HFP fluorinated cationic lipids for enhanced lipoplex stability and gene delivery

Article abstract of DOI:10.1021/bc900469z

Although a great number of cationic lipids have been designed and evaluated as gene delivery systems, there is still a need for improvement of nonviral vectors. Recently, cationic lipids incorporating terminal fluoroalkyl segments ( FHP lipids) have been described to display remarkable transfection potency. Here, we describe the synthesis of a new family of fluorinated triblock cationic lipids in which a fluorous segment lays between the cationic and the lipophilic parts of the molecule ( HFP lipids). The compounds were designed so their self-assembly would offer enhanced resistance toward the host's degradation mechanisms mediated by lipophilic insertion. Self-assembly properties of these cationic lipids were evaluated at the air-water interface where they collapse in a highly ordered liquid phase. The HFP lipids efficiently condense DNA, and the resulting lipoplexes display enhanced resistance to amphiphilic agents when compared to nonfluorinated or FHP cationic lipids. Transfection properties of the fluorinated vectors, alone or as mixtures with different helper lipids (DOPE and a fluorinated analogue of DOPE), were then investigated on different cell lines (BHK-21, HepG2, and HeLa) and compared to those of the reference cationic lipid DOTAP. Data show that impermeabilization of the lipidic phase by fluorous segments alter significantly the gene transfection activities. Remarkably, incorporation of DOPE within the lipoplexes provides the particles with high gene transfection activity without reducing their resistance to amphiphilic agents.

Full text of DOI:10.1021/bc900469z

360
Bioconjugate Chem. 2010, 21, 360–371
“HFP” Fluorinated Cationic Lipids for Enhanced Lipoplex Stability and
Gene Delivery
Emmanuel Klein,† Miahala Ciobanu,† Je´roˆme Klein,† Vale´rie Machi,† Christian Leborgne,‡ Thierry Vandamme,†
Benoˆıt Frisch,† Franc¸oise Pons,† Antoine Kichler,‡ Guy Zuber,† and Luc Lebeau*,†
Laboratoire de Conception et Application de Mole´cules Bioactives, CNRSsUniversite´ de Strasbourg, 74 route du Rhin, BP
´
60024, 67401 Illkirch Cedex, France, and Ge´ne´thon-FRE 3087 CNRS, 1 rue de l’Internationale, BP 60, 91002 Evry, France.
Received October 28, 2009; Revised Manuscript Received January 5, 2010
Although a great number of cationic lipids have been designed and evaluated as gene delivery systems, there is
still a need for improvement of nonviral vectors. Recently, cationic lipids incorporating terminal fluoroalkyl segments
(“FHP” lipids) have been described to display remarkable transfection potency. Here, we describe the synthesis
of a new family of fluorinated triblock cationic lipids in which a fluorous segment lays between the cationic and
the lipophilic parts of the molecule (“HFP” lipids). The compounds were designed so their self-assembly would
offer enhanced resistance toward the host’s degradation mechanisms mediated by lipophilic insertion. Self-assembly
properties of these cationic lipids were evaluated at the air-water interface where they collapse in a highly ordered
liquid phase. The HFP lipids efficiently condense DNA, and the resulting lipoplexes display enhanced resistance
to amphiphilic agents when compared to nonfluorinated or FHP cationic lipids. Transfection properties of the
fluorinated vectors, alone or as mixtures with different helper lipids (DOPE and a fluorinated analogue of DOPE),
were then investigated on different cell lines (BHK-21, HepG2, and HeLa) and compared to those of the reference
cationic lipid DOTAP. Data show that impermeabilization of the lipidic phase by fluorous segments alter
significantly the gene transfection activities. Remarkably, incorporation of DOPE within the lipoplexes provides
the particles with high gene transfection activity without reducing their resistance to amphiphilic agents.
hand, the cationic residual charge induces a poor circulation
time of lipoplexes in blood, mostly due to their interaction with
serum components. Many strategies have been developed to
improve gene transfer mediated by cationic lipids. Those aiming
at improving lipoplex circulation time are essentially based on
the use of a poly(ethylene glycol) (PEG) coating of the cationic
particles. However, introduction of PEG at the lipoplex surface
unexpectedly does not highly increase the residential time of
particles in the blood, which may be related to the drastic
increase of the critical micelle concentration (cmc) of a lipid
provoked by PEGylation (by a ca. 106-fold factor) (8). In
addition, it may have rather adverse effects by inhibiting gene
transfer in some cases (9, 10). A related strategy proposed by
Mignet and Scherman results from the combination of cationic
lipoplexes and PEGylated anionic liposomes (11). These pH-
sensitive PEGylated anionic particles display interesting DNA
delivery properties and are supposed to reverse to non-
PEGylated cationic ones in the acidic endosome compartment.
Behr and Vierling developed an alternative strategy to improve
lipoplex circulation time by introducing a highly hydrophobic
fluoroalkyl short segment at the extremity of the fatty acid chains
of the cationic lipids (12-17). Owing to their increased
hydrophobic character, these lipids (further named “FHP”
fluorinated lipids to indicate that the fluoroalkyl moiety, F, is
separated by an alkyl segment, H, from to the polar head, P)
condense DNA into lipoplexes with higher stability than
conventional cationic lipids. FHP lipid assemblies, however, are
not expected to efficiently prevent anchoring/insertion of
amphiphilic or lipophilic compounds to/within the bilayer
structure, with such an event most often being a prelude to
lipoplex destruction (i.e., lipid solubilization, recruitment of
amphiphilic proteins triggering particle elimination by the
reticuloendothelial system, activation of the complement system
and opsonization, etc.). Importantly, FHP helper lipids have been
INTRODUCTION
The delivery of a “medicine” gene in somatic cells involves
the use of vectors, the function of which is to help the therapeutic
gene cross the biological barriers and reach the nucleus of the
target cells where it will be processed into mRNA and, later
on, translated into proteins. Due to their high transfection
efficiency, viral vectors such as adenoviruses, adeno-associated
viruses (AAVs), or retroviruses are the most widespread vectors
used in the laboratory or in clinical trials (1). However, their
performance cannot overcome several major side effects, which
include adverse immunogenic reactions, insertional mutagenesis,
and sometimes fatal toxicity (2, 3). An additional limitation of
these vectors lies in the restricted size of the DNA fragments
they can introduce into a cell (less than 5 Kbp). The nonviral
vectors, including cationic lipids, present interesting activity in
vitro, but always appeared rather disappointing when evaluated
in vivo. However, these vectors are devoid of some of the
limitations of viruses (size restriction of the nucleic acid to be
transfected, difficulty of production and purification, immuno-
genicity, etc.) and allow for specialized delivery options such
as time-enhanced circulation, time-dependent release, and
targeted delivery (4).
Since the first studies were conducted, hundreds of cationic
lipids have been synthesized as candidates for nonviral gene
delivery (5-7). Cationic lipids interact with negatively charged
DNA through electrostatic interactions leading to condensed
nucleic acid particles (lipoplexes). Optimum transfection efficacy
invariably requires excess of cationic lipid for full DNA
condensation and protection from serum nuclease. On the other
* Corresponding author. Tel.: +33(0)3 6885 4303, Fax: +33(0)3
6885 4306. E-mail address: llebeau@unistra.fr.
† CNRSsUniversite´ de Strasbourg.
‡ Ge´ne´thon-FRE 3087 CNRS.
10.1021/bc900469z  2010 American Chemical Society
Published on Web 01/25/2010

“HFP” Fluorinated Cationic Lipids
Bioconjugate Chem., Vol. 21, No. 2, 2010 361
described to enhance in vitro and in vivo gene delivery by
cationic lipids (18, 19).
Syrian hamster kidney cells (BHK-21), and immortalized human
epithelial cells (HeLa) were from ATCC-LGC (Molsheim,
France). pSMD2-Luc∆ITR (7.6 kpb) or pEGFP-Luc (6.4 kpb)
expression plasmids (BD Biosciences Clontech, Franklin Lakes,
NJ, USA) were used as reporter genes to monitor transfection
activity. Both plasmids encoded the firefly luciferase gene under
the control of a strong promoter. HepG2 cells were transfected
with the pSMD2-Luc and the other cell lines with the pEGFP-
Luc.
Preparation of Cationic Lipids. 2,3-Bis(3,3,4,4,5,5,6,6,7,7,8,8-
dodecafluorotetradecanoyloxy)-propyl-1-trimethylammonium Chlo-
ride (1a). The acid 7a (209 mg, 0.47 mmol) in anhydrous CH2Cl2
(2 mL) was stirred at room temperature with oxalyl chloride
(0.2 mL, 2.29 mmol) and a catalytic amount of DMF (5 µL)
for 4 h. Volatiles were removed under reduced pressure, and
the residue was coevaporated twice with anhydrous toluene. The
crude acid chloride was dissolved in anhydrous CH2Cl2 (2 mL),
and the resulting solution was added dropwise to a mixture of
anhydrous diol 8 (32 mg, 0.19 mmol) and pyridine (38 µL, 0.47
mmol) in THF/CH2Cl2 (2:1 v/v, 3 mL). The resulting suspension
was sonicated for 2 h and stirred overnight at room temperature.
Solvent was removed under reduced pressure at room temper-
ature, and the residue was purified by flash chromatography over
silica gel (CHCl3/MeOH 100:0 to 60:40) to yield compound
1a (51 mg, 26%; containing 37% of elimination byproduct) as
a white wax.
2,3-Bis(3,3,4,4,5,5,6,6-octafluorohexadecanoyloxy)-propyl-1-tri-
methylammonium Chloride (1b). Compound 1b (79 mg, 24%;
containing 18% of elimination byproduct) was obtained as a
white wax starting from compound 7b following the same
procedure as described for the preparation of 1a.
2,3-Bis(3,3,4,4,5,5,6,6,7,7,8,8-dodecafluorooctadecanoyloxy)-
propyl-1-trimethylammonium Chloride (1c). Compound 1c (54
mg, 28%; containing 11% of elimination byproduct) was
obtained as a white wax starting from compound 7c following
the same procedure as described for the preparation of 1a.
2,3-Bis(3,4,4,5,5,6,6,7,7,8,8-undecafluorotetradecen-2-oyloxy)-
propyl-1-trimethylammonium Chloride (2a). Compound 1a (25
mg, 24.5 µmol) in CH3CN/CH2Cl2 (2:1 v/v, 3 mL) was stirred
for 2 h with 2 M triethylamine in THF (36 µL, 62.0 µmol) at
room temperature. The reaction mixture was filtered over a pad
of finely powdered NaHCO3, and the filtrate was reduced under
vacuum to yield 2a (20 mg, 84%) as a white wax.
2,3-Bis(3,4,4,5,5,6,6-heptafluorohexadecen-2-oyloxy)-propyl-1-
trimethylammonium Chloride (2b). Compound 2b (6 mg, 88%)
was prepared from 1b following the same procedure as described
for 1a.
2,3-Bis(3,4,4,5,5,6,6,7,7,8,8-undecafluorooctadecen-2-oyloxy)-
propyl-1-trimethylammonium Chloride (2c). Compound 2c (10
mg, 86%) was prepared from 1c following the same procedure
as described for 1a.
1,2-Di[(Z)-3,3,4,4,5,5,6,6,7,7,8,8-dodecafluorohexadec-9-enoyl]-
sn-glycero-3-phosphatidylethanolamine (3). Triphenyl phosphine
(4.1 mg, 15.7 µmol) and azido compound 15 (6.0 mg, 5.2 µmol)
in THF/CH2Cl2/CF3CH2OH/HCl 2% (3:1:1:0.1 v/v, 3 mL) were
stirred for 48 h at room temperature. Volatiles were removed
under reduced pressure, and the residue was purified by silica
gel chromatography (CHCl3/MeOH/H2O 10:0:0 to 10:6:1). The
fluorinated analogue of DOPE was obtained as a colorless oil
(3.0 mg, 51%; mixture of isomers, Z/E ) 85:15).
3,3,4,4,5,5,6,6,7,7,8,8-Dodecafluoro-1,10-diiodotetradecyl Ben-
zoate (4a). Sodium dithionite (3.14 g, 17.8 mmol) was slowly
added to a mixture of 1,6-diiodoperfluorohexane (5.00 g, 9.0
mmol), vinyl benzoate (1.25 mL, 9.0 mmol), 1-hexene (1.12
mL, 9.0 mmol), and NaHCO3 (1.91 g, 18.0 mmol) in CH3CN/
H2O (8:7 v/v, 18.4 mL) at 0 °C. The resulting solution was
stirred for 50 min at 0 °C, brought to pH 5-7 with HCl 3N,
Recently, some of us showed that phospholipids that are
fluorinated close to their polar head spread at the air-water
interface into monolayers which are stable for several hours in
the presence of high concentrations of detergents (20, 21). In
the same conditions, conventional phospholipid monolayers
hardly resist a few seconds. This remarkable property only partly
results from the high hydrophobicity of fluorinated species as
is the case with FHP lipids. Indeed, displaying the fluoroalkyl
segment close to the lipid polar head likely induces a stabiliza-
tion of higher magnitude, which may be explained by the
interactions between adjacent fluorine atoms in the same
perfluorinated chain that cause the chain to twist into a rigid,
helical conformation (22). Consequently, close packing of the
rigid fluoroalkyl segments does substantiate a kind of fluorous
repelling shield that is highly impermeable to nonfluorinated
hydrophilic, lipophilic, as well as amphiphilic compounds (23).
Thus, by preventing or reducing insertion of surface active
compounds within the lipid layer it is possible to efficiently
prevent the destruction of the lipid assembly. We made the
hypothesis that cationic lipids displaying such characteristics
would condense DNA into fluorinated lipoplexes with original
capabilities in gene transfer experiments. By resisting serum
component adsorption (and subsequent destruction triggered by
insertion of hydrophobic anchors of recruited proteins or other
destruction mechanisms) (24), such lipoplexes are expected to
present an extended half-life time, and possibly a higher
transfection efficiency, however keeping in mind that intracel-
lular trafficking of the delivery system requires at some points
disassembly of the lipid-DNA complexes. Therefore, the
immediate goal of this study was to prepare cationic lipids
fluorinated close to their polar head (further named “HFP”
fluorinated lipids, a terminal alkyl chain, H, being separated by
a fluoroalkyl segment, F, from the polar head, P) and to evaluate
(i) their capability to form complexes with DNA, (ii) the stability
of the resulting lipoplexes, and (iii) the gene delivery efficacy
of the latter in vitro. In the following, we describe the design,
synthesis, characterization, and evaluation of a series of
unprecedented HFP fluorinated triblock cationic lipids.
EXPERIMENTAL PROCEDURES
Chemicals and Materials. Unless otherwise stated, all chemi-
cal reagents were purchased from Alfa Aesar (Bischeim, France)
and used without purification. R,ω-Diiodoperfluoroalkanes were
from Apollo Scientific Ltd. (Bredbury, Cheshire, UK). When
required, solvents were dried by standard procedures just before
use (25). Products were purified by chromatography over silica
gel (Silica Gel 60, 40-63 µm, Merck, Darmstadt, Germany).
Reverse-phase filtration was carried out with Sep-Pack classic
short-body C-18 cartridges (Waters, Division of Millipore,
Milford, MA, USA).
N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium chlo-
ride (DOTAP chloride), 1,2-dioleoyl-sn-glycero-3-phosphoet-
hanolamine (DOPE), sodium n-dodecyl sulfate (SDS), 3-[(3-
cholamidopropyl)dimethylammonio]-1-propanesulfonate(CHAPS),
sodium cholate (NaCh), cetyltrimethylammonium bromide
(CTAB), Tween 80, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT), penicillin, streptomycin, L-
glutamine, D-glucose, and tryptose phosphate broth were from
Sigma-Aldrich (Saint-Quentin Fallavier, France). Culture media
Dulbecco’s Modified Eagle Medium Glutamax (DMEM) and
Minimum Essential Eagle Medium (MEM) were from GIBCO-
BRL (Cergy-Pontoise, France). Fetal calf serum (FCS) was from
Perbio (Brebie`res, France). Lysis and luciferin solutions for
monitoring luciferase activity were purchased from Promega
(Charbonnie`res, France). Human hepatocarcinoma cells (HepG2),

362 Bioconjugate Chem., Vol. 21, No. 2, 2010
Klein et al.
and stirred for an additional 20 min period at room temperature.
Water was added, and the solution was extracted with CH2Cl2.
The organic layer was dried over MgSO4, filtered, and reduced
under vacuum. The residue was purified by silica gel chroma-
tography (C6H14/CH2Cl2 95:5) to yield benzoate 4a (2.03 g,
28%) as a slightly yellow waxy solid.
3,3,4,4,5,5,6,6-Octafluoro-1,8-diiodohexadecyl Benzoate (4b).
Compound 4b (2.46 g, 29%) was prepared from 1,4-diiodop-
erfluorobutane, vinyl benzoate, and 1-decene following the same
procedure as described for 4a.
3,3,4,4,5,5,6,6,7,7,8,8-Dodecafluoro-1,10-diiodooctadecyl Ben-
zoate (4c). Compound 4c (1.53 g, 33%) was prepared from 1,6-
diiodoperfluorohexane, vinyl benzoate, and 1-decene following
the same procedure as described for 4a.
3,3,4,4,5,5,6,6,7,7,8,8-Dodecafluorotetradecyl Benzoate (5a).
Tributyltin hydride (173 µL, 0.64 mmol) was added to com-
pound 4a (100 mg, 0.13 mmol) and AIBN (2 mg, 0.01 mmol)
in Et2O (3 mL). The reaction mixture was refluxed under the
light of a domestic halogen lamp (I.CEM Zu¨blin AG, type 0201,
500 W) for 15 h. Solvent was removed under reduced pressure
and the residue was purified by flash chromatography over silica
gel (n-C6H14/CH2Cl2 9:1) to yield benzoate 5a (61 mg, 90%)
as a white waxy solid.
3,3,4,4,5,5,6,6-Octafluorohexadecyl Benzoate (5b). Compound
5b (1.38 g, 96%) was prepared from 4b following the same
procedure as described for 5a.
3,3,4,4,5,5,6,6,7,7,8,8-Dodecafluorooctadecyl Benzoate (5c). Com-
pound 5c (1.49 g, 90%) was prepared from 4c following the
same procedure as described for 5a.
3,3,4,4,5,5,6,6,7,7,8,8-Dodecafluorotetradecanol (6a). Com-
pound 5a (3.0 g, 5.61 mmol) was stirred in methanolic 1 M
LiOH (20 mL) for 3 h at room temperature. Solvent was
removed under vacuum, and the residue was dissolved in
CH2Cl2, then washed with water and brine. The organic layer
was dried over MgSO4 and evaporated, and compound 6a (2.21
g, 91%) was obtained as a white waxy solid after purification
over silica gel (n-C6H13/CH2Cl2 1:1).
3,3,4,4,5,5,6,6-Octafluorohexadecanol (6b). Compound 6b (0.60
g, 86%) was prepared from 5b following the same procedure
as described for 6a.
3,3,4,4,5,5,6,6,7,7,8,8-Dodecafluorooctadecanol (6c). Com-
pound 6c (0.47 g, 77%) was prepared from 5c following the
same procedure as described for 6a.
3,3,4,4,5,5,6,6,7,7,8,8-Dodecafluorotetradecanoic Acid (7a). Al-
cohol 6a (1.0 g, 2.32 mmol) was dissolved in acetone (40 mL)
and water (0.5 mL). Jones’ reagent was added dropwise at room
temperature until a persistent red-brown solution was obtained.
The reaction mixture was stirred for 1 h, added with i-PrOH (2
mL), and stirred for an additional 15 min period. Solvent was
then removed under reduced pressure, water (5 mL) was added,
and the resulting mixture was extracted with Et2O. The organic
layer was washed with H2O, brine, dried over MgSO4, filtered,
and reduced under vacuum to yield acid 7a (0.93 g, 90%) as a
white waxy solid.
3,3,4,4,5,5,6,6-Octafluorohexadecanoic Acid (7b). Compound
7b (0.37 g, 92%) was prepared from 6b following the same
procedure as described for 7a.
3,3,4,4,5,5,6,6,7,7,8,8-Dodecafluorooctadecanoic Acid (7c). Com-
pound 7c (1.64 g, 89%) was prepared from 6c following the
same procedure as described for 7a.
N-[1-(2,3-Dihydroxy)propyl]-N,N,N-trimethylammonium Chlo-
ride (8). (()-Glycidol (2 mL, 30.1 mmol), trimethylamine
hydrochloride (2.88 g, 30.1 mmol), and N,N-diisopropylethy-
lamine (5.25 mL, 30.1 mmol) in methanol (20 mL) were stirred
for 12 h at room temperature. Solvent was removed under
reduced pressure, and compound 8 (3.65 g, 71%) was purified
by recrystallization in EtOH/acetone (1:1 v/v).
Benzyl 2-Bromoethyl Phosphite (9). N,N-Diisopropylethylamine
(1.0 mL, 5.73 mmol) was added dropwise to phosphorus
trichloride (0.5 mL, 5.73 mmol) in anhydrous THF (10 mL).
The reaction mixture was cooled down to -78 °C, and benzyl
alcohol (0.6 mL, 5.73 mmol) in THF (20 mL) was added with
a syringe driver under vigorous stirring over a 3 h period. The
resulting suspension was stirred for 30 min, and a second portion
of N,N-diisopropylethylamine (1.0 mL, 5.73 mmol) was added.
Then, freshly distilled 2-bromoethanol (0.4 mL, 5.73 mmol) in
THF (20 mL) was added dropwise over a 3 h period. Stirring
was prolonged for 90 min at -78 °C before water (7 mL) was
added. The reaction mixture was warmed to room temperature,
THF was removed under vacuum, and the residue was washed
twice with AcOEt. The organic layer was dried over MgSO4,
reduced under vacuum, and purified over silica gel (n-C6H14/
AcOEt 5:5) to yield compounds 9 (1.08 g, 67%) as a slightly
yellow oil.
Benzyl 2-Bromoethyl (2,2-Dimethyl-1,3-dioxolan-4-yl)methyl
Phosphate (10). Sulfuryl chloride (4.3 mL, 53.5 mmol) was
added dropwise at 0 °C to phosphite 9 (5.0 g, 17.9 mmol) in
dry CCl4 (50 mL). Stirring was prolonged 30 min at 0 °C and
10 min at room temperature. Volatile was removed under
vacuum, and the residue was coevaporated twice with anhydrous
toluene. Benzyl 2-bromoethyl chlorophosphate was obtained as
a colorless oil and was used without purification. (R)-(-)-2,2-
Dimethyl-1,3-dioxolane-4-methanol (2.37 g, 17.9 mmol) in
anhydrous THF (200 mL) was treated with sodium hydride (60%
oil suspension, 0.86 g, 21.5 mmol) at 0 °C for 30 min. Then,
previously prepared chlorophosphate was added, and the reaction
mixture was stirred for 3 h at room temperature. THF was
removed under vacuum, saturated aqueous NH4Cl (100 mL) was
added, and the resulting solution was extracted with AcOEt.
The organic layer was washed with H2O, brine, dried over
MgSO4, and reduced under vacuum. The residue was purified
over silica gel (n-C6H14/Et2O 5:5 to 8:2) to yield 10 (4.6 g, 63%,
mixture of diastereomers) as a yellow oil.
2-Azidoethyl Benzyl (2,2-Dimethyl-1,3-dioxolan-4-yl)methyl Phos-
phate (11). Compound 10 (0.50 g, 1.22 mmol) and sodium azide
(0.10 g, 1.54 mmol) were stirred in anhydrous DMF (6 mL)
for 2 h at 50 °C. Water (15 mL) was added, and the resulting
solution was extracted with Et2O. The organic layer was dried
over MgSO4, reduced under vacuum, and purified by flash
chromatography (n-C6H14/AcOEt 5:5 to 8:2) to yield 11 (0.34
g, 75%, mixture of diastereomers) as a yellow oil.
2-Azidoethyl Benzyl 2,3-dihydroxypropyl Phosphate (12). Com-
pound 11 (118 mg, 0.32 mmol) was stirred in EtOH (8 mL)
with strongly acidic cation exchange resin Dowex 50 × 8 (H+
form) for 12 h at room temperature. The resin was filtered off,
solvent was removed under vacuum, and the residue was purified
by flash chromatography (AcOEt/EtOH 10:0 to 8:2) to yield
12 (69 mg, 64%) as a yellow oil.
(Z)-3,3,4,4,5,5,6,6,7,7,8,8-Dodecafluorohexadec-9-enoic Acid
(13). Unsaturated acid 13 (0.14 g, 99%, Z/E ) 85:15) was
obtained as a colorless oil starting from alcohol 17 and following
the same procedure as described for 7a.
2-Azidoethyl Benzyl 3-{1,2-di[(Z)-3,3,4,4,5,5,6,6,7,7,8,8-dode-
cafluorohexadec-9-enoyl]-sn-glycero} Phosphate (14). Unsaturated
acid 13 (250 mg, 0.53 mmol) in anhydrous CH2Cl2 (2 mL) was
stirred at room temperature for 4 h with oxalyl chloride (0.25
mL, 2.86 mmol) and DMF in catalytic amount. Volatiles were
removed under reduced pressure, and the residue was coevapo-
rated twice with anhydrous toluene and dissolved in CH2Cl2 (2
mL). Then, diol 12 (68 mg, 0.21 mmol) and pyridine (45 µL,
0.56 mmol) in anhydrous THF (3 mL) were added, and the
reaction mixture was stirred at room temperature for 8 h.
Saturated aqueous NH4Cl was added, and the mixture was
extracted with Et2O. The organic layer was dried over MgSO4,

“HFP” Fluorinated Cationic Lipids
Bioconjugate Chem., Vol. 21, No. 2, 2010 363
filtered, and reduced under vacuum. The residue was purified
by flash chromatography (n-C6H14/CH2Cl2/AcOEt 6:2:2 to 0:0:
10) to yield 15 (172 mg, 68%; mixture of isomers, Z/E ) 85:
15) as a colorless oil.
2-Azidoethyl 3-{1,2-di[(Z)-3,3,4,4,5,5,6,6,7,7,8,8-dodecafluoro-
hexadec-9-enoyl]-sn-glycero} Hydrogen Phosphate (15). Bromot-
rimethylsilane (35 µL, 265 µmol) was added to compound 14
(84 mg, 68 µmol) in anhydrous CH2Cl2 (5 mL). The reaction
mixture was stirred for 3 h at room temperature and moist THF
is added. Solvent was removed under vacuum, and the residue
was purified over silica gel (CHCl3/MeOH 10:0 to 6:4) to yield
compound 15 (74 mg, 95%; mixture of isomers, Z/E ) 85:15)
as a colorless oil.
vortex, the solution was added to the plasmid DNA (6 µg) in
10 mM Hepes pH 7.5, 150 mM NaCl (150 µL). The solution
was vortexed again and incubated for 20 min at room temper-
ature before addition to the cells. For gel retardation analysis,
the same concentration and procedure were used except that
the amount of DNA per sample was 1 µg.
Dynamic Light Scattering Measurements. The average
particle size and the polydispersity of the lipoplexes were
measured by using photon correlation spectroscopy (Malvern
Zetasizer nanoZS, Malvern Instruments Inc., UK) using the
multimodal analysis and the automatic mode. Lipoplex solutions
(100 µL, vide supra) were diluted 10 times, in DMEM medium
unless stated otherwise.
DNA Retardation Assays. After 15 min incubation, com-
plexes were analyzed by electrophoresis through a 1% agarose
gel. The gel was run in a 40 mM Tris-acetate-EDTA buffer pH
8.0 (TAE). DNA was then visualized with ethidium bromide
solution at 0.5 µg/mL.
3,3,4,4,5,5,6,6,7,7,8,8-Dodecafluoro-1,10-diiodotetradec-9-
enyl Benzoate (16). A degassed mixture of 1,6-diiodoperfluoro-
hexane (200 mg, 0.36 mmol), 1-octyne (80 µL, 0.54 mmol),
vinyl benzoate (75 µL, 0.54 mmol), and AIBN (12 mg, 0.07
mmol) was stirred for 15 h at 80 °C in a sealed tube. The
resulting mixture was cooled down to room temperature,
volatiles were removed under reduced pressure, and the residue
was purified by silica gel chromatography (n-C6H14/CH2Cl2 9:1
to 0:10) to yield compound 16 (105 mg, 36%, Z/E ) 15:85) as
a yellow waxy solid.
(Z)-3,3,4,4,5,5,6,6,7,7,8,8-Dodecafluorohexadec-9-enyl Alcohol
(17). Diiodoester 16 (354 mg, 0.44 mmol) in anhydrous THF
(4 mL) was added dropwise to a suspension of LiAlH4 (100
mg, 2.63 mmol) in refluxing THF (8 mL). The resulting mixture
was stirred at 66 °C for 30 min and cooled down at room
temperature, and excess reagent was slowly decomposed by
addition of solid NH4Cl. Ether (15 mL) was then added, and
the organic layer was washed with water and brine, dried over
MgSO4, filtered, and reduced under vacuum. The crude residue
was purified over silica gel (n-C6H14/CH2Cl2 5:5 to 0:10) to yield
alcohol 17 (135 mg, 68%, Z/E ) 88:12) as a colorless oil.
Film Formation and Surface Pressure Measurements. Mono-
layer experiments were performed on a computer-controlled
Langmuir film balance KSV 3000 (KSV, Helsinki, Finland).
Surface pressure isotherms were recorded on a Teflon trough
(570 × 150 mm) with a Wilhelmy plate coupled to a linear
transducer. Monolayers were spread from 0.5 mM lipid solutions
in hexane/chloroform (1/1: v/v). Volumes ranging from 100 to
300 µL were delivered at several locations across the water
surface with a Hamilton microsyringe. At least 15 min were
allowed for the spreading solvent to evaporate before monolayer
compression. The subphase consisted of purified water (pH 5.35,
Millipore filtration system, Barnstead, NANO pure 11) with a
resistivity of 18.2 ·106 Ω cm. Compressions were realized using
a hydrophilic Delrin barrier (polyacetal). Surface pressure vs
molecular area curves were recorded at 21.0 ( 0.5 °C with a
compression speed of 2-5 Å2 per lipid molecule per minute.
All measurements were carried out in triplicate. Specific areas
As, lift-off areas Alo, and collapse pressures πc were determined
as previously described (26).
Cell Culture. Human hepatocarcinoma cells (HepG2) were
cultured in DMEM supplemented with 100 units/mL penicillin,
100 µg/mL streptomycin, 292 mg/L L-glutamine, 4.5 g/L
D-glucose, and 10% heat-inactivated FCS. The syrian hamster
kidney fibroblast cell line (BHK-21) was cultured in MEM
supplemented with 100 units/mL penicillin, 100 µg/mL strep-
tomycin, 10 mg/mL L-glutamine, 50 mg/mL tryptose, and 10%
FCS. The HeLa cell line was cultured in MEM supplemented
with 100 units/mL penicillin, 100 µg/mL streptomycin, 292
mg/L L-glutamine, and 10% FCS.
Transfection Procedure. Cells were plated in 24-well plates
one day before transfection in order to obtain 60-80% confluent
cultures at the time of the experiment. Transfection mixture (110
µL containing 2 µg of DNA plasmid, vide supra) was deposited
in each well of the triplicate. After a 24 h incubation period,
cells were harvested in lysis buffer (250 µL: 8 mM MgCl2, 1
mM DTT, 1 mM EDTA, 1% Triton X-100, 15% glycerol, in
25 mM Tris-phosphate buffer pH 7.8). The cell lysate was
centrifuged for 5 min at 10 000 g to pellet cell debris. Luciferase
light units were measured in a 96-well plate format with a PhL
luminometer (Mediators Diagnostika) from an aliquot of the
supernatant (50 µL) with 10 s integration after automatic
injection of assay buffer (100 µL: lysis buffer without Triton
X-100 but supplemented with 2 mM ATP) and 167 µM luciferin
(100 µL). The protein content of each cell lysate was measured
using the BioRad protein assay. The transfection efficiency was
expressed as relative light units/10 s/mg protein (RLU/mg
protein) after subtraction of background luciferase value from
each sample value. Values of each sample are the means of
triplicate determinations.
Lipoplex Cytotoxicity Assessment. Lipoplex cytotoxicity was
assessed using a tetrazolium-based assay. Cells were seeded in
24-well culture plates at a density of 5.0 × 104 cells per well
and cultured at 37 °C for 24 h. On the next day, culture medium
was changed to fresh DMEM medium supplemented with 10%
FCS before addition of lipoplexes (100 µL) prepared as
previously described. After a 24 h incubation period at 37 °C,
culture supernatant was removed, cells were carefully washed
with PBS, and 0.5 mg/mL MTT (1 mL) in complete culture
medium was added. After a 2 h incubation period at 37 °C,
MTT solution was removed and DMSO (1 mL) was added to
lyse cells and dissolve reduced MTT. Intensity of MTT
reduction was then evaluated by measuring absorbance at 570
nm. Viability of cells treated with lipoplexes was expressed as
a percentage of the absorbance measured in untreated cells. Each
treatment was assessed in triplicate, and data are presented as
means ( sem of n ) 3 values.
RESULTS AND DISCUSSION
Molecular Design and Chemistry. To investigate the
influence of fluoroalkyl moieties located in the vicinity of the
headgroup of a cationic lipid on its transfection efficiency, we
first need a reference compound and selected the widely used
DOTAP (Figure 1). The introduction of fluoroalkyl segments
in its structure must fulfill some preliminary requirements in
terms of chemical stability. Considering the high reactivity of
R,R′-difluoroesters toward water with half-life time for hydroly-
sis in the range 0.1-5 min (27), we decided on structures in
which one methylene group lies in between the fluoroalkyl chain
Lipoplex Preparation. For transfection experiments, cationic
lipid (30 µL of a 1.8 mM solution in ethanol) and DOPE (6 µL
of a 18 mM solution in ethanol) were mixed and added to 10
mM Hepes pH 7.5, 150 mM NaCl (150 µL). After stirring by

364 Bioconjugate Chem., Vol. 21, No. 2, 2010
Klein et al.
Scheme 1. Synthesis of Compounds 1a-c and 2a-ca
a Conditions: (i) NaHCO3, Na2S2O4; (ii) Bu3SnH, AIBN; (iii) LiOH;
(iv) CrO3, H2SO4; (v) (COCl)2, N-[1-(2,3-dihydroxy)propyl]-N,N,N-
trimethylammonium chloride (8), py; (vi) Et3N.
The synthesis of compounds 1a-c was realized starting from
R,ω-diiodoperfluoroalkanes according to Scheme 1. Hemiflu-
orinated fatty acids 7a-c were key intermediates. Their
preparation involved the well-described radical addition of a
iodoperfluoroalkane to an alkenyl compound (33). However,
the homobifunctional fluorinated starting material introduced
some difficulty, as heterofunctionalization was required. The
reaction of a large excess of diiodoperfluoroalkane with 1 equiv
of alkene allows high yield but low conversion and is not
acceptable due to high cost of the fluorinated reagents. When
the reaction was carried out with diiodo and alkenyl compounds
in stoichiometric amounts, the proportion of monoalkylated
product obtained was close to 50%, which is in agreement with
statistics. Nevertheless, the process of the radical reaction
remained difficult to monitor and reduction compounds RF-H
rapidly appeared as alkene concentration decreased in the
reaction mixture. Consequently, the heterobifunctionalization
of the diiodo starting material was carried out using a one-step
procedure involving stoichiometric amounts of both alkenyl
reagents. Perfluoroalkyl iodide addition to alkene can be
promoted in many ways (by photolysis, pyrolysis, electrolysis,
free radical initiator, enzymes, metals, and transition metal
complexes). In that work, radicals were generated under the
conditions described by Rong and Keese, and using sodium
dithionite and sodium carbonate in CH3CN/H2O (34). The yield
of the double transformation was close to 30% and was higher
than that by any multistep procedure we have experienced.
Efforts to run the reaction under anhydrous conditions with
triethylborane (35) did not give better results either. Radical
reduction of compounds 4a-c using Bu3SnH and AIBN (36)
proved efficient and 5a-c were obtained in high yield. Benzoate
hydrolysis and Jones oxidation of the resulting alcohols 6a-c
afforded hemifluorinated fatty acids 7a-c (37, 38). Further
conversion into acyl chlorides allowed direct coupling with N-[1-
(2,3-dihydroxy)propyl]-N,N,N-trimethylammonium chloride 8
straightforwardly prepared from glycidol. Fluorinated cationic
lipids 1a-c were obtained in 26-28% yield. Important to note
is the high sensitivity of compounds 1a-c that very easily
eliminate HF even at room temperature. As a consequence, they
were obtained as a mixture with eliminated byproducts that were
very difficult to separate (1a, 37% elimination byproduct; 1b,
18% elimination byproduct; 1c, 11% elimination byproduct).
Figure 1. Structures of DOTAP, DOPE, and fluorinated cationic lipids
developed.
and the carboxyl group. We thus designed compounds 1a-c
with modulated lengths for fluoroalkyl and terminal alkyl
moieties. In order to target compounds with hydrophobicity
tentatively comparable with that of DOTAP, we varied the
number of methylene (m) and difluoromethylene (n) groups in
the chains, considering the “1 CF2 ) 1.5 CH2” rule (28, 29)
and its variations (1.5 CH2 e1 CF2 e 4 CH2) (30). Under mild
basic conditions, ꢀ,ꢀ′-difluoroesters easily eliminate HF, and
compounds 2a-c result from a double elimination in 1a-c (vide
infra).
A number of transfection protocols with cationic lipids use
dioleoylphosphatidylethanolamine (DOPE) as a helper lipid
(Figure 1). The role of DOPE in transfection assays has been
extensively studied (31, 32). The mechanism of action of that
lipid has been attributed to its capacity to induce transition of
a lipid bilayer from the liquid-crystalline lamellar phase into
the inverted hexagonal HII phase. Vierling et al. examined the
use of terminal fluorinated glycerophosphoethanolamines ana-
logues of DOPE as helper lipids in nonfluorinated cationic lipid-
mediated gene transfer (18, 19). A fluorinated segment was
introduced at the extremity of the ester at C1 (or C1 and C2) on
the glycerol backbone, and the resulting increase in transfection
efficiency by cationic lipids was attributed to the pronounced
cone-shaped geometry of the molecule, displaying a greater
tendency to promote a transition from the lamellar phase into
the inverted hexagonal HII phase. Considering the results
obtained with lipoplexes prepared from mixtures of 1a-c and
DOPE (vide infra), we were especially interested in evaluating
a fluorinated analogue of DOPE displaying two fluorous
segments close to the lipid polar head. Consequently, we
designed HFP compound 3 that displays all structural features
of DOPE and displays a fluorous segment in between the double
bond and the carboxyl group of the two fatty acid chains. Chain
length is less than C18 to compensate for enhanced hydrophobic-
ity due to fluorine. Stereochemistry of the double bond is Z in
order to tentatively preserve the original chain conformation of
oleoyl chains. Last, C2 stereochemistry on glycerol backbone
was imposed to fit with that in natural phospholipids.

“HFP” Fluorinated Cationic Lipids
Bioconjugate Chem., Vol. 21, No. 2, 2010 365
Scheme 2. Synthesis of the Fluorinated Analogue of DOPE 3a
Scheme 3. Synthesis of Unsaturated Hemifluorinated Fatty Acid
13a
a Conditions: (i) Na2CO3, Na2S2O4, AIBN. (ii) LiAlH4. (iii) CrO3,
H2SO4.
the fluorous part opposite the carboxyl group proved inadequate
(44, 45). Reduction using nBu3SnH-AIBN (46) gave a mixture of
cis and trans alkenes (Z:E ) 60:40) revealing extended isomer-
ization of the intermediate vinyl radical. More satisfactorily,
treatment with lithium aluminum hydride allowed extensive
reduction of compound 16 into 17 in better favor of the cis isomer
(47). Under these conditions, reduction proceeded with full retention
of the configuration (Z:E ) 88:12), and concomitant reduction of
the benzoate moiety occurred with release of the free primary
hydroxyl group. The last step consisted of oxidation of alcohol 17
that was quantitatively transformed into unsaturated hemifluorinated
fatty acid 13 upon treatment with CrO3-H2SO4.
Self-Assembly Properties of the Fluorinated Cationic
Lipids. Self-assembly properties of fluorinated cationic lipids
were investigated at the air/water interface (due to their high
content in elimination byproduct, samples 1a and 1b were not
subjected to monolayer study). Compounds were spread from
a hexane/chloroform solution, and the surface pressure π vs area
A was recorded. All the compounds analyzed exhibited similar
behavior (see Supporting Information). The pressure regularly
increased up to collapse pressure at 28-34 mN/m. No surface
pressure decrease was recorded at high lateral pressure (π < 30
mN/m) when the compression barrier was stopped, indicating
that the monolayers formed at the air/water interface were stable.
The values for specific area As, lift-off area Alo, and collapse
pressure πc for compounds 1c and 2a-c are presented in
Supporting Information.
Monolayer spread from saturated lipid 1c (containing 11%
of elimination products) collapsed in a more condensed state
than those from unsaturated compounds 2a-c. This indicates
that unsaturation to some extent opposes close packing of the
compounds. That is further supported by the lower value for
the lift-off area recorded for 1c when compared to those of
2a-c. However, the Z configuration of the double bonds in
2a-c seems to be contradictory to these results. Indeed, it is
expected that 1c and the Z conformer of 2c display comparable
monolayer properties and more particularly similar specific areas
(mean molecular area in the more condensed state of the
monolayer). The large difference in specific area between the
two compounds could then reflect some unusual conformation
of the glycerol backbone in 2c, and computational modeling
would be necessary to get a deeper insight in the packing
properties of the compounds. The measured value for specific
area of lipid 1c at the air/water interface was close to twice
that of a section of crystalline fluoroalkyl chain (2 × 24.9 Å2)
(48). That indicates that compound 1c collapsed in a highly
ordered liquid phase, as no phase transition could be detected
through isothermal monolayer compression. The same held for
compounds 2a-c though slightly higher As values were
recorded. For comparison, the As value for DOTAP is 50 Å2,
which is larger than twice the section of alkyl chain (16 Å2)
and accounts for a higher free volume within the lipidic phase.
That may be correlated to susceptibility to detergents (vide
infra).
a Conditions: (i) Et3N, BrC2H4OH, H2O; (ii) SO2Cl2, (S)-2,2-
dimethyl-1,3-dioxolane-4-methanol, NaH; (iii) NaN3; (iv) Dowex 50
× 8 (H+ form); (v) 13, (COCl)2, py; (vi) TMSBr, H2O; (vii) PPh3,
H2O.
In order to tentatively assume better control of the composition
and properties of later lipoplexes, compounds 1a-c were
quantitatively transformed into the corresponding unsaturated
cationic lipids 2a-c upon treatment with Et3N. The unsaturated
double bonds are in the Z configuration as was determined from
3
the coupling constant JH-F ≈ 29 Hz, a value which is
characteristic of the trans position between fluorine and hydro-
gen on ethylenic double bonds (39, 40). Lipid 1c, however, was
considered for further biophysical and biological investigations
(vide infra), as it was obtained at the lower contamination rate.
Synthesis of the fluorinated analogue of DOPE 3 was
achieved according to Scheme 2. Benzyl 2-bromoethyl phosphite
9 was prepared in one step from phosphorus trichloride.
Transformation of 9 into corresponding chlorophosphate allowed
condensation with (S)-2,2-dimethyl-1,3-dioxolane-4-methanol.
Resulting phosphotriester 10 was obtained in 53% yield.
Bromide nucleophilic displacement with sodium azide provided
compound 11, and the isopropylidene protecting group was
removed upon treatment with a strongly acidic cation exchange
resin. Resulting diol 12 was then acylated with unsaturated
hemifluorinated fatty acid 13 to give 14 in 68% yield. Interest-
ingly, elimination byproducts were not obtained or could not
be detected. In the preparation of compounds 2a-c, due to the
amphiphilicity of the compounds, the crude reaction mixture
was not submitted for aqueous work up but reduced in vacuo
and directly purified by silica gel chromatography. This could
possibly indicate that HF elimination occurs during concentra-
tion of the crude reaction mixture and may be avoided by
aqueous workup prior to solvent evaporation. Debenzylation of
14 was achieved with bromotrimethylsilane (41, 42). The final
step was reduction of the azide group in 15 via a Staudinger
reaction affording 3 in 51% yield.
Unsaturated fluorinated fatty acid 13 was a key compound
for the preparation of the fluorinated DOPE analogue. It was
prepared in three steps (Scheme 3). The first one involved the
reaction of RFI with an alkynyl compound (43-45). It was
similar to the preparation of 4a-c except that 1-alkene was
replaced with 1-octyne. The double radical oxidative addition
of 1,6-diiodoperfluorohexane on a stoichiometric mixture of
vinylbenzoate and alkyne furnished vinyl iodide 16 as E/Z
mixture (E:Z ) 85:15). Reduction of compound 16 under the
conditions employed by Takagi (nBuLi, Et2O, -78 °C) for
the synthesis of fluorinated analogues of oleic acid displaying

366 Bioconjugate Chem., Vol. 21, No. 2, 2010
Klein et al.
Table 1. Semiquantitative Evaluation of Lipoplex Stability in the
Presence of Surface Active Compounds
As already observed with SDS and sodium cholate, cationic
lipids 1c and 2c were revealed to be more potent, likely due to
their greater hydrophobic character (longer fatty acid chains,
higher fluorine content). The zwitterionic detergent CHAPS,
though neutral, exhibited very different behavior and destabilized
lipoplexes poorly. Most of them survived to 10-50 mM, and
once again, 1c-DNA and 2c-DNA were characterized by
higher stability. Lastly, cationic CTAB proved highly detri-
mental to DOTAP-DNA complexes but affected fluorinated
lipoplexes only weakly. Thus, even if a superficial positive
charge of the lipoplexes may shield the particles from other
cations, 1 mM CTAB revealed enough to fully destabilize
DOTAP-DNA complexes, whereas fluorinated lipoplexes mostly
resisted up to 150 mM. It is interesting to note that the stability
of 1c-DNA complexes was invariably higher than for
2c-DNA, especially in the presence of Tween 80. The
difference in lipoplex stability between 1c-DNA and 2c-DNA
probably cannot be explained just considering fluorine content
(24 vs 22 F atoms). More likely, chain conformation imposed
by unsaturation in 2c thwarts close packing of helical fluorinated
chains (vide supra), resulting in higher free volume in the
external lipid layer of the lipoplex and consequently higher
permeability. Such a permeability increase facilitates access of
detergent molecules to the condensed DNA core and favors
lipoplex destruction.
As DOPE was used as the lipid helper (vide infra), we
characterized the mixed lipoplexes (lipid/DOPE/DNA) and
looked at their stability. Considering DOTAP and DOTAP/
DOPE lipoplexes, no significant difference was observed in
detergent resistance. As DOTAP and DOPE are more likely
similarly solubilized by detergents, behavior of a mixed lipoplex
was indeed expected to be the same as for the pure DOTAP
lipoplex. For mixed fluorinated lipid/DOPE lipoplexes, we could
possibly anticipate some differences. Actually, no such deviation
was observed, and DNA condensed particles resisted detergents
in the same concentration range as with pure fluorinated cationic
lipids. Whether DOPE remained associated to the lipoplexes
or was depleted via micelle formation was not checked.
detergent resistance (mM)b
cationic lipida
SDS
(1)c
1 (10)c 100
NaCh
Tween 80 CHAPS
CTAB
DOTAP
1c
2a
2b
2c
10
<1
150
<1
<1
1
50
1
150
150
1
10
10 (50)c 150
1
1
1
10
10
50
10
150
50
10
50
1 (150)c
50 (150)c
10
10 (150)c
1
DOTAP/DOPEd
1c/DOPEd
2a/DOPEd
2b/DOPEd
2c/DOPEd
<1
<1
<1
<1
<1
(1)c
1
100
10
1 (150)c
1 (150)c
1
1
10
1
150
150
a Lipoplexes were prepared as indicated in the experimental section,
with N/P ) 3. b Detergent was added to reach 1, 10, 50, 100, and 150
mM final concentration (concentration of cationic lipid: 0.18 mM).
Concentration reported corresponds to the higher one tested at which
lipoplexes were 100% preserved. c At that detergent concentration, only
partial release of plasmid DNA ((10%) is observed. d Cationic lipid/
DOPE was in the ratio 1/2.
Characterization of Lipid-DNA Complexes. The ability
of the fluorinated cationic lipids to interact electrostatically with
DNA was studied by conventional electrophoretic DNA retarda-
tion assays in which a full retard of plasmid DNA is observed
at a lipid/DNA phosphate ratio (N/P) corresponding to electro-
neutrality. As expected, all the fluorinated lipids led to a full
complexation of the plasmid at N/P 1, indicating that the
fluorous block does not impact on the ability of the cationic
headgroup to interact with the DNA phosphate groups (data
not shown).
Cationic lipoplexes destruction may occur essentially through
ionic competition or detergence of the lipidic phase (49-52).
Both cause DNA to dissociate from the complex and release
nucleic acids then being vulnerable to nuclease attack. We
therefore evaluated the resistance of fluorinated lipoplexes in
the presence of various surfactants: anionic, cationic, or neutral
ones (Table 1). Plasmid DNA was first condensed with
fluorinated lipids 1c and 2a-c at N/P 3 to form overall cationic
complexes with putative transfection activity. Complexes were
then treated with increasing amounts of surfactant. The resulting
mixtures were homogenized and analyzed by gel electrophoresis
to allow semiquantitative evaluation of lipoplex destruction by
monitoring DNA release. Results indicate that, regardless of
the cationic lipid, lipoplexes hardly resisted to anionic surfactant
SDS. This is consistent with an effect mediated by electrostatic
competition between sulfate and phosphate for the cationic lipid
headgroup, coupled to high lipid detergence by the alkyl chain.
Sodium cholate (NaCh), the other anionic detergent tested, is a
poorer competitor of phosphate for ammonium complexation
(due to a higher pka value) and lipoplexes resisted better.
Integrity of DOTAP, 2a, and 2b lipoplexes was respected to
up to 10 mM cholate. Remarkably, the higher fluorinated
homologues 1c and 2c were revealed to form highly stable
complexes with DNA that resisted 150 mM sodium cholate,
suggesting that these lipids may be impermeable to the
hydrophobic cholyl moiety. A similar but less pronounced effect
was reported by Vierling’s group with FHP lipospermine-DNA
complexes that resist a taurocholate concentration twice as high
as the corresponding nonfluorinated lipoplex (17.5 mM vs. 10
mM) (13, 16). Next, and to minimize destabilization by anionic
contribution, we evaluated the stability of DNA lipoplexes in
the presence of neutral (Tween 80), zwitterionic (CHAPS), and
cationic (CTAB) detergents. A little surprisingly, neutral sur-
factant Tween 80 showed drastic effects on lipoplex stability.
DOTAP-DNA complexes did not resist much and were fully
destroyed even at the lowest detergent concentration investigated
(1 mM). The same held for 2a-DNA and 2b-DNA complexes.
From that series of experiments, we conclude that the fluorous
segments within the lipid structure indeed increase the stability
of the lipoplexes in the presence of surfactants, and the longer
the fatty acid chains and the higher the fluorine content, the
more stable the lipoplexes are.
It is well-known that the size of transfecting complexes is an
important parameter that impacts the cell entry mechanism of the
particles. Most cationic particles establish electrostatic interaction
with proteoglycans and enter adherent cells entrapped into endo-
somal vesicles. Escape from these recycling compartments occurs
only for complexes incorporating molecules with endosomolytic
activity and is the result of the specific activity of such endoso-
molytic element(s) (53, 54). Efficiency of current and multipurpose
lipid or polymeric nucleic acid delivery agents seems to rely on
the preparation of “large” complexes with diameter in the 100-1000
nm range for optimal cell anchorage and subsequent intracellular
routing activity. Such complexes offer excellent transfection
efficiency for adherent cell lines for which multitissular organization
does not impede accessibility of large particles to cell surface
receptors (55-60). Even if particles smaller than 100 nm might
be more suited for in vivo administration, their efficiency seems
to rely on the use of endosomolytic agents with high activity (61),
and large delivery systems have shown some effectiveness upon
regional administration (62, 63).
Classically, optimal gene transfection efficacies are obtained
when DNA lipoplexes are prepared in 150 mM NaCl at N/P
charge ratio of 3 and above. In these conditions, DNA plasmids
assemble with cationic lipids to form cationic aggregates of
optimal size. We therefore carried out dynamic light scattering

“HFP” Fluorinated Cationic Lipids
Bioconjugate Chem., Vol. 21, No. 2, 2010 367
Figure 2. TEM images of 1c/DOPE (A) and 1c/DOPE-DNA (B). Figure
shows negatively stained samples (uranyle acetate) obtained with
fluorinated lipid/DOPE molar ratio 1/2 and total lipid concentration of
1.16 mM (A) and 0.42 mM (B, see Experimental Procedures). Charge
ratio N/P is 3. Scale bars correspond to 200 nm.
Figure 4. Expression of luciferase in HepG2 cells treated with DNA
complexes with cationic lipids DOTAP (N/P 4) and 2a-c/DOPE (1/1
molar ratio, N/P 3) in serum-free conditions (gray) or in the presence
of 25% FCS (black).
One serious shortcoming of gene delivery mediated by
cationic lipids is that the transfection mostly requires serum-
free conditions to be effective. Serum proteins are negatively
charged and interact with cationic liposomes, thereby competing
with DNA for cationic lipids (50). Untimely DNA decomplex-
ation even to a low extent usually results in transfection
inhibition. To investigate the effect of serum, we then tested
transfection activity of our fluorinated lipoplexes by direct
addition of DNA complexes to cells in serum-free conditions
and in the presence of 25% fetal calf serum (Figure 4).
Lipoplexes were incubated with cells for a 4 h period before
the cell culture medium, which contained either too much or
too little FCS for optimal cell growth, was replaced with DMEM
containing 10% serum. Luciferase expression was monitored
24 h later. Whereas DOTAP transfection efficiency was invari-
ably reduced by 30-40% in the presence of serum, that of
fluorinated lipoplexes was preserved or even increased. This
might be tentatively interpreted considering that lower adsorp-
tion of plasma elements onto fluorinated particles would not
significantly provoke their degradation (as is the case with
DOTAP particles) but rather would induce or amplify sedimen-
tation, which would result in improved cellular internalization.
Having shown that the transfection activity of fluorinated
lipoplexes is not impaired by a high concentration of FCS, we
simplified the gene transfection procedure. The following
experiments were conducted on the BHK-21 cell line, which is
more convenient to culture and to handle than HepG2. DNA
lipoplexes were directly added to cells in culture medium
containing 10% serum, and transfection/toxicity activities were
measured 24 h later without any medium change. The effect of
chain unsaturation in fluorinated cationic lipids is presented in
Figure 5. At optimal charge ratio N/P 3, DOTAP/DOPE, 1c/
DOPE, and 2c/DOPE (in a 1/2 molar ratio) roughly displayed
similar transfection efficiency in BHK-21 cells. An increase of
charge ratio provoked a rapid loss of transfection activity with
saturated lipid 1c, whereas activity remained much better
preserved with unsaturated compound 2c or DOTAP.
Figure 3. Expression of luciferase in HepG2 cells treated with DNA
complexes with DOTAP and fluorinated 2a-c with various composi-
tions. Lipids were mixed with DOPE in different molar ratios (lipid/
DOPE molar ratio: white, 1/0; light gray, 1/1; deep gray, 1/2; black,
1/3). Lipoplexes were prepared in 150 mM NaCl, 10 mM Hepes, pH
7.5, at charge ratio N/P 4.
measurements for evaluating the size of lipoplexes DOTAP/
DOPE-DNA, 1c/DOPE-DNA, and 2c/DOPE-DNA, assembled
in 150 mM NaCl (cationic lipid/DOPE 1/2, N/P 3). In these
conditions, DOTAP-based lipoplexes show a mean particle size
of 140 nm. Lipoplexes prepared from 1c and 2c appear as larger
particles with mean diameter of 250 and 400 nm, respectively.
Lipoplexes were analyzed as well by electron microscopy. No
significant difference was observed between DOTAP and
fluorinated particles, which presented similar globular morphol-
ogy, except that negative staining of the latter was more difficult
and provided lower contrast (Figure 2). That last point is
consistent with some exacerbation of the peripheral hydropho-
bicity of the particles due to the presence of fluorous segments
very close to the surface exposed to the hydrophilic staining
material (UO22+).
Transfection Biology. The transfection activity of fluorinated
lipoplexes was first evaluated on HepG2 cells. The activity of
different formulations of lipids 2a-c is shown in Figure 3.
Experiments were carried out at a charge ratio commonly used
with cationic lipids (N/P 4), and DOTAP was used as a control.
Lipoplexes were classically incubated with cells in serum-free
conditions for a 4 h period before addition of serum-
complemented culture medium. The expression level of lu-
ciferase was low for 2a-c/DNA complexes. Transfection
efficiency was, however, drastically increased upon addition of
the helper lipid DOPE. With 2/DOPE molar ratio between 1/2
and 1/3, transfection efficacy of most fluorinated lipoplexes was
comparable to that of DOTAP. Further optimization of the
charge ratio (in the N/P range 1-6) indicated that the trans-
fection efficacy reached a plateau for N/P 3-4, a behavior that
was already noticed for classical cationic lipids (64).
The efficiency of DOPE as a helper lipid in fluorinated
lipoplexes was somehow uncertain, as DOPE and fluorinated
cationic lipids are supposed to segregate. In fact, DOPE was
revealed to be even more potent when mixed with compound
1c than when mixed with DOTAP (Figure 6). DOPE increased
DOTAP transfection efficiency by a 40-fold factor, whereas that
of 1c gained up to 4 orders of magnitude. From there, the
evaluation of a HFP fluorinated analogue of DOPE appeared
inevitable to us. We assumed that the mixture of 1c and
fluorinated DOPE analogue 3 would exhibit different aggrega-
tion properties than 1c and DOPE. Properties of the resulting
lipoplexes should thus differ from and, possibly, outdo those

368 Bioconjugate Chem., Vol. 21, No. 2, 2010
Klein et al.
Table 2. Mean Size of Lipoplexes in Different Media as Determined
by Light Scattering Experimentsa
DOTAP/DOPE-DNA (nm)b 1c/DOPE-DNA (nm)b
incubation medium
- FCS
+ FCS
- FCS
+ FCS
waterc
100 (0.1)
140 (0.08)
95 (0.11)
180 (0.22)
225 (0.24)
175 (0.22) 1200 (0.55) 1015 (0.47)
2080 (0.93) 675 (0.35) 680 (0.93)
731 (0.25) 1635 (1.00)
255 (0.38) 400 (0.68)
150 mM NaClc
5% glucosec
MEM
1400 (0.2)
a Complexes were prepared as described for gene transfection assays
(cationic lipid/DOPE 1/2, charge ratio N/P 3) and incubated in the
absence or presence of 10% FCS. b Polydispersity index is indicated into
brackets. c Solution contains 10 mM Hepes, pH 7.5.
Figure 5. Expression of luciferase in BHK-21 cells treated with DNA
complexes with DOTAP (white) and saturated and unsaturated fluori-
nated lipids 1c and 2c (gray and black, respectively). Cationic lipids
were mixed in a 1/2 molar ratio with DOPE. Lipoplexes were prepared
in 150 mM NaCl, 10 mM Hepes, pH 7.5. Transfection was performed
in cell culture medium containing 10% FCS.
Figure 7. Transfection efficiency of DOTAP/DOPE (gray) and 1c/
DOPE (black) in a 1/2 molar ratio as a function of medium used for
lipoplex preparation (for composition, see text). Lipoplexes were
prepared at charge ratio N/P 3, and BHK-21 cells were transfected in
the presence of 10% FCS.
lipid exchange from lipoplexes to lipid bilayer of the endosomes.
Consequent rupture of the endosome and DNA escape are
therefore reduced.
Transfection with cationic lipids is known to be significantly
affected by the procedure and medium used for the preparation
of lipoplexes. Differences probably may be interpreted by
considering the size of the resulting complexes, but not
exclusively. Therefore, we investigated the influence of the
lipoplex preparation medium on the size of the particles (Table
2) and on the level of luciferase expression in BHK-21 cells
(Figure 7). Dried lipid mixtures (DOTAP/DOPE and 1c/DOPE
in 1/2 molar ratio) were hydrated in different solutions ((i) 10
mM Hepes buffer, pH 7.5; (ii) 150 mM NaCl, 10 mM Hepes,
pH 7.5; (iii) 5% glucose, 10 mM Hepes, pH 7.5; (iv) MEM)
before DNA condensation at N/P 3. The size and gene delivery
efficacy of these complexes were then evaluated. With the
DOTAP-based lipoplex, the mean particle size was in the
100-255 nm range except in MEM (>1 µm). In the presence
of 10% FCS, size of the lipoplexes was increased by ca.
50-80%, and the larger particles (obtained in MEM) showed
poor transfection efficacy, likely because micrometric size
impairs particle endocytosis. The same was observed with
fluorinated lipoplexes unless smaller particles were produced
in 150 mM NaCl and larger in water or glucose 5%. The
fluorinated segment, however, has a significant effect on the
assembly process, and 1c/DOPE-DNA complexes formed in 150
mM NaCl were roughly twice as large as DOTAP/DOPE-DNA
complexes. Obtainment of these larger complexes resulted in
enhanced transfection activity for reasons discussed earlier. It
is important to note, in identical conditions, that the size of 2c/
DOPE-DNA complexes was 690 and 400 nm with and without
FCS, respectively, indicating that unsaturation does not signifi-
cantly alter the assembly process. The larger size of lipoplexes
and associated transfection efficacies measured when they were
prepared in the medium with higher ionic strength (150 mM
NaCl, 10 mM Hepes, pH 7.5) may be explained by considering
Figure 6. Transfection efficiency of DOTAP and fluorinated cationic
lipid 1c, pure (white) and mixed with DOPE (gray) and its fluorinated
analogue 3 (black) in a 1/2 molar ratio. Lipoplexes were prepared at
charge ratio N/P 3, in 150 mM NaCl, 10 mM Hepes, pH 7.5. BHK-21
cells were transfected in the presence of 10% FCS.
of 1c/DOPE-DNA particles. Results did not completely meet
our expectations, as 3 only improved transfection efficiency of
1c by 1 order of magnitude. However, the same quantitative
effect was observed with DOTAP and a similar effect was
observed with FHP DOPE analogues (18, 19). Although they
are surprising at first sight, these results may be rationalized
and attributed to the introduction of some lipid heterogeneity
in membranes upon fusion/internalization of liposomes/li-
poplexes and possibly modifying the internalization pathway
and intracellular trafficking. On one hand, mixed lipoplexes,
i.e., those containing both fluorinated and nonfluorinated lipids
(DOTAP/3-DNA and 1c/DOPE-DNA), most likely exhibit a
heterogeneous lipid distribution resulting from partial segrega-
tion of the fluorinated and nonfluorinated components. Li-
poplexes may thus display discrete nonfluorinated lipid domains
at their periphery. These domains are brought into close contact
with the negatively charged membrane of cells through elec-
trostatic interactions and may trigger the fusion/internalization
process. Incorporation of some fluorinated lipid components into
the cell membrane is supposed to introduce additional hetero-
geneity in the lipid bilayer, possibly resulting in endosome
destabilization. On the other hand, fusion of homogeneous fully
fluorinated lipoplexes (1c/3-DNA) with cell membrane is made
difficult due to the low miscibility of fluorocarbon-hydrocarbon
mixture. Though contact between the fluorinated cationic surface
of lipoplexes and the negatively charged cell membrane may
be tight, most probably it does not suffice to provoke efficient

“HFP” Fluorinated Cationic Lipids
Bioconjugate Chem., Vol. 21, No. 2, 2010 369
Figure 8. Expression of luciferase in cells treated with pEGFP-Luc
complexed by DOTAP/DOPE, 1c/DOPE, and 2c/DOPE (1/2 molar
ratio) at N/P 3. Three cell lines were investigated: HepG2 (white), BHK-
21 (gray), and HeLa (black). Transfection experiments were carried
out in the presence of 10% FCS.
Figure 9. Cytotoxicity of DOTAP/DOPE-DNA (gray) and 1c/DOPE-
DNA (black) on different cell lines. Experiments were conducted at
N/P 3 in culture medium supplemented with 10% FCS.
lipoplexes strongly interact with negatively charged cell mem-
branes through electrostatic forces, with potent negative impact
on cell membrane integrity and therefore cell homeostasis. In
the case of DOTAP lipoplexes, 36% cytotoxicity was observed
at N/P 6, and a similar tendency was observed with fluorinated
lipoplexes, although these complexes exhibited a limited effect,
with cytotoxicity not exceeding 10-20% at N/P 6. In another
set of experiments, cytotoxicity of 1c/DOPE-DNA was evalu-
ated at N/P 3 on three different cell lines, BHK-21, HepG2,
and HeLa (Figure 9). No real difference was observed between
DOTAP and fluorinated lipoplexes, except for the HeLa cell
line. In this cell line, whereas DOTAP lipoplexes provoked 30%
cytotoxicity, viability was fully maintained in the presence of
1c/DOPE-DNA complexes.
that increasing ionic strength exacerbates hydrophobic interac-
tions and shields electrostatic repulsions. Such conditions
allowed kinetically formed and thermodynamically unstable
cationic lipoplexes to equilibrate toward lower-energy systems
(aggregation). As two fluoroalkyl segments increase hydropho-
bicity in 1c, exacerbation of cohesive interactions by ionic
strength is stronger than with DOTAP-based particles, provoking
a stabilizing effect on the lipid phase and micellar aggregation
of higher magnitude. The behavior of 1c/DOPE-DNA in low
ionic strength media is interesting. The absence of salts is
supposed to stabilize the colloidal solution to small sizes by
favoring electrostatic repulsions (56) and large sizes were
observed. One possible explanation is that hydration of the
fluorinated lipid is much more sensitive to the medium
composition than that of classical lipids. When NaCl was
substituted for glucose, transfection efficiency was close to that
observed when lipoplexes were prepared in 10 mM Hepes buffer
pH 7.5, which is consistent with the previous results. The
negative influence of MEM on transfection efficiency might be
interpreted in terms of both equilibration between phosphate
(DNA) and carboxylate (MEM amino acids, 5-6 mM) for
ammonium complexation and possible insertion of the side chain
of the more hydrophobic amino acids into the lipid phase of
the particles. That could account for both destabilization of the
DOTAP-based particles and stabilization of 1c-based ones, the
latter being more resistant to both phenomena (vide supra) and
taking advantage of ionic strength increase to a greater extent.
Transfection efficiency mediated by nonviral vectors is
strongly affected by the cell type. We investigated three different
cell lines: HepG2, BHK-21, and HeLa (respectively hepatoma,
kidney, and epithelial cells). These cells were treated with mixed
1c/DOPE, 2c/DOPE, or DOTAP/DOPE lipoplexes in the
presence of FCS. Results are shown in Figure 8. In the BHK-
21 cell line, fluorinated lipoplexes appeared more potent than
DOTAP-based lipoplex by 1 to 2 orders of magnitude. In HeLa
cells, 2c/DOPE proved superior by almost an order of magnitude
to the other formulations. Formulations 2c/DOPE and DOTAP/
DOPE displayed same transfection efficiency in hepatoma cells
HepG2, whereas 1c/DOPE appeared less effective.
CONCLUSIONS
Although a great number of cationic lipids have been designed
and evaluated as gene delivery systems, there is still a need for
improvement in the design of synthetic lipids for gene transfer.
Novel cationic lipids with triblock structure were designed with
the purpose of achieving optimal condensation of DNA and
enhancing transfection performances. Cationic lipids displaying
a fluorous moiety close to their ammonium headgroup were
prepared. Self-assembly properties of these cationic lipids were
evaluated. They revealed efficient DNA condensing agents, and
the resulting lipoplexes displayed enhanced resistance to am-
phiphilic agents. Transfection data obtained from the fluorinated
vectors, alone or as a mixture with different helper lipids, on
different cell lines and at various charge ratio revealed in vitro
activities that are comparable to that of parent DOTAP, with
similar or lower associated toxicity. Due to the fluorous
segments within the lipid structure, lipoplexes displayed im-
proved stability, and gene delivery appeared to be regardless
of the presence of serum. On the other hand, in vitro transfection
efficiency appeared to be very sensitive to the preparation
protocol when compared to DOTAP-based lipoplexes. This work
highlights the very atypical properties of these fluorinated
triblock cationic lipids. Although their in vitro use did not lead
to a significant improvement of transfection efficiency, this result
does not prejudge their behavior in vivo, as many additional
variables, and especially resilience of delivery systems, may
induce changes in the behavior of lipoplexes in vivo. Effective
impermeabilization of the lipid phase, combined with a pre-
served ability to deliver genes across endosomal membrane,
make these molecules potent building blocks for the preparation
of novel gene delivery systems with improved stability and
resilience in an in vivo context. In vivo evaluation is currently
underway to establish the utility of these new vectors in gene
therapy.
To complete our evaluation of fluorinated lipoplexes, we
assessed their cytotoxicity as a function of charge ratio and cell
type. In a first set of experiments, BHK-21 cells were treated
with 1c/DOPE-DNA or 2c/DOPE-DNA complexes at various
charge ratios (cationic lipid/DOPE 1/2, molar ratio) and using
the same amount of DNA plasmid (2 µg) when compared to
gene transfection assays (see Supporting Information). Cell
viability generally tends to decrease with increasing N/P ratio.
That may be explained by considering that positively charged

370 Bioconjugate Chem., Vol. 21, No. 2, 2010
Klein et al.
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ACKNOWLEDGMENT
This work was supported by Association Franc¸aise contre
les Myopathies (AFM, France) and Agence Nationale de la
Recherche (ANR, France). We wish to thank Centre National
de la Recherche Scientifique (CNRS, France) and Re´gion Alsace
for a grant to J.K.
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Supporting Information Available: Characterization of
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and 2c. This material is available free of charge via the Internet
at http://pubs.acs.org.
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