Chemistry and Physics of Lipids 127 (2004)

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1 Chemistry and Physics of Lipids 127 (2004) The kinetics and mechanism of the formation of crystalline phase of dipalmitoylphosphatidylethanolamine dispersed in aqueous dimethyl sulfoxide solutions Lin Chen a, Xin Xie a, Zhi-Wu Yu a,, Peter J. Quinn b a Laboratory of Bioorganic Phosphorous Chemistry and Chemical Biology, Department of Chemistry, Tsinghua University, Beijing , China b Department of Life Sciences, King s College London, 150 Stamford Street, London SE1 9NN, UK Received 30 September 2003; received in revised form 7 October 2003; accepted 8 October 2003 Abstract The phase transition kinetics and mechanism of formation of a lamellar-crystalline phase of dipalmitoylphosphatidylethanolamine (DPPE) dispersed in different concentrations of aqueous dimethyl sulfoxide (DMSO) during cooling have been examined by differential scanning calorimetry and synchrotron X-ray diffraction techniques. In dispersions containing mole fractions of DMSO (x <0.22), the phase transition sequence of the phospholipid is from lamellar liquid-crystal phase to lamellar-gel phase. Increasing the mole fraction of DMSO to 0.22 <x<0.50 induced the formation of a lamellar crystal phase from the gel phase. The proportion of gel phase converted to the lamellar crystal phase increased with DMSO concentration. Dispersion of phospholipid in aqueous DMSO solutions when x>0.5 resulted in a direct transition from liquid-crystal phase to lamellar crystal phase with no detectable intermediate gel phase. A temperature versus DMSO concentration phase diagram was constructed based on calorimetric data with phase assignments made using synchrotron X-ray diffraction measurements. The non-isothermal formation kinetics of the lamellar crystal phase, which is expressed as the half time of the transformation process, was found to depend on DMSO concentration. The inducement of lamellar crystal phase in DPPE by DMSO is discussed in terms of the dehydration effect of DMSO and competitive molecular interactions between DMSO, water, and the phospholipid Elsevier Ireland Ltd. All rights reserved. Keywords: Kinetics; DPPE; Crystalline phase; Dimethylsulphoxide; Half time; DSC 1. Introduction Phosphatidylethanolamines (PEs) and phosphatidylcholines (PCs) are major constituents of most mammalian plasma membranes. Knowledge of the Corresponding author. Tel.: ; fax: address: yuzhw@mail.tsinghua.edu.cn (Z.-W. Yu). phase behaviour of phospholipids helps to understand the biofunctions of lipid assemblies in living cells. Undoubtedly, lamellar liquid-crystal phase (L )isthe predominant state of lipid assembly in membranes (Singer and Nicolson, 1972). Other forms of the lipid polymorphisms, however, may be also of importance. For example, non-lamellar phases of lipids are believed to relate to the membrane fusion process; and the formation of crystalline phase could be /$ see front matter 2003 Elsevier Ireland Ltd. All rights reserved. doi: /j.chemphyslip

2 154 L. Chen et al. / Chemistry and Physics of Lipids 127 (2004) correlated with cryoinjury when living organisms are exposed to low temperatures (Yu and Quinn, 1998). Dimethylsulfoxide (DMSO), as a kind of widely used cryoprotectant (Karlsson et al., 1993) and membrane permeabilizer (Hsieh, 1994), will affect the stability of membranes and particularly the phase behaviour of model membranes comprised of phospholipids (Cheng and Caffrey, 1996; Yu and Quinn, 1998). Its interactions with model membranes have been extensively investigated in order to elucidate the mechanism of physiological action of the compound at the molecular level (Kinoshita et al., 2001). The interaction mechanism of DMSO molecules with the phospholipids has been much discussed from different angles, but it still remains elusive (Vaismann and Berkowtiz, 1992; Gordeliy et al., 1998; Chang and Dea, 2001). Knowledge of the kinetics of phase transitions, particularly that of the formation of lamellar-crystal phase, would be very useful for revealing the solvent interaction mechanism with phospholipids (Grabitz et al., 2002). The existence of various crystalline phases is one of the features of PE molecules (Takahashi et al., 1997). It has been reported that at least three distinct types of microcrystalline solids are formed for dried diacyl-pes (Lewis and McElhaney, 1993). For the fully hydrated PEs, except for PEs with short-chains, the formation of the crystalline phases usually requires a long incubation time at low temperatures (Kodama et al., 1995). For example, it takes 3 4 weeks incubation at temperatures near 10 C for the formation of the stable crystalline phase of a fully hydrated dimyristoylphosphatidylethanolamine, and the formation rate can be accelerated by the method of two-step annealing, which will be finished less than 12 h. In other reports, the relaxation time from lamellar-gel (L ) to crystalline phase (L c ) of distearoylphosphatidylethanolamine dispersed in glycerol was found to be about several days at room temperature (Williams et al., 1991), whereas the transformation could complete in about half an hour at the incubation of 75 C(Chen et al., 2001). These suggest that the formation of the L c phase for a given lipid is comparatively a very slow process, which is temperature- and time-dependent. In addition, small molecules will also have much effect on the crystalline phase formation rate. The cholesterol facilitates the formation of the crystalline phase in phosphatidylethanolamine bilayers, whereas the formation of such phase in phosphatidylcholine bilayers is inhibited by the presence of cholesterol (McMullen et al., 2000). Inappropriate control of experimental conditions would result in inconsistent results. The question of how the formation rate of crystalline phase is affected by the surrounding conditions including temperature, solvent, and small molecules and the nature of the interaction mechanism is of interest to us. The present work aims to provide more insights into the effect of DMSO on the phase behaviour of DPPE. Focus has been on the kinetics and mechanism of the formation of crystalline phase upon cooling. 2. Materials and methods Dipalmitoylphosphatidylethanolamine (DPPE) in lyophilized form was purchased from Sigma Chemical Co. (Louis, MO, USA). Its purity was claimed to be better than 99% and it was used without further purification. Dimethyl sulfoxide (DMSO) was purchased from Yili Fine Chemical Co. (Beijing, China), and was of AR grade. Deionized water was used in all the experiments. Mole fractions of aqueous DMSO solutions were prepared between and 1, and used to disperse DPPE to give a lipid/solvent ratio of 1:3 (w/v) to ensure an excess of solvent. The lipid was solvated by at least three heating-cooling cycles between 35 and 100 C. A Mettler-Toledo DSC821 e differential scanning calorimeter was used to examine thermotropic phase behaviour of the lipid dispersions. The signal time constant was 3 s and resolution is better than 0.7 W with noise (RMS) less than 1 W. The thermograms of the samples were recorded after cooling down from 95 C at a scan rate of 1 C/min. Phase transition temperatures were determined as onset temperatures. X-ray diffraction was conducted at Station 8.2 of the Synchrotron Radiation Source of the SERC Daresbury Laboratory, using a method described previously (Feng et al., 2002). Briefly, the lipid samples were mounted in a cell of 1 mm thickness and sealed with thin mica windows. Measurements during heating/cooling were performed at a rate of 5 C/min. Only cooling process data is used in this presentation to compare with the calorimetric results. The small-angle (SAXS) and wide angle X-ray scattering (WAXS) patterns were recorded simultaneously.

3 L. Chen et al. / Chemistry and Physics of Lipids 127 (2004) Experimental data were processed using the OTOKO software package (EMBL, Hamburg, Germany). 3. Results and discussion 3.1. Phase behaviour of DPPE in aqueous DMSO solutions upon cooling The DSC thermograms of DPPE in excess aqueous DMSO solutions recorded during cooling scans from 85 to 55 C recorded at a scan rate of 1 C/min are shown in Fig. 1. The thermograms can be divided into three categories reflecting the effects of different DMSO concentrations. The first category covers the dispersions in the mixed solvents with mole fraction of DMSO greater than 0.5. Only a single exothermic peak in each curve is seen. The onset temperatures do not change significantly with different mole fractions of DMSO in the range 0.6 <x<1 and are about 75 C. The phases of the samples were also monitored during cooling by recording real-time X-ray scattering intensity patterns. Representative data are presented in Fig. 2, which show a sequence of small-angle (SAXS) and wide-angle (WAXS) X-ray scattering patterns recorded from a dispersion of DPPE in DMSO Heat Flow/a.u Temperature/ o C Fig. 1. Thermograms recorded during cooling of DPPE dispersed in different aqueous DMSO solutions from 85 to 55 C at a scanning rate of 1 C/min. DMSO mole fraction is shown on the right of each thermogram. 0 Fig. 2. Real-time synchrotron X-ray scattering intensity profiles of DPPE dispersed in DMSO recorded simultaneously at small-angle (left) and wide-angle (right) during a cooling scan at 5 C/min, showing a phase transition from liquid-crystalline to crystalline phase. Arrows indicate the positions of diffraction peaks. during a cooling scan from 100 to 36 C at a scan rate of 5 C/min. The SAXS patterns show that a single diffraction peak centred at about 3.51 nm is transformed at about 75 C into two scattering maxima located at spacings of 5.10 nm (S nm 1 ) and 2.45 nm (S nm 1 ), which indicate a lamellar structure. The corresponding WAXS patterns indexing the hydrocarbon chain spacings show that, at 100 C, there is a single broad scattering peak centred at about nm, which is typical of liquid-crystalline phase. Because higher order diffractions were missing in the SAXS region at the temperature, it is impossible to assign the phase as a lamellar or non-lamellar phase. As a result, we denote the liquid-crystal phase as phase in this paper. The broad diffraction band is replaced at about 75 C by a series of sharp diffraction bands, the major peak of which is centred at about nm, with two additional peaks at about and nm, respectively. The transition is assigned as L c. The position of the three peaks is in close agreement with a lamellar-crystalline phase reported by Williams and co-workers (1991): 0.460, 0.395, and nm, respectively. The conclusion from the SAXS and WAXS results is that the phase transition sequence on cooling DPPE dispersions in aqueous DMSO solutions is

4 156 L. Chen et al. / Chemistry and Physics of Lipids 127 (2004) L c when mole fraction of DMSO is high (x > 0.5) and the transition temperature is about 75 C. The lamellar repeat spacings are much shorter than the corresponding repeat spacings of DPPE dispersed in water, which gave values of 5.22 nm for L phase and 5.54 nm for L c phase (Yao et al., 1992). This is consistent with the effect of DMSO on the repeat spacings of phospholipid multilayer structures at both gel and liquid-crystalline phase (Yu and Quinn, 1998, 2000). The formation of an intermediate L phase from phase upon cooling could not be detected by DSC or X-ray diffraction methods employed in the present experimental conditions. A second category of transition behaviour of DPPE is observed when the mole fraction of DMSO is less than Again only one exothermic peak is detected (Fig. 1). From synchrotron X-ray scattering data, the phase transition is a lamellar liquid-crystalline phase to lamellar-gel phase, the same sequence as that of DPPE dispersed in water (Yao et al., 1992). The onset temperature was found to increase from 64 to 71 C with increasing concentration of DMSO (Fig. 3). The third category is recognised in dispersions of DPPE in mole fractions of DMSO in the range 0.22 < x<0.50. This group is characterised by two exothermic peaks in the thermograms (Fig. 1). The transition temperatures of the first peak during cooling are between 69 and 71 C and are assigned from synchrotron X-ray data as a transition from to L phase, similar to those seen in DPPE dispersions in lower concen- Temperature/ o C L β II L β α III L c DMSO Mole Fraction Fig. 3. Partial phase diagram of DPPE dispersed in aqueous DMSO solutions upon cooling at a scan rate of 1 C/min. Phase boundaries were determined from calorimetry and phase assignments from X-ray diffraction data: ( ) L,( ) L L c,( ) L c. L c I Fig. 4. A plot of the enthalpy ratio of the phase transition (L L c )/( L ) of DPPE dispersed in the mediate DMSO mole fraction range recorded during cooling scans. trations of DMSO. The second exothermic peak is attributed to a transition from L to L c phase, similar to that seen at higher DMSO concentrations. The temperature of this phase transition increases progressively from 61.7 to 71.2 C with increasing DMSO concentration. It is thus clear that this is a two-step process L L c and the phase transition temperature of the former is not influenced as significantly as that of the latter with increasing DMSO mole fraction. The ratio of the enthalpy values of these two phase transitions, the latter to the former, is presented in Fig. 4. It is found to increase progressively from 0.4 to 0.8 over the range of DMSO mole fractions Assuming that enthalpy is directly proportional to the fraction of a dispersion that has undergone a phase transition, the data in Fig. 4 suggests that a greater proportion of L phase transforms to the L c phase with higher DMSO concentration within the cooling time span. Thus, it is not surprise that further increase in DMSO concentration induces the formation of the L c phase directly from liquid-crystal phase upon cooling, as in the first category of the dispersions. The amount of the L c phase formed depends on the DMSO concentration in a limited time span The non-isothermal kinetics of the phase transition of DPPE upon cooling Based on calorimetry, the phase transition fraction θ(t) at different DMSO mole fractions can be calculated, and results for DPPE dispersed in water ( L ) and DMSO ( L c ) are presented in Fig. 5. This

5 L. Chen et al. / Chemistry and Physics of Lipids 127 (2004) Fig. 5. Development of phase transition fraction θ(t) as a function of time for the non-isothermal phase transition process of DPPE dispersed in water ( )and DMSO ( ). shows the relationships of θ(t) with time for representative dispersions of DPPE in the low and high DMSO concentration solutions, that is the second and the first categories of phase transition sequences referring to Fig. 3. The overall transition from L c takes about 30 min, but the L transition is markedly faster (about 3 min), indicating that the rate-limiting step is the formation of the L c phase. It is believed that the formation of L c phase involves more significant rearrangements of the molecules and proceeds by nucleation and growth processes. The half-time (t 1/2 ) of phase transition can be used to describe the rate of transformation, which is defined as the time taken from the onset of the transformation until 50% completion. The value can be extracted directly from a plot of θ(t) versus time (Liu et al., 2003). The dependence of t 1/2 of different phase transformations on the concentration of DMSO is shown in Fig. 6. The half time of the phase transformation L at low DMSO mole fractions was found to be a constant of about 30 s. This suggests that the changing rate of the phase transition of DPPE dispersed in aqueous DMSO solutions is not markedly affected by the concentration of DMSO. When the mole fraction is less than 0.22, the transition from L to L c cannot be obtained by calorimetry under present experimental conditions. During subsequent heating scans, the lamellar-gel phase transforms directly into the liquid-crystalline phase. Incubation of the L phase at appropriate temperatures appears to be required for efficient crystallisation of the lipid dispersion (Chen et al., 2001). Fig. 6. Plots of the half time (t 1/2 ) of the different phase transformations plotted as a function of DMSO mole fraction for the dispersion of DPPE in aqueous DMSO solution. The significance of symbols used here are the same as that described in Fig. 3. When the DMSO mole fraction in aqueous DPPE dispersions increases from 0.22 to 0.5, two exothermic peaks in the DSC thermograms were detected upon cooling. The half time of the transformation L is still about 0.5 min, whilst the half time of the transformation from L to L c decreases from about 8 to 0.5 min. If the L phase does not transform completely to L c phase during cooling, the process can still continue slowly on storage at low temperatures or proceed rapidly during subsequent heating. The formation rate of L c phase from L phase in this concentration range was found to increase with increasing DMSO mole fraction. This is consistent with an earlier study (Ruocco and Shipley, 1982), which concluded that the main driving force for the creation of L c phase is dehydration of the polar head group. Increasing mole fraction of DMSO reduces the amount of water in the lipid solvent interface because of the dehydration effect of DMSO (Shashkov et al., 1999). The overall process during cooling from liquid-crystalline to gel and then from gel to crystalline can be regarded as successive two-step physical changes. When DPPE is dispersed in aqueous DMSO solutions with mole fractions greater than 0.5, no L phase can be distinguished by calorimetry even if the scan rate is reduced to 0.1 C/min (data not shown). Fig. 6 shows that the half time of the transformation L increases with increasing DMSO mole fraction. This means that the formation rate of the L c phase of DPPE decreases with increasing DMSO concentration, an opposite effect in comparison with that when

6 158 L. Chen et al. / Chemistry and Physics of Lipids 127 (2004) DMSO mole fraction is less than 0.5. Such interesting and apparently controversial results can be explained as follows. When the content of DMSO is less than equimolar in the mixed solvents, the dehydration process dominates, due to probably stronger interactions between DMSO and water than that between water and the polar head groups of phospholipids (Shashkov et al., 1999; Yu et al., 2002). When the content of DMSO is greater than equimolar in the mixed solvents, the excess DMSO molecules tend to replace water and form hydrogen bonds with the head groups of DPPE molecules, because the hydrogen bonds formed between DMSO and DPPE are stronger than between water and DPPE (Yu et al., 2002). The formation of a hydrogen bond network at the lipid solvent interface acts to prevent the reorientation of hydrocarbon chains of DPPE molecules that makes it increasingly difficult to rearrange into the ordered L c phase (Gordeliy et al., 1998). As a result, the half time of the transformation from L c increases with increasing DMSO concentration. 4. Conclusions (a) The lamellar-crystalline phase (L c ) of DPPE dispersed in aqueous DMSO solutions can be observed during cooling at a scan rate of 1 C/min when the mole fraction of DMSO is greater than (b) DMSO does not have a significant effect on the rate of the transformation from liquid-crystalline to lamellar-gel phase. The half time of this transformation is about 0.5 min. (c) The rate of formation of the L c phase depends markedly on the DMSO concentration when the DMSO mole fraction is greater than The rate initially increases with DMSO concentration until the mole fraction reaches about 0.5, above which the rate decreases. This is believed to be due to different modes of hydrogen bond formation resulting from competitive interactions among head groups of DPPE, water and DMSO. Acknowledgements Partial support by a grant from the Natural Science Foundation of China ( ) and that from the TRAPOYT program of the Ministry of Education of China are gratefully acknowledged. References Chang, H.H., Dea, P.K., Insights into the dynamics of DMSO in phosphatidylcholine bilayers. Biophys. Chem. 94, Chen, L., Yu, Z.W., Quinn, P.J., Kinetic phase behavior of distearoylphosphatidylethanolamine dispersed in glycerol. Biophys. Chem. 89, Cheng, A.C., Caffrey, M., Interlamellar transition mechanism in model membranes. J. Phys. Chem. 100, Feng, Y., Yu, Z.W., Quinn, P.J., Effect of urea, dimethylurea, and tetramethylurea on the phase behavior of dioleoylphosphatidylethanolamine. Chem. Phys. Lipids 114, Gordeliy, V.I., Kiselev, M.A., Lesieur, P., Pole, A.V., Teixeira, J., Lipid membrane structure and interactions in dimethylsulfoxide/water mixtures. Biophys. J. 75, Grabitz, P., Ivanova, V.P., Heimburg, T., Relaxation kinetics of lipid membranes and its relation to the heat capacity. Biophys. J. 82, Hsieh, D.S. (Ed.), Drug Permeation Enhancement, Theory and Applications. Marcel Dekker, Dortmund. Karlsson, J.O.M., Cravalho, E.G., Rinkes, I.H.M.B., Nucleation and growth of ice crystals inside cultured hepatocytes during freezing in the presence of dimethyl sulfoxide. Biophys. J. 65, Kinoshita, K., Li, S.J., Yamazaki, M., The mechanism of the stabilization of the hexagonal II (H II ) phase in phosphatidylethanolamine membranes in the presence of low concentrations of dimethyl sulfoxide. Eur. Biophys. J. Biophys. 30, Kodama, M., Inoue, H., Tsuchida, Y., The behavior of water molecules associated with structural changes in phosphatidylethanolamine assembly as studied by DSC. Thermochim. Acta 226, Lewis, R.N.A.H., McElhaney, R.N., Calorimetric and spectroscopic studies of the polymorphic phase behavior of a homologous series of n-saturated 1,2-diacyl phosphatidylethanolamines. Biophys. J. 64, Liu, M.Y., Zhao, Q.X., Wang, Y., Zhang, C.G., Mo, Z.S., Cao, S.K., Melting behaviors, isothermal and non-isothermal crystallisation kinetics of nylon Polymer 44, McMullen, T.P.W., Lewis, R.N.A.H., McElhaney, R.N., Differential scanning calorimetric and Fourier transform infrared spectroscopic studies of the effects of cholesterol on the thermotropic phase behavior and organization of a homologous series of linear saturated phosphatidylserine bilayer membranes. Biophys. J. 79, Ruocco, M.J., Shipley, G.G., Characterization of the subtransition of hydrated dipalmitoylphosphatidylcholine bilayers: kinetic, hydration and structural study. Biochim. Biophys. Acta 691, Shashkov, S.N., Kiselev, M.A., Tioutiounnikov, S.N., Kiselev, A.M., Lesieur, P., The study of DMSO/water and DPPC/DMSO/water system by means of the X-ray, neutron small-angle scattering, calorimetry and IR spectroscopy. Physica B 271,

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