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

Abstract

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 β-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 (≤20 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.

Optimization of an ex vivo wound healing model in the adult human skin: Functional evaluation using photodynamic therapy

“…This was subsequently illuminated with a PDT machine (Omnilux PDT, Photo Therapeutics, PA), with an arrangement of red light‐emitting diodes (LEDs) of 633 ± 3 nm wavelength. The power intensity of the PDT machine was adjusted to 20 J/cm 2 and calibrated with an IL1700 radiometer (International Light Technologies Inc, Peabody, MA) . PDT treatment was given once on the third day after collection.…”

“…PDT works by energy transfer from illumination to molecular oxygen through the action of a photosensitizer, 5‐aminolevulonic acid (5‐ALA), as precursor of protoporphyrin IX. Cellular effects of PDT depend on tissue type, oxygen tension and dosage, resulting in necrosis, apoptosis, and autophagy …”

Photodynamic therapy in dermatology beyond non-melanoma cancer: An update

“…Keloids are a type of scar formed from fibroproliferative dermal tissue characterized by the excessive proliferation of fibroblasts. Mendoza et al [47] evaluated the cytotoxic effects of PDT using MAL and ALA on KD fibroblasts compared to normal skin fibroblasts. Li et al [48] investigated the effects of ALA-PDT and showed that hypertrophic scar-derived fibroblasts accumulated more PpIX.…”

Strategic management of keloid disease in ethnic skin: a structured approach supported by the emerging literature

“…74 This study showed that the outcome of PDT was dependent on the photosensitizer precursor, the fluence and the location of fibroblasts in the lesion. A positive effect of PDT was initially reported in a patient with a persistent keloid that had not responded to a number of routine therapies.…”