Membrane interactions and photodynamic activity of 1,9-dimethyl methylene blue in oropharyngeal carcinoma models

The growing demand for alternative cancer treatments has intensified interest in photodynamic therapy (PDT), a minimally invasive approach that combines light irradiation with photosensitizing agents (PSs) to achieve localized cytotoxicity. Phenothiazine derivatives are well established as antimicrobial and anticancer PSs, positioning 1,9-dimethyl methylene blue (DMMB) as a promising candidate. However, the photodynamic performance of DMMB against oropharyngeal carcinoma, particularly as a function of photosensitizer concentration and light dose, remains poorly explored. Here, we investigate the molecular interactions and photodynamic effects of DMMB using membrane-mimetic systems based on lipid extracts from human oropharyngeal carcinoma cells (HEp-2), complemented by in vitro cellular assays. Langmuir monolayers revealed strong membrane affinity of DMMB, leading to pronounced monolayer expansion (up to ∼90% at 1.0 μmol/L) and changes in elastic properties. Upon photoactivation, DMMB induced concentration- and light dose-dependent reductions in monolayer surface area, consistent with light-triggered photodynamic effects at the membrane interface. At 0.1 μmol/L, a modest decrease of ∼3.5% was observed, whereas at 1.0 μmol/L the surface area reduction increased from 6.9% to 13.4% as the light dose rose from 1 to 18 J/cm². FTIR spectroscopy of Langmuir-Schaefer films revealed limited spectral changes mainly associated with interfacial lipid regions, particularly carbonyl hydration and relative intensity variations, while phosphate and acyl-chain regions were largely preserved. Cellular assays were consistent with the interfacial findings. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and LDH (lactate dehydrogenase) assays demonstrated pronounced phototoxicity in HEp-2 cells with no detectable dark toxicity, yielding IC₅₀ values that decreased from 0.82 μmol/L at 1 J/cm² to 0.35 μmol/L at 18 J/cm². Increased LDH release and confocal fluorescence microscopy revealed plasma membrane disruption and intracellular damage consistent with light-induced membrane perturbation inferred from the monolayer studies, which may involve lipid oxidation-related processes. Overall, these results support an association between DMMB-membrane interactions and cellular phototoxicity, highlighting how photosensitizer concentration and light dose modulate DMMB photodynamic effects under the investigated experimental conditions. DMMB thus emerges as a promising candidate for further investigation in oropharyngeal carcinoma PDT models.