*2.9. Proinflammatory Cytokine Release Quantification: TNF-*α*, IL-1*β *and IL-6*

DHAH-NLCs antiinflammatory effect against LPS (lipopolysaccharide) was carried out in microglia primary cells (Figure 3A). To perform the assay, cells were pretreated for 24 h with DHAH-NLCs and Mygliol-NLCs at different concentrations selected from studies previously carried out in Section 2.6 (50 µM, 25 µM and 12.5 µM) or just media change. After that treatment, media were removed and cells were incubated for another 24 h with LPS 50 ng/mL and the different concentration of the nanoparticles. After that 24 h, the cell media supernatant was collected and stored at −80 ◦C. The levels of TNF-α, IL-1β and IL-6 were analyzed with ELISA assay (Peprotech, London, UK). The total amount of cytokine release was normalized according to cell viability measured with CCK-8 assay at the same time point.

**Figure 2.** (**A**) Schematic representation of dopaminergic cell-based assays to evaluate the neuroprotective effects of DHAH-NLCs and Mygliol-NLCs. This figure was created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License (https://smart.servier.com). (**B**) Graphic representation of dopaminergic neuron viability after 24 h of incubation with different doses of 6-OHDA (\*\*\*\**p* < 0.0001 C+ vs C<sup>−</sup>, 500 µM, 100 µM and 25 µM \*\*\* *p* < 0.001 C+ vs 10 µM); one-way ANOVA. (**C**) Graphic representation of the neuroprotective effect of DHAH-NLCs (\*\* *p* < 0.01 25 µM 6-OHDA vs 50 µM DHAH-NLCs and 25 µM DHAH-NLCs, \* *p* < 0.05 25 µM 6-OHDA vs 12.5 µM DHAH-NLCs); one-way ANOVA. (**D**) Representative **Figure 2.** (**A**) Schematic representation of dopaminergic cell-based assays to evaluate the neuroprotective effects of DHAH-NLCs and Mygliol-NLCs. This figure was created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License (https: //smart.servier.com). (**B**) Graphic representation of dopaminergic neuron viability after 24 h of incubation with different doses of 6-OHDA (\*\*\*\* *p* < 0.0001 C<sup>+</sup> vs C−, 500 µM, 100 µM and 25 µM \*\*\* *p* < 0.001 C<sup>+</sup> vs 10 µM); one-way ANOVA. (**C**) Graphic representation of the neuroprotective effect of DHAH-NLCs (\*\* *p* < 0.01 25 µM 6-OHDA vs 50 µM DHAH-NLCs and 25 µM DHAH-NLCs, \* *p* < 0.05 25 µM 6-OHDA vs 12.5 µM DHAH-NLCs); one-way ANOVA. (**D**) Representative fluorescence images of the neuroprotective assay with DAPI staining. The scale bar indicates 50 µM.

Section 2.6 (50 µM, 25 µM and 12.5 µM) or just media change. After that treatment, media were removed and cells were incubated for another 24 h with LPS 50 ng/mL and the different concentration of the nanoparticles. After that 24 h, the cell media supernatant was collected and stored at −80 °C.

DHAH-NLCs antiinflammatory effect against LPS (lipopolysaccharide) was carried out in

fluorescence images of the neuroprotective assay with DAPI staining. The scale bar indicates 50 µM.

*2.9. Proinflammatory Cytokine Release Quantification: TNF-α, IL-1β and IL-6* 

The levels of TNF-α, IL-1β and IL-6 were analyzed with ELISA assay (Peprotech, London, UK). The total amount of cytokine release was normalized according to cell viability measured with CCK-8

**Figure 3.** (**A**) Schematic representation of the antiinflammatory assay with DHAH-NLCs and Mygliol-NLCs. This figure was created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License (https://smart.servier.com). (**B**) Graphic representation of TNF-α values (pg/mL) for all the different tested concentrations and formulations (\*\*\*\* *p* < 0.0001 C+ vs C<sup>−</sup> and 25 µM DHAH-NLCs, \*\*\* *p* < 0.001 C+ vs 50 µM DHAH-NLCs,\*\* *p* < 0.01 C+ vs 12.5 µM DHAH-NLCs); one-way ANOVA. (**C**) Graphic representation of IL-6 values (pg/mL) for all the different tested concentrations and formulations (\*\* *p* < 0.01 C+ vs C− and 50 µM DHAH-NLCs, \* *p* < 0.05 C+ vs 25µM DHAH-NLCs and 12.5µM DHAH-NLCs); one-way ANOVA. (**D**) Graphic **Figure 3.** (**A**) Schematic representation of the antiinflammatory assay with DHAH-NLCs and Mygliol-NLCs. This figure was created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License (https://smart.servier.com). (**B**) Graphic representation of TNF-α values (pg/mL) for all the different tested concentrations and formulations (\*\*\*\* *p* < 0.0001 C <sup>+</sup> vs. C<sup>−</sup> and 25 µM DHAH-NLCs, \*\*\* *p* < 0.001 C<sup>+</sup> vs. 50 µM DHAH-NLCs, \*\* *p* < 0.01 C <sup>+</sup> vs. 12.5 µM DHAH-NLCs); one-way ANOVA. (**C**) Graphic representation of IL-6 values (pg/mL) for all the different tested concentrations and formulations (\*\* *p* < 0.01 C<sup>+</sup> vs. C<sup>−</sup> and 50 µM DHAH-NLCs, \* *p* < 0.05 C<sup>+</sup> vs. 25µM DHAH-NLCs and 12.5µM DHAH-NLCs); one-way ANOVA. (**D**) Graphic representation of IL-1β values (pg/mL) for all the different tested concentrations and formulations (\*\* *p* < 0.01 C<sup>+</sup> vs. C<sup>−</sup> and \* *p* < 0.05 C<sup>+</sup> vs. 5 µM DHAH-NLCs); one-way ANOVA.

#### representation of IL-1β values (pg/mL) for all the different tested concentrations and formulations (\*\* *2.10. Statistical Analysis*

*p* < 0.01 C+ vs C<sup>−</sup> and \* *p* < 0.05 C+ vs 5 µM DHAH-NLCs); one-way ANOVA. All results are expressed as mean ± SD. The results obtained from the cell culture have been performed in *n* = 3 biological replicates for all the experiments described in this article. Experimental data were analyzed using the computer program GraphPad Prism (v. 6.01, GraphPad Software, San Diego, CA, USA). One-way ANOVA was used for analyzing all the data represented in this research article. *p* values <0.05 were considered significant.
