SUPPORTING INFORMATION. Characterization of Aqueous Oleic Acid/Oleate Dispersions by

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1 SUPPORTING INFORMATION Characterization of Aqueous Oleic Acid/Oleate Dispersions by Fluorescent Probes and Raman Spectroscopy Keishi Suga, Dai Kondo, Yoko Otsuka, Yukihiro Okamoto, and Hiroshi Umakoshi* Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyamacho, Toyonaka, Osaka , Japan * Corresponding author (H. U.): TEL, ; , b-ice@cheng.es.osaka-u.ac.jp KEYWORDS: Oleic acid, Vesicle, Raman spectroscopy, Microviscosity, Phase behaviors S-1

2 Figure S1. Modified phase diagram of OA/sodium oleate/water, reported by Edwards. 1 In this diagram, relatively-diluted conditions have not described clearly (total amphiphile concentration <100 mm). 1, reverse micelle phase (O/W emulsion); 2, cubic phase; 3, reverse hexagonal phase; 4, lamellar phase (vesicle); 5, hexagonal phase (cylindrical micelle); and 6, spherical micelle phase. S-2

3 Figure S2. (a) ph titration curve of OA/Oleate assemblies. The OA/Oleate suspensions in distilled water were obtained by adding NaOH (1 M). The ph titration curve was obtained by adding HCl (1 M) at room temperature, as described previously. 2 Initial concentration of amphiphile was 100 mm, and the sample volume was 10 ml. The captions (i), (ii), (iii), and (iv) indicate the inflection point in the titration curves or turbidity curves: ph(i)=10.1, ph(ii)=9.6, ph(iii)=8.0, and ph(iv)=7.4. (b) Turbidities (OD400) of OA/Oleate dispersions measured by using UV vis spectroscopy. (c) Protonation degree (PD) at each ph. The data was calculated on the basis of Henderson-Hasselbalch equation 3 described as eq 1 in the manuscript. S-3

4 Figure S3. Reversibility of R values. The OA/Oleate suspensions was first prepared at ph 8.5 (I), by ph titration method. Then, the solution ph was changed by adding 88 mm of NaOH solution ((II), (III)). The ph was finally titrated at around 8.5 by adding 100 mm of HCl (IV). Arrows indicate the operating sequence. Total concentration of amphiphile was 100 mm. The measurement was carried out at 25 C. S-4

5 Figure S4 Dielectric dispersion analysis for OA/oleate assemblies. Relaxation frequency ( ) was plotted against ph. Total amount of OA molecules was 100 mm. The measurement was carried out at 25 C. S-5

6 Figure S5. Photo of OA/Oleate assembly at ph 7.6. The sample solution was prepared by ph titration method, and stirred for 3 days at room temperature. The total concentration of amphiphile was 100 mm. In cases of the OA/Oleate suspensions prepared with different ph conditions, no increase of the solution viscosity was observed. S-6

7 Figure S6. (a) π-a isotherms of oleic acid measured in the subphase with different ph. (b) Elastic modulus of oleic acid at different ph. Symbols indicate ph 6.9 (circle), ph 7.5 (diamond), ph 7.8 (triangle), ph 8.5 (reverse triangle), ph 9.7 (ribbon), and ph 10.6 (square), respectively. -A isotherm studies of OA/Oleate assemblies at different ph. π-a isotherms were measured by KSV film balance (trough dimensions: width 60 mm, length 190 mm). The surface pressure was measured with use of a platinum plate as a pressure probe. Monolayers were prepared by spreading an aliquot (5 μl) of the OA dissolved in chloroform (total concentration: 1 mg/ml) onto an aqueous subphase. Monolayers of the OA were spread on the trough with an initial surface of 150 cm 2, at different subphase ph. Film compression was started 15 min after spreading, at the rate of 100 cm 2 /min. Experiments were repeated at least three times at room temperature. Elastic compressibility (Cs -1 ) was calculated from the pressure versus area isotherm using the follows: 4,5 Cs -1 = -A(dA/d ) where Cs -1 is elastic modulus, A is the area, is surface pressure. In general, a lower Cs -1 value means an increase in the monolayer compressibility, while a higher Cs -1 value means less compressibility in the monolayer. S-7

8 Supporting References (1) Edwards, K.; Silvander, M.; Karlsson, G.; Aggregate structure in dilute aqueous dispersions of oleic acid/sodium oleate and oleic acid/sodium oleate/egg phosphatidylcholine. Langmuir 1995, 11, (2) Suga K.; Yokoi T.; Kondo D.; Hayashi K.; Morita S.; Okamoto Y.; Shimanouchi T.; Umakoshi H. Systematical characterization of phase behaviors and membrane properties of fatty acid/didecyldimethylammonium bromide vesicles. Langmuir 2014, 30, (3) Rendo n, A.; Carton, D. G.; Sot, J.; García-Pacios, M.; Montes, L. R.; Valle, M.; Arrondo, J. L.; Gon i, F. M.; Ruiz-Mirazo, K. Model systems of precursor cellular membranes: Long-chain alcohols stabilize spontaneously formed oleic acid vesicles. Biophys. J. 2012, 102, (4) Smaby, J.M.; Kulkarni, V.S.; Momsen, M.; Brown, R.E. The interfacial elastic packing interactions of galactosylceramides, sphingomyelins, and phosphatidylcholines. Biophys. J. 1996, 70, (5) Sabatini, K.; Mattila, J.-P.; Megli, F.M.; Kinnunen, P.K.J. Characterization of two oxidatively modified phospholipids in mixed monolayers with DPPC. Biophys. J. 2006, 90, S-8