58 Vol. 62, No. 1Chem. Pharm. Bull. 62(1) 58–63 (2014)

© 2014 The Pharmaceutical Society of Japan

Thermotropic Phase Behavior of Hydrogenated Soybean Phosphatidylcholine–Cholesterol Binary Liposome MembraneHiroki Kitayama,a Yuki Takechi,a Nobutake Tamai,b Hitoshi Matsuki,b Chikako Yomota,c and Hiroyuki Saito*,a

a Institute of Health Biosciences and Graduate School of Pharmaceutical Sciences, The University of Tokushima; 1–78–1 Shomachi, Tokushima 770–8505, Japan: b Department of Life System, Institute of Technology and Science, The University of Tokushima; 2–1 Minamijosanjima-cho, Tokushima 770–8506, Japan: and c National Institute of Health Sciences; 1–18–1 Kamiyoga, Setagaya-ku, Tokyo 158–8501, Japan.Received July 26, 2013; accepted October 9, 2013

By combination of differential scanning calorimetry (DSC) and fluorescence spectroscopy of 6-propi-onyl-2-(dimethylamino)naphthalene (Prodan), we elucidated the thermotropic phase behavior of hydroge-nated soybean phosphatidylcholine (HSPC)–cholesterol binary liposome membrane which has similar lipid composition to Doxil®, the widely used liposome product in treatment of various tumors. We found that the characteristic points at cholesterol mole fraction (Xch)=0.023 and 0.077 correspond to the hexagonal lat-tice, in which cholesterol molecules are considered to be regularly distributed in all regions of HSPC lipid bilayer with 1 : 42 and 1 : 12 units, respectively, as static averaged structures. Apparent endothermic peak disappeared at Xch=0.40 in the DSC thermograms, indicating the existence of single liquid ordered phase at Xch>0.40. In addition, fluorescence measurements of Prodan and its lauroyl derivative in poly(ethylene gly-col) (PEG)-modified liposomes indicated that PEG modification has a negligible effect on the phase behavior of HSPC–cholesterol binary liposome membrane. These results may provide useful information in developing novel liposome products whose stability and encapsulated drug release are controlled.

Key words liposome; cholesterol; phase diagram; differential scanning calorimetry; Prodan

Liposomes are used as drug delivery systems because of their high biocompatibility and ability to encapsulate a large amount of pharmaceuticals inside the vesicle.1) Thermody-namic states of liposome membrane are considered as an important factor in terms of particle stability and encapsulated drug release. Therefore, phase transition temperatures and phase states of lipid membranes are important physicochemi-cal index in assessing the liposomal product.

Liposomes are mainly composed of phospholipids in the la-mellar phase such as dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and hydrogenated soybean phosphatidylcholine (HSPC). Because all these lipids have saturated fatty acids, they have relatively high phase transition temperatures2) and therefore, are used in prepara-tion of stable liposomes. The commercial liposomal products such as Doxil® and AmBisome® are composed of HSPC and cholesterol in which the incorporation of cholesterol into the lipid bilayer modulates the liposome membrane fluidity.3,4) Poly(ethylene glycol) (PEG)-lipid is also a fundamental com-ponent in Doxil® to extend the circulation time in plasma. In theory, HSPC and cholesterol can be either domain segregat-ed, randomly distributed, or regularly distributed depending upon the energy function.5) However, the relationship between the phase states of HSPC–cholesterol liposome membrane and lateral distribution of cholesterol in the membrane has not been fully elucidated. Optimal modulation of miscibility be-tween lipid molecules is of fundamental importance to control drug release from liposomes.6)

The phase diagram for lipid mixtures shows several differ-ent regions of phase coexistence,7) including lamellar gel (Lβ or Lβ′), ripple gel (Pβ′), liquid crystalline (Lα) and liquid-ordered (Lo) phases. At the pretransition, the flat Lβ′ phase, where acyl

chains of phospholipid molecule in all-trans conformation are tilted at about 30° to the membrane normal, changes to the wavy Pβ′ phase.8–10) At the main transition, the Pβ′ phase changes to the fluid-like Lα phase, where the acyl chains are disordered due to high degree of trans–gauche isomerization and do not show any tilt.11) The Lo phase, which is character-ized by high conformational order in the hydrocarbon chains of phospholipid like the gel state and relatively fast lateral diffusion, appears when cholesterol content in the membrane increases.12–14) Identification and characterization of respective phases and their location in the compositional diagram are im-portant to understand the behavior of lipid mixtures.

In the present study, we examined the thermotropic phase behavior of HSPC–cholesterol binary liposome mem-brane by high-sensitivity differential scanning calorimetry (DSC) and fluorescence spectroscopy using 6-propionyl-2-(dimethylamino) naphthalene (Prodan). Prodan molecule is lo-cated in the surface region of lipid membrane, providing criti-cal information about the microscopic change near the hydro-philic surface of the membrane.15) In terms of interpretation of the phase behavior, we explained the lateral distribution of cholesterol molecules in the binary liposome membrane based on a modified superlattice model.16) We also investigated the effect of PEG modification on the phase diagram of the HSPC–cholesterol binary liposome membrane.

ExperimentalMaterials HSPC was kindly provided from NOF cor-

poration (Tokyo, Japan). Cholesterol was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). N-(Carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG2000) was obtained from NOF corporation. Prodan and 6-lauroyl-2-(dimethylamino)-naphthalene (Laurdan) were from Life Technologies (Carlsbad,

Regular Article

* To whom correspondence should be addressed. e-mail: hsaito@tokushima-u.ac.jp

The authors declare no conflict of interest.

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CA, U.S.A.).Liposome Preparation For multilamellar vesicle (MLV)

preparation used in DSC measurements, the mixture of HSPC and cholesterol was dissolved in a chloroform–methanol mix-ture (2 : 1, v/v) and dried to a thin film under a stream of ni-trogen gas, followed by incubation overnight under vacuum to remove residual solvent. The thin lipid film was hydrated with 10 mM Tris–HCl buffer (pH 7.4) in a water bath above 60°C. In fluorescence measurements, the mixture of HSPC and cho-lesterol containing 0.2 mol% of Prodan or 1 mol% of Laurdan was used for liposome preparation and the liposome suspen-sion was passed through a Mini-extruder (Avanti, Alabaster, AL, U.S.A.) equipped with 0.2 µm polycarbonate filter. For PEG-modified liposome, the mixture of HSPC, cholesterol, and DSPE-PEG2000 was premixed in a chloroform–metha-nol solution and PEG-liposome was prepared similarly as described above. The phospholipid concentration was deter-mined by the Bartlett method.17)

DSC Measurements DSC measurements were carried out by a high-sensitivity differential scanning calorimeter MicroCal MCS DSC. The heating rate was 45°C/h and the temperature range was from 30 to 70°C. The midpoint tem-

perature and the enthalpy associated with phase transitions were determined from endothermic peaks in the thermograms using MicroCal ORIGIN software.

Fluorescence Studies All fluorescence measurements were performed using a Hitachi F-4500 fluorescence spec-trophotometer. The excitation wavelengths for Prodan and Laurdan were 361 and 350 nm, respectively, and the emission spectra were recorded from 370 to 600 nm. The general-ized polarization (GP) values for Prodan and Laurdan were calculated from the emission intensities using the equation GP=(IB−IR)/(IB+IR), where IB and IR are the emission intensi-ties at the blue (440 nm) and red (490 nm) edges of the emis-sion spectrum, respectively.18)

ResultsDSC Measurements for HSPC–Cholesterol Binary Lipo-

some Membrane Figure 1A shows DSC thermograms of HSPC–cholesterol liposome membrane at different Xch. The thermograms in the vicinity of the pretransition temperature are magnified in Fig. 1B. HSPC liposome exhibited two endo-thermic peaks corresponding to the pre- and main transitions at 47.8°C and 53.6°C, respectively. The endothermic peak of the main transition became smaller and broader with increas-ing Xch, and no endothermic peak was observed at Xch=0.40 (Fig. 1A). The pretransition peak also became smaller with increasing Xch and disappeared at Xch=0.03 (Fig. 1B).

Fig. 1. (A) DSC Thermograms of HSPC–Cholesterol Liposome Mem-brane at Different Xch from 0 to 0.40　Xch=0 (1), 0.05 (2), 0.15 (3), 0.25 (4), 0.35 (5), 0.40 (6)　(B) DSC Thermograms in the Vicinity of the Pretransition Temperature Xch=0 (1), 0.01 (2), 0.02 (3), 0.03 (4)　(C) Enthalpy Change of HSPC–Cholesterol Liposome Membrane as a Func-tion of Xch for the Main Transition Peak

Fig. 2. (A) Fluorescence Spectra of Prodan in HSPC Liposome Mem-brane (Xch=0) from 38 to 64°C　(B) Temperature Dependence of Wave-length at Maximum Fluorescence Intensity and GP Value of Prodan for HSPC Liposome Membrane

Prodan concentration was 2 µM.

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In Fig. 1C, the enthalpy change (ΔH) for the main transition is plotted as a function of Xch. The ΔH values decreased lin-early with increasing Xch and reached zero at about Xch=0.40. The ΔH is attributable to the required energy for the trans–gauche conformational change of the hydrocarbon chains of phospholipids (i.e., chain melting) in the liposome membrane. The incorporation of cholesterol into the gel phase increases the fluidity of liposome membrane, resulting in the decrease in ΔH of the main transition.19) Taking into consideration that en-dothermic peak disappears at about Xch=0.40, it is suggested that all HSPC molecules are conformationally restrained and form the Lo phase at Xch>0.40. As for the pretransition, the ΔH values became zero at Xch>ca. 0.02 (Fig. 1B), correspond-ing to the conversion of both Lβ′ and Pβ′ phases to the Lβ phase.

Prodan Fluorescence Measurements for HSPC Liposome Membrane Figure 2A shows the 3-D image of Prodan emis-sion spectra in HSPC liposome membrane at temperatures from 38 to 64°C. It is known that the wavelength of fluo-rescence maximum (λmax) or GP value of Prodan varies de-pending on the dielectric constant of the solvent. The dipole moment increases upon excitation and causes reorientation of the solvent water. The solvent reorientation decreases the excited state energy of Prodan molecule, which is reflected in a continuous red shift of the steady-state fluorescence spec-trum.20–22) Therefore, the shift of λmax or change in GP value of Prodan in HSPC liposome membrane with increasing tem-perature shown in Fig. 2A are attributed to the change in the polarity around Prodan molecules.

Figure 2B shows the changes in λmax or GP value of Prodan in HSPC liposome with temperature, which were constructed on the basis of the spectra shown in Fig. 2A. The λmax values of 440 and 490 nm correspond to the gel and liquid crystal-line phases of liposome membrane, respectively.15) A two-step change in λmax with increasing temperature was seen: the first step is from ca. 440 to ca. 470 nm at 45−47°C and the sec-ond from ca. 470 to ca. 490 nm at 50−53°C. Such a two-step change was also seen in the GP value and is expected to result from the change in the vertical location of Prodan molecules with the thermotropic phase transition of liposome membrane; that is, the pretransition from the Lβ′ to the Pβ′ phase and the main transition from the Pβ′ to the Lα phase.23) We found that the lower and higher temperatures for abrupt changes in λmax or GP value of Prodan are close to those in DSC thermograms showing the pre- and main transitions at 47.8°C and 53.6°C, respectively (Fig. 1A). These results indicate that the phase state of the liposome membrane can be detected by the change in λmax or GP value of Prodan embedded in the membrane surface.

Prodan Fluorescence Measurements for HSPC–Choles-terol Binary Liposome Membrane Figure 3A shows the temperature dependence of Prodan λmax for HSPC–cholesterol binary liposome membrane. The pretransition became obscure at Xch=0.05 and the stepwise shift of λmax disappeared at Xch=0.30. During this range of Xch, the continuous increase in temperature for abrupt change in λmax was observed with increasing Xch. For example, at Xch=0.25, at least about 20°C rise in temperature was needed for the λmax value to reach the plateau region at 490 nm from 440 nm. At Xch≥0.30, no significant increase in λmax was observed (data not shown). In contrast, GP values for HSPC–cholesterol binary liposome membrane gradually decreased with increasing temperature (Fig. 3B). Interestingly, the temperature at which the gradual decrease in the GP value starts is almost constant independent of Xch, and close to the transition temperature observed in the DSC measurements (Fig. 1A).

Phase Diagram of HSPC–Cholesterol Binary Liposome Membrane In Fig. 4, the phase transition temperatures determined from DSC measurements (closed circle) and the temperatures at which Prodan λmax reaches about 490 nm (open square) indicative of the completion of the transition to

Fig. 3. Temperature Dependence of Wavelength at Maximum Fluores-cence Intensity (A) and GP Value (B) of Prodan for HSPC–Cholesterol Binary Liposome Membranes at Different Xch from 0.05 to 0.30

Prodan concentration was 2 µM.

Fig. 4. Phase Diagram for HSPC–Cholesterol Binary Liposome Mem-brane. Open and Closed Symbols Designate the Results from Prodan Fluorescence and DSC Measurements, Respectively

(I) Lα, (II) Lα+Pβ′, (III) Lβ (1 : 42)+Pβ′, (IV) Lβ (1 : 42)+Lβ′, (V) Lα+Lβ (1 : 12), (VI) Lβ (1 : 42)+Lβ (1 : 12), (VII) Lα+Lβ (1 : 12), (VIII) Lα+Lo, (IX) Lβ (1 : 12)+Lo, (X) Lo. See Results section for details.

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the Lα phase are plotted as a function of Xch to construct the temperature–Xch phase diagram for HSPC–cholesterol binary liposome membrane. As seen from the shape of the phase diagram, the range between both main-transition temperatures determined from the DSC measurements and the Prodan λmax behavior arises from the two-phase coexistence of the gel and Lα phases or the Lo and Lα phases. Note that we use a term of Lα phase as a synonym of liquid disordered (Ld) phase, which is generally used for representing the phase state of binary or ternary membranes containing cholesterol.

We assigned the phase states for each area in the phase diagram (Fig. 4) on the basis of the above consideration and also thermodynamics of solid–liquid equilibria as follows: (I) Lα, (II) Lα+Pβ′, (III) Lβ (1 : 42)+Pβ′, (IV) Lβ (1 : 42)+Lβ′, (V) Lα+Lβ (1 : 12), (VI) Lβ (1 : 42)+Lβ (1 : 12), (VII) Lα+Lβ (1 : 12), (VIII) Lα+Lo, (IX) Lβ (1 : 12)+Lo, (X) Lo. The ex-planation on the phase assignment is given in the following section. In these assignments, Lβ (1 : 42) phase is a gel phase composed of a single type of unit which includes one choles-terol molecule and 42 surrounding HSPC molecules within the next–next–next nearest neighbor sites (Fig. 5A). Lβ (1 : 12) phase is similarly depicted as one cholesterol molecule and 12 surrounding HSPC molecules within the next–next nearest neighbor sites (Fig. 5B). In these figures, an open and a closed circles on each lattice point represent an HSPC (i.e., DSPC or DPPC) and a cholesterol molecules, respectively. Here, we should note that these schematic illustrations are given only to explain the range of cholesterol effects presumed as static averaged structures from bulk thermodynamic properties in terms of the formation of several types of complexes with dif-ferent stoichiometries, and not to claim that rigid and highly ordered structures are formed within the HSPC–cholesterol bilayer depending on Xch. The phase diagram indicates that four kinds of phase separation can occur in the membranes below the main transition temperature and at Xch<0.40 (i.e.,

areas III, IV, VI, IX in Fig. 4). This means that the respective phase-separation regions differ in the combination of coexist-ing two phases depending upon Xch. For example, at Xch=0.05 in area VI, the present liposome membrane consists of the two different types of regions, namely Lβ (1 : 42)-phase region and Lβ (1 : 12)-phase region.

Effect of PEG Modification on Prodan and Laurdan Fluorescence in Liposomes To investigate the effect of PEG modification on the phase diagram of HSPC–cholesterol bina-ry liposome membrane, the fluorescence measurements using Prodan were conducted for PEG-modified liposomes contain-ing 5.6 mol% PEG-lipid. This composition is similar to that in Doxil®. The temperatures at which the λmax of Prodan in PEG-modified liposomes or unmodified liposomes begins to rise from about 440 nm and reaches about 490 nm are shown in Table 1. The temperature dependence of Prodan λmax for PEG-liposomes is similar to that for unmodified liposomes, indicating that the PEG modification has an insignificant effect on the phase behavior of HSPC–cholesterol binary lipo-some membrane.

Laurdan, a fluorescent probe with the same fluorophore as Prodan, is similarly sensitive to the polarity of its environ-ment. These two probes differ only in the length of their acyl residue: Laurdan has a lauroyl chain, composed of 12 carbon atoms, while Prodan has a propionyl chain. Thus, as compared to Prodan, Laurdan is tightly anchored in the hydrophobic core by the cooperative van der Waals interactions between the lauroyl chain and lipid hydrocarbon chains. Thus, the GP values for each probe allow a quantitative estimation of local membrane polarity, related to local membrane fluidity.18,24) As shown in Fig. 6A, the GP values for Prodan in the PEG-modified liposomes were significantly lower than those for unmodified liposomes at all temperatures, suggesting that the incorporation of PEG-lipids into the liposome membranes increases the membrane fluidity of the hydrophilic region in

Fig. 5. Schematic Illustration for Regular Distribution of Cholesterol in Binary Liposome Membrane Based on the Hexagonal Lattice ModelAn open and a closed circles represent HSPC (DSPC or DPPC) and cholesterol molecules, respectively. A fundamental unit is indicated as hexagonal area around cho-

lesterol molecule in each panel. (A) Unit (1 : 42) includes 42 phospholipid molecules in Lβ (1 : 42) state (Xch=0.023); (B) unit (1 : 12) includes 12 phospholipid molecules in Lβ (1 : 12) state (Xch=0.077).

Table 1. Effects of PEG Modification on the Transition Temperature of Onset (ca. 440 nm) and Completion (ca. 490 nm) in Prodan Fluorescence

Xch

DSPE-PEG2000 0 mol% DSPE-PEG2000 5.6 mol%

Onset temperature (°C) Completion temperature (°C) Onset temperature (°C) Completion temperature (°C)

0.06 50.5 54.6 50.0 53.50.10 50.1 57.9 48.5 57.30.25 54.3 68.4 54.7 66.5

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which Prodan is located. In contrast, it is only above the main transition temperature that the GP values for Laurdan in the PEG-liposomes were lower than those in the unmodified lipo-somes (Fig. 6B). This result is consistent with the hypothesis that a part of PEG-chain inserts into the fluid-phase phospho-lipid bilayers.25)

DiscussionIn this study, we elucidated the thermotropic phase behavior

of HSPC–cholesterol binary liposome membrane by combina-tion of the Prodan fluorescence and DSC measurements. In addition, by use of the superlattice model,16) we theoretically explained the interaction with HSPC and cholesterol mol-ecules in gel phases.

We constructed the comprehensive phase diagram of HSPC–cholesterol binary liposome membrane (Fig. 4) by plot-ting the phase transition temperature from the DSC thermo-grams (Fig. 1) as well as the temperature at which the Prodan λmax reaches about 490 nm (Figs. 2B, 3A) as a function of Xch. The resultant phase diagram resembles that of the DSPC–cholesterol binary liposome membrane.26) HSPC primarily contains 18 : 0-PC (approx. 85% DSPC and 15% DPPC). One pretransition and one main transition were observed for the HSPC liposome, and these phase transitions occurred at slightly lower temperatures compared to those for the DSPC liposome. This indicates that DSPC and DPPC molecules are homogeneously distributed in the HSPC liposome membrane, which is consistent with the previous study which demon-strated that the main transition temperature of the liposomes composed of DPPC and HSPC can be controlled by the molar ratio of the two phospholipids.27) At Xch=0.40, which is a cho-

lesterol composition similar to that in Doxil®, no endothermic peak was observed in DSC thermograms (Fig. 1A), indicating that only the single Lo phase exists at Xch>0.40. This agrees with the fact that Doxil® is thermally stable liposome com-posed of the Lo phase. In contrast, the appearance of the single Lo phase in the DPPC–cholesterol and the DSPC–cholesterol binary liposome membranes was observed at Xch>0.30 and 0.50, respectively.26,28) These results indicate that the requisite cholesterol contents for the appearance of the single Lo phase differs depending upon the sort of the constituent phospho-lipid.

The phase diagram of the HSPC–cholesterol bilayer mem-brane shown in Fig. 4 may seem to be complicated but is very similar to a typical solid–liquid phase diagram for a eutectic binary mixture which exhibits congruent melting behavior (e.g. benzene–aniline binary mixture). In general, the presence of a congruent melting point indicates that the two compo-nents react to produce a new solid compound with a specific stoichiometry, and its stoichiometry well corresponds to the composition of the congruent melting point. Taking this into consideration, the fact that the congruent melting point is ob-served at Xch=0.077 for the HSPC–cholesterol bilayer suggests that a cholesterol molecule interacts with 12 HSPC molecules around the cholesterol molecule to form a 1 : 12-complex of cholesterol and HSPCs (Fig. 5B). This is also explained rea-sonably by the superlattice view proposed by Somerharju et al.16) That is, when the bilayer surface is entirely occupied with the units (1 : 12) composed of one cholesterol molecule and 12 surrounding HSPC molecules within the next–next nearest neighbor sites, the Xch value equals to 0.077 (Fig. 5B). This Xch value is close to the composition of the congruent melting point on the phase diagram (Fig. 4). Similarly, another characteristic composition of Xch ca. 0.02 corresponding to the composition at which the pretransition is abolished (Fig. 1B), can be well explained by the unit (1 : 42) in terms of the super-lattice view, as shown in Fig. 5A. The abolition of the pretran-sition is generally thought to be attributed to the induction of an Lβ-like molecular orientation by the presence of cholesterol, in which phospholipid molecules are arranged almost paral-lel to the bilayer normal. Therefore, this cholesterol effect of inducing such non-tilted molecular orientation is presumed to prevail among 42 HSPC molecules around a cholesterol mol-ecule on average. Finally, the phase assignment as described above was derived from the consideration on the identifica-tion of each phase state, including Lo phase, and from the general thermodynamic knowledge on the solid–liquid phase equilibria for a eutectic mixture exhibiting congruent melting behavior. For example, it is understandable that all the looped areas above the eutectic temperature on the phase diagram correspond to the two-phase regions composed of a solid and a liquid phase, and also that all the solid-phase regions below the eutectic temperature are basically two-phase regions made of two different types of solids, because all the components including a new compound composed of the two components of the mixture are immiscible in a solid state.

As shown in Table 1, the PEG modification has no effect on the Prodan λmax, indicating that the thermotropic phase be-havior of the PEG-liposome membrane is similar to that of the unmodified liposome membrane. However, recent molecular dynamics simulations and Langmuir monolayer measurements suggested that PEG-lipids may insert their polyether moiety

Fig. 6. (A) Temperature Dependence of Prodan GP Values for PEG-Modified Liposomes and Unmodified Liposomes　(B) Temperature Dependence of Laurdan GP Values for PEG-Modified Liposomes and Unmodified Liposomes

Prodan and laurdan concentrations were 2 µM and 1 µM, respectively.

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into the fluid phase phospholipid bilayers.25) In this regard, the quantitative assessment of liposome membrane fluidity by using the GP values for Prodan (Fig. 6A) indicates that the hydrophilic region in liposome membrane appears to be somewhat perturbed by incorporated PEG-lipids. In addition, Fig. 6B suggests that some of PEG polymers interact with the glycerol backbone region of the liposome membrane above the main transition temperature, which is consistent with the finding that the PEG chain triggers the release of drug in lipo-somal lumen above the transition temperature.29)

In summary, the phase diagram of the HSPC–cholesterol binary liposome membrane was successfully obtained by com-bination of the high-sensitivity DSC and Prodan fluorescence measurements. In the phase diagram, the lipid miscibility and the characteristic compositions were explained by the hex-agonal lattice model. The present results may provide useful information on the development of novel liposome products whose stability and encapsulated drug release are controlled.

Acknowledgment This work was supported in part by Health Labour Sciences Research Grant from the Ministry of Health, Labour and Welfare of Japan.

References 1) Samad A., Sultana Y., Aqil M., Curr. Drug Deliv., 4, 297–305

(2007). 2) Huang C., Li S., Biochim. Biophys. Acta, 1422, 273–307 (1999). 3) Demel R. A., De Kruyff B., Biochim. Biophys. Acta, 457, 109–132

(1976). 4) Ladbrooke B. D., Williams R. M., Chapman D., Biochim. Biophys.

Acta, 150, 333–340 (1968). 5) Chong P. L., Zhu W., Venegas B., Biochim. Biophys. Acta, 1788,

2–11 (2009). 6) Karve S., Bandekar A., Ali M. R., Sofou S., Biomaterials, 31,

4409–4416 (2010). 7) Vist M. R., Davis J. H., Biochemistry, 29, 451–464 (1990). 8) Janiak M. J., Small D. M., Shipley G. G., Biochemistry, 15, 4575–

4580 (1976). 9) Smith G. S., Sirota E. B., Safinya C. R., Clark N. A., Phys. Rev.

Lett., 60, 813–816 (1988).10) Wack D. C., Webb W. W., Phys. Rev. A, 40, 1627–1636 (1989).11) Petrache H. I., Dodd S. W., Brown M. F., Biophys. J., 79, 3172–3192

(2000).12) Thewalt J. L., Bloom M., Biophys. J., 63, 1176–1181 (1992).13) Nielsen M., Miao L., Ipsen J. H., Zuckermann M. J., Mouritsen O.

G., Phys. Rev. E Stat. Phys. Plasmas Fluids Relat. Interdiscip. Top-ics, 59 (5 Pt B), 5790–5803 (1999).

14) Ohvo-Rekilä H., Ramstedt B., Leppimaki P., Slotte J. P., Prog. Lipid Res., 41, 66–97 (2002).

15) Krasnowska E. K., Gratton E., Parasassi T., Biophys. J., 74, 1984–1993 (1998).

16) Somerharju P., Virtanen J. A., Cheng K. H., Biochim. Biophys. Acta, 1440, 32–48 (1999).

17) Bartlett G. R., J. Biol. Chem., 234, 466–468 (1959).18) Parasassi T., De Stasio G., d'Ubaldo A., Gratton E., Biophys. J., 57,

1179–1186 (1990).19) Hinz H. J., Sturtevant J. M., J. Biol. Chem., 247, 3697–3700 (1972).20) Chong P. L., Biochemistry, 27, 399–404 (1988).21) Zeng J. W., Chong P. L., Biochemistry, 30, 9485–9491 (1991).22) Bondar O. P., Rowe E. S., Biophys. J., 71, 1440–1449 (1996).23) Krasnowska E. K., Bagatolli L. A., Gratton E., Parasassi T., Bio-

chim. Biophys. Acta, 1511, 330–340 (2001).24) Parasassi T., Giusti A. M., Gratton E., Monaco E., Raimondi M.,

Ravagnan G., Sapora O., Int. J. Radiat. Biol., 65, 329–334 (1994).25) Stepniewski M., Pasenkiewicz-Gierula M., Rog T., Danne R., Or-

lowski A., Karttunen M., Urtti A., Yliperttula M., Vuorimaa E., Bunker A., Langmuir, 27, 7788–7798 (2011).

26) Tamai N., Uemura M., Takeichi T., Goto M., Matsuki H., Kaneshina S., Biophys. Chem., 135, 95–101 (2008).

27) Chen J., Cheng D., Li J., Wang Y., Guo J. X., Chen Z. P., Cai B. C., Yang T., Drug Dev. Ind. Pharm., 39, 197–204 (2013).

28) Tamai N., Uemura M., Goto M., Matsuki H., Kaneshina S., Colloids Surf. B Biointerfaces, 65, 213–219 (2008).

29) Li L., ten Hagen T. L., Schipper D., Wijnberg T. M., van Rhoon G. C., Eggermont A. M., Lindner L. H., Koning G. A., J. Controlled Release, 143, 274–279 (2010).