**1. Introduction**

Melanins are the primary determinants of skin, hair and exoskeletal pigmentation in mammals, birds and insects [1–6], and their importance has been growing over the past few years not only from a biological point of view, related to their role in the human body, but also for the exploitation of their unique properties in biomedicine or in the cosmetic and health sectors [7–16]. These pigments are biosynthesized in melanocytes, starting with the oxidation of tyrosine to dopaquinone catalyzed by the enzyme tyrosinase (Figure 1) [1,17,18]. Dopaquinone may then undergo cyclization leading, after a further oxidation step, to 5,6-dihydroxyindole (DHI) or 5,6-dihydroxyindole-2-carboxylic acid (DHICA) whose oxidative polymerization ultimately leads to melanin pigments brown or dark in color, known as eumelanins [1,9,18]. On the other hand, entrapment of dopaquinone by cysteine, a process which is under genetic control, gives rise to isomeric cysteinyldopas whose polymerization is responsible for the biosynthesis of the reddish-brown pigments known as pheomelanins, typical of the red hair phenotype [1,19,20] (Figure 1).

**Figure 1.** Biosynthetic pathways leading to eumelanins and pheomelanins.

Traditionally, eumelanins have been attributed a role as antioxidant and photoprotective agents in dark-skinned phenotypes, whereas pheomelanins have been implicated in the enhanced susceptibility to skin cancer of individuals belonging to the red-hair phenotype due to their photosensitizing and pro-oxidant properties [1,9,21–26]. Among eumelanins, bionspired synthetic pigments obtained by oxidative polymerization of DHICA have shown remarkable antioxidant properties, and have been proposed as a plausible explanation for the high content of DHICA-related units in natural eumelanins [27,28]. Indeed, DHICA-melanin is able to act as a potent hydroxyl radical scavenger in the Fenton reaction [29] and has been found to act as an efficient antioxidant and radical scavenger also in the 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2--azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and nitric oxide scavenging assays [30]. DHICA-melanin also exhibited inhibition properties against in vitro lipid peroxidation [31], whereas silica/DHICA-melanin hybrid nanoparticles exerted protecting effects against hydrogen peroxide-induced cytotoxicity [32]. More recently, the higher antioxidant activity of DHICA-melanin compared to DHI-melanin has also been confirmed by the Folin–Ciocalteu assay [33].

The antioxidant properties of DHICA-melanin seem to also play a role in the maintenance of immune hyporesponsiveness to melanosomal proteins of relevance for the onset of autoimmune

vitiligo [34]. Recently, a natural pigment isolated from marine *Aspergillus nidulans* and tentatively identified as a DHICA-melanin exhibited protective e ffects against Ultraviolet B (UVB)-induced oxidative stress in cellular and mice models [35,36]. UV radiations are known to be very harmful for the human skin, as they induce reactive oxygen species (ROS) production. Both Ultraviolet A (UVA) and UVB are able to induce DNA damages [37,38]. UVB radiations induce DNA dimerization reactions between adjacent pyrimidine bases, whereas UVA radiations, weakly absorbed by DNA, can excite endogenous chromophores, leading to mispairing of DNA bases with consequent translation of mutated proteins [39].

In this context, the remarkable antioxidant properties of DHICA-melanin and its chromophoric characteristics, allowing significant absorption in the UVA region [1,9,30], would sugges<sup>t</sup> its use in dermo-cosmetic formulations with photoprotective action.

Yet, full exploitation of DHICA-melanin has so far been hampered by the low solubility in lipophilic or hydroalcoholic solvents usually employed in cosmetics, and the relatively high susceptibility to (photo)degradation [40,41]. On these bases, we recently developed a variant of DHICA-melanin which was obtained by oxidative polymerization of the methyl ester of DHICA (MeDHICA-melanin) and shown to consist of a collection of intact oligomers from the dimer up to the heptamer by MALDI-MS analysis (Figure 2) [42]. The material was characterized by an intense and broad absorption band centered at 330 nm and proved to be soluble in water miscible organic solvents. Moreover, MeDHICA-melanin retained the antioxidant properties of DHICA-melanin, proving indeed even more active. It was also stable to prolonged oxidation or exposure to a solar simulator [42].

**Figure 2.** Structure proposed for the methyl ester of 5,6-dihydroxyindole-2-carboxylic acid (MeDHICA)-melanin based on MALDI-MS analysis [42].

Here, we report the protective e ffect of MeDHICA-melanin, prepared by aerial oxidation of MeDHICA, on oxidative photodamage of immortalized human keratinocytes (HaCaT) induced by UVA-exposure. Keratinocytes represent the most exposed cellular layer in the epidermis, functioning as a protective barrier from environmental stimuli, pathogens and radiation, and it is now generally recognized that molecules endowed with antioxidant activity, especially polyphenols, can strengthen the barrier function of keratinocytes from photoaging [43,44].

#### **2. Materials and Methods**
