Nano-silymarin combined with methylene blue photodynamic therapy versus monotherapies of head and neck squamous cell carcinoma: in-vitro study

Head and neck squamous cell carcinoma (HNSCC) presents significant therapeutic challenges due to its aggressiveness and the morbidity of conventional treatments. Photodynamic therapy (PDT) offers a localized approach, but its efficacy can be limited. This study investigated the potential of nano-silymarin, a bioactive flavonolignan complex with known anticancer properties, in combination with methylene blue-mediated PDT (MB-PDT) in HNSCC cells. The cytotoxic, apoptotic, and anti-migratory effects of various treatments were assessed in HN5 cells. Groups included controls, individual agents (nano-silymarin, MB, laser), dual therapies, and the triple combination (nano-silymarin + MB + laser). Cell viability was measured using the MTT assay, apoptosis by flow cytometry, and migration by the scratch assay. Results were analyzed using one-way ANOVA with Tukey’s post hoc test. Synergy was quantified using the Bliss independence, Loewe additivity, and zero interaction potency models. The triple combination (nano-silymarin + MB-PDT) demonstrated the most potent effect, synergistically reducing cell viability to 40.63% (compared with 69.65% for nano-silymarin and 77.32% for MB-PDT). It induced the highest level of total apoptosis (42.93%) and nearly completely inhibited cell migration (97.2% wound openness at 72 h). This anti-migratory effect was significantly greater than the sum of the effects of the individual monotherapies, indicating a synergistic blockade of cellular invasion. Synergy analysis confirmed a strong synergistic interaction, with a Combination index of 0.682. Nano-silymarin significantly potentiates the anticancer efficacy of MB-PDT against HNSCC cells through strong synergistic interactions, thereby enhancing cytotoxicity, apoptosis, and migration inhibition. These findings support further development of nano-silymarin in combinatorial PDT regimens for HNSCC.