## 3 Results and Discussion
### Physicochemical Stability  
Stability is the crucial parameter of any nanocarrier in which several aspects must be understood carefully to produce a future commercial product. During the design and the first steps of the production of the nanocarrier, various factors must be considered to obtain a stable nanocarrier [69]. The preparation method, such as the component choice, should be selected to optimize droplet size distribution since it strongly affects stability behavior. In particular, regarding composition, the surfactant employed, its concentration, and oil type, are of great importance [70]. Moreover, the hydrodynamic diameter (i.e., systems with droplets diameter smaller than 200 nm usually have a homogeneous and stable structure) together with an opportune \(\zeta\)-potential value (able to preserve the coalescence phenomena) could affect the stability of the nanocarriers [71]. The stability of nanocarriers is determined by the balance between electrostatic repulsion due to their surface charge and short-range attractive interactions between nanoparticles [72]. Z-potential plays a crucial role in the stability of formulated NEs. As reported in the literature, high negative values of \(\zeta\)-potential prevent aggregation or precipitation phenomena [73]. In an ideal scenario, nanocarriers need to remain stable, avoid aggregation, and retain the drug until they reach the target site. Instability can negatively impact their effectiveness in vivo. Consequently, evaluating the stability of nanocarriers is a crucial aspect of their characterization. Various techniques exist to predict the stability of nanocarriers in different environments--physical, chemical, and physiological--depending on the specific nature of the nanocarriers. Freezing and lyophilization are widely employed for the long-term storage of several nanoparticles. Nevertheless, these processes can induce stresses from crystallization and vacuum dehydration that may damage macromolecules, reducing the stability of the nanoparticles. To mitigate this issue, suitable cryoprotectants could be employed but the added components could affect the drug carrier's features [74]. In the present study, the nanocarriers' stability in aqueous condition has been evaluated. To assess the stability over time of empty and loaded NEs, all the samples were stored at room temperature and 4 \({}^{\circ}\)C for 90 days and hydrodynamic diameter and \(\zeta\)-potential were evaluated. Analyzing the obtained results reported in Figure 8, it is possible to affirm that all samples preserved their size and \(\zeta\)-potential at both 4 \({}^{\circ}\)C and room temperature for up to 90 days, with only a slight and not significant increase of a few nanometers in particle size observed in both empty and loaded NEs. Probably, the samples are characterized by a significant stability over time due to (i) negative \(\zeta\)-potential values (\(-24\) mV/ \(-23\) mV, respectively, for empty and loaded NEs) representing the ability to prevent aggregation or precipitation phenomena; (ii) dimensions smaller than 200 nm; and (iii) appropriate preparation method (high-energy method, with specific and selected sonication parameters) able to control NEs droplet size [75]. These results suggest that an aqueous condition is a suitable way for long-term storage of NEs.