Foscan (mTHPC) photosensitized macrophage activation: enhancement of phagocytosis, nitric oxide release and tumour necrosis factor-alpha-mediated cytolytic activity.

Photodynamic activation of macrophage-like cells contributes to an effective outcome of photodynamic therapy (PDT) treatment. The possibility for an enhancement of macrophage activity by photosensitization with meta-tetra(hydroxyphenyl)chlorin (mTHPC) (1 microg ml(-1)) was studied in U937, monocyte cell line differentiated into macrophages (U937) cells). Phagocytic activity of U937phi cells was evaluated by flow-cytometry monitoring of ingestion of fluorescein-labelled Escherichia coli particles. Increase in irradiation fluence up to 3.45 mJ cm(-2) (corresponding irradiation time 15 s) resulted in significant increase in fluorescence signal (145%), while at higher light fluences the signal lowered down to the untreated control values. A light energy-dependent production of tumour necrosis factor-alpha (TNF-alpha) by photosensitized macrophages was further demonstrated using the L929 assay. The maximum TNF-alpha mediated cytolysis was observed at 28 mJ cm(-2) and was 1.7-fold greater than that in control. In addition, we demonstrated a fluence-dependent increase in nitric oxide (NO) production by mTHPC-photosensitized macrophages. NO release increased gradually and reached a plateau after irradiation fluence of 6.9 mJ cm(-2). Cytotoxicity measurements indicated that the observed manifestations of mTHPC-photosensitized macrophage activation took place under the sublethal light doses. The relevance of the present findings to clinical mTHPC-PDT is discussed.

Meta-tetra(hydroxyphenyl)chlorin-sensitized photodynamic damage of cultured tumor and normal cells in the presence of high concentrations of alpha-tocopherol.

Alpha-tocopherol at low concentrations protects biosubstrates from oxidative damage, while at high concentrations it may become toxic. Certain lines of tumor cells are reported to contain higher levels of vitamin E than normal cells. In our study alpha-tocopherol was successfully incorporated by cultured HT29 adenocarcinoma cells, but not by MRC-5 normal fibroblasts. At high concentrations (0.3-1 mM) alpha-tocopherol enhanced meta-tetra(hydroxyphenyl)chlorin (mTHPC)-sensitized photoinactivation of HT29 cells (415 nm), but not that of normal fibroblasts. At none of the concentrations used (0.001-1 mM) did alpha-tocopherol protect cells from photokilling, indicating that lipid peroxidation is of minor importance in mTHPC photoactivity. Our findings encourage the in vivo testing of phenolic antioxidants for selective enhancement of PDT-damage in tumors.

Characterization of photodegradation of meta-tetra(hydroxyphenyl)chlorin (mTHPC) in solution: biological consequences in human tumor cells.

The photobleaching of meta-tetra(hydroxyphenyl)chlorin (mTHPC) (irradiation wavelength 413 nm) in protein-containing solution was evaluated by decay in absorbance in Soret band and in fluorescence (lambda exc = 423 nm, lambda em = 655 nm). Light exposure resulted in a decrease in absorption throughout the spectrum and simultaneous appearance of new absorption bands in the spectral region 325-450 nm. The rate of mTHPC photodegradation, followed by decay in absorbance, was 15-fold lower than that observed in fluorescence. This fact reflects the photobleaching of presumably monomeric, fluorescing species of mTHPC. In order to determine the consequences of photobleaching of fluorescing mTHPC material on cellular uptake and photocytotoxicity, human HT29 colon adenocarcinoma cells were incubated with photobleached mTHPC during 5 h with or without following irradiation with the fixed fluence. Surprisingly, but up to the time when the fluorescence decreased by 50%, only a slight decrease in photocytotoxicity was detected. Either aggregated forms that have been taken up undergo intracellular monomerization (but we did not observe increase in fluorescence in living cells) or the photodynamic activity is mostly due to aggregates. The discrepancy of mTHPC-photodynamic therapy (PDT) effect and fluorescence measurements may suggest that aggregated mTHPC plays an important role in mTHPC-PDT.

Spectroscopic and biological testing of photobleaching of porphyrins in solutions.

The photobleaching of protoporhyrin IX (PP IX) and hematoporphyrin derivative (HpD) solutions was followed using three different methods: spectrophotometry, fluorometry and photodynamically induced cytotoxicity. The latter entails photoirradiation of HT29 human colon adenocarcinoma cells in the presence of preirradiated solutions of HpD and PP IX (lambda < or = 415 nm). The highest cytotoxicity was observed in the presence of unirradiated dye and decreased with the time of preirradiation. This decay in photocytotoxicity was further used to determine the porphyrin photobleaching kinetics in solution. For both sensitizers, quantum yields of photobleaching obtained by matching fluorescence were higher than that obtained from absorbance measurements (10 and 11 times for HpD and PP IX, respectively). This difference reflects preferential photobleaching of photolabile monomeric forms compared to aggregated. The highest quantum yield was obtained in the biological test (decay in cytotoxicity) which was 14 times higher for HpD and 30 times higher for PP IX than the quantum yield obtained from absorbance measurements. The absence of correlation between biological and fluorescence measurements has to be taken into account in the in vivo situation. Dark storage of preirradiated sensitizers (37 degrees C, 24 h) completely restored photocytotoxity for PP IX but only partially for HpD, whereas fluorescence patterns were partially restored for both sensitizers.