Micelles of lipid-oligonucleotide conjugates: Implications for membrane anchoring and base pairing

Article abstract of DOI:10.1021/jp031188m

This report examines the organization properties of new fluorescent DNA-lipids, either alone in water or in interaction with 1-octyl-?2-D- glucopyranoside micelles or egg phosphatidylcholine vesicles. We first describe the design and the syntheses of the

Full text of DOI:10.1021/jp031188m

J. Phys. Chem. B 2004, 108, 6485-6497
6485
Micelles of Lipid-Oligonucleotide Conjugates: Implications for Membrane Anchoring and
Base Pairing
C. Gosse,*,†,‡,§ A. Boutorine,| I. Aujard,‡ M. Chami,#, A. Kononov,‡ E. Cogne´-Laage,‡
J.-F. Allemand,*,†,‡ J. Li,‡ and L. Jullien*,‡
Laboratoire de Physique Statistique CNRS UMR 8550, Ecole Normale Supe´rieure, 24 rue Lhomond,
F-75231 Paris Cedex 05, France, De´partement de Chimie CNRS UMR 8640, Ecole Normale Supe´rieure,
24 rue Lhomond, F-75231 Paris Cedex 05, France, Laboratoire de Biophysique CNRS/MNHN UMR 5153,
Muse´um National d’Histoire Naturelle, 43 rue CuVier, F-75231 Paris Cedex 05, France, and Laboratoire de
Physico-Chimie CNRS UMR 168, Institut Curie, 26, rue d’Ulm, 75248 Paris Cedex 05, France
ReceiVed: October 27, 2003; In Final Form: February 23, 2004
This report examines the organization properties of new fluorescent DNA-lipids, either alone in water or in
interaction with 1-octyl-â-D-glucopyranoside micelles or egg phosphatidylcholine vesicles. We first describe
the design and the syntheses of the conjugates. Then, we use UV-Vis absorption, steady-state fluorescence
emission, electron microscopy, and fluorescence correlation spectroscopy after two-photon excitation to show
that these DNA-lipids form spherical micelles in aqueous solution and incorporate much better in micelles
than in vesicles. We also investigate the significance of the lipophilic chains of these DNA-lipids on the
melting behavior of the double-stranded hybrids: in water melting curves are broadened whereas in amphiphilic
assemblies duplexes melt as the unconjugated controls. This work is expected to be useful for improving the
rational design of antisense medicines.
Introduction
cultures, but the mechanisms ruling drug action are still
unclear.15,21 Indeed, cholesterol-DNA was found to bind to low-
density lipoproteins receptors, allowing an alternative internal-
ization route to passive membrane anchoring followed by
pinocytosis.14 Moreover, drug activity does not always correlate
with a specific sequence, suggesting mechanisms other than
antisense inhibition.15,21
Although lots of efforts have been devoted to lDNA synthesis
and pharmaceutical characterization, data concerning the phys-
icochemical properties of such amphiphiles are scarce.17,18 Con-
sequently, the present report investigates several features of
newly synthesized lDNAs bearing a fluorescent label in their
hydrophobic moiety. The manuscript first exposes the design
and the syntheses of the fluorescent lDNAs. Subsequently, the
phase behavior of these conjugates, alone in aqueous solution,
or in the presence of organized assemblies such as micelles or
vesicles, was studied by fluorescence spectroscopy and electron
microscopy. Finally, we tried to analyze the influence of the
location of the lipophilic chains, i.e., in water or in amphiphilic
assemblies, on the melting behavior of the double-stranded
hybrids.
DNA oligonucleotides recently emerged as useful building
blocks to obtain self-assembled arrays such as large organized
molecular crystals.1 Indeed, they only exhibit strong intermo-
lecular association when their sequences are complementary.
In view of these attractive features, we have been interested to
use the DNA strategy to design bidimensional polymeric
networks2,3 reminiscent of biological structures.4-7 To restrict
cross-linking in two dimensions, we decided to rely on a 2D
template,7-12 i.e., the surface of lipid vesicles, to polymerize
amphiphilic monomers made of oligonucleotides.
The prerequisite to achieve the preceding goal was to design
and synthesize DNA-lipids (lDNAs) able to incorporate within
bilayer cores. Such an issue is closely related to the improvement
of the cellular uptake of antisense oligonucleotides by lipophilic
modification.13-15 According to this therapeutic strategy, short
single-stranded DNA (ss-DNA) first bind to cellular membrane
because of their hydrophobic moiety; then, after internalization,
they hybridize to the targeted mRNA by sequence recognition
and consequently prevent the translation of the associated
protein. Oligonucleotides have been conjugated to numerous
small lipophilic molecules including the widely used choles-
terol,13,16-18 alkyl chain,16,19 di-o-alkyl-rac-glycerol,15,16 fullerene,20
and adamantane.15 Biological effects such as inhibition of viral
protein expression by lipid-conjugates could be observed on cell
Results and Discussion
Design and Syntheses of DNA-Lipids. In relation with the
initial goal of building a 2D-polymeric network, we considered
the stepwise temperature-controlled polycondensation of a DNA
network on a lipid bilayer. Each monomer was designed as a
core DNA duplex bearing a lipophilic anchor and three single-
stranded dangling oligonucleotides at its extremities (see Sup-
porting Information; Figure 1S). More precisely, such an
assembly was aimed to be obtained by hybridization between a
long unmodified strand (ACA or A′CA′) and a lipophilic
conjugate (lGeB or lGeB′; see Scheme 2 for conjugate formula
and Experimental Section for detailed sequences). Central core
sequences (C and G) were chosen to pair at any temperature
below 50 °C and distal sequences (A and A′ or B and B′) to
reticulate complementary monomers only around 30 °C. In the
* To whom correspondence should be addressed. E-mail: charlie.
gosse@lpn.cnrs.fr. Tel: 33 1 69 63 61 55. Fax: 33 1 69 63 60 06; E-mail:
Jean-Francois.Allemand@ens.fr. Tel: 33 1 44 32 34 92; Fax: 33 1 44 32
34 33. E-mail: Ludovic.Jullien@ens.fr. Tel: 33 1 44 32 33 33; Fax: 33 1
44 32 33 25.
† CNRS UMR 8550, Ecole Normale Supe´rieure.
‡ CNRS UMR 8640, Ecole Normale Supe´rieure.
| CNRS/MNHN 5153, Muse´um National d’Histoire Naturelle.
# CNRS UMR 168, Institut Curie.
§ Present address: CNRS/LPN, Laboratoire de Photonique et de Nano-
structures, Route de Nozay, 91460 Marcoussis, France.
Present address: M. E. Mu¨ller Institute (MSB), Biozentrum, University
of Basel, Klingelbergstr. 70 CH-4056 Basel, Switzerland.
10.1021/jp031188m CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/28/2004

6486 J. Phys. Chem. B, Vol. 108, No. 20, 2004
Gosse et al.
SCHEME 1a
a (a) K2CO3, n-BuOH, 110 °C, 72 h; (b) POCl3, DMF, 100 °C, 3 h; (c) K2CO3, KI, n-BuOH, 110 °C, 12 h; (d) KOH, EtOH/CH2Cl2 1/1, 35 °C,
48 h; (e) P(Ph)3, imidazole, CCl4 reflux, 3 h; (f) NaI, 2-butanone reflux, 48 h; (g) NaN3, DMF, 80 °C, 12 h; (h) P(Ph)3, H2O, THF, 24 h.
TABLE 1: Solvatochromism of the Chalcone Chromophorea
the condensation of acetophenone derivatives on aldehydes. It
is generally performed in alcohols sometimes mixed with water.
Basic conditions are generally used for the condensation.24,25
Catalytic amounts of bases were surprisingly not sufficient to
afford satisfactory yields; instead, stoichiometric amounts were
used as already reported.25,26 Aldehydes parasubstituted with
electron donating groups such as amine are deactivated and lead
to modest yields as observed in the present case in agreement
with the literature.26,27 Thus, 4 and 7 were condensed under
basic conditions (progressive addition of potassium hydroxide)
in a mixture of ethanol and methylene chloride to yield the
chalcone hydroxy-terminated lipid 8. The latter was then
converted into the corresponding chloride 9 using triphen-
ylphosphine and imidazole in carbon tetrachloride.28 9 was
eventually transformed either into the iodide derivative 10 by
refluxing in butanone in the presence of potassium iodide, or
into the amino derivative 12 after triphenyl phosphine reduction
of the azido derivative 11 resulting from substitution of chloride
by sodium azide in DMF.
chalcone dielectric
λmax
λ
em (λexc)
(nm)
solvent
substrate constantb (nm)
ethyl acetate
8
6
8
416
408
424
418
427
425
430
430
429
419
492 (426)
484 (418)
518 (434)
513 (428)
556 (437)
523 (435)
533 (440)
559 (430)
560 (430)
530 (430)
551 (430)
555 (430)
tetrahydrofurane
methylene chloride
acetone
8
8
9
8
21
25
37
46
78
78
ethanol
8
dimethylformamide
dimethyl sulfoxide
H2Oc
8
8
lGeB
lGeB′
lGeB′
lGeB
lGeB′
H2Oc
OG micelles in H2Od
EPC vesicles in H2Oe
EPC vesicles in H2Oe
a Wavelengths of the maxima of absorption (λmax) and emission (λem
;
excitation at λexc) in different solvents. Measurements were performed
at 298 K at micromolar concentrations. b Values extracted from ref 30.
c 100 mM NaCl, 10 mM Na(CH3)2AsO2-HCl, pH 7 buffer. d [OG] )
20 mM in 100 mM NaCl, 10 mM Na(CH3)2AsO2-HCl pH 7 buffer.
e [EPC] ) 1.2 mM in 100 mM NaCl, 10 mM Na(CH3)2AsO2-HCl pH
7 buffer.
lDNAs were obtained from coupling between the appropriate
oligonucleotides and the fluorescent lipid backbone. Two
different coupling procedures were investigated (Scheme 2).29
In the first, conjugate is obtained by reaction between the
activated 5′ terminal phosphate of the oligonucleotide and the
amino lipid 12. The second way of synthesis included attache-
ment of a cysteamine moiety to the terminal 5′ phosphate,
reduction of the product by dithiothreitol to generate a terminal
sulfhydryl group, and alkylation of this group by the iodide
chalcone 10. The presently investigated lDNA, lGeB and lGeB′,
were obtained using the second procedure; however, no essential
differences were found between conjugates whatever the
synthetic route followed. Moreover, we would like to point out
that classical acrylamide gel or reverse phase HPLC purifications
were not possible due to the aggregation of lDNA in micelles
(vide infra). Consequently, as for fullerene conjugates,20 mixed
previous notations, l is a chalcone fluorophore-bearing lipid,
and e is a tri(ethylene-glycol) spacer that had to be introduced
for preventing interactions between 3′ and 5′ dangling strands
facing each other at the duplex end (Table 1S). The chalcone
motif was selected as a polarity fluorescent reporter in relation
to its favorable photophysical properties and easy synthetic
access (vide infra).
The fluorescent lipid backbone l was first synthesized
(Scheme 1). Dialkylation of aniline 1 was performed by
bromohexadecane 2 in n-butanol.22 The resulting N,N-dihexa-
decyl aniline 3 was converted to 4-N,N-dihexadecylaminoben-
zaldehyde 4 by the Vilsmeier-Haack reaction.23 The acetophen-
one unit 7 was prepared from alkylation of the para-hydroxy-
acetophenone 5 with 2-[2-(2-chloroethoxy)ethoxy]-ethanol 6 in
n-butanol in the presence of potassium carbonate and potassium
iodide. The key reaction to build the chalcone chromophore is

Micelles of Lipid-Oligonucleotide Conjugates
J. Phys. Chem. B, Vol. 108, No. 20, 2004 6487
SCHEME 2a
a (a) P(Ph)3, dipyridyl disulfide, DMAP, DMSO, 15 min; (b) RNH2, TEA, DMSO, 2 h; (c) DTT, Tris HCl pH 7.5, 1 h; (d) RI, TEA, pyridine,
12 h. The lipid backbone is designated by the short-hand l in the text. See Experimental Section for detailed oligonucleotide sequences.
Triton X-100/lDNA micelles were purified on agarose, resulting
in a relatively low overall yield (∼10%).
Investigation of the Photophysical Properties of the
Chalcone Chromophore as a Fluorescent Label. The signifi-
cance of solvatochromism on absorption and emission features
of the chalcone chromophore was first examined on the model
chalcone lipid 8 (Table 1). The organic solutions of 8 are yellow
(absorption in the 400-430 nm range) and emit a strong green
fluorescence (480-560 nm). The observed positive solvato-
chromism, i.e., the spectra red shift with rising solvent polarity,
is indicative of an increase of the dipolar molecular moment
upon light absorption;30 it conforms to the reported features from
different 4-amino phenyl-substituted chalcones.31 This behavior
made suitable the use of the synthesized chromophore as a
polarity-sensitive fluorescent label32 for investigating the parti-
tion properties of lGeB and lGeB′ between the aqueous phase
and organized assemblies.33
Supramolecular Organization of DNA-Lipids in Aqueous
Solution. In the micromolar range, lGeB and lGeB′ gave yellow
nondiffusing aqueous solutions at room temperature in 100 mM
NaCl, 10 mM Na(CH3)2AsO2-HCl pH 7 buffer. The red-shifted
maxima for the chalcone absorption and emission (Table 1)
suggested that the chromophore was surrounded by a polar
medium. These observations supported at first sight that these
lDNAs were monomeric in water. To control the latter, a 2.5
µM lGeB solution was examined by electron microscopy (EM).
Numerous small spherical aggregates (diameter range: 8-15
nm) were distinctly observed (Figure 1b), suggesting the
presence of small micelles made of pure lGeB. Because the
conditioning of the samples for EM observation could promote
the formation of the micelles, we independently investigated
the structure of the lGeB solution by measuring the lateral
Figure 1. Titration of 2.5 µM lGeB by EPC vesicles as observed by
electron microscopy. (a) control experiment: EPC vesicles alone ([EPC]
) 18 mM); (b) [EPC] ) 0 mM; (c) [EPC] ) 18 mM.
diffusion coefficient of the conjugate in diluted solutions. Indeed,
diffusion coefficients are sensitive to the shape and size of the
diffusing objects; thus, they can be fruitfully used to evidence
the formation of any aggregates.34,35 In view of the low amounts
of available material and of the intrinsic fluorescence of the
present chalcone, fluorescence correlation spectroscopy (FCS)36
was the technique selected for this experiment.37 The diffusion

6488 J. Phys. Chem. B, Vol. 108, No. 20, 2004
Gosse et al.
coefficients measured at 293 K were converted into hydrody-
namic radii by using the Stokes law (see Experimental Section,
eq 8) that accounts for an isotropic reorientation of the objects.
At 10 nM, the apparent hydrodynamic radius of lGeB in 100
mM NaCl, 10 mM Tris-HCl pH 7 buffer was found to be equal
to 11 ( 2 nm using the reported 2.7 nm hydrodynamic radius
of 1-octyl-â-D-glucopyranoside (OG) micelles38 for calibration
(vide infra). This value strongly exceeded the molecular size
of lGeB but was in fair agreement with the size of the aggregates
observed by EM. It supported that lGeB spontaneously forms
stable micelles at concentrations as low as a 10 nM. Assuming
that nucleotidic strands are spheres, approximately 2 nm in
radius,37 which are compactly arranged at the aggregate surface,
we can estimate a crude average number of lDNAs per micelle
of about 20. In relation to solvatochromism, the large polarity
of the micellar core of lGeB could express a poor packing of
alkyl chains and a resulting hydrophobic center rather open to
the penetration of water molecules.
Despite the large volume of the DNA headgroup and the
possible attractive interactions between the dangling nucleic
bases and the alkyl chains, lGeB gives micelles exhibiting a 10
nM upper limit of the critical micellar concentration (CMC).
Studies on cholesterol conjugated 10mers reported either a 150
µM CMC17 or no CMC at all below 1 mM.18 This large
difference with our CMC value can be interpreted as a
consequence of the marked hydrophobic nature of the chalcone
lipid used here. Indeed, if we roughly consider that lipid 8 is
equivalent to a cholesterol molecule plus two C16 alkyl chains,
the two CMC should differ, following theoretical derivations,
by a factor 216 i.e., ∼65000.39
At 293 K, the equilibrium constant Kwfpm characterizing
lGeB partition between the buffer and the pure lGeB micellar
phase (respectively denoted w and pm in subscript):
lGeB (buffer) ) lGeB (lGeB micelles)
(1)
Figure 2. Titration of lGeB′ (450 µL; initial concentration 3 µM) by
1-octyl-â-D-glucopyranoside (titrating solution: 200 mM) in 100 mM
NaCl, 10 mM Na(CH3)2AsO2-HCl pH 7 buffer at 293 K. (a) Evolution
of the absorption spectra after addition of 0 µL (thin solid line), 20 µL
(thin dotted line), 40 µL (thin solid line), and 60 µL (thick dotted line;
corresponds to OG CMC) of titrating solution; (b) Evolution of the
emission spectra (λexc ) 430 nm) after addition of 0 µL (thin solid
line), 20 µL (thin dotted line), 40 µL (thin solid line), 60 µL (thick
dotted line; corresponds to OG CMC) of titrating solution.
is thus larger than CMC-1 39 that is 108.40 This result bears much
,
significance for optimizing the partition coefficients of lDNAs
between the aqueous phase and organized assemblies such as
vesicles. In fact, anchoring of the lipid backbone within
hydrophobic cores was anticipated as the major driving force
for favoring extraction from water. However, in the present
system, lipid chains are buried inside micelles, already kept out
of contact with water molecules. Consequently, weaker entropic
considerations arising from lateral steric interactions between
headgroups of lDNAs and involving the curvature of the
organized medium will probably represent the only driving force
that controls incorporation in other organized assemblies.
Partition of DNA-Lipids between Water and 1-Octyl-â-
D-glucopyranoside Micelles. The interaction between the
lDNAs and OG was first approached by titration of 3 µM lGeB′
by 200 mM OG in 100 mM NaCl, 10 mM Na(CH3)2AsO2-
HCl pH 7 buffer at 293 K. Figure 2 display the results of this
experiment followed by UV-Vis absorption and steady-state
fluorescence emission spectroscopies. The absorbance of lGeB′
decreased and became blue-shifted after addition of the first
aliquot of OG solution; then, one observed a monotonic increase
of extinction at larger OG concentrations. Meanwhile, the
fluorescence emission first increased and became blue-shifted
up to OG concentrations lying in the range of its critical micellar
concentration (25 mM in water at 298 K38); beyond this
concentration, it did not evolve anymore. Similar observations
were made with lGeB. They can be explained on the basis of
the phase behavior of OG molecules that, below their CMC, at
least partially anchor into micelles initially made of pure lGeB′.
Following this assumption, the consequences should be 2-fold.
First, the average distance between lDNA molecules is in-
creased, leading to decrease the short-range strong electronic
coupling41 existing between chalcone chromophores initially at
close contact. Although difficult to quantitatively evaluate in
the absence of a satisfactory description of the relative orienta-
tion and distance between the transition dipole moments in initial
micelles, the absorption alteration in the very first stages of the
present titration (blue- and hypsochromic shift) could be
typically observed.34 Second, addition of cone-shaped OG
molecules is anticipated to render the micellar core less
permeable to water molecules, and thus more hydrophobic, by
better accommodating smooth micellar shapes. In view of the
solvatochromic chalcone behavior, blue-shifts in absorption and
emission should be observed as in the present case (Table 1).
In addition, the corresponding increase of fluorescence quantum
yield of the chalcone chromophore during titration conforms
to the reported behavior of 4-dimethylaminochalcone interacting
with several detergents.42 Finally, above OG CMC, increasing
amounts of OG micelles are formed; they induce light scattering,
promoting the increase of extinction observed in Figure 2a. In

Micelles of Lipid-Oligonucleotide Conjugates
J. Phys. Chem. B, Vol. 108, No. 20, 2004 6489
performed on OG micelles labeled with 8 at 10 nM provided
the same value of the hydrodynamic radius as the asymptotic
one observed in this second regime. Consequently, the whole
R([OG]) curve was calibrated using reported values of the
hydrodynamic radius for OG micelles.38
The present observations suggest that mixtures of lGeB′ and
OG molecules give homogeneous lyotropic organized as-
semblies in the whole range of the investigated concentrations.
Small micelles are present at both extremities of the corre-
sponding phase diagram. In the intermediate regime in which
OG is present below its CMC, one observes the formation of
large assemblies43 whose core is suggested to be more hydro-
phobic than the one of the initial lGeB′ micelles, indicating a
better packing of the hydrophobic moieties of the lipids. The
absence of heterogeneity supported by FCS observations at 10
nM lDNA concentration allows us to evaluate an upper limit
of 108 for the equilibrium constant Kpmfmm at 293 K of lGeB′
partition between the pure (pm) and the mixed micellar (mm)
phases:
lGeB′ (lGeB′ micelles) ) lGeB′ (OG-lGeB′ micelles) (2)
Partition of DNA-Lipids between Water and EPC Large
Unilamellar Vesicles. We used large unilamellar vesicles (LUV)
made from egg phosphatidylcholine (EPC) to evaluate the
partition of the present lDNAs between water and lipid bilayers.
In a first series of experiments, we titrated 0.8 µM lGeB by
increasing amounts of 3.6 mM EPC LUV in 100 mM NaCl, 10
mM Na(CH3)2AsO2-HCl pH 7 buffer at 293 K. Both the
intensity and the steady-state anisotropy r of fluorescence
emission of the chalcone lipid were recorded as a function of
EPC concentration up to 1.5 mM (Figure 4). As observed during
the titration of the lDNAs by OG, addition of EPC vesicles
promoted at the same time a blue-shift and an increase of the
fluorescence emission of the chalcone. These features are in
line with an increase of hydrophobicity of the chromophore
surroundings upon titration. In addition, the steady-state emission
anisotropy r increased in the presence of EPC vesicles. This
trend can be interpreted as a local increase in viscosity due to
interaction between lDNA and EPC assemblies. Indeed, emis-
sion anisotropy r expresses the depolarizing motions occurring
during the lifetime τ of the excited state.32 As an example, the
Perrin’s relation
Figure 3. (a) Autocorrelation curve recorded at 293 K from a solution
10 nM in lGeB and 23 mM in OG (λexc ) 780 nm) leading to τD ) 36
µs; (b) Evolution of the hydrodynamic radius RH as extracted from the
titration by FCS of lGeB (10 nM) by 1-octyl-â-D-glucopyranoside
(titrating solution: 200 mM) in 100 mM NaCl, 10 mM Tris-HCl/Tris
pH 7 buffer.
contrast, no major evolution is observed in fluorescence emission
(Figure 2b), in line with the absence of influence of light
scattering on the fluorescence intensity in the investigated OG
concentration range.
1
r
1
r0
τ
τc
)
1 +
(3)
(
)
The titration of a 10 nM lGeB′ solution by 200 mM OG in
100 mM NaCl, 10 mM Na(CH3)2AsO2-HCl pH 7 buffer was
also followed by FCS at 293 K. Figure 3a displays a typical
autocorrelation curve. Satisfactory fits were obtained during the
whole titration by assuming a single purely diffusive behavior
obeying eq 6 (see Experimental Section). This observation
strongly suggested that the structures in solution were rather
homogeneous at any step of the titration. The characteristic
diffusion time τD for crossing the excitation volume was
extracted from each experimental curve by fitting, and it was
converted after viscosity correction into a hydrodynamic radius
RH according to eqs 7 and 8 (see Experimental Section and
Supporting Information, Figure 5Sa). Figure 3b displays the
evolution of the hydrodynamic radius of lGeB′-containing
organized assemblies as a function of OG concentration. The
behavior is nonmonotonic: the hydrodynamic radius first
increases sharply up to 4 mM in OG with spherical micelles of
pure lGeB′ being transformed in much larger assemblies. Then,
in a second regime, the hydrodynamic radius drops quickly and
stabilizes above OG CMC. An independent measurement
applies for isotropic rotations where r0 designates the funda-
mental anisotropy characteristic of the chromophore and τc the
molecular correlation time. The quantum yield of fluorescence
was larger in the presence of EPC LUV, implying a longer
lifetime of the excited state (those two parameters are propor-
tional). Since both r and τ increased, we conclude that the
correlation time of the chalcone chromophore also strongly
increased in the presence of EPC LUV; the surrounding medium
of the fluorescent label became more viscous upon vesicle
addition. The same type of titration was performed with 7.1
mM EPC LUV labeled with 2% (mol/mol) rhodamine-phos-
phatidylethanolamine. Because of energy transfer of electronic
excitation from the chalcone to the rhodamine, a decrease of
the chalcone emission and an increase of the rhodamine one
were simultaneously anticipated if the distance between both
chromophores would drop sufficiently during the experiment.32
Such a behavior was indeed observed (see Supporting Informa-
tion, Figure 2Sa,b). It supports that mixed assemblies from lGeB

6490 J. Phys. Chem. B, Vol. 108, No. 20, 2004
Gosse et al.
TABLE 2: Affinity Constants Kmix of the Conjugates for
EPC Vesicles and Alteration of the Emissive Properties of
the Chalcone Fluorophore during Fluorescence-Based
Titration by LUVs (λexc ) 430 nm)a
IF(∞)/IF(0) (λobs in nmc)
b
lDNA
Kmix
lGeB
1300 ( 500d
400 ( 200e
1300 ( 500f
800 ( 400d
2.0 (535)d
3.4 (535)e
0.6 (540)f
1.8 (535)
lGeB′
a See text and Experimental Section. b See eq 9; standard state: ideal
solute at 1 M for the infinitely diluted solution; data collected at 293
K. c Wavelength at which data analysis was performed according to
eq 9. d As evaluated from the evolution of the fluorescence intensity
of the chalcone lDNA-lipid as a function of R. See eq 9. e As evaluated
from the evolution of the steady-state emission anisotropy r of the
chalcone lDNA-lipid as a function of R. See eq 9. f As evaluated from
the evolution of the fluorescence intensity of the chalcone lDNA-lipid
as a function of R in the presence of Rh-PE-containing vesicle. See eq
9.
coexistence of spherical micelles and vesicles was continuously
observed. Using EM photographs from the titrating vesicle
suspension and from the initial micellar solution of lGeB as
references (respectively, Figure 1, panels a and b), we concluded
that (i) lGeB did not promote any vesicle breakage; (ii) the
concentration in micelles did not considerably drop during the
titration until 2-3 mM of EPC were added. These observations
led us to mainly interpret the process observed by steady-state
emission of fluorescence in the sub-millimolar EPC concentra-
tion range as a scrambling of lipids among present micelles and
vesicles. Such exchanges of lipids at constant morphologies have
been already reported between vesicles.45 Above [EPC] ≈ 2-3
mM, the concentration in micelles noticeably drops, and, at 20
mM, one essentially only distinguished vesicles on EM photo-
graphs (Figure 1c). The EM investigation thus suggested that
the value of the equilibrium constant Kmfv associated to the
reaction:
Figure 4. Titration of 0.8 µM lGeB by 3.6 mM EPC LUV at 293 K.
(a) Collection of steady-state emission spectra corrected from dilution
upon excitation at 430 nm as a function of the EPC concentration of
vesicle suspension; (b) Normalized fluorescence intensity IF(430, 535)
(empty circles) and steady-state emission anisotropy r (filled circles)
at 535 nm as a function of the ratio R ) [EPC]tot/[lGeB]tot. The fits
according to eq 9 are respectively displayed as dotted (IF(430, 535))
and solid ( r ) lines. λexc ) 430 nm. See Experimental Section.
lGeB (micelles) + EPC (vesicles) ) lGeB-EPC (vesicles)
(5)
was at room temperature in the 100 range (roughly the inverse
of the EPC concentration where half the micelles had disap-
peared).
To obtain a better estimate of Kmfv without possible
interference associated with sample conditioning for electron
microscopy, the titration of 10 nM lGeB by 9.3 mM EPC LUV
was followed by FCS in 100 mM NaCl, 10 mM Tris-HCl pH
7 buffer at 293 K. The autocorrelation curves collected during
the titration displayed a biphasic behavior that was more readily
observed in log-log plots such as in Figure 5a. Under the
assumption, supported by EM observations, of the coexistence
of two different fluorescent species, micelles and vesicles, these
curves were analyzed according to eq 11 to extract the two
prefactors gmicelles and gvesicles (see Experimental Section). The
average concentration of chalcone-containing LUVs was then
computed for each titration point and finally fitted according
to eqs 14 and 15 (Figure 5b) to obtain 20 as an estimate for
Kmfv at 293 K, which is in line with the order of magnitude
derived from EM observations.
and EPC LUV were formed. The experimental data were
quantitatively analyzed by modeling the interaction between
lGeB and EPC by the complexation reaction of equilibrium
44
constant Kmix
:
lGeB (micelles) + EPC (vesicles) )
lGeB-EPC (mixed assembly) (4)
Similar observations were made with lGeB′, and Table 2 sums
up the results for both lDNAs. Association constants Kmix in
the 500-1500 range were found at 293 K. Hence, starting from
a solution of micelles of lDNAs in the micromolar range, these
evaluations suggested that incorporation in EPC vesicles would
mainly occur over by [EPC] ≈ 2-3 mM.
During our experiments, drawbacks of steady-state fluores-
cence spectroscopy were 2-fold: it could not be used at EPC
concentrations larger than a few millimolar due to light
scattering by LUVs, and it did not provide any global structural
information on the sample. Therefore, we independently used
electron microscopy to investigate lDNA partition. The mor-
phology of lDNA-EPC mixed assemblies was examined upon
titration of 2.5 µM lGeB by EPC LUV up to [EPC] ) 25 mM:
The present series of experiments demonstrates that the
mixture of lDNA and EPC is heterogeneous in a large interval
of EPC concentration (0-2 mM) where vesicles and micelles
coexist while essentially exchanging lipids. Larger concentra-
tions of EPC are required to promote complete incorporation
of lDNA within vesicle walls. This result is in marked contrast

Micelles of Lipid-Oligonucleotide Conjugates
J. Phys. Chem. B, Vol. 108, No. 20, 2004 6491
TABLE 3: Melting Temperatures of the ds-DNA and
ds-lDNA Duplexes as Measured from Denaturation
Experiments Followed by Absorption at 260 nm or by
BOBO3 Fluorescencea
conjugated oligonucleotidec
ds-DNA
sequences
nonconjugated
oligonucleotide
without
Triton X-100
with
Triton X-100
G/C
53, 56b
54
GeB/C
51 (b)
50
GeB′/C
GeB/ACA
GeB′/A′CA′
53
50 (b)
51
51
51
?
?
52 (b)
50 (b)
a Buffer 100 mM NaCl, 10 mM Na(CH3)2AsO2-HCl pH 7; concen-
trations of 2 µM per strand). When present, Triton X-100 was at 0.36
mM concentration. See text and Experimental Section. b Measured by
the assay relying on BOBO3® fluorescence. c (b) refers to a broad
transition, ? denotes that it was impossible to determine the melting
temperature during the corresponding experiment.
Figure 5. (a) Autocorrelation curve recorded at 293 K from a solution
Figure 6. Experimental DNA denaturation curves from different
experiments involving G/C pairing: GeB′/C (circles), lGeB′/C (squares),
lGeB′/C + 0.36 mM Triton X-100 (diamonds). 2 µM concentration
for each DNA single strand. 100 mM NaCl, 10 mM Na(CH3)2AsO2-
HCl pH 7 buffer. To improve reading, a vertical offset was added.
10 nM in lGeB and 0.85 mM in EPC (λexc ) 780 nm) leading to τD
micelles
) 38 µs and τDvesicles ) 7.8 ms; (b) Evolution of 1/[gvesicles(1 + (gmicelles
/
g
vesicles))2] as a function of R ) [EPC]tot/[lGeB]tot during the titration
by FCS of 10 nM lGeB by 9.3 mM EPC vesicles in 100 mM NaCl, 10
mM Tris-HCl/Tris pH 7 buffer at 293 K. See text and Experimental
Section.
ing self-aggregation that hindered incorporation of conjugates
into vesicle bilayers. In relation with the initial goal and with
potential use as antisense drug, we were also concerned with
the possible interference of lDNA alkyl chains in the pairing
process between complementary oligonucleotides. In fact, the
lipid backbone was anticipated to interact with the nucleic bases
either in the single or in the double-stranded DNA form.
We first measured the melting temperatures (Tm) for different
combinations of complementary strands (G/C, GeB/C, GeB′/
C, GeB/ACA, GeB′/A′CA′). Either the absorbance at 260 nm
or the fluorescence emission from the intercalating agent
BOBO3 were recorded as a function of the temperature (See
Experimental Section). Tm values ranging between 50 and 55
°C, associated to sharp transitions, were typically obtained
(Table 3 and Table 1S). In contrast, when lGeB and lGeB′ were
used for pairing, we observed either broad transitions (lGeB/C
and lGeB′/C) or no transition at all (lGeB/ACA and lGeB′/
A′CA′; Figure 6). After addition of the detergent Triton X-100
above its CMC, the transitions arising from the same combina-
tions of complementary strands became much more visible
around 50-52 °C: sharp for lGeB/C and lGeB′/C, broad for
lGeB/ACA and lGeB′/A′CA′.
with the one obtained for the largely favored transfer of the
conjugate into OG micelles. In fact, anchoring ss-DNA at a
bilayer surface has an entropic cost because of the polymer
confinement in a half-space; meanwhile, changing the hydro-
phobic environment of the chalcone (from a micellar core to
phospholipidic chains) does not necessarily lead to an important
enthalpic gain. Comparatively, the transfer into the small
ellipsoidal OG micelles38 occurs at nearly constant surface
curvature, whereas the micellar core becomes more hydrophobic
due to the presence of numerous small detergent molecules.
Finally, we can notice that the insertion in LUVs is more
difficult for chalcone conjugate than for cholesterol ones. From
the literature18 and on the basis of a simple 1:1 complexation
model,44 we could evaluate an underestimate equal to 103 for
the partition coefficient of cholesterol-DNA between the solution
and vesicles. The latter value is about hundred times larger than
Kmfv. Our interpretation for this difference of behavior relies
on the less pronounced hydrophobicity of cholesterol with regard
to the present chalcone moiety: cholesterol-DNA anchors more
easily in bilayers because they do not form stable micelles in
water.
Pairing Properties of lDNAs. The preceding paragraphs
emphasized the significance of the hydrophobic tail in determin-
Terminal lipophilic groups have variously been reported to
decrease,15,17,21 or have no effect at all16,21 on stability of

6492 J. Phys. Chem. B, Vol. 108, No. 20, 2004
Gosse et al.
oligonucleotide duplexes. The Tm variations were always small
(less than 5 °C), and the present data are thus in line with
previously published articles. However, the corresponding
melting curves are scarce in the literature and we cannot evaluate
whereas the broadening effect observed here is common.
In our experiment, the chalcone-DNA duplexes seem to start
melting at a lower temperature than the nonconjugated ones.
The denaturation curves cannot be fitted anymore by a simple
all-or-none two-state model,46 either because of a polydispersity
effect or because of a more complex melting mechanism. Such
observations demonstrate that the presence of the hydrophobic
tail and the subsequent conjugate aggregation hinder at least
partially hybridization between complementary strands. In fact,
interaction between the alkyl chains and the nucleic bases is
expected to decrease the affinity for pairing. Consequently, the
recovery of the duplex melting properties when lDNA is mixed
with Triton X-100 can be interpreted as follows: like when
OG is present the lipophilic chains are buried into the micelle
core; hence, they cannot interact anymore with the aromatic
bases of DNA.
plates. Commercially available reagents were used as obtained.
Dry solvents were distilled according to classical procedures.
N,N-Dihexadecylaniline (3). A suspension of freshly distilled
aniline 1 (5 mL, 54 mmol, 1 equiv), 1-bromohexadecane 2 (50
mL, 162 mmol, 3 equiv), dry potassium carbonate (22.4 g, 162
mmol, 3 equiv) and dry potassium iodide (10.75 g, 65 mmol,
1.2 equiv), in dry n-butanol (100 mL) was vigorously stirred
under nitrogen at 110 °C for 72 h. After cooling to room
temperature, the mixture was filtered and the n-butanol was
evaporated under vacuum. The residue was dissolved in ether
(100 mL) and washed with distilled water (3 × 50 mL). The
organic layer was dried over potassium carbonate and concen-
trated under reduced pressure. Recrystallization in absolute
ethanol gave 3 as white crystals (25.0 g, 85% yield). 1H NMR
(CDCl3, 200 MHz, δ, ppm): 7.24-7.16 (m, 3H), 6.63 (m, 2H,
J ) 8.0 Hz), 3.24 (t, 4H, J ) 7.6 Hz), 1.60-1.20 (m, 56H),
0.89 (t, 6H, J ) 6.4 Hz); 13C NMR (CDCl3, 50 MHz, δ, ppm):
148.0, 129.0, 114.9, 111.5, 50.9, 31.8, 29.5, 29.5, 29.5, 29.5,
29.4, 29.2, 27.1, 27.0, 22.5, 14.0; C38H71N (541.99): Anal.
Calcd., C,84.21; H,13.20; N, 2.58; Found, C,83.93; H,13.63;
N, 2.61.
Conclusion
4-N,N-Dihexadecylaminobenzaldehyde (4). Phosphoryl chlo-
ride (2 mL, 21.6 mmol, 1.2 equiv) was added to a solution of
dry N,N-dimethylformamide (50 mL, 0.64 mol, 35 equiv) at 0
°C under nitrogen. The mixture was warmed to 50 °C for 10
min when a yellow-orange color appeared. A solution of 3 (9.75
g, 18 mmol, 1 equiv) in dry N,N-dimethylformamide (30 mL)
was added and the mixture was warmed to 100 °C for 3 h. After
cooling to room temperature, the mixture was quenched by
adding water (50 mL), neutralized with a saturated aqueous
solution of NaHCO3 and extracted with cyclohexane (100 mL).
The organic layer was washed with distilled water (3 × 50 mL),
dried over potassium carbonate, and concentrated under reduced
pressure. After recrystallization in absolute ethanol, white
crystals of 4 were obtained (8,7 g, 85% yield). 1H NMR (CDCl3,
200 MHz, δ, ppm): 9.70 (s, 1H), 7.70 (d, 2H, J ) 8.9 Hz),
6.64 (d, 2H, J ) 8.9 Hz), 3.34 (t, 4H, J ) 7.6 Hz), 1.60-1.20
(m, 56H), 0.89 (t, 6H, J ) 6.4 Hz); 13C NMR (CDCl3, 50 MHz,
δ, ppm): 189.8, 152.5, 132.0, 124.4, 110.6, 51.0, 31.9, 29.6,
29.6, 29.6, 29.5, 29.5, 29.4, 29.3, 27.1, 27.0, 22.6, 14.1; C39H71-
NO (570.00): Anal. Calcd., C, 82.17; H, 12.55; N, 2.45; Found,
C, 82.02; H, 12.57; N, 2.40.
Aiming to investigate the insertion of lipid-oligonucleotides
into model bilayer, we synthesized conjugates labeled in their
hydrophobic moiety by a solvatochromic chalcone. These
derivatives were found to form micelles in aqueous solution
with a critical micellar concentration lower than 10 nM. The
implications related to the behavior of these DNA lipids were
2-fold. First, the gain in Gibbs free energy upon transferring
the present DNA lipids from the pure micellar phase to the
bilayer of small unilamellar vesicles was as low as a few kBT.
Partition between pure micelles and EPC vesicles was therefore
not favorable except at the largest vesicle concentrations.
Second, the aromatic nucleic bases probably interact with the
aggregate lipophilic core and hybridization to complementary
strands became less efficient with a decrease and a broadening
of the melting transition.
The present results bear much significance for designing
efficient strategies both for using bilayers scaffold to self-
assembled lipids with large hydrophilic headgroups and for
improving the activity of antisense medicines. Comparing these
results to the ones reported for cholesterol conjugates (CMC
above 100 µM, insertion in bilayers favored and melting
behavior nearly unchanged), we can indeed put forward that a
too lipophilic group can be detrimental to the anchoring and
the pairing properties of DNA lipids. In fact, such lipid moiety
will not enhance the partition between the aqueous solution and
the bilayer. In contrast, it will promote the formation of stable
micelles impeding any further transfer to organized assemblies.
4-{2-[2-(2-Hydroxy-ethoxy)-ethoxy]-ethoxy}acetophenone (7).
A suspension of 4-hydroxyacetophenone 5 (3.9 g, 30 mmol, 1
equiv), 2-[2-(2-chloroethoxy)ethoxy]ethanol 6 (5.2 mL, 36
mmol, 1.2 equiv), dry potassium carbonate (6.2 g, 45 mmol,
1.5 equiv) and dry potassium iodide (6.5 g, 39 mmol, 1.3 equiv)
in dry n-butanol (60 mL) was vigorously stirred under nitrogen
at 110 °C for 12 h. After cooling to room temperature, the
reaction mixture was filtered and the n-butanol was evaporated
under vacuum. The residue was dissolved in dichloromethane
(100 mL). The resulting organic solution was washed with
sodium hydroxide 5% (50 mL) and with distilled water (3 ×
50 mL). The organic layer was dried over potassium carbonate
and concentrated under reduced pressure to give a pale yellow
Experimental Section
Chemical Synthesis. General Procedures. Microanalyses
were performed by the Service de Microanalyses de l’Universite´
Pierre et Marie Curie (Paris). Melting points were determined
with a Bu¨chi 510 capillary apparatus. 1H and 13C NMR spectra
were recorded at room temperature on a Bruker AM 200 SY or
an Avance Bruker DRX 400 spectrometer; chemical shifts are
reported in ppm using the solvent as internal reference (1H,
CHCl3 in CDCl3 7.27 ppm; 13C, 13CDCl3 in CDCl3 77.0 ppm);
coupling constants J are given in Hz. Mass spectra (chemical
ionization with NH3 or FAB positive) were performed by the
Service de Spectrome´trie de Masse de l’ENS (Paris). Column
chromatography was performed on silica gel 60 (0.040-0.063
mm) Merck. Analytical or preparative thin-layer chromatography
(TLC) was conducted on Merck silica gel 60 F254 precoated
1
oil (7.7 g, 95% yield). H NMR (CDCl3, 200 MHz, δ, ppm):
7.92 (d, 2H, J ) 8.9 Hz), 6.95 (d, 2H, J ) 8.9 Hz), 4.20 (t, 2H,
J ) 4.3 Hz), 3.86 (t, 2H, J ) 4.3 Hz), 3.80-3.55 (m, 8H), 2.52
(s, 3H); 13C NMR (CDCl3, 50 MHz, δ, ppm): 196.7, 162.4,
130.4, 130.3, 114.1, 72.3, 70.7, 70.1, 69.3, 67.3, 61.5, 26.2;
C14H20O5 (268.31): Anal. Calcd., C,62.67; H,7.51; Found, C,-
62.59; H,7.65.
4-N,N-Dihexadecylamino-4′-{2-[2-(2-Hydroxy-ethoxy)-ethoxy]-
ethoxy}chalcone (8). A solution of 4 (569 mg, 1 mmol, 1 equiv)

Micelles of Lipid-Oligonucleotide Conjugates
J. Phys. Chem. B, Vol. 108, No. 20, 2004 6493
and 7 (268 mg, 1 mmol, 1 equiv) in absolute ethanol/
dichloromethane: 1/1 (3 mL) was stirred at 35 °C. Solid
potassium hydroxide was progressively added to the reaction
flask: 280 mg (5 mmol, 5 equiv), then 280 mg (5 mmol, 5 equiv)
after stirring for 2 h, and 840 mg (15 mmol, 15 equiv) 1 h latter.
After the sample was stirred for 36 h at 35 °C, the reaction
mixture was cooled to room temperature and quenched by
adding distilled water (100 mL) and dichloromethane (100 mL).
The organic layer was washed with a saturated aqueous solution
of NaCl, dried over sodium sulfate, and concentrated under
reduced pressure. Purification by column chromatography (silica
gel; ethyl acetate) yielded 8 as a yellow solid (154 mg, 63%).
1H NMR (CDCl3, 200 MHz, δ, ppm): 8.01 (d, 2H, J ) 8.8
Hz), 7.78 (d, 1H, 15.4 Hz), 7. 52 (d, 2H, J ) 8.8 Hz), 7.32 (d,
1H, J ) 15.4 Hz), 6.98 (d, 2H, J ) 8.8 Hz), 6.62 (d, 2H, J )
8.8 Hz), 4.21 (t, 2H, J ) 4.6 Hz), 3.89 (t, 2H, J ) 4.6 Hz),
3.80-3.55 (m, 8H), 3.31 (t, 4H, J ) 7.3 Hz), 1.60-1.20 (m,
56H), 0.89 (t, 6H, J ) 6.4 Hz); 13C NMR (CDCl3, 50 MHz, δ,
ppm): 188.8, 161.9, 149.8, 145.0, 132.1, 130.5, 130.4, 121.8,
115.8, 114.1, 111.2, 72.4, 70.8, 70.3, 69.5, 67.4, 61.7, 51.0, 31.8,
29.6, 29.6, 29.6, 29.5, 29.4, 29.3, 27.2, 27.0, 22.6, 14.0; C53H89-
NO5 (820.30): Anal. Calcd., C,77.61; H, 10.94; N, 1.71; Found,
C, 77.43; H, 11.09; N, 1.58.
0.88 (t, 6H, J ) 6.5 Hz); 13C NMR (CDCl3, 100 MHz, δ, ppm):
188.8, 162.0, 149.9, 145.0, 132.1, 130.5, 130.4, 121.8, 115.8,
114.2, 111.2, 71.9, 70.8, 70.2, 69.6, 67.5, 51.0, 31.9, 29.6, 29.6,
29.5, 29.4, 29.3, 27.2, 27.0, 22.6, 14.1, 2.8; MS (CI, NH3); m/z
930.7 (calc.avg.mass for C53H88INO4 + H: 931.2); C53H88INO4
(930.18): Anal. Calcd., C,68.44; H, 9.54; N, 1.51; Found, C,
68.39; H, 9.60; N, 1.43.
4-N,N-Dihexadecylamino-4′-{2-[2-(2-Azido-ethoxy)-ethoxy]-
ethoxy}chalcone (11). A solution of 9 (427 mg, 0.5 mmol, 1
equiv) and sodium azide (65 mg, 1 mmol, 2 equiv) in dry N,N-
dimethylformamide (5 mL) was stirred at 80 °C for 12 h. After
cooling to room temperature, the mixture was quenched with
distilled water (20 mL) and extracted with ethyl ether (4 × 20
mL). The combined organic layers were washed with brine,
dried over sodium sulfate, and concentrated under reduced
pressure. After the residue was purified by column chromatog-
raphy (silica gel; cyclohexane/ethyl acetate 85/15), 11 was
obtained as a viscous oil (389 mg, 90% yield). 1H NMR (CDCl3,
200 MHz, δ, ppm): 8.01(d, 2H, J ) 8.8 Hz), 7.78 (d, 1H, J )
15.4 Hz), 7.52 (d, 2H, J ) 8.8 Hz), 7.32 (d, 1H, J ) 15.4 Hz),
6.98 (d, 2H, J ) 8.8 Hz), 6.62 (d, 2H, J ) 8.8 Hz), 4.21 (t, 2H,
J ) 4.7 Hz), 3.90 (t, 2H, J ) 4.7 Hz), 3.80-3.60 (m, 6H),
3.42-3.27 (m, 6H), 1.60-1.20 (m, 56H), 0.88 (t, 6H, J ) 6.4
Hz); 13C NMR (CDCl3, 50 MHz, δ, ppm): 188.7, 162.0, 149.8,
145.0, 132.0, 130.4, 130.3, 121.7, 115.8, 114.1, 111.2, 70.8,
70.6, 70.0, 69.6, 67.4, 50.9, 50.6, 31.8, 29.6, 29.6, 29.6, 29.5,
29.4, 29.3, 27.2, 27.0, 26.8, 22.6, 14.0; MS (FAB, MB); m/z
845.8 (calc. avg. mass for C53H88N4O4: 845.31).
4-N,N-Dihexadecylamino-4′-{2-[2-(2-Chloro-ethoxy)-ethoxy]-
ethoxy}chalcone (9). A mixture of 4 (260 mg, 0.32 mmol, 1
equiv), triphenylphosphine (320 mg, 1.22 mmol, 3.8 equiv) and
imidazole (83 mg, 1.22 mmol, 3.8 equiv) in carbon tetrachloride
(5 mL) was stirred vigorously at reflux under nitrogen for 3 h.
The mixture was cooled to room temperature, and the solvent
was evaporated under vacuum. The residue was dissolved in
ether (50 mL) and washed with water (3 × 50 mL). After the
organic layer was dried over potassium carbonate and solvent
was evaporated, the crude residue was purified by column
chromatography (silica gel; cyclohexane/ethyl acetate 9/1) to
4-N,N-Dihexadecylamino-4′-{2-[2-(2-Amino-ethoxy)-ethoxy]-
ethoxy}chalcone (12). A mixture of 11 (359 mg, 0.425 mmol,
1 equiv), triphenylphospine (167 mg, 0.64 mmol, 1.5 equiv)
and 2 drops of distilled water in tetrahydrofuran (15 mL) was
stirred at room temperature for 24 h. The solution was dried
over potassium carbonate and concentrated under reduced
pressure. Purification of the residue by column chromatography
(silica gel; chloroform/methanol/distilled water 15/5/0.5) as
1
yield 9 as an orange solid (210 mg, 80% yield). H NMR
(CDCl3, 200 MHz, δ, ppm): 8.01 (d, 2H, J ) 8.8 Hz), 7.78 (d,
1H, J ) 15.4 Hz), 7.52 (d, 2H, J ) 8.8 Hz), 7.32 (d, 1H, J )
15.4 Hz), 6.98 (d, 2H, J ) 8.8 Hz), 6.62 (d, 2H, J ) 8.8 Hz),
4.21 (t, 2H, J ) 4.5 Hz), 3.89 (t, 2H, J ) 4.5 Hz), 3.80-3.55
(m, 8H), 3.31 (t, 4H, J ) 7.3 Hz), 1.60-1.20 (m, 56H), 0.89
(t, 6H, J ) 6.4 Hz); 13C NMR (CDCl3, 50 MHz, δ, ppm): 188.7,
162.0, 149.8, 145.0, 132.1, 130.4, 130.3, 121.8, 115.8, 114.1,
111.2, 71.3, 70.8, 70.6, 69.6, 67.4, 51.0, 42.6, 31.8, 30.1, 29.6,
29.6, 29.6, 29.5, 29.4, 29.3, 27.2, 27.0, 22.6, 14.0; C53H88ClNO4
(838.74): Anal. Calcd., C,75.90; H, 10.57; N, 1.67; Found, C,
75.73; H, 10.64; N, 1.57.
1
eluent gave 12 as a yellow solid (310 mg, 90%). H NMR
(CDCl3, 200 MHz, δ, ppm): 8.01 (d, 2H, J ) 8.8 Hz), 7.78 (d,
1H, J ) 15.4 Hz), 7.52 (d, 2H, J ) 8.8 Hz), 7.32 (d, 1H, J )
15.4 Hz), 6.98 (d, 2H, J ) 8.8 Hz), 6.62 (d, 2H, J ) 8.8 Hz),
6.34 (brs, 2H, NH2), 4.21 (t, 2H, J ) 4.4 Hz), 3.87 (t, 2H, J )
4.4 Hz), 3.75-3.60 (m, 6H), 3.34 (t, 4H, J ) 7.3 Hz), 3.08 (t,
2H, CH2NH2), 1.60-1.20 (m, 56H), 0.88 (t, 6H, J ) 6.4 Hz);
13C NMR (CDCl3, 50 MHz, δ, ppm): 188.7, 161.8, 149.8, 145.1,
132.0, 130.5, 130.3, 121.7, 115.6, 114.2, 111.2, 70.5, 70.0, 69.3,
68.1, 67.3, 50.9, 39.9, 31.8, 29.6, 29.6, 29.5, 29.5, 29.4, 29.2,
27.2, 27.0, 22.6, 14.0; MS (CI, NH3); m/z 819.9 (calc.avg.mass
for C53H90N2O4: 819.31).
4-N,N-Dihexadecylamino-4′-{2-[2-(2-Iodo-ethoxy)-ethoxy]-
ethoxy}chalcone (10). A suspension of 9 (226 mg, 0.27 mmol,
1 equiv) and sodium iodide (1.2 g, 8 mmol, 30 equiv) in
2-butanone (20 mL) freshly dried over aluminum oxide was
stirred at reflux for 48 h. After the sample was cooled to room
temperature and concentrated under reduced pressure, the
residue was dissolved in dichloromethane (100 mL). The organic
phase was washed with a saturated aqueous solution of sodium
thiosulfate (2 × 50 mL) and with distilled water (2 × 50 mL).
After the organic layer was dried over sodium sulfate and the
solvent was evaporated, the crude residue was purified by
column chromatography (silica gel; dichloromethane/absolute
Synthesis of Derivatized Oligonucleotides. Coupling Reac-
tion. Commercially available reagents were used as obtained.
Water is of Millipore 18 MΩcm-1 quality. Triethylamine was
distilled over acetic anhydride. The oligonucleotides were
purchased from Eurogentec (Seraing, Belgium) and their 5′ to
3′ sequences can be summarized as follow: A ) TCG TTC
AAT TC; A′ ) GAA TTG AAC GA; B ) ACA ATT CGA T;
B′ ) ATC GAA TTG T; C ) ACG CGT AGG ACC; G )
GGT CCT ACG CGT; e designates a triethyleneglycol spacer.
The derivatization of the commercial oligonucleotides requires
different standard protocols that are described in a paper of
Grimm et al.29 First, oligonucleotides with a terminal 5′
phosphate group were precipitated by hexadecyltrimethylam-
monium bromide (CTAB), dissolved in dimethyl sulfoxide
(DMSO) and activated by a triphenylphosphine/dipyridyl dis-
ulfide/dimethyl-aminopyridine mixture.
1
ethanol 97/3) to give 10 as a yellow solid (208 mg, 81%). H
NMR (CDCl3, 250 MHz, δ, ppm): 8.01 (d, 2H, J ) 8.8 Hz),
7.78 (d, 1H, J ) 15.4 Hz), 7.52 (d, 2H, J ) 8.8 Hz), 7.32 (d,
1H, J ) 15.4 Hz), 6.99 (d, 2H, J ) 8.8 Hz), 6.62 (d, 2H, J )
8.8 Hz), 4.22 (t, 2H, J ) 4.7 Hz), 3.91 (t, 2H, J ) 4.7 Hz),
3.80-3.60 (m, 6H), 3.40-3.20 (m, 8H), 1.70-1.20 (m, 56H),

6494 J. Phys. Chem. B, Vol. 108, No. 20, 2004
Gosse et al.
For attachment of amino-modified chalcone 12, the solution
of chromophore in DMSO (1 mg/50 µL) was directly added to
activated oligonucleotide and incubated at least 2 h, preferably
overnight. The yield of the conjugate was between 40 and 85%.
Then the product was precipitated by ethanol/sodium acetate
solution and purified on 1.5% agarose gel in the presence of
0.1% Triton X-100. Formation of micelles did not permit us to
purify the conjugate on HPLC or by denaturing polyacrylamide
gel electrophoresis, as it is normally done.29
Fluorescence Correlation Spectroscopy (FCS) Measure-
ments. FCS Setup. The two-photon excitation FCS setup
consists of a laser source, a home-built microscope, and two
avalanche photodiodes (ADP; see Supporting Information,
Figure 3S). The laser source is a mode-locked Ti: Sapphire laser
(Mira 900, Coherent, Auburn, CA) pumped by a solid-state laser
at 532 nm (Verdi, Coherent). Pumped at 5 W, the Ti:Sapphire
laser generates 200 fs pulses at 80 MHz with about 700 mW
average power output at 800 nm. The whole tunable spectral
range covers the 700-1000 nm region. The power is adjusted
by means of neutral filters. The laser beam is expanded 3 times
at the entrance of the microscope in order to reach a diameter
of approximately 7 mm to fill up the back entrance of the
employed objective. After the beam expander, the light is passed
through a dichroic mirror and focused into the sample with a
60× water-immersion objective (NA ) 1.2, UPlanApo, Olym-
pus). The sample is contained within a 1-mm square glass
capillary (VitroCom, Mountain Lakes, NJ). The fluorescence
is collected through the same objective, reflected by the dichroic
mirror and filtered through four short pass filters to absorb any
possible diffused infrared light. The light is then separated by
a cube beam-splitter with a 1:1 ratio and focused on the 200
µm2 working surfaces of two APDs (SPCM-AQR-14, Perkin-
Elmer, Vaudreuil, Canada). The signal output of the APDs
modules (TTL pulses) are acquired by a digital autocorrelator
module (ALV-6000, ALV, Langen, Germany) which computes
on-line the cross-correlation function of the fluorescence
fluctuations. Using two APDs and cross-correlation function
reduces significantly the afterpulsing noise that is strongly
correlated in the range shorter than 10 µs. The resulting function
is formally similar to the autocorrelation function. The data are
then stored in the computer and analyzed by a home-written
routine which allows us to (i) average chosen successive
acquisitions; (ii) make a least-squares fit for both single- and
two-components cross-correlation functions with diffusion in
two- or three-dimensions; (iii) calculate the associated standard
deviations.
For attachment of iodoalkyl derivative of the chalcone, a
slightly longer procedure was used. The activated oligonucleo-
tide was precipitated by LiClO4/acetone solution, washed by
acetone, and quickly dissolved in the water solution of cystamine
(5 mg/50 µL) together with 5 µL of triethylamine. After
incubation of the sample at least for 2 h at room temperature,
the derivative was precipitated by ethanol/sodium acetate,
redissolved in water, and reduced by 0.1 M dithiothreitol to
afford cysteamino-modified oligonucleotide phosphoroamidate
with a terminal -SH group. After precipitation of the sample
by CTAB, the dried derivative was dissolved in DMSO,
alkylated by the iodoalkyl-chalcone 10 (10-100 fold molar
excess) in the presence of triethylamine, reprecipitated by
ethanol/acetate, and purified on 1.5% agarose gel in the presence
of 0.1% Triton X-100. Yields were typically in the 10% range.
Purification by Agarose Gel Electrophoresis. The electro-
phoresis was performed with a 1.5% GTG NuSieve agarose gel
(FMC, Rockland, ME; buffer 10 mM BisTris 1 mM EDTA pH
6.5). The band corresponding to the desired derivatized oligo-
nucleotide was submitted to the action of â-agarase (New
England Biolabs, Beverly, MA) according to manufacturer’s
recommendations. To remove remaining oligosaccharides, the
derivate was again precipitated by CTAB, the supernatant was
discarded, and the pellet was dissolved in methanol and
centrifuged. Nonsoluble particles were discarded, and the
dissolved derivate was precipitated by ethanol/sodium acetate.
The final product was dissolved in water and stored at -20 °C.
The sample concentration was evaluated from the UV absorption
spectrum using the following values of the molar absorption
coefficients given by Eurogentec: ꢀGeB(260 nm) ) 210 600
M-1cm-1; ꢀGeB′(260 nm) ) 209 800 M-1cm-1. For lDNA
originating from the conjugation of thiol-terminated oligonucleo-
tides, the latter values made it possible to derive the molar
absorption coefficient corresponding to the chalcone moiety.
In 100 mM NaCl, 10 mM Na(CH3)2AsO2-HCl pH 7 buffer,
one obtained: ꢀlGeB(λmax ) 433 nm) ) 24 000 M-1 cm-1 and
Calibration of the FCS Setup. The fluctuations of fluorescence
intensity I from a volume V containing freely diffusing
fluorescent molecules (average concentration Ch) were analyzed
with the correlation function G(τ) given by:
δI(t)δI(t + τ)
G(τ) - 1 )
)
2
I
1
1
1
wxy
(6)
1/2
ChV
τ
τD
2 τ
ꢀlGeB′(λmax ) 433 nm) ) 32 000 M-1 cm-1 47
.
1 +
1 +
(
) (
(
)
)
wz τD
UV/Vis Absorption and Steady-State Emission Spec-
troscopies. UV/Vis absorption spectra were recorded on a
Uvikon-930 spectrophotometer (Kontron, Zu¨rich, Switzerland).
Corrected fluorescence spectra and excitation polarization
spectra were obtained with a LPS 220 spectrofluorometer (PTI,
Monmouth Junction, NJ). Steady-state fluorescence anisotropies
defined as r ) (I| - I )/(I| + 2I ) were determined by the
G-factor method (I|, respectively I , being the fluorescence
intensity observed with vertically polarized excitation light and
vertically, respectively horizontally, polarized emissions).48 The
temperature was regulated at 0.1 °C and directly measured
within the cuvettes.
where τD is the diffusion time through a 3D Gaussian excitation
profile characterized by Iex ) I0 exp[-2(x2 + y2)/wxy - 2z2/
2
wz2]. In the case of two-photon excitation,
τD ) wxy2/8D
(7)
where D is the diffusion coefficient of the fluorescent species.
Consequently, fitting the G(τ) - 1 curve for species of known
diffusion coefficient allows us to calibrate the beam-waists wxy
and wz of our experimental setup. Fluorescein labeled beads of
0.02 µm radius RH (Molecular Probes, Eugene, OR) were used
under the assumption that
Electron Microscopy (EM). Samples containing EPC vesicles
were prepared by dialysis. After staining of the samples with
2% uranyl acetate, they were observed on a Philips CM208
transmission electron microscope operating at 80 kV. Electron
micrographs were recorded at a nominal magnification of
40 000.
D ) kB
T/6πηRH
(8)
(with kB the Boltzmann constant, T the absolute temperature,
and η the solvent viscosity); we found wxy ≈ 0.3 µm and (wxy/

Micelles of Lipid-Oligonucleotide Conjugates
J. Phys. Chem. B, Vol. 108, No. 20, 2004 6495
wz)2 ≈ 0.01. The wxy value corresponds to the expected one for
the objective used (wxy ) 0.61λex/NA ≈ 0.4 µm) and the
excitation volume can be then calculated as V ) πwxy4/λex,49
typically in the femtoliter range.
Preparation by the Dialytic Detergent RemoVal Technique.51
The resulting solution was transferred to dialysis tubing
(Viskase, MW cut off 12000-14000). Detergent was removed
by dialysis at room temperature; typically 2 mL of detergent/
lipid solution was dialyzed against 1 L of buffer solution and
the dialysis solution was changed three times over 2 days.
Preparation Relying on Detergent RemoVal with Bio-Beads.52
The suspension resulting from addition of buffer (2 mL) and
previously conditioned53 Bio-Beads SM2 (BioRad; 30 mg) was
vortexed at room temperature for 2 h. Fresh Bio-Beads SM2
(60 mg) were added and the mixture was vortexed again for 1
h. This operation was repeated once with 30 mg of beads. The
resulting vesicular suspension was filtered and immediately used.
In the case of RhPE-containing vesicles, Rh-PE (Molecular
Probes) was added together with EPC before chloroform
evaporation.
Determination of lDNA:EPC Association Constants. The
simple two-state models eqs 4 and 5 were introduced to describe
the association between the lDNA and EPC LUV.44 In these
equations, the lDNA is assumed to give a 1:1 “complex” with
an EPC molecule. The corresponding association constants (Kmix
and Kmfv) were evaluated either by steady-state emission of
fluorescence, electron microscopy, or fluorescence correlation
spectroscopy.
Power-Squared Dependence of Two-Photon-Excited Fluo-
rescence. The molecules under study were excited through the
two-photon absorption process at λexc ) 780 nm. For FCS
applications, it is essential to be in a regime in which the
concentration of excited molecules is proportional to the square
of the excitation power; otherwise, eqs 6 and 11 cannot be used
for treating experimental data.50 We investigated the dependence
of the fluorescence as a function of the excitation power for all
the compounds studied, and we chose the power such as to
remain in the regime of power-squared dependence (see for
instance Supporting Information, Figure 4S; as a rule, the power
before the entrance of the beam expander was set less than 60
mW). With those experimental conditions, no contribution of
the triplet state to the experimental FCS curves was observed
either.
Calibration of Medium Viscosity during Titrations by Micelles
and Vesicles. The diffusion time of fluorescent species extracted
from FCS depends on solution viscosity (see eqs 7 and 8). Thus,
to interpret the diffusion times only in terms of structural
changes occurring during the subsequent titrations, it is neces-
sary to calibrate medium viscosity as a function of the
concentrations both in OG and in EPC vesicles to perform the
relevant corrections. By recording, in a series of independent
experiments, the G(τ) curves originating from 10 nM fluorescein
solutions in 100 mM NaCl, 10 mM Tris-HCl/Tris pH 9 buffer,
we evaluated the variation of medium viscosity as a function
of the concentrations in OG or in EPC vesicles. In the course
of the corresponding studies, the experimental G(τ) curves were
satisfactorily fitted by eq 6, suggesting no significant interaction
to arise between fluorescein and the organized media. We also
observed that G(0) decreased and τD increased during the
titration by micelles or by concentrated vesicles. We checked
that the corresponding observation was not related to changes
of refractive index: titrating a 10 nM fluorescein solution in
100 mM NaCl, 10 mM Tris-HCl/Tris pH 9 buffer with
concentrated sucrose led to the expected observations of no
change in G(0) and an increase in τD linked to viscosity.
Therefore, we assume light diffusion by micelles and vesicles
to expand the excitation profile without strongly altering its
geometry, leading to the subsequent decrease in G(0). Conse-
quently, the calibration of viscosity was performed from τD
EValuation by Steady-State Emission of Fluorescence. In a
first step, the fluorescence data were corrected from the dilution
arising from vesicle addition. They were subsequently analyzed
from the easily derived eq 9:54
IF(0) - IF(∞)
1
Kmix[1DNA]tot
1/2
IF([EPC]) ) IF(0) -
1+ R +
{(
)
2
2 - 4R
(9)
1
-
1 + R +
(
)
}
[
]
Kmix[lDNA]tot
where IF(0) and IF(∞) are the initial fluorescence intensity and
the asymptotic limit of the fluorescence intensity at large EPC
concentrations, [lDNA]tot is the total concentration of lDNA
during the titration,55 and R is the ratio [EPC]tot/[lDNA]tot with
[EPC] tot referring to the total EPC concentration. The fit of the
experimental data according to eq 9 provides orders of
magnitude for IF(∞)/IF(0) and for Kmix. Then, numerical calcula-
tions were further performed to refine the latter values:
IF([EPC]) values were calculated from different {IF(∞)/IF(0),
Kmix} sets closely located around the previously derived orders
of magnitude, and the final selected set was the one that gave
the best simulation of the experimental data.
EValuation by Electron Microscopy. In the concentration
regime used for this series of experiments in which [EPC]tot
always exceeds [lDNA]tot, one has:
G(0)-1 instead of τ : both functions are linearly dependent
x
D
2
on viscosity, but the latter is also proportional to wxy (from
eqs 7 and 8), whereas the former does not depend on wxy (from
eqs 6-8 with V ) πwxy4/λexc). The reported value of viscosity
for 100 mM NaCl at 293 K was used for determining the
[lDNA]tot
[lDNA] )
(10)
proportionality factor linking η to τD G(0)-1, yielding to the
x
1 + Kmfv[EPC]tot
curves displayed in Supporting Information, Figure 5Sa,b. One
notices that the viscosity of the OG solutions strongly increases
above OG CMC (Supporting Information, Figure 5Sa), whereas
the viscosity of suspensions of EPC vesicles remains essentially
unaltered in the investigated range of EPC concentrations
(Supporting Information, Figure 5Sb).
In particular, Kmfv[EPC]tot ) 1 for [lDNA] ) [lDNA]tot/2. The
titration of 2.5 µM lGeB by EPC vesicles in 100 mM NaCl, 10
mM Na(CH3)2AsO2-HCl pH 7 buffer was performed up to
[EPC]tot ) 20 mM. The association constant was crudely
evaluated as 1/[EPC]tot when half the initial lGeB micelles had
disappeared from the EM picture.
EValuation by FCS. The evaluation relies on the determination
of the molar fractions of fluorescent lDNA in vesicles as a
function of the total EPC concentration. Such amounts can be
extracted from experiments devoted to measure diffusion
coefficients since vesicles and micelles significantly differ in
size.
Large Unilamellar Vesicles (LUVs) Preparation. A solution
containing R-L-phosphatidylcholine (100 mg mL-1 in hexane;
50 µL, 5 mg, 7 µmol), and 1-octyl-â-D-glucopyranoside (10.4
mg; 36 µmol, 5 equiv) in chloroform (0.5 mL) was evaporated
to dryness under reduced pressure at room temperature. The
film was then dissolved after addition of the suitable buffer (2
mL).

6496 J. Phys. Chem. B, Vol. 108, No. 20, 2004
Gosse et al.
In a regime of slow exchange of lDNA between the micelles
and the vesicles, the correlation function of fluorescence
intensity G(τ) can be satisfactory analyzed by eq 11, which
accounts for two different species crossing a 2D-Gaussian
excitation beam:56
Then, the 1/[gvesicles(1 + (gmicelles/gvesicles))2] curve was plotted
as a function of R and fitted according to eq 15 leaving the
prefactor [lDNA]totV/2Q2
and Kmfv as floating param-
vesicles
eters. Error on Kmfv is evaluated to be 50%.
DNA Melting Curves. The 100 mM NaCl, 10 mM Na(CH3)2-
AsO2-HCl pH 7 buffer was chosen for the low dependence of
its pH with temperature.57
-1
τ
τ
G(τ) - 1 ) gmicelles 1 +
-1 + gvesicles 1 +
(
τD
)
(
τD
)
Assay Relying on UV Absorption. The experiments were
performed with an Uvikon spectrophotometer using the Macrotm
software. Except for the samples containing micelles where the
heating cycle was first applied, the absorbance at 260 nm was
first recorded during a cooling cycle from 84 to 4 °C (0.1-0.2
°C min-1) and then during a heating cycle from 4 to 84 °C
(0.1-0.2 °C min-1). No hysteresis was ever detected, suggesting
that equilibrium is reached at each temperature. Half-dissociation
temperatures (Tm) were determined from the denaturation curves
with a precision equal to 1 °C.46
Assay Relying on Emission from an Intercalating Agent. We
designed this assay to measure the melting temperature of ds-
DNA in vesicle suspensions. Indeed, the traditional method
relying on recording the absorbance of DNA at 260 nm was
not appropriate in a scattering medium where the extinction
signal originating from particles strongly overcame the DNA
absorption. The fluorescence emission of the intercalant BOBO3
(Molecular Probes) is strongly enhanced in the presence of ds-
DNA and remains essentially unaffected in the presence of ss-
DNA. Figure 6Sa (Supporting Information) displays, as a
function of temperature, the collection of BOBO3 emission
spectra (0.67 µM) in 100 mM NaCl, 10 mM Na(CH3)2AsO2-
HCl pH 7 buffer in the presence of C/G (2 µM). As anticipated,
the emission drops when the temperature is increased. More
micelles
vesicles
(11)
(12)
with
Q2micellesNhmicelles
(Q2micellesNhmicelles + Qv2esiclesNhvesicles
gmicelles
)
2
)
and
Q2vesiclesNhvesicles
(Q2micellesNhmicelles + Qv2esiclesNhvesicles
gvesicles
)
(13)
2
)
In the above equations, τDmicelles and τDvesicles are the characteristic
diffusion times for crossing the excitation volume respectively
associated with micelles and vesicles, Nhmicelles and Nhvesicles the
respective average numbers of labeled particles contained within
the excitation volume V, Qmicelles, and Qvesicles the respective
particles fluorescent brightness.
Both diffusion times were first measured independently.
τD
was obtained from analyzing the correlation function
micelles
G(τ) arising from a 10 nM lGeB solution in 100 mM NaCl, 10
mM Tris-HCl/Tris pH 7 buffer according to eq 6. τD was
vesicles
similarly obtained from analyzing the correlation function G(τ)
arising from a vesicular suspension labeled with 8 ([8] ) 10
nM) in 100 mM NaCl, 10 mM Tris-HCl/Tris pH 7 buffer. 50
precisely, the plot of BOBO3 emission (λexc ) 530 nm, λem
)
µs and 10 ms were typically found for τD
and τD
micelles
vesicles
600 nm) as a function of temperature exhibits two different
trends (Supporting Information; Figure 6Sb). First, the emission
exhibits a smooth decrease over the whole temperature range
as expected from the contribution of nonradiative desexcitation
pathways of fluorescent probes that increases with the temper-
ature. Second, emission suddenly drops between 40 and 65 °C,58
generating a transition whose width is larger than the one
observed during the classical assay. Inflection occurs at 56 °C,
which compares satisfactorily with the 53 °C melting temper-
ature measured by UV absorption. The apparent upward shift
suggests that BOBO3 intercalation weakly stabilizes ds-DNA.
Similar results were obtained in the presence of EPC vesicles,
despite a smoother continuous decrease of fluorescence emission
may be due to a direct interaction between BOBO3 and bilayers.
respectively. We already demonstrated that medium viscosity
did not change significantly upon vesicles additions and that
the sizes of micelles and vesicles remained constant during all
the titration (see EM); consequently, τD
and τD
were
micelles
vesicles
anticipated to remain in the same order of magnitude whatever
the amount of LUVs introduced.
The titration of a 10 nM lGeB solution by 9.3 mM EPC
vesicles in 100 mM NaCl, 10 mM Tris-HCl/Tris pH 7 buffer
was then performed. We checked that the extracted τDmicelles and
τD
values for each EPC concentration were in the range
Vesicles
determined during the first series of experiments, confirming
that the morphology of the two phases, micelles and vesicles,
remained essentially the same during the whole titration.
Therefore, the process (5) and eq 10 were considered to
satisfactorily model the phenomena during the whole titration.
Then one can easily derive from eq 11 and conservation laws
Acknowledgment. We are indebted to D. Bensimon who
encouraged us to design 2D polymers relying on polyconden-
sation between oligonucleotides. We also thank O. Ruel and
J.-B. Baudin for advice concerning organic synthesis, L. Lacroix
and J.-L. Mergny for introducing us to melting experiments,
and T. Garestier and C. He´le`ne for helpful discussions.
the following equations that allows for extracting Kmfv
:
2
gmicelles
1
) NhvesiclesQ2vesicles 1 +
(14)
(
)
gvesicles
gvesicles
[lDNA]totV
Supporting Information Available: This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
1
Nhvesicles
)
1 + R +
-
[
(
)
2
Kmfv[lDNA]tot
1 + R +
2 - 4R (15)
1
References and Notes
(
)
]
Kmfv[lDNA]tot
x
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