**4. Conclusions**

UVA radiations are highly harmful as they can penetrate the skin, crossing the epidermis and reaching the dermis. They are responsible for a variety of physiopathological conditions, ranging from inflammation to premature skin aging and skin cancer development [45,57,58]. On the other hand, the antioxidant and photoprotective properties of eumelanins and model pigments from DHICA are well-established [29,30,41,42].

In this paper, MeDHICA-melanin, previously shown to possess marked in vitro antioxidant activity and favorable solubility properties for dermo-cosmetic applications [42], was demonstrated to exert protective effects on a cellular model of immortalized keratinocytes (HaCaT) exposed to UVA radiations. All the endpoint parameters of oxidative stress, i.e., ROS, lipid peroxidation, and intracellular oxidized glutathione levels, were significantly inhibited in cells incubated with MeDHICA-melanin at concentrations compatible with cell viability. Moreover, a comparison with related findings recently reported in the literature on natural phenols or phenol-rich extracts further highlighted the advantages of MeDHICA-melanin.

Similarly to other natural antioxidants [58], MeDHICA-melanin also proved to provide protection by activating the Nrf-2 pathway. Indeed, the nuclear translocation of Nrf-2 was effective, as demonstrated by the activation of downstream genes. To our knowledge, this is the first report on the correlation between the antioxidant activity of melanins and the Nrf-2 pathway. We also demonstrated that MeDHICA-melanin was able to enter keratinocytes, although, as expected, only

Low molecular weight components such as dimeric compounds were appreciably internalized after 1 h incubation. These data provide further confirmation to biological studies indicating that keratinocytes are able to internalize melanins as the result of a cross-talk with melanocytes, mediated by the protease-activated receptor 2 (PAR-2) and the Ras-related protein Rab11 [59,60]. Overall, our findings demonstrate that MeDHICA-melanin is able to enter the cells and activate the antioxidant system to protect skin cells from UVA-induced damage, encouraging its use as an effective component in dermo-cosmetic formulations for the treatment of skin damage, photoaging and skin cancers.

**Figure 7.** HPLC and LC-MS analysis of cell lysate. (**A**) HPLC profile of acetylated cell lysate (blue trace) and acetylated lysate from control cells (not incubated with MeDHICA-melanin) (red trace). (**B**) LC-MS extracted ion chromatogram (*m*/*z* 581). (**C**) MS spectrum of the compound eluted at 19 min.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3921/9/4/270/s1, Figure S1: Lipid peroxidation levels evaluated by TBARS assay.

**Author Contributions:** Conceptualization, D.M.M., L.P. and A.N.; methodology, D.M.M., L.P. and A.N.; validation, D.M.M., L.P. and A.N.; investigation, D.L and M.L.A.; data curation, D.M.M., L.P. and A.N.; writing—original draft preparation, D.L., M.L.A, D.M.M and L.P.; writing—review and editing, D.M.M., L.P. and A.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded in part by Kao Corporation.

**Conflicts of Interest:** The authors declare no conflict of interest.
