28

Phase Behavior of Phospholipid-

Cholesterol Liposomes Stabilized
With Trehalose
S. Ohtake, C. Schebor, and J.J. de Pablo

28.1. Introduction
Achieving long-term stability in biological systems has been a long-standing
goal of the food, pharmaceutical, and biomedical industries. Avoiding the
need for refrigeration would reduce production and storage costs drastically.
The desiccation of phospholipidic vesicles has been studied in an effort to
understand biological membranes under low water content conditions (Crowe
and Crowe, 1988; Ohtake et al., 2004).
Trehalose is effective in protecting biological membranes upon freeze-drying,
and it has been widely used to preserve the integrity of phospholipid liposomes
(Crowe and Crowe, 1988; Ohtake, et al., 2004; Crowe et al., 1986). Despite
the abundance of cholesterol in mammalian plasma membranes (Rouser, et al.,
1968), studies examining the effects of dehydration on cholesterol-containing
model membranes are scarce (Van Winden and Crommelin, 1999; Harrigan, et al.,
1990). Furthermore, the ability of well known lyoprotectants, such as trehalose, to
stabilize cholesterol-containing liposomes has not been examined in detail. The
aim of this work is to understand how cholesterol containing liposomes behave
upon lyophilization.

28.2. Methodology
Liposomes were obtained by extruding (through 100 nm membrane filters)
mixtures consisting of 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC),
1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine (DPPE) and cholesterol
(Ch) at a temperature above the phase transition temperature, Tm, of the respec-
tive mixtures. Cholesterol molar ratios ranged from 0% to 41%. Samples were
freeze-dried for 48 h in a Virtis Genesis 12EL (New York, USA) freeze-dryer at a
pressure of 30 mtorr and a condenser temperature of −80 °C. The samples under
vacuum were transferred to a glove box (Vac, Nexus One, Hawthorne, CA) and
then loaded into pre-weighed DSC pans and sealed for calorimetric analysis.

383
384 S. Ohtake et al.

Fully hydrated samples were also analyzed by DSC, for which the DSC pans
were loaded immediately following extrusion.
DSC was used to determine the Tm of the phospholipids. Tm represents the
peak temperature of the endotherm for the lipid gel-to-fluid phase transition
recorded during the heating scan. The instrument used was a TA Q100 DSC (New
Castle, DE). All measurements were made at 10 °C/min, using sealed aluminum
pans (crimped pans, TA), and an empty pan was used as a reference. The average
value of at least three replicate samples was reported. Data was analyzed using
Universal Analysis.
Residual water contents for the dehydrated samples were analyzed using a Karl
Fisher Coulometer Metrohm, Model 737 (Herisau, Switzerland).

28.3. Results and Discussion

The thermotropic phase behavior of liposomes composed of DPPC, DPPE
and their mixtures with cholesterol was studied using differential scanning
calorimetry (DSC). Thermograms of fully hydrated DPPC-cholesterol liposomes
containing cholesterol at molar ratios ranging from 0% to 41% (molar) are
shown in Fig. 28.1. The phase transition temperature (Tm) of DPPC in the fully
hydrated state is 42 °C, which is in agreement with previous reports (McMullen
et al., 1993; Ohtake et al., 2005). The addition of cholesterol to DPPC liposomes
did not change the Tm significantly. The enthalpy of the transition, however,
decreased considerably, until it became negligible at 41% cholesterol, consist-
ent with literature data (McMullen et al., 1993; Vist and Davis, 1990). Addition
of trehalose did not significantly alter the melting behavior of cholesterol-
containing liposomes in the fully hydrated state (data not reported). The addition
of cholesterol to DPPE and DPPE-DPPC mixtures exhibited behavior similar to

41%
9 33%
Heat Flow (W/g)

23%
1
9%
0%
−7
Exo >

−15
35 37 39 41 43 45
Temperature (⬚C)

Fig. 28.1. Thermograms of fully hydrated DPPC-cholesterol mixtures at the indicated

cholesterol molar ratios
 28. Phase Behavior of Phospholipid-Cholesterol Liposomes 385

that observed for DPPC, namely a decrease in the enthalpy of transition. The tran-
sition temperature decreased for DPPE (data not reported), as was also reported
previously (Blume, 1980).
Figure 28.2 shows the thermograms of freeze-dried DPPC-cholesterol lipo-
somes with (solid lines) and without (dotted lines) trehalose. The Tm of DPPC
increased from 42 °C to 105 °C upon dehydration, in accordance with previous
studies (Crowe and Crowe, 1988; Ohtake et al., 2004). The increase in phase
transition temperature was caused by the decrease in the spacing between the
headgroups, which allowed for increased van der Waals interactions between
the lipid hydrocarbon chains (Crowe et al., 1998). The transition peak observed
at 105 °C for dehydrated DPPC decreased dramatically, both in magnitude and
in temperature, as the proportion of cholesterol in the liposomes increased
(Fig. 28.2, dotted lines). The intercalation of cholesterol between the phos-
pholipids destabilized their packing in the gel phase, leading to a depression in
the Tm of the dehydrated samples. A second broad endothermic transition,
having a main peak at 70 °C, became observable at 23% cholesterol (Fig.
28.2, dotted line). The peak corresponding to this transition increased as the
cholesterol proportion increased, and at 41% cholesterol the two transitions
merged. The presence of the two endothermic peaks can be explained by the
existence of Ch-rich and Ch-poor domains, as described for these same sys-
tems in the fully hydrated state (McMullen et al., 1993; Vist and Davis, 1990).
In the presence of trehalose, the phase transition temperature of dehydrated
DPPC dropped by 80 °C, to 25 °C (Fig. 28.2, solid line), as was reported pre-
viously (Crowe and Crowe, 1988; Ohtake et al., 2004). This Tm depression
has been ascribed to hydrogen bond formation between the sugar molecules
and the phospholipid headgroups, allowing the phospholipids to maintain an
intermolecular spacing similar to that present in the hydrated state (Crowe

2.5
41%
0.5
Heat Flow (W/g)

33%
−1.5
23%
−3.5
9%
−5.5
Exo >

0%
−7.5
10 25 40 55 70 85 100 115
Temperature (⬚C)

Fig. 28.2. Thermograms of lyophilized DPPC-cholesterol mixtures in the presence (solid

lines) and absence (dotted lines) of trehalose. The cholesterol molar ratios (%) in the lipo-
somes are indicated in the figure. The samples contain less than 1.5 % (wt) water
386 S. Ohtake et al.

and Crowe, 1988; Ohtake et al., 2004; Tsvetkova et al., 1998). In the presence
of trehalose, cholesterol addition did not have a significant effect on the Tm,
although the enthalpy of the transition decreased monotonically as the propor-
tion of cholesterol increased (Fig. 28.2, solid lines), as was observed in the
fully hydrated state (Fig. 28.1).
Figure 28.3 shows the thermograms of freeze-dried DPPE–Ch mixtures
with (solid lines) and without (dotted lines) trehalose. Two transitions at 52 °C
(corresponding to the gel-to-liquid crystalline phase transition) and 94 °C (cor-
responding to the crystal-to-liquid crystalline phase transition) are observed for
dehydrated DPPE (Fig. 28.3, bottom dotted line), which are in accordance with
previously reported data (Handa et al., 1985). All of the DPPE–Ch mixtures
exhibit multiple transitions: (i) the phase transition at approximately 94 °C was
always present, irrespective of the proportion of cholesterol; (ii) the transition at
52 °C decreased both in magnitude and in temperature with increasing choles-
terol content; and (iii) a new transition at approximately 72 °C appeared upon the
addition of cholesterol, and was present at all of the cholesterol concentrations
examined in this work. The consistency of the peak observed at 94 °C suggests
that the crystalline phase of DPPE does not incorporate cholesterol. The gel
phase, however, is more prone to interact with cholesterol, as the enthalpy of the
peak at 52 °C diminished considerably with increasing cholesterol proportion.
We speculate that the transition at 72 °C represents an intermediate DPPE phase
(between gel and crystal) enriched in cholesterol. In the presence of trehalose, only
one transition can be observed, and the Tm reduced from 52 °C to 39 °C for pure
DPPE (Fig. 28.3, bottom solid line). The Tm further reduced upon the addition
of cholesterol to approximately 21 °C (Fig. 28.3, solid lines). Tm corresponds to
the melting of neighboring phospholipid molecules. As previously described,

1 41%
Heat Flow (W/g)

0 33%
−1
23%
−2
9%
−3
Exo >

−4 0%

20 35 50 65 80 95 110
Temperature (°C)

Fig. 28.3. Thermograms of lyophilized DPPE-cholesterol mixtures in the presence (solid

lines) and absence (dotted lines) of trehalose. The cholesterol molar ratios (%) in the lipo-
somes are indicated in the figure. The samples contained less than 1.5 % (wt) water
 28. Phase Behavior of Phospholipid-Cholesterol Liposomes 387

cholesterol addition can affect its surrounding environment, which in turn can
affect the Tm. The decrease in Tm observed for DPPE-Ch mixtures suggests that
trehalose is better able to interact with the PE molecules upon the intercalation of
cholesterol between phospholipid molecules, which most likely results from the
perturbation of the PE–PE hydrogen bonding network by cholesterol. This is in
contrast to DPPC–Ch mixtures, in which the PC–PC headgroup interactions are
not significantly modified with cholesterol incorporation. Trehalose interaction
with DPPC molecules, hence, is not affected by the presence of cholesterol
(Fig. 28.2, solid lines).
A freeze-dried DPPE–DPPC mixture (1:1 molar ratio) exhibited a single
transition at 83 °C (Fig. 28.4, bottom dotted line). In the hydrated state, the
DPPE–DPPC mixtures were miscible, exhibiting a single transition peak (54 °C)
(Blume, 1980; Blume and Ackerman, 1974). This suggests that the two phospholi-
pids remain miscible upon dehydration. Upon the addition of cholesterol, the
Tm at 83 °C decreased both in temperature and in magnitude. A new transition
at 60 °C appeared above 9 mol% Ch and grew in magnitude with increasing
cholesterol content (Fig. 28.4, dotted lines). As was previously described for
DPPC–Ch systems (Fig. 28.2), the two peaks can be ascribed to Ch-rich and
Ch-poor regions. It has been reported that cholesterol does not interact prefer-
entially with either PE or PC in the DPPE–DPPC–Ch mixtures in excess water
(Blume and Ackerman, 1974). As we did not observe phase separation of DPPC
from DPPE upon dehydration, we expect the two peaks in the DPPE–DPPC–Ch
mixtures to contain a constant DPPE/DPPC ratio (although containing different
amounts of cholesterol). In the presence of trehalose, a decrease in Tm can be
observed, from 83 °C to 21 °C (Fig. 28.4, bottom solid line). Note that DPPE–
DPPC–Ch mixtures show a more pronounced depression of Tm by the addition

−3
41%
−6
33%
Heat Flow (W/g)

−9 23%

−12 9%

0%
−15
Exo >

−18
10 25 40 55 70 85 100 115
Temperature (°C)

Fig. 28.4. Thermograms of lyophilized DPPE-DPPC-cholesterol mixtures in the presence

(solid lines) and absence (dotted lines) of trehalose. The cholesterol molar ratios (%) in
the liposomes are indicated in the figure. The samples contained less than 1.5 % (wt)
water
388 S. Ohtake et al.

of cholesterol (Fig. 28.4, solid lines) than that observed for DPPC (Fig. 28.2,
solid lines), but less than that observed for DPPE (Fig. 28.3, solid lines). This
suggests that DPPC is able to disrupt the strong PE–PE interaction, thereby
facilitating the interaction with trehalose.

28.4. Conclusion
The phase behavior of various freeze-dried mixtures of DPPE, DPPC, and cho-
lesterol has been analyzed. As the cholesterol proportion is increased, the phase
behavior becomes more complex owing to the inhomogeneous mixing of cho-
lesterol with the phospholipids. The effects of trehalose addition have also been
examined. The main focus of the use of trehalose has been to reduce the Tm of
phospholipids upon dehydration. This is an important aspect, since liposomes
undergoing phase transition during rehydration can lead to leakage of encapsu-
lated components. In this study we show that for dehydrated systems containing
cholesterol, trehalose is also necessary to limit phase separation. Despite the
abundance of reports examining the Ch-containing liposomes in the fully hydrated
state, there is a shortage of studies on these systems in the dried state. Examination
of cholesterol-containing model membranes may offer a more realistic view of
how mammalian cells behave upon dehydration, and may aid in evaluating the
effectiveness of lyoprotectants in conferring stability.

Acknowledgements The authors are thankful for financial support from the
National Science Foundation and the MRSEC program. Carolina Schebor thanks
CONICET (PIP5977) and UBACYT X226.

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