Photodynamic Activity of Acridine Orange in Keratinocytes under Blue Light Irradiation

Acridine orange (AO) is a metachromatic fluorescent dye that stains various cellular compartments, specifically accumulating in acidic vacuoles (AVOs). AO is frequently used for cell and tissue staining (in vivo and in vitro), mainly because it marks different cellular compartments with different colors. However, AO also forms triplet excited states and its role as a photosensitizer is not yet completely understood. Human immortalized keratinocytes (HaCaT) were incubated for either 10 or 60 min with various concentrations (nanomolar range) of AO that were significantly lower than those typically used in staining protocols (micromolar). After incubation, the cells were irradiated with a 490 nm LED. As expected, cell viability (measured by MTT, NRU and crystal violet staining) decreased with the increase in AO concentration. Interestingly, at the same AO concentration, altering the incubation time with HaCaT substantially decreased the 50% lethal dose (LD₅₀) from 300 to 150 nM. The photoinduced cell death correlated primarily with lysosomal disfunction, and the correlation was stronger for the 60 min AO incubation results. Furthermore, the longer incubation time favored monomers of AO and a distribution of the dye to intracellular sites other than lysosomes. Studies with mimetic systems indicated that monomers, which have higher yields of fluorescence emission and singlet oxygen generation, are favored in acidic environments, consistent with the more intense emission from cells submitted to the longer AO incubation period. Our results indicate that AO is an efficient PDT photosensitizer, with a photodynamic efficiency that is enhanced in acidic environments when multiple intracellular locations are targeted. Consequently, when using AO as a probe for live cell tracking and tissue staining, care must be taken to avoid excessive exposure to light to avoid undesirable photosensitized oxidation reactions in the tissue or cell under investigation.