In vitro Plasma Protein Binding and Cellular Uptake of ATX‐S10(Na), a Hydrophilic Chlorin Photosensitizer

ATX‐S10(Na), a hydrophilic chlorin photosensitizer having an absorption maximum at 670 nm, is a candidate second‐generation photosensitizer for photodynamic therapy (PDT) for cancer treatment. In this study, we examined plasma protein binding, cellular uptake and subcellular targets of ATX‐S10(Na) in vitro. Protein binding ratios of 50 μg/ml ATX‐S10(Na) in rat, dog and human plasma were 73.0%, 87.2% and 97.7%, respectively. Gel filtration chromatography revealed that 1 mg/ml ATX‐S10(Na) bound mainly to high‐density lipoprotein (HDL) and serum albumin at the protein concentration of 0.4%, with binding ratios of 46% and 36%, respectively. The free form of ATX‐S10(Na) was mostly incorporated into T.Tn cells, and its cellular uptake was partially but significantly inhibited by endocytosis inhibitors such as phenylarsine oxide, chloroquine, monensin and phenylglyoxal, and by chilling the cells to 4°C. However, ouabain, harmaline, sodium cyanide, probenecid and aspartic acid did not influence the uptake of ATX‐S10(Na), suggesting that cellular uptake of ATX‐S10(Na) was not related to sodium‐potassium pump activity, sodium‐dependent transporter activity, mitochondrial oxidative respiration, organic anion transporter activity or aspartic acid transporter activity. By fluorescence microscopy, lysosomal localization of ATX‐S10(Na) was observed in T.Tn cells. However, electron microscopic observation revealed that many subcellular organelles such as mitochondria, endoplasmic reticulum, ribosomes, Golgi complex and plasma membrane were damaged by PDT using 25 μg/ml ATX‐S10(Na) soon after laser irradiation at 50 J/cm(2), and tumor necrosis was rapidly induced. This result indicated that ATX‐S10(Na) was widely distributed within the cell.