Phylloerythrin: Mechanisms for cellular uptake and location, photosensitisation and spectroscopic evaluation

Abstract

AIM: To elucidate the photobiological behaviour of phylloerythrin by studying the cellular uptake and intracellular localisation pattern of phylloerythrin and its spectral properties in Chinese hamster lung fibroblast cells (V79). METHODS: Phylloerythrin was diluted in dimethylsulfoxide (DMSO). Fluorescence emission and excitation spectra were measured using a luminescence spectrometer equipped with a red-sensitive photomultiplier. V79 cells were cultured in monolayers and labelled with 0.25 µg/ml phylloerythrin for uptake, cell survival and intracellular localisation studies. For cell survival and intracellular localisation studies, cells were subsequently exposed to blue light at a fluence rate of 9.0 mW/cm2. RESULTS: The fluorescence excitation spectrum of phylloerythrin in DMSO was characterised by a Soret band exhibiting a maximum peak at 418 nm. The fluorescence emission spectrum had peaks at 643 and 706 nm. The corresponding spectra in cells were red-shifted to 422, 650 and 712 nm, respectively. The cellular uptake of phylloerythrin was complete after about 10 h of incubation. The uptake together with the activation energy and analysis of cells incubated with phylloerythrin at 37°C and 0°C using fluorescence microscopy indicated that the dye is taken up into cells via a diffusion-mediated pathway. Measurements of subcellular marker enzymes were performed immediately afterlight exposure of phylloerythrin-treated cells. The mitochondrial marker enzyme, cytochrome-c oxidase, and the marker enzyme for the Golgi apparatus, UDP galactosyl transferase, but not those for lysosomes, β-N-acetyl-D-glucosaminidase (β-AGA), and endoplasmic reticulum, NADPH cytochrome-c reductase, were inactivated upon photodynamic treatment. CONCLUSION: These results indicate that phylloerythrin is located mainly in the Golgi apparatus and mitochondria of V79 fibroblasts cells.