Cutaneous melanoma (CM) represents the malignant neoplasm that originates from the cancerous switch of epidermal melanocytes due to aberrant molecular and biochemical changes. Despite being the third most frequent skin malignancy after basal cell and squamous cell carcinomas, accounting for less than 5% of all cases, CM remains the deadliest and the most aggressive cutaneous cancer, having a high metastatic potential and a mortality rate that exceeds 10% (Coricovac et al., 2018; Davey et al., 2021). Epidemiologically, the overall global incidence of CM presents an alarming annual increase, surpassing the rate of any other cancer type (Leonardi et al., 2018). Numerous environmental and genetic risk factors have been correlated with CM oncogenesis (Conforti and Zalaudek, 2021). Although there is a certain interplay between these factors that contributes to CM arising, the excessive exposure to ultraviolet radiation (UVR) emanated by natural or artificial sources remains the greatest trigger for the development of the “cancer that rises with the Sun” (Coricovac et al., 2018; Burns et al., 2019; Davey et al., 2021). Additionally, cutaneous pigmentation–mainly caused by the production and accumulation of melanin (MEL) within the epidermal level of the skin, plays an indisputable key role in the susceptibility of melanocytes to malignant transformation (Conforti and Zalaudek, 2021; Moreiras et al., 2021).

MEL is a generic term defining a widely spread class of pigments with diverse structure and origin, synthesized by melanocytes through a unique and strictly-controlled process known as melanogenesis, via the oxidation and polymerization of tyrosine (Mostert, 2021; Marcovici et al., 2022). This process results in the obtainment of two distinct MEL subtypes: the brown-to-black eumelanin comprising 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) moieties, and the yellow-to-red pheomelanin containing sulphurous benzothiazine and benzothiazole units (Moreiras et al., 2021; Marcovici et al., 2022). Melanogenesis is regulated by several factors acting at the systemic, tissue, cellular, and subcellular levels. UVR was established as the primordial exogenous factor regulating cutaneous melanogenesis, nonetheless, skin pigmentation is also influenced by other factors (e.g., nutritional, endocrine, paracrine, autocrine, intracrine) that involve the interaction between melanocytes and keratinocytes (Slominski et al., 2022). Both melanocytes and CM cells present the ability of producing MEL, and thus causing pigmentation (Bratu et al., 2022). However, melanogenesis is highly dysfunctional in CM cells, which can shift from pigmented to non-pigmented states (Sarna et al., 2019). Therefore, based on the presence or absence of this pigment, CM has been classified as melanotic (pigmented) and amelanotic (unpigmented) (Bratu et al., 2022). Up to date, the ability of MEL to prevent CM development and progression has been documented. The pigment acts as a an UVR-absorbing and photoprotective agent that shields epidermal cells from radiation-induced DNA damage, and oxidative stress, this activity being attributed to the eumelanin subtype (Solano, 2020; Cabaço et al., 2022; Slominski et al., 2022). The presence of endogenous MEL was also correlated to impaired CM metastasis and invasiveness under both in vitro and in vivo conditions, by enhancing the stiffness and attenuating the elasticity of CM cells, while also diminishing their spread in nude mice (Sarna et al., 2018; 2019; Cabaço et al., 2022). Moreover, amelanotic CM was associated with a lower patient survival rate and higher metastatic potential compared to melanotic CM (Sarna et al., 2019).

Recently, MEL has garnered significant attention from the scientific community owing to its numerous therapeutic properties (e.g., antioxidant, anti-hemolytic, immunomodulatory, anti-neoplastic), exceeding conventional drugs in terms of safety profile and broad activity spectrum (Marcovici et al., 2022). An increasing importance has been appointed to the anti-carcinogenic effect of MEL, its potential efficacy in the treatment of several cancers (e.g., colorectal, leukemia) being explored in previous preclinical studies (Öberg et al., 2009; Al-Obeed et al., 2020; El-Obeid et al., 2020). Additionally, MEL was also found to exert anti-melanoma properties, presenting cytotoxic and pro-apoptotic effects in CM cells (Rudrappa et al., 2023). The abrupt and continuous expansion of nanotechnology, as well as the fascinating functionalities of the natural pigment (e.g., biocompatibility, broadband light absorption, chelating ability, radical-scavenging effects, etc.), fueled the strategic design of versatile MEL-based nanoplatforms for various biomedical applications (Kohri, 2019; Liu et al., 2020; Marcovici et al., 2022). The majority of the as-made MEL-like nanostructures were developed from polydopamine (PDA), the artificial analogue of the natural eumelanin in terms of precursors, synthesis pathways, and structure-property relationship, which has gained a noteworthy popularity especially due to its diverse functions in nanotechnology-based cancer diagnosis and treatment, either as a platform with intrinsic antineoplastic properties, or as a carrier for anti-cancer agents (Liang et al., 2023; Liu et al., 2020; Marcovici et al., 2022). Despite these previous findings, the therapeutic potential of MEL/PDA nanoparticles in CM management remains insufficiently explored nowadays, urging further investigations in this research area.

Therefore, driven by the innate ability of CM in producing MEL, the known implications of the pigment in CM prevention, the inherent anti-melanoma effects of MEL, the unprecedented outburst in MEL/PDA-based nanotechnological advancements and the recent studies of the anti-tumor activity of MEL and MEL/PDA nanoparticles, the present study endeavors to conduct a preclinical evaluation of the anti-cancer properties retained by artificially produced bioinspired MEL nanoparticles (MEL-NPs) as a potential platform with applications in CM therapy. Debuting with the physicochemical characterization of the obtained MEL-NPs, the study continues by exploring their safety profile in healthy cutaneous cells (human melanocytes - HEMa, human keratinocytes - HaCaT, murine epidermal cells - JB6 Cl 41-5a) and anti-neoplastic properties in CM cells (human melanoma cells - SH-4, murine melanoma cells - B164A5) and ends with the investigation of their irritant potential and anti-angiogenic effect in ovo, furnishing novel perspectives to the applications of MEL-like nanostructures in CM treatment.

CM remains a leading contributor to the global increases in mortality rates caused by skin cancers, owing to its resistance to therapy, aggressive and metastatic nature, but also to the severe adverse effects and lack of tumor-specificity of the current treatment approaches (Cassano et al., 2021; Dhanyamraju and Patel, 2022). Thus, the modern pharmaceutical and biomedical research has prioritized the development of state-of-the-art site-specific therapeutics, the applications of nanotechnology promptly expanding within the area of CM diagnosis and treatment (Chen et al., 2013; Cassano et al., 2021). The outstanding properties and innate biocompatibility of MEL and PDA hastened the obtainment of MEL/PDA-based nanomaterials for cancer therapy (Qi et al., 2019; Zheng et al., 2022), being able to trigger therapeutic cellular responses per se, in the absence of drug cargoes (Liang et al., 2023). However, their applications in CM management lack a thorough investigation at present, requiring supplemental research. In the light of these data, the current work proposed the synthesis, physicochemical characterization, and preclinical pharmaco-toxicological screening of bioinspired MEL-NPs as a potential nanomedicine for CM, revealing several novelties, as follows: (i) the obtained MEL-NPs present a size of around 85.61 nm, a sphere-like shape, and a strong antioxidant potential; (ii) MEL-NPs retain a proper cytocompatibility in healthy skin cells (HEMa, HaCaT, and JB6 Cl 41-5a) at concentrations up to 75 µg/mL; (iii) MEL-NPs exert potent anti-tumor and anti-migratory activities in SH-4 and B164A5 CM cells after 24 h of treatment; (iv) the anti-neoplastic effect of MEL-NPs in CM cells is related to their efficacy in modulating the EMT, triggering apoptosis, and generating intracellular oxidative stress; and (v) MEL-NPs are non-irritant on the CAM and suppress angiogenesis in ovo.

The first point of interest in the current study was the physicochemical characterization of MEL-NPs (Figure 1) which were artificially produced through the controlled oxidative polymerization of dopamine in alkaline conditions, a method routinely employed for the obtainment of this type of nanoparticles which generally present a round morphology and a size up to 500 nm (Lemaster et al., 2019; Mavridi-Printezi et al., 2020; Marcovici et al., 2022). The as-synthesized MEL-NPs exhibited a clear sphere-like shape, and an average size of 85.61 nm with a small population of MEL-NPs presenting a dimension higher than 100 nm. The specific characteristics of their UV-VIS spectrum included a gradual decrease of absorbance from 200 nm to 1,000 nm, which is in accordance with the optical features of eumelanin (Ju et al., 2011), and other studies reporting the UV-VIS spectra of MEL or MEL-NPs (Li et al., 2018; Madkhali et al., 2019).

Oxidative stress plays a crucial role in different phases of melanomagenesis from tumor initiation and progression, to metastasis onset and chemoresistance development, CM exhibiting particularly elevated ROS levels compared to other solid cancers (Pizzimenti et al., 2021; Becker and Indra, 2023). Thus, antioxidants have become a promising strategy for CM management, with several radical-scavenging products showing potential applications in CM prevention and progression blockage (Becker and Indra, 2023). Based on these aspects, the study also included the assessment of the antioxidant activity retained by MEL-NPs at five increasing concentrations of interest – 10, 25, 50, 75, and 100 µg/mL, by applying the DPPH assay, defined as a simple, reliable, and widely used in vitro non-cellular test (Semenescu et al., 2023). MEL-NPs showed a concentration-dependent scavenging potential of the DPPH radical that was comparable to the antioxidant efficacy of ascorbic acid and gradually increased from 18.27% (at 10 µg/mL) to 84.17% (at 100 µg/mL), results that confirm previous reports, as MEL-like (PDA) nanostructures have been employed and thoroughly studied before as efficient oxidative stress scavengers (Yang et al., 2020). Lou et al. demonstrated the ROS-suppressing effects of PDA nanoparticles which showed a concentration-dependent clearance of O₂−, OH, and DPPH radicals (Lou et al., 2021). Similarly, Ju and colleagues showed that the antioxidant activity of MEL-NPs increased dose-dependently up to 85%, stressing the importance of particle size in the DPPH scavenging ability of MEL-NPs: the smallest nanoparticles (68 ± 21 nm) exerting almost the same scavenging activity as ascorbic acid, while the ones with the highest size (291 ± 57 nm) presenting the lowest efficacy (Ju et al., 2011). The obtained results can be also correlated with the antioxidant ability of the natural pigment resulting from its efficacy in binding metal ions or quenching excited molecules and free radicals due to the chemical structure formed of phenol and indole groups, and especially to the eumelanin-specific DHICA unit that comprises of an extra carboxylate group (Caldas et al., 2020; Yang et al., 2020).

The second aspect investigated herein was the biocompatibility of MEL-NPs in cutaneous non-tumorigenic HEMa, HaCaT and JB6 Cl 41-5a cell lines, selected as representative in vitro models for cutaneous toxicity evaluation due to their specific characteristics. HEMa are human primary epidermal melanocytes presenting a needle-like, stellar, multipolar, or dendritic aspect and applications in skin diseases such as CM (https://www.atcc.org/). HaCaT represent spontaneously immortalized human keratinocytes (KCs) that contain similar surface markers and functionalities as isolated KCs and are employed in epidermal homeostasis and pathophysiology studies (Colombo et al., 2017; Blanchard et al., 2022). The murine JB6 Cl 41-5a epidermal cells exhibit an epithelial morphology and are currently employed in skin toxicity investigations (https://www.atcc.org/; Tewari-Singh et al., 2010). MEL-NPs are generally considered as highly biocompatible due to their similarity to the natural pigment which is produced within melanocytes in a nanosized form (Marcovici et al., 2022). The results obtained during this study (Figure 2) indicated that these nanoparticles triggered a concentration-dependent decline in the viability of skin-derived cells. Thus, MEL-NPs lacked cytotoxic potential in HEMa, HaCaT and JB6 Cl 41-5a cells up to the concentration of 75 µg/mL, while at 100 µg/mL a significant decline in cell viability (under 70%) was noticed. Moreover, at the lowest concentration, 10 µg/mL, MEL-NPs exerted a stimulatory effect on the viability of all healthy cell lines. In a recent report, PDA nanosheets presented a low cytotoxicity in L929 fibroblasts after 24, 72, and 144 h of treatment, the cells’ viability remaining over 90% (Chen et al., 2021). A significant decrease in the viability of mesenchymal stem cells’ viability was obtained only after their 24 h stimulation with MEL-NPs at a high concentration (200 µg/mL), while at lower ones (20 and 40 µg/mL), a stimulatory effect was noted (Tang et al., 2022).

The third interest of this research was the intrinsic anti-melanoma properties exerted by MEL-NPs that were explored using two melanogenic CM cell lines–SH-4 and B164A5. SH-4 is a human MEL-producing BRAF-mutated CM cell line formed of a mixture of spindle- and epithelial-shaped cells (https://www.atcc.org/; Bratu et al., 2020; Gambichler et al., 2023). B164A5 is a fibroblast-like highly-aggressive murine CM cell line capable of producing MEL in very high quantities (Danciu et al., 2013; Danciu et al., 2015), which was previously selected as an in vitro model for the investigation of the anti-tumor effects of various nanoplatforms (Soica et al., 2014; Danciu et al., 2019). According to Figure 2, MEL-NPs reduced the viability of SH-4 and B164A5 cells in a dose-dependent trend, a significant loss of viability being obtained starting with 25 µg/mL. Nonetheless, cytotoxicity, evidenced by a decline of viability under 70% according to the ISO Standard 10993-5:2009 (Marcovici et al., 2024), was obtained starting with the concentration of 50 µg/mL in CM cells compared to 100 µg/mL in healthy cutaneous cells. A previous study conducted by Nieto et al. described PDA as being an antineoplastic system by inducing cytopathic effects in BT4T4 and HCT116 cancer cells after 24, 48, and 72 h of treatment (Nieto et al., 2018), as well as to the findings of Perring and colleagues who described an increase in MO59K, RH30, RD, and U87 cancer cell death exposed to MEL-NPs for 24 h, at concentrations up to 200 µM (Perring et al., 2018). The cytocidal properties of MEL-NPs were associated with a significant shape change in SH-4 and B164A5 cells which became round and shrinked following treatment (Figure 3). One factor ensuring the successful application of nanomaterials in biomedicine is their interactions with target cells, resulting in cytoplasmic internalization and consequent cellular responses (Kim et al., 2012; Donahue et al., 2019). After 24 h of treatment, both SH-4 and B164A5 cells presented signs of pigmentation, evidencing the ability of MEL-NPs to enter and reside within their intracellular space and adopt a specific perinuclear localization. Nonetheless, a stronger pigmentation degree was noticed in B164A5 CM cells, indicating a potential more effective accumulation of MEL-NPs compared to the SH-4 cell line. The efficacy of the initial nanoparticle-cell interactions and cellular nanoparticle uptake depends on the properties of nanoparticles such as size, morphology, aggregation state, density, and sedimentation rate, but also on the cell phenotype and cell cycle phase (Kim et al., 2012; Donahue et al., 2019), which could partially explain the differences in the ability of SH-4 and B164A5 cells to internalize MEL-NPs. Moreover, as demonstrated in preceding studies, the PDA-NPs-cell interactions are facilitated by the dopamine receptors placed within the cell membrane and is performed through caveolae- and Rab34-mediated endocytosis, these nanoplatforms presenting an increased structural integrity within the intracellular space (Liang et al., 2023). Despite the observed differences in MEL-NPs uptake, their cytotoxic potential was similar in both SH-4 and B164A5 CM cells. The potential cell death behind the anti-CM properties of MEL-NPs was another aspect of interest for this study. Considering that apoptosis is distinguished from other cell death types due to its unique morphological features such as cell shrinkage and blebbing, nuclear constriction, cleavage of chromatin into pyknotic bodies, actinomyosin ring contraction and microtubule reorganization (Povea-Cabello et al., 2017; Yan et al., 2020), the aspect of the nuclei and cytoskeletal filaments in the CM cells treated with MEL-NPs was explored. These nanoparticles were found to produce several apoptotic-like alterations in cell nuclei indicated by a visible size reduction, and a massive chromatin condensation at all tested concentrations, and changes in cytoskeletal dynamics such as tubulin and F-actin constriction associated with cell dysmorphology, rounding and blebbing (Figure 4). The study continued with an assessment of the potential ability of MEL-NPs to trigger oxidative stress in SH-4 and B164A5 CM cells considering that redox homeostasis targeting was described as an appealing therapeutic strategy for CM (Becker and Indra, 2023) and that one potential mechanism underlying the anti-tumor activity of PDA nanoparticles is ROS production (Nieto et al., 2018). The findings presented in Figure 5 indicated an increased but statistically non-significant level of ROS in both CM cell lines treated with MEL-NPs (10, 50 and 100 µg/mL) for 24 h, with higher oxidative stress being caused in B164A5 cells, which can be related to the more pronounced internalization of MEL-NPs in this cell line compared to SH-4 cells as illustrated in Figure 3. The observed double-edge properties of these nanostructures that, on one hand scavenge oxidants (Figure 1E) while on the other hand trigger oxidative stress (Figure 5), were previously explained by Liu et al. who found that PDA is redox-active and has electron-donating abilities, being able to donate electrons not only to O₂ to generate ROS, but also to free radicals to quench them (Liu et al., 2019).

At the lowest non-cytotoxic concentration of 10 µg/mL, MEL-NPs significantly suppressed the motility of both SH-4 and B164A5 CM cells, causing a reduction in migration rate following treatment (Figure 6). In a previous study, it was found that PDA-NPs associated with a mild photothermal effect hindered the migration of CM and breast cancer cells without causing cell death. The observed effects were associated with the ability of PDA-NPs to efficiently target the transmembrane MUC18 marker highly expressed in metastatic cancers and subsequently alter cytoskeletal actin dynamics, as well as cell morphology and stiffness (Liu et al., 2021). The impaired SH-4 and B164A5 cell migratory ability caused by MEL-NPs 10 µg/mL was also accompanied by repressed clonogenic properties and inhibited colonization (Figure 7), a process described as micrometastasis formation (Werner-Klein et al., 2018), although the inhibitions were not significant compared to control. The EMT is a complex biologic process through which tumor cells lose their epithelial character, acquire a mesenchymal phenotype and consequently gain enhanced migratory and invasive capacity along with elevated resistance to apoptosis and therapy (Pedri et al., 2022). EMT is characterized by an increase in the expression of mesenchymal proteins such as vimentin and matrix metalloproteinases (MMPs), as well as a loss in the expression of proteins involved in epithelial integrity maintenance (e.g., E-cadherin, Occludins, Claudins, etc.) (Pearlman et al., 2017; Dongre and Weinberg, 2019). Its contribution to melanoma aggressiveness has been extensively documented up to date, therefore becoming a potential therapeutic target for diminishing its invasion and metastasis formation abilities (Pearlman et al., 2017). As presented in Figure 8, after a 24 h treatment of SH-4 CM cells, MEL-NPs 10 µg/mL modulated the mRNA expressions of several markers involved in EMT by significantly elevating E-cadherin and inhibiting vimentin, MMP-2 and MMP-9. E-cadherin represents a well-studied type-I classical cadherin and a strong tumor suppressor which plays a crucial role in maintaining the cell epithelial phenotype (Loh et al., 2019), its loss of expression in melanoma cells being associated with increased proliferation and mobility (D’arcy and Kiel, 2021). Vimentin is a key EMT biomarker present in mesenchymal cells and usually overexpressed during cancer metastasis (Usman et al., 2021), while MMP-2 and MMP-9 are peptidases involved in extracellular matrix remodeling and tumor invasive processes, favoring melanoma spreading and metastasis (Napoli et al., 2020). A recently published study reported the development of N-cadherin targeted MEL-NPs able to reverse the EMT in vascular endothelial cells and thus potentially slow cancer progression (Liu et al., 2024). According to the results from Figure 8, at a high and cytotoxic concentration (50 µg/mL), MEL-NPs were also found to trigger apoptosis by increasing pro-apoptotic Bad, Bak, and Bax and suppressing anti-apoptotic Bcl-XL, specific biomarkers belonging to the B-cell lymphoma 2 (Bcl-2) superfamily of proteins that regulates the intrinsic mitochondrial apoptosis pathway and is highly dysfunctional in different human cancers, including melanoma (Coricovac et al., 2021). However, statistical significance was obtained only in the case of Bad and Bcl-XL markers. This apoptosis-inducing effect of MEL-NPs relates to previous findings on the cell death mechanisms of MEL in cancer cells. For instance, Al-Obeed et al. previously showed the pro-apoptotic effect of herbal MEL in colorectal carcinoma cells which was associated with increased cytochrome c release, inhibition of Bcl-2 proteins and Caspase −3/−7 activation (Al-Obeed et al., 2020).

The last point of interest in the pharmaco-toxicological screening of MEL-NPs was the in ovo evaluation of their potential vascular toxicity and angio-inhibitory effect using the CAM, a vascularized extraembryonic membrane with versatile applications in the evaluation of nanomaterials in terms of mucosal irritant potential and impact on vascularization, among others (Buhr et al., 2020). As presented in Figure 9 and Table 2, at the concentrations of 10, 50, and 100 µg/mL, MEL-NPs lacked irritant activity on the CAM, inducing no significant alterations in the structure of the blood vessels and presenting an IS score lower than 0.9. Angiogenesis, the complex process leading to the formation of new blood vessels, is crucial for the occurrence and development of CM that requires a sustained supply of nutrients and oxygen to grow. Therefore, angiogenesis inhibition using anti-angiogenic drugs has emerged as a potential therapeutic measure for CM (Wu et al., 2022a). As highlighted in Figure 10, MEL-NPs exerted an angiostatic effect in ovo by suppressing CAM neovascularization, significant reductions in the total vascular area (to 87.78% and 84%) and number of vascular branching points (to 86.65% and 74.78%) being obtained at 50 and 100 µg/mL. By investigating similar concentrations as the ones tested herein, namely 25, 50, and 100 µg/mL, a recent study has demonstrated the ability of PDA-PEG nanoparticles to inhibit osteoclast-related angiogenesis, and block blood vessel formation in vivo (Wu et al., 2022b).

Although the present study provided novel perspectives on the innate anti-tumor properties of MEL-NPs describing their promising application in CM treatment, some limitations should be considered in future studies. On one hand, the pro-apoptotic effect of MEL-NPs in CM cells was assessed by evaluating the specific hallmarks of apoptosis such as nuclear and cytoskeletal morphological changes and the expression of apoptosis markers, while additional methods are necessary to fully capture the complexity of the cell death underlying the observed effects of MEL-NPs in CM cells. On the other hand, the full understanding of the potential utilization of MEL-NPs in CM treatment in terms of safety and efficacy should be further extended by validating these in vitro and in ovo findings through in vivo studies.

Built upon previous contributions, this study furthers the current understanding on the anti-tumor activity of MEL-NPs and their utilization in cancer therapy, opening a new avenue for the therapeutic approach of CM by redirecting the applications of MEL-NPs towards the management of skin malignancies. The findings related herein show that MEL-NPs selectively target CM cells, while also presenting a proper, but concentration-dependent cytocompatibility in healthy cutaneous cells, illustrate the specific intracellular localization of MEL-NPs in the perinuclear area of CM cell lines, and demonstrate the innate anti-melanoma activity of MEL-NPs which was correlated to their ability to trigger apoptosis-related cytotoxicity, cause oxidative stress and block cell motility by targeting the EMT. The conducted in ovo screenings also classified MEL-NPs as a non-irritant nanoplatform with angio-inhibitory properties.