10300
J. Phys. Chem. B 1999, 103, 10300-10310
Thermotropic Phase Behavior of Cationic Lipid-DNA Complexes Compared to Binary
Lipid Mixtures
Roman Zantl,† Laura Baicu,† Franck Artzner,†,§ Irene Sprenger,† Gert Rapp,‡ and
Joachim O. Ra1 dler*,†
Lehrstuhl für Biophysik, E22, Technische UniVersität München, James-Franck-Str. 1,
D-85748 Garching, Germany, and European Molecular Biology Laboratory, EMBL c/o DESY,
Notkestrasse 85, D-22603, Hamburg, Germany
ReceiVed: May 18, 1999; In Final Form: August 16, 1999
The thermotropic phase behavior of zwitterionic/cationic binary lipid mixtures is investigated and compared
to its corresponding lipidic phase diagram of mixtures complexed with DNA. We focus on isoelectric cationic
lipid-DNA condensates where the number of cationic lipids equals the number of phosphate groups on the
DNA. Using differential scanning calorimetry, X-ray scattering, freeze fracture electron microscopy, and
film balance, we studied mixtures of di-myristoyl-phosphatidyl-choline (DMPC) and the cationic lipid, dimyristoyl-tri-methyl-ammonium-propane (DMTAP). The lipid phase diagram shows the well-known LR, Lβ′,
and Pβ′ ripple phase with peritectic behavior at a low molar fraction of cationic lipid, χTAP < 0.12. Beyond
χTAP ) 0.8 crystalline phases appear. A systematic variation in the hydrocarbon chain tilt in the prevailing Lβ′
phase is measured by wide-angle X-ray scattering. Most importantly, the Lβ′ phase shows strong nonideal
mixing with an azeotropic point at about 1:1 molar stoichiometry. This finding is related to the reduced
headgroup area for equimolar mixtures found in monolayer pressure-area isotherms. The intercalation of
DNA in cationic lipid-DNA complexes affects the lipid-phase behavior 2-fold: (i) the chain-melting transition
temperature shifts to higher temperatures and (ii) a demixing gap with coexistence of lipid vesicles and lipidDNA complexes arises at a low cationic fraction, χTAP < 0.25. In agreement with experiments we present a
thermodynamic model that describes the shift of the melting transition temperatures by DNA-induced
electrostatic screening of the cationic membrane.
Introduction
Cationic liposomes have found widespread application in
molecular cell biology as transfection agents. When a protocol,
known as lipofection, is used, plasmid DNA is delivered into
eucaryotic cells by means of lipid mixtures of usually zwitterionic and cationic lipids. Cationic liposomes aggregate with
DNA and enhance the gene delivery due to electrostatic
attraction to the cell surface.1 During the last few years cationic
lipids have also been tested in clinical trials as possible
candidates for gene transfer in human gene therapy. For this
purpose many new cationic amphiphiles have been synthesized
and screened for their efficacy (for reviews see refs 2-4).
However, at present there is little understanding, and much less
ability to predict, gene-transfer efficiencies on a molecular level.
It became apparent that, besides cytotoxicity and cell-specific
interactions, also nonspecific physico-chemical properties of
the lipid-DNA constructs play an important part in gene
delivery. This concerns the structure, stability, and surface
properties of the gene delivery complexes. The aggregates that
form when cationic liposomes and plasmid DNA are initially
mixed are condensed lipid-DNA composite phases with unique
liquid-crystalline structures. When small-angle X-ray scattering
(SAXS) was used, lamellar and hexagonal mesomorphic phases
* To whom correspondence should be addressed. Phone: (49) 89 2891
2539. Fax: (49) 89 2891 2469. E-mail: [email protected].
† Technische Universität München.
‡ EMBL.
§ Permanent address: Faculté de Pharmacie, UMR8612, CNRS, 5 rue
J.B. Clement, 92296 Chatenay-Malabry Cedex, France.
with alternating DNA and lipid layers were discovered.3,5-7
Recent theoretical work is able to model lipid-DNA complexes
within the framework of well-known lipid-phase polymorphism.8,9
The present work is an attempt to contribute to the understanding of the thermotropic phase behavior of mixed phospholipid-cationic liposomes and their corresponding DNAcomplexed mesomorphic aggregates. Specifically, we ask the
question, what are the corresponding lipid phases in condensed
lipid-DNA aggregates and what can we learn from changes in
the lipid-phase behavior upon complexation with DNA? To this
end, we investigate cationic lipids with saturated alkyl chains.
We choose DMPC/DMTAP as a model phospholipid-cationic
lipid (PL/CL) system. From the large number of available
bilayer-forming cationic amphiphiles, or cationic lipids, DMTAP
may be considered as one of the most natural cationic derivatives
of DMPC. As seen in the chemical structure shown in Figure
1. The zwitterionic phosphatidyl-choline is simply replaced by
propane-trimethylamine. The latter is a fully dissociated cationic
quaternary amine.
DMPC/DMTAP mixtures form lamellar lipid-DNA complexes analogous to the previously studied unsaturated DOPC/
DOTAP-DNA complexes.5,10 In DMPC/DMTAP-DNA complexes a low-temperature lamellar lipid gel phase and a hightemperature fluid phase exist as evidenced by recent small- and
wide-angle X-ray scattering (SAXS and WAXS) experiments.11
Here, we present, for the first time, the full thermotropic phase
diagram of this cationic lipid-DNA complex. The study is,
10.1021/jp991596j CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/30/1999
Behavior of Lipid-DNA and Binary Lipid Mixtures
J. Phys. Chem. B, Vol. 103, No. 46, 1999 10301
characterize each phase by means of electron microscopy,
SAXS, and WAXS. In the discussion section the thermodynamic
features of the cationic lipid-phase diagrams are analyzed. In
particular, we recall the regular solution theory of azeotropic
lipid mixtures to explain the elevated transition temperatures
around equimolarity, and focus on the shift in the transition
temperature between the pure lipid phases and the DNA
complexed phases. We present a modified theory based on the
work by Träuble and Eibl,16 which is found to describe the
experimentally determined shifts quantitatively. Finally, an
Appendix is added to describe in detail the derivation of the
C
phase by analysis of the powder line shapes
chain tilt of the Lβ′
of the WAXS experiments.
Materials and Methods
Figure 1. (a) Structures of di-myristoyl-phosphatidyl-choline (DMPC)
showing a zwitterionic headgroup and di-myristoyl-tri-methyl-ammonium-propane (DMTAP) with a quaternary amine group. (b)
Schematic picture of the self-assembled supramolecular arrangement
C
in DMPC/DMTAP-DNA complexes. Both the Lβ′
gel state and LCR
fluid phase exhibit intercalated lamellar order.
however, limited to the quasi-binary-phase diagram of isoelectric
complexes, where the amount of DNA is fixed by the mole
fraction of cationic lipid. For simplicity the DNA-bound binary
phases are indexed with an additional “C”, for “condensed” or
“complexed”. In the literature few purely lipidic phospholipid/
cationic lipid mixtures have been studied. In a seminal paper,
Silvius investigated the phase behavior of zwitterionic and
anionic lipids mixed with cationic amphiphiles and found that
DPPC/DHDAC exhibits strongly nonideal mixing with pronounced maxima in the solidus and liquidus lines around
equimolar mole fractions.12 Similar maxima were also found
in other binary cationic mixtures.13-15 Our findings will be
consistent with these phase diagrams. Moreover, we show that
the complexation with DNA leads to surprisingly small effects
on the overall phase behavior. The effects are 2-fold: first, the
main transition shifts to higher temperatures and second a
demixing gap appears for small mole fractions of cationic lipid.
We relate this phase behavior to structural investigations, in
C
particular, of the Lβ′
phase, that reveals a continuous variation
in tilt. Eventually, an untilted LCβ phase is located at χTAP )
0.25, remarkably close to the demixing gap.
The article is structured as follows. First, we present the
calorimetric studies of binary DMPC/DMTAP mixtures. From
the signatures of the differential scanning calorimetry (DSC)
scans we derive the binary-phase diagram of DMPC/DMTAP.
In parallel, we present the calorimetric measurements of
isoelectric DMPC/DMTAP-DNA complexes and the corresponding pseudo-binary-phase diagram of the latter. We then
Materials and Sample Preparation. Di-myristoyl-phosphatidyl-choline (DMPC) and di-myristoyl-tri-methyl-ammoniumpropane (DMTAP) were purchased from Avanti Polar Lipids
Inc. (Alabaster, AL) in chloroform. The lipids were mixed in
the required ratios, dried under nitrogen flow, and subsequently
stored in vacuum for more than 12 h. Multilamellar vesicles
(MLVs) were prepared at a concentration of 5.0 mg/mL using
Millipore water and vortex mixing every 20 min for 2 h at 60
°C. The resulting suspension was diluted to 2.5 mg/mL lipid
concentration and divided into two parts. One part was used as
pure lipid sample. The second half was complexed with an
isoelectric amount of calf thymus DNA, i.e., a charge-neutral
ratio (1:1) of DNA-phosphate groups to DMTAP headgroup.
All samples were stored at 4 °C for 2 weeks to ensure
thermodynamic equilibrium. Calf thymus DNA sodium salt
(Sigma Aldrich Chemie GmbH, Deisenhofen, Germany) was
dissolved in Millipore water and the concentration determined
by 260/280 UV absorption. SUVs (small unilamellar vesicles)
of DMPC and DMTAP were prepared by solvent evaporation
under nitrogen flow, vacuum drying, resuspension in Millipore
water at 70 °C for 4 h and 10 min of sonication. For X-ray
measurements the lipid vesicle solutions containing a concentration of ca. 150 mg/mL were filled into quartz capillaries
(Hilgenberg, Malsfeld, Germany). The lipid-DNA complexes
were prepared by adding a 50 mg/mL vesicle suspension onto
the freeze-dried DNA inside the quartz capillaries. The samples
were annealed several times between 10 and 60 °C and allowed
to equilibrate for more than 7 days.
Electron Microscopy. A small amount (1 µL) of the sample
was squeezed between two Balzers gold holders (sandwich
technique) and then quickly frozen in nitrogen-cooled propane
at room temperature (freezing velocity, 1000-3000 °C/s)
following standard protocols.17,18 The freeze-fracture procedure
was carried out in a Balzers HVA BAF 400 T at a pressure of
10-6 mbar and a temperature of approximately -170 °C. The
shadowing was done with electron beam evaporators; the one,
Pt/C, at an angle of 45° and the other, C, horizontally to stabilize
the metal film.19,20 The replica was floated on a water-acetone
surface (100:5). No further cleaning was necessary. The replica
was picked up with 400-mesh copper grids. Pictures were taken
with a Philips CM 100 at 100 kV with Agfa Scientia electron
microscopy films.
Calorimetry. DSC measurements were performed using a
MC-2 differential scanning calorimeter (MicroCal, Northampton,
MA) at heating rates of 20 °C/h. Tests with slower scanning
rates obtained transition temperatures in accordance of 0.1 °C
and transition peaks with widths deviating less than 5%. All
samples contained a lipid concentration of 2.5 mg/mL and DNA
concentrations between 0 and 1.35 mg/mL. Measuring and
10302 J. Phys. Chem. B, Vol. 103, No. 46, 1999
Zantl et al.
reference cells were held under 3 bar using pressurized nitrogen.
The solidus (respectively, liquidus) temperature was determined
by the intersection of a tangent at the low- (respectively, high-)
temperature inflection point and the base line. Tm was determined as the maximum of the cP curves. The transition
enthalpies were determined by numerical integration of the base
line-corrected DSC curves. The estimated error of the transition
enthalpy for vesicle solutions is about 6%, taking a 0.2 °C
temperature precision and a weighing failure of 5%. Problems
occurred because of the size (≈1 mm) and stickiness of lipidDNA aggregates. An exact control of the total amount of
complexes transferred inside the DSC cell proved to be
impossible. The reproducibility of DSC data was scrutinized
for selected molar ratios by scanning different preparations more
than five times.
X-ray Diffraction. SAXS and WAXS was carried out in the
EMBL synchrotron-beamline X13. A detailed description of the
setup used is given by Rapp et al.21 The resolution in the
reciprocal space was better than 0.002 Å-1 for the SAXS and
0.0055 Å-1 for the WAXS as measured by the HWHM of the
lamellar peaks of silver behenate at q ) 0.108 Å-1 and of
parabromobenzoe acid at q ) 1.34 Å-1. The investigated q range
for SAXS was 0.03-0.35 Å-1 and for WAXS 1.0-1.5 Å-1.
The temperature was controlled within 1 °C by a peltier-water
bath setup.
Film Balance. A Langmuir-type film balance was used as
described in ref 22. The lipid was applied from a 10 mg/mL
chloroform stock solution. The maximal monolayer area was
900 cm2 with a maximal compression ratio of 3:1. Typical
sweeping rates were about 100 µm/s. All scans were performed
on a Millipore subphase with the temperature stabilized at 20
°C.
Results
Differential Scanning Calorimetry. Baseline-corrected DSC
scans of DMPC/DMTAP MLVs and DMPC/DMTAP-DNA
complexes are shown in Figure 2a. The data are stacked as a
function of the increasing mole fraction of cationic lipid, χTAP.
The bottom scan corresponds to pure DMPC lipid with the wellknown gel-to-liquid crystalline transition at Tm ) 24.5 °C. With
increasing cationic mole fractions, this main transition shifts to
higher transition temperatures and exhibits a high-temperature
shoulder. At χTAP ) 0.45 the transition temperature reaches a
maximum and decreases from there to about 20 °C. Samples
with very high DMTAP content exhibit a pronounced hysteresis
in the heat-cooling cycles and dependence on sample history
and preparation. The hatched triangle in the lipid phase diagram
in Figure 3a indicates the region where enthalpic transitions
were detected by DSC. Furthermore the DSC scans exhibit a
small enthalpic pretransition at low TAP concentrations. The
pretransition peaks are enlarged in Figure 2b. The transition
temperature increases with the cationic mole fraction and merges
P
) 0.18. The transition
with the main transition at χTAP
enthalpies of the endothermic transitions are summarized in
Table 1. Note that the values for the enthalpies of the lipidDNA complexes in italic letters are lower estimates because in
these cases loss of material could not be avoided during the
transfer into the calorimeter measuring cell.
In the case of the cationic lipid-DNA complexes we find
similar behavior of the main transition, which also increases
with increasing χTAP. However, at low DMTAP concentrations
(Figure 2b), in contrast to the pure binary lipid mixtures we
observe a second transition at about 40 °C besides the
pretransition and the main transition. The area of the high-
Figure 2. (a) Calorimetric heating scans of DMPC/DMTAP liposome
suspensions (left) and DMPC/DMTAP/DNA complexes (right). The
molar fraction of cationic lipid, χTAP, increases from bottom to top.
All peaks are normalized to the same amplitude for clarity. Samples
were prepared and equilibrated in Millipore water. (b) Magnified
selection of calorimetric scans displaying the evolution of the pretransition at low DMTAP mole fraction. The purely lipidic mixtures (left)
show peritectic behavior, while cationic lipid-DNA complexes (right)
exhibit a demixing gap.
temperature transition peak increases at the expense of the area
of the low-temperature peak. Both transitions do not significantly
shift their position within this coexistence regime, χTAP < 0.32.
The shape of the main transition peak at χTAP > 0.32 is,
furthermore, strongly asymmetric. Beyond the composition χTAP
) 0.45 the transition temperature decreases monotonically. For
mole fractions of 0.73 < χTAP < 1.0 we find a new lowtemperature transition with increasing enthalpy but constant
transition temperature of about 20 °C.
Construction of the Phase Diagrams. On the basis of the
calorimetric data, we construct the phase diagrams as shown in
Figure 3a,b. The DMPC/DMTAP mixtures are presented as
purely binary systems given that the pressure is constant and
that excess water can be neglected as a third component. In the
case of the ternary DMPC/DMTAP/DNA complexes we present
a two-dimensional cut through the truly three-dimensional phase
diagram, which corresponds to stoichiometrically charged
neutral complexes with a fixed DNA-to-cation ratio. The latter
Behavior of Lipid-DNA and Binary Lipid Mixtures
Figure 3. (a) Phase diagram of the DMPC/DMTAP system derived
from calorimetry and SAXS. The highest transition temperature is
reached at mole fraction cationic lipid, χmax
TAP ) 0.45. Here, the
coexistence region is of minimal width. At low DMTAP concentrations
the phase diagram shows peritectic behavior of the adjacent Lβ′ and
Pβ′ gel phases. At high DMTAP concentrations there are two distinguishable solid phases, S1 and S2. In the hedged region DSC scans were
not reproducible. (b) In comparison, the pseudobinary thermotropic
phase diagram DMPC/DMTAP mixtures complexed with an isoelectric
amount of calf thymus DNA. The transition temperature maximum is
max
Tmax
m ) 41.6 °C at about χTAP ) 0.42. At low χTAP values a demixing
gap with coexisting vesicles and lipid-DNA complexes. The “stretched”
peritectic behavior of the high chain melting transition in this region
indicates the existence of DMTAP-depleted vesicles and DMTAPenriched lipid-DNA complexes. On the right side of the diagram simple
C
eutectic behavior of the complexed Lβ′
and one crystalline DMTAPc
DNA S phase is observed.
TABLE 1: Summary of Transition Temperatures and
Enthalpies for the Lipidic and Lipid-DNA Mixtures
DMPC/DMTAP MLVs
χTAP
Tm
(°C)
Tpre
(°C)
0.00
0.04
0.09
0.18
0.27
0.35
0.43
0.51
0.53
0.59
0.67
0.76
0.84
0.92
1.00
24.5
25.8
26.8
31.3
34.6
36.3
37.1
36.7
36.9
36.1
34.6
32.6
22.4
20.7
18.9
12.7
19.0
20.7
28.2
33.8
∆H
(kcal/mol)
6.2
6.5
6.8
8.9
6.6
6.9
5.4
6.8
6.1
7.1
7.3
16.6 6.9
17.0 ca. 5
18.2 ca. 3
17.6
DMPC/DMTAP-DNA complexes
Tm
(°C)
Tpre
(°C)
∆H
(kcal/mol)
24.45
32.25
37.35
39.35
40.55
41.15
41.85
40.25
39.25
39.35
36.65
34.45
29.05
25.46
12.65
19.85
25.85
27.45
6.2
>5.9
>4.1/0.7
>1.9/2.4
>0.5/2.5
>1.9
>1.6
>2.0
>5.0
>1.7
>3.9
>0.2/3.2
>2.2/2.4
>3.1/0.1
>8.3
23.25
23.55
20.95.
22.45
can be called a pseudo-binary lipid-phase diagram of DNAcomplexed cationic lipid mixtures. For this reason we simply
J. Phys. Chem. B, Vol. 103, No. 46, 1999 10303
furnish the lipid-phases denotation with an additional index “C”
for “complexed” or “condensed”. Solidus and liquidus lines for
each phase diagram were constructed from the intersections of
lateral peak tangents and the base line of the DSC scans
respectively. To illustrate true broadening of the transition in
the coexistence regions, the solidus and liquidus lines were
corrected by the width of pure DMPC as suggested by Mabrey
et al.23
The dominant phases in the binary DMPC/DMTAP phase
diagram are the Lβ′ gel phase and the liquid-crystalline LR phase.
Both phases are separated by narrow coexistence regions (drawn
in gray) in the range 0.25 < χTAP < 0.75. Most striking is the
fact that the chain-melting transition temperature reaches a
pronounced maximum at about χmax
TAP ) 0.45. At low cationic
lipid mole fractions we find a Pβ′ ripple phase. The ripple phase
P
)
exhibits peritectic behavior with a peritectic point at χTAP
0.18. At this point the LR, Lβ′, and additionally the ripple phase
(Pβ′) coexist. The pretransition temperature, Tpre, given by the
maxima of the low-temperature peaks in Figure 2b increases
from Tpre ) 12.5 °C of DMPC to TP ) 30 °C. At χTAP > 0.75
two different crystalline phases, S1 and S2, are found from two
enthalpic transitions in the DSC spectra, which we will
corroborate later in this article by two distinguishable WAXS
reflexes. X-ray patterns will also reveal the coexistence of two
Lβ′ WAXS peaks for samples containing more than 91% DMPC.
Consequently, the low-temperature enthalpic transition detected
by DSC is interpreted as the transition from S1 to S2, the middletemperature peak as the transition from S2 to LR, and the hightemperature peak as the transition from Lβ to LR. The lateralphase separation of the solid phases and the miscibility of the
high-temperature phases correspond to eutectic behavior.
In Figure 3b the phase diagram of the cationic lipid-DNA
mixtures is shown. Again, the diagram is dominated by the
C
and LCR phases. The most remarkable differcorresponding Lβ′
ence is a demixing gap at low mole fractions of cationic lipid
where excess lipid mixtures coexist with CL-DNA. DSC
detects the vesicle pretransition and main transition and at higher
C
-LCR transition of the complexes. The
temperatures the Lβ′
phase behavior of the lipid vesicles is very similar to that of
pure lipid mixtures, but shifted toward that of lower TAP
concentration inside the lipid vesicles. The majority of the
cationic lipids are bound in CL-DNA complexes because of
electrostatic effects. The high χTAP values in the complexes also
lead to a relatively high transition temperature. With increasing
χTAP the area of the transition peak increases at the expense of
the pure vesicle peak because of the growing amount of CLDNA. We point out that the exact composition in the left-hand
corner of the phase diagram (Figure 3b) cannot be derived by
a simple lever rule as in the binary case. The coexistence of
lipid-DNA complexes and binary excess liposomes is possible
because of the additional degree of freedom in the ternary
system. Samples containing more than 20% of DMTAP form
only lipid-DNA complexes of one kind. In this case the lipidphase behavior in the DNA-complexed form is pseudo-binary.
The first DSC-detectable transition of CL-DNA (χTAP )
0.09) shows already a relatively high Tm of 39 °C that is slowly
)
increasing to the maximum transition temperature of Tmax
m
max
) 0.4.
41.60 °C at the corresponding TAP mole fraction TTAP
Tmax
m of CL-DNA is consequently 5 °C higher than that for the
pure lipid vesicles shown in Figure 3a. The thermotropic
behavior of the CL-DNA at χTAP > 0.75 shows behavior
similar to the lipid vesicles. The chain-melting temperature, Tm,
of the complexes is close to that of binary lipid vesicles of
corresponding composition. However, thermodynamically and
structurally, only one solid phase, S, is observed with the onset
10304 J. Phys. Chem. B, Vol. 103, No. 46, 1999
Zantl et al.
Figure 4. Freeze fracture electron micrographs of binary D1MPC/DMTAP vesicles. (a) The ripple phase of pure DMPC vesicles compared to (b)
vesicles containing 8% DMTAP (the insets are magnified 5×). The ripple repeat distance is about 20 nm in both cases. (c) Vesicles in the Lβ′ gel
phase. (d) Vesicles quenched from the Lβ′-LR transition temperature, Tm ) 36.5 °C. (e) Pure DMTAP vesicles in the solid state and (f) in the liquid
crystalline LR state.
C
of the Lβ′
-S coexistence region being shifted to higher χTAP
values. Interestingly, no hysteresis due to sample history is found
anymore for complexes at low temperatures.
Freeze-Fracture Electron Microscopy. The different phases
of binary lipid vesicles are visualized by freeze-fracture electron
micrographs. The rippled surface of a multilamellar vesicle in
the ripple phase at 21.6 °C is clearly seen for pure DMPC
vesicles (Figure 4a) and vesicles containing 8% DMTAP (Figure
4b), χTAP ) 0.08. The ripple wavelengths, λ ) 20.7 and 20.4
nm, respectively, are similar. At χTAP ) 0.48 no ripples were
found at any temperature on these vesicles. In the gel Lβ′ phase
(T ) 34 °C) the vesicle surface is totally smooth (Figure 4c).
In the middle of the Lβ′-LR phase coexistence region at Tm )
36.5 °C the vesicle surface exhibits a surface relief which might
Behavior of Lipid-DNA and Binary Lipid Mixtures
J. Phys. Chem. B, Vol. 103, No. 46, 1999 10305
Figure 5. Freeze fracture electron micrograph of isoelectric, lamellar cationic lipid-DNA complexes containing a cationic mole fraction, χTAP )
0.5, quenched from 20 °C. The extended layered structure is in agreement with the structure derived from X-ray scattering shown in Figure 1b. The
DNA strands cannot be resolved by freeze fracture electron microscopy.
correspond to membrane deformations caused by the coexistence
of Lβ′ and LR domains (Figure 4d).24 Pure DMTAP vesicles
quenched from 26 °C show a characteristic surface structure as
well as smooth regions on the surface (Figure 4e). Possibly
C
and the solid phase S is
lateral phase separation of Lβ′
observed. In Figure 4f, finally, it is shown that DMTAP vesicles
have smooth surfaces at 36 °C corresponding to the liquidcrystalline phase LR. The fluid DMTAP vesicles with diameters
smaller than 2 µm are somewhat smaller than vesicles of
DMPC-DMTAP mixtures, where diameters up to 30 µm are
found.
In the case of lipid-DNA complexes electron microscopy
reveals the existence of condensed multilamellar aggregates. As
an example a freeze-fracture micrograph of aggregates with
equimolar lipid composition is shown in Figure 5. The sample
was quenched from 20 °C to room temperature, corresponding
C
to the Lβ′
state of the membranes. More than a hundred
membranes in parallel order can be counted in one stack.
However, a parallel arrangement of the DNA rods, as detected
by SAXS, is not observable. The expected repeat distance of
about 4 nm is indeed too small to be resolved by freeze fracture.
Wide-Angle X-ray Scattering of the Gel Phases. Figure 6
shows wide-angle X-ray diffraction data of purely lipidic
multilamellar vesicles (MLVs) and lipid-DNA complexes. The
well-known wide-angle reflections of the LR, Pβ′, and Lβ′ phase
of pure DMPC is given as a reference on the left side of the
panel. The DMPC Lβ′ peak shows a sharp, resolution-limited,
peak superimposed with a broadened peak at higher q values.25
The latter arises from powder averaging of the (1,1) reflection
of a distorted hexagonal lattice with chains tilted toward the
next neighbors. We compare the line shape of the WAXS peak
of the binary DMPC/DMTAP mixtures. The latter exhibit a
broadened reflection centered around 1.48 Å-1 at low temperatures. The broadening is characteristic for a Lβ′ phase with
hydrocarbon chain tilt between the nearest neighbors.26,27 In
particular we can exclude the possibility of a rippled Pβ′
Figure 6. (a) Wide-angle X-ray scattering (WAXS) of DMPC
multilamellar vesicles (MLVs) in the Lβ′, Pβ′, and LR phase. (b) WAXS
signal of the low-temperature Lβ′ phase for binary DMPC/DMTAP
mixtures with varying mole fractions of cationic lipid. (c) DMTAP
shows two different solid phases, S1 and S2, at low temperatures and a
fluid LR phase at high temperatures. (d) In comparison the WAXS signal
of lipid-DNA complexes for different mole fractions of DMTAP and
(e) DMTAP/DNA complexes is shown. Note that the shape of the
WAXS reflexes are indicative of different tilts of the hydrocarbon chains
with respect to the bilayer normal as described in detail in the Appendix.
10306 J. Phys. Chem. B, Vol. 103, No. 46, 1999
Zantl et al.
Figure 7. Small-angle X-ray scattering at the chain-melting transition.
C
The coexistence of Lβ′
and LCβ is seen by two sets of integral Bragg
reflections according to two lamellar spacings. (inset) Fractional
conversion shown by the fraction gel phase, fraction liquid crystalline
phase as a function of temperature calculated from the normalized
C
second Bragg reflection (left inset) and from the Lβ′
-WAXS-peak
intensity (right inset).
Figure 8. (a) Pressure-area diagram of monolayers of pure DMPC,
DMTAP, and an equimolar mixture of DMPC/DMTAP at the airwater interface. The inset shows the average headgroup area as a
function of cationic mole fractions for two constant pressures corresponding to the crystalline and liquid expanded phases, respectively.
(b) Schematic drawing of the headgroup arrangement in the fluid and
frozen state at 1:1 stoichiometry. In the gel state the quaternary amine
(TAP) and phosphate group get into closer proximity, which might be
responsible for the overall attractive headgroup interaction.
configuration because the electron microscopy study clearly
showed a flat membrane. In the Appendix we describe in detail
how the wide-angle reflections of Lβ′ phases characterize
different structures, depending on the tilt and tilt direction of
the chains with respect to the in-plane orientation. In our data
we see a slight tendency of increasing tilt angle with increasing
mole fraction, χTAP, cationic lipid.
The same binary lipid mixtures complexed with DNA exhibit
similar wide-angle diffraction. The line shape is in agreement
C
with a tilted Lβ′
phase, where the tilt direction points again
between next neighbors. However, the tilt angle increases with
increasing molar fraction, χTAP, as can be seen from the apparent
broadening of the width of the reflection. In fact, the chain tilt
seems to almost vanish close to χTAP ) 0.25, where the width
is as narrow as expected for an untilted Lβ phase. As a general
observation, we find that the tilt of all binary lipid mixtures
decreases with the addition of DNA.
On the right-hand side of Figure 6 two solid phases, S1 and
S2, and the liquid-crystalline LR phase of pure DMTAP are
shown. In the case of pure DMTAP the complexation with DNA
leads to a new solid phase, S, that is slightly different from the
S1 and S2. Also, WAXS showed a strong thermotropic polymorphism of DMPC-DMTAP vesicles as well as for the lipidDNA complexes at high χTAP, i.e., the hatched region in the
phase diagrams shown in Figure 3. Fast cooling of the fluid
phases revealed retarded crystallization and consequently supercooled LR and metastable Lβ phases. This explains the
problem of reproducibility of the DSC-derived phase transition
temperatures in this region of the phase diagram.
Small-Angle X-ray Scattering at the Chain-Melting Transition. Figure 7 shows the SAXS signal of lipid-DNA
complexes at the chain-melting transition. The top scan corresponds to the liquid-crystalline LCR state and the bottom scan to
C
gel state as sketched schematically in Figure 1b. CLthe Lβ′
DNA in the LCR phase consists of lipid bilayers in the liquidcrystalline state with intercalated parallel-ordered DNA double
C
helices.5 In the case of the Lβ′
phase the lipid membrane is gellike with a charge-density-dependent chain tilt angle.11 Both
scans show a set of equally spaced Bragg reflections corresponding to the lamellar stacking of the lipid membrane. In both
scans an additional broad peak is seen, which corresponds to
the lattice constant of the intercalated DNA lattice. The DNA
C
gel
lattice constant of the LCR phase is larger than that in the Lβ′
state, while the interlamellar distance is larger in the latter. The
coexistence of two phases is clearly seen in the temperature
interval around Tm. While the position of the lamellar Bragg
reflections remains constant, the amplitudes increase and
decrease, respectively. The melting transition was found to be
fully reversible in all cases.
SAXS may be employed to determine the relative fractions
C
phases and allow one to determine the solidus
of the LCR and Lβ′
and liquidus temperatures. The normalized intensities of the
C
second Bragg peaks (wide-angle Lβ′
peaks) are plotted versus
temperature in the left (right) inset of Figure 7.
Headgroup Area Studied by Film Balance. In an attempt
to understand the TAP-PC headgroup interaction, we examined
a series of pressure-area isotherms of DMPC/DMTAP monolayers using a standard Langmuir film balance. Figure 8a shows
the pressure-area diagrams of pure DMPC, pure DMTAP, and
an equimolar mixture of both lipids. We plotted the headgroup
area, aH, at two constant pressures corresponding to the liquid-
Behavior of Lipid-DNA and Binary Lipid Mixtures
J. Phys. Chem. B, Vol. 103, No. 46, 1999 10307
expanded and -crystalline phase (Figure 8 inset) as a function
of the cationic mole fraction. In the liquid-expanded phase, the
area decreases monotonically from the larger PC headgroup size
down to the TAP headgroup. In contrast, in the ordered phase
the headgroup shows a minimum at about the equimolar ratio.
Discussion
Nonideal Mixing of Cationic-Zwitterionic Lipid Mixtures. The most striking feature in the binary-phase diagram
of DMPC/DMTAP (Figure 3a) is the maximum in the gel-toliquid crystalline transition temperature at a molar ratio of
max
) 0.45. Similar nonideal mixing behavior was previously
TTAP
reported for related cationic amphiphiles.12,13 This phase
behavior of cationic-zwitterionic alloys is particularily distinct
from anionic-zwitterionic systems such as DMPC/DMPS or
DMPC/DMPG, which show almost ideal mixing.28 A thermodynamic description of nonideal mixing is given qualitatively
by simple regular solution theory. For a regular solution the
excess free energy is written
gE ) ΩXAXB
(1)
where Ω is a parameter independent of temperature and
composition and XA and XB the molar fractions of components
A and B. To obtain phase diagrams like Figure 3a we have to
assume an attractive intermolecular interaction, Ω, which is
stronger in the gel state compared to that in the fluid state. As
suggested by Silvius, the elevation of the main phase transition
in these mixtures is largely due to electrostatic- rather than
hydrogen-bonding interactions.12 Furthermore, the maximum
around equimolar composition indicates the formation of
interacting units with 1:1 stoichiometry. The latter are often
called complexes in the literature. We avoid the term complex,
which here is reserved for lipid-DNA aggregates, but ask the
question, if stoichiometric units do exist in the lipidic binary
system. In alloy systems maxima in binary-phase diagrams are
nearly always associated with the existence of an intermetallic
phase.29 However, our electron microscopy studies as well as
the SAX and WAX scattering data provide no indication of a
distinct stoichiometric-phase and macroscopic-phase separation.
Nonetheless, it is most likely that stoichiometric units, such
as PC-TAP pairs, do form. These congruently melt at the gelto-liquid crystalline transition. We found that the headgroup
areas of the binary mixtures at the air-water interface exhibit
a minimum at a 1:1 PC-TAP ratio (Figure 8a). A molecular
interpretation is given in Figure 8b. The small headgroup of
the cationic amphiphiles are able to add onto the phosphate
group and hence the PC-TAP headgroup interaction may be
attractive. Alternating PC and TAP groups are able to pack
tightly and will lead to a reduction of the average headgroup
area. In fact, it was shown by NMR measurements that cationic
amphiphiles can change the orientation of the choline dipole
by more than 30° toward the water phase.15 The rotation of the
N+ end of the choline group further out into the bulk water
will reduce the electrostatic self-energy of this group. TAPPC units can be thought to exhibit a chain-packing density
resembling that of phosphatidyl-ethanolamines. Recent Monte
Carlo simulations of TAP-PC headgroup arrangements provide
evidence for the existence of stoichiometric domains that can
be imagined as clusters with increased TAP-PC correlations
mixed into an otherwise disordered PC-TAP gel phase.30 Our
strongest experimental arguments for the existence of such
stoichiometric units are, firstly, the narrowing of the transition
width compared to the pure DMPC and, secondly, the decrease
TAP
< 1/2. (aPC
of the headgroup area a(1:1)
H
H + aH ) in the
crystalline phase of monolayers.
Shift in the Transition Temperature for Lipid-DNA
Complexes. Condensation of cationic lipid vesicles and DNA
results in a substantial increase of the lipid chain-melting
transition temperature. The first quantitative study of the melting
transition in charged binary lipid membranes was carried out
by Träuble and Eibl.16 In general, charging the membrane lowers
the lipid chain-melting transition temperature. The decrease in
transition temperature, ∆Tm, with increasing surface charge
arises from an additional electrostatic free energy change, ∆Gel,
due to the change in surface area associated with the gel-toliquid crystalline-phase transition. The lateral electrostatic
pressure, Πel, favors an increase in the lipid headgroup area, A.
The transition temperature of a charged versus neutral lipid
membrane shifts by
∆Tm )
∆Gel Πel∆A
)
∆S*
∆S*
(2)
where ∆S* denotes the transition entropy of the chain melting.
Assuming full dissociation of the cationic lipid and large surface
potential eΨ . kT, the electrostatic pressure can be calculated
using classical Gouy-Chapman theory:16,31
∆Tm )
2RT ∆A
- (200RTc)1/2(40RT/e∆S*)∆A
∆S* A
(3)
The first term is the maximum electrostatic shift due to the
introduction of surface charge. The second term comprises the
entropic contribution of the diffuse layer of counterions. The
latter screens the electrostatic effect in the presence of salt. For
the lipids investigated here, we may insert ∆A/A ) 0.19 as
measured from the repeat distance of the intercalated DNA
lattice DMPC/DMTAP-DNA11 and ∆S* ) 2 × 1022 kT/mol
°C as known for pure DMPC.31 Equation 3 enables us to predict
the temperature shift in the presence of salt. For example, in
the case of equimolar mixtures of DMPC/DMTAP we measured
∆Tm ) 2.6 °C for 10 mM NaCl and ∆Tm ) 4.3 °C for 100
mM NaCl, in good agreement with eq 3 (see Figure 9a).
However, if we neutralize cationic liposomes by an isoelectric
quantity of DNA, one might expect that the total shift in the
transition temperature is simply given by the first term of eq 3,
yielding ∆Tm ) 10.8 °C. This means that we should find a
transition temperature Tmax
m + ∆Tm ) 47.8 °C, which is higher
than that of the experiment shown in Figure 9a.
To correctly describe the shift in the transition temperature
of DMPC/DMTAP in the presence of intercalated DNA, we
will proceed analogously to Träuble and Eibl. In brief, we will
show that the adsorbed DNA acts like a condensed twodimensional counterion lattice, which contributes to the change
in free energy by its electrostatic pressure. We take advantage
of the fact that the structure and molecular arrangment of the
lipid/DNA complexes are known from previous X-ray studies.5,11 DNA strands are intercalated between lamellar galleries
of lipid bilayers as shown in Figure 1b. In isoelectric complexes
the spacing between adjacent DNA strands, d, is fixed by the
charge density of the membrane. An expansion around the
spacing, d1/2, for χTAP ) 0.5 yields an approximately reciprocal
dependence on the mole fraction of the cationic lipid:11,32
d≈
d1/2
2χTAP
(4)
10308 J. Phys. Chem. B, Vol. 103, No. 46, 1999
Zantl et al.
Figure 9. (a) Comparison of the DSC main transition peaks of an
equimolar mixture of DMPC/DMTAP with and without DNA. A similar
shift to higher transition temperature as induced by DNA is observed
by the addition of salt (- - -). (b) The shift in the chain-melting transition
temperature of DMPC/DMTAP mixtures due to DNA condensation.
The straight line in the middle region indicates the theoretical prediction
according to eq 8.
In addition, the DNA lattice exhibits a lateral compressibility
modulus, BDNA
el , which is determined by the electrostatic
repulsion between the strands:33,34
BDNA
)
el
λ2
6πd
(5)
Here, ) 0r denotes the dielectric constant of water and λ
) e/1.7 Å, the line charge density of phosphate groups along
the DNA. The two-dimensional compressional modulus has
units of energy/area and was measured by analysis of the thermal
diffuse scattering of the DNA X-ray reflection.33,34
At the chain-melting transition the lipid membrane expands
laterally and the DNA lattice spacing increases with it. The gain
in electrostatic free energy of the DNA lattice is approximated
by ∆F/A ) ∆[1/2B(d/d0)2] ≈ B(∆d/d). Hence, the shift in
transition temperature is obtained using the first term in eqs 3
and 5, accounting for the fact that the intercalated DNA lattice
(i) fully compensates the surface charge and (ii) has a finite
lateral electrostatic compressibility.
∆Tm )
A DNA∆d
2RT ∆A
B
∆S* A
∆S* el d
(6)
Equation 6 compares well with experiment as shown in Figure
9b. Because of the dependence of BDNA
on χTAP via eqs 4 and
el
5, the temperature shift, ∆Tm ) TA - TBχTAP, is a linear function
of χTAP. For d1/2 ) 36 we obtain B ) 2 × 10-2 kT/Å2 from eq
5 and yield TA ) 10.8 °C and TB ) 7.6 °C, assuming ∆A/A )
∆d/d ) 0.19 and ∆S* ) 2 × 1022 kT/mol °C as used before.
The experimental results suggest TA ) 11 °C and TB ) 13 °C
in the middle χTAP regime. This experimental result is matched
by the theory with even better precision, if we take the
experimentally determined value for the compressional modulus,
B ) 2.8 × 10-2 kT/Å2 at d1/2 ) 36.33,34 As seen in Figures 9b
and 3b the theory will not be applicable at a low cationic mole
fraction, where we found a demixing gap, as well as at the far
right side of the phase diagram, where the existence of
ill-defined dehydrated solid phases impairs the comparison.
The agreement of our simple theory is remarkable considering
the apparent complexity of the system. However, the lipidDNA complexes are special in that all counterions are released
from the complex and the DNA effectively plays the role of a
two-dimensional layer of rodlike counterions. Secondly, the
DNA strands are separated from the bilayer by a thin hydration
layer, approximately 2 Å according to the X-ray results, and
are well-separated from each other because of the constraint to
preserve local charge neutrality. Hence, no short-range forces
become effective. Finally, the effect of monovalent salt on our
particular binary lipid system is well-described by the GouyChapman model, which is a prerequisite that the electrostatic
theory can be extended to the case of an adsorbed DNA lattice.
In this context it is also interessting to note that DMPE exhibits
a chain-melting temperature, Tm ) 48 °C, that is comparable
to a completely neutralized equimolar PC-TAP mixture, as
described by the first term in eq 3. This indicates that the state
of hydration and chain packing of PC-TAP units is similar to
that of DMPE, which is very intuitive, when looking at the
molecular packing model given in Figure 8b. The total enthalpy
change of the melting transition due to DNA complexation could
not be measured accurately because of problems with material
transfer. However, as indicated in Table 1 and Figure 9a, the
enthalpy change decreases from 6 kcal/mol to about 3-5 kcal/
mol. Similar enthalpy changes are known for negatively charged
membranes with adsorbed proteins like cytochrome c.35 Note
that here the change of the enthalpy change is discussed. A more
important quantity in this context might be the enthalpy of
lipid-DNA complex formation. The latter has been measured,
for example, for CTAB and DNA using titration calorimetry.36
Finally, we like to comment on the distinct shape of the DSC
peak of DNA-lipid complexes compared to the sharp peak of
equimolar lipidic mixtures (compare Figure 9a). It is reasonable
to assume that DNA-induced demixing is responsible for the
observed broadening. Lateral redistribution of cationic lipids
in the presence of DNA was shown to be expected by recent
theoretical studies.8,9
Conclusion
The phase behavior of the zwitterionic-cationic mixture
DMPC/DMTAP was studied with focus on the thermodynamic
effect of DNA complexation. We found strongly nonideal
mixing expressed in an elevated azeotropic point at almost
equimolar mole fractions. The formation of stoichiometric PCTAP units is assumed on the basis of the observed reduction in
the PC-TAP headgroup area in lipid monolayers as well as
the higher cooperativity of the main-phase transition for
equimolar mixtures. The complexation of DMPC/DMTAP
liposomes with DNA results in the formation of condensed
lipid-DNA composite aggregates. These aggregates have a
lamellar liquid-crystalline structure, which retains in the gel and
Behavior of Lipid-DNA and Binary Lipid Mixtures
J. Phys. Chem. B, Vol. 103, No. 46, 1999 10309
Figure 10. Schematic presentation of the WAXS powder signal arising from hexagonal-packed alkyl chains. From top to bottom a real space
picture of tilted rod lattices, the representation in Fourier space and the powder-averaged signal is shown. The WAXS peak exhibits characteristic
line shapes depending on the tilt direction: (a) tilt toward next neighbors, LβI, (b) tilt between next neighbors, LβF, and (c) no tilt, Lβ.
liquid-crystalline phase. WAXS gave evidence for variations
in the lipid chain tilt throughout the Lβ′ phase. The presence of
DNA alters the lipidic thermotropic phase behavior 2-fold: we
found (i) a systematic increase in the main transition temperature
and (ii) a demixing gap at low TAP concentration. The shift in
the transition temperature is in quantitative agreement with a
modified Gouy-Chapman theory that treats the DNA as an
adsorbed rod lattice.
The results presented here should be more generally valid
for other cationic lipids. Similar behavior could be expected,
e.g., for DODAB, DOTMA, and double-chain amphiphiles with
small cationic headgroups. For transfection applications saturated
lipids are usually avoided because of the presence of lipid-phase
transitions. However, in view of the present study, saturated
cationic lipids might show an even broader spectrum of
properties and need not be ruled out a priori. For example,
isothermally induced transitions such as pH- or ionic-induced
transitions could act synergetically in the course of gene
delivery.
Acknowledgment. We thank E. Sackmann for support and
critical comments on the phase diagrams. We enjoyed helpful
discussions with D. Pink, A. Tardieu, P. Garidel, and Th.
Heimburg and furthermore benefited from a constructive referee
report. F.A. gratefully acknowledges a European TMR postdoctoral scholarship. This work was supported by BMBF Grant
03-SA5TU1-0.
Appendix: Tilt Direction Analysis by Wide-Angle X-ray
Scattering
In this Appendix we will shortly recall the reciprocal lattice
of Lβ′ phases and discuss the analysis of the tilt direction in the
Lβ′ phases from powder-averaged line shapes as proposed by
Tardieu and Luzzatti26 and Levelut.27
In the chain frozen Lβ′ phase the hydrocarbon chains pack in
an all-trans configuration in hexagonal, or rather distorted
hexagonal, local order. These phases may have different tilt as
experimentally observed in X-ray scattering experiments by
Smith et al. using oriented freely suspended DMPC films.37
Following the nomenclature of these authors, we denote the Lβ
phases as hexatic phases with (a) tilt toward the nearest neighbor
LβI or (b) tilt between next neighbors LβF. Intermediate directions
are known as LβL phases.37,38 In the Lβ gel phase the chain
director is normal to the bilayer as shown in Figure 10c.
The electron density of the chain lattice can be described by
a product of a hexagonal lattice of infinite rods and the form
factor of the layer, i.e., the electronic density perpendicular to
the bilayer. Thus, in reciprocal space the lattice is given by the
convolution product of a hexagonal two-dimensional point lattice
perpendicular to the rods and the rodlike form factor resulting
from the Fourier transform of the bilayer. The three cases are
shown in Figure 10. The shape of the rodlike form factor is
related to the bilayers thickness. Here, it is approximated by a
Gaussian profile.
The WAXS line shape depends on the position of the rod
center vs the equatorial plane (qx, qy) (Figure 10): (a) If the
center is located in the equatorial plane, the powder averaging
yields an asymmetric resolution-limited peak with a foot at wide
angle, indicating that the rod is tangential to the q sphere. (b)
If the center is out-of-plane, the powder averaging yields a
symmetric broadened peak and its width is increasing with qz.
In the Lβ gel phase (c), all the centers are located in-plane and
the WAXS reflection is sharp. In the first Lβ′ gel phase, two
centers are in-plane and four centers are out-of-plane so that
the WAXS is the sum of a sharp peak and a broadened peak.
In the second Lβ′ gel phase all the peaks are out-of-plane at qz
and 2qz and the peak is the superposition of two broadened
peaks. The line shapes of the WAXS peaks are clearly
distinguishable, depending on the direction of the tilt.
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