Behavior of Self-Organized Molecular Assemblies Composed of

Sep 13, 2000 - Composed of Phosphatidylcholines and Synthetic. Triple-Chain Amphiphiles in Water. Yasushi Sumida,† Araki Masuyama,‡ Mayuko Takasu,...
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Langmuir 2000, 16, 8005-8009

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Behavior of Self-Organized Molecular Assemblies Composed of Phosphatidylcholines and Synthetic Triple-Chain Amphiphiles in Water Yasushi Sumida,† Araki Masuyama,‡ Mayuko Takasu,‡ Toshiyuki Kida,‡ Yohji Nakatsuji,‡ Isao Ikeda,*,‡ and Masatomo Nojima‡ Cosmetic Laboratory, Kanebo Corporation, Kotobuki-cho 5-3-28, Odawara, Kanagawa 250-0002, Japan, and Department of Applied Chemistry, Faculty of Engineering, Osaka University, Yamadaoka 2-1, Suita, Osaka 565-0871, Japan Received May 19, 2000. In Final Form: July 6, 2000 The enhancing effect of synthetic triple-chain amphiphiles on the stability of a vesicle made from phosphatidylcholine was investigated in terms of the leakage of the inner marker. The addition of amphiphiles bearing three hydrophobic chains and two carboxylate groups to the vesicle membrane with a phosphatidylcholine base contributed effectively to strengthening the barrier effect of the membrane of the vesicle. The leakage of entrapped materials was suppressed with increasing ratio of the triple-chain amphiphile in the membrane. To elucidate the factors which bring about the high barrier effect of the triple-chain amphiphile, the ζ potential of the vesicle, microfluidity of the bilayer membrane, pressurearea (π-A) isotherms of the monolayer membrane, and differential scanning calorimetry of the amphiphiles were studied. Both the increasing surface charge of the vesicle and the enhancement of the hydrophobic interaction near the hydrophilic moiety of the bilayer membrane were considered to contribute to the stability of the vesicle containing the triple-chain amphiphile. It was also found that the microfluidity of the bilayer membrane made from triple-chain amphiphiles was less sensitive to temperature change than that of the bilayer membrane made from phosphatidylcholine.

Introduction Phospholipids are typical “double-chain” amphiphiles and treated as functional materials as a result of possessing an ability to form structures similar to biomembranes. The molecular assemblies of phospholipids in water have been attracting attention for more than 20 years as biomembrane model systems or carriers of great promise in drug delivery systems (DDSs). One of the most substantial subjects for the practical use of liposomes or vesicles as a carrier of DDSs is the exploration of appropriate methods to suppress or control leakage of the entrapped materials during storage. Up to now, numerous attempts have been made to construct stable liposomes or vesicles. For example, the addition of other materials, such as cholesterol1 or cholesterol polysaccharides,2 to bilayer systems and the introduction of special group(s) or structures, such as a polymerizable functional group3 or phytanyl moiety,4 into the hydrophobic chains of the amphiphile have been carried out as conventional approaches. Our strategy for stabilizing vesicles is a strengthening of the bilayer structure by enhanced hydrophobic interac* To whom correspondence should be addressed. Fax: +81-6-6879-7359. Phone: +81-6-6879-7356. E-mail: ikeda@ ap.chem.eng.osaka-u.ac.jp. † Kanebo Corporation. ‡ Osaka University. (1) Seeling, A.; Seeling, J. Biochemistry 1974, 13, 4839. (2) Takada, M.; Yuzuriha, T.; Katayama, K.; Iwamoto, K.; Sunamoto, J. Biochim. Biophys. Acta 1984, 802, 237. (3) (a) Fendler, J. H. Ind. Eng. Chem. Prod. Res. Dev. 1985, 24, 107. Regen, S. L. In Liposomes: From Biophysics to Therapeutics; Ostro, M. J., Ed.; Marcel Dekker: New York, 1987. (b) Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. Engl. 1988, 27, 114. (4) (a) Yamauchi, K.; Sakamoto, Y.; Moriya, A.; Yamada, K.; Hosokawa, T.; Higuchi, T.; Kinoshita, M. J. Am. Chem. Soc. 1990, 112, 3188. (b) Nishikawa, N.; Mori, H.; Ono, M. Chem. Lett. 1994, 767.

tion between amphiphiles, that is, close packing of the hydrophobic chains of the amphiphile that constitutes the vesicle. So far, we have designed and prepared a series of novel amphiphiles bearing three hydrophobic alkyl chains and two ionic headgroups. From the results of the study on the interfacial properties of these compounds, it was found that these “triple-chain” compounds showed greater ability to lower surface tension and to form micelles at lower concentrations than the corresponding “double-chain” surfactants.5 Especially, these “triple-chain” compounds were also found to form a highly packed monolayer at the air-water interface.6 In our previous communication,7 we reported that vesicles made from triple-chain amphiphiles bearing two carboxylate groups (2 and 3) showed much higher stability toward the leakage of the entrapped marker inside the vesicle in comparison with the vesicles made from phosphatidylcholine. But many kinds of phospholipids are commercially available. This is of great advantage in the choice of a fundamental component of bilayer membranes when artificial vesicle systems having special functions are designed. In this connection, we devised a means to improve the stability of vesicles mainly made from phosphatidylcholines by addition of triple-chain bis(carboxylate) amphiphiles as the second component. The latter compounds will be expected to impart a pH-sensitive character to the vesicles. In this study, we investigated the effect of triple-chain amphiphiles on the stabilization of the vesicle with a phosphatidylcholine base in terms of the leakage of the (5) Zhu, Y.-P.; Masuyama, A.; Kirito, Y.; Okahara, M.; Rosen, M. J. J. Am. Oil Chem. Soc. 1992, 69, 626. (6) Sumida, Y.; Oki, T.; Masuyama, A.; Maekawa, H.; Nishiura, M.; Kida, T.; Nakatsuji, Y.; Ikeda, I.; Nojima, M. Langmuir 1998, 14, 7450. (7) Sumida, Y.; Masuyama, A.; Maekawa, H.; Takasu, M.; Kida, T.; Nakatsuji, Y.; Ikeda, I.; Nojima, M. Chem. Commun. 1998, 2385.

10.1021/la000694p CCC: $19.00 © 2000 American Chemical Society Published on Web 09/13/2000

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Chart 1

inner marker. In addition, we will explain the mechanism of the stabilizing effect by addition of the triple-chain amphiphile from the results of the ζ potential of the vesicle, microfluidity of the bilayer membrane, pressure-area (πA) isotherms of the monolayer membrane, and differential scanning calorimetry (DSC) of the amphiphiles. The triple-chain compounds bearing two carboxylate groups (1-3) and phosphatidylcholines mentioned in this paper are listed in Chart 1. Results and Discussion Preparation of Vesicles. The aqueous dispersion of phosphatidylcholines and/or triple-chain amphiphiles 1-3 were prepared according to the procedures mentioned in the Experimental Section. There are some methods to confirm the formation of vesicles prepared by the sonication method. We have already employed a wellestablished gel-filtration method8 in our previous communication.7 In this work, we additionally measured the trapping efficiency of these aggregates by the reported method.9 As a result, the aqueous dispersion of the triple-chain compound bearing three octadecyl chains (3) showed much larger trapping efficiency (3.3%) than that of distearoylphospatidycholine (DSPC) (1.4%). It is also obvious from these results that both amphiphiles form an aggregate which has an inner aqueous phase. Ability To Keep the Entrapped Marker inside the Vesicles. Figure 1 shows the released percentage of 5(6)carboxyfluorescein (CF) from vesicles made from DSPC and/or the triple-chain amphiphile 3 during storage at 40 °C along with the results of a dipalmitoylphosphatidylcholine (DPPC) vesicle under the same conditions. The DPPC vesicle released the entrapped CF much faster than the others. In our previous communication,7 we have already found that the vesicle made from compound 3 had higher ability to suppress the leakage of trapped CF than vesicles made from DSPC. The former vesicle released less than 10% of CF after 1 year under the experimental conditions used in this work. In regard to the mixed systems of DSPC and 3, the leakage of CF was suppressed with increasing ratio of 3 in the mixture. We have surmised that the long-term stability of vesicles made from triple-chain amphiphiles resulted from the much closer packing of alkyl chains in the bilayer membrane, as compared to phosphatidylcholines.7 Taking the above result of the mixed system of triple-chain amphiphiles and phosphatidylcholines into consideration, it is obvious that the following two factors contribute to the high barrier effect of triple-chain amphiphile 3 on the leakage of the entrapped marker: One is an increase of (8) Sunamoto, J.; Iwamoto, K.; Kondo, H. Biochem. Biophys. Res. Commun. 1980, 94, 1367. (9) Oku, N.; Kendall, D. A.; MacDonald, R. C. Biochim. Biophys. Acta 1982, 691, 332.

Figure 1. Release (%) of 5(6)-carboxyfluorescein (CF) trapped inside vesicles composed of DSPC and/or triple-chain amphiphile 3 as a function of storage time (days) at 40 °C along with the data of DPPC.

the surface charge of vesicles, suppressing aggregation and successive fusion of vesicles. The other is enhancement of the hydrophobic interaction in the bilayer membrane. Concerning the former subject, the ζ potential of the vesicle was measured. ζ potential values of vesicles made from DSPC, compound 3, and their mixed system (DSPC/3 ) 1/1) at pH 7.5 were -2, -61, and -55 mV, respectively. DSPC vesicles had no apparent surface potential because of the formation of an inner salt at this pH. On the other hand, vesicles made from compound 3 showed high negative value, indicating that its hydrophilic group dissociated into ions. In the case of the mixed system (DSPC/3 ) 1/1), the value was slightly shifted to a neutral direction as compared with that of the compound 3 vesicle but was still negative. These results indicate that vesicles including 3 were reluctant to fuse with each other because of electrostatic repulsion between the surface of the vesicles as compared with vesicles made from DSPC. It is well-known that the leakage of entrapped materials occurs in the process of the fusion of vesicles.10 In other words, inhibition of the fusion can be considered as one factor for suppressing the leakage of entrapped materials. The microfluidity of the bilayer membrane was estimated by using 2-(9-anthroyloxy)stearic acid (2-AS) and 12-(9-anthroyloxy)stearic acid (12-AS) in terms of the fluorescent polarization (P). The 2-AS has an anthroyloxy group at the 2-position of stearic acid so that it can reflect the fluidity of the vicinity of the hydrophilic moiety in the membrane. On the other hand, the 12-AS has the group at the 12-position of stearic acid and, therefore, gives information on the central part of the hydrophobic moiety in the membrane.11 Figure 2 shows the relation between the microfluidity (P) of membranes composed of DSPC and compound 3 measured by using 2-AS or 12-AS and the temperature. No great difference was observed in the P value of the 12-AS system between DSPC and compound 3 at 40 °C, which is the storage temperature of vesicles in this work. On the other hand, the P value of the compound 3 membrane in the 2-AS system in the vicinity of 40-50 °C was higher than that of the DSPC membrane, and the difference between these two systems could be regarded as significant. This result will be attributed to the closer (10) Crowe, J. H.; Crowe, L. M.; Carpenter, J. F.; Rudolph, A. S.; Wistrom, C. A.; Spargo, B. J.; Anchordoguy, T. J. Biochim. Biophys. Acta 1988, 947, 367. (11) Inoue, T.; Matsuoka, Y.; Fukushima, K.; Shimozawa, R. Chem. Phys. Lipids 1988, 46, 107.

Amphiphile Effect on Phosphatidylcholine Vesicles

Figure 2. Effect of temperature on the fluorescent polarization of 12-AS and 2-AS buried in the bilayer membrane of vesicles made from DSPC or compound 3.

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Figure 4. Release (%) of 5(6)-carboxyfluorescein (CF) trapped inside vesicles made from DPPC, DSPC, or compound 2 or 3 as a function of temperature.

Figure 3. π-A isotherms for DSPC and compound 3.

packing of the hydrophobic moiety near the hydrophilic moieties of vesicles made from compound 3 than that of vesicles made from DSPC. Furthermore, for the purpose of evaluating the packing manner of these bilayers, the pressure-area (π-A) isotherms were measured at 25 °C. As can be seen in Figure 3, both DSPC and compound 3 are found to form a liquidcondensed monolayer without a liquid-expanded state. The limiting areas (A∞), a parameter approximating the area occupied by one molecule on the surface at zero pressure, of DSPC and 3 obtained from Figure 3 are 47 and 67 Å2, respectively. If each A∞ value is converted to the limiting occupation area per hydrophobic alkyl chain in a molecule [A∞′/Å2 (alkyl chain)-1], almost the same values are found for DSPC and compound 3 [ca. 23 and 22 Å2 (alkyl chain)-1, respectively]. Taking into account the structural difference in the hydrophilic groups between DSPC and 3, that is, amphoteric/anionic and one/two hydrophilic groups, it would be predicted that the occupation area per hydrophobic alkyl chain of compound 3 will be much larger than that of DSPC. The experimental data showed, however, almost the same values for the A∞′ of each molecule. This result supports the speculation that this triple-chain structure of compound 3 has a pronounced effect on the close packing of its membrane. In connection with this relationship between the molecular structure and hydrophobic interaction, we have already reported6 that triple-chain amphiphiles bearing a glycerol backbone showed unusually tight packing at the air-water interface. The rigidity near the hydrophilic moiety of the bilayer membrane clarified by the measurement of the microfluidity of membranes also supports this speculation regarding the packing manner.

Figure 5. DSC thermograms of the single and mixed systems of DSPC and compound 3.

Leakage of the Entrapped Marker inside a Vesicle Made from Phosphatidylcholine and a Triple-Chain Amphiphile: Dependence on Temperature. Figure 4 shows the leakage percentage of CF from vesicles made from DPPC, DSPC, 2, or 3 as a function of temperature. Vesicles made from the triple-chain amphiphiles were more stable against increasing temperature than vesicles made from phosphatidylcholines bearing the same number of carbon atoms in one hydrophobic chain (2 vs DPPC, 3 vs DSPC, respectively). While the phosphatidylcholine vesicle released the inner marker steeply at a certain temperature, the triple-chain amphiphile vesicle released them gradually with increasing temperature. To investigate the temperature dependence of the behavior of the aggregates, DSC of a series of amphiphiles dispersed in water was measured. Figure 5 depicts the DSC thermograms of the single and mixed systems of DSPC and compound 3. The phase transition, which is attributed to the transition from gel to liquid crystal states, was detected for each case besides the pre-phase transition in the case of DSPC only.12 The peak-top temperature (Tc) and the heat of endothermic transition (∆H) are listed in Table 1. Each Tc value was compatible with the temperature at which leakage of the entrapped marker occurred drastically from the vesicle made from DSPC or compound (12) Chapman, D.; Williams, R. M.; Ladbrooke, B. D. Chem. Phys. Lipids 1967, 1, 445.

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Figure 6. Effect of temperature on the fluorescent polarization of DPH buried in the bilayer membrane composed of DMPC, DPPC, DSPC, or compound 1, 2, or 3.

Figure 7. Effect of temperature on the fluorescent polarization of DPH buried in the bilayer membrane of the single and mixed systems of DSPC and compound 3.

Table 1. Phase Transition Temperature from the Gel to Liquid Crystal Phase (Tc) and Heat of the Transition (∆H) of the Mixed System of Amphiphiles Measured by DSC

is less sensitive to temperature changes than that composed of phosphatidylcholines. The P values of DPH in the mixed system of DSPC and compound 3 as a function of temperature are also shown in Figure 7. The slope of the P value of the mixed system near its Tc was more moderate than that of the compound 3 system, and the curve of the mixed systems shifted to higher temperature. Furthermore, as can be seen in Figure 2, the fluidity at different depths of the membrane measured by using 2-AS and 12-AS exactly reflects the difference in the property between membranes of vesicles made from DSPC and compound 3. Thus, the P value for the DSPC vesicle showed a steep decrease at its Tc, while that for vesicles made from 3 showed a gradual decrease. Especially, reflecting the fluidity of the vicinity of the hydrophilic moieties in the membrane, the difference in the P values by 2-AS between DSPC and compound 3 vesicles was clear. These results regarding the microfluidity of the membrane are compatible with the temperature dependence of the leakage behavior of vesicles mentioned above. In addition, it is surmised that the difference of the temperature dependence between DSPC and compound 3 was mainly due to the fluidity of the vicinity of the hydrophilic moieties in the membrane because the sharpness of DSC thermograms was not so different between DSPC and compound 3. In conclusion, the addition of amphiphiles bearing three hydrophobic chains and two carboxylate groups to vesicles with a phosphatidylcholine base improved the stability toward the leakage of entrapped materials. The leakage of entrapped materials was suppressed with increasing ratio of triple-chain amphiphiles in the mixture. Both increasing the surface charge of the vesicle and enhancement of the hydrophobic interaction near the hydrophilic moieties of the bilayer membrane were considered to contribute to the high barrier effect of triple-chain amphiphiles. Furthermore, it was found that a vesicle made from triple-chain amphiphiles was more stable against increasing temperature than a vesicle made from phosphatidylcholine. This observation agreed with the result that the microfluidity of the bilayer membrane composed of triple-chain amphiphiles was less sensitive to temperature change than that composed of phosphatidylcholines.

molar ratio ∆H/of DSPC/3 Tc/°C (mJ mol-1) 100/0 75/25 50/50

56.7 56.5 57.1

26 33 42

molar ratio ∆H/of DSPC/3 Tc/°C (mJ mol-1) 25/75 0/100

58.7 59.5

48 66

3. In addition, it was found that the ∆H value of the phase transition of compound 3 was much larger than that of DSPC and the ∆H values of the mixed system increased with increasing mixing ratio of 3. The Tc values shifted to higher temperature with increasing mixing ratio of 3. The ∆H value is considered as a reliable indication of the tightness of the hydrophobic interaction in the aggregates. Even if the difference in the number of hydrophobic chains between DSPC and compound 3 is taken into account, the ∆H value of the phase transition of compound 3 is much larger than that of DSPC. This result supports the speculation that the hydrophobic chains of compound 3 pack more tightly than those of DSPC do. However, the sharpness of the DSC thermogram of the triple-chain compound is not in accord with the peculiar gradual release of the entrapped marker from vesicles made from this compound above its Tc shown in Figure 4. The large value of ∆H for triple-chain compounds alone cannot explain this gradual release. So, we evaluated the microfluidity of the vesicle membrane in terms of the fluorescent polarization (P) with increasing temperature by using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a fluorescent marker.13 Figure 6 shows the fluorescence polarization of DPH embedded inside the vesicle bilayer membrane composed of triple-chain amphiphiles or phosphatidylcholines bearing hydrophobic chains of different lengths (C14, C16, and C18) as a function of temperature. In all the vesicles, the decrease in the P value was observed around each Tc. Upon comparing the results for the triple-chain amphiphiles with those for the corresponding phospholipids bearing the same number of carbon atoms in one hydrophobic chain [1 vs DMPC, 2 vs DPPC, 3 vs DSPC, respectively], the P value of triple-chain amphiphiles decreased more gradually than that of phospholipids around each Tc. This means that the microfluidity of the bilayer membrane composed of triple-chain amphiphiles (13) Shinitzky, M.; Barenholz, Y. Biochim. Biophys. Acta 1978, 515, 367.

Experimental Section Materials. Triple-chain amphiphiles 1-3 were synthesized and purified by the previously reported method.5 The structure and purity of newly prepared compound 1 were confirmed using

Amphiphile Effect on Phosphatidylcholine Vesicles the corresponding dimethyl esters because bis(carboxylate) compound 1 was hygroscopic. The properties of compounds 2 and 3 have already been reported.7 Data for {2-[2-(2-Methoxycarbonylmethoxy-3-tetradecyloxypropoxy)-1-tetradecyloxymethyl-ethoxy]-1tetradecyloxymethylethoxy}acetic Acid Methyl Ester (the Corresponding Dimethyl Ester of 1). Oil; FAB-MS m/e 973 [(M + 1)+, 100]; 1H NMR (400 MHz, CDCl3) δ 0.87 (t, 9 H), 1.20-1.35 (m, 66 H), 1.51-1.59 (m, 6 H), 3.32-3.65 (m, 21 H), 3.72 (s, 6 H), 4.29-4.33 (m, 4 H). Anal. Calcd for C57H112O11: C, 70.33; H, 11.60. Found: C, 70.53; H, 11.81. Phosphatidylcholines (DMPC, DPPC, and DSPC) were purchased from Nippon Fine Chemical Co., 99.8% purity, and used without further purification. Water-soluble fluorescent marker Calcein was purchased from Tokyo Kasei Co. and purified by dissolving in 0.1 M aqueous NaOH solution, acidifying the solution with 1 M hydrochloric acid after addition of methanol, recrystallization in cooling condition, and finally drying in a desiccator. CF was purchased from Eastman Kodak and purified by dissolving in 1 M aqueous NaOH solution, treating with active carbon, acidifying the filtered solution with 1 M hydrochloric acid, centrifuging, washing the resulting precipitate with distilled water, and finally drying in a desiccator. Fluorescent probes 2-AS and 12-AS were purchased from Molecular Probes, Inc., and DPH was from Wako Pure Chemical Industries, Ltd., and used without further purification. All other chemicals were commercial products of reagent grade. Methods. The aqueous dispersion of phosphatidylcholines and/or triple-chain amphiphiles 1-3 were prepared by the following procedures: A film of lipid and/or amphiphile (total 20 µmol) was prepared on the inside wall of a test tube by evaporation of its CHCl3 solution and was stored in a desiccator overnight under reduced pressure. After addition of 2 mL of a Tris-HCl buffer solution (20 mM, pH 7.5) containing 100 mM NaCl to the test tube, the mixture was vortex-mixed for 10 min, and successively sonicated for 5 min at about 10 °C higher than its Tc (mentioned later) using a probe-type sonicator under a stream of nitrogen. Then, a translucent liquid containing the aggregates of lipid and/or amphiphile was obtained. The trapping efficiency of these aggregates was estimated as follows: A film of phosphatidylcholine or the triple-chain amphiphile (20 µmol) was dispersed in 2 mL of a Tris-HCl buffer (10 mM, pH 7.3) containing 0.1 mM bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein (Calcein). The dispersion was sonicated for 5 min at about 10 °C higher than its Tc using a probe-type sonicator under a stream of nitrogen. After dilution of 40 µL of the dispersion with 2 mL of a Tris-HCl buffer solution (10 mM, pH 7.3), the fluorescence intensity at 530 nm was measured using an excitation wavelength at 490 nm (Ft). The fluorescence intensities were measured successively after addition of 20 µL of an aqueous solution (10 mM) of CoCl2 (Fin), followed by addition of 20 µL of 20% Triton X-100 (Fq). The trapping efficiency was calculated by means of eq 1.

trapping efficiency (%) ) (Fin - Fq)/(Ft - Fq) × 100 (1) Preparation of the vesicles containing CF was carried out as follows: A film of lipid and/or amphiphile (40 µmol) was prepared on the inside wall of a test tube by evaporation of its CHCl3 solution and stored in a desiccator overnight under reduced pressure. After addition of 4 mL of a Tris-HCl buffer (20 mM,

Langmuir, Vol. 16, No. 21, 2000 8009 pH 7.5) containing 100 mM CF to the test tube, the mixture was vortex-mixed for 10 min and successively sonicated for 5 min at about 10 °C higher than its Tc using a probe-type sonicator under a stream of nitrogen. Small unilamellar vesicles containing trapped CF were separated from untrapped CF by eluting the vesicle dispersion through a Sephadex G-50 gel column with 20 mM Tris-HCl buffer containing 100 mM NaCl (pH 7.5). Then the vesicle fraction was diluted to 1 mM lipids and/or amphiphiles with the same buffer solution as an eluent and subjected to measurement of the leakage of CF from the vesicles. The amount of CF released (%) from the vesicles was calculated by means of eq 2,where I0 is the fluorescence intensity of the vesicle suspension containing CF at initial time,

CF released (%) ) (Ix - I0)/(It - I0) × 100

(2)

Ix is the intensity of the suspension after a definite period of storage at definite temperature, and It is the fluorescence intensity after addition of an aqueous solution of Triton X-100 (100 g L-1) to the suspension. The fluorescence intensity at 530 nm was measured at 25 °C using an excitation wavelength at 490 nm. The ζ potential of the vesicle was measured using an ELS-800 (Ohtsuka Electronics Co., Ltd.), which is a laser doppler electrophoresis apparatus. For this measurement, vesicles without entrapped materials were prepared in Tris-HCl buffer (20 mM, pH 7.5) containing 10 mM NaCl under the same conditions, as mentioned above. Measurement of the microfluidity of bilayer membranes was carried out as follows: A fluorescent probe, 2-AS or 12-AS, was dissolved in CHCl3/CH3OH (1/1) together with phosphatidylcholines and/or amphiphiles. The molar ratio of the probe to lipids and/or amphiphiles was 1/100. After evaporation of the solvents, the vesicle dispersion was prepared according to the same procedures. The microfluidity of the vesicle was determined by fluorescence polarization (P), which was calculated by means of eq 3, where IP and IV are the fluorescence intensities of the

P ) (IP - GIV)/(IP + GIV)

(3)

emitted light polarized parallel and vertical to the exiting light, respectively, and G is the grating correction factor.11 Excitation and emission wavelengths were 365 and 439 nm, respectively. In the case of fluorescence measurement using DPH, DPH solution in THF was ultimately added to the vesicle dispersion prepared by the above procedures. The molar ratio of DPH to lipids and/or amphiphiles was 1/1000. Then, the vesicle dispersion was kept for 1 h at about 10 °C higher than its Tc. The P values of DPH were also calculated by means of eq 3. Excitation and emission wavelengths were 360 and 428 nm, respectively. The π-A isotherms were recorded with a computer-controlled film balance system (Nippon Laser & Electronics Laboratory type NL-LB80S-MTC). Surface pressure (π) as a function of molecular area (A) was measured in an equilibrium-relaxation compression mode at 25 °C. DSC measurements were carried out using DSC-20 (Seiko Instruments & Electronics Ltd.). Two milligrams of lipids and/ or amphiphiles was put in a sampling pan made of Al2O3, and then 10 µL of distilled water was added, followed by the pan being sealed. After these samples were held at 80 °C for 1 h, the measurement was carried out with a 2 °C min-1 scanning rate. The heat of endothermic transition (∆H) was calculated from the peak area.

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