Comprehensive characterization of temperature- and pressure-induced bilayer phase transitions for saturated phosphatidylcholines containing longer chain homologs

Colloids and Surfaces B: Biointerfaces 128 (2015) 389–397

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Comprehensive characterization of temperature- and pressure-induced bilayer phase transitions for saturated phosphatidylcholines containing longer chain homologs Masaki Goto a,1 , Takuya Endo b , Takahiro Yano b , Nobutake Tamai c , Joachim Kohlbrecher a , Hitoshi Matsuki c,∗ a

Laboratory for Neutron Scattering and Imaging, Paul Scherrer Institute, CH-5232 Villigen PSI, Switzerland Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, 2-1 Minamijosanjima-cho, Tokushima 770-8506, Japan c Department of Life System, Institute of Technology and Science, The University of Tokushima, 2-1 Minamijosanjima-cho, Tokushima 770-8506, Japan b

a r t i c l e

i n f o

Article history: Received 4 September 2014 Received in revised form 6 January 2015 Accepted 17 February 2015 Available online 25 February 2015 Keywords: Bilayer membrane Diacylphosphatidylcholine Interdigitation Phase transition Pressure Subgel phase

a b s t r a c t Complete elucidation of the phase behavior of phospholipid bilayers requires information on the subtransition from the lamellar crystal (Lc ) phase to the gel phase. However, for bilayers of saturated diacylphosphatidylcholines (CnPCs), especially longer chain homologs, equilibration in the Lc phase is known to be very slow. In this study, bilayer phase transitions of three CnPCs with longer acyl chains, C19PC, C20PC and C21PC, were observed by differential scanning calorimetry under atmospheric pressure and by light-transmittance measurements under high pressure. Using lipid samples treated by thermal annealing enabled the observation of the sub-, pre- and main transitions of the C19PC and C20PC bilayers under atmospheric pressure. Only the pre- and main transitions could be observed for the C21PC bilayer due to very slow kinetics of the Lc phase formation for lipids with long acyl chains. The temperature and pressure phase diagrams constructed and phase-transitions quantities (enthalpy, entropy and volume changes) evaluated for these bilayers were compared with one another and with those of bilayers of the CnPC homologs examined in previous studies. These results allowed us (1) to clarify the temperatureand pressure-dependent phase sequence and phase stability of the CnPC (n = 12–22) bilayers as a function of the hydrophobicity of the molecules, (2) to prove the presence of a shorter and a longer limit (n = 13 and 21) in the acyl chain length for the pressure-induced bilayer interdigitation and (3) to reveal the chain-length dependence of the thermodynamic quantities of the subtransitions including the volume change. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Phosphatidylcholines are major components of biological membranes in eukaryotic organisms. Bilayers of saturated diacylphosphatidylcholines (CnPCs) containing two identical saturated fatty acids have been frequently used as model membranes for lifescience studies and simultaneously their properties have been examined by several physico-chemical techniques [1–3]. One notable feature of CnPC bilayers is the gel-phase polymorphism. CnPC bilayers undergo a thermotropic phase transition between

∗ Corresponding author. Tel.: +81 88 656 7513; fax: +81 88 655 3162. E-mail address: [email protected] (H. Matsuki). 1 Visiting Scientist; on leave from The University of Tokushima, Japan. http://dx.doi.org/10.1016/j.colsurfb.2015.02.036 0927-7765/© 2015 Elsevier B.V. All rights reserved.

the gel phases below the main-transition temperature due to the bulky choline head group of the PC molecule. This transition is known as the pretransition from the lamellar gel (L␤ ) phase with flat bilayer sheets composed of tilted PC molecules to the ripple gel (P␤ ) phase with undulated bilayer sheets. In addition to these gel phases, another gel phase appears in CnPC bilayers. It is well known that the fully interdigitated gel (L␤ I) phase of CnPC bilayers is induced by various additives such as polyols, short-chain alcohols and anesthetics under atmospheric pressure [4–9]. On the other hand, the L␤ I phase is also induced by an application of pressure. It was found that bilayers of CnPCs, such as dimyristoyl-PC (DMPC: C14PC) and dipalmitoyl-PC (DPPC: C16PC), form the fully L␤ I phase under high pressure [10–18]. In the L␤ I phase, hydrocarbon chains of the CnPC molecules in one of the monolayers constituting a bilayer extend beyond the region of the bilayer midplane and

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alternately interpenetrate into the other opposing monolayer. Most cases where the pressure-induced interdigitation can be observed involve phospholipids with saturated linear acyl chains like CnPC [19–25]. Another feature of the CnPC bilayers is the formation of the lamellar crystal (Lc ) phase, which is often referred as the subgel phase. It is known that the formation of the Lc phase of the CnPC bilayer is slow [26–30]. The time required to completely form the Lc phase increases with acyl chain length [31]. Hence, it is difficult to determine the Lc -phase boundary on a phase diagram for longer-chain CnPC bilayer. The slow kinetics of the formation of the Lc phase markedly affects phase stability (i.e., stable, unstable and metastable) of the CnPC bilayers. Although the various phases are observed in the CnPC bilayers, the L␤ , P␤ and L␤ I phases are polymorphs of the gel (L␤ ) phases. The fundamental phases of the CnPC bilayers are the Lc , L␤ and liquid crystalline (L␣ ) phases. Therefore, the information of the Lc phase depending on temperature and pressure is indispensable for the construction of the complete phase diagrams of the CnPC bilayers, in other words, the full elucidation of their phase behavior. The full description of the phase behavior of the CnPC bilayers gives us not only detailed knowledge of lipid molecular interactions in the bilayer but also useful information to understand the environmental adaptation of organisms, food and cosmetics processing, biologically relevant phenomena such as pressure reversal of anesthesia and mechanosensing. Recently, we have found in the temperature (T)–pressure (p) phase diagrams of the CnPC (n = 14–18) bilayers that a minimum interdigitation pressure (MIP), which is the minimum pressure required for the bilayer interdigitation, decreases with an elongation of the acyl chains [32–34]. From the acyl chain-length dependence of the MIP values, it is expected that in the bilayer of certain CnPC with longer acyl chains the enhanced interaction may induce the bilayer interdigitation under atmospheric pressure by only hydration. Previously we demonstrated this on the T–p phase diagram of the dibehenoyl-PC (C22PC) bilayer by methods of small angle neutron scattering (SANS) and fluorometry [35]. At the same time, it was suggested that the dihenearachidoyl-PC (C21PC) is the longest chain CnPC that requires pressure to induce the bilayer interdigitation. However, the bilayer phase behavior of CnPCs between C18PC and C22PC, that is, dinonadecanoyl-PC (C19PC), diarachidoyl-PC (C20PC) and C21PC, have not yet been clarified. In addition to the pressure-induced bilayer interdigitation for bilayers of longer chain homologs, it is interesting to investigate their Lc phase formation, the process that seems to require a long time. By investigating all the phase transitions including the subtransition for longer-chain CnPC bilayers, we can compare the T–p phase diagrams and the thermodynamic quantities of the bilayer phase transitions for a series of CnPCs to consider the phase transitions of the CnPC bilayers as a function of the acyl chain length n, or hydrophobicity, in a wider n-range [36–40]. Therefore, in the present study, the bilayer phase transitions of C19PC, C20PC and C21PC are observed under atmospheric and high pressures to comprehensively understand the bilayer properties of CnPCs as a function of n. To construct complete phase diagrams containing all phase transitions (sub-, pre-, main transitions and interdigitation) of the CnPCs bilayers, we also identify the boundaries of the Lc phase of the CnPC bilayers (n < 18). First, the temperatureand pressure-induced phase transitions of the C19PC, C20PC and C21PC bilayers are discussed in terms of the obtained T–p phase diagrams and thermodynamic quantities. Next, the effect of the acyl chain length on membrane properties of the fully hydrated CnPC bilayers (n = 12–22) are considered systematically by comparing the phase diagrams and thermodynamic quantities of the C19PC, C20PC and C21PC bilayers to those of the CnPC homolog

(C12PC–C18PC, C22PC) bilayers from previous studies [32–34,41] together with data on their subtransition. 2. Experimental 2.1. Materials and sample preparation Synthetic phospholipids, 1,2-dioctadecanoyl-sn-glycero-3phosphocholine (C18PC), 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (C19PC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (C20PC) and 1,2-dihenearachidoyl-sn-glycero-3-phosphocholine (C21PC), were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA) and/or Sigma Chemical Co. (St. Louis, MO, USA). Other phospholipids, CnPC (n < 18), were also obtained from both reagent companies. They were used as received. Water used was distilled twice from a dilute alkaline permanganate solution. The multilamellar vesicle dispersions of 1.0 mmol kg−1 concentration were prepared for each phospholipid by sonicating for a few minutes with a sonifier at a temperature several degrees above the main-transition temperature. The vesicle dispersions of C19PC, C20PC and C21PC were annealed by at least 180 repeated thermal cycles to form the subgel phase [34]; 1 thermal cycle comprises freezing storage at –15 ◦ C for 23 h, and at –30 ◦ C for 1 h and cold storage at 5 ◦ C for one day, each of which corresponds to conditions that the complete freeze of bulk water, the complete freeze of freezable interlamellar water and the complete melting of all kinds of water (freezable and nonfreezable interlamellar water and bulk water), respectively [42,43]. The repeating of these conditions as a freeze and thaw cycle promotes the dehydration around the head group, then results in the formation of Lc phase. For bilayers of CnPCs for n < 18, we performed the above thermal annealing treatment repeatedly until the constant values of the subtransition enthalpy were obtained: 7 cycles for C13PC, 14 for C14PC and C15PC, 21 for C16PC, 70 for C17PC and C18PC. 2.2. Phase-transition observation The phase transitions of the CnPC bilayers under atmospheric pressure were observed by a high-sensitivity differential scanning calorimeter MicroCal VP-DSC (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The heating rate was 0.75 ◦ C min−1 . For determination of phase transition below 10 ◦ C, another calorimeter, SSC 5200-DSC 120 calorimeter (SII Nanotechnology Co. Ltd, Chiba, Japan), was used with a lipid concentration of 10–20 mmol kg−1 . The enthalpy changes of the phase transitions were determined from the endothermic peak areas as average values over several measurements. The DSC thermograms were analyzed by use of software Origin 7.0 (Lightstone Corp., Tokyo, Japan). The phase transitions of the CnPC bilayers under high pressure were observed by a light-transmittance technique using high-pressure cell assemblies PCI-400 and PCI-500 (Syn. Corp., Kyoto, Japan) attached to spectrophotometers U-3010 and U-3900 (Hitachi High-Technology Corp., Tokyo, Japan), respectively. The apparatus can detect light-transmittance changes under an isobaric thermotropic condition. The measurements were repeated at least three times at the same pressure to judge the phase stability. The phase-transition temperatures determined by this technique under atmospheric pressure were in good agreement with those by the DSC. The heating rate was 0.33 or 0.50 ◦ C min−1 and the pressure range was 0.1–200 MPa except the C21PC bilayer, the range of which was 0.1–100 MPa. The detailed procedure for the measurements under high pressure was described elsewhere [33,34,44,45]. The phase assignment for each CnPC bilayer was done by checking the phase-transition behavior (appearance mode, magnitude, cooperativity, etc.) obtained from both DSC and light transmittance

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Fig. 1. (A) DSC thermograms of C19PC bilayer: (1) 1st heating scan, (2) 2nd heating scan. (B) Light-transmittance curves of C19PC bilayer: (1) 68 MPa (annealed), (2) 118 MPa (annealed), (3) 25 MPa and (4) 60 MPa.

measurements and by referring to the previous data obtained from small and wide angle X-ray diffraction measurements (lamellar repeated distance and the chain–chain distance in the CnPC bilayers) [46–48]. 3. Results and discussion 3.1. Thermotropic and barotropic phase transitions The DSC thermogram of the C19PC bilayer obtained by heating scans is depicted in Fig. 1A. For the bilayer treated by thermal annealings (curve 1), three peaks were observed at 35.4, 56.6, and 60.5 ◦ C, which indicates that it undergoes three kinds of endothermic transitions in the first heating scan. The enthalpy changes (H) of the transitions were determined to be 26.8, 5.0, and 46.1 kJ mol−1 , respectively, which were consistent with the previous report except for the H value of the transition at the lowest temperature [31]. The three phase transitions correspond to the subtransition from the Lc phase to the L␤ phase, the pretransition from the L␤ phase to the P␤ phase and the main transition from the P␤ phase to the L␣ phase, in turn. In the subsequent heating scan (second scan), where the lipid sample was reheated immediately after cooling, it exhibited only the pretransition and main transition (curve 2). This thermal behavior signifies that it takes a long time to form the Lc phase and the transformation into the Lc phase requires an appropriate annealing treatment to the sample before experiments. Since it has been reported that the period of the annealing required to form the Lc phase increases with the elongation of the acyl chains of the CnPC molecule [31], the lower H value of the subtransition as compared with the previous study (44.8 kJ mol−1 ) may be due to insufficient annealing even though the annealing of 180 cycles (360 days) was applied to the sample. The DSC thermogram of the C20PC bilayer (supplementary figure 1A (Fig. S1A)) also showed that the bilayer undergoes three endothermic transitions, which was similar to that of the C19PC bilayer. The subtransition temperature of 32.7 ◦ C was lower than the previous reported value of 37.8 ◦ C [31], and the H value was also considerably smaller. It is known that the subtransition temperature increases with increasing number of the annealing cycles, signifying that the Lc phase is converted to the most stable phase through several metastable states. We judged from the consistency and inconsistency of the subtransition temperatures of the C19PC and C20PC bilayers with the reported values [31] that the C19PC bilayer formed the most stable Lc phase while the C20PC bilayer did not completely form the

stable Lc phase in our experimental conditions. On the other hand, we could not observe the subtransition in the C21PC bilayer under our experimental conditions (Fig. S1B). Actually, Lewis et al. [31] barely managed to observe the subtransition of the C21PC bilayer, and thus formation of the metastable Lc phase, after annealing of the sample for 16 months. Supplementary Fig. S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.02.036. The isobaric thermotropic phase transitions of the C19PC bilayer were detected by light-transmittance measurements, as shown in Fig. 1B. Here the phase transitions under high pressures were identified by tracking the transitions identified under atmospheric pressure as pressure was raised in small increments, and both annealed and unannealed lipid samples were used to construct boundaries for the stable and metastable phases in the phase diagrams. The bilayer treated with the thermal annealing exhibited two kinds of phase transitions at 68 MPa: the subtransition from the Lc phase to the pressure-induced L␤ I phase and the main transition from the L␤ I phase to the L␣ phase (curve 1). The temperature of the Lc /L␤ I phase transition increased by the application of pressure (curve 2). For the unannealed C19PC bilayer, the pre-(L␤ /P␤ ) and main transitions were found at 25 MPa (curve 3), and the L␤ /L␤ I phase transition was observed in addition to the pre-(L␤ I/P␤ ) and main transitions at 60 MPa (curve 4). The phase-transition behavior of the C20PC bilayer (Fig. S2A) in the light-transmittance curves was similar to that of the C19PC bilayer and also to that of the C16PC bilayer in the previous report [19]. On the other hand, the C21PC bilayer showed three kinds of phase transitions, the L␤ /L␤ I, pre-(L␤ I/P␤ ) and main transitions at 19 MPa (curve 1 in Fig. S2B) for both annealed and unannealed samples while the L␤ /L␤ I and main transitions were found at 28 MPa (curve 2 in Fig. S2B) due to the disappearance of the P␤ phase. Supplementary Fig. S2 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.02.036.

3.2. Temperature–pressure phase diagrams The T–p phase diagrams of C19PC, C20PC and C21PC bilayers were constructed by employing the phase-transition temperatures and pressures obtained by DSC and light-transmittance measurements. The resulting phase diagrams of the CnPC bilayers are shown in Fig. 2. Here, we reconstructed the T–p phase diagram of the C18PC bilayer by identifying the phase boundary of the Lc phase to

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Fig. 2. Temperature–pressure phase diagrams for bilayers of (A) C18PC, (B) C19PC, (C) C20PC and (D) C21PC. Bilayer phases are assigned as the liquid crystalline (L␣ ), lamellar gel (L␤ ), ripple gel (P␤ ), interdigitated gel (L␤ I) and subgel (Lc ) phases. Bilayer phases in parentheses refer to metastable phases. Schematic illustration of each phase is indicated in Fig. 2A.

confirm the consistency of the phase-transition data between the present and previous studies [32], and the diagram is also included in the figure together with schematic illustration of each phase. The temperatures of the pre- and main transitions of all CnPC bilayers increased with increasing pressure. The slopes of each phase

boundary (dT/dp) have similar values for all CnPC bilayers depending on the kind of phase transition as shown in Table 1 (see also Fig. 4 below). In all CnPC bilayers except the C22PC bilayer (Fig. S3) [35], the L␤ I phase was induced in the high-pressure region in between the L␤ and P␤ phases. The triple point at which the

Table 1 Thermodynamic properties of phase transitions for PC bilayer membranes obtained from DSC and light-transmittance measurements. Lipid

Transition

Temp. (◦ C)

Temp. (K)

dT/dp (K MPa−1 )

H (kJ mol−1 )

S (J K−1 mol−1 )

V (cm3 mol−1 )

C18PC C19PC C20PC C21PC C22PC

P␤ /L␣ P␤ /L␣ P␤ /L␣ P␤ /L␣ L␤ I/L␣

55.6b 60.5 ± 0.01a 65.1 ± 0.02a 69.6 ± 0.03a 73.4c

328.8b 333.7 338.3 342.8 346.6c

0.230b 0.240 0.260 0.266 0.254c

45.2b 46.1 ± 1.04a 48.7 ± 1.15a 55.1 ± 0.86a 65.4c

137b 138.2 144.0 160.7 188.7c

31.6b 33.2 37.4 42.8 47.9c

C18PC C19PC C20PC C21PC C22PC

L␤ /P␤ L␤ /P␤ L␤ /P␤ L␤ /P␤ L␤ /P␤

50.9b 56.6 ± 0.32a 63.2 ± 0.21a 67.9 ± 0.11a –

324.1b 329.8 336.4 341.1 –

0.14b 0.17 0.15 0.18 –

5.0b 5.0 ± 0.30a 4.3 ± 0.48a 5.7 ± 0.50a –

15b 15.2 12.8 16.7 –

C18PC C19PC C20PC C21PC C22PC

Lc /P␤ Lc /L␤ Lc /L␤ Lc /L␤ Lc /L␤

31.0 ± 0.02a 35.4 ± 0.17a 32.7 ± 0.23a – –

304.2 308.6 305.9 – –

0.22d 0.22 0.25 – –

28.6 ± 0.14a 26.8 ± 0.27a 11.2 ± 0.83a – –

94.0 86.8 36.6 – –

a b c d

Each value is denoted as (average ± standard deviation) over at least 4 measurements. Data from Ichimori et al. [32]. Data from Goto et al. [35]. Data from Goto et al. [33].

2.2b 2.6 1.9 3.0 – 20.7 19.1 9.2 – –

M. Goto et al. / Colloids and Surfaces B: Biointerfaces 128 (2015) 389–397

Fig. 3. Minimum interdigitation pressure of CnPC bilayers as a function of carbon number of acyl chain.

three gel (L␤ , P␤ and L␤ I) phases can coexist was determined for these bilayers, respectively: 58 ◦ C, 59 MPa for C18PC, 62 ◦ C, 35 MPa for C19PC, 64 ◦ C, 15 MPa for C20PC and 69 ◦ C, 8 MPa for C21PC. The pressure of the triple point corresponds to the MIP because it is the minimum pressure required for the bilayer interdigitation. Supplementary Fig. S3 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.02.036. The slope of the L␤ /L␤ I transition of the unannealed CnPC bilayers changed from a negative value to a positive value with increasing pressure. The dT/dp value is related to the Clapeyron equation (dT/dp = V/S = TV/H) thermodynamically. At ambient and low pressures, it has been reported that the volume changes (V) for the transition from the L␤ phase to the L␤ I phase for C16PC and C18PC bilayers are negative (V < 0) [49] and the corresponding H value is positive (H > 0) [50]. The dT/dp value should thus be negative and this coincides with the observed behavior. The L␤ and L␤ I phases have different compressibilities and the L␤ phase is expected to have the larger compressibility based on comparison of the molecular packing in both phases. At high pressures, the molar volume of the L␤ phase may be smaller than that of the L␤ I phase. Then the V value of the L␤ /L␤ I transition might transformed from a negative to a positive value, the dT/dp value becoming positive. This behavior was also observed in the heptadecanoyl-PC (C17PC) bilayer [33]. The subtransition was observed for the annealed C18PC, C19PC and C20PC bilayers over the whole range of pressure. The T–p curves of the Lc /L␤ and L␤ /L␤ I transitions intersected each other in the high-pressure region for the C18PC and C19PC bilayers. Similar intersections of both curves were found for the C16PC [19] and C17PC [33] bilayers. Interestingly, the T–p curves for the Lc /L␤ and L␤ /L␤ I transitions of the C20PC bilayer do not intersect but touch with each other at about 100 MPa as seen in Fig. 2C. The figure indicates that the stable L␤ phase can exist also above the pressure. Thermodynamically, this means that the L␤ phase is the stable phase in the whole pressure range and there is no metastable region of the L␤ I phase. Since the difference in temperature between the subtransition and the other transitions (main transition and transition between gel phases) increases with the chain elongation [33,34,41], it is expected that the L␤ I phase of the C21PC as well as the C20PC bilayer might become a stable phase in the whole range of pressure even if the subtransition of the bilayer could be observed. Further, it should be also noted that the slight pressure (8 MPa) is needed to induce the L␤ I phase in the C21PC bilayer. In Fig. 3, the

393

MIP values of the present bilayers are plotted against the acyl chain length together with the previous results [32–35]. The MIP values of the CnPC bilayers decreased with an increase in acyl chain length in a smooth non-linear manner. Recently, we have demonstrated that the C22PC bilayer forms the L␤ I phase by only hydration under atmospheric pressure by SANS under atmospheric pressure and fluorometry under high pressure [35]. We expected in this study that the C21PC bilayer may be the longest chain PC that requires pressure to induce bilayer interdigitation. The MIP value of the C21PC bilayer in Fig. 3 agreed with our expectation. This signifies that the ease with which the interdigitation is induced is well correlated to the magnitude of cohesive interaction between acyl chains. On the other hand, the MIP value increased with decreasing acyl chain length and the value became ca. 300 MPa for the C14PC bilayer. We have shown [51] from volumetric consideration of pressureinduced interdigitation by using temperature and pressure (MIP) at the triple point that the pressure-induced interdigitation substantially does not occur for the bilayers of CnPC for n < 14. Therefore, we can say that the pressure-induced interdigitation of the CnPC bilayers occurs at limited acyl chain lengths from 14 to 21: only eight kinds of CnPC bilayers can form the structure under high pressure. The decrease in the MIP value with the chain length definitely shows that the bilayer interdigitation is caused by not only the repulsive interaction of the head groups but also the attractive interaction of the acyl chains.

3.3. Thermodynamic quantities of phase transitions The thermodynamic quantities of phase transitions for the present CnPC bilayers under atmospheric pressure are summarized in Table 1 together with those of the C18PC [32] and C22PC bilayers [35]. Here the entropy changes (S) were calculated from the equation of S = H/T using the H and T values from DSC, and the V values were from the Clapeyron equation using the H values and the dT/dp values from the T–p curves of the phase boundary [32]. Reflecting the similar phase behavior of the CnPC bilayers, their dT/dp values were similar depending on the kind of the phase transition: 0.22–0.25 K MPa−1 for subtransition; 0.14–0.18 for pretransition; and 0.23–0.27 K MPa−1 for main transition, although these values were slightly higher than the corresponding values of the bilayers of CnPC for n = 12–17 [32–34,41]. The pre- and maintransition temperatures of the CnPC bilayers increased with an increase in acyl chain length, and the difference between them decreased with the chain length. In contrast with this, the subtransition temperature of the CnPC bilayers seemed to have a maximum at the C19PC bilayer: the subtransition temperature of the C20PC bilayer was lower than that reported previously [31]. Considering that the Lc phase of the C20PC bilayer was not completely formed under the condition of this experiment as mentioned above, the subtransition detected for the C20PC bilayer is not necessarily the transition between stable phases under the condition. If the completely stable Lc phase of the C20PC bilayer is obtained, the subtransition temperature is expected to be higher than that of the C19PC bilayer. The thermodynamic quantities (H, S and V) for the main transition for the CnPC bilayers all increased with increasing the acyl chain length. The H, S and V values of the C22PC bilayer were larger than those expected from the chain-length dependence (refer to Figs. 5B and C). This presumably originated from the fact that the main-transition quantities of the C22PC bilayer were estimated using the L␤ I/L␣ transition rather than the P␤ /L␣ transition because endothermic peaks of the L␤ I/P␤  and the P␤ /L␣ transitions almost entirely overlap each other [35]. In addition, the dT/dp had a slightly different value from that expected from the chain-length dependence. On the other hand, the pretransition quantities were

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Fig. 4. (A) Overlays of temperature–pressure phase diagrams for bilayers of (1) C14PC, (2) C16PC, (3) C18PC, (4) C20PC and (5) C22PC. Open and closed symbols indicate phase transitions between gel phases and main transition, respectively. (B) Pressure dependence of temperature of transition between subgel and lamellar (or ripple) gel phases for CnPC bilayers: (1) C13PC, (2) C14PC, (3) C15PC, (4) C16PC, (5) C17PC, (6) C18PC, (7) C19PC and (8) C20PC.

much smaller than the main-transition quantities and nearly constant irrespective of the acyl chain length. For the subtransition quantities, although the values of the C20PC and C21PC bilayers could not be obtained because of very slow kinetics of the Lc phase formation, those of the C18PC and C19PC bilayers could be determined and they were relatively large (about 60% of the main transition quantities). 3.4. Comparison of bilayer phase transitions among the homologs of CnPC Since we have elucidated the thermotropic and barotropic phase transitions of the CnPC bilayers for n = 12–18 in the previous study [32–34,41], we can compare the acyl chain-length dependence on the phase transitions of the CnPC bilayers for n = 12–22 comprehensively. The phase diagrams of the CnPC bilayers for n = 12–17 are given in Fig. S4, where the diagram of the C12PC bilayer was obtained in 50 wt% ethylene glycol solutions to avoid the freezing of the solvent water and draw the complete diagram including the Lc phase [41]. Here the phase boundary of the Lc phase was added to each diagram to compare the complete phase diagrams containing all phase transitions (sub-, pre-, main transitions and interdigitation) of the CnPCs bilayers. The thermodynamic quantities of the main transition, pretransition and subtransition for the CnPC bilayers for n = 12–18 are given in supplementary Tables 1–3 (Tables S1–S3). Fig. 4A depicts the overlays of the phase boundary curves between the gel and L␣ phases in the T–p phase diagrams of the bilayers of CnPC with acyl chain of even carbon number n = 14, 16, 18, 20, 22 [32–35]. It was found in the diagram that the increases in the phase-transition temperatures by the enhanced cohesive interaction between the acyl chains due to the chain elongation were further reinforced by pressure. Namely the interaction greatly stabilized the gel phases as compared with L␣ phase. It should be noted that the region of the L␤ I phase expanded the most greatly among the three gel phases with the chain elongation and almost all the gel-phase region became the L␤ I-phase region for the C22PC bilayer because of the disappearance of the L␤ phase and the extreme shrinkage of the P␤ -phase region [refer to Fig. S3]. The packing of the L␤ I phase is the densest and thickest of the gel phases because the molar volume of the CnPC in the bilayer increases in the order of the L␤ I, L␤ and P␤ phases [16]. Thus, the L␤ I phase has the most compact packing of all gel phases, which is consistent with the tendency for pressure to promote the formation of the L␤ I phase. The enhanced cohesive interaction between the PC molecules with an

increase in acyl chain length makes the bilayer packing tighter in that order and further reduces the free energy of the L␤ I phase, as a result, the L␤ I-phase region is also extended with the chain length. Supplementary Fig. S4 and Tables S1–S3 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.colsurfb.2015.02.036. The overlays of the subtransition curves of the bilayers of CnPC for n = 13–20, which were taken up from the respective T–p phase diagrams [33,34,41], are shown in Fig. 4B. Here the subtransitions of the C13PC bilayer at all pressures and C14PC bilayer at low pressures are the Lc /P␤ transition while that of the C14PC bilayer at high pressures and those of the rest of the CnPC bilayers are the Lc /P␤ transition. The dT/dp values of the subtransition for the C13PC and C14PC bilayers were somewhat smaller than those for the other CnPC bilayers, which may originate from the fact that the transitions are different. Moreover, the subtransition temperature under atmospheric pressure and dT/dp value of the C20PC bilayer were, respectively, lower and higher than those of the C19PC bilayer. This is because the C20PC bilayer did not completely transform into the stable Lc phase, as mentioned above. With increasing acyl chain length, the subtransition curve shifted to higher temperature and the range of the Lc phase was gradually extended, indicating that enhancement of the cohesive interaction between the acyl chains increases the stability of the Lc phase. Compared with the temperature shift of phase boundary curves between the gel and L␣ phases resulting from an increase of chain length in Fig. 4A, the shift of the subtransition curve by the chain elongation is much smaller (Fig. 4B) and, as a result, the phase behavior of the C14PC and C15PC bilayers [33] becomes complicated as seen in Fig. S4: the subtransition curve intersects the phase boundary between the gel phases at two points. Since the gap between both phase boundary curves is increased with the chain elongation, the phase behavior of the bilayers of CnPC for n > 16 resembles one another. Three thermodynamic properties of the CnPC bilayers for n = 12–22 under atmospheric pressure, namely transition temperature, H and V, are plotted against n in Fig. 5 [32–35,41]. Again the values of the C12PC bilayer were obtained in 50 wt% ethylene glycol solutions. The three kinds of transition temperatures except the subtransition temperature of the C20PC bilayer increased with increasing acyl chain length in a non-linear manner, and the oddeven effect was not seen in the acyl chain-length dependence (Fig. 5A). The pre- and main-transition temperatures increased more significantly with the chain length than the subtransition temperature. The difference in temperature between pre- and main

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Fig. 5. (A) Phase-transition temperatures, (B) enthalpy change (H) and (C) volume change (V) of CnPC bilayers as a function of carbon number of acyl chain: (open circle) main transition, (open triangle) pretransition, (closed circle) subtransition. Closed square refers to H value for Lc /Lx transition of C12PC bilayer.

transitions reduced with the chain length and became almost zero at the C22PC bilayer, and at the same time, the pretransition converted from the L␤ /P␤ transition to the L␤ I/P␤ transition. On the other hand, the order of the sub- and pretransition temperatures was reversed between the C14PC and C15PC bilayers, suggesting that the L␤ phase of CnPC bilayer for n ≤ 14 under atmospheric pressure becomes the metastable gel phase. Furthermore, the order of the sub- and main-transition temperatures was also reversed between the C12PC and C13PC bilayers. This fact means that the CnPC bilayers for n ≥ 13 undergo the main transition from the gel (L␤ or P␤ ) phase to the L␣ phase while the C12PC bilayer undergoes the transition from the Lc phase to the L␣ phase directly: the gel phase itself is the metastable phase. However, the Lc /L␣ transition does not occur in the C12PC bilayer because a new phase called Lx phase, which has intermediate properties of the gel and L␣ phases [41], appears. Then, the Lc /Lx transition is observed instead of the Lc /L␣ transition for the C12PC bilayer. This transition with a large H value (closed square in Fig. 5B) can be clearly observed at a higher temperature than the main transition in the 50 wt% ethylene glycol solutions by DSC [41]. In the case of the bilayers of CnPC for n < 12, the main transition itself does not occur as described below. The main transition corresponds to the chain melting of the acyl chains. Hence, the thermodynamic quantities increase with increasing acyl chain length like the melting temperature and heat of long-chain compounds. The H and V values of the main transition for all CnPC bilayers increased with acyl chain length in a non-linear manner (Fig. 5B and C). In our previous study [32], we have considered the chain-length dependence of H and V for the CnPC bilayers by using the perturbation parameter introduced by Mason and Huang [52] for a CnPC molecule. The parameter is defined by taking account of the bilayer interface region and conformationally inequivalent terminal ends of the fatty acyl chains.

We also applied the parameter to the present bilayers. The resulting H and V versus perturbation parameter plots are given in Fig. S5. An almost linear correlation between the perturbation parameter and the H or V values was obtained for acyl chain length from 12 to 21. Therefore, we can say that the hydrophobic contribution of a methylene group in the parameter to the quantities of the main transition is nearly equivalent among the CnPC bilayers. Further, it is evident from the plots that the bilayers of CnPC for n ≤ 11 do not undergo the main transition, which suggests that there appears no gel-phase region in the diagram for CnPCs with shorter chains (n ≤ 11). Supplementary Fig. S5 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.02.036. By contrast, no acyl chain-length dependence was found in the H and V values of the pretransition for all CnPC bilayers, and they were considerably smaller. The L␤ /P␤ transition corresponds to the conversion from the flat bilayer to the undulated bilayer due to the change in packing of the CnPC molecules with all-trans conformations maintained. Their small and almost constant values irrespective of the acyl chain length indicate that the pretransition occurs with a smaller energy change, which is not influenced by the chain length. Regarding the H and V values of the subtransition, both values were relatively large (about 60% of those for the main transition) and definitely dependent on the acyl chain length, although those of the C20PC were small due to the incomplete Lc phase formation as mentioned above. It has been considered that the subtransition from the Lc phase to the gel (L␤ or P␤ ) phase is closely related to the hydration change among the polar head group of the CnPC molecules. Kodama and Aoki [53] have shown from the detailed analysis of ice-melting peaks of the C16PC bilayer dispersions by DSC that the ratio of nonfreezable to freezable water molecules in interlamellar water changes at the subtransition. On

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the other hand, considerably large values and the acyl chain-length dependence of the H and V values cannot be explained by only the hydration change near the head group. Recently, we have found [54] that bilayers of some asymmetric PCs with different linear acyl chains in length are suitable for a study of the Lc phase formation because they form the Lc phase in a much shorter time than CnPCs, and because the formation is further accelerated by the application of pressure. Our previous high-pressure fluorometric study on a myristoylpalmitoylphosphatidylcholine bilayer suggested that the asymmetric PC molecules in the Lc phase are probably arranged in staggered structure where polar head groups of the molecules are alternately protruding with respect to the membrane surface. Considering the similarity of the bilayer phase behavior between asymmetric PCs and CnPCs [33,34], it is highly probable that the CnPC molecules also form the staggered structure in the Lc phase. PC is an intramolecularly neutral lipid with a positive charge of a nitrogen atom in the choline group and a negative charge in the phosphate group. Then, considerably large thermodynamic quantities of the subtransition are understandable by taking into account the electrostatic interactions among the PC molecules in the Lc phase with the enhanced cohesive interaction of the molecules [29]. Furthermore, the stronger cohesive interaction between acyl chains hampers the formation of the staggered molecular arrangement in the Lc phase, which may bring about the extremely slow transformation from the L␤ phase to the Lc phase in proportion to the acyl chain length of CnPC. 4. Conclusions The temperature- and pressure-induced phase transitions of the bilayers of CnPC with longer acyl chains, the subtransitions of which are difficult to observe, were investigated. The pre- and main transitions and interdigitation were observed for all bilayers. However, the subtransition was only observed for the annealed lipid sample of the C19PC bilayer (perfectly) and the C20PC bilayer (imperfectly) while it was not observed for that of the C21PC bilayer. By combining the T–p phase diagrams constructed for these bilayers and those obtained for a series of CnPC bilayers in the previous results together with the subtransition data, the complete phase diagrams of the CnPC (n = 12–22) bilayers, that is, the temperature- and pressure-dependent phase states, phase sequence and phase stability of the CnPC bilayers as a function of the hydrophobicity of the molecules were elucidated. It was found that the phase sequence and stability with respect to temperature and pressure markedly changes at the acyl chain length between 12 and 15 in contrast with the fact that the long-chain PC bilayers have almost the same phase sequence. Furthermore, it was revealed that the pressure-induced interdigitation occurs only in the bilayers of CnPCs for n = 14–21. From the comprehensive comparison of thermodynamic quantities, we concluded that the subtransition is an acyl chain-length dependent transition with slow kinetics, the pretransition is an acyl chain-length independent transition with fast kinetics and the main transition is an acyl chain-length dependent transition with fast kinetics, respectively. The interaction between CnPC molecules in the bilayer was also deduced from the systematic consideration on the thermodynamic quantities of three phase transitions. Acknowledgments This study was supported in part by a Grant-in Aid for Scientific Research (C) (24550157 and 26410016) from Japan Society for the Promotion of Science (JSPS). Authors wish to thank the professor emeritus Shoji Kaneshina at The University of Tokushima for his helpful comments and suggestions through the phase-transition research of the CnPC bilayers.

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