Relation of early Photofrin uptake to photodynamically induced phototoxicity and changes of cell volume in different cell lines

Abstract

For efficacy of photodynamic therapy, selective uptake and retention of photoactive substances has been postulated. Therefore, measurements were performed to find out whether the photosensitiser Photofrin is taken up differently in malignant and non-malignant cells in vitro. In addition, the sensitivity of malignant cells and non-malignant cells to photodynamic exposure was investigated, by quantifying viability and volume alterations of the cells. Bovine aortic endothelial cells, mouse fibroblasts and amelanotic hamster melanoma cells were suspended in a specially designed incubation chamber under controlled conditions (e.g. pH, pO2, pCO2 and temperature). After establishing constant baseline conditions, the cellular fluorescence intensity per cell volume, indicative of the uptake of Photofrin, and cell volume were assessed by flow cytometry, and cell viability was quantified by the trypan blue exclusion test. Photodynamic exposure of cells was performed using an argon-pumped dye laser system via a 600 microns optical fibre at energy density of 4 Joules at the cell surface (40 mW/cm2, 100 s). In comparison to endothelial and fibroblast cells, the melanoma cells exhibited no increased uptake of Photofrin, and no enhanced sensitivity to photodynamic therapy (PDT). However, the fluorescence intensity/volume of endothelial cells was two to three times higher at each concentration of the photosensitiser. Following PDT, reduction in cell viability was dependent on the concentration of Photofrin, and directly correlated with fluorescence intensity per cell volume. In addition, the cells of all three lines, treated by PDT, revealed dose-dependent changes in cell volume. Melanoma cells exhibited the most excessive increase. It is suggested that selective uptake of photosensitiser in vitro is not characteristic for tumour cells. The high uptake of Photofrin by endothelial cells may indicate that the vascular endothelium is a major target for PDT, leading to cessation of tumour blood flow and subsequent destruction of tumour tissue. In addition, PDT-induced swelling of tumour cells might represent and effect synergistically impairing tumour perfusion, and thereby promoting tumour death.