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Effect of trehalose on the contributions to the dipole potential of lipid monolayers
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Chemistry and Physics of Lipids 150 (2007) 117–124
Effect of trehalose on the contributions to the dipole potential of lipid monolayers Fabiana Lairion, E. An´ıbal Disalvo ∗ Laboratorio de Fisicoqu´ımica de Membranas Lip´ıdicas y Liposomas, C´atedra de Qu´ımica General e Inorg´anica, Facultad de Farmacia y Bioqu´ımica, Universidad de Buenos Aires, Jun´ın 956 2o Piso (1113), Capital Federal, Argentina Received 31 May 2007; received in revised form 27 June 2007; accepted 28 June 2007 Available online 1 July 2007
Abstract The dipole potential and the area changes induced by trehalose on dimyristoyl phosphatidylcholine (DMPC), 1,2-diO-tetradecyl-sn-glycero-3-phosphocholine (dietherPC), dimyristoyl phosphatidylethanolamine (DMPE), 1,2-di-O-tetradecyl-snglycero-3-phosphoethanolamine (dietherPE) monolayers have been studied at different temperatures. The insertion of trehalose into DMPC monolayers in the fluid and gel states requires of the presence of carbonyl groups. The area increase observed at 0.15 M trehalose is congruent with the decrease in the dipole potential. However, in dietherPC, in which trehalose does not affect the area, a decrease in the dipole potential is also observed. This is interpreted as a result of the displacement of water from the phosphate groups exposed to the aqueous phase. In DMPE, trehalose also decreases the dipole potential without affecting the area of saturated monolayers and in dietherPE no effect on dipole potential and area was observed. It is concluded that the spacer effect of trehalose depends on the specific interaction with CO, which is modulated by the strength of the interaction of the PO groups with lateral NH groups. However, it is not the only contribution to the dipole potential decrease. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Lipid monolayers; DMPC; DMPE; DietherPC; DietherPE; Trehalose; Dipole potential; Area per lipid; Surface pressure
1. Introduction The understanding of the interfacial properties of lipid membranes requires information on the arrangement and properties of water localized at the polar head group region. It is known that phosphatidylcholines hydrates with 14–18 water molecules per lipid the fluid state and with around 7–8 in the gel state. The first 14 tightly bound water molecules are considered a hard-core hydration shell, as derived from studies in reversed micelles and molecular simulation (Essman et al., 1995; Nagle and ∗
Corresponding author. Fax: +54 11 49648274. E-mail address: [email protected] (E.A. Disalvo).
Tristam-Nagle, 2000; Luzardo et al., 2000; Lairion et al., 2002). In this regard, several H bonding compounds, such as sugars, polyphenols, and phenolic sugars, have been used to study the distribution of water around the hydration sites, i.e. carbonyl and phosphate groups, and its effects on the interfacial properties, such as dipole potential and area per lipid (Disalvo et al., 2002, 2004; Lairion and Disalvo, 2007; Fr´ıas et al., 2006). Among sugars, trehalose has been extensively investigated due to its special properties for the preservation of the structure of lipid membranes, in comparison to others. For these reasons, trehalose–membrane interaction is an interesting system to study, due to its potential technological applications as a preservative of cellular structures and functions in
0009-3084/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2007.06.221
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freeze-drying and heat-drying processes (Crowe et al., 1984, 1987, 1997). The studies carried out until now, in relation to the effects of trehalose on lipid membranes and its protective properties can be divided in three groups. One of them focuses on the structural properties of the lipid membranes dried in the presence of trehalose (Crowe et al., 1987; Crowe and Crowe, 1995; Sun et al., 1996). In this condition, the transition temperature of the solid is greatly decreased and a new phase has been described (Tsvetkova et al., 1998). Another approach has dealt with the properties of bilayers in the gel state, formed after the rehydration of lipids dried in the presence of trehalose (Viera et al., 1992). Hydrophobic probes as Merocyanine 540 gave, in this condition, a peak at 570 nm, characteristic of membranes in the fluid state, at temperatures corresponding to the gel state. The results of these studies concluded that trehalose remains anchored in the lipid membrane after rehydration, giving a gel phase with particular surface properties. As the magnitude of the peak ascribed to fluid phases appeared together with those featuring the gel state, it was argued that trehalose was able to expand some regions of the lipid interphase acting as a spacer of the lipid head groups. A third line of research has studied the effect of trehalose in lipids dispersed in aqueous solutions without further dehydration and rehydration. In this condition, two experimental models have been employed: the multi and unilamelar vesicles and lipid monolayers spread on the air–water interface. FTIR measurements in MLV’s, showed that trehalose interacts strongly with the phosphate and the carbonyl groups of phosphatidylcholines (Luzardo et al., 2000). Concomitant with this interaction, a decrease in the amount of water molecules per lipid was calculated, which decreased from about 18 to 10. It was suggested that, at the concentration used, trehalose was not able to displace the water strongly bound to the first shell of hydration, i.e. the first 10 water molecules, probably bound to the phosphate. In addition, the measurements in PC monolayers showed a decrease of the dipole potential congruent with the water displacement caused by the interaction with the hydration sites PO and CO groups (Luzardo et al., 2000). Trehalose insertion has also been studied by simulation techniques (Skibinsky et al., 2005; Sum et al., 2003; Villarreal et al., 2004). All of them show that this sugar intercalates between the phospholipid head groups concerting H-bonds with the head groups in the absence of water. However, different features merge from the different studies. Skibinsky et al. (2005) predicts an expansion of the area per lipid, Sum et al. (2003) claims for no
changes and Villarreal et al. (2004) suggest a small decrease. In spite of the differences in the simulation conditions, several of these results are coincident with experimental findings in monolayers and bilayers. The calculations of Skibinsky et al. (2005) are in nice agreement with the evaluation of the number of water molecules determined by water activity in DMPC (Luzardo et al., 2000; Disalvo et al., 2002). They found that trehalose is able to replace the same number of hydrogen bonds than those concerted by the displaced water molecules. Thus, around 10 trehalose molecules displace 14 water molecules (Skibinsky et al., 2005; Pereira and Hunenberger, 2006). The data of water activity in DMPC, at different trehalose ratio, indicates that four trehalose molecules per lipid displace eight water molecules per lipid. In contrast to these predictions and experimental results, the simulations carried out by Sum et al. (2003) showed that the addition of trehalose to DPPC bilayers does not alter the bilayer structure. The dissimilar consequences were explained suggesting that trehalose may adopt several dynamical spatial conformations that allow it to conform to the topology of the nearest lipids. Villarreal et al. (2004) suggest an intercalation of the sugar with its main axis parallel to the normal of the bilayer, thus suggesting a small decrease in the dipole potential. Although it is accepted that trehalose intercalates in the polar head group region causing a partial displacement of water, details of the effects on the dipole potential in relation to the topological features of carbonyl and phosphate groups, the effects on area and the hydration properties have not been reported. In order to compare the different contributions to the changes in the dipole potential, mainly: hydration of constitutive dipoles, presence/absence of constitutive dipoles and area per lipid (amount of dipoles per area), we have undertaken a comparative study between DMPC and DMPE and its ether derivatives ditetradecyl-PC (dietherPC) and ditetradecyl-PE (dietherPE). The studies using these lipids would allow the inspection of how the lateral interaction due to the presence of CO, PO, choline and amine groups can affect the trehalose interaction with lipids, giving place to different interfacial properties. The influence of CO and PO on the insertion of polyhydroxilated compounds have been reported with other polyhydroxilated compounds, such as arbutin and phloretin (Lairion and Disalvo, 2004, 2007). In contrast, to our knowledge, studies of the dipole potential and the area changes of lipid interfaces, in which the accessibility of CO and the PO groups are modulated, have
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not been reported for trehalose. For this reason, in the present work, the effects of trehalose on dipole potential is analyzed by determining the changes produced by trehalose in monolayers of the DMPC, dietherPC, DMPE and dietherPE, at different phase states. 2. Materials and methods Dimyristoylphosphatidylcholine (DMPC), 1, 2-di-Otetradecyl-sn-glycero-3-phospho choline (dietherPC), dimyristoyl phosphatidylethanolamine (DMPE), 1, 2-di-O-tetradecyl-sn-glycero-3-phosphoethanolamine (dietherPE) were obtained from Avanti Polar Lipids, Inc (Alabaster, AL). The purity of lipids was checked by thin layer chromatography using chloroform:methanol:water mixtures as running solvent and used without further purification. d-(+)-Trehalose dihydrate was obtained from Fluka and purity was checked according to previous procedures (Alonso-Romanowski et al., 1989). Chloroform and KCl were analytical grade. Water was MilliQ quality. 2.1. Determination of the dipole potentials in monolayers The values of interfacial potential (Vsurf ) were determined through a circuit of high impedance by means of an ionizing electrode above the monolayer and a reference electrode in the aqueous sub phase (KCl 1 mM) using the following expression: Vsurf = VAg/AgCl − Vgrd = Vsolution − Vgrd where VAg/AgCl is the potential of the reference electrode and Vgrd the potential of the shield covering the ionizing electrode. Temperature was set at the values indicated in each assay (18 and 28 ◦ C) and measured with a calibrated thermocouple immersed in the sub phase and maintained within ±0.5 ◦ C.
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The dipole potential of the monolayers (Ψ D ) was evaluated as: ΨD = Vsurf + Vlip where Vsurf is the potential of the clean surface (without lipids) and Vlip the potential after the monolayers were formed. To attain the formation of the monolayers, aliquots of a chloroform solution of lipids were spread upon the interface of the aqueous subphase, exhaustively cleaned by suction, or with the addition of 0.15 M trehalose, until a constant potential was reached (see next section). 2.2. Measure of surface pressures and areas per lipid in monolayers The saturation point of monolayers was monitored by measurements of the surface pressure of the different lipid monolayers in Kibron Trough S equipment, at constant temperature and area of the trough. Aliquots of a chloroform solution of lipids were spread on a clean aqueous surface of water or aqueous solutions with 0.15 M trehalose, and left to reach constant surface pressure, until no changes were observed with further additions of lipids (saturation). Results of surface pressure were expressed in mN/m. All dipole potential measurements were assayed at this saturation condition. With this methodology, the dipole potential is determined at the surface pressure attained with lipids in the monolayer in equilibrium with lipids in the sub phase (Mac Donald and Simon, 1987). In this procedure, the lipid conformations are stabilized spontaneously according to the aqueous solution properties, without forcing the lipids by the lateral pressure, as it would be the case in the determinations of the dipole potential along the surface pressure/area curve. The saturation point of the different monolayers, in the conditions assayed, were determined considering the standard deviation of the results. Those points for which,
Table 1 Surface pressures and areas of saturation of monolayers of acyl and alkyl PC’s and PE’s at different temperatures Water
(18 ◦ C)
DMPC DMPC (28 ◦ C) ePC (28 ◦ C) DMPE (28 ◦ C) ePE (28 ◦ C)
Tre 150 mM
Lipid (nmol)
Π (saturation)
˚ A/molec
Lipid (nmol)
Π (saturation)
˚ A/molec
6 5 4 6 6
48 47.5 48 45 44.5
56.3 (±4.7) 67.5 (±6.7) 84.7 (±10.6) 56.1 (±4.7) 56.3 (±4.7)
3 2.5 4 6 6
46.5 48 47.5 45 44.5
112.6 (±18.7) 135.1 (±27) 84.7 (±10.6) 56.1 (±4.7) 56.3 (±4.7)
Lipid (nmol) = nanomoles of lipids added to reach the saturation surface pressure of the monolayers. Π (surface pressure) is expressed in mN/m. ˚ ˚ 2 per molecule of the different phospholipids in the saturated monolayer (see Section 2). A/molec = area in A
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the difference with the mean point of saturation were higher than the standard deviation were not considered as first point of saturation. With these criteria, areas per lipid, shown in bold in Table 1, were calculated with the first point of the saturation plateau of a curve of monolayer surface pressure versus nmol of lipid added to a constant area of the trough. Considering that each aliquot corresponds to 1 nmol, each determination is affected by an error in the area corresponding to ±0.5 nmol, for each lipid (see Table 1). 3. Results and discussion The consequences of the presence of the different polar groups on the interfacial properties can be put into relevance by analyzing the values of the dipole potentials and the area per lipid at constant surface pressure achieved at the saturated conditions of the monolayer, in the absence of the sugar. The dipole potential of the different lipid monolayers and the effect of 0.15 M trehalose on them are shown in Figs. 1 and 4. Figs. 2, 3, 5 and 6 show the data of surface pressures at these conditions. The area per lipid obtained and the data of dipole potential, at the corresponding saturation surface pressures, are resumed in Table 1. It denotes that the surface pressure of the different lipid monolayers is not affected significantly by the presence of trehalose in comparison to that obtained without the sugar.
Fig. 1. Decrease of the dipole potential of DMPC and dietherPC monolayers at 28 ◦ C by 0.15 m trehalose. , DMPC; , DMPC + 0.15 M trehalose; , dietherPC; , dietherPC + 0.15 M trehalose.
Fig. 2. Changes in the surface pressure by addition of DMPC to pure water and trehalose solutions. DMPC on water (18 ◦ C), ♦, DMPC on 0.15 M trehalose (18 ◦ C); , DMPC on water (28 ◦ C); , DMPC on 0.15 M trehalose (28 ◦ C).
The dipole potential of dietherPC, is lower in 90 mV in comparison to that of DMPC at the same temperature and surface pressure (Fig. 1), in agreement with the absence of the CO groups and the water polarized by them. The values of the dipole potentials were obtained when the lipid monolayer was formed by the titration of the surface of pure water, to reach saturation at a given temperature (i.e. constant surface pressure) in a
Fig. 3. Changes in the surface pressure by addition of dietherpc to pure water and trehalose solutions. dietherPC on water (28 ◦ C), , dietherPC on 0.15 M trehalose (28 ◦ C).
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trough of known area. With this methodology a value of the area per lipid in each condition can be obtained. This direct methodology gives area per lipid values in good agreement with those reported in literature and evaluated by means of indirect ones (Nagle and TristamNagle, 2000). As denoted in Fig. 2, the area per lipid of DMPC in the fluid state is higher than that in the gel state, which is the main cause of the decrease in the dipole potential (Lairion and Disalvo, 2004). In the same direction, the larger area per lipid of the alkyl derivatives of PC’s in comparison to the acyl ones, would explain the decrease in the dipole potential (Fig. 2). In both cases, the decrease in the dipole potential can be explained by the area increase due to a greater hydration of the polar head groups. The larger area in dietherPC in comparison to DMPC can be interpreted considering the proposal that the glycerol backbone is parallel to the membrane plane in the alkyl derivatives, in contrast to the acyl phospholipids, in which it lies along the bilayer normal. This change in conformation would be the reason for which the hydration of the PO groups of the alkyl derivatives is higher, as inferred from the fact that the antisymmetric stretching mode of PO group of the dietherPC is centered at a lower frequency in comparison to the phosphate of DMPC (Lewis et al., 1994; Shinoda et al., 2004). In conclusion, both changes in the dietherPC (absence of constitutive and adsorbed dipoles and area expansion) explain the decrease in the dipole potential. The presence of trehalose decreases the dipole potential (Fig. 1) and affects significantly the area per lipid in DMPC, both in condensed (18 ◦ C) and in liquid expanded (28 ◦ C) monolayers (Fig. 2). The surface pressure at the saturation is achieved with 3 and 2.5 nmol respectively, when the monolayer is formed on the surface of a 0.15 M trehalose solution, in comparison to the 6 and 5 nmol of lipid (in condensed and expanded state, respectively) required when the monolayer is saturated in water. This means that the area per lipid is larger in the presence of trehalose than in water. The ˚ 2 per molecule corresponding values of area are 56.3 A 2 ˚ for pure DMPC in water and 112.6 A per molecule in the presence of trehalose, at 18 ◦ C. At 28 ◦ C, the respec˚ 2 per molecule (Table 1). tive areas are 67.5 and 135.1 A Therefore, the expansion of the monolayer by the insertion of trehalose would be one of the reasons for the dipole potential decrease. However, although significantly lower, 0.15 M trehalose decreases the dipole potential of dietherPC monolayers in about 35 mV, in comparison to the 60 mV observed in DMPC in the same condition. In this case, the presence of trehalose does not affect the area per lipid of dietherPC (Fig. 3) which was found to be equal to
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˚ 2 per molecule, at 28 ◦ C. This means that the area 84.7 A expansion is not the only contribution to the dipole potential decrease. The absence of the CO groups in dietherPC gives no effect on the area per lipid, but does not hinder the interaction of trehalose affecting the dipole potential (Table 1). The decrease in the dipole potential induced by 0.15 M trehalose in DMPC, at 28 ◦ C, has been also ascribed to a decrease in the hydration from 18 to 10 water molecules per lipid (Luzardo et al., 2000). This dehydration can be correlated to the binding of trehalose by H-bonding, to CO and PO groups, as indicated by FTIR experiments (Luzardo et al., 2000; Disalvo et al., 2004) showing that the phosphate and carbonyl frequencies are shifted to lower values. The effect can be explained assuming that trehalose inserts in the plane of the membrane, acting as a spacer between the lipid head groups, confirming previous results with Merocyanine 540 (Viera et al., 1992). However, the decrease in dipole potential is modest (60 mV), in comparison to other compounds, such as arbutin or phloretin (180 and 120 mV, respectively) at similar molar ratios (Lairion and Disalvo, 2004, 2007). It would be expected that due to the dehydration and the area expansion, the decrease of the dipole potential would be more significant in comparison to the other compounds in which the dehydration is lower or absent. This can be explained by suggesting that trehalose inserts into the lipid interphase by opposing its own dipole to those of the membrane. This explanation is in accordance to the results in simulation obtained by Skibinsky et al. (2005). Although trehalose reduces the contribution of the water to the dipole potential, as expected from the activity measures (Luzardo et al., 2000), it may contribute to a negative term by its own, giving a small reduction of the dipole potential with respect to the pure system. The simulations of Villarreal et al. (2004) also revealed a small reduction in the dipole potential, although smaller than those reported by Skibinsky et al. (2005). This calls the attention to the sensibility of the potential values to the simulation details which may be related to small changes in the lipid and trehalose orientation. In dialkyl PC, in absence of carbonyls, the number of water dipoles that trehalose may displace is lower than in the acyl PC (Disalvo et al., 2002) and no effect on the area per lipid was observed, as mentioned above. Thus, in this case, two possible contributions to the dipole potential changes, produced by trehalose, are absent or reduced: the area expansion and the water displaced from the carbonyls. From the comparison of the effect of trehalose on both lipids it may be concluded that, in the absence of CO groups, trehalose is not able to interca-
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Fig. 4. Decrease of the dipole potential of DMPE and dietherPE monolayers at 28 ◦ C by 0.15 m trehalose., DMPE; , DMPE + 0.15 M trehalose; , dietherPE; , dietherPE + 0.15 M trehalose.
late deeply between the lipids (probably with its principal axis parallel to the membrane surface) and thus, it does not change the area per lipid. Despite this, as in both cases the dipole potential decreases, it is concluded that trehalose-phosphate interaction would be contributing to the dipole potential. In this sense, it is of interest to evaluate furthermore what happens when POs are less accessible due to strong interaction with neighboring ethanolamines. In the case of pure DMPE monolayers, the dipole potential is higher than that of DMPC, in the same conditions (Fig. 4). This, again, could be, at first glance, ascribed to the lower area per lipid of the PE. As expected, dietherPE has a lower dipole potential due to the absence of carbonyl dipoles. The decrease in dipole potential caused by trehalose on DMPE is comparable to that on DMPC (that is, around 56 mV, Fig. 4). In the absence of carbonyls, trehalose does change neither the area per lipid nor the dipole potential of dietherPE (Figs. 4 and 5). However, a significant difference between DMPC and DMPE (both with ester carbonyls) is that trehalose does not affect the area per lipid, at the saturation point, of PE (Fig. 6). This could be explained because, in DMPE, the strong interaction of phosphates and ethanolamines makes difficult the entrance of trehalose in the carbonyl region, but it does not hinder the decrease in dipole potential. In other words, the dipole potential can be changed without an effect in the area. In this case, the effect on dipole potential would be a consequence of the interaction with the accessible PO groups, which would be
Fig. 5. Changes in the surface pressure by addition of dietherpe to pure water and trehalose solutions. , dietherPE on water (28 ◦ C); ♦, dietherPE on 0.15 M trehalose (28 ◦ C).
even less accessible in dietherPE. A possibility is that the packing resulting of the strong P–N + interaction in PE’s restricts partially the contact of the CO groups with the water phase. However, this interaction would not be as strong as in the absence of carbonyls, since trehalose affects the dipole potential of DMPE but not that of dietherPE. The simulation results have also shown that trehalose inserts deeply in the membrane, at the level of the car-
Fig. 6. Changes in the surface pressure by addition of dmpe to pure water and trehalose solutions. , DMPE on water (28 ◦ C); , DMPE on 0.15 M trehalose (28 ◦ C).
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bonyl region (Sum et al., 2003; Villarreal et al., 2004). These predictions are congruent with the present experimental finding that trehalose does not affect the area of lipids in which the carbonyls are absent (dietherPC and PE), or severely hidden by the packing of the membrane (DMPE) (Table 1). The influence of packing in the effect of trehalose on DMPE, can be visualized at lower surface pressures (Fig. 6). At surface pressures less than 20 mN/m, trehalose causes a slight spacer effect on DMPE. That is, the same surface tension is achieved at higher areas with trehalose, indicating a spacing effect. At higher packing of the monolayers (saturation), achieved by the addition of more lipids to the monolayer, trehalose has no effect on the area. This suggests that the lipid–lipid interaction is strong enough to displace trehalose moieties from its interaction with the COs or, at least, to induce an orientation normal to the bilayer surface (instead of parallel), with no effect on the area. At constant lipids (2 nmol DMPE), the surface pressure in trehalose is higher than in pure water. This denotes that the surface tension of the monolayer is decreased by the addition of trehalose to the subphase. This is in agreement with the predictions of Skibinsky et al. (2005), although they made the simulation on DPPC. The present series of experiments, done with DMPC in 0.15 M trehalose at 18 ◦ C, show the same trend in the data of area increase as those reported by Lambruschini et al. (2000) at 22 ◦ C for DPPC, with 0.1 M trehalose and predicted by Skibinsky et al. (2005). In a previous study in our laboratory, it was shown that trehalose, at a much lower concentration (0.05 M), may reduce the area per lipid of DMPC, with no effect on dietherPC. This was interpreted as a consequence of the elimination of water from the hydration shell of the polar head groups and an insertion of the trehalose with the axis of the molecule normal to the bilayer, as suggested by the simulation of Villarreal et al. (2004). Probably, the decrease in the dipole potential may be achieved by the elimination of the so-called polarized water molecules, thus reducing the excluded volume. This might produce, as an ultimate consequence, a closer approach of the membrane lipids reducing the area per lipid. However, at higher concentrations, trehalose can oppose to this by inserting into the lipid interphase as a spacer, increasing the area per lipid. From the balance of these two opposing effects, the changes in the area per lipid would remain constant or increase. A possible reason for this difference could be that the hysteresis of the lipids to stabilize at the interphase might be affected by the trehalose concentration. Although the importance of carbonyl in the PCs for the trehalose inser-
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tion was confirmed, the opposite results in area to the present results and those reported by Lambruschini et al. (2000) would be worth of a more detailed study. 4. Conclusions The insertion of trehalose into DMPC monolayers in the fluid and gel state requires of the presence of carbonyl groups in order to affect the area of the lipids. The area change that appears at 0.15 M is congruent with the decrease in the dipole potential. However, the presence of CO groups in PE’s is not significant for the trehalose insertion, since no changes in area are observed. In dietherPC, in which trehalose does not affect the area, a decrease in the dipole potential is also observed. This suggests that trehalose is interacting with and displacing water from the phosphate groups, too. In DMPE, trehalose also decreases the dipole potential without affecting the area, at the higher surface pressure in saturated monolayers. In dietherPE no effect is observed, since no carbonyls are present and the phosphates are severely hidden to concert H bonds with trehalose. In accordance, the spacer effect of trehalose depends on the interaction with CO and it depends on the strength of the interaction of the PO groups with lateral NH groups, modulated by the surface pressure. Finally, the effect of trehalose on dipole potential is a consequence of different mechanisms which must be considered in the different situations, mainly: dehydration of constitutive dipoles of the interphase, spacer effect and opposition of its own dipole to the dipole of the monolayer. Acknowledgements This work was supported with funds from Agencia Nacional de Promoci´on Cient´ıfica y Tecnol´ogica, PICT 0324, CONICET (PIP 5476) and UBACyT (B047). EAD is a member of the Research Career of CONICET (National Research Council, Argentina). References Alonso-Romanowski, S., Biondi, A.C., Disalvo, E.A., 1989. Effect of carbohydrates and glycerol on the stability and surface properties of lyophilized liposomes. J. Membr. Biol. 108 (1), 1–11. Crowe, J.H., Crowe, L.M., Carpenter, J., Prestrelski, F.S., Hoekstra, F.A., et al., 1997. Anhydrobiosis: cellular adaptation to extreme dehydration. In: Dantzler II, W.H. (Ed.), Handbook of Physiology. Oxford Univ. Press, Oxford, pp. 1445–1478. Crowe, J.H., Crowe, L.M., Carpenter, J.F., Wistrom, C.A., 1987. Stabilization of dry phospholipid bilayers and proteins by sugars. Biochem. J. 242, 1–10.
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Crowe, J.H., Whittam, M.A., Chapman, D., Crowe, L.M., 1984. Interactions of phospholipid monolayers with carbohydrates. Biochim. Biophys. Acta 769, 151–159. L.M. Crowe J.H. Crowe, Freeze-dried liposomes, in: Peuisieux, Couvreur, Delattre, Devissaguet (Eds.), Liposomes, New Systems and New Trends in their Applications, Paris, 1995, pp. 237–272. Disalvo, E.A., Lairion, F., Diaz, S., Arroyo, J., 2002. Physical chemistry of lipid interfaces: states of hydration, topological and electrical properties. In: Condat, C.A., Baruzzi, A. (Eds.), Recent Research Developments in Biophysical Chemistry, pp. 181–197. Disalvo, E.A., Lairion, F., Martini, F., Almaleck, H., Diaz, S.B., Gordillo, G., 2004. Water at biological Interfaces: does it play a functional role? J. Argent. Chem. Soc. 92, 1–22. Essman, U., Perera, L., Berkowitz, M., 1995. The origin of the hydration interaction of lipid bilayers from MD simulation of dipalmyitoylphosphatidylcholine membranes in gel and liquid crystalline phase. Langmuir 11, 4519–4531. Fr´ıas, M., D´ıaz, S., Ale, N., Ben Altabef, A., Disalvo, E.A., 2006. FTIR analysis of the interaction of arbutin with dimyristoyl phosphatidylcholine in anhydrous and hydrated states. Biochim. Biophys. Acta 1758, 1823–1829. Lairion, F., Filler, R., Disalvo, E.A., 2002. Reversed micelles as model systems to study interfacial properties of lipid bilayers. Colloids Surf. B: Biointerfaces 25, 369–371. Lairion, F., Disalvo, E.A, 2004. Effect of phloretin on the dipole potential of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol monolayers. Langmuir 20, 9151–9155. Lairion, F., Disalvo, E.A., 2007. Effect of arbutin on the dipole potential and area per lipid of ester and ether phosphatidylcholine and phosphatidylethanolamine monolayers. Biochim. Biophys. Acta 1768, 450–456. Lambruschini, C., Relini, A., Ridi, A., Cordone, L., Gliozzi, A., 2000. Trehalose interacts with phospholipid polar heads in langmuir monolayers. Langmuir 16, 5467. Lewis, N.A., McElhaney, R.N., Pohle, W., Mantsch, H., 1994. Components of the carbonyl stretching band in the infrared spectra of
hydrated 1,2-diacylglycerol bilayers: a re-evaluation. Biophys. J. 67, 2367–2375. Luzardo, M., del, C., Amalfa, F., N´un˜ ez, A.M., Diaz, S., Biondi de Lopez, A.C., Disalvo, E.A., 2000. Effect of trehalose and sucrose on the hydration and dipole potential of lipid bilayers. Biophys. J. 78 (5), 2452–2458. Mac Donald, R.C., Simon, S.A., 1987. Lipid monolayer states and their relationships to bilayers. Proc. Natl. Acad. Sci. U.S.A. 84, 4089. Nagle, J., Tristam-Nagle, S., 2000. Structure of lipid bilayers. Biochim. Biophys. Acta 1469, 159–195. Pereira, C.S., Hunenberger, P.H., 2006. Interaction of the sugars trehalose, maltose and glucose with a phospholipids bilayer: a comparative molecular dynamics study. J. Phys. Chem. B, Condens. Matter Surf. Interfaces Biophys. 110 (31), 15572–15581. Shinoda, K., Shinosa, W., Baba, T., Mikami, M., 2004. Comparative molecular dynamics study of ether- and ester-linked phospholipid bilayers. J. Chem. Phys. 121 (19), 9648–9654. Skibinsky, A., Venable, R.M., Pastor, R.W., 2005. A molecular dynamics study of the response of lipid bilayers and monolayers to trehalose. Biophys. J. 89, 4111–4121. Sum, A.K., Faller, R., de Pablo, J.J., 2003. Molecular simulation study of phospholipid bilayers and insights of the interactions with disaccharides. Biophys J. 85 (5), 2830–2844. Sun, W.Q., Leopold, A.C., Crowe, L.M., Crowe, J.H., 1996. Stability of dry liposomes in sugar glasses. Biophys. J. 70, 1769– 1776. Tsvetkova, N.M., Phillips, B.L., Crowe, L.M., Crowe, J.H., Risbud, S.H., 1998. Effect of sugars on headgroup mobility in freeze-dried dipalmitoylphosphatidylcholine bilayers: solid-state 31P NMR and FTIR studies. Biophys. J. 75 (6), 2947–2955. Viera, L.I., Alonso-Romanowski, S., Borovyagin, V., Feliz, M.R., Disalvo, E.A., 1992. Properties of gel phase lipid-trehalose bilayers upon rehydration. Biochim. Biophys. Acta 1145, 157–166. Villarreal, M., D´ıaz, S.B., Disalvo, E.A., Montich, G., 2004. Molecular dynamics simulation study of the interaction of trehalose with lipid membranes. Langmuir 20, 7844–7851.
Chemistry and Physics of Lipids 150 (2007) 117–124
Effect of trehalose on the contributions to the dipole potential of lipid monolayers Fabiana Lairion, E. An´ıbal Disalvo ∗ Laboratorio de Fisicoqu´ımica de Membranas Lip´ıdicas y Liposomas, C´atedra de Qu´ımica General e Inorg´anica, Facultad de Farmacia y Bioqu´ımica, Universidad de Buenos Aires, Jun´ın 956 2o Piso (1113), Capital Federal, Argentina Received 31 May 2007; received in revised form 27 June 2007; accepted 28 June 2007 Available online 1 July 2007
Abstract The dipole potential and the area changes induced by trehalose on dimyristoyl phosphatidylcholine (DMPC), 1,2-diO-tetradecyl-sn-glycero-3-phosphocholine (dietherPC), dimyristoyl phosphatidylethanolamine (DMPE), 1,2-di-O-tetradecyl-snglycero-3-phosphoethanolamine (dietherPE) monolayers have been studied at different temperatures. The insertion of trehalose into DMPC monolayers in the fluid and gel states requires of the presence of carbonyl groups. The area increase observed at 0.15 M trehalose is congruent with the decrease in the dipole potential. However, in dietherPC, in which trehalose does not affect the area, a decrease in the dipole potential is also observed. This is interpreted as a result of the displacement of water from the phosphate groups exposed to the aqueous phase. In DMPE, trehalose also decreases the dipole potential without affecting the area of saturated monolayers and in dietherPE no effect on dipole potential and area was observed. It is concluded that the spacer effect of trehalose depends on the specific interaction with CO, which is modulated by the strength of the interaction of the PO groups with lateral NH groups. However, it is not the only contribution to the dipole potential decrease. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Lipid monolayers; DMPC; DMPE; DietherPC; DietherPE; Trehalose; Dipole potential; Area per lipid; Surface pressure
1. Introduction The understanding of the interfacial properties of lipid membranes requires information on the arrangement and properties of water localized at the polar head group region. It is known that phosphatidylcholines hydrates with 14–18 water molecules per lipid the fluid state and with around 7–8 in the gel state. The first 14 tightly bound water molecules are considered a hard-core hydration shell, as derived from studies in reversed micelles and molecular simulation (Essman et al., 1995; Nagle and ∗
Corresponding author. Fax: +54 11 49648274. E-mail address: [email protected] (E.A. Disalvo).
Tristam-Nagle, 2000; Luzardo et al., 2000; Lairion et al., 2002). In this regard, several H bonding compounds, such as sugars, polyphenols, and phenolic sugars, have been used to study the distribution of water around the hydration sites, i.e. carbonyl and phosphate groups, and its effects on the interfacial properties, such as dipole potential and area per lipid (Disalvo et al., 2002, 2004; Lairion and Disalvo, 2007; Fr´ıas et al., 2006). Among sugars, trehalose has been extensively investigated due to its special properties for the preservation of the structure of lipid membranes, in comparison to others. For these reasons, trehalose–membrane interaction is an interesting system to study, due to its potential technological applications as a preservative of cellular structures and functions in
0009-3084/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2007.06.221
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freeze-drying and heat-drying processes (Crowe et al., 1984, 1987, 1997). The studies carried out until now, in relation to the effects of trehalose on lipid membranes and its protective properties can be divided in three groups. One of them focuses on the structural properties of the lipid membranes dried in the presence of trehalose (Crowe et al., 1987; Crowe and Crowe, 1995; Sun et al., 1996). In this condition, the transition temperature of the solid is greatly decreased and a new phase has been described (Tsvetkova et al., 1998). Another approach has dealt with the properties of bilayers in the gel state, formed after the rehydration of lipids dried in the presence of trehalose (Viera et al., 1992). Hydrophobic probes as Merocyanine 540 gave, in this condition, a peak at 570 nm, characteristic of membranes in the fluid state, at temperatures corresponding to the gel state. The results of these studies concluded that trehalose remains anchored in the lipid membrane after rehydration, giving a gel phase with particular surface properties. As the magnitude of the peak ascribed to fluid phases appeared together with those featuring the gel state, it was argued that trehalose was able to expand some regions of the lipid interphase acting as a spacer of the lipid head groups. A third line of research has studied the effect of trehalose in lipids dispersed in aqueous solutions without further dehydration and rehydration. In this condition, two experimental models have been employed: the multi and unilamelar vesicles and lipid monolayers spread on the air–water interface. FTIR measurements in MLV’s, showed that trehalose interacts strongly with the phosphate and the carbonyl groups of phosphatidylcholines (Luzardo et al., 2000). Concomitant with this interaction, a decrease in the amount of water molecules per lipid was calculated, which decreased from about 18 to 10. It was suggested that, at the concentration used, trehalose was not able to displace the water strongly bound to the first shell of hydration, i.e. the first 10 water molecules, probably bound to the phosphate. In addition, the measurements in PC monolayers showed a decrease of the dipole potential congruent with the water displacement caused by the interaction with the hydration sites PO and CO groups (Luzardo et al., 2000). Trehalose insertion has also been studied by simulation techniques (Skibinsky et al., 2005; Sum et al., 2003; Villarreal et al., 2004). All of them show that this sugar intercalates between the phospholipid head groups concerting H-bonds with the head groups in the absence of water. However, different features merge from the different studies. Skibinsky et al. (2005) predicts an expansion of the area per lipid, Sum et al. (2003) claims for no
changes and Villarreal et al. (2004) suggest a small decrease. In spite of the differences in the simulation conditions, several of these results are coincident with experimental findings in monolayers and bilayers. The calculations of Skibinsky et al. (2005) are in nice agreement with the evaluation of the number of water molecules determined by water activity in DMPC (Luzardo et al., 2000; Disalvo et al., 2002). They found that trehalose is able to replace the same number of hydrogen bonds than those concerted by the displaced water molecules. Thus, around 10 trehalose molecules displace 14 water molecules (Skibinsky et al., 2005; Pereira and Hunenberger, 2006). The data of water activity in DMPC, at different trehalose ratio, indicates that four trehalose molecules per lipid displace eight water molecules per lipid. In contrast to these predictions and experimental results, the simulations carried out by Sum et al. (2003) showed that the addition of trehalose to DPPC bilayers does not alter the bilayer structure. The dissimilar consequences were explained suggesting that trehalose may adopt several dynamical spatial conformations that allow it to conform to the topology of the nearest lipids. Villarreal et al. (2004) suggest an intercalation of the sugar with its main axis parallel to the normal of the bilayer, thus suggesting a small decrease in the dipole potential. Although it is accepted that trehalose intercalates in the polar head group region causing a partial displacement of water, details of the effects on the dipole potential in relation to the topological features of carbonyl and phosphate groups, the effects on area and the hydration properties have not been reported. In order to compare the different contributions to the changes in the dipole potential, mainly: hydration of constitutive dipoles, presence/absence of constitutive dipoles and area per lipid (amount of dipoles per area), we have undertaken a comparative study between DMPC and DMPE and its ether derivatives ditetradecyl-PC (dietherPC) and ditetradecyl-PE (dietherPE). The studies using these lipids would allow the inspection of how the lateral interaction due to the presence of CO, PO, choline and amine groups can affect the trehalose interaction with lipids, giving place to different interfacial properties. The influence of CO and PO on the insertion of polyhydroxilated compounds have been reported with other polyhydroxilated compounds, such as arbutin and phloretin (Lairion and Disalvo, 2004, 2007). In contrast, to our knowledge, studies of the dipole potential and the area changes of lipid interfaces, in which the accessibility of CO and the PO groups are modulated, have
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not been reported for trehalose. For this reason, in the present work, the effects of trehalose on dipole potential is analyzed by determining the changes produced by trehalose in monolayers of the DMPC, dietherPC, DMPE and dietherPE, at different phase states. 2. Materials and methods Dimyristoylphosphatidylcholine (DMPC), 1, 2-di-Otetradecyl-sn-glycero-3-phospho choline (dietherPC), dimyristoyl phosphatidylethanolamine (DMPE), 1, 2-di-O-tetradecyl-sn-glycero-3-phosphoethanolamine (dietherPE) were obtained from Avanti Polar Lipids, Inc (Alabaster, AL). The purity of lipids was checked by thin layer chromatography using chloroform:methanol:water mixtures as running solvent and used without further purification. d-(+)-Trehalose dihydrate was obtained from Fluka and purity was checked according to previous procedures (Alonso-Romanowski et al., 1989). Chloroform and KCl were analytical grade. Water was MilliQ quality. 2.1. Determination of the dipole potentials in monolayers The values of interfacial potential (Vsurf ) were determined through a circuit of high impedance by means of an ionizing electrode above the monolayer and a reference electrode in the aqueous sub phase (KCl 1 mM) using the following expression: Vsurf = VAg/AgCl − Vgrd = Vsolution − Vgrd where VAg/AgCl is the potential of the reference electrode and Vgrd the potential of the shield covering the ionizing electrode. Temperature was set at the values indicated in each assay (18 and 28 ◦ C) and measured with a calibrated thermocouple immersed in the sub phase and maintained within ±0.5 ◦ C.
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The dipole potential of the monolayers (Ψ D ) was evaluated as: ΨD = Vsurf + Vlip where Vsurf is the potential of the clean surface (without lipids) and Vlip the potential after the monolayers were formed. To attain the formation of the monolayers, aliquots of a chloroform solution of lipids were spread upon the interface of the aqueous subphase, exhaustively cleaned by suction, or with the addition of 0.15 M trehalose, until a constant potential was reached (see next section). 2.2. Measure of surface pressures and areas per lipid in monolayers The saturation point of monolayers was monitored by measurements of the surface pressure of the different lipid monolayers in Kibron Trough S equipment, at constant temperature and area of the trough. Aliquots of a chloroform solution of lipids were spread on a clean aqueous surface of water or aqueous solutions with 0.15 M trehalose, and left to reach constant surface pressure, until no changes were observed with further additions of lipids (saturation). Results of surface pressure were expressed in mN/m. All dipole potential measurements were assayed at this saturation condition. With this methodology, the dipole potential is determined at the surface pressure attained with lipids in the monolayer in equilibrium with lipids in the sub phase (Mac Donald and Simon, 1987). In this procedure, the lipid conformations are stabilized spontaneously according to the aqueous solution properties, without forcing the lipids by the lateral pressure, as it would be the case in the determinations of the dipole potential along the surface pressure/area curve. The saturation point of the different monolayers, in the conditions assayed, were determined considering the standard deviation of the results. Those points for which,
Table 1 Surface pressures and areas of saturation of monolayers of acyl and alkyl PC’s and PE’s at different temperatures Water
(18 ◦ C)
DMPC DMPC (28 ◦ C) ePC (28 ◦ C) DMPE (28 ◦ C) ePE (28 ◦ C)
Tre 150 mM
Lipid (nmol)
Π (saturation)
˚ A/molec
Lipid (nmol)
Π (saturation)
˚ A/molec
6 5 4 6 6
48 47.5 48 45 44.5
56.3 (±4.7) 67.5 (±6.7) 84.7 (±10.6) 56.1 (±4.7) 56.3 (±4.7)
3 2.5 4 6 6
46.5 48 47.5 45 44.5
112.6 (±18.7) 135.1 (±27) 84.7 (±10.6) 56.1 (±4.7) 56.3 (±4.7)
Lipid (nmol) = nanomoles of lipids added to reach the saturation surface pressure of the monolayers. Π (surface pressure) is expressed in mN/m. ˚ ˚ 2 per molecule of the different phospholipids in the saturated monolayer (see Section 2). A/molec = area in A
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the difference with the mean point of saturation were higher than the standard deviation were not considered as first point of saturation. With these criteria, areas per lipid, shown in bold in Table 1, were calculated with the first point of the saturation plateau of a curve of monolayer surface pressure versus nmol of lipid added to a constant area of the trough. Considering that each aliquot corresponds to 1 nmol, each determination is affected by an error in the area corresponding to ±0.5 nmol, for each lipid (see Table 1). 3. Results and discussion The consequences of the presence of the different polar groups on the interfacial properties can be put into relevance by analyzing the values of the dipole potentials and the area per lipid at constant surface pressure achieved at the saturated conditions of the monolayer, in the absence of the sugar. The dipole potential of the different lipid monolayers and the effect of 0.15 M trehalose on them are shown in Figs. 1 and 4. Figs. 2, 3, 5 and 6 show the data of surface pressures at these conditions. The area per lipid obtained and the data of dipole potential, at the corresponding saturation surface pressures, are resumed in Table 1. It denotes that the surface pressure of the different lipid monolayers is not affected significantly by the presence of trehalose in comparison to that obtained without the sugar.
Fig. 1. Decrease of the dipole potential of DMPC and dietherPC monolayers at 28 ◦ C by 0.15 m trehalose. , DMPC; , DMPC + 0.15 M trehalose; , dietherPC; , dietherPC + 0.15 M trehalose.
Fig. 2. Changes in the surface pressure by addition of DMPC to pure water and trehalose solutions. DMPC on water (18 ◦ C), ♦, DMPC on 0.15 M trehalose (18 ◦ C); , DMPC on water (28 ◦ C); , DMPC on 0.15 M trehalose (28 ◦ C).
The dipole potential of dietherPC, is lower in 90 mV in comparison to that of DMPC at the same temperature and surface pressure (Fig. 1), in agreement with the absence of the CO groups and the water polarized by them. The values of the dipole potentials were obtained when the lipid monolayer was formed by the titration of the surface of pure water, to reach saturation at a given temperature (i.e. constant surface pressure) in a
Fig. 3. Changes in the surface pressure by addition of dietherpc to pure water and trehalose solutions. dietherPC on water (28 ◦ C), , dietherPC on 0.15 M trehalose (28 ◦ C).
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trough of known area. With this methodology a value of the area per lipid in each condition can be obtained. This direct methodology gives area per lipid values in good agreement with those reported in literature and evaluated by means of indirect ones (Nagle and TristamNagle, 2000). As denoted in Fig. 2, the area per lipid of DMPC in the fluid state is higher than that in the gel state, which is the main cause of the decrease in the dipole potential (Lairion and Disalvo, 2004). In the same direction, the larger area per lipid of the alkyl derivatives of PC’s in comparison to the acyl ones, would explain the decrease in the dipole potential (Fig. 2). In both cases, the decrease in the dipole potential can be explained by the area increase due to a greater hydration of the polar head groups. The larger area in dietherPC in comparison to DMPC can be interpreted considering the proposal that the glycerol backbone is parallel to the membrane plane in the alkyl derivatives, in contrast to the acyl phospholipids, in which it lies along the bilayer normal. This change in conformation would be the reason for which the hydration of the PO groups of the alkyl derivatives is higher, as inferred from the fact that the antisymmetric stretching mode of PO group of the dietherPC is centered at a lower frequency in comparison to the phosphate of DMPC (Lewis et al., 1994; Shinoda et al., 2004). In conclusion, both changes in the dietherPC (absence of constitutive and adsorbed dipoles and area expansion) explain the decrease in the dipole potential. The presence of trehalose decreases the dipole potential (Fig. 1) and affects significantly the area per lipid in DMPC, both in condensed (18 ◦ C) and in liquid expanded (28 ◦ C) monolayers (Fig. 2). The surface pressure at the saturation is achieved with 3 and 2.5 nmol respectively, when the monolayer is formed on the surface of a 0.15 M trehalose solution, in comparison to the 6 and 5 nmol of lipid (in condensed and expanded state, respectively) required when the monolayer is saturated in water. This means that the area per lipid is larger in the presence of trehalose than in water. The ˚ 2 per molecule corresponding values of area are 56.3 A 2 ˚ for pure DMPC in water and 112.6 A per molecule in the presence of trehalose, at 18 ◦ C. At 28 ◦ C, the respec˚ 2 per molecule (Table 1). tive areas are 67.5 and 135.1 A Therefore, the expansion of the monolayer by the insertion of trehalose would be one of the reasons for the dipole potential decrease. However, although significantly lower, 0.15 M trehalose decreases the dipole potential of dietherPC monolayers in about 35 mV, in comparison to the 60 mV observed in DMPC in the same condition. In this case, the presence of trehalose does not affect the area per lipid of dietherPC (Fig. 3) which was found to be equal to
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˚ 2 per molecule, at 28 ◦ C. This means that the area 84.7 A expansion is not the only contribution to the dipole potential decrease. The absence of the CO groups in dietherPC gives no effect on the area per lipid, but does not hinder the interaction of trehalose affecting the dipole potential (Table 1). The decrease in the dipole potential induced by 0.15 M trehalose in DMPC, at 28 ◦ C, has been also ascribed to a decrease in the hydration from 18 to 10 water molecules per lipid (Luzardo et al., 2000). This dehydration can be correlated to the binding of trehalose by H-bonding, to CO and PO groups, as indicated by FTIR experiments (Luzardo et al., 2000; Disalvo et al., 2004) showing that the phosphate and carbonyl frequencies are shifted to lower values. The effect can be explained assuming that trehalose inserts in the plane of the membrane, acting as a spacer between the lipid head groups, confirming previous results with Merocyanine 540 (Viera et al., 1992). However, the decrease in dipole potential is modest (60 mV), in comparison to other compounds, such as arbutin or phloretin (180 and 120 mV, respectively) at similar molar ratios (Lairion and Disalvo, 2004, 2007). It would be expected that due to the dehydration and the area expansion, the decrease of the dipole potential would be more significant in comparison to the other compounds in which the dehydration is lower or absent. This can be explained by suggesting that trehalose inserts into the lipid interphase by opposing its own dipole to those of the membrane. This explanation is in accordance to the results in simulation obtained by Skibinsky et al. (2005). Although trehalose reduces the contribution of the water to the dipole potential, as expected from the activity measures (Luzardo et al., 2000), it may contribute to a negative term by its own, giving a small reduction of the dipole potential with respect to the pure system. The simulations of Villarreal et al. (2004) also revealed a small reduction in the dipole potential, although smaller than those reported by Skibinsky et al. (2005). This calls the attention to the sensibility of the potential values to the simulation details which may be related to small changes in the lipid and trehalose orientation. In dialkyl PC, in absence of carbonyls, the number of water dipoles that trehalose may displace is lower than in the acyl PC (Disalvo et al., 2002) and no effect on the area per lipid was observed, as mentioned above. Thus, in this case, two possible contributions to the dipole potential changes, produced by trehalose, are absent or reduced: the area expansion and the water displaced from the carbonyls. From the comparison of the effect of trehalose on both lipids it may be concluded that, in the absence of CO groups, trehalose is not able to interca-
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Fig. 4. Decrease of the dipole potential of DMPE and dietherPE monolayers at 28 ◦ C by 0.15 m trehalose., DMPE; , DMPE + 0.15 M trehalose; , dietherPE; , dietherPE + 0.15 M trehalose.
late deeply between the lipids (probably with its principal axis parallel to the membrane surface) and thus, it does not change the area per lipid. Despite this, as in both cases the dipole potential decreases, it is concluded that trehalose-phosphate interaction would be contributing to the dipole potential. In this sense, it is of interest to evaluate furthermore what happens when POs are less accessible due to strong interaction with neighboring ethanolamines. In the case of pure DMPE monolayers, the dipole potential is higher than that of DMPC, in the same conditions (Fig. 4). This, again, could be, at first glance, ascribed to the lower area per lipid of the PE. As expected, dietherPE has a lower dipole potential due to the absence of carbonyl dipoles. The decrease in dipole potential caused by trehalose on DMPE is comparable to that on DMPC (that is, around 56 mV, Fig. 4). In the absence of carbonyls, trehalose does change neither the area per lipid nor the dipole potential of dietherPE (Figs. 4 and 5). However, a significant difference between DMPC and DMPE (both with ester carbonyls) is that trehalose does not affect the area per lipid, at the saturation point, of PE (Fig. 6). This could be explained because, in DMPE, the strong interaction of phosphates and ethanolamines makes difficult the entrance of trehalose in the carbonyl region, but it does not hinder the decrease in dipole potential. In other words, the dipole potential can be changed without an effect in the area. In this case, the effect on dipole potential would be a consequence of the interaction with the accessible PO groups, which would be
Fig. 5. Changes in the surface pressure by addition of dietherpe to pure water and trehalose solutions. , dietherPE on water (28 ◦ C); ♦, dietherPE on 0.15 M trehalose (28 ◦ C).
even less accessible in dietherPE. A possibility is that the packing resulting of the strong P–N + interaction in PE’s restricts partially the contact of the CO groups with the water phase. However, this interaction would not be as strong as in the absence of carbonyls, since trehalose affects the dipole potential of DMPE but not that of dietherPE. The simulation results have also shown that trehalose inserts deeply in the membrane, at the level of the car-
Fig. 6. Changes in the surface pressure by addition of dmpe to pure water and trehalose solutions. , DMPE on water (28 ◦ C); , DMPE on 0.15 M trehalose (28 ◦ C).
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bonyl region (Sum et al., 2003; Villarreal et al., 2004). These predictions are congruent with the present experimental finding that trehalose does not affect the area of lipids in which the carbonyls are absent (dietherPC and PE), or severely hidden by the packing of the membrane (DMPE) (Table 1). The influence of packing in the effect of trehalose on DMPE, can be visualized at lower surface pressures (Fig. 6). At surface pressures less than 20 mN/m, trehalose causes a slight spacer effect on DMPE. That is, the same surface tension is achieved at higher areas with trehalose, indicating a spacing effect. At higher packing of the monolayers (saturation), achieved by the addition of more lipids to the monolayer, trehalose has no effect on the area. This suggests that the lipid–lipid interaction is strong enough to displace trehalose moieties from its interaction with the COs or, at least, to induce an orientation normal to the bilayer surface (instead of parallel), with no effect on the area. At constant lipids (2 nmol DMPE), the surface pressure in trehalose is higher than in pure water. This denotes that the surface tension of the monolayer is decreased by the addition of trehalose to the subphase. This is in agreement with the predictions of Skibinsky et al. (2005), although they made the simulation on DPPC. The present series of experiments, done with DMPC in 0.15 M trehalose at 18 ◦ C, show the same trend in the data of area increase as those reported by Lambruschini et al. (2000) at 22 ◦ C for DPPC, with 0.1 M trehalose and predicted by Skibinsky et al. (2005). In a previous study in our laboratory, it was shown that trehalose, at a much lower concentration (0.05 M), may reduce the area per lipid of DMPC, with no effect on dietherPC. This was interpreted as a consequence of the elimination of water from the hydration shell of the polar head groups and an insertion of the trehalose with the axis of the molecule normal to the bilayer, as suggested by the simulation of Villarreal et al. (2004). Probably, the decrease in the dipole potential may be achieved by the elimination of the so-called polarized water molecules, thus reducing the excluded volume. This might produce, as an ultimate consequence, a closer approach of the membrane lipids reducing the area per lipid. However, at higher concentrations, trehalose can oppose to this by inserting into the lipid interphase as a spacer, increasing the area per lipid. From the balance of these two opposing effects, the changes in the area per lipid would remain constant or increase. A possible reason for this difference could be that the hysteresis of the lipids to stabilize at the interphase might be affected by the trehalose concentration. Although the importance of carbonyl in the PCs for the trehalose inser-
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tion was confirmed, the opposite results in area to the present results and those reported by Lambruschini et al. (2000) would be worth of a more detailed study. 4. Conclusions The insertion of trehalose into DMPC monolayers in the fluid and gel state requires of the presence of carbonyl groups in order to affect the area of the lipids. The area change that appears at 0.15 M is congruent with the decrease in the dipole potential. However, the presence of CO groups in PE’s is not significant for the trehalose insertion, since no changes in area are observed. In dietherPC, in which trehalose does not affect the area, a decrease in the dipole potential is also observed. This suggests that trehalose is interacting with and displacing water from the phosphate groups, too. In DMPE, trehalose also decreases the dipole potential without affecting the area, at the higher surface pressure in saturated monolayers. In dietherPE no effect is observed, since no carbonyls are present and the phosphates are severely hidden to concert H bonds with trehalose. In accordance, the spacer effect of trehalose depends on the interaction with CO and it depends on the strength of the interaction of the PO groups with lateral NH groups, modulated by the surface pressure. Finally, the effect of trehalose on dipole potential is a consequence of different mechanisms which must be considered in the different situations, mainly: dehydration of constitutive dipoles of the interphase, spacer effect and opposition of its own dipole to the dipole of the monolayer. Acknowledgements This work was supported with funds from Agencia Nacional de Promoci´on Cient´ıfica y Tecnol´ogica, PICT 0324, CONICET (PIP 5476) and UBACyT (B047). EAD is a member of the Research Career of CONICET (National Research Council, Argentina). References Alonso-Romanowski, S., Biondi, A.C., Disalvo, E.A., 1989. Effect of carbohydrates and glycerol on the stability and surface properties of lyophilized liposomes. J. Membr. Biol. 108 (1), 1–11. Crowe, J.H., Crowe, L.M., Carpenter, J., Prestrelski, F.S., Hoekstra, F.A., et al., 1997. Anhydrobiosis: cellular adaptation to extreme dehydration. In: Dantzler II, W.H. (Ed.), Handbook of Physiology. Oxford Univ. Press, Oxford, pp. 1445–1478. Crowe, J.H., Crowe, L.M., Carpenter, J.F., Wistrom, C.A., 1987. Stabilization of dry phospholipid bilayers and proteins by sugars. Biochem. J. 242, 1–10.
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