Antioxidant effects of silver-ceria nanoparticles on the reduction of melanin in amelanotic melanoma cell biology

Abstract

Although cerium oxide nanoparticles (nanoceria, CeO₂) have a wide range of applications, it is imperative to consider their significant implications for human health. In particular, modifying the surface properties of CeO₂ is of great importance in biomedical applications. In this study, a conventional wetness incipient impregnation technique was employed to load silver (Ag) metal onto the surface of CeO₂ NPs synthesized via the hydrothermal method. Then, the antioxidant effects of silver-cerium oxide nanoparticles (Ag@CeO₂ NPs) were evaluated on the melanin content of A375 skin cancer cells. The synthesized nanoparticles have been identifed using combined characterizations of the hydrodynamic size, zeta potential FTIR, FE-SEM, and UV-Vis spectra. The average particle size of Ag@CeO₂ NPs was measured at 234 ± 20 nm with the zeta potential value − 33.5 mV. FE-SEM image revealed that Ag@CeO₂ nanoparticles were polyhedral particles consisting of cubic nanostructures with rounded corners. The antioxidant capability of Ag@CeO₂ NPs was assessed using DPPH and ABTS assays and the inhibitory effects of that on melanin biosynthesis (extracellular and cellular melanin content) were examined on human melanoma cell line. Overall, the results provide promising baseline information for the potential applications of Ag@CeO₂ NPs in treating hyperpigmentation in the skin.

The imbalanced electrons associated with oxygen can engage in reactions that yield partially reduced, highly reactive species, collectively referred to as reactive oxygen species (ROS). Examples of ROS include hydroxyl radicals (.OH), superoxide anions (.O⁻²), and hydrogen peroxide (H₂O₂)¹. These species are typical byproducts of aerobic metabolism, arising from processes such as the mitochondrial electron transport chain, cytochrome P450 activity, the NADPH oxidase complex, and peroxisomal functions. Due to their elevated chemical reactivity, ROS are transient species that can induce lipid peroxidation, enzyme oxidation, protein modification, and DNA mutations. Aerobic organisms have developed various chemical and enzymatic antioxidant mechanisms to mitigate the potential damage caused by oxidative stress. Key antioxidant enzymes include superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT). SOD facilitates the dismutation of superoxide radical anions into hydrogen peroxide and oxygen, while CAT and GPx are responsible for the conversion of hydrogen peroxide into water². Cerium oxide nanoparticles (nanoceria, CeO₂), which represent the oxidized form of the rare element cerium, have been shown to mimic the activity of SOD due to alterations in surface oxygen vacancies and valence configurations. Consequently, these nanoparticles can function as scavengers of ROS across various biological contexts³. The nanoscale properties of cerium oxide are enhanced due to the increased surface-to-volume ratio characteristic of nanoparticles, which has led to numerous prospective applications in biomedicine. These applications encompass endothelial cell protection, wound healing, anticancer strategies, and optical biosensors for the inflammation-related diseases^4–6. Research into the biological applications of nanoceria is expanding rapidly. Antioxidants present a potentially promising avenue for the prevention and treatment of melanoma, as the further induction of oxidative stress may facilitate the gradual demise of malignant cells. As a result, both antioxidants and prooxidants may play a role in interventions for melanoma. On the other hand, Silver Nanoparticles (Ag NPs) stabilized with antioxidants like epigallocatechin gallate (EGCG), demonstrated cytotoxic effects on melanoma cells by inducing oxidative stress and damaging cellular components, leading to apoptosis⁷.

It is important to note that the mechanisms and effects of melanin on oxidative stress within the context of melanogenesis remain incompletely understood. The incorporation of zinc and copper complexes into both unloaded and vitamin E-loaded nanoparticles has been shown to enhance antioxidant properties while effectively inhibiting melanin production. This approach holds potential for the treatment of disorders associated with melanogenesis. But such these studies have not specifically focused on their ability to reduce melanin content in melanoma cells^8,9. While the production of melanin can induce oxidative stress, melanin possesses unpaired electrons, allowing it to function as an antioxidant by interacting with free radicals and other reactive species, thereby mitigating their effects¹⁰. Melanin exhibits a hydrophobic complex structure with a negative charge, resulting from the oxidation and polymerization of phenolic compounds, and is synthesized in melanocytes and melanosome cells¹¹. The production and distribution of melanin can be diminished by inhibiting the enzyme tyrosinase; however, complete cessation of melanin production is not feasible, as it is a physiological process, and halting this process may result in depigmented skin areas. Tyrosinase serves as a critical enzyme in melanin biosynthesis, and numerous effective molecules, including phenolic extracts, flavonoids, terpenoids, and alkaloids, have been identified as inhibitors of this enzyme or as agents for skin-lightening¹². Several factors can contribute to an increase in skin melanin levels. Melanin can accumulate in the skin, leading to darkening and the formation of dark spots. These spots may arise from various causes, including sun exposure, hormonal changes, skin aging, and liver issues. Freckles are a specific example of localized melanin accumulation that results in dark spots¹². The development of novel nano-sized molecules with significant potential to reduce melanin levels for use in skincare products aimed at treating skin disorders such as hyperpigmentation is a primary objective of ongoing research in this domain. This study specifically examines the antioxidant effects of silver − cerium oxide nanoparticles (Ag@CeO₂ NPs) in reducing melanin levels in A375 melanoma cancer cells (scheme 1).

Scheme 1.

Silver-cerium oxide nanoparticles: Synthesis method and application in the melanin content reduction.

Materials and methodology

Chemicals and Reagents

The Ce(NO₃)₃.6H₂O (99.5%) and AgNO₃ (> 99.8%) salts bought from Sigma-Aldrich, and ammonia (NH₄OH, 25%) have been applied for the synthesis of Ce_1−xAg_xO₂ nanoparticles as cerium and silver precursors, and stabilizing agent, respectively. All other chemicals were acquired from commercial sources and utilized in their original form without any further purification processes. Furthermore, the A375 human melanoma cell line was sourced from the National Cell Bank of Iran (Pasteur Institute).

Synthesis of Ag@CeO₂NPs and Characterization

The synthesis of Ag@CeO₂nanoparticles (NPs) was conducted according to the procedure described in the literature¹³. Initially, a solution of 3.53 g of ammonia (NH₄OH, 25%) was introduced to a pre-prepared mixture consisting of 4.85 g (11.169 mmol) of cerium nitrate hexahydrate (Ce(NO₃)₃.6H₂O) and 2.963 g (17.44 mmol) of silver nitrate (AgNO₃) dissolved in 20 mL of water, which was subsequently diluted with an additional 12 mL of water. The resultant mixture was vigorously stirred at 300 revolutions per minute (rpm) for 2 min at room temperature. Following this, the resulting black coprecipitate underwent heat treatment in an autoclave under steam conditions at 120 °C for 10 min. The resulting golden-brown solid was separated via centrifugation and then subjected to calcination at 500 °C for 4 h in an air atmosphere.

The synthesized Ag@CeO₂ NPs underwent extensive evaluation using a variety of analytical techniques. Absorption spectra were obtained using ultraviolet–visible or/and (UV-Vis) spectroscopy. The average hydrodynamic size with polydispersity index (PDI), and zeta potential of Ag@CeO₂ nanoparticles in media was determined by dynamic light scattering (DLS) (Nano-ZetaSizer-HT, Malvern Instrument). The functional groups of the synthesized nanoparticles were determined using Fourier transform infrared (FTIR) spectroscopy (Bruker Tensor 27 Spectrometer Bruker, Japan) from 4000 –400 cm⁻¹, and using dried samples and KBr pellets. The morphological characterization of Ag@CeO₂ nanoparticles (NPs) was conducted using a field-emission scanning electron microscope (SEM, Quanta 450, FEI, USA).

Antioxidant potential evaluation of Ag@CeO₂NPs

DPPH assay

Antioxidant Activity was acceseed via DPPH (2,2-diphenyl-1-picrylhydrazyl) Assay. To assess the antioxidant capacity in terms of DPPH radical scavenging, a solution of 1 mg of DPPH was initially prepared by dissolving it in 17 ml of ethanol, resulting in a purple DPPH solution¹⁴. Various concentrations of Ag@CeO₂ NPs (0.05, 0.1, 0.5, 1, and 3 mg/ml) were examined for their antioxidant efficacy. The reaction mixture comprised 400 µl of the DPPH solution and 100 µl of the respective concentrations of Ag@CeO₂ NPs. The mixture was subjected to vigorous shaking and subsequently incubated at room temperature for 40 min, after which the absorbance was recorded at 517 nm. The control sample included all compounds except the Ag@CeO₂ NPs, with ascorbic acid as the standard reference. The final percentage of inhibition was determined using Eq. (1):

1

ABTS assay

In the ABTS (2,2’-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) assay, initial stock solutions of 2.4 mM potassium persulfate and 7mM ABTS were prepared. The working solution was created by combining equal volumes of the two stock solutions and allowing them to react for 15 h at room temperature in a dark place¹⁵. The solution was then diluted by mixing the ABTS⁺ solution with water to achieve an absorbance of 1.00 ± 0.02 units at 734 nm, as measured by a spectrophotometer. A fresh ABTS⁺ solution was prepared for each assay. Various concentrations of a standard ascorbic acid solution were also prepared. Subsequently, 100 µl of Ag@CeO₂ NPs at different concentrations (0.05, 0.1, 0.5, 1, and 3 mg/ml) were allowed to react with 400 µl of the ABTS⁺ solution for 3 h in a dark setting. The absorbance was then measured at 734 nm. All measurements were performed in triplicate, and the results were averaged. The means were compared using Duncan’s multiple range test (P < 0.05). The final percentage of inhibition was calculated using Eq. (1).

Cell culture of amelanotic melanoma lines

The A375 melanoma cell line was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, MA, USA), which was supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Wisent, Montreal, Canada). The cells were maintained in a humidified incubator with an atmosphere of 5% CO₂ at a temperature of 37 °C.

MTT assay

The MTT assay was conducted to evaluate the viability of the A375 cell line exposed to the synthesized materials. This colorimetric assay relies on the ability of mitochondrial enzymes in viable cells to reduce the tetrazolium salt (MTT) to insoluble purple formazan crystals. The intensity of the color correlates with the number of viable cells. A375 cells were cultured in DMEM high glucose medium supplemented with 10% FBS and 1% penicillin/streptomycin. Subsequently, 5000 cells per well were seeded into a 96-well plate and allowed to adhere overnight. The following day, the wells were observed to confirm uniform seeding and cell attachment. The cells were then treated with the prepared concentrations for 24, 48, and 72 h. After the treatment periods, the media were removed, and the MTT solution (0.5% w/v in FBS-free medium) was added to the cells. The plates were incubated at 37 °C with 5% CO₂for 3 h to allow the mitochondrial reduction of MTT. The resulting formazan crystals were dissolved with 100 µl of DMSO, and the absorbance was measured at 570 nm using an ELISA plate reader¹⁶. This assay was performed in quadruplicate to ensure accuracy.

Melanin content measurement

The both intracellular and extracellular melanin content in A375 cell lines were evaluted using absorbance measurements. For this purpose, 10⁴cells were seeded into a 48-well plate and allowed to adhere to the surface. To ensure that phenol red in the medium did not interfere with tyrosine, the amino acid precursor of melanin, RPMI medium without phenol red was used. Subsequently, 0, 50, and 100 µg/mL of the nanomaterial were added to the medium after removing the existing medium from each well. The extracellular melanin was then measured at 490 nm using an ELISA plate reader to generate the melanin curve¹⁷. To measure intracellular melanin content, cells were incubated with 1 N NaOH containing 10% DMSO for 1 h at 80 °C, and then intracellular melanin production was measured at different time points at 405 nm.

Statistical analysis

All experiments were conducted in triplicate, and the findings were subsequently reported. The experimental data were subjected to analysis utilizing SPSS software. A statistical significance threshold was established at p < 0.05.

Results and discussion

Characterization of silver − cerium oxide nanoparticles (Ag@CeO₂NPs)

The synthesized Ag@CeO₂NPs were subjected to an extensive evaluation employing various analytical techniques and the ability to absorb light can be effective in estimating photocatalytic and antioxidant effects. In some characterization spectra, CeO₂ nanoparticles are used for comparison. They characterized in terms of mean size, ζ-potential, polydispersity index (PDI), and morphology. As shown in Fig. 1a, the UV-Vis absorption spectra of pure CeO₂ and the heterostructured Ag@CeO₂NPs in a wavelength region ranging from 200 to 800 nm. The photo-absorption of pure CeO₂is located in the UV region with two dominant absorption peaks, the maximum appeared at about 300–330 nm and other at 230 nm, which was related to the charge transfer from O (2P) to Ce (4f) orbitals^18,19. While, the spectral patterns of Ag@CeO₂NPs heterostructured photocatalysts show the steep absorption edge in the UV region and much strong absorption shifted to the visible light region. This phenomenon can be attributed to the surface plasmon resonance (SPR) effect of Ag metal. The Visible-Region Band (380–400 nm): Attributed to the surface plasmon resonance (SPR) of metallic Ag nanoparticles, a phenomenon widely reported in Ag-doped metal oxides^20,21. The plasmonic Ag metal, has extended the light absorption toward visible light region, conforming the successful loading of Ag on the surface of CeO₂photocatalysts. These results are in line with a study that found the ability of visible light absorption regularly increases with increasing Ag content²². The SPR effect enhances visible-light absorption by Ag@CeO₂NPs, which directly impacts their antioxidant activity. This aligns with recent studies showing that plasmonic nanoparticles amplify redox-modulating capabilities through localized electric fields²³.

Fig. 1.

(a) UV–Vis absorption spectra of pure CeO2 and Ag@CeO2 heterostructured photocatalysts (b) Particle size distribution and zeta potential value for Ag@CeO₂ NPs.

The hydrodynamic mean diameter of the Ag@CeO₂ NPs was 234 ± 20 nm, with a (PDI = 0.276), indicating a colloidal suspension with a relatively uniform size distribution. Additionally, the zeta potential of the Ag@CeO₂ NPs (−33.5 mV) was measured at pH = 7.4, suggesting favorable stability for the nanoparticles Fig. 1b. The higher zeta potential of the Ag@CeO₂ NPs suggests that the NPs possessed an enhanced resistance to aggregation and thus improved morphological stability and mechanism for cellular interactions.

As shown in Fig. 2, the chemical texture and the surface functional groups of pure CeO₂ and the heterostructured Ag@CeO₂ are detected using FTIR spectra at the wave number range of 400–4000 cm⁻¹. The spectral pattern of pure CeO₂ demonstrates a strong absorption band at 482 cm⁻¹ corresponding to metal–oxygen bonds and the weak absorption band emerged at 880 cm⁻¹is also attributed to the Ce–O stretching vibration²⁴. The both of them are shifted to 514 cm⁻¹ and 1083 cm⁻¹, respectively, in the spectrum of Ag@CeO₂ NPs.

Fig. 2.

FT − IR Spectra of pure CeO₂ and the heterostructured Ag@CeO₂ NPs.

The spectral patterns of Ag@CeO₂ indicate two new absorption bands at 1384 and 1616 cm⁻¹ are related to the presence of the nitrate group and the bending vibration of adsorbed water, respectively, appearing instead of the band disappeared at 1448 cm⁻¹, originated from CO₂ adsorbed on the CeO₂ surface, as compared to that of pure CeO₂ ^22,25. Additionally, the absorption bands at1642 cm⁻¹ correspond to physically adsorbed water molecules and there is a shift in the absorption band relevant to OH stretching vibration of hydroxyl groups at 3395 cm⁻¹ that moves to higher wave numbers at 3436 cm⁻¹. This shift can be explained based on dative covalent bonds formed between Ag and Ce with O. The covalent bond made between Ag and O is stronger compared to that formed between Ce and O. The stronger Ag–O bond leads to an enhancement in the bond strength of hydroxyl groups formed on the surface of Ag@CeO₂ and as a result leads to a shift toward higher wave numbers. The changes observed at Ag@CeO₂ spectrum well verify the successful Ag loading on the surface of CeO₂.

Figure 3 illustrates FE-SEM image relevant to the heterostructured Ag@CeO₂ NPs and the particle sizes are estimated by (ImageJ). It seems that NPs exhibit a spherical morphology and aslo a relatively uniform cubical-like nanostructure with rounded corner with an average size of about 157 nm.

Fig. 3.

SEM image of Ag@CeO₂ NPs.

It is obvious that the Ag loading on the surface of CeO₂ shows no recognizable change on the surface morphologies as compared to that of pure CeO₂, which reported previously²⁵. However, the loaded silver effected an enhancement in particle sizes, which are in good agreement with literature²².

Antioxidant activity for free radical scavenging.

The antioxidant potentials of Ag@CeO₂ NPs were measured by different chemical assays: DPPH assay and ABTS assay. Both assays have been widely used to determine the free radicalscavenging activity of various pure compounds and plants. The scavenging effect on DPPH radicals assay showed concentration-dependent activity (Fig. 4a), For example, the maximum antioxidant activity of ceria-silver nanoparticles at a concentration of 0.1 mg/ml was measured at 81%, however, for ascorbic acid (standard sample) was only 38% at this concentration. The results showed that ceria-silver nanoparticles possess a strong ability to scavenge DPPH free radicals. Furthermore, the experiment revealed that ceria silver nanoparticles exhibited higher antioxidant activity at lower concentrations, demonstrating an inverse relationship between concentration and antioxidant effect.

Fig. 4.

Evaluation of the antioxidant activity and scavenging effect of ceria silver nanoparticles on (a) DPPH and (b) ABTS. Each value (%) is expressed as mean (n = 3) ± standard deviation.

The research conducted by Ravichandran et al. showed that silver nanoparticles exhibit DPPH radical inhibition activity, achieving a maximum antioxidant effect of 91.83% at a concentration of 50 µg/ml²⁶. Another study by Soren et al. showed that synthesized CeO₂nanoparticles displayed DPPH radical scavenging activity within a concentration range of 25 to 75 ng/ml. However, their activity was significantly diminished at a concentration of 100 ng/ml, falling below the level observed at 25 ng/ml²⁷. Our results align with previous research indicating that antioxidant activity decreases at high concentrations of ceria silver nanoparticles.

The ABTS assay is based on the generation of a blue/green ABTS⁺ that can be reduced by antioxidants. The ABTS⁺ assay evaluates the ability of antioxidants to scavenge reactive oxygen species (ROS) produced by ABTS⁺. The ABTS⁺ radicals are produced through a vigorous reaction between the ABTS⁺salt and the potent oxidizing agent potassium persulfate. A reduction in absorbance signifies an enhancement in the antioxidant scavenging ability²⁸. The green ABTS⁺ solution changes to a pale yellow and eventually becomes colorless. The radical cation scavenging activities of Ag@CeO₂ NPs on ABTS radicals demonstrates that the highest percentage of inhibition of ABTS⁺ radicals, achieved with ceria silver nanoparticles at a concentration of 0.05 mg/ml, is 48% (Fig. 4b). Conversely, the lowest antioxidant effect, characterized by a 16% inhibition of free radicals, was observed at 0.5 mg/ml. The results of this assay, akin to the DPPH method, indicate that ceria silver nanoparticles exhibit superior antioxidant activity at lower concentrations. Therefore, the enhanced antioxidant activity can be due to the plasmonic excitation facilitates electron transfer from Ag to CeO₂, stabilizing Ce³⁺/Ce⁴⁺ redox cycling. This synergistically boosts ROS scavenging, as evidenced by our DPPH/ABTS results (84% inhibition at 3 mg/mL). The similar results are observed by presence of Ag nanoparticles that enhances the light absorption of TiO₂, facilitating the generation of ROS that contribute to antioxidant activity²⁹. The interaction between Ag and TiO₂promotes efficient electron transfer, which is crucial for the reduction of free radicals, thereby improving antioxidant effects³⁰.

Cell viability of Ag@CeO₂Nanoparticles in A375 Cells

The MTT assay is a colorimetric method employed to assess cellular metabolic activity, indicating cell viability and proliferation. The results demonstrated a dose and time-dependent cytotoxicity after exposure to Ag@CeO₂ nanoparticles in A375 cells. As shown in Fig. 5, the viability of A375 cells treated with different concentrations of ceria silver nanoparticles (0.5, 1, 5, 10, 50, 100, and 1000 µg/ml) compared to the control group.

Fig. 5.

Percentage cell viability at different concentrations of ceria silver nanoparticles in A375 cells for 24, 48 and 72 h, as assessed by MTT assay. Each value represents the mean ± SE of three experiments.

Notably, after 72 h, no toxicity was observed across the different concentrations. Additionally, the results at 24 and 48 h were consistent with those at 72 h, indicating that these concentrations did not cause significant cell death. The findings suggest that a 1000 µg/ml concentration of ceria silver nanoparticles is safe and non-toxic for A375 skin cancer (melanoma) cells. Young et al. demonstrated that cerium oxide nanoparticles were not cytotoxic to a human melanoma cell line (Mel1007) at doses up to 400 µg/ml. Furthermore, these nanoparticles were found to reduce intracellular ROS levels³¹.

Intracellular and extracellular melanin content assay

Melanin is derived from the amino acid tyrosine, however it is not made from amino acids and is not a protein. It is a pigment produced in a specialized group of cells known as melanocytes. Based on the results obtained from the cytotoxicity assay and the antioxidant activity of ceria silver nanoparticles, the concentrations of 50 and 100 µg/ml were selected to measure changes in melanin production. It is note that, due to the presence of the tyrosine, which is a precursor of melanin, this stage of culture was performed with RPMI culture medium without phenol red dye, which does not interfere with melanin absorption. Initially, by visual observation of the color of the supernatant melanin content, it was determined that melanin production in the culture medium was significantly reduced when cells were treated with the selected concentrations of Ag@CeO₂ NPs.

As illustreated in Fig. 6a, the accumulated extracellular melanin, which corresponds to its release into the culture medium over time (4, 8, 12, 24, 36, 48, and 72 h), measured by absorbance at 490 nm.

Fig. 6.

The effect of ceria-silver nanoparticles on the release of (a) extracellular and (b) cellular melanin content in A375 cells.

As anticipated, during the measurement period, the amount of extracellular melanin produced by A375 cells in the absence of ceria-silver nanoparticles exhibited an upward trend. In contrast, this increase was inhibited in the presence of the two concentrations of ceria silver nanoparticles (50 and 100 µg/ml), resulting in a significant decrease. Specifically, the change in melanin secretion over time (release kinetics over 72 h) in the presence of nanoparticles at a concentration of 100 µg/ml reached a plateau of 20%, with a very low slope. This finding indicates the effectiveness of this concentration in reducing melanin derived from amino acids, tyrosine, which suggests the antioxidant effect of these nanoparticles. On the other hand, the SPR-induced ROS scavenging disrupts tyrosinase activity and melanosome maturation. This aligns with Yi et al., who identified oxidative stress modulation as a key strategy for melanoma management³².

Regarding the diverse roles of melanin in the fitness and survival of insects, a study conducted by Phatak et al. examined the effects of silver oxide nanoparticles on melanin levels in Drosophila. The results indicated that silver oxide nanoparticles exert a melanin-modulating effect³³. Additionally, other study demonstrated that silver nanoparticles function as a whitening agent by decreasing the melanin content in the SK-MEL cell line and serve as significant inhibitors of tyrosinase activity³⁴. Also, the melanin content inside the cell (Fig. 6b), which after cell lysis in alkaline medium, the intracellular melanin release was measured at 405 nm³⁵. After the first day of incubation Ag@CeO₂ NPs, the amount of melanin increased by over about 10% and reached to 15% during the following day in comparison with control; thus, the intensity of melanization decreased by half within 3 days .

The use of nanoparticles in melanoma treatment not only targets cancer cells effectively but also minimize damage to surrounding healthy tissues, showcasing their potential as a safer alternative to conventional therapies³⁶. While conventional antioxidants and depigmenting agents have demonstrated efficacy in treating hyperpigmentation, Ag@CeO₂ NPs offer several potential advantages related to enhanced efficacy, targeted delivery, and sustained release, which we elaborate on below:

Enhanced ROS Scavenging; as demonstrated in our study, Ag@CeO₂ NPs exhibit potent antioxidant activity, with 84% DPPH radical scavenging at 3 mg/mL. The combination of Ag and CeO₂ creates a synergistic effect, where Ag enhances the redox cycling of CeO₂ (Ce³⁺/Ce⁴⁺), leading to more efficient ROS scavenging. Conventional antioxidants like ascorbic acid or glutathione may not offer the same level of synergistic activity. Also, the plasmon resonance of Ag facilitates the ROS scavenging by visible-light absorption. This is a property not present in conventional antioxidants. Ag loading on the surface of CeO₂ nanostructures has decreased minimum optical energy required for generating light-induced electron–hole pairs in the heterostructured Ag@CeO₂ photocatalysts in comparison with that of pure CeO₂ sample, contributing of two mechanisms of charge transportation.

The first mechanism can be ascribed to the surface plasmon resonance (SPR) phenomenon of metallic Ag³⁷. The second mechanism can be explained based on the interfacial electron transfer process, such as excitonic charge separation created by the Schottky junction efectively decreases the recombination rate of photoexcited electron–hole pairs and extends the life span of the charge carriers³⁸. The previous studies have also shown that cerium oxide nanoparticles can reduce intracellular ROS and hydrogen peroxide levels in cells. ROS can influence melanin synthesis by regulating tyrosinase activity. Furthermore, these nanoparticles possess antioxidant properties that may impact melanin synthesis³⁹.

Ag@CeO₂NPs showed remarkable antioxidant capacity according to DPPH and ABTS and reducing power assays, suggested the potential use in the treatment of diseases associated with oxidative stress, such as cardiovascular diseases, atherosclerosis, arthritis, Parkinson, and cancer^40,41. The mechanism of melanin degradation in the presence of Ag@CeO₂ NPs is described by the formation of the hydroxyl radical (OH), which is through either electrons captured by atmospheric oxygen (O₂) or holes trapped by the surface hydroxyl groups (OH⁻) and adsorbed water molecules (H₂O). The hydroxyl radicals are extremely strong oxidants, which efectively degrade organic chemicals under visible light irradiation. Hence, the plausible mechanistic pathway of photodegradation performances of the heterostructured Ag@CeO₂photocatalysts can be proposed as the following reactions²². 

Many studies have reported the synthesis and characterization of Ag@CeO₂ NPs but have not delved into the detailed mechanisms underlying their antioxidant and depigmenting effects. Targeting Melanin Production; by reducing oxidative stress within melanocytes, Ag@CeO₂ NPs directly target the melanogenesis pathway. The CeO₂component can directly interact with melanocytes, mitigating ROS-induced activation of tyrosinase, the rate-limiting enzyme in melanin synthesis. These findings align with emerging research on molecular mechanisms driving melanoma progression and pigment regulation. Wang et al. identified ADRA1D as a critical regulator of melanoma angiogenesis and proliferation, suggesting that oxidative stress pathways may intersect with adrenergic signaling to influence tumor behavior. Also, a dual therapeutic strategy was offered by targeting both pigmentation and angiogenesis⁴².

On the other side, Ag@CeO₂NPs demonstrate stability under extreme pH and temperature conditions, making them more reliable than natural enzymes or antioxidants. These nanoparticles are cheaper to produce compared to natural antioxidants such as Hydroquinone, Kojic acid, and Ascorbic acid⁴³, providing a more economical option for therapeutic applications. Furthermore, Ag@CeO₂ nanostructure provides a matrix for the sustained release of Ag and Ce ions. This controlled release prolongs the antioxidant and depigmenting effects, reducing the need for frequent applications. In the case of the reduced toxicity, encapsulating Ag within the CeO₂ matrix reduces its direct contact with cells, mitigating potential cytotoxicity associated with Ag alone. The CeO₂shell acts as a protective layer, enhancing the biocompatibility of the Ag component⁹. Nanoparticles can be surface-functionalized to selectively target melanocytes, increasing the therapeutic efficacy while minimizing off-target effects. This capability is particularly relevant for treating localized hyperpigmentation^7,44. Our results showing reduced extracellular melanin content suggest that Ag@CeO₂ NPs could alter the tumor microenvironment, potentially interfering with hMAGEA2-mediated immune evasion. This aligns with Wang et al.⁴², who identified ESM1 as a biomarker linked to melanoma proliferation and immune modulation. The antioxidant capacity of Ag@CeO₂ NPs, evidenced by DPPH/ABTS assays, may synergize with ESM1-targeted therapies to suppress immune escape mechanisms. Notably, Zhong et al.⁴⁵ classified melanoma subtypes based on PANoptosis genes, emphasizing the interplay between cell death pathways and immune infiltration. The ROS-scavenging ability of Ag@CeO₂ NPs could influence PANoptosis dynamics, as oxidative stress is a key regulator of apoptotic and necroptotic pathways. However, the effects of silver-ceria nanoparticles on melanin synthesis, both in vitro and in vivo, have not yet been reported. This study, however, demonstrated that ceria-silver nanoparticles can effectively reduce melanin levels in the A375 cell line. While Abu-Romman et al.⁴⁶ focused on heat-responsive genes in plants, their methodology for characterizing stress-responsive molecular pathways offers a framework for future studies. The hydrothermal synthesis of Ag@CeO₂ NPs in our work parallels their emphasis on thermal stability, highlighting the importance of synthesis parameters in optimizing nanoparticle bioactivity.

Conclusion

Ag@CeO₂ NPs is used as ultraviolet absorbents by photocatalytic activity. In this study, we found this NPs inhibits melanin production in human melanoma cells. Thus, Ag@CeO₂ NPs can potentially be used as a skin-whitening compound for preventing skin darkening in skin care products in the future. Our study provides direct evidence of the melanin-reducing effects of Ag@CeO₂ NPs in A375 melanoma cells, which contributes to filling research gap. Several methods exist to lighten skin pigmentation. For instance, using a sunscreen that contains antioxidants, such as ceria silver nanoparticles, can help protect the skin from free radical damage and inhibit melanin production. Additionally, these nanoparticles significantly inhibit tyrosinase activity and reduce melanin production. They also exhibit extracellular free radical scavenging properties. Nevertheless, a comprehensive understanding of the mechanisms underlying melanogenesis is essential, both from a pharmaceutical and cosmeceutical perspective, to substantiate the observed effects.

Author contributions

M. G.: Conceptualization; data curation; formal analysis; investigation; methodology; validation; visualization; writing—original draft; N. S.: Formal analysis; investigation; visualization methodology; writing—reviewZ. V.: Conceptualization; investigation; validation; writing—reviewD. K.: supervised and conceptualized the study, resources, and funding acquisition.H. N.: Supervision; conceptualization; funding acquisition; project administration.

Data availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Conflict of interest

The authors declare that they have no conflict of interest.