# Hydrothermal Preparation of Faceted Vesicles Made of Span 40 and Tween
## 3 Results
### Characterization of Faceted Vesicles  
#### 3.3.1 Membrane Properties  
Based on the results of the previous section, the membrane properties of faceted vesicles were evaluated in terms of zeta potential, fluorescent probe motility, and the polar environment around the fluorescent probe [7; 8; 9; 10; 19].  
Figure 6a shows the \(\zeta\)-potential values of the samples obtained from the various preparation conditions for the SCW/SD method and the conventional method. The \(\zeta\)-potential is the potential on the slipping surface, and the larger the \(\zeta\)-potential value, the stronger the electrostatic repulsion between the particles and the higher the dispersibility [10]. The faceted vesicles prepared by the conventional method indicated the \(\zeta\)-potential of \(-\)45.5 \(\pm\) 5 mV. The \(\zeta\)-potential of each sample prepared using the SCW/SD method was approximately \(-\)30 to \(-\)60 mV, regardless of the lipid concentration. Therefore, the obtained samples including the faceted vesicles, were considered highly dispersible and stable, regardless of the preparation method.  
Figure 6b shows membrane fluidity, which is the motility of the fluorescent probe DPH in the membrane. DPH motility was measured using the depolarization method. The higher the (1/P)\({}_{\text{DPH}}\) value (i.e., the higher the mobility of the DPH molecules), the higher the membrane fluidity. The faceted vesicles obtained by the conventional method indicated a (1/P)\({}_{\text{DPH}}\) value of 3.5 \(\pm\) 0.4, which was lower than that of the spherical vesicles ((1/P)\({}_{\text{DPH}}\)-10 [10]). This strongly suggests that the faceted vesicles have rigid membranes. Span 40/Tween 40 (Span 40 80-100 wt%) was considered to be rigid at room temperature  
Figure 6: Comparison of various properties (**a**) \(\zeta\)-potential, (**b**) mobility of DPH, and (**c**) generalized polarization of Laurdan. (**d**) Calcein leakage from faceted vesicle. (**d**) Calcein leakage behavior of vesicles prepared by the SCW/SD and conventional method. Conv.: conventional preparation method of faceted vesicles (see the Section 2.3). The error bar represents the standard error. All the experiments were at least triplicated.  
owing to its phase transition temperature (40-50 \({}^{\circ}\)C) [8]. Next, the sample (0.25 wt%) had the lowest (1/P)\({}_{\text{DPH}}\) (6 to 7) of the samples prepared by the SCW/SD method, which was the closest value to the conventionally obtained faceted vesicles. The (1/P)\({}_{\text{DPH}}\) values 0.05 and 0.1 wt% were approximately 10-15, which corresponded to the observed emulsion.  
Figure 6c shows the general polarization GP\({}_{\text{340}}\) value for each sample obtained using Laurdan. The GP\({}_{\text{340}}\) value is an index of the polar environment around the fluorescent probe, Laurdan. The larger the GP\({}_{\text{340}}\) value, the more hydrophobic the surrounding environment. The conventionally prepared faceted vesicles had a GP\({}_{\text{340}}\) value of 0.4 \(\pm\) 0.03. This suggests that the membrane structure was hydrophobic. On the other hand, the GP\({}_{\text{340}}\) values of samples prepared by the SCW/SD method (0.05-0.4 wt%) ranged from 0.1 to 0.3. Therefore, the membrane became more hydrophilic than the conventional faceted vesicles.  
From the above results, the sample prepared with a lipid concentration of 0.25 wt% had the membrane properties closest to the conventionally prepared faceted vesicles.