## 3 Results and Discussion
### Characterization of Nanoparticles  
Nanotechnology was used to increase the stability of active compounds, to modulate their release, as well as to improve the solubility of poorly water-soluble compounds [1; 32; 33]. Thus, two different polymeric NPs for skin delivery were selected: PLGA and PCL NPs polymers [12; 34]. Figure 4 shows the results of mean size, PdI, pH, and zeta potential of NPs suspension over time and at different storage temperatures: Refrigerated Conditions (RC), Room Temperature (RT), and Accelerated Conditions (AC). Figure 3: Comparison of chromatographic profile of a MeOH reference with 3025 ppm and a sample of extract as generated by SPME-GC-MS.  
Figure 2: MRM chromatograms of rutin and naringenin in elderflowers’ methanolic extract. The DLS analysis demonstrated that empty PLGA and PCL NPs were stable over time for all parameters in the three different tested temperatures. After loading, the mean size of extract-loaded PLGA NPs at RC and AC decreased from month zero (\(471.3\pm 169.9\) and \(480.0\pm 150.8\) nm) to month 12 (\(164.2\pm 6.4\) and \(180.8\pm 78.0\) nm). However, at RT this effect was not observed. In this sense, the results suggest that extract-loaded PLGA NPs are stable over time, mainly at RT. An increase of size after extract encapsulation was observed; however, this change is less pronounced over time for all temperatures. In the case of extract-loaded PCL NPs, at RC and RT, an increase of size values from \(242.7\pm 33.7\) nm and \(245.5\pm 35.3\) nm (0 months) to \(882.1\pm 189.4\) nm and \(451.6\pm 147.9\) nm (12 months), respectively, was observed. However, at AC, they presented similar values over time from \(192.7\pm 83.8\) nm (0 months) to \(173.6\pm 12.2\) nm (12 months). A slight increase of size was observed after extract encapsulation; however, this increase is less pronounced over time for all temperatures. Concerning the extract-loaded PCL NPs, the influence of temperature on NPs means that size was temperature-dependent, decreasing with the increase of the temperature. To conclude, the extract's encapsulation caused an increase in the particle's size, which is in line with what the literature mentions for other or similar compounds of the same type of NPs [5; 13]. However, PCL NPs presented similar sizes, with only a slight decrease, which is also in line with the literature, for this type of NPs [12]. In contrast with empty NPs, the extract-loaded PLGA NPs revealed a decrease in size at the different temperatures tested over 12 months, while the extract-loaded PCL NPs presented an increase in size for RC and RT and a decrease in size at AC. The decrease of PLGA NPs' can be associated to the intrinsic nature of the polymer; PCL presents a higher hydrophobicity that can also contribute to this change [35]. Moreover, as it was verified by the TEM and AFM analysis, the PCL NPs presented an higher agglomeration tendency when compared to the PLGA NPs, which is also supported by the literature [36]. Figure 4: Mean size, PdI, pH, and zeta potential of empty PLGA NPs (white columns), empty PCL NPs (light grey columns), extract-loaded PLGA NPs (black columns) and extract-loaded PCL NPs (dark grey columns) suspension over time: mean size (**a–c**), PdI (**d–f**), pH (**g–i**), and zeta potential (**j–l**). (**a**) size at RC, (**b**) size at RT, (**c**) size at AC, (**d**) PdI at RC, (**e**) PdI at RT, (**f**) PdI at AC, (**g**) pH at RC, (**h**) pH at RT, (**i**) pH at AC, (**j**) zeta potential at RC, (**k**) zeta potential at RT, and (**l**) zeta potential at AC (mean \(\pm\) S.D., \(n=9\)). Refrigerated Conditions—RC, Room Temperature—RT, and Accelerated Conditions—AC. Particle's size is considered as a key factor for skin delivery, although the ideal size of NPs is still not consensual. According to the literature, particles between 300 and 500 nm can penetrate through intact skin [37]. However, the administration of NPs can be affected by skin barrier changes such as scrapes, wounds, dermatitis conditions, etc.