## 3 Results and Discussion
### Characterization of Nanoparticles
Moreover, factors, such as age, gender, skin thickness and vascularity, density of hair follicles, mechanical trauma (integrity of the skin), and diseases can also have an impact on topical administration [1]. In addition to the particle's size, it is also important to have monodisperse batches in order to better predict the stability, as well as the in vivo behavior of any nanoproduct. The PdI values for extract-loaded PLGA NPs presented the same statistical difference for the three temperature conditions tested. In general, they presented monodisperse populations over time, except at time 0 for the three temperatures and after 12 months under RT. Furthermore, under AC the results suggest that those NPs present higher polydisperse populations which were stable over time, mainly at RT. Although, after encapsulation, an increase of the PdI value was observed, this improvement was less pronounced over time for RC and RT. The extract-loaded PCL NPs presented polydisperse populations, mainly at RC and RT. The major difference between the values of time 0 and time 12 was verified at RT, followed by RC and finally the AC. These results suggest that these NPs are stable over time, mainly at AC, where the values of PdI are closer to monodisperse populations (<0.25, as previously mentioned). The extract-loaded PCL NPs presented a decrease of PdI, that it was only maintained under AC. To conclude, there was a reduction of PdI of extract-loaded PLGA NPs, while extract-loaded PCL NPs exhibited an increase of PdI at RC and RT, with a decrease at AC. This means that PLGA NPs might be more stable. The addition of extract to the PLGA NPs displayed an increase in size, which is in line with the literature for other similar compounds in the same type of NPs [5; 13]. However, concerning the PCL NPs, they presented similar sizes with a slight decrease, which is also in line with the literature [12]. Nevertheless, the overall assessment of PdI confirms the physical stability of the formulations throughout the study. The addition of extract to the formulation resulted in an initial decrease of PdI. pH was also assessed as a good stability prediction parameter. Concerning pH values, the extract-loaded PLGA NPs' suspensions presented the same statistical difference of \(p\) < 0.001 over time, and at the different analyzed temperatures, which suggests that the variation of temperature has not influenced pH values. However, the major difference between the values at time 0 and time 12 was observed at AC, followed by RT and finally by RC. In this sense, the results suggest that the extract-loaded PLGA NPs are stable over time, mainly at RC. After encapsulation, an increase of pH value of NPs suspension was observed, which was maintained over time, probably due to the PLGA hydrolysis to lactic and glycolic acids. The extract-loaded PCL NPs presented the same statistical difference over time at the three different temperatures, which suggests that the change of temperature was not influenced by the statistical difference of pH values of NPs suspension. However, the major difference between the values at time 0 and time 12 was verified at RT, followed by RC and AC. From that point of view, these results suggest that the extract-loaded PCL NPs are stable over time, mainly at AC. Similarly to what was observed in PLGA NPs, the extract's encapsulation into the PCL NPs increased the pH values of the respective suspension. Both values observed throughout the study are compatible to skin application [32], where the ideal range of pH is 3.5-5.0 [38; 39]. After encapsulation, the extract-loaded formulation displayed an increase of pH value, which is in line with the literature in the same type of NPs, PLGA [5] and PCL [12]. The zeta potential analysis revealed that all the extract-loaded NPs (PLGA and PCL) were negatively charged. Both are stable over time, mainly at RC and AC, respectively.