Flow cytometric studies of cellular uptake of PII, m-THPP and m-THPC: kinetics and cell density dependence

The uptake of the photosensitizers Photofrin II (PII), meso- tetra-hydroxyphenyl-porphyrin (m-THPP) and meso-tetra- hydroxyphenyl-chlorin (m-THPC) in REH-cells was studied by flow cytometry (FCM). The uptake kinetics were studied with and without metabolic inhibitors. The uptake of PII after 60 min incubation was roughly 50% lower in the presence of the metabolic inhibitors, suggesting a partly active uptake mechanism for PII in REH-cells. The accumulation of m-THPP and m-THPC was not reduced by the metabolic inhibitors. Incubating PII-loaded cells for 60 minutes in sensitizer- free medium reduced the fluorescence intensity by approximately 60%, but had only a minor impact on the fluorescence intensity of cells incubated with m-THPP or m- THPC, even when the washing medium contained 10% serum. The uptake per cell was 9.5, 16, and 11 times higher at 105 than at 107 cells ml-1 for PII, m-THPP and M- THPC in PBS, respectively. This was not solely due to depletion of dye from the medium, since the extracellular sensitizer-concentrations decreased less than 50%. Medium from suspensions of high cell density inhibited the uptake of the sensitizers in low cell density suspensions, indicating that the inhibition may be due to factors secreted by the cells.