1.75 (tt, 3J ) 7.5 Hz, 3JHF ) 19.0 Hz, 4H, CF2CH2); 1.90-2.12 (m, 4H, CH2C(O)); 2.75-2.96 (m, 2H, CH2N); 3.48-4.18 (m, 6H, CH2OPOCH2, C(O)OCH2); 5.06-5.21 (m, 1H, CH). 13C NMR (CDCl3/CD3OD): δ 19.6 (t, 3JCF ) 4 Hz, CF2CH2CH2); 24.5 (CH2CH2CO); 28.7, 28.8, 30.0 ((CH2)6(CH2)2CO); 30.4 (t, 2JCF ) 22 Hz,

952 Bioconjugate Chem., Vol. 12, No. 6, 2001

CF2CH2); 33.6, 33.7 (CH2C(O)); 40.1 (d, 3JCP ) 6 Hz, CH2N); 61.3 (d, 2JCP ) 5 Hz, POCH2CH2N); 62.2 (C(O)OCH2); 63.5 (d, 2JCP ) 5 Hz, CHCH2OP); 70.1 (d, 3JCP ) 8 Hz, CH); 173.5, 173.8 (CO). 19F NMR (CD3OD): δ -82.5 (3F, CF3); -115.9 (2F, CF2CH2); -125.7, -127.4 (4F, CF3(CF2)2). 31P NMR{1H} (CDCl3/CD3OD): δ -0.2 (s). Anal. (C35H52F18NO8P + 1H2O) Calcd: C, 41.80; H, 5.21; N, 1.39. Found: C, 41.78; H, 5.17; N, 1.38. rac-3-[11-(F-Octyl)undec-10-enyl]-2-(hexadecyl)glycero-1-phosphoethanolamine, [F8E11][C16]OPE. rac-1-[11-(F-Octyl)undec-10-enyl]-2-(hexadecyl)glycero-3-phospho(tert-butoxycarbonyl)ethanolamine, 7b (70% yield): TLC (CHCl3/MeOH: 7:3, v:v; molybdenum blue): Rf ) 0.60. 1H NMR (CDCl3/CD3OD): δ 0.85 (t, 3J ) 6.1 Hz, 3H, CH3); 1.05-1.35 (s large, 38H, CH3(CH2)13, (CH2)6(CH2)2O); 1.35-1.63 (m, 13H, CH2CH2O, CH3(BOC)); 2.10-2.28 (m, 2H, CHdCHCH2); 3.17-3.32 (m, 2H, CH2N); 3.32-3.65 (m, 7H, CH2OCH2, CH2OCH); 3.73-3.95 (m, 4H, CHCH2OP, POCH2CH2N); 5.42-5.72 (m, 1H, CF2CHdCH); 6.38-6.50 (m, 1H, CF2CH)CH). 13C NMR (CDCl3/CD3OD): δ 13.6 (CH3); 22.4 (CH3CH2); 25.9 (CH2CH2CH2O); 28.0 (CH3 (BOC)); 27.8, 28.8, 29.2, 29.4, 29.6, 30.0 (CH2CH2O, CH3(CH2)2(CH2)10, CHdCHCH2(CH2)5); 31.8, 31.9 (CH3CH2CH2, dCHCH2); 40.8 (d, 3JCP ) 6 Hz, CH2N); 64.3 (d, 2JCP ) 5 Hz, POCH2CH2N); 65.2 (d, 2JCP ) 5 Hz, CHCH2OP); 70.1, 70.5, 71.6 (CH2OCH2, CH2OCH); 77.7 (CH); 79.2 (C(CH3)3); 116.7 (t, 2JCF ) 23 Hz, CF2CHdCH); 143.0 (t, 3JCF ) 9 Hz, CF2CHdCH trans); 145.0 (CF2CHdCH cis); 157.2 (C(O)). 19F NMR (CDCl3/CD3OD): identical to that of 6. 31P{1H} NMR (CDCl /CD OD): δ 2.1 (s). 3 3 [F8E11][C16]OPE, 2.3 TFA (white powder; 75% yield from 7b): TLC (CHCl3/MeOH/NH4OH 8:2:0.2, molybdenum blue and H2SO4): Rf ) 0.30. 1H NMR (CDCl3/ CD3OD): δ 0.85 (t, 3J ) 6.1 Hz, 3H, CH3); 1.10-1.45 (broad s, 38H, CH3(CH2)13, (CH2)6(CH2)2O); 1.45-1.67 (m, 4H, CH2CH2O); 2.10-2.30 (m, 2H, CHdCHCH2); 3.053.27 (m, 2H, CH2N); 3.35-3.70 (m, 7H, CH2OCH2, CH2OCH); 3.80-4.01 (m, 2H, CHCH2OP); 4.01-4.21 (m, 2H, POCH2CH2N); 5.40-5.74 (m, 1H, CF2CHdCH); 6.35-6.50 (m, 1H, CF2CHdCH). 13C NMR (CDCl3/ CD3OD): δ 13.6 (CH3); 22.4 (CH3CH2); 25.9 (CH2CH2CH2O); 27.8, 28.8, 29.2, 29.4, 29.6, 30.0 (CH2CH2O, CH3(CH2)2(CH2)10, CHdCHCH2(CH2)5); 31.8 (CH3CH2CH2, dCHCH2); 40.2 (d, 3JCP ) 6 Hz, CH2N); 61.5 (d, 2JCP ) 5 Hz, POCH2CH2N); 65.2 (d, 2JCP ) 5 Hz, CHCH2OP); 70.1, 70.5, 71.6 (CH2OCH2 , CH2OCH); 77.7 (d, 3JCP ) 8 Hz, CH); 116.7 (t, 2JCF ) 23 Hz, CF2CHdCH); 143.0 (t, 3JCF ) 9 Hz, CF2CHdCH trans); 145.0 (CF2CHdCH cis). 19F NMR (CDCl3/CD3OD): δ -72.6 (1.3F, CF3COOH); -81.3 (3F, CF3); -107.2 (0.2F, CF2CH cis); -112.7 (1.8F, CF2CH trans); -122.0, -122.5, -123.2, -124.0 (2F, 4F, 2F, 2F, CF3CF2(CF2)5); -126.6 (2F, CF3CF2). 31P {1H} NMR (CDCl3/CD3OD): δ 1.0 (s). Anal. (C40H65F17NO6P + 1.3 CF3COOH + 2H2O) Calcd: C, 42.64; H, 5.55; N, 1.16. Found: C, 42.56; H, 5.45; N, 1.31. rac-3-[11-(F-Octyl)undecyl]-2-(hexadecyl)glycero1-phosphoethanolamine, [F8C11][C16]OPE. rac-1[11-(F-Octyl)-10-undecyl]-2-(hexadecyl)glycero-3phospho(tert-butoxycarbonyl)ethanolamine,7c: (40% yield) TLC (CHCl3/MeOH: 7:3 v/v; molybdenum blue, ninhydrine): Rf ) 0.65. 1H NMR (CDCl3/CD3OD): δ 0.85 (t, 3J ) 6.1 Hz, 3H, CH3); 1.04-1.37 (s large, 40H, (CH2)7(CH2)2O, CH3(CH2)13); 1.37-1.70 (m, 15H, CH2CH2O, CF2CH2CH2, CH3(BOC)); 2.04 (tt, 3J ) 7.5 Hz, 3JHF ) 19.0 Hz, 2H, CF2CH2); 3.12-3.35 (m, 2H, CH2N); 3.35-3.69 (m, 7H, CH2OCH2, CH2OCH); 3.73-4.03 (m, 4H, CH2OPOCH2). 13C NMR (CDCl3/CD3OD): δ 13.9 (CH3); 20.1 (t, 3JCF ) 4 Hz, CF2CH2CH2); 22.6 (CH3CH2); 26.1

Gaucheron et al.

(CH2CH2CH2O); 28.3 (CH3 (BOC)); 29.1, 29.3, 29.4, 29.5, 29.6, 29.7, (CH2CH2O, CH3(CH2)2(CH2)10, CF2(CH2)2(CH2)6); 30.9 (t, 2JCF ) 22 Hz, CF2CH2); 31.9 (CH3CH2CH2); 40.9 (d, 3JCP ) 6 Hz, CH2N); 64.8 (d, 2JCP ) 5 Hz, POCH2CH2N); 65.0 (d, 2JCP ) 5 Hz, CHCH2OP); 70.6, 70.7, 71.7 (CH2OCH2, CH2OCH); 77.1 (CH); 79.0 (C(CH3)3); 157.2 (C(O)). 19F NMR (CDCl3/CD3OD): identical to that of 2c. 31P {1H} NMR (CDCl3/CD3OD): δ - 2.5 (s). [F8C11][C16]OPE, 2.4 TFA (white powder; 80% yield from 7c): TLC (CHCl3/MeOH/NH4OH 8:2:0.2, molybdenum blue and H2SO4): Rf ) 0.36. 1H NMR (CDCl3/ CD3OD): δ 0.80 (t, 3J ) 6.1 Hz, 3H, CH3); 1.02-1.40 (broad s, 40H, (CH2)7CH2CH2O, CH3(CH2)13); 1.40-1.67 (m, 6H, CH2CH2O, CF2CH2CH2); 1.97 (tt, 3J ) 7.5 Hz, 3 JHF ) 19.0 Hz, 2H, CF2CH2); 2.99-3.19 (m, 2H, CH2N); 3.38-3.64 (m, 7H, CH2OCH2, CH2OCH); 3.70-3.91 (m, 2H, CHCH2OP); 3.91-4.12 (m, 2H, POCH2CH2N). 13C NMR (CDCl3/CD3OD): δ 13.8 (CH3); 20.1 (t, 3JCF ) 4 Hz, CF2CH2CH2); 22.5 (CH3CH2); 25.9 (CH2CH2CH2O); 29.0, 29.2, 29.3, 29.6, 29.9 (CH2CH2O, CH3(CH2)2(CH2)10, CF2(CH2)2(CH2)6); 30.9 (t, 2JCF ) 22 Hz, CF2CH2); 31.8 (CH3CH2CH2); 40.2 (d, 3JCP ) 6 Hz, CH2N); 61.7 (d, 2JCP ) 5 Hz, POCH2CH2N); 65.2 (d, 2JCP ) 5 Hz, CHCH2OP); 70.1, 70.6, 71.76 (CH2OCH2, CH2OCH), 77.7 (d, 3JCP ) 8 Hz, CH). 19F NMR (CDCl3/CD3OD): δ -74.6 (0.2F, CF3COOH); -79.6 (3F, CF3); -113.3 (2F, CF2CH2); -120.7 (6F, (CF2)3CF2CH2); -121.6 (2F, CF3(CF2)2CF2); -122.4 (2F, CF3CF2CF2); -125.0 (2F, CF3CF2). 31P NMR (CDCl3/CD3OD): δ -3.6 (s). Anal. (C35H67F18NO8P + 0.2 CF3COOH + 1H2O) Calcd: C, 46.07; H, 6.43; N, 1.33. Found: C, 46.09; H, 6.58; N, 1.41. Differential Scanning Calorimetry (DSC). For DSC measurements in the +4 to 95 °C temperature range, the samples were prepared by weighing the powdered phospholipid (∼10 mg) into pierced aluminum pans and adding a weighed amount of deionized water to obtain a water concentration of 60% (w/w). DSC measurements were carried out at a rate of 10 °C/min with a Mettler-Toledo DSC 821e apparatus which was previously calibrated using indium, zinc, and lead standards. The samples were sealed and heated in a static atmosphere. Transition enthalpies and temperatures were determined after several heating/cooling cycles. The reported values of the Tc represent the temperature at maximum excess heat capacity. Preparation of Complexes Composed of the Cationic Lipids, the Helper Lipids and Plasmid pTG11033. The plasmid pTG11033 was produced by Transge`ne. The endotoxin content of the plasmid preparation was checked using a Limulus Amebocyte Lysat kit (Biogenic, Maurin, 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 compounds used were calculated according to the desired DNA concentration of 0.1 mg/mL, the N/P ratio, the molar weight, and the number of positive charges in the selected cationic lipid (CL). The N/P ratio of 10, for example, corresponds to the molar amount of CL necessary to have a ratio of 10 amino group nitrogens (for 1 mol of CL) per one phosphate in the DNA (330 Da mean Mw), as described elsewhere (1, 22, 27). The DNA/CL: colipid(s) complex is formulated by adding a desired volume of the liposomal CL:colipid(s) preparation at a CL concentration of 10 mg/mL in 5% glucose to the desired volume of DNA solution to reach a DNA concentration of 0.1 mg/mL. Thus for the preparation of the DNA/DOGS:DOPE (1:1 mol) complex at N/P ratio 5 and 0.1 mg/mL DNA, the desired volume of DOPE (10 mg/ mL chloroform solution) to get mol:mol DOGS:DOPE was


added to 50 µL of DOGS solution (10 mg/mL in EtOH), and then the mixture was transferred to a borosilicate glass tube (16 × 100 mm). The solvent was evaporated in Rotavap evaporation system (45 °C, 30 pm, 0.2 bar, 40 min). 50 µL of 5% glucose were added to the film obtained. The preparation was vortexed for 2 h and then sonicated for 5-10 min to yield a liposomal preparation of 50-100 nm mean size as measured by photon correlation spectroscopy (see below). Then, 47.9 µL of this preparation was added to 952.1 µL of DNA solution [(100 µL DNA (1 mg/mL) diluted with 852.1 µL of 5% glucose)]. This preparation was vortexed for 10 s and was used within 1 h for the particle size measurements and the in vitro transfection experiments. Measurement of the Size of the Lipoplexes. The sample was diluted with 5% glucose in the measurement tube and homogenized, and the average sizes were measured by photon correlation spectroscopy using a Coulter N4Plus particle size analyzer, as described in ref 5. The formulations and analyses were reproduced twice. Agarose Gel Electrophoresis. Each sample was analyzed and plasmid integrity in each sample was confirmed by electrophoresis after decomplexing the lipoplex with sodium dodecyl sulfate, following the procedures described in ref 5. In Vitro Transfection of A549 Cells. Twenty-four hours before transfection, A549 cells (epithelial cells derived from human pulmonary carcinoma) were grown in Dulbeco-modified Eagle culture medium (DMEM) (GIBCO-BRL, Life Technologies, Cergy Pontoise, France), containing 10% fetal calf serum, FCS (SIGMA, Saint Quentin Fallavier, France), in 96-well plates (2 × 104 cells per well), in a wet (37 °C) and 5% CO2/95% air atmosphere. Volume of DNA/CL:colipid(s) (1:1 mol) or (1:2 mol) lipoplex (5 and/or 1 µL) was diluted to 100 µL in DMEM supplemented with 10% FCS in order to obtain various amounts of DNA (0.5 and/or 0.1 µg, respectively) in the preparation. The culture medium was removed and replaced by 100 µL of DMEM supplemented with 10% FCS and containing the desired amount of DNA. After 4 and 24 h, 50 µL and 100 µL of DMEM supplemented with 30 and 10% FCS, respectively, were added. Forty-eight hours after transfection, the culture medium was discarded, and the cells were washed twice with 100 µL of PBS and then lysed with 50 µL of lysis buffer (Promega, Charbonnie`res, France). The lysates were frozen at -40 °C awaiting analysis of luciferase activity. These measurements were done for 10 s on 10 µL of the lysis mixture in a Berthold LB96P luminometer in dynamic mode, using the “Luciferase” determination system (Promega) in 96-well plates. The total protein concentration per well was determined using conventional techniques (BCA test, Pierce, Montluc¸ on, France). For cells grown in the absence of lipoplexes, a well contains around 30 to 50 µg of proteins. The percentage of cell viability of the lipoplexes was calculated as the ratio of the total protein amount per well of the transfected cells relative to that measured for untreated cells × 100%. The given means ( SEM were calculated from four independent experiments. Statistical Analyses. Statistical analyses were performed using a Student t test. The difference between two means was considered as statistically significant when p is e 0.05. RESULTS AND DISCUSSION

Synthesis. The synthesis of the fluorocarbon DF4C11PE, [F8E11][C16]OPE, and [F8C11][C16]OPE


phosphoethanolamines from their respective glycerol precursors 2a-c was performed according to well documented procedures that have been published for the preparation of DF4C11OPE (19). These procedures, illustrated in Scheme 2 part C, involved phosphorylation of these 2a-c synthons with POCl3, condensation with N-BOC-ethanolamine, hydrolysis, and then BOC-deprotection using trifluoroacetic acid (TFA), affording the expected F-PEs as TFA salts (15-50% yields from 2ac). The ester-linked glycerol derivative 2a was obtained in two steps from benzylglycerol which included conventional acylation and benzyl deprotection by hydrogenolysis (Scheme 2, part A). The more demanding preparation of the mixed fluorocarbon/hydrocarbon 1,2-di-ether linked glycerol derivatives 1b,c (Scheme 2, part B) was best performed from 1,3-O-benzylideneglycerol 3 using a strategy developed for close analogues (19). In this cyclicstrained protected glycerol, the secondary hydroxyl function has been shown to be more accessible and easier to alkylate than in acyclic protected analogues (23). Alkylation of 3 performed under phase catalysis conditions using hexadecyl bromide, followed by the reductive cleavage of the 1,3-benzylidene-protecting group with BH3/THF (23) afforded derivative 4 (70% yield). Subsequent alkylation of the hydroxyl thus liberated in 4 using 11-undecenyl bromide yielded the benzyl-protected derivative 5 (70% yield). The addition of the linear perfluorooctyliodide (C8F17I) on the terminal CH2dCHdouble bond of 5 was performed using the modified method of Burton and Kehoe (24): this two-step one-pot reaction led to the perfluorooctyl unsaturated derivative 6, in 75% yield. According to 19F NMR which showed more particularly the presence of two resonances for the CF2 in R to the double bond, the isolated compound consisted in a mixture of E- and Z-isomers. The most abundant one was the E-isomer (at least 85%). These products were also contaminated by a compound (less than 5%) which possesses a CF2-CH2-CHdCH- sequence, as a result of a β,γ-HI elimination during the perfluoroalkylation step (19, 24). This was evident from the presence of, respectively, a doublet of triplets (3JHH ) 7 Hz, 3JHF ) 18 Hz) at 2.7 ppm, a triplet (2JCF ) 23 Hz) at 34.7 ppm for the CH2 group and a CF2 resonance at -115.8 ppm, in the 1H, 13C, and 19F NMR spectra of 6. Selective benzyl-deprotection was performed by action of excess FeCl3 in anhydrous CH2Cl2 followed by hydrolysis (25). This procedure afforded the mixed fluorocarbon/ hydrocarbon unsaturated 1,2-glycerol derivative 2b in 75% yield. The saturated and benzyl-deprotected analogue 2c was obtained in nearly 60% yield by simultaneous benzyl-deprotection and double-bond hydrogenation using high-pressure hydrogenolysis conditions (40 atm H2 in the presence of 10% Pd/C). Under milder hydrogenolysis conditions (1 atm H2), double-bond hydrogenation but only partial debenzylation was observed. Thermotropic Phase Behavior. The thermotropic phase behavior of samples consisting of hydrated powders of the F-PEs was investigated by DSC. Table 1 collects the thermodynamic parameters (Tc, gel (crystalline) to liquid-crystalline main phase transition and/or lamellar to inverted hexagonal phase transition, see hereafter, and their associated ∆H and ∆S) determined on these dispersions by DSC. The phase behavior of hydrated DF4C11OPE has been investigated in detail by X-ray diffraction, DSC, and optical microscopy with polarized light (26). This latter study, performed as a function of


Gaucheron et al.

Scheme 2a-j

a (a) F(CF ) (CH ) COCl/pyridine/Et O; (b) H /Pd/C/THF; (c) CH (CH ) Br/phase transfer catalysis; (d) BH /THF; e) NaH/toluene, 2 4 2 10 2 2 3 2 15 3 then CH2dCH(CH2)9Br; (f) C8F17I/CuCl/NH2(CH2)2OH/t-BuOH; (g) anhydrous FeCl3/CH2Cl2 then H2O; (h) H2 (40 atm)/Pd/C/EtOH/ AcOH; (i) 1. POCl3/NEt3/THF; 2. HO(CH2)2NHBOC/CHCl3/pyridine; 3. H2O; (j) TFA/CH2Cl2.

Table 1. Thermodynamic Parameters of the Lamellar to Inverted Hexagonal HII Phase Transition for the Fluorinated Glycerophosphoethanolamines (F-PEs). DSC Experiments Were Performed in Deionized Water (60% w:w) F-PE compound

Tc ((1 °C) (∆T1/2)a

∆H ((5%) (kJ/mol)

∆S (J/mol‚K)

DF4C11PE [F8E11][C16]OPE [F8C11][C16]OPE DF4C11OPEb

25 (3) 38 (4) 45 (5) 32 (2)

36.4 2.9 27.7 20.2

122 9.3 87 66.2

a ∆T 1/2 is the transition width at half-maximal excess specific heat capacity b Data from ref 26.

temperature, showed that DF4C11OPE, in excess water, forms a hexagonal phase above 28 °C, (i.e., below but close to the phase transition temperature measured by DSC). It showed also that the unique and broad phase transition detected by DSC consisted in a superposition of transitions, which included the lamellar gel to liquidcrystalline phase transition followed, almost simultaneously, by a transition to a hexagonal phase. Owing to their very close molecular structures, one can therefore assume that the unique and broad phase transition detected by DSC for the other fluorinated PEs is also most likely a transition to a hexagonal phase. The presence of a HII phase at a temperature above the phase transition temperature detected by DSC was further attested by optical microscopy with polarized light. Lipoplex Formation and Characterization. The capability of the various F-PEs as colipid of cationic lipids, CL () DOGS, pcTG90 or DOTAP) to form lipoplexes was analyzed for various CL:DOPE:F-PE molar ratios [1:(1 - x):x with x ) 0.5, 0.75, or 1, and 1:(2 - y):y with y ) 1, 1.5, or 2], and for various N/P ratios (10, 5, 2.5, 1.25, and 0.8), as compared to CL:DOPE (1:1) or (1:2

mol) control lipoplexes. These studies were performed with pTG11033 plasmid, also used for the in vitro transfection assays (see next section). The procedure applied for the lipoplex preparation relies on the dilution of a liposomal solution obtained from CL and the colipid(s) in 5% glucose with the DNA solution in 5% glucose, using N/P ratios of 10, 5, 2.5, 1.25, and 0.8 (N ) number of lipid cations; P ) number of DNA phosphates (22, 27)). These formulations were analyzed by agarose gel electrophoresis which showed the absence of , free . plasmid for N/P ratios from 10 to 1.25, DNA being totally protected from ethidium bromide interaction. By contrast, the plasmid was accessible to ethidium bromide in the case of N/P 0.8 formulations. These formulations were also analyzed by light scattering spectroscopy (LSS) for size determination (which is an important parameter to control when in vivo uses are contemplated or when their in vitro transfection efficiency is evaluated (16, 22)). For the CL:colipid(s) (1:1 mol) formulations, and irrespective of the nature of CL and colipid(s) and of colipid composition, one observed most often lipoplexes of mean size in the 50-180 nm range for an excess of cationic charges (N/P g 2.5) and in the 150-400 nm range for N/P ) 0.8 while precipitates were systematically detected for N/P ) 1.25 (data not shown). Concerning the pcTG90: colipid(s) (1:2 mol) formulations, a very similar behavior was observed, i.e., lipoplexes of mean size in the 50-150 nm range for N/P g 2.5, in the 150-300 nm range for N/P ) 0.8, and precipitates for N/P ) 1.25 except when co-formulated with DF4C11OPE or DF4C11PE (data not shown). This latter result is noteworthy as most often precipitates for a N/P ratio of 1.25 (which corresponds to almost neutraly charged lipoplexes) were reported in the literature (4, 5, 28). Concerning the N/P 0.8, 2.5, 5, and 10 formulations, our results are also close to those



Figure 1. A: Luciferase expression (bars) and cell viability (points) in A549 cells of the lipoplexes prepared in 5% glucose and made of CL() pcTG90, DOGS, or DOTAP):DOPE:[F8E11][C16]OPE (1:1 - x:x with x ) 0.5, 0.75, or 1) and plasmid pTG11033 (DNA) for various N/P ratios, as compared to control DOPE lipoplexes (x ) 0). The given means ( SEM were calculated from four independent experiments. B: Transfection efficiency of the [F8E11][C16]OPE-based lipoplexes vs that of their corresponding DOPE control lipoplexes. The luciferase level ratio LLR is the ratio of luciferase amount measured for the fluorinated formulation vs that measured for its corresponding DOPE control. The efficiency is significantly higher if LLR is g 5, significantly lower if LLR e 0.2, or comparable if 0.2 < LLR < 5. When outside this range, p < 0.01 were carried out in a pairwise comparison. The values given as statistics correspond to the number of formulations that satisfy the given LLR condition vs the total number of formulations investigated, and to the respective percentages. *The luciferase expression levels (in the 2 × 103 to 7 × 103 fg luciferase per mg protein range) corresponding to the F-PE and control N/P 0.8 pcTG90-based formulations are not shown.

reported for other lipoplexes, i.e., stable dispersions for “negatively” (N/P ) 0.8) and positively charged lipoplexes (N/P g 2.5) (4, 5, 28). As compared with DOPE alone, no impact of the F-PE’s nature or of their molecular structure on lipoplex size could be evidenced. Our results indicate that the fluorinated PEs are as effective colipids as DOPE in terms of compacting DNA with DOGS, pcTG90 or DOTAP into small-sized particles. These data suggest further that the lamellar to hexagonal phase transition temperature of the F-PEs, and the expected lower miscibility between the fluorinated colipids and the cationic lipids as compared with DOPE (fluorinated amphiphiles are usually not miscible with hydrocarbon ones (29)) are not determinant for the DNA compaction process when using cationic lipids in combination with such colipids.

In Vitro Transfection. To evaluate the contribution of the various F-PEs to in vitro lipoplex-mediated gene transfer, we examined the transfection potential of the above-described lipoplexes formulated with these F-PEs (alone or in combination with DOPE) as colipids of pcTG90, DOGS, or DOTAP. Both CL:colipid(s) (1:1 mol) and (1:2 mol) formulations were investigated as the pcTG90:colipid(s) (1:1 mol) and (1:2 mol) lipoplexes proved to be more efficient in vitro and in vivo transfection assays, respectively (8, 14, 15). Lipofection was assayed in vitro on lung epithelial A549 cells, from human pulmonary carcinoma. These assays were performed using the luciferase reporter plasmid pTG11033 (pCMV-intronHMG-luciferase-SV40pA, 9572 bps) in the presence of 10% fetal calf serum for 48 h. All the lipoplex formulations described in the precedent section were


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Figure 2. A: Luciferase expression (bars) and cell viability (points) in A549 cells of the lipoplexes prepared in 5% glucose and made of CL() pcTG90, DOGS, or DOTAP):DOPE:[F8C11][C16]OPE (1:1 - x:x with x ) 0.5, 0.75, or 1) and plasmid pTG11033 (DNA) for various N/P ratios, as compared to control DOPE lipoplexes (x ) 0). The given means ( SEM were calculated from four independent experiments. B: Transfection efficiency of the [F8E11][C16]OPE-based lipoplexes vs that of their corresponding DOPE control lipoplexes. *The luciferase expression levels (in the 2 × 103 to 7 × 103 fg luciferase per mg protein range) corresponding to the F-PE and control N/P 0.8 pcTG90-based formulations are not shown. For more details concerning LLR, and the statistics, see caption of Figure 1. When LLR is outside the 0.2-5 range, p < 0.01 in a pairwise comparison.

tested, except those that precipitated. The transfection efficiency of the lipoplexes, expressed in femtograms (fg) of luciferase/mg of protein, was evaluated for a DNA amount of 0.5 µg/well: in our transfection protocol, a plateau of luciferase expression was generally obtained for such a DNA amount. For comparison, naked DNA and the control lipoplexes based on DOPE as sole colipid were also tested. 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 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 30-50 µg/well range). The transfection and cell viability results for the lipoplexes based on‚[F8E11][C16]OPE, [F8C11][C16]OPE, DF4C11OPE or DF4C11PE are illustrated in Figures 1A

to 4A for the CL:colipid(s) (1:1 mol) formulations and in Figure 5A for the pcTG90:colipid(s) (1:2 mol) ones. These results indicate that the “fluorinated” lipoplexes formed from any of these F-PEs (used as sole colipid or in combination with DOPE) and any of the cationic lipids (pcTG90, DOGS, or DOTAP) do transfect cells to a larger extent than naked DNA. Concerning the CL:colipid(s) (1:1 mol) formulations (Figures 1A to 4A), the highest luciferase expression levels (LEL) were most frequently observed for N/P ratios of 2.5, 5, and 10 in the case of pcTG90, of 2.5 and 5 in the case of DOGS (LEL g 106 fg per mg protein), and of 0.8, 2.5, and 5 in the case of DOTAP (LEL g 105 fg per mg protein). A much lower plasmid expression was evidenced for the N/P 0.8 lipoplexes formulated with pcTG90 (2 × 103 < LEL < 7 × 103 fg per mg protein, data not shown) or DOGS (104 < LEL e 106 fg per mg



Figure 3. A: Luciferase expression (bars) and cell viability (points) in A549 cells of the lipoplexes prepared in 5% glucose and made of CL() pcTG90, DOGS, or DOTAP):DOPE:DF4C11OPE (1:1 - x:x with x ) 0.5, 0.75, or 1) and plasmid pTG11033 (DNA) for various N/P ratios, as compared to control DOPE lipoplexes (x ) 0). The given means ( SEM were calculated from four independent experiments. B: Transfection efficiency of the DF4C11OPE-based lipoplexes vs that of their corresponding DOPE control lipoplexes. *The luciferase expression levels (in the 2 × 103 to 7 × 103 fg luciferase per mg protein range) corresponding to the F-PE and control N/P 0.8 pcTG90-based formulations are not shown. For more details concerning LLR, and the statistics, see caption of Figure 1. When LLR is outside the 0.2-5 range, p < 0.01 in a pairwise comparison.

protein). Concerning the pcTG90:colipid(s) (1:2 mol) formulations (Figure 5A), the highest LEL (g 105 fg per mg protein) were observed for N/P 5 and 2.5 while for N/P 1.25 and 0.8, LELs remained below 104 fg per mg protein (results not shown). The lower transfection efficiency of the N/P 0.8 and 1.25 lipoplexes is most probably related to a lower cellular uptake of these “anionic” and “neutral” formulations as compared with that of the more cationic lipoplexes owing to expected lower lipoplex-cell electrostatic interactions. The mechanism of cellular endocytic uptake of the lipoplexes is indeed mainly governed by electrostatic interactions between the DNA complexes and the anionic proteoglycans expressed at the surface of these adherent cells (30, 31), this uptake increasing further with raising the N/P ratio of the lipoplexes (32). However, lower luciferase expression was evidenced for some of the N/P

10 lipoplexes as compared with that of their corresponding N/P 5 and 2.5 ones (and even of the N/P 0.8 ones in the case of DOTAP). Concomitantly, cell viability of the N/P 10 lipoplexes (and more particularly for pcTG90 or DOGS) was improved, indicating that cytotoxicity induced by these lipoplexes was not responsible for the lower gene expression. That the transfection efficiency of the lipoplexes does not necessarily increase with a N/P ratio increase was recently shown to be related to significant differences in intracellular distribution and trafficking (probably release from the endosomes or lysosomes) of the lipoplexes between these N/P ratios (32). As illustrated in Figure 6, it appears, for N/P g 2.5 and irrespective of the fluorinated colipid and of the F-PE:DOPE molar ratio, that most of the “fluorinated” pcTG90- and DOGS-based formulations (90 and 79% of


Gaucheron et al.

Figure 4. A: Luciferase expression (bars) and cell viability (points) in A549 cells of the lipoplexes prepared in 5% glucose and made of CL () pcTG90, DOGS, or DOTAP):DOPE:DF4C11PE(1:1 - x:x with x ) 0.5, 0.75, or 1) and plasmid pTG11033 (DNA) for various N/P ratios, as compared to control DOPE lipoplexes (x ) 0). The given means ( SEM were calculated from four independent experiments. B: Transfection efficiency of the DF4C11PE-based lipoplexes vs that of their corresponding DOPE control lipoplexes. *The luciferase expression levels (in the 2.103 to 7.103 fg luciferase per mg protein range) corresponding to the F-PE and control N/P 0.8 pcTG90-based formulations are not shown. For more details concerning LLR, and the statistics, see caption of Figure 1. When LLR is outside the 0.2-5 range, p < 0.01 in a pair-wise comparison.

48 formulations, respectively) were at least 10-fold more efficient (p < 0.01) for transfecting A549 cells than their corresponding DOTAP lipoplexes. In a pairwise comparison, two formulations were considered to have comparable transfection efficiency when the LLR ratio of their respective LEL was within the 0.2-5 range, and of significantly different efficiency if this ratio is outside this range, providing its associated p value is e0.05. One should further underscore the higher transfection potential of the pcTG90-based lipoplexes when compared with the DOGS ones: if 60% of the 48 pcTG90 formulations investigated were found to lead to comparable LELs to those of their corresponding DOGS complexes, 35% of the 48 pcTG90 formulations led however to LELs that were at least 5-fold higher (p < 0.05). The transfection efficacy sequence evidenced for the F-PE-based lipoplexes (i.e., pcTG90 g DOGS > DOTAP) for N/P g 2.5 is further

identical to that observed for the lipoplexes based on DOPE as sole colipid. The higher in vitro transfection efficiency of the pcTG90 and DOGS lipopolyamines as compared with that of DOTAP can be attributed to their endosomolytic activity related to the presence of protonable amine functions of low pKa (“proton sponge” effect) (2, 14, 15). However, for the “negatively charged” lipoplexes (N/P 0.8), the opposite tendency was found for both the F-PEs and DOPE, as shown in Figure 6. Their transfection efficiency was indeed seen to decrease along the sequence DOTAP > DOGS > pcTG90. Concerning the fluorinated lipoplexes, 63 and 100% of the 16 DOTAPbased formulations were significantly more efficient than their corresponding DOGS- (at least 10-fold, p < 0.01) or pcTG90-based lipoplexes (at least 10- to 100-fold, p < 0.01), respectively, and 69% of the 16 DOGS-based lipoplexes were significantly more efficient (at least 10-



Figure 5. A: Luciferase expression (bars) and cell viability (points) in A549 cells of the lipoplexes prepared in 5% glucose and made of pcTG90:DOPE:F-PE (1:2 - y:y with y ) 1, 1.5, or 2) and plasmid pTG11033 (DNA) for N/P 2.5 and 5, as compared to control DOPE lipoplexes (y ) 0). The given means(SEM were calculated from four independent experiments. B: Transfection efficiency of the F-PE-based lipoplexes vs that of their corresponding DOPE control lipoplexes. For more details concerning LLR, and the statistics, see caption of Figure 1. When LLR is outside the 0.2-5 range, p < 0.01 in a pair-wise comparison.

fold, p < 0.01) than the pcTG90-based ones. As for the cationic lipids, the different tendency observed for their N/P 0.8 lipoplexes as compared with that found for their more cationic (N/P g 2.5) ones is most likely attributable to significant differences in the mechanism of cellular uptake and of intracellular traffic of the lipoplexes between these cationic lipids and N/P values. Irrespective of the nature of the F-PE and of the DOPE: F-PE molar ratio, increasing the colipid content from 50 to 66.7% mol (1:1 to 1:2 formulations) led, as shown in Figure 7, most often for the N/P 5 lipoplexes to a significant decrease in transfection (67% of the 12 F-PE formulations) while comparable and higher LELs were observed for 67 and 33% of the 12 N/P 2.5 F-PE formulations, respectively. These differences can tentatively be attributed to differences in intracellular distribution and

trafficking of the lipoplexes between these N/P and pcTG90:colipid molar ratios (32). One can further observe that, whatever the N/P ratio, [F8C11][C16]OPE constituted the sole colipid (among the F-PEs and DOPE) for which transfection was not affected by or increased with this CL:colipid molar ratio increase. The main objectives of this study were aimed at examining the lipid helper effect on lipofection of the F-PEs as compared with that of DOPE and at determining whether their lipid helper potential was cationic lipid specific or not. Our results indicate that the F-PEs display such a lipid helper effect that is most often comparable with that of DOPE. However, their helper effect shows some dependency on the cationic lipid. This is strongly supported by the statistics deduced from panels B of Figures 1 to 4 and of Figure 5 for the (1:1)


Gaucheron et al.

Figure 6. Dependence of the F-PE lipid helper effect with the cationic lipid. The luciferase level ratios, LLR, correspond to the ratios of luciferase amount measured for the formulations based on [F8E11][C16]OPE (A), [F8C11][C16]OPE (B), DF4C11OPE (C), or DF4C11PE (D) and (i) on pcTG90 vs that measured for their corresponding DOGS (upper section) or DOTAP (middle section) lipoplexes, or (ii) on DOGS vs that measured for their corresponding DOTAP (lower section) lipoplexes. For more details concerning LLR, and the statistics, see caption of Figure 1. When LLR is outside the 0.2-5 range, p < 0.01 in a pair-wise comparison.

and (1:2 mol) formulations, respectively. These panels display the luciferase level ratios (LLR) measured for the formulations based on‚[F8E11][C16]OPE, [F8C11][C16]OPE, DF4C11OPE, or DF4C11PE, respectively, vs that measured for their corresponding control lipoplexes (which contain DOPE as sole colipid). The “helper” effect related to the presence of F-PE (in place of part of or of all DOPE) is significantly higher or lower to that of DOPE if this ratio is higher or lower than 5 or 0.2, respectively, providing its associated p value is e 0.05, and this effect is comparable with that of DOPE if this ratio is within the 0.2-5 range. Irrespective of the F-PE chemical structure, of the colipid mixture composition, and of the N/P ratio, we found indeed that, when using one of the F-PEs as colipid in place of part of or of all DOPE, comparable transfection levels were most frequently measured for the fluorinated lipoplexes and their respective control DOPE lipoplexes. This is the case for 75100% of the 12 pcTG90-based (1:1 mol) formulations investigated for each F-PE, for 83% of the 24 pcTG90based (1:2 mol) formulations, and for 75-50% of the 12 DOGS-based formulations investigated for each F-PE. However, a significant proportion of fluorinated DOGSbased lipoplexes (8-33% of the 12 formulations investigated for each F-PE) were found to display a higher transfection efficiency than their DOPE control. By contrast for the DOTAP-based lipoplexes, the opposite tendency was evidenced: if statistically comparable

transfection levels were obtained for 75-25% of the 12 formulations investigated for each F-PE, the complementary 25-75% ones displayed significantly lower transfection levels than their respective DOPE control lipoplexes. The dependence of the lipid helper potential of the F-PEs on the cationic lipid can be tentatively explained by considering the geometrical complementarity between the cationic lipids and the colipids, which might have an effect on the packing within the lamellar phases formed between each of the CLs and each of the F-PEs or DOPE, and, consequently, on the physicochemical and biological properties of the DNA lipoplexes they form, and on transfection. Of the three cationic lipids investigated, it is DOGS with its branched polar head which displays the most-pronounced “direct” cone-shaped geometry (pure DOGS was shown to form mainly direct micelles when dispersed in water (33)). The F-PEs displaying a morepronounced “inverted” cone-shaped geometry than DOPE, one therefore expects a higher degree of geometrical complementarity between the F-PEs and DOGS, which should result in more tightly packed (or more stable) lamellar phases than between DOPE and DOGS or than between any of the F-PEs and any of the two other cationic lipids. Consequently, one expects that, among the different combinations, more stable lipoplexes are formed from the F-PEs and DOGS, and thus a more pronounced helper effect for the F-PEs when used with


Figure 7. Effect of increasing the colipid content on luciferase expression in A549 cells of the pcTG90:colipid(s) lipoplexes (from 1:1 to 1:2). The luciferase level ratio LLR is the ratio of luciferase amount measured for the 1:2 mol formulation vs that measured for its corresponding 1:1 mol formulation. For more details concerning LLR, and the statistics, see caption for Figure 1. When LLR is outside the 0.2-5 range, p < 0.01 in a pair-wise comparison.

DOGS. Conversely, DOTAP which, of the three cationic lipids, displays a geometry that is the closest to a cylinder, forms lamellar phases which when combined with the F-PEs are expected to be substantially less tightly packed (or more destabilized) than those formed between DOTAP and DOPE. Owing to the lower degree of geometrical complementarity between DOTAP and the F-PEs, one expects a helper effect of the F-PEs that is below that of DOPE, as observed. Where the helper effect on transfection resulting from the composition of the colipid mixture (DOPE:F-PE molar ratio) is concerned, it seems most preferable to use a DOPE:F-PE (1:3 mol) colipid combination than F-PE alone or than a F-PE:DOPE (1:1 mol) mixture, whatever the cationic lipid and the F-PE. If a preference for a specific DOPE:F-PE combination could not be evidenced among the few (eleven) CL:colipid(s) (1:1 mol) formulations that led to significantly higher transfection levels than their respective DOPE controls (see Figure 1B to 4B), this is however not the case for the 36 formulations that displayed a significantly lower transfection level than the DOPE controls. Indeed, of these 36 less efficient formulations, only 7 (19%) concerned the DOPE:F-PE (1:3 mol) colipid combination while 12 (33%) and 17 (48%) concerned lipoplexes based on the (1:1 mol) colipid mixture and on F-PE as sole colipid, respectively, indicating


Figure 8. Panel A: Effect of the presence of a fluorinated tail on one or on both hydrophobic chains on lipofection. LLR represents the ratio of luciferase amount measured for the [F8E11][C16]OPE-based formulation vs that measured for its corresponding DF4C11OPE-based one. Panel B: Effect of the presence of a double bond in the hydrophobic chain on lipofection. LLR represents the ratio of luciferase amount measured for the unsaturated [F8E11][C16]OPE-based formulation vs that measured for its corresponding saturated [F8C11][C16]OPEbased one. Panel C: Impact of the chemical connexion (ester vs ether) on lipofection. LLR represents the ratio of luciferase amount measured for the ester DF4C11PE-based formulation vs that measured for its corresponding ether DF4C11OPE-based one. For more details concerning LLR, and the statistics, see caption of Figure 1. When LLR is outside the 0.2-5 range, p < 0.01 in a pair-wise comparison.

that transfection was likely optimal for a DOPE:F-PE (1:3 mol) colipid combination. The aim of this study was also to highlight the impact of the F-PE structural elements (one fluorinated tail on one or on both hydrophobic chains, saturated or unsaturated hydrophobic chains, ester or ether-linkage) on their helper potential. Concerning the effect of the presence of a fluorinated tail on one or on both hydrophobic chains (as in [F8E11][C16]OPE or DF4C11OPE, respectively), a statistical analysis performed on the 33 [F8E11][C16]OPE-based formulations as compared with their respective DF4C11OPE ones did not allow to distinguish between these two F-PEs (Figure 8, panel A). This analysis demonstrated comparable, higher and lower transfection levels for 65, 15, and 20% of the formulations, respectively. Although of very different structures, these two F-PEs contain almost the same number of fluorine atoms (17 vs 18). Both compounds display furthermore a phase transition temperature which is below or close to the incubation temperature. One expects therefore a similar hydrophobic and lipophobic character,


and comparable interactions with the cationic lipids, serum proteins, and cellular constituents that have no consequences on transfection. The presence of a double bond in the hydrophobic chain, although not determinant, was found to favor transfection, as supported by the statistical analysis performed on the 33 formulations containing [F8E11][C16]OPE as compared with their respective [F8C11][C16]OPE ones. This analysis (panel B of Figure 8) showed that, irrespective of the cationic lipid, the composition of the colipid mixture, the CL:colipid(s) molar ratio, and the N/P ratio, higher and comparable transfection levels were measured for 21 and 70% of these formulations, respectively. This result is in line with literature which showed that optimal transfection activity was preferably achieved when using cationic lipids and/ or colipids containing hydrophobic chains that organize as “fluid” lamellar phases (e.g., lipids with olefinic chains such as [F8E11][C16]OPE which displays a phase transition temperature of 38 °C) rather than “gel“ lamellar ones (e.g. lipids with long saturated chains such as [F8C11][C16]OPE which displays a phase transition temperature of 45 °C) (3, 10, 11, 34-37). Finally, a comparable helper potential was evidenced for the ester DF4C11PE and ether DF4C11OPE: indeed 85% of the 33 formulations containing DF4C11PE displayed similar transfection levels than their corresponding ones based on DF4C11OPE (panel C of Figure 8). This result is not surprising as both colipids possess a very close thermotropic phase behavior and is in line with data from literature concerning DOTAP and its ether analogue DOTMA (17). One should underscore that, under the conditions of transfection used, the chemical nature of the linkage had no impact on lipoplex cell viability. CONCLUSION

The present work indicates that “fluorinated” lipoplexes formulated with fluorinated helper lipids and conventional cationic lipids are definitely very attractive candidates for gene delivery, highlighting the diversity of lipids leading to efficient transfection. The remarkable in vitro and in vivo transfection potency found for such fluorinated lipoplexes (8) and that reported for fluorinated lipoplexes formulated with fluorinated lipospermines (6, 7) confirms further that lipophobicity and restricted miscibility of the lipoplex lipids with the endogenous lipids does not preclude efficient gene transfer and expression. Their transfection potency is rather attributable to their unique lipophobic and hydrophobic character (resulting from the formulation of DNA with fluorinated lipids), thus preventing DNA to some extent from interactions with lipophilic and hydrophilic biocompounds, and from degradation (7). Further experiments aimed at evaluating the in vivo helper transfection potential of the fluorinated PEs are currently underway. ACKNOWLEDGMENT

We wish to thank Drs. H.V.J. Kolbe and O. Meyer (Transge`ne) for their interest in this project. Many thanks to Dr. B. Cavallini (Transge`ne) for supplying the plasmid. Supporting Information Available: Two tables listing the mean sizes of the CL() pcTG90, DOGS, DOTAP: DOPE:F-PE (1:1 - x:x mol) and pcT9O:DOPE:F-PE (1:2 - y:y mol) lipoplexes formed with plasmid pTG11033, respectively, as determined by light scattering spectros-

Gaucheron et al.

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In Vitro Cationic Lipid-Mediated Gene Delivery with Fluorinated

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