Oleate Dispersions by

Article pubs.acs.org/Langmuir

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 560-8531, Japan S Supporting Information *

ABSTRACT: Oleic acid (OA) and oleates form selfassembled structures dispersible in aqueous media. Herein, the physicochemical properties of OA/oleate assemblies were characterized using fluorescent probes and Raman spectroscopy, under relatively high dilution ( 11), the sample solutions were titrated by adding 100 mM HCl with stirring at room temperature. The solution pH was measured by using alkali-resistant pH electrode (LAQUA, Horiba, Ltd., Kyoto, Japan). For the analyses of membrane properties (DPH and Laurdan measurements), OA was dispersed in distilled water including 50 mM bicine. The sample solutions were once saponified with NaOH (88 mM) and titrated to arbitrary pH by adding HCl (100 mM). After 1 week incubation with stirring at room temperature, the sample solutions were applied to measurements (turbidity and Raman). The turbidity of the sample solution was monitored by UV−vis spectroscopy (UV-1800, Shimadzu Corporation, Kyoto, Japan), on the basis of literature.8 The pKa values of OA was defined as the pH that the half of OA was in ionized state.21 According to Henderson− Hasselbalch equation,22 the molar fraction of the protonated OA (PD) was calculated as follows:

XHA + X A − = 1 pH = pK a + log10 PD = XHA =

X A− XHA

1 1 + 10 pH − pKa

(1)

where XHA and XA− represent the molar fraction of protonated OA and oleate (ionized OA), respectively. Measurement of Microviscosity Using DPH. The fluorescence anisotropy (r) of DPH was calculated on the basis of literature.23 The OA/oleate suspension (total amphiphile: 100 mM) was first prepared by the method described in above. Volumes of 250 μL of OA/oleate suspension and 8 μL of DPH solution (250 μM, in ethanol) were diluted by 50 mM bicine buffer (pH 8.5), and the solution pH was titrated by addition NaOH or HCl. The total concentrations of amphiphile and DPH were 5 mM and 0.4 μM (molar ratio, amphiphile/DPH = 12 500/1; total volume, 5 mL), to reduce the influence of solution turbidity during measurement. The samples were incubated at least 1 h in the dark before measurement. Fluorescence intensities of DPH were stable at least 3 h, independent to the surrounding pH (pH 6.9−10.6). The samples were excited with vertically polarized light (360 nm), and emission intensities both perpendicular (I⊥) (0°, 0°) and parallel (I∥) (0°, 90°) to the excited light were recorded at 430 nm. The anisotropy (r) of DPH was then calculated using the following equations:23

r = (I − GI⊥)/(I + 2GI⊥)

(2)

G = i⊥/i

(3)

(5)

R = I2850/I2930

(6)

where S and R indicate the degree of chain torsion and chain packing, respectively.26 Statistical Analysis. Results are expressed as mean ± standard deviation. All experiments were performed at least three times. The distribution of data was analyzed, and statistical differences were evaluated by use of Student’s t test. A P-value of pH(i)), vesicle−micelle coexistence (pH(i) > pH > pH(ii)), vesicle (pH(ii) > pH > pH(iii)), vesicle-oil-in-water (O/W) emulsion coexistence (pH(iii) > pH > pH(iv)), and O/W emulsion (pH < pH(iv)).8,27 Considering their protonation degrees (PD) (Figure S2c), sodium oleate molecules are the dominant components at pH > 8.0, while protonated OA molecules are dominant at pH < 8.0, whereby the apparent pKa of OA is 8.0.21 Depending on the OA/oleate ratio, the assembly size could be varied:28,29 micelle (3 days) increased the viscosity of the OA/oleate suspension prepared at pH 7.6 (Figure S5). The PD value for the OA/oleate assembly at pH 7.5 was 0.76 (Figure S2), which is consistent with the OA/oleate molar ratio of the cubic phase27 and suggests the occurrence of a dispersed bicontinuous cubic phase (cubosome) in this pH region. Hence, it can be concluded that the OA/oleate assemblies have relatively ordered phase states at pH 7.5, 8.5, and 9.7. Discussion for the Metastable Phases of OA/Oleate Assemblies. Generally, the critical packing parameter (γ) is employed to describe the type of amphiphilic molecule assembly in aqueous media. This dimensionless parameter is defined as follows:

γ = V /lca

(7)

where V, lc, and a represent the volume, hydrocarbon chain length, and molecular headgroup area, respectively.41,42 In general, the phase states of amphiphilic molecules vary with γ: spherical micelles at γ ≤ 1/3; cylindrical micelles at 1/3 < γ ≤ 1/2; lamellar structure (e.g., bilayer vesicle) at 1/2 < γ ≤ 1; and reverse micelles at γ > 1.41 This γ theory, however, does not distinguish between reverse micelle geometries (e.g., spherical reverse micelle and cubic phase). Although an accurate measurement of the headgroup area of oleic acid at different pH values might be difficult due to its solubility43 in the subphase, measurements of S and R values by Raman spectroscopy could provide insight into the packing states of amphiphilic molecules. Increased S values lead to a decrease in relative lc values; thus, γpH=9.7 > γpH=10.6. Considering vesicle formation at pH 8.0−9.7, the apparent packing parameter at pH 9.7 is γpH=10.6(micelle) < γpH=9.7 < γpH=8.5(vesicle). The speculated γpH=9.7 could be in agreement with the γ value of (dispersed) cylindrical micelles: 1/3 < γ ≤ γpH=8.5(vesicle), 1/3 < γpH=8.5(vesicle) ≤ 1/2). The π−A isotherm measurements of oleic acid at different pH values imply a decrease in the molecular headgroup area at lower pH (Figure S6a). Vesicles are a kind of lamellar phase. When the vesicle diameter is small, the internal and external curvatures are opposite. The elastic or strain energy is one of the driving forces that can stabilize selfassemblies.43,44 Since the S and R values of the OA/oleate

Figure 3. S values and R values of OA/oleate assemblies. Total concentration of amphiphile was 100 mM. The measurement was carried out at 25 °C.

vesicles (e.g., 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles in gel phases, S > 1.0).8,31 Since I1124 and I1096 originate from C−Ctrans and C−Cgauche, respectively, the steric conformation of the hydrocarbon chain could be gauche-rich in the vesicle state, revealed by higher membrane fluidity of OA vesicles at pH 8.5.8 Dielectric dispersion analysis results show that the relaxation frequency peak position was pH-dependent, with the peak maximum observed at around pH 8.5 (Figure S4). Because relaxation is due to the dipole moment of a charged lipid (i.e., sodium oleate), it is assumed that the vesicle at pH 8.5 is stabilized by dipolar interaction between amphiphiles. These results show that the membrane properties of OA/oleate vesicles (pH 8.5) are similar to those of the phospholipid vesicles in liquid-disordered phases.40 In the pH range 6−10, OA/oleate assemblies at pH 7.5 and 9.7 were also in a highly ordered state (O/W emulsion-vesicle coexistence D

DOI: 10.1021/acs.langmuir.6b02257 Langmuir XXXX, XXX, XXX−XXX

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assemblies at pH 7.5 and 8.5 were almost equal, the differences between their self-assembly types can be difficult to distinguish. However, analysis of the elastic modulus (compressibility) suggests that oleic acid at pH 7.5 was less compressive compared to at pH 8.5 (Figure S6b). This could be due to an increased PD value at pH 7.5 compared to that at pH 8.5, which could increase the negative curvature of the OA/oleate membrane, probably resulting in dispersed cubic phase (cubosome) formation. Based on the aforementioned results, the type and characteristics of OA/oleate assemblies are summarized in Figure 4.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b02257. Modified phase diagram of OA/sodium oleate/water; pH titration curves of OA/oleate assemblies; reversibility of R values; dielectric dispersion analysis for OA/oleate assemblies; photo of OA/oleate assembly at pH 7.6; π−A isotherms of oleic acid measured in the subphase with different pHs; and elastic modulus of oleic acid at different pHs (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-6-6850-6287. E-mail: [email protected]. jp. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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Figure 4. Summary of this study. The phase behaviors of OA/oleate assemblies at diluted conditions are estimated as follows: spherical micelle at pH 10.6; cylindrical micelle at pH 9.7; vesicle at pH 8.5; cubosome at pH 7.5; and O/W emulsion at pH 6.9.

ACKNOWLEDGMENTS The authors are grateful to Keysight Technologies for providing a probe tip in dielectric dispersion measurements. The authors are also thank to Malvern Instruments Ltd for zeta potential measurements. This work was supported by the Funding Program for Next Generation World-Leading Researchers of the Council for Science and Technology Policy (CSTP; GR066), Japan Society for the Promotion of Science (JSPS) KAKENHI Grant number JP26249116, JP25889039, JPT15K14204, and JP16K18279.

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CONCLUSIONS The physicochemical properties of OA/oleate assemblies were characterized using fluorescent probes (DPH and Laurdan) and Raman spectroscopy. The microviscosity of the OA/oleate assembly at pH 7.5 increased, suggesting the occurrence of another self-assembly type. Raman spectroscopy revealed that the chain torsion (S) and chain packing (R) values of OA/ oleate assemblies were maximal at pH 7.5, 8.5, and 9.7, showing that dispersed cubic phases (cubosomes) and dispersed cylindrical micelles, composed of OA and oleate, could be formed even at relatively high dilution (100 mM amphiphile). Because the S and R values were reversible during pH titration, the phase behavior of OA/oleate assemblies could be analyzed by Raman spectroscopy. In diluted systems (>95 wt % water), transitions from one type of fatty acid/ionized fatty acid structure to another can be easily induced by changing the pH.45 Considering the fact that OA is used in nanoparticle synthesis/stabilization,46,47 it is assumed that the self-assembled structure of fatty acid molecules can be used as a nanostructural template for controlling the size of nanoparticles and their assemblies. Although further investigations of the morphology of OA/oleate assemblies are needed, this is the first report systematically characterizing the phase behavior of OA/oleate assemblies by using Raman spectroscopy. The dispersed cubic phase or cubosome of FA assemblies are usually found in a limited area of phase diagrams and require longer incubation times.48,49 The fatty acid assemblies with ordered membrane properties can be used as a platform for drug delivery, modeling cell membranes, and handling membrane proteins.

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