Cationic Liposomes in Mixed Didodecyldimethylammonium Bromide
and Dioctadecyldimethylammonium Bromide Aqueous Dispersions
Studied by Differential Scanning Calorimetry, Nile Red Fluorescence,
Eloi Feitosa,*,†Fernanda Rosa Alves,†Anna Niemiec,‡M. Elisabete C. D. Real Oliveira,§
Elisabete M. S. Castanheira,§and Adelina L. F. Baptista§
Physics Department, Sa ˜o Paulo State UniVersity, Sa ˜o Jose ´ do Rio Preto, SP, and Instituto de Quı ´mica,
UniVersidade Estadual de Campinas, Campinas, SP, Brazil, and Physics Department, UniVersity of Minho,
Campus de Gualtar, 4710-057 Braga, Portugal
ReceiVed NoVember 30, 2005. In Final Form: February 14, 2006
The thermotropic phase behavior of cationic liposomes in mixtures of two of the most investigated liposome-
forming double-chain lipids, dioctadecyldimethylammonium bromide (DODAB) and didodecyldimethylammonium
bromide (DDAB), was investigated by differential scanning calorimetry (DSC), turbidity, and Nile Red fluorescence.
The dispersions were investigated at 1.0 mM total surfactant concentration and varying DODAB and DDAB
dispersions are around 16 and 43 °C, respectively, and we aim to investigate the Tmbehavior for mixtures of these
but the Tmof DDAB is roughly independent of the DODAB concentration. Both DSC and fluorescence measurements
show that, within the mixture, at room temperature (ca. 22 °C), the DDAB-rich liposomes are in the liquid-crystalline
state, whereas the DODAB-rich liposomes are in the gel state. DSC results point to a higher affinity of DDAB for
phase behavior. Fluorescence measurements also show that the presence of a small amount of DODAB in DDAB-rich
liposomes causes a pronounced effect in Nile Red emission, due to the increase in liposome size, as inferred from
since it can be used to monitor the structure and phase behavior
technology; such applications may require sample preparation
with well-controlled properties.1-5The mixture of lipids in
solution can thus be used to modify the phase behavior of the
individual lipids by varying the lipid composition in the mixture
and monitoring some physical properties of these mixtures.
of the aggregates by investigating the interaction with other
systems. For example, to overcome the precipitation problems
charged lipids, a colipid with neutral or opposite charge is often
size and architecture and chain conformation in the lipid
yldimethylammonium bromide (DDAB) have been some of the
most investigated cationic lipids,6and liposome formation in
aqueous solutions of these lipids is extensively reported,7-9but
the literature is scarce on the liposome formation in aqueous
mixtures of these surfactants. The difference in chain length of
these lipids (C18and C12) yields interesting characteristics and
behavior for mixed solutions of these lipids, as shown in this
reduce10whereas cationic surfactants tend to increase11the Tm
of DODAB. Cationic liposomes also interact with DNA to form
attraction between the positively charged liposome and the
negatively charged DNA molecule.12-15
Steady-state fluorescence spectroscopy has been used to
characterize the properties of cationic liposomes, such as
* To whom correspondence should be addressed. Phone: +55 17 3221
22 40. Fax: +55 17 3221 22 47. E-mail: email@example.com.
†Sa ˜o Paulo State University.
‡Universidade Estadual de Campinas.
§University of Minho.
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(2) Fendler, J. H. Membrane Mimetic Chemistry; Wiley-Interscience: New
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Polymers in Aqueous Solution; John Wiley & Sons: New York, 1998.
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(5) Rosoff, M., Ed. Vesicles; Marcel Dekker: New York, 1996.
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J. Phys. Chem. 1990, 94, 3722.
(9) Proverbio, Z. E.; Schulz, P. C.; Puig, J. E. Colloid Polym. Sci. 2002, 280,
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M.; Northrop, J. P.; Ringold, G. M.; Danielsen, M. Proc. Natl. Acad. Sci. U.S.A.
1987, 84, 7413.
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M. C. Biochim. Biophys. Acta 1999, 1418, 71.
Langmuir 2006, 22, 3579-3585
10.1021/la053238f CCC: $33.50© 2006 American Chemical Society
Published on Web 03/17/2006
for this work, Nile Red, is hydrophobic with low solubility and
solvatochromic, and both steady-state and time-resolved fluo-
rescence emission properties are strongly medium dependent.
Nile Red usually exhibits an increase in fluorescence yield with
decreasing solvent polarity accompanied by a blue shift in the
DSC has also widely been employed to investigate the phase
behavior of liposomes both in the absence and in the presence
of additives.25-31Thus, the combination of these two methods
may give important information about liposome systems, such
as the DODAB/DDAB mixed system investigated here.
of cationic liposome dispersions in mixtures of DDAB and
DODAB by using differential scanning calorimetry (DSC) and
formation of two populations of liposomes with distinctive
properties. The turbidity of the mixture dispersion is dominated
by the gel state, rather than by the liquid-crystalline state of the
Materials. DODAB, DDAB, and Nile Red (9-(diethylamino)-
5H-benzo[R]phenoxazin-5-one) were supplied by Aldrich. All
chemicals were used as received. Scheme 1 shows the molecular
similar to that of DODAB, except for the shorter C12double chain,
instead of the C18DODAB.
Liposome Preparation. Stock solutions of DODAB and DDAB
liposome dispersions were prepared by simple dilution of 5.0 mM
lipid in water at room temperature (ca. 22 °C). The DODAB/water
mixture was then warmed to 60 °C (safely above the DODAB Tm
Tmis lower than room temperature, this lipid was mixed with the
aqueous solvent at room temperature and kept standing for at least
24 h. After solubilization, both lipid dispersions were kept standing
at room temperature and stored. Mixed DODAB/DDAB aqueous
dispersions were prepared by mixing properly 1.0 mM aqueous
dispersions of DODAB and DDAB to have 1.0 mM total lipid
concentration and varying the individual lipid concentrations.
For fluorescence measurements, the DODAB/DDAB aqueous
mixtures were sonicated at 60 °C in a bath-type sonicator (Heat
Systems W-225R), to obtain optically clear dispersions with the
probes incorporated within the liposome bilayer.20,22A final
concentration of 3 µM Nile Red was introduced in the liposome
dispersions by injection of 10 µL of a 10-3M stock solution of this
probe in ethanol. The liposome dispersions were cooled to room
temperature and stored for 24 h prior to the measurements.
DODAB and DDAB liposomes, prepared by simply mixing the
mixtures at different fractions of each lipid.
The DODAB molar fraction, xDODAB, in the dispersions is given
by eq 1 such that xDODAB+ xDDABequals unity.
High-Sensitivity Differential Scanning Calorimetry Measure-
ments. A VP-DSC (MicroCal, Northampton, MA) calorimeter
equipped with 0.542 mL twin cells for the reference and sample
solutions was used. The measurements for 1.0 mM total lipid
concentration and varying DODAB and DDAB concentration were
of 5-80 °C. This method is useful for observing, for example, the
gel to liquid crystal phase transition temperature that takes place in
the system. Moreover, it enables one to find Tm, which is the
temperature at the peak maximum. The enthalpy change associated
curves using the equipment software (MicroCal Origin, v. 5.0).
Further details on the DSC methods and setup can be found in
performed using a Spex Fluorolog 2 spectrofluorimeter. Polarized
emission spectra were recorded using Glan-Thompson polarizers.
Turbidity Measurements. Turbidity data were collected using
a Cary 100 Scan spectrophotometer equipped with a quartz cuvette
of 1.0 cm path length.
Results and Discussion
DSC Results. The DSC traces for neat DODAB and DDAB
in water and mixtures of these surfactants, for selected
concentrations such that the total lipid concentration equals 1.0
mM, are shown in Figures 1 and 2, respectively, for the first and
second upscans. In dilute dispersions the Tm of these neat
surfactants is constant (results not shown). All DSC traces were
repeated twice, and the first and second upscans recorded. The
prescanning time, that is, the waiting time to start the second
upscan after the sample being cooled, was 20 min. At 1.0 mM
the DSC traces for the neat DDAB and DODAB dispersions
exhibit a single narrow peak, characteristic of the main gel to
liquid crystalline phase transition (melting) temperature. The
to as the lower and higher main transition temperatures, Tm(1)
and Tm(2), for the DDAB/DODAB system. The parameters Tm,
∆T1/2, and ∆H obtained from these traces are summarized in
Tables 1 and 2, respectively, for the first and second upscans.
(16) Zuidam, N. J.; Barenholz, Y. Biochim. Biophys. Acta 1997, 1329, 211.
M. G.; Burrows, H. D. J. Phys. Chem. B 2002, 106, 4061.
(19) Greenspan, P.; Fowler, S. D. J. Lipid Res. 1985, 26, 781.
(20) Krishnamoorthy, I. G. J. Phys. Chem. B 2001, 105, 1484.
(21) Coutinho, P. J. G.; Castanheira, E. M. S.; Rei, M. C.; Real Oliveira, M.
E. C. D. J. Phys. Chem. B 2002, 106, 12841.
(22) Hungerford, G.; Castanheira, E. M. S.; Baptista, A. L. F.; Coutinho, P.
J. G.; Real Oliveira, M. E. C. D. J. Fluoresc. 2005, 15, 835.
(23) Krishna, M. M. G. J. Phys. Chem. A 1999, 103, 3589.
(24) Ghoneim, N. Spectrochim. Acta, A 2002, 56, 1003.
(25) Feitosa, E.; Barreleiro, P. C. A.; Olofsson, G. Chem. Phys. Lipids 2000,
(26) Benatti, C. R.; Feitosa, E.; Fernandez, R. M.; Lamy-Freund, M. T.Chem.
Phys. Lipids 2001, 111, 93.
(27) Benatti, C. R.; Tiera, M. J.; Feitosa, E.; Olofsson, G. Thermochim. Acta
1999, 328, 137.
(28) Marques, E. F.; Khan, A.; Lindman, B. Thermochim. Acta 2002, 394, 31.
(29) Schulz, P. C.; Puig, J. E.; Barreiro, G.; Torres, L. A. Thermochim. Acta
1994, 231, 239.
(30) Blandamer, M. J.; Briggs, B.; Cullis, P. M.; Kirby, S. D.; Engberts, J. B.
F. N. J. Chem. Soc., Faraday Trans. 1997, 93, 453.
Scheme 1. Molecular Structures of Nile Red and DODAB
[DDAB] + [DODAB]
3580 Langmuir, Vol. 22, No. 8, 2006 Feitosa et al.
Note that there is no peak for the neat DDAB in the second
upscan (Figure 2). This is because a longer prescanning time
long enough for the surfactants to rearrange into the gel state of
the liposome bilayer to attain a kinetic equilibrium. Of course,
this time is system dependent. Accordingly, Figure 3 shows the
effect of the prescanning time of 0, 0.4, 0.5, 1.0, 2.0, and 4.0 h
on the DSC traces for 5.0 mM DDAB. For 1.0 mM DDAB, the
peak intensity is even lower, and the prescanning time required
to have a measurable peak is even larger (larger than 4 h, results
not shown). The Tmfor 1.0 and 5.0 mM neat DDAB equals 16.2
the main transition peak does not come up, owing to kinetic
reasons as proposed by Blandamer and co-workers;30that is, the
bilayer in the gel (more rigid) state of the chains. Interestingly,
the prescanning time affects only the peak intensity and the area
(proportional to the melting enthalpy of the surfactant chains),
but not to the peak position (related to the surfactant Tm). Table
3 summarizes the Tm, ∆T1/2, and ∆H for DDAB at increasing
prescanning time up to 4 h. These results indicate that Tmand
∆T1/2are roughly constant, but ∆H increases roughly exponen-
tially with the prescanning time (curve not shown).
Depending on the lipid relative concentration, the DODAB/
DDAB mixtures exhibit one or two main peaks in the DSC
thermograms (additional peaks may appear, owing to the
pretransition or posttransition observed for the neat DODAB, as
discussed below). The main peaks for the mixtures approach the
when the molar fraction of the other surfactant goes to zero, and
the peak position varies systematically (or not) with surfactant
for 1.0 mM total lipid concentration. Numbers beside the curves
correspond to the DODAB molar fraction, xDODAB.
Figure 2. Second DSC upscan for the mixture DODAB/DDAB/
water, for 1.0 mM total lipid concentration. Numbers beside the
curves correspond to the DODAB molar fraction, xDODAB.
Table 1. Parameters Obtained from the First DSC Upscan
0.2 15.65 3.81.06
0.3 15.59 3.3 0.80
0.415.75 3.0 0.79
0.7 15.581.0 1.06
Figure 3. Effect of the prescanning time on the DSC thermograms
for a 5.0 mM DDAB aqueous solution. Numbers beside the curves
indicate the prescanning time in hours.
Table 2. Parameters Obtained from the Second DSC Upscan
0.2 15.85 1.6 0.79
0.515.62 0.3 0.79
Table 3. Parameters Obtained from the First DSC Upscan, for
Different Prescanning Times (Figure 3)
Cationic Liposomes in DDAB and DODAB Dispersions Langmuir, Vol. 22, No. 8, 2006 3581
composition, as Figures 4 and 5 indicate. In fact, the lower Tm(1)
but Tm(2)decreases from Tmof neat DODAB when xDDABis
around 0.2-0.8 (or 0.2-0.7 in the second upscan) two main
peaks are present, indicating the presence of two different
populations of DDAB-rich and DODAB-rich liposomes, with
well-defined melting temperatures, Tm(1)and Tm(2).
are related to the pretransition (Ts≈ 33.2 °C) and posttransition
(Tp≈ 53 °C) temperatures, as reported before.25In the presence
of a small amount of DDAB, the posttransition temperature
vanishes, but the pretransition temperature remains; upon
and the main peak approaches and overlaps the pretransition
of solutes and cosurfactants on the pre- and posttransition
temperatures of DODAB liposomes have been reported, and it
is shown that these peaks are sensitive to cosolutes and
cosurfactants, in addition to the surfactant concentration itself
(they tend to increase intensity with surfactant concentration).31
due to kinetic control; in this case, as discussed above, a longer
prescanning time is required,30or alternatively the system may
not be reversible, giving different traces. After the first cooling,
before being rescanned, as Figure 3 suggests. This can explain
the absence of a peak in the second DSC upscan for DDAB/
water shown in Figure 2. Note that these traces give slightly
higher Tm) 16.2 °C relative to that reported in Table 1 (Tm)
°C for Tm. Since this was a new sample preparation, we may
infer that the actual Tmfor DDAB lies in the range 15.6-16.2
°C, in good accord with literature data.28The mean Tmobtained
from these two values is 15.9 °C.
First Upscan. According to Figure 4, the lower Tm(1)for the
neat DDAB is 15.6 °C as obtained by the first DSC upscan, in
xDODAB(thus decreasing xDDAB), Tm(1)remains roughly constant
and equals Tmfor the neat DDAB, showing that DODAB does
not affect the DDAB Tm until xDODAB ≈ 0.8, when the
corresponding transition peak vanishes, probably because of the
low intensity of the DSC signal owing to the low xDDAB(∼0.2),
that is, partially solubilized within the bilayers of DODAB
liposomes. When xDODABexceeds 0.2, a second peak appears
around 30 °C, corresponding to the upper melting temperature
Tm(2). As xDODABis further increased, Tm(2)increases linearly to
attain the Tmfor 1.0 mM neat DODAB (xDODAB) 1). These
results indicate that the DDAB incorporation into DODAB
liposomes yields a higher bilayer fluidity of DODAB-rich
liposomes, leading to a decrease in the DODAB melting
temperature Tm(2), as xDDABis increased. DODAB, on the other
hand, poorly incorporates into the DDAB-rich liposomes, thus
leaving the DDAB Tm roughly constant. Overall, the results
suggest that DODAB and DDAB do not mix ideally to get
It seems that DDAB has more affinity for DODAB than the
reverse, as can be inferred by the monotonic decrease in Tm(2)
upon increasing xDDABand constancy of Tm(1)upon increasing
xDDAB. Probably this is related to the shorter chain length of
DDAB relative to DODAB, which favors the binding of DDAB
to the DODAB liposomes.
Tables 1 and 2 summarize the parameters Tm, ∆T1/2, and ∆H
upscans (discussed below). Accordingly, as xDODABincreases,
the cooperativity (as measured by the peak width) of the lower
but that for the upper transition tends to decrease (∆T1/2(2)
increases). This indicates a higher solubility of DDAB into
a less cooperative melting transition relative to a neat lipid. The
decreases for the lower transition, after an initial steep increase
should note that the values of the melting enthalpy for DDAB
and DODAB shown in Tables 1 and 2 were calculated per mole
should be calculated per mole of lipids within each population
of liposomes, but this is an unknown parameter. Thus, we refer
to this calculated enthalpy as an apparent enthalpy, since we
Figure 4. Effect of DODAB or DDAB concentration on Tmof the
DSC data were obtained from the first upscan.
Figure 5. Effect of DODAB or DDAB molar fraction on Tmof the
DSC data were obtained from the second upscan.
3582 Langmuir, Vol. 22, No. 8, 2006 Feitosa et al.
Second Upscan. The second upscan gives roughly the same
result as the first scan, except for the lower Tm(1)that decreases
slightly with xDODAB(instead of being constant as observed for
liposomes takes place, Tm(1)decreasing slightly. Alternatively,
the decrease in Tm(1)may be due to kinetic effects as discussed
above, but this is less probable, since Tmdoes not depend on the
of the liposome Tm to vary with the concentration of added
toward DDAB liposomes relative to that suggested by the first
upscan is probably related to an additional amount of DODAB
upscan. Also, the decrease in Tm(1)upon addition of DODAB
indicates that a few more DODAB molecules are solubilized
into DDAB-rich liposomes, to form DODAB-in-DDAB lipo-
an increase in Tm(1)would be observed, since the surfactant Tm
larger than Tm(1).32
Grossly the same behavior was observed for the parameters
obtained from the second upscan (Table 2) relative to those
obtained from the first upscan (Table 1); an exception is for the
lower Tm(1), which decreases slightly instead of being constant,
overlap, and we used the deconvolution method to calculate the
for this upper transition. The apparent ∆H(2)was calculated per
Turbidity Results. Figure 6 shows the effect of xDODABon
the turbidity, measured at 320 nm and 22 °C, for mixed DDAB/
molar fraction is increased the turbidity increases smoothly up
to xDODAB≈ 0.9. Then the turbidity increases steeply to attain
a change of size or liposome structure.
The hydrodynamic radius (Rh) of the cationic liposomes
investigated here was reported before.33-36For DDAB and
DODAB spontaneously formed liposomes, Rh) 350 Å33and
3370 Å,34respectively. After bath sonication of the dispersion,
Rhof the DODAB liposomes drops to 22 Å,35or 325 Å after tip
Fluorescence Results. Nile Red in the Neat DODAB and
DDAB Liposomes. Nile Red is a well-known solvatochromic
probe, which in polar media exhibits a red shift in the emission
maximum, together with fluorescence quenching. Owing to its
capability to establish H-bonds with protic solvents, Nile Red
fluorescence in water is very weak and red shifted (λmax≈ 660
nm).18Nile Red has been used as a lipid probe, due to its
Nile Red incorporated into pure DODAB liposomes at different
temperatures. Accordingly, the fluorescence intensity increases
Tmof neat DODAB, and decreases thereafter.
A significant blue shift in the emission spectrum is also
(until ca. 45 °C), giving an indication that Nile Red feels a more
hydrophobic environment when the temperature is raised. This
(due to the increase of the competitive nonradiative processes).
At low temperature (10 °C), the Nile Red emission maximum
(λmax≈ 650 nm) is close to the value in water,18indicating that
the probe is located in a water-rich environment. In the fluid
(lower emission intensity and a small red shift).
For DDAB liposomes, the Nile Red fluorescence intensity
decreases with increasing temperature (Figure 8), as expected
from the increase of nonradiative pathways. A monotonic red
shift is observed when the temperature increases. In the
temperature range investigated (10-50 °C), Nile Red seems not
see Table 1).
(32) Feitosa, E.; Jansson, J.; Lindman, B. Chem. Phys. Lipids, in press.
Chem. 1999, 103, 8353.
(34) Feitosa, E.; Karlsson, G.; Edwards, K. Chem. Phys. Lipids, in press.
(35) Feitosa, E.; Brown, W. Langmuir 1997, 13, 4810.
(36) Cuccovia, I. M.; Feitosa, E.; Chaimovich, H.; Sepulveda, L.; Reed, W.
J. Phys. Chem. 1990, 94, 3722.
for the mixture DODAB/DDAB/water and 1.0 mM total lipid
temperatures (λexc) 550 nm).
Cationic Liposomes in DDAB and DODAB Dispersions Langmuir, Vol. 22, No. 8, 2006 3583
Nile Red Fluorescence in DDAB/DODAB Mixed Systems.
Figure 9 shows the emission spectra for DDAB/DODAB mixed
In all cases, the emission spectrum is a broad structureless band,
but the relative fluorescence yield of Nile Red depends on the
surfactant molar fraction. The lower fluorescence emission of
Nile Red in neat DODAB liposomes (xDODAB) 1) compared to
that for the neat DDAB (xDODAB) 0) indicates that Nile Red is
more exposed to the water phase in the neat DODAB liposomes
(which are in the gel phase, i.e., below Tm(2)≈ 43 °C) relative
i.e., above Tm(1)≈ 16 °C). When the DDAB fraction increases
(DODAB decreases), the emission intensity of Nile Red also
increases (except for the neat DDAB). This indicates that when
can penetrate deeper into the liposome bilayer, thus being less
exposed to water. This is probably caused by the increasing
fluidity of the membrane upon DDAB addition. In neat DDAB
than for xDODAB ) 0.2, indicating that DDAB liposomes are
Figure 10 shows the average steady-state anisotropy of Nile
Red in DODAB/DDAB liposomes with increasing temperature
by eq 2, where IVVand IVHare the emission spectra obtained
with vertical and horizontal polarization, respectively (for
correction factor, given by
and horizontal polarization (for excitation with horizontally
The steady-state anisotropy has a pronounced decrease upon
that the DODAB transition temperature is lower in the mixed
removed for lower fractions of DODAB (xDODABe 0.4).
(Itotal ) IVV + 2GIVH) with temperature for several lipid
concentrations. For neat DODAB liposomes (xDODAB ) 1) a
maximum is observed around the phase transition temperature
DODAB concentrations (xDODAB> 0.5), a similar but weaker
trend can be seen, with the emission maximum now being
observed around 40 °C.
For higher DDAB concentrations (xDODABe 0.6), a slight
emission maximum is observed at 10-15 °C. This can be an
indication of the presence of quasi-neat DDAB liposomes, as
inferred from DSC results. Above 15 °C and xDODABe 0.4, the
rich in DDAB are mainly in the liquid-crystalline phase above
15 °C (DDAB Tm).
The DODAB-rich systems (xDODABg 0.6) are predominantly
in the gel phase until ca. 35 °C, with the transition temperature
depending on the DODAB content (ca. 40 °C for 75% DODAB
and between 35 and 40 °C for 60% DODAB). These figures are
in good agreement with those obtained by DSC reported above.
temperatures (λexc) 550 nm).
Figure 9. Nile Red emission spectra in DODAB/DDAB mixed
liposomes, at several DODAB molar fractions, at 25 °C (λexc) 550
Figure 10. Nile Red steady-state anisotropy in DODAB/DDAB
mixed liposomes with increasing temperature for several DODAB
G ) IHV/IHH
3584 Langmuir, Vol. 22, No. 8, 2006Feitosa et al.
Conclusions Download full-text
The melting temperature (or gel to liquid-crystalline phase
transition temperature) of mixed cationic liposomes composed
of DDAB and DODAB were investigated by DSC and
fluorescence of Nile Red. Depending on the surfactant composi-
tion, the system exhibits one or two melting temperatures, Tm(1)
and Tm(2), owing to the existing two liposome populations with
very distinctive characteristics. At room temperature, the
population of DODAB-rich liposomes is in the gel state while
the other population of DDAB-rich liposomes is in the liquid-
crystalline state. Accordingly, the upper Tm(2) of DDAB-in-
DODAB liposomes decreases with xDDAB, owing to the incor-
poration of DDAB into the DODAB bilayer, and the mixed
liposomes are in the gel state at room temperature. Therefore,
DDAB increases the fluidity of DDAB-in-DODAB liposomes.
to less extent into the DDAB bilayer, probably due to its longer
These results suggest that there is more affinity of DDAB for
DODAB liposomes than the reverse, resulting in a higher
incorporation of DDAB into DODAB-rich liposomes than the
Nile Red fluorescence results corroborate that DDAB-rich
systems are mainly in the gel phase, the transition temperature
depending on the DDAB content.
of DODAB into DDAB vesicles may exist, but without any
that the presence of a small fraction of DODAB in DDAB-rich
liposomes causes a pronounced effect in Nile Red emission.
Further investigation on this system and other similar systems
is under way by our group.
Acknowledgment. We thank Dr. W. Loh (Unicamp) for
kindly supplying the micro DSC equipment for the calorimetric
experiments. F.R.A. thanks CNPq for a Ph.D. grant.
Figure 11. Variation of Nile Red total fluorescence intensity in
DODAB/DDAB mixed liposomes with increasing temperature for
several DODAB molar fractions.
Cationic Liposomes in DDAB and DODAB Dispersions Langmuir, Vol. 22, No. 8, 2006 3585