Simple H-bond aggregates of isolated indole monomers (Dreyer et al., 2012) have a very low conjugation degree, and thus the broad-band absorption spectra of eumelanin cannot be easily explained. Massive chromophore stacking and π-interactions occur in aromatic compounds either in solution or solid state (e.g., tri- and macrocyclic dyes, as well as stacked base-pairs in nucleic acids), but they have no broad-band absorption but well-structured spectra (Stockert and Blázquez-Castro, 2017). Therefore, models of eumelanin only based on stacking of isolated indole units seem unlikely (Stockert, 2021).

IQ cyclization gives the 2-7 IQ tetramer or benzoquinone-porphyrin (BQP) (Kaxiras et al., 2006; Meng and Kaxiras, 2008). However, it is again not expected that only stacking of these structures without extensive covalent conjugation could explain spectral properties. Indeed, if the molecular structure of eumelanin is non-covalent and only based on hydrophobic and/or H-bonding forces, then solubilization and bleaching of the pigment should occur by treatment with organic solvents and H-bond disrupting agents (e.g., urea, formamide), and this is not the case.

It must be noted that instead of planar stacking of cyclic IQ tetramers, helical stacking of a linear continuous polymer has been suggested as an alternative melanin model (Meng and Kaxiras, 2008). The helix is formed by connecting successive tetramers through 2-7 bonds, with the fifth monomer stacked directly above the first, but without an adequate scaffold, the formation of this helical model is rather difficult to explain. Other planar cyclic or irregular oligomers containing 5-8 indole units have been also proposed as eumelanin models (Zajac et al., 1994; Arzillo et al., 2010; Chen et al., 2014). A tetra-indole model formed by two DHI and two IQ monomers linked by two amide groups between N1 and C2’ has been suggested for eumelanin (Schroeder et al., 2015).

On the other hand, a mixed model combining stacked monomers (Dreyer et al., 2012) and covalent chains (d’Ischia et al., 2015) has become fashionable. In this model, a stacked DA-DHI-DA physical trimer together with two linear zig-zag 2-2, 4-7 DHI trimers was shown as the molecular structure of PDA-melanin (Hong et al., 2012; Hong et al., 2018, Hauser et al., 2020). Again, it seems difficult that this model could explain the main properties of the synthetic polymer, as mentioned above.

A melanin-like, zig-zag chain of 2-2,3-3 indoles, named “indole black”, was suggested by Berlin (quoted by Nicolaus (1997)), but on account of steric hindrance, torsional angles of indole rings have 58° and the polymer is not planar. However, it is worth to note that free rotation of the 2-2 and 3-3 bonds can just form the BQPo ring. Likewise, a flexible 3-7 IQ chain has been proposed for eumelanin (Raghavan et al., 1990), with dihedral angle of ~20°.

In the case of 2-4,2-7 DHI or IQ polymers, the chain takes up a zig-zag way with a dihedral angle of ~18° between indole rings, allowing considerable π-stacking and almost a planar configuration (Micillo et al., 2016; Panzella et al., 2018). Linear poly 4-7 IQ and poly 4-7 IQCA chains are the most frequently shown flexible models (Liebscher et al., 2013; d’Ischia et al., 2015; Micillo et al., 2016; Panzella et al., 2018), with an angle of ~40° between IQ units, which becomes smaller (~20°) in the first excited state, allowing greater conjugation (Blázquez-Castro and Stockert, 2021).

Regarding the still poorly known 3D organization of these eumelanin models, there are different views according to the polymer. Stacking of flexible linear or zig-zag chains (d’Ischia et al., 2015; Liebscher et al., 2013), or bundling arrays of flexible linear chains have been proposed (Micillo et al., 2016; Panzella et al., 2018), but these models do not agree with the graphitic structure detected by both X-ray crystallography and electron microscopy.

Fused indole rings have been described to form a possible rigid polymer. A curved planar eumelanin model based in the formation of 2-7 and 3-4 bonds, followed by decarboxylation at 5 and 6 positions has been proposed by Swift (2009). An intriguing feature of this fused model is the absence of catechol or quinone groups, which do not allow the typical redox possibilities of eumelanin. A rigid oligomer based on a 1-7,3-4 IQ (see Fig. 1(B)) with a planar structure has been also suggested as a eumelanin model (Meredith and Sarna, 2006). In this case, a polymeric structure with suitable stacking and extended π conjugation is possible.

A very attractive double IQ chain for eumelanin was early suggested by Olivieri and Nicolaus (1999). This structure can be formulated as poly 2-2,3-3,4-4,7-7 IQ, and then the unit of this polymer is a tetra-benzoquinone (BQ) derivative of porphycene (Po) (see Fig. 1(C), and Fig. 3(A)). Therefore, this model can be simply named poly-BQPo. The Po ring and derivatives, as well as several metal complexes (e.g., Ni, Cu, Zn, Pd) are planar unsaturated macrocycles (Arad et al., 2002; Stockert et al., 2007).

The possibility that a planar BQPo unit could be the precursor of both natural and synthetic eumelanins is amazing (Stockert, 2021). Some authors have already indicated that a component of eumelanin could be BQPo (Bridelli et al., 1990) and similar indole derivatives (Zajac et al., 1994; Arzillo et al., 2010). In contrast with linear or zig-zag flexible chains, the BQPo model fulfills the main characteristics of eumelanin as a supramolecular solid (stacked multilayered graphite-like material), explaining its broad-band absorption, photoconductivity, photothermal decay with efficient heat production, and crystallographic and electron microscopical features. Therefore, it is tempting to assume that among the speculative models suggested for eumelanin, poly-BQPo could be the most plausible (Stockert, 2021), showing similar structure and properties to those of graphene oxide and graphite oxide (Dreyer et al., 2010).

The brown-black color of eumelanin is a direct evidence of its efficient light absorption (Cavallini et al., 2020). Spectral absorption of mammalian, invertebrate (cuttlefish), and synthetic melanins, as well as carbon black (e.g., China ink) presents similar features, showing a broad-band photonic absorption with exponential decay from the ultraviolet (UV) to visible and near-infrared (NIR) region (Tran et al., 2006; Plaetzer et al., 2009; Micillo et al., 2016; Micillo et al., 2017; Mostert, 2021). This feature reminds more of graphitic materials and inorganic semiconductors with a small Eg (~0.5–1.5 eV) than of organic chromophores with structured absorption peaks, which are typically associated to transitions from π bonding to antibonding π* localized orbitals.

In keeping with this, eumelanins are supramolecular amorphous semiconductors characterized by valence and conduction bands (Nicolaus, 1997; Mavridi-Printezi et al., 2020). These features are also found in graphene and pyrolyzed PDA, as well as carbon spheres and films (Liu et al., 2014). In addition to UV-visible-NIR radiation, eumelanin is capable of absorbing X- and γ-rays (Hill, 1992; Krol and Liebler, 1998), and ultrasound in the MHz range (Kono et al., 1979; Kono et al., 1981; McGinness et al., 1976).

Taking into account the stimulating effect of UV radiation on melanogenesis, UV treatments have been applied to mitigate depigmentation in vitiligo patients. Using several types of phototherapy (psoralen–UVA, narrow-band UVB) has not yet produced a definite cure, although prolonged phototherapy with the latest modality appears encouraging for face and neck vitiligo lesions (Bae et al., 2017).

The amplitude of the Eg in oligomers and unstacked eumelanins is relatively wide, but it diminishes when the conjugation (number of repetitive units) increases, and also when stacking occurs. In this way, the Eg of the pigment reaches the typical narrow values of semiconductor materials (Fig. 10).

Although lower than in monomer precursors, the Eg of oligomers with few indole units is still considerable (Fig. 10(A)). However, with increasing units the separation between the VB and CB becomes smaller and within the semi-conductivity range (Eg ≤ ~4 eV). In the case of oligo-BQPo with ether bridges (BQPoe), Eg depends on the number of bridges. The same behavior of Eg occurs in the case of allomelanin materials (Fig. 10(B)).

Therefore, melanins are similar to conductive and semi-conductive organic polymers such as poly acetylene, poly p-phenylene, poly aniline, poly thiophene, and poly pyrrole (with Eg of 2 eV and 3 eV for the two latter compounds, respectively), co-planarity of aromatic units being a basic requisite (Gardini and Berlin, 1991; Nicolaus, 2005a). Linear semi-conductive polymers, either flexible or rigid, is based on a high π-electron delocalization, and this is just what occurs in the planar poly-BQPo model (see Fig. 3).

Synthetic and natural eumelanins act as an amorphous semiconductor threshold switch. Switching occurs reversibly at potential gradients two to three orders of magnitude lower than reported for inorganic films (McGinness et al., 1974; Filatovs et al., 1976; Nicolaus, 1997; Jastrzebska et al., 2002). In a post-publication note (Mott, 2001), a flash of light (clearly electro-luminescence) was described when melanins switch. The weak electrical conductivity (σ = 10^–11 to 10^–7 Ω^–1cm^–1) is increased by formation of charge-transfer complexes, metal doping and included agents, presence of counterions, hydration, light and temperature.

Threshold conductivity switching, photo-conductivity, stable EPR signal, photo-voltaic effect, etc., are conspicuous features but small variations can be found according to the method of synthesis and purification used (Nicolaus, 1997). Black, solid-state amorphous semiconductors have a very narrow Eg (0.1–1.7 eV). An example is the broad-band absorbing fullerene C60 with Eg = 1.7 eV. NIR radiation of 808 nm corresponds to an energy of 1.53 eV.

Eumelanins are capable of dissipating >99.9% of absorbed UV-visible radiation through a non-radiative decay mode (Meredith and Riesz, 2004). Since the radiative decay of excited eumelanin is nearly zero, it is expected that its fluorescence emission should be negligible, as was early pointed out (Thompson, 1966). However, an intriguing autofluorescence has been assigned to melanin (Fellner et al., 1979; Gallas and Eisner, 1987), with an excitation peak at 450 nm and emission from 440 nm to >800 nm. Fluorescence lifetime imaging for ophthalmoscopy, thermophoresis assays of melanin-binding drugs, and detection of melanin in pigmented cells have been reported and assigned to autofluorescence (Fernandes et al., 2016; Dysli et al., 2017; Hellinen et al., 2020).

Although under UV or short visible light, melanin has negligible fluorescence, under 785-nm NIR excitation it shows a very weak broad-band emission between 820 and 920 nm, which is superimposed with the Raman scatter at 880 and 890 nm (Huang et al., 2006). In contrast, a strong yellow autofluorescence of melanins is induced by partial degradation after H₂O₂ oxidation or UV/violet (330–380 nm, 400–440 nm) radiation on natural and synthetic eumelanin, neuromelanin, and pheomelanin (Kayatz et al., 2001; Elleder and Borovanský, 2001). Interestingly, fluorescent quantification of melanin can be made after degradation under oxidative conditions (heating in alkaline H₂O₂) (Rosenthal et al., 1973; Fernandes et al., 2016).

Likewise, the fluorescence of opio-melanins at 440 and 520 nm, is due to low- and middle-molecular weight fractions, respectively, formed during oxidative photo-bleaching (Mosca et al., 1999), whereas the high molecular weight fraction does not fluoresce. A similar process occurs for the eumelanin-like, non-fluorescent, oxidized brown-black DAB polymer. After immunoperoxidase-DAB staining and irradiation with UV light for 2–4 min, positive cells develop a strong yellow fluorescence that is due to the cleavage of the DAB polymer into smaller fluorescent products (Grube, 1980).

On the other hand, a formaldehyde-induced fluorescence (FIF) in human melanocytes was early described and identified as due to the reaction of the aldehyde with DOPA, yielding an isoquinoline derivative (Falck et al., 1965; Rost and Polak, 1969). A green FIF was found in melanin-containing cells from the tapetum lucidum layer of the cat eye (Büssow et al., 1980). In this case, the exc/em maxima (430/490 nm) corresponded to those from 5-S-cysteinyl-DOPA component of pheomelanin rather than DOPA. The levels of 5-S-cysteinyl-DOPA in serum and urine were also a sensitive diagnostic method for detection of melanoma metastasis.

Studies using the FIF method (Paul, 1984) indicated that nevus cells are possibly derivatives of normal melanocytes, still showing a dendritic pattern. Photo-historic analysis also showed that the initial growth of melanoma was very protracted, and mistaken for nevi. Although dendritic tumor cells often occur in all types of melanoma, a great number are only visible in lentigo maligna.

In contrast, fluorescence quenching by melanin is now used for reducing the background autofluorescence of Xenopus laevis oocytes, thus improving specific signals from fluorescent-labeled probes after injection of mRNA (Lee and Bezanilla, 2019). Two methods were suitable, either using the drug, HG 9-91-01, to stimulate melanin production, or direct injection of synthetic PDA-melanin within oocytes.

Mammal-, sepio- and DHI-melanin have a radical-cation nature. About one cationic center for every 6-8 indole units was calculated from chlorine values. Melanins also contain one stable radical (unpaired electron) per 200–300 units, which gives an intense electron paramagnetic (spin) resonance (EPR) signal (Blois et al., 1964; Nicolaus, 2005a). Likewise, synthetic fungal DHN-melanin shows higher paramagnetism and stronger radical scavenging capacity than eumelanin (Longuet-Higgins, 1960).

A method for the unambiguous identification of melanin is just based on the detection of radicals by EPR spectroscopy, and it can be applied for the recognition of microscopically undetectable melanin in melanomas (Sarna and Swartz, 1978). It is also known that the strong binding of NM to Fe³⁺ and other metals enables both EPR and nuclear magnetic resonance (NMR) detection in the living brain (Sulzer et al., 2018).

In the NM–Fe complex the neuromelanin component contains a stable radical associated with the catechol semi-quinone group, and a high spin Fe³⁺. The two species closely interact, forming a complex used for magnetic resonance imaging (MRI). Sequestering radical-containing NM in an autophagic organelle could be protective against radical damage, whereas the synthesis of NM is thought to be neuroprotective as it removes excess cytosolic dopamine (Sulzer et al., 2018). Among the most versatile platforms, SiO₂ shell nanoparticles coated with Gd³⁺-chelated synthetic dopa-melanin have been applied for in vivo MRI-fluorescence imaging, as well as to induce antitumoral photothermal effect (Cho et al., 2016).

Metal-chelation by oxygen ligands (e.g., catechol, carbonyl, carboxyl) from melanins allows binding between polymer chains, Mg²⁺ and Ca²⁺ ions being the main cations. Histochemical methodology using selective binding of Ca²⁺ by catechol- or quinone-containing reactive dyes are well known (Stockert and Blázquez-Castro, 2017). It is accepted that the high adhesivity of melanins are based on the presence of catechol and quinone groups (Scognamiglio et al., 2017; Ruiz-Molina et al., 2018; Cavallini et al., 2020; Hauser et al., 2020). In the case of melanin models, side-to-side dimers of different structures (e.g., 1-7, 3-4 IQ or DHI, and BQPo) can be easily formed by edge-to-edge binding of metal cations (Sulzer et al., 2018; Cavallini et al., 2020) (see Fig. 2(H)), in stacked planar, cyclic or spiral configurations.

Native sepiomelanin appears as a Ca and Mg salt. Binding to metal cations include alkali, alkali earth, transition metals, and lanthanides (e.g., Na, K, Mg, Ca, Mn, Fe, Cu, Zn, Cd, Sr, Pb, La, Gd, etc.) (Zecca et al., 2008: d’Ischia et al., 2015; Cho et al., 2016; di Mauro et al., 2017; Sulzer et al., 2018; Cavallini et al., 2020). The amount and binding affinity of melanin can be quite large, binding to Mg²⁺, Ca²⁺, Sr²⁺, and Cu²⁺ being 5, 4, 14 and 34 times stronger than EDTA. For Ca, Mg, Fe, Cu o Zn, the saturation levels of binding are ~3-4 indole units per ion (Mostert, 2021).

Metal-chelation by N ligands in porphyrin-like regions (e.g., cyclic IQ tetramer, porphycene) is possible for the corresponding melanin models. Porphycenes form metal complexes with Al³⁺, Fe²⁺, Fe³⁺, Mn²⁺, Ni²⁺, Cu²⁺, Co²⁺, Zn²⁺, Pd²⁺, etc. (Rubio et al., 2005; Stockert et al., 2007). Doping of eumelanin and synthetic melanin with metal ions allows metal-to-ligand charge transfer complexes (e.g., TiO₂ with the indole unit DHICA), producing nanoplatforms for multimodal imaging and therapeutic applications, enhancing anti-inflammatory and antibacterial activity, and improving photocatalysis, light absorption, and photothermal effect of melanins (Cho et al., 2016; di Mauro et al., 2017; Cavallini et al., 2020).

Although eumelanin is a dark pigment, its light microscopic visualization depends on its concentration in the melanosome. Melanin can also modify the color of bound dyes. Unfortunately, cultured melanomas often show weakly or non-pigmented cells. The well-known B16 melanoma can differ significantly in pigmentation, minimal tumorigenic dose, expression of antigens, growth rate in mice, metastatic potential, etc. (Overwijk and Restifo, 2000). Microscopic analysis of cultured cells should allow the observation of melanosomes (Benito-Martínez et al., 2020), but morphological studies are often not performed or the absence of melanosomes is overlooked.

Although at present, many immunohistochemical methods for diagnosis and prognosis of melanomas are available (Hessler et al., 2020), the classical reaction for detection of melanin by means of silver staining remains useful. In the Fontana–Masson method (Thompson, 1966), the silver diammine cation (Ag[NH₃]₂)⁺ from an ammoniacal silver nitrate solution is reduced by melanin to colloidal metallic Ag⁰ (Bancroft and Gamble, 2008). This argentaffin reaction does not require any developer, and the dark color of the reduced silver directly demonstrates the presence of melanin. In addition, chromaffin cells and lipofuscin pigments are also revealed by this method. On account of their catechol groups, melanin also reduces ferric ferricyanide to Prussian blue at acidic pH (Thompson, 1966), and under acid conditions, the bound Fe³⁺ forms Prussian blue with added potassium ferrocyanide (Perls reaction, Lillie, 1977; Sulzer et al., 2018). Melanoma cells, additionally, can be histochemically detected by tyrosinase assays (Angeletti et al., 2004).

Eumelanin is negatively charged at pH values above 4 (Ball, 2010), and then some staining reactivity is related to its polyanionic nature. After common hematoxylin-eosin staining of tissue sections, melanosomes are visualized by staining with the cationic blue aluminum-hematein, which depends on the binding of Al ions to anionic sites. Other staining reactions are based on the high affinity of eumelanin for metal cations and cationic dyes (basophilia). An example is the iron uptake reaction giving a blue-green color (Thompson, 1966), which is typical of ferric-catechol complexes (Lillie, 1977; Sulzer et al., 2018).

Numerous cationic dyes can stain eumelanin. Examples are methylene blue and toluidine blue that stain the pigment in blue. Nile blue stains melanin dark green but also lipofuscin deposits in a blue color (Lillie, 1977). Pinkus’ Giemsa stains melanin in a green-brown color (Thompson, 1966). The precise binding mechanism of cationic dyes to eumelanin has been rather overlooked. Since Lerman’s formulation of the intercalative binding mode into DNA (Lerman, 1964), numerous dyes have been found to bind into nucleic acids by intercalation (Stockert, 1985), using strong face-to-face hydrophobic interactions between planar dyes and base pairs.

Therefore, on account of the aromatic and anionic character of eumelanin, intercalation of planar cationic dyes should be the preferential binding mode, as occurs with similar substrates stained by acridine, thiazine, azine, oxazine, and xanthene dyes. The emission of fluorochromes bound or near to eumelanin is quenched (Lee and Bezanilla, 2019), as occurs in the presence of graphene and graphene oxide, which severely quench the emission of attached fluorophores (He et al., 2010).

The copper-phthalocyanine (CuPc) macrocycle is an interesting model dye regarding binding to eumelanin. The CuPc dye Alcian blue 8GX has been used in TEM studies of PDA-melanin (Chen et al., 2013), but a rather poor increase of electron contrast occurs over the natural contrast of a graphitic structure. Using this dye, a new composite was formed by layer-by-layer film deposition of PDA-melanin and Alcian blue 8GX, which displayed an electrical conductivity 5 orders of magnitude higher than that of pure melanin films (Chen et al., 2013).

In addition to π–π stacking, electrostatic interactions with this and other cationic dyes should also play a role, because of the negative charge of eumelanin at neutral pH. Obviously, positive charges on the phthalocyanine ring itself (e.g., cuprolinic blue and its analogous zinc complex, Scott, 1980; Tas et al., 1983; Juarranz et al., 1987; Tempesti et al., 2008), or on the side chains (e.g., Alcian blue 8GX, Juarranz and Stockert, 1982; Scott, 1996) could be advantageous but not essential for intercalative binding and staining of eumelanin.

On the other hand, the acid dye CuPc tetrasulfonate was found to form stacked aggregates on the external surface of multiwall carbon nanotubes (Hatton et al., 2007). Therefore, three binding modes of CuPc dyes on aromatic polymers could occur, namely (a) intercalation between aromatic planes, (b) aggregation on aromatic surfaces, and (c) mixed binding modes. Distinct spectroscopic features should be observed for each case. Intercalation of inorganic cations (Li⁺, K⁺) and anions (PF₆^-, BH₄^-) between graphite layers is also possible (Gao et al., 2021; Ko et al., 2021).

PDT is an antitumor treatment based on the selective uptake of a photosensitizer within tumor cells followed by a suitable light irradiation, which generates reactive oxygen species (ROS) and/or radicals inducing cell death (Stockert et al., 2004; Plaetzer et al., 2009; Zhang et al., 2018; Baskaran et al., 2018; Wang et al., 2021). PDT and PTT protocols for melanomas can be now used. The strong light absorption of melanin and its anti-oxidant and radical-scavenger capacity could hinder PDT effects on melanoma cells, but advances in PDT applications in melanoma cultures or tumors have been described (Barbazetto et al., 2003; Sheleg et al., 2004; Kolarova et al., 2007; Skidan et al., 2008; Chen et al., 2008; Cook-Moreau et al., 2010; Maduray et al., 2011; Radzi et al., 2012; Baldea and Filip, 2012; Rapozzi et al., 2014; Naidoo et al., 2018; Pereira et al., 2018; Valli et al., 2019; Akasov et al., 2019; Pan et al., 2021).

Recently, Pires et al. (2020) applied dual PDT for murine B16-F10 pigmented and B78-H14 non-pigmented melanomas using cellular-PDT with Photodithazine, and vascular-PDT with Visudyne, followed by irradiation with 670 and 690 nm, respectively, with good results. Here, a novel approach was the topical use of optical clearing agents before PDT to improve the light penetration in melanoma tissue.

A recent addition to the stockpile of PDT is upconversion PDT (UC-PDT). This approach takes advantage of the upconversion phenomenon: the emission of more energetic photons (UV-VIS) after two or three less energetic photons (NIR) are absorbed and their energies summed up by certain rare-earth doped materials (Qiu et al., 2018). This UC-PDT has been successfully applied in experimental models of melanoma PDT, both in vitro (Lee et al., 2020) and in vivo (Idris et al., 2012). However, it must be noted that upconversion in general and, by extension, UC-PDT rely on light intensities (0.5 Wcm^–2 and above) that can lead to a direct photothermal action, particularly when a melanotic tumor is the target. Indeed, this is the case for some of the results reported. Idris et al. (2012) found that laser illuminating the melanotic tumors without UC nanoparticles induced a notable tumor toxicity. This could only happen due to a photothermal effect, probably driven by melanin absorption of the 980 nm photons employed. In fact, UC nanoparticles have been recently employed to enhance the photothermal effect in a melanoma model due to the efficient NIR-to-heat conversion of these nanoparticles (Krylov et al., 2020). Even more recently, another group has advanced a complementary UC-PDT/UC-PTT approach to maximize the damaging action upon melanoma tumors (Zhong et al., 2021). In our opinion, the intense light fluxes (around Wcm^–2) necessary to produce the UC-PDT preclude their successful application in melanotic melanomas because the NIR absorption of melanin will provoke an unavoidable thermal shock in the tissue.

In contrast with the photochemical principle supporting PDT, PTT is an antitumoral therapy based on the physical photothermal effect (light-to-heat conversion) (Parrish et al., 1983; Jori and Spikes, 1990; Camerin et al., 2009). In this case, the generation of ROS is not required for tumor cell death. An efficient photothermal effect requires fast conversion of excited electrons to vibrational excitation states, which then decay with heat production inducing denaturation of macromolecules, vaporization, and acoustic shock-waves.

Eumelanin is practically the ideal photothermal sensitizer, and the massive vibrational decay from photo-excited electrons in melanotic melanomas induces an efficient heating response that results in coagulative necrosis of the tumor (Kostenich et al., 2000; Colombo et al., 2019; Blázquez-Castro and Stockert, 2021). In the case of melanin-PTT, the endogenous chromophore eumelanin is the selective PTT agent for NIR irradiation (Fig. 11). Control B16-F10 melanoma cells appear with numerous melanosomes showing variable size and shape, whereas after NIR irradiation, massive coagulative necrosis, cell debris, and a large amount of round melanin-containing macrophages (melanophages) are observed, allowing repetitive PTT treatments. Necrotic tumor cells actively attract macrophages through a phosphatidyl-serine-exposure mechanism (Li et al., 2015). This approach has been applied in Au-SiO₂ nanoshells-loaded macrophages for NIR-PTT of tumors (Madsen et al., 2015).

Melanin-like materials, colloidal PDA-melanin, and PDA-coated nanoparticles are suitable NIR photothermal agents for in vivo cancer therapy, and have increasing interest for applications in nanomedicine (Liu et al., 2013; Yue and Zhao, 2021). The retinal melanin-pigmented epithelium has been proposed as a suitable PTT target for treatment of macular diseases (Roider et al., 2000). An enhanced PTT also occurs after metal binding to melanin (Cavallini et al., 2020).

On account of an adequate ratio between light absorption by PTT agents and light penetration into tissues, the use of 800-nm NIR-laser irradiation is very suitable to induce successful PTT effects (Weissleder, 2001). Glycerol can be applied to reduce light dispersion by the skin over the tumor (Blázquez-Castro et al., 2018). As glycerol is a strong protecting agent against cell hyperthermia (Henle and Warters, 1982), application of a glycerol drop on the depilated skin also avoids the undesired heating and damage of healthy tissues.

In addition to melanins, carbon materials, black pigments and dyes, mixed-valence compounds, and metal nanoparticles show relevant photothermal activity under NIR illumination (Jiang et al., 2013), and could be used to reinforce melanin-PTT. In the case of black pigments, successful PTT responses have been obtained by using graphitic materials such as carbon dots, nanotubes, etc. (Fisher et al., 2010; Zheng et al., 2015; Chen et al., 2016). Simple China ink (carbon black) is a highly effective PTT agent (Blázquez-Castro et al., 2018). Commercial black toners could be suitable, but they have little carbon content, and large quantities of plasticizer additives (styrene acrylate copolymer and polyester resin), and are not adequate for in vivo applications. Carbon quantum dots (<10 nm, obtained from microwave treatment of lemon juice, as well as citric acid, urea, amino acids, etc.) consist of graphitic cores and hydrophilic functionalized shells (Ross et al., 2020), and are very appropriate for PTT of tumors (Sharma and Das, 2019), and sensing toxic metals by fluorescence quenching (Tadesse et al., 2020).

Some Cu(II)-, Ni(II)-, and Pd(II)-containing dyes, such as Cu-hematoporphyrin (Soncin et al., 1999), Ni-octabutoxy-naphthalocyanine (Busetti et al., 1999), and Pd-octabutoxy-naphthalocyanine (Diddens et al., 2003), show NIR-induced photothermal effects. Extensive chemical and mechanical cell damages are caused by the photo-generation of an acoustic shock wave from clusters of aggregated dye within cells (Camerin et al., 2005). Conventional black dyes with canonical absorption spectra, such as Sudan black B, organol black R, amidoblack 10B, aniline blue black, and nigrosine WS (Lillie, 1977; Horobin and Kiernan, 2002) are potential candidates to be used as photothermal agents. The same would be expected for mixed-valence compounds such as ruthenium red, phosphomolybdic blue, Prussian blue, and amylose-iodine inclusion complex (Robin and Day, 1967; Lillie, 1977; Clark, 1984).

Metal and oxide nanoparticles are also currently studied for diagnostic and therapeutic applications (Shi et al., 2015; Li et al., 2018). Administration of gold nanoshells composed of silica cores covered by Au layers to murine colon carcinoma cells followed by 808-nm irradiation resulted in an excellent PTT response (O’Neal et al., 2004). Among other processes, the plasmonic effect induces a photothermal response useful for antitumoral therapy (Carrasco et al., 2020). NIR-laser irradiation of gold (Au) nanoparticles (mainly nanorods, both in vitro and in vivo, either after intratumoral or intravenous injection) generates a plasmonic photothermal effect (PPTT), which results in cell apoptosis and tumor destruction (Huang and El-Sayed, 2011).

An interesting approach for selective targeted PTT of melanoma cells was described by Lu et al. (2009), using hollow Au nanospheres attached to a ligand peptide (melanocyte-stimulating hormone analog) for receptor-mediated PTT of the B16/F10 melanoma subjected to 808-nm irradiation. The combination of spherical shape, small size (average diameter ~40 nm), absence of silica core, and strong absorption bands in the NIR region makes these nanoparticles ideally suited for PTT.

The analysis of chemical candidates to quickly evaluate a possible PTT effect can be performed using “spot test” on paper strips, followed by NIR laser irradiation. Ignition of blotted papers occurs after few seconds, when temperature reaches 218–246°C, and it is a clear end point of the photothermal reaction. The temperature increase of aqueous solutions of photothermal candidates subjected to irradiation can be also recorded by using a simple mercury thermometer or a thermocouple to assess photothermal responses (Blázquez-Castro et al., 2018; Colombo et al., 2019). Useful biological materials to study mechanisms and applications of melanin-based PTT include bacterial biofilms, 3D melanoma spheroids, and whole organisms such as black amphibian eggs and embryos (Rhinella, Bufo), black insects (Aedes aegipty), plant beans (Phaseolus vulgaris), and black seeds (Sesamum indicum, Salvia hispanica, etc.).

In addition to the biomedical use of ultrasounds (US) in echography, US applications include imaging methods for microscopy, antitumoral high-intensity focused US therapy (HIFU), and sonodynamic therapy. The US microscope uses frequencies near 1,000 MHz, which display viscoelastic properties with a resolution comparable to the light microscope. Sections of normal human retina revealed acoustic attenuation (absorption) in tissue structures such as cell nuclei, rod and cone outer segments, melanin, and red blood cells (Marmor et al., 1977). Comparison of tissue from albino and pigmented rabbits showed that melanin was a particularly strong acoustic attenuator. Photoacoustic signals can also be used to reveal circulating melanoma cells by flow cytometry (O’Brien et al., 2012; Viator et al., 2020).

At present, photoacoustic imaging (PAI) has evolved into a 3D imaging modality (Kratkiewicz et al., 2021). The sample to be imaged is optically excited, leading to a transient temperature rise, with very fast (ps-ns) thermoelastic expansion of the chromophore followed by emission of an acoustic wave. The absorber agent can be endogenous (melanin, hemoglobin, myoglobin, lipid, bilirubin) or exogenous agents such as dyes. Acoustic waves from the absorber are detected by US transducers, generating the absorption map of the tissue. As acoustic waves can traverse longer distances than photons, sensitivity of PAI in deep tissues is orders of magnitude higher than that of pure optical imaging modalities.

In the case of HIFU, a direct and lethal heating of water is obtained focusing high-intensity US into tumor tissue (Lee et al., 2006). However, the melanin content of melanomas strongly absorbs US across several frequencies, and can be used as specific sono-sensitizer (SS). The absorption at 4 MHz has been assigned to molecular motions of a melanin protoparticle with an average molecular weight of ~1.4 × 10⁴ Da (Kono et al., 1981). Interestingly, this value agrees with the molecular weight of a protoparticle modeled as a 4-turn spiral BQPoe (~1.3 × 10⁴ Da). Likewise, the diameter of a protoparticle of PDA-melanin by X-ray diffraction studies is ~30 Å (Chen et al., 1994; Bridelli, 1998), which also corresponds closely to the diameter of a 4-turn spiral BQPoe model (~34.3 Å). In addition, a resonance at 250 MHz was found in melanin associated with stacking of the indole monomer (Kono et al., 1981).

An emerging approach for the treatment of atherosclerosis and invasive tumors is sonodynamic therapy (SDT), which involves the use of a SS followed by exposure of the labeled tissue to low intensity ultrasound (US) (Kou et al., 2017; Li et al., 2020; An et al., 2021). Because US has deeper tissue penetration than light, SDT has advantages over PDT and PTT methods. It is accepted that after SDT, tumor cell damage and death are due to generation of ROS and radicals, and then this modality should be better named “sono-electrochemical therapy” (SET), to avoid the misleading use of the term “dynamic”. Employed USs are mainly 1–2 MHz and 0.5–10 W/cm² (Abrahamse and Hamblin, 2016). Natural SSs such berberine, curcumin, hypericin, and protoporphyrin IX have now increasing importance (Kou et al., 2017; An et al., 2021). Significant regression of experimental melanomas has been achieved using several SSs such as TiO₂ nanoparticles, chloroaluminum phthalocyanine disulfonate, and nickel ferrite/carbon nanocomposites (NiFeO/C), which indicates that SET may be more effective than PDT in treating advanced melanotic melanomas (Li et al., 2020). It is noteworthy that the strong US-absorbing eumelanin from melanomas is just a highly suitable endogenous SS for SET.

Natural and synthetic melanins are capable of binding other molecules with high dielectric constants (water, dimethyl sulfoxide, formamide, methanol), inducing reversible conductivity changes of as much as ten orders of magnitude (Filatovs et al., 1976). The resistivity of hydrated DOPA-melanin complexed with diethylamine and subjected to US of 10 kHz reduces by ~6 orders of magnitude (Corry et al., 1976). The high affinity of sepia and hair melanins for organic compounds allows to form charge-transfer complexes between the pigment (electron acceptor) and the included compound (electron donor), resulting in a strongly increased conductivity (Nicolaus, 1997).

Both US absorption by melanin, and the number of dead cells do increase sharply with temperature from 7 to 37°C. US absorption is the initial step of the cytotoxic events that convert the phonon energy into cytotoxic products through phonon-electron interactions within the melanosome (Kono et al., 1979). Melanin-binding drugs and dyes induce toxicity in melanocytes subjected to US. Cultured melanotic tumor cells treated with chlorpromazine are preferentially killed by 10 kHz US irradiation (Corry et al., 1976), with radical production appearing involved in DNA damage. Likewise, 1 MHz-US induces melanin degradation and killing of melanin-containing cells, which are strongly potentiated by previous treatment with melanin-binding drugs, such as chlorpromazine and kanamycin (McGinness et al., 1976; Kono et al., 1979).

On the other hand, copper phthalocyanine (CuPc) dyes (e.g., Alcian blue 8GX, Alcian blue pyridine variant, cuprolinic blue, etc.) stain chromatin DNA, and intercalate into nucleic acids duplexes, triplexes and quadruplexes (Juarranz and Stockert, 1982; Stockert, 1985; Juarranz et al., 1987; Scott, 1996; Macii et al., 2020). Therefore, intercalative binding of these dyes with other aromatic substrates such as eumelanin would be expected to occur, as is the case of Alcian blue 8GX binding to PDA-melanin (Chen et al., 2013). Fig. 12 illustrates this possible binding mode, in which a close molecular fitting occurs between the aromatic CuPc chromophore and two aromatic BQPoe units (Figs. 12(A) and 12(B)), showing fused LUMOs of the intercalation complex (Figs. 12(C)). Intercalative binding of dyes appears as a striking feature of eumelanin, which does not involve structural changes in the X-ray diffraction of the pigment (Thathachari and Blois, 1969), because the dye represents just another aromatic plane.

The high binding affinity of eumelanin for drugs and dyes was found as an unexpected adverse effect of some neuroleptic and antimalarial drugs. Chronic administration of antidepresive phenothiazines (Kinross-Wright, 1956; Burian and Fletcher, 1958) and high-dose chloroquine therapy (Hobbs et al., 1959) produced chorioretinopathy, suggesting an association between toxic effects of some drugs and affinity for eumelanin. Binding of drugs to eumelanin was proved in silico by simple free-energy methods, and was also implicated in ototoxicity and disturbances of the skin and hair pigmentation (Raghavan et al., 1990).

Typical dyes and drugs that bind to eumelanin are acridine orange, aflatoxin B₁, p-aminobenzoic acid, aminoglycoside and tetracycline antibiotics, bisazo dyes, carcinogenic hydrocarbons, chloroquine, chlorpromazine, dexamethasone, diclofenac, fluorocinolone, fluoro-quinolones, herbicides, iodoquine, methotrexate, papaverine, psychotropic and ophthalmic drugs, quinidine, thiazine dyes, thiouracil, thioureas, trimethyl-psoralen, etc. (Potts, 1962; Blois, 1965; Blois and Taskovich, 1969; Lindquist and Ullberg, 1972; Blois, 1972; Lindquist, 1973; Swan, 1974; Larsson, 1991; Larsson, 1993; Lowrey et al., 1997; Jakubiak et al., 2019; Hellinen et al., 2020; Kowalska et al., 2020; Rimpelä et al., 2020). Interestingly, binding of radionuclide-labeled dyes and drugs to eumelanin has been applied for melanoma diagnosis or therapy (Lindquist and Ullberg, 1972; Panasiewicz et al., 1978; Link and Łukiewicz, 1982; Napolitano et al., 1996; Chen et al., 2020).

In the intercalative binding mode, the planar ligand slips between the aromatic units of eumelanin layers from a side edge, and remains trapped as an inclusion complex or “graphitic sandwich”, allowing both linear and stacked fused LUMOs (Fig. 13). Numerous dyes and drugs may be used as photo- and sono-sensitizers to enhance the antitumoral activity against melanoma cells, by generating increased and selective photothermal and US electrochemical cytotoxicity, respectively. Suitable dyes for intercalation between aromatic eumelanin layers would be planar vital probes such as acridines, thiazines, porphyrins, phthalocyanines, porphycenes, bisazo dyes, etc.

Acridine orange, toluidine and methylene blue, thionine, Nile blue, TMPyP, ZnTPP, ZnPc, TPPo, PdTPPo, etc., accumulate in endosome-lysosome organelles (Stockert et al., 2004; Stockert et al., 2007), to which also belong melanosomes (Orlow, 1995; Raposo and Marks, 2007). The selective metachromatic staining of skin melanosomes from Eubalaena australis with the thiazine dye, toluidine blue, is illustrated in Fig. 14. It is worthy of remark that photo- and sono-active melanin-binding dyes could be the same vital probes commonly used for lysosomes. New developments are expected to occur in these innovative fields of melanosome labeling and physical melanoma therapy.

In all these cases, very small Eg values of dye/drug-melanin complexes allow efficient energy coupling (Corry et al., 1976; McGinness et al., 1976: Crippa et al., 1991), which will result in improved photothermal and US-induced cytotoxicity in melanoma cells. A schematic drawing of possible photon-electron-phonon coupling involving NIR and US radiation followed by thermal and electrochemical responses is shown in Fig. 15.

As US in the MHz range has considerable tissue penetration and is also strongly absorbed by eumelanin, appropriate ligand-eumelanin complexes would give an improved and deeper US therapeutical response, likely due to ROS and radical production (Corry et al., 1976). Therefore, this drug-enhanced SET may represent a more effective and non-invasive treatment for melanoma.