*2.8. Neuroprotective Assay*

After 7–10 days of maintaining a dopaminergic culture, a neuroprotective assay was carried out with the developed nanocarriers (Figure 2A). The media were removed and different concentrations of DHAH-NLCs and Mygliol NLCs were added to the cells (50, 25 and 12.5 µM) 24 h before the 6-OHDA neurotoxin was added to the culture. 24 h after, the media were removed and fresh media were added, with a final concentration of 25 µM 6-OHDA neurotoxin and the previously tested concentrations for NLCs (50, 25 and 12.5 µM). To assess the neuroprotective effect of DHAH-NLCs against 6-OHDA neurotoxin, the media were removed 24 h later and cells were fixed with 3.7% paraformaldehyde (PFA) for 10 min and, then, washed three times in PBS. DAPI staining (1:10,000) in PBS for 15 min was used to determine viable cells. For cell viability quantification, fluorescence microscopy images were obtained by means of an inverted microscope (Nikon TMS, Hampton, NH). Two images per well and three wells were used for each group; in total, six images were taken per group. Dopaminergic cells were scored as positive if they exhibited defined nuclear counterstaining. The data are expressed as the percentage of the C<sup>+</sup> group with no treatment but just media change, which was set as 100%.
