Differential cytotoxic response in keloid fibroblasts exposed to photodynamic therapy is dependent on photosensitiser precursor, fluence and location of fibroblasts within the lesion

Treatment of keloid disease (KD) is ill-defined and remains challenging. We previously reported successful clinical application of photodynamic therapy (PDT) in KD. The aim here was to evaluate cytotoxic effect of PDT using methyl aminolevulinate (M-ALA) and 5-aminolevulinic acid (5-ALA) on keloid fibroblasts (KF) (n = 8) from different lesional sites (top, middle and margin) as compared to normal skin fibroblasts (n = 3). The effect of protoporphyrin IX (PpIX) precursors was evaluated by fluorescence emission, LDH and WST-1 assay, reactive oxygen species (ROS) generation and qRT-PCR analysis. Apoptosis/necrosis differentiation and senescence were studied by fluorometric staining with Hoechst 33258/propidium iodide and beta-galactosidase activity, respectively. Three hours post incubation with 4 mM precursors of photosensitisers, PpIX accumulation was site specific and higher with M-ALA. Cytotoxicity was also site specific (higher in fibroblasts from middle of the keloid as compared to cells from other sites) and increased proportionately to fluence rates post-PDT. Additionally, cytoproliferation was significantly decreased post-PDT depending on the light energy. Fluorescence analysis revealed that M-ALA instigated higher KF cytotoxicity at lower fluence (a parts per thousand currency sign20 J/cm(2)) while 5-ALA instigated higher KF cytotoxicity at higher fluence, except in cells derived from middle of the keloid. ROS-mediated cytotoxicity was light energy dependent. Senescence was not observed at higher light energies (> 10 J/cm(2)). Compared to other sites, fibroblasts from the middle were more prone to cell death post 5-ALA treatment. We conclude that cytotoxicity post-PDT in KD fibroblasts is dependent on the lesional site, precursor of intracellular photosensitiser and fluence. Thus, PDT may be used for site-targeted therapy of KD.