PG fruits were purchased from the western part of Romania (Timișoara, coordinates: 45°44′58″ N latitude, 21°13′38″ W longitude) and identified at the Faculty of Pharmacy, “Victor Babeș” University of Medicine and Pharmacy Timișoara. First, the pomegranate fruits were washed several times with ultrapure water from the Milli-Q^® Integral Water Purification System (Merck Millipore, Darmstadt, Germany) to avoid contamination. After thorough washing and drying, the peel was carefully separated, oven-dried at 23 ± 1 °C for 5 days, and ground into a fine powder.

For the preparation of the PG ethanolic extract, 50 g of fine powder was mixed with 250 mL of 95% ethanol and left to macerate for 2 weeks. Then, the mixture was ultrasonicated (50% amplitude) for 1 h to extract more polyphenols from the pomegranate peel using a QSonica 700W ultrasonic processor. Finally, the ethanolic extract was filtered through filter paper.

To obtain PG-AgNPs via the green method, 100 mL of pomegranate peel ethanolic extract was stirred using a magnetic stirrer. At 60 °C, under stirring at 500 rpm, an aqueous solution of 1 M AgNO₃ (50 mL) was added to a thin wire, and the mixture was stirred for another two hours. After 15 min, the light red-orange mixture turned reddish-orange to reddish-brown, and after 30 min, it became reddish-black. After two hours, the color of the mixture changed to brown-black, which confirmed the reduction of the AgNO₃ solution to AgNPs. Thus, pomegranate AgNPs synthesized by the green method were obtained.

In vitro analyses were performed using reagents and equipment obtained from certified commercial suppliers. From Sigma Aldrich, Merck KGaA (Darmstadt, Germany), the following reagents were purchased: phosphate-buffered saline (PBS) solution and an antibiotic mixture of penicillin/streptomycin. From Roche Holding AG (Basel, Switzerland) the MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was obtained and used for cell viability assays. From Thermo Fisher Scientific (Waltham, MA, USA), the nuclear dye Hoechst 33342 and the mitochondrial marker MitoTracker™ Red CMXRos were used for cell staining and visualization; from PAN-Biotech GmbH (Aidenbach, Germany), trypsin–EDTA, fetal bovine serum (FBS), and the culture medium EMEM (Eagle’s Minimum Essential Medium) were obtained. For extract preparation, 96% ethanol was acquired from Chemical Company SA (Iasi, Romania).

Regarding instrumentation, all devices and software used were provided by BioTek Instruments Inc. (Winooski, VT, USA), including a Cytation 5 microplate reader, a Lionheart FX automated microscope, and Gen5™ software (version 3.14) used for data acquisition and analysis.

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to assess cell viability, following the protocol described by Romanu et al. [12]. Experiments were performed after RPMI-7951 cells were exposed for 24 h to PG-AgNPs at concentrations varying from 10 to 30 μg/mL (10, 15, 20, 25, and 30 μg/mL). Cells were cultured in 96-well flat-bottomed plates at a density of 1 × 10⁴ cells/well and treated with the test substance. At the end of the 24 h incubation period, the culture medium was replaced with fresh medium, and 10 μL of MTT kit 1 was added to each well. Cells were then incubated for 3 h at 37 °C in a 5% CO₂ atmosphere. Subsequently, 100 μL of MTT kit two was added, and the plates were left at room temperature for 30 min. The absorbance was measured at wavelengths of 570 nm and 630 nm using a Cytation 5 instrument delivered by BioTek Instruments Inc. (Winooski, VT, USA).

To evaluate the effects of PG-AgNPs on the cell morphology of RPMI-7951 cells, they were cultivated in 96-well plates (1 × 10⁴ cells/well). To assess the possible morphological changes, images were taken at 20× magnification under bright-field illumination using the Lionheart FX automated microscope and processed with Gen5 Microplate Data Collection software, version 3.14 (from BioTek Instruments Inc., Winooski, VT, USA).

To further investigate the effect of PG-AgNPs on cell nuclei, the Hoechst staining method was used,following the protocol described by Dan et al. [13]. Cells were cultured in 12-well plates at a density of 1 × 10⁵ cells/well and treated with PG-AgNPs at the highest concentration of 30 µg/mL for 24 h under standard incubation conditions. After this period, protected from light, the culture medium was removed, and the Hoechst solution obtained by 1:2000 dilution in PBS was added to each well. The plate was then incubated for 5–10 min in a dark place. Subsequently, the staining solution was removed, and the wells were washed three times with PBS. Representative images of the nuclei were captured using the Lionheart FX automated microscope at 20X magnification, and image analysis was performed using Gen5™ Microplate Data Collection and Analysis software (version 3.14) from BioTek Instruments Inc. (Winooski, VT, USA).

To assess mitochondrial morphology, RPMI-7951 cells were cultured in 12-well plates at a density of 1 × 10⁵ cells/well and allowed to adhere until optimal confluence was achieved, and the experiments were performed following a previously published protocol [13]. The cells were then treated for 24 h with PG-AgNPs at the highest concentration of 30 μg/mL. The MitoTracker dye was initially dissolved in DMSO at a stock concentration of 1 mM and subsequently diluted in the culture medium (EMEM 10%) to a final concentration of 300 nM. Following a 30 to 45 min incubation period with the staining solution under standard culture conditions, the cells were thoroughly washed with PBS to remove any remaining dye. For evaluation, images were captured using a Lionheart FX automated fluorescence microscope at 20× magnification, and the data were analyzed with Gen5™ Microplate Data Collection and Analysis software (version 3.14, BioTek Instruments Inc., Winooski, VT, USA).

All results are presented as mean ± standard deviation (SD). Data distribution was confirmed to be normal using the Shapiro–Wilk test. Therefore, a one-way ANOVA followed by Dunnett’s multiple comparisons test was applied for statistical analysis. The analysis was performed using GraphPad Prism version 10.2.3 (GraphPad Software, San Diego, CA, USA; www.graphpad.com). Statistical significance was indicated as follows: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

The MTT assay was employed to assess the cytotoxic potential of PG-AgNPs on human melanoma RPMI-7951 cells at concentrations of 10, 15, 20, 25, and 30 μg/mL (Figure 1). A dose-dependent decrease in cell viability was observed following exposure to the nanoparticles. At the lowest tested concentration (10 μg/mL), cell viability was 95.06% compared to the control group, gradually decreasing to 88.85% at 15 μg/mL, 84.38% at 20 μg/mL, 81.06% at 25 μg/mL, and reaching 79.43% at 30 μg/mL. These findings indicate a moderate cytotoxic effect of AgNPs on melanoma cells, indicating a precise dose–response relationship across the tested concentration range.

In line with these results, Fernandes et al. demonstrated that green-derived (with pomegranate extract) silver nanoparticles exhibited low cytotoxicity on mouse fibroblast cells (L929), maintaining a viability of about 80% at 50 µg/mL. In contrast, conventional nanoparticles were found to be toxic at 6.25 µg/mL, with viability below 20%. This offers a promising perspective for the use of green synthesis nanoparticles in biomedical applications due to their better profile [19]. Moreover, similar concentration-dependent decreases in cell viability have been reported in other cell lines, including cervix adenocarcinoma (HeLa), larynx carcinoma (HEp-2), liver carcinoma (Hep-G2), prostate adenocarcinoma (PC3), and kidney epithelial cells (Vero), confirming the generalized nature of this biological response [20]. According to another study by Badawi et al., solid lipid nanoparticles or PG formulations exhibit more potent cytotoxicity against cancer cell lines such as human mammary carcinoma (MCF-7), human prostate carcinoma (PC-3), and human hepatocellular carcinoma (HepG2); their nanoencapsulation further enhances this selectivity [21]. In line with these findings, the present results on RPMI-7951 melanoma cells also demonstrated dose-dependent reductions in viability and morphological alterations, supporting the anticancer potential of pomegranate-based nanostructures.

Investigating the impact of natural compounds on human cells and epithelial structures is essential, as morphological evaluation provides insight into cellular responses and potential therapeutic applications [22]. To further investigate the cytotoxic effects observed in the MTT assay, the impact of PG-AgNPs on the morphology of RPMI-7951 human melanoma cells was evaluated after 24 h of treatment at concentrations of 10, 15, 20, 25, and 30 μg/mL (Figure 2). A progressive, dose-dependent reduction in cell confluency was noted, with pronounced morphological alterations at higher concentrations, particularly at 25 and 30 μg/mL.

To gain a more detailed understanding of the mechanism of action of PG-AgNPs, the nuclear morphology of RPMI-7951 cells was assessed using a staining method. Hoechst staining is a fluorescent method used to observe apoptotic nuclear changes, such as chromatin condensation and fragmentation. It is widely applied for in vitro studies due to its DNA specificity and low toxicity [23]. According to Figure 3, after 24 h of treatment with the highest tested concentration (30 μg/mL), mild changes in nuclear morphology—highlighted by yellow arrows—were observed compared to the untreated control. The nuclei appeared slightly rounded and reduced in number, and in some cells, chromatin condensation was noticeable, a typical feature associated with apoptotic-like changes.

To assess the effect of PG-AgNPs on mitochondrial morphology, RPMI-7951 cells were treated for 24 h with the highest tested concentration (30 μg/mL), followed by immunofluorescence staining with MitoTracker (Figure 4). Compared to untreated control cells, the treatment induced mild morphological alterations at the mitochondrial level, including mitochondrial condensation (indicated by yellow arrows) and a decrease in mitochondrial confluency.

Previous studies have demonstrated that AgNPs can induce cytotoxic effects through molecular pathways such as reactive oxygen species (ROS) generation, mitochondrial dysfunction, and the activation of caspase-dependent apoptosis [24,25,26]. The nuclear condensation and mitochondrial alterations observed in our study on the RPMI-7951 cells are therefore in line with these reported mechanisms, suggesting that a similar mechanism may underlie the effects of PG-AgNPs in this model.

One of the limitations of this study is the lack of detailed physicochemical characterization of PG-AgNPs (e.g., particle size distribution, morphology, and stability), which will be addressed in future research to enhance reproducibility and interpretation of the results.