Thymoquinone (TQ), a bioactive compound found in Nigella sativa seeds, possesses diverse therapeutic properties for skin conditions. However, formulating TQ presents challenges due to its hydrophobic nature and chemical instability, which hinder its skin penetration. Transethosomes, as a formulation, offer an environment conducive to enhancing TQ’s solubility, stability, and skin permeation. To optimize TQ transethosomal formulations, we introduced a combination of ionic and nonionic surfactants, namely Tween 20 and sodium lauryl sulfate (SLS) or sodium lauroyl glutamate (SLG). Surfactants play a crucial role in stabilizing the formulation, reducing aggregation, improving biocompatibility, and minimizing potential toxicity. We fine-tuned the formulation composition and gained insights into its interfacial behavior using the Langmuir monolayer technique. This method elucidated the interfacial properties and behavior of phospholipids in ethosome and transethosome formulations. Our findings suggest that monolayer studies can serve as the initial step in selecting surfactants for nanocarrier formulations based on their interfacial dilational rheology studies. It was found that the addition of surfactant to the formulation increased the elasticity considering the capability of transethosomes to significantly decrease their radius when permeating the skin barrier. The results of the dilational rheology experiments were most relevant to drug permeation through the skin for the largest amplitude of deformation. The combination of Tween 20 and SLS efficiently modified the rheological behavior of lipids, increasing their elasticity. This conclusion was supported by in vitro studies, where formulation F2 composed of Tween 20 and SLS demonstrated the highest permeation after 24 h (300.23 µg/cm²). Furthermore, the F2 formulation showed the highest encapsulation efficiency (EE) of 94%, surpassing those of the control and ethosomal formulations. Additionally, this transethosomal formulation exhibited antimicrobial activity against S. aureus, with a zone of inhibition of 26.4 ± 0.3 mm. Importantly, we assessed the cytotoxicity of both ethosomes and transethosomes at concentrations ranging from 3.5 µM to 50 µM on HaCaT cell lines and found no cytotoxic effects compared to TQ hydroethanolic solution. These results suggest the potential safety and efficacy of TQ transethosomal formulations.

Combining anti-cancer agents in cancer therapies is becoming increasingly common because of their improved efficacy, reduced toxicity, and decreased risk of resistance development. Melanoma, a highly aggressive form of skin cancer characterized by limited treatment options due to chemoresistance, poses a considerable challenge for effective management. Here, we test the hypothesis that dietary supplements such as thymoquinone (TQ) and curcumin (CU) cooperatively modulate cancer-associated cellular mechanisms to inhibit melanoma progression. Through a series of in vitro experiments utilizing the A375 melanoma cell line, including assessments of cell viability, apoptosis, multicellular tumor spheroid models, reactive oxygen species (ROS) quantification, metabolomics analysis, and RNA sequencing, we established that the combined application of TQ and CU exhibited superior anti-tumor effects compared to their individual use. Our results indicate that the combination treatment significantly inhibited cell viability and induced apoptosis more effectively than either agent alone, with optimal synergy observed at concentrations of 25 µM CU and 10 µM TQ against A375 cells. Additionally, the combination treatment markedly elevated ROS levels, selectively activating the mitochondrial apoptotic pathway via caspase-9. Differential gene expression analysis further revealed a unique synergistic effect of the combination treatment, with enhanced regulation of genes related to oxidative stress and apoptosis. Notably, pathways such as mitochondrial apoptotic signaling and redox homeostasis were more effectively influenced by the combination, with genes such as GPX3, CYP4F11, and HSPB8 cooperatively regulated. Overall, the findings suggest that, in combination, TQ and CU acts synergistically against melanoma; however, further experimental and clinical studies are required to confirm its therapeutic potential.