# Hydrothermal Preparation of Faceted Vesicles Made of Span 40 and Tween
## 4 Discussion  
The present preparation method resulted in the formation of vesicles or emulsions (Figure 5a-c). This propensity to form might depend on the state of the coarse emulsion. Generally, the homogenization of water/decane/surfactant mixtures at a high water content might favor a coarse O/W emulsion. After hydrothermal emulsification, O/W emulsions 100 nm in size were produced, as shown in Figure 5a. This was because (1) the amount of surfactant covering the surface of the W/O/W emulsions was insufficient, or (2) because the water content of solution I was too high to form an O/W emulsion rather than a W/O emulsion. The latter case might be that a reduced-size O/W emulsion obtained via hydrothermal treatment. Therefore, the successful formation of vesicles would require an adequate water-to-decane volume ratio and a sufficient concentration of surfactants. Whether a mixture of Span 40/Tween 40 (80/20) possesses the ability to form the faceted shape in the SCW/SD method is herein discussed. The W/O/W emulsion before the SD step indicated that there were not definite faceted emulsion droplets (e.g., Figure 5a). It was considered that Span 40 and Tween 40 might be insufficient for covering W/O/W emulsions. Previously, faceted O/W emulsion droplets made of cationic surfactant, octadecyltrimethylammonium bromide plus salt were stable, owing to the formation of a frozen interfacial layer at the oil-water interface [24]. It is, therefore, considered that Span 40/Tween 40 can form the stiff interface-like frozen interfacial layer because of the phase-transition temperature: Span 40 (40-50 \({}^{\circ}\)C) and Tween 40 (<10 \({}^{\circ}\)C) [8]. Figure 5a shows that it was unclear whether O/W emulsion droplets had a faceted shape. This might result from insufficient concentration of Span 40 and Tween 40 for a coverage of emulsions after a SD step. In the SD step, lateral diffusion of surfactants was altered in the process of the diffusion of decane from W/O/W emulsions. From the observations of faceted vesicles (Figure 5b,c), the pair of curvatures were produced, accompanied with the phase separation between Span 40 and Tween 40. According to the literature [8], this process originated from the difference between their headgroup structures (see Figure 2). [PERSON] et al. have reported that the lateral heterogeneity in the interface of O/W emulsions resulted from the different structures of surfactants [25], which supports the possible mechanism mentioned above. Thus, a mixture of Span 40/Tween 40 (80/20) led to the shape of faceted in the SCW/SD method. In the latter period of the SD step, the rims should occur at the surface of the vesicles. Tween 40 might be localized, especially at the rims, owing to a phase separation which resulted from a difference in headgroup structure between Span 40 (cylinder type, \(a_{0}\) = 0.37 nm\({}^{2}\)) and Tween 40 (cone type, \(a_{0}\) = 0.533 nm\({}^{2}\)) [26]. This mechanism is similar to the mechanism that cationic surfactants with large headgroups locate at the outer rims and anionic surfactants with smaller headgroups locate at the inner rims by flip-flop [27]. However, such a heterogeneity of Tween 40 could not be detected from the cryo-TEM observations (e.g., Figure 5b,c) because oligoethylene glycol, that is the hydrophilic head of Tween 40, could not be observed by cryo-TEM. This is because there is no significant difference in electron density compared to water [10; 28]. For this reason, another scenario is also noted: that a formation of faceted vesicles is a result of the difference in lipids between the outer and inner leaflet of a gel-phase interdigitated membranes [11; 18]. Next, the facets seemed to consist of Span 40, and were at a gel state because of its phase-transition temperature (40-50 \({}^{\circ}\)C) being greater than the SD temperature (4 \({}^{\circ}\)C or 25 \({}^{\circ}\)C) [8].