## 3 Results and Discussion
### NEs Design and Physicochemical Characterization  
To select the appropriate NEs in terms of composition, a ternary phase diagram of LO, Brij O10 and Hepes buffer was developed. Several formulations were prepared with different amounts of each component. The samples were analyzed by visual inspection to identify homogeneous dispersions or not homogeneous ones. These results have been useful to ternary phase diagram construction (Figure 6), in which the light blue region represents the homogenous region, and the blue zone is the non-homogenous one. Figure 5: Percentage areas of the sesquiterpenes detected through HS-SPME-GC-MS analyses. Figure 6: Ternary phase diagram of LO, Brij O10, and Hepes Buffer. The resulting phases were the homogeneous phase (light blue area) and the not homogeneous phase (dark blue area). NEs composition has been placed into the homogeneous region. To prepare NEs with suitable physical-chemical features, all samples of the light blue region area were sonicated (under the conditions described in Section 2). The selection was made taking into consideration several parameters such as hydrodynamic diameter and \(\zeta\)-potential. The composition of the chosen sample (NEs) has been detailed in Table 2 and its features (DLS results and anisotropy) outlined in Table 2.  
In particular, NEs were characterized by a hydrodynamic diameter of 165.7 nm and a size distribution that was nearly monodisperse (PDI \(\leq\) 0.3) [63]. Visual inspection of selected NEs by TEM revealed that both NEs and NEsL samples were spherical vesicles with dimensions comparable to DLS measurements. Empty NEs showed tighter aggregation than NEsL due to the droplet dehydration effect due to the TEM procedure, and appeared with smaller size than LVX-loaded vesicles (Figure 7A). Furthermore, LVX loading does not appear to be able to modify the size and ultrastructure of the NEs (Figure 7B). The E.E. % value of LVX NEs (NEsL) is shown in Table 2. First of all, it is observable that the EE% value of the sample is 100%, suggesting the oil capability to solubilize the drug [64]. Therefore, it is possible to affirm that NEs formulation is able to greatly improve the water solubility of this hydrophobic molecule. Moreover, the drug inclusion inside the nanocarriers does not affect the size and PDI values with respect to empty NEs (Table 2). Both formulations were characterized by similar negative \(\zeta\)-potential values able to prevent vesicle aggregation during the storage due to the superficial electrostatic repulsion. In fact, according to [PERSON] et al., 2020 [65], a \(\zeta\)-potential value bigger than \(\pm\)20 mV assures sample stability. Furthermore, \(\zeta\)-potential contribution could also affect drug delivery performance. In fact, as described by [PERSON] et al., 2021 negative values of \(\zeta\)-potential could ensure better interaction and penetration within the bacterial biofilm, due to their capability to prolong the NEs/biofilm interaction time [66]. The oil droplet rigidity plays a critical role in the retention and release of the loaded drug in the nanocarriers, and the rigidity also affects the in vivo circulation time and the interaction capability of NEs with membranes or cells [67]. DPH experiments allow to collect information on oil droplet anisotropy that is correlated with the rigidity of the \begin{table}
\begin{tabular}{c c c c c c} \hline  & **Hydrodynamic** & **PDI \(\pm\) SD** & \(\zeta\)**-Potential \(\pm\) SD** & **EE (\%)** & **Anisotropy A.U.** \\  & **Diameter \(\pm\) SD (nm)** & **PDI \(\pm\) SD** & **(mV)** & **(Fluidity)** \\ \hline NEs & 165.7 \(\pm\) 2.5 & 0.22 \(\pm\) 0.02 & \(-\)24.3 \(\pm\) 0.2 & - & 0.24 \\ NEsL & 152.6 \(\pm\) 2.9 & 0.21 \(\pm\) 0.01 & \(-\)22.9 \(\pm\) 1.2 & 100 & 0.26 \\ \hline \end{tabular}
\end{table} 
Table 2: Physico-chemical characterization of samples in terms of hydrodynamic diameter, polydispersity index (PDI), \(\zeta\)-potential, LVX entrapment efficiency percentage (EE %), and anisotropy (fluidity).