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 max imum at 670 nm, is a candidate second-generation photosensitizer for photod ynamic therapy (PDT) for cancer treatment. In this study, we examined plasm a protein binding, cellular uptake and subcellular targets of ATX-S10(Na) i n vitro. Protein binding ratios of 50 mu g/ml ATX-S10(Na) in rat, dog and h uman plasma were 73.0%, 87.2% and 97.7%, respectively, Cel Filtration chrom atography revealed that 1 mg/ml ATX-S10(Na) bound mainly to high-density li poprotein (HDL) and serum albumin at the protein concentration of 0.4%, wit h 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 parti ally but significantly inhibited by endocytosis inhibitors such as phenylar sine oxide, chloroquine, monensin and phenylglyoxal, and by chilling the ce lls to 4 degrees 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 pum p activity, sodium-dependent transporter activity, mitochondrial oxidative respiration, organic anion transporter activity or aspartic acid transporte r activity. By fluorescence microscopy, lysosomal Localization of ATX-S10(N a) was observed in T.Tn cells. However, electron microscopic observation re vealed that many subcellular organelles such as mitochondria, endoplasmic r eticulum, ribosomes, Golgi complex and plasma membrane were damaged by PDT using 25 mu g/ml ATX-S10(Na) soon after laser irradiation at 50 J/cm(2), an d tumor necrosis was rapidly induced. This result indicated that ATX-S10(Na ) was widely distributed within the cell.