*3.6. DHAH-NLCs Decreased Cytokine Proinflammatory Release*

In order to assess the antiinflammatory effect of our DHAH-enriched nanoparticles (DHAH-NLCs), we performed the ELISA technique from a cell culture supernatant, as described in the Materials and Methods section. The results obtained from the cell supernatants for TNF-α, IL-1β and IL-6 cytokines are summarized in Figure 3B–D.

Regarding TNF-α ELISA (Figure 2B), the basal levels of TNF-α, C<sup>−</sup> (204.0 ± 23.96) statically increased (\*\*\*\* *p* < 0.0001) after LPS treatment (50 ng/mL), named C<sup>+</sup> (879.4 <sup>±</sup> 204.0). In addition, although DHAH-NLCs were not able to obtain control levels of this cytokine, it is noteworthy that the effect of this treatment decreases this proinflammatory cytokine release, as seen in Figure 3B. This effect can be seen in all DHAH-NLCs concentrations: 50 µM DHAH-NLCs (581.0 ± 100.1, \*\*\* *p* < 0.001), 25 µM DHAH-NLCs (532.1 ± 186.0, \*\*\*\* *p* < 0.0001) and 12.5 µM DHAH-NLCs (609.2 ± 55.1, \*\* *p* < 0.01), without any statistically significant differences between the different doses. In the case of Mygliol-NLCs, we could not see any positive effect decreasing cytokine levels for any of the tested concentrations (*p* > 0.05).

For the second tested cytokine, IL-6 (Figure 3C), we also observed the increase of basal levels (213.7 ± 47.3) up to almost double concentration (365.1 ± 125.9, \*\*\* *p* < 0.001) after the incubation with LPS. In this case, we could also see the decrease of this proinflammatory cytokine after the treatment with DHAH-NLCs. All the tested concentrations demonstrated a positive effect, decreasing IL-6 levels; indeed, 50 µM DHAH-NLCs (219.3 ± 29.3, \*\* *p* < 0.01), 25 µM DHAH-NLCs (232.7 ± 40.8, \* *p* < 0.05) and 12.5 µM DHAH-NLCs (237.2 ± 15.4, \* *p* < 0.05) were the obtained values. However, no statistically significant differences were observed between the different concentrations. Regarding Mygliol-NLCs, no antiinflammatory effect was obtained after the treatment with these nanoparticles for any of the tested concentrations (*p* > 0.05).

Finally, we analyzed the levels of IL-1β proinflammatory cytokine (Figure 3D). In this case, the LPS stimuli also increased the basal values (457.4±125.8) more than three-fold, as we can see for the C<sup>+</sup> values (1495.0 ± 518.1, \*\* *p* < 0.01). As seen before with the other two proinflammatory cytokines, the treatment with DHAH-NLCs decreased IL-1β values. Actually, the values for DHAH-NLCs were the following: 50 µM DHAH-NLCs (726.2 ± 256.6, \* *p* < 0.05), 25 µM DHAH-NLCs (948.7 ± 301.6) and 12.5 µM

DHAH-NLCs (985.6 ± 301.0). Although in this case only the group treated with 50 µM DHAH-NLCs showed a statistically significant difference, the trend to decrease this proinflammatory cytokine is notorious in all DHAH-NLCs concentrations. As in the previously analyzed proinflammatory cytokines, the levels of IL-1β remained elevated for the cells treated with Mygliol-NLCs, for any of the tested conditions, showing values similar to the positive control.
