*3.3. In Vitro Cell Viability Study*

In order to assess the cytocompatibility of the nanoparticles, they were incubated with the dopaminergic and microglia cell cultures. After 24 and 48 h, cell viability was measured through the CCK-8 assay. Figure 7A,B illustrates the results obtained in the CCK-8 assay after incubating NLCs with a dopaminergic cell culture. None of the concentrations for the nanoformulations tested in dopaminergic cell cultures were cytotoxic, showing percentages of cell viability >70%. Indeed, as shown in Figure 7B, at 48 h, cell viability for the cultures treated with DHAH-NLCs was slightly better at any of the tested concentrations compared to Mygliol-NLCs treated cells. The results of the viability assay for the microglia cell culture are, to some extent, different. As shown in Figure 7C,D, the tested concentrations of 100 and 75 µM for the Mygliol-NLC formulation decreased cell viability below 70% at both tested time points; this effect was not seen with DHAH-NLC for any of the tested time points. This beneficial effect for DHAH-NLC was seen in any of the tested conditions, being more notorious at 50, 25 and 12.5 µM concentrations (Figure 7C,D). *Pharmaceutics* **2020**, *12*, x 15 of 23

**Figure 7.** (**A**) Dopaminergic neuron viability at 24 h. (**B**) Dopaminergic neuron viability at 48 h. (**C**) Microglia cell viability at 24 h. (**D**) Microglia cell viability at 48 h. In all cases, different concentrations of DHAH-NLCs and Mygliol-NLCs were tested (the graphs show the results of three biological **Figure 7.** (**A**) Dopaminergic neuron viability at 24 h. (**B**) Dopaminergic neuron viability at 48 h. (**C**) Microglia cell viability at 24 h. (**D**) Microglia cell viability at 48 h. In all cases, different concentrations of DHAH-NLCs and Mygliol-NLCs were tested (the graphs show the results of three biological replicates media ± SD).

#### replicates media ± SD). *3.4. 6-OHDA Neurotoxin E*ff*ect on Dopaminergic Culture*

with 87.17 ± 18.02 remaining living cells.

*3.5. DHAH-NLCs Exhibited A Neuroprotective Effect* 

*3.4. 6-OHDA Neurotoxin Effect on Dopaminergic Culture*  The incubation of dopaminergic neurons with 6-OHDA decreased cell viability in a dosedependent manner (Figure 2B). The incubation of cells with 500 µM of 6-OHDA resulted in only 6.78 ± 4.11 (\*\*\*\* *p* < 0.0001) remaining living cells, which is similar to the values obtained after the The incubation of dopaminergic neurons with 6-OHDA decreased cell viability in a dose-dependent manner (Figure 2B). The incubation of cells with 500 µM of 6-OHDA resulted in only 6.78 ± 4.11 (\*\*\*\* *p* < 0.0001) remaining living cells, which is similar to the values obtained after the incubation with our C<sup>−</sup> (DMSO 10%) (5.45 ± 5.2, \*\*\*\* *p* < 0.0001). In the following test concentrations, the values for 100

In the case of 25 µM, the remaining living cells were around 50% (49.05 ± 7.79, \*\*\*\* *p* < 0.0001), and, for 10 µM, it was slightly higher (60.37 ± 24.33, \*\*\* *p* < 0.001). Finally, the incubation of dopaminergic cells with 5 µM 6-OHDA neurotoxin led to no statistical difference when compared to the C+ group,

After selecting the dose for generating a cell death of 50% after 24 h of incubation with 6-OHDA, 25 µM, we tried to demonstrate the neuroprotective effect of our DHAH-NLCs. The incubation of the dopaminergic neuron culture with DHAH-NLCs before and after 6-OHDA addition resulted in a neuroprotective effect in comparison to the neurotoxin itself (Figure 2C). The treatment with Mygliol-NLCs did not increase cell viability, with remaining living cell values similar to those obtained with just the neurotoxin incubation. The values for 50 µM Mygliol NLCs (38.35 ± 33.45), 25 µM Mygliol

and 50 µM were below 50% of the remaining living cells; more specifically, for 100 µM (13.82 ± 8.24, \*\*\*\* *p* < 0.0001) and for 50 µM (33.37 ± 11.87, \*\*\*\* *p* < 0.0001), the values were obtained. In the case of 25 µM, the remaining living cells were around 50% (49.05 ± 7.79, \*\*\*\* *p* < 0.0001), and, for 10 µM, it was slightly higher (60.37 ± 24.33, \*\*\* *p* < 0.001). Finally, the incubation of dopaminergic cells with 5 µM 6-OHDA neurotoxin led to no statistical difference when compared to the C<sup>+</sup> group, with 87.17 <sup>±</sup> 18.02 remaining living cells.
