Localization and distribution of pyropheophorbide-a derivatives in vitro and in vivo

Previously Pandey et al. synthesized a congeneric series of pyropheophorbide-a derivatives with alkyl ether chains three to twelve carbons in length. Henderson et al. performed an in vivo QSAR study with these derivatives and reported a parabolic photodynamic activity relationship versus carbon chain length that peaked at the hexyl and heptyl derivatives. In contrast the amount of material accumulated by tumor tissue increased linearly with carbon chain length when administered at equivalent does. To accommodate both results Henderson suggested that there may be a susceptible subcellular target to which optimally active derivatives localize with high affinity. The current study was performed to find a susceptible subcellular target, as was proposed by Henderson et al. Secondary to this goal, experiments were then performed to determine how the carbon chain influenced localization to the target site. Fluorescence microscopy was used to show that mitochondria are the putative PDT target predicted by Henderson et al. For the most hydrophobic congeners lysosomal localization correlated with decreased PDT activity confirming other reports showing that they are less sensitive to PDT damage. In vitro localization, efficiency of intracellular accumulation, and photodynamic activity were inversely proportional with alkyl chain length. Since aggregation was proportional with alkyl chain length it was hypothesized that free monomers are the active fraction that is photodynamically active. All of the derivatives were equally active in vitro, at equal intracellular concentrations, indicating that the compounds have little inherent specificity for the mitochondria. However free monomers appear to be more easily internalized, thus a disaggregated compound is more photodynamically efficient than a highly aggregated compound in vitro. Photodynamic activity, efficiency of cellular accumulation, and localization Pyropheophorbide- a derivatives serum proteins was also shown to be affected by serum proteins. The compounds were found to have a high affinity for serum proteins, especially the lipoproteins. In vitro serum proteins competed with tumor cells for available compound leading to the conclusion that serum proteins are a sink which lowers the total monomer concentration. Furthermore lysosomal localization was found to occur when the compounds bound strongly to lipoproteins, especially low density lipoprotein (LDL), which is accumulated to lysosomes by LDL-receptor-mediated endocytosis. From these data it was concluded that free monomers are the active fraction of the pyropheophorbide- a derivatives and any factor (aggregation, serum protein binding, clearance) which reduces the available monomer pool reduces their photodynamic activity.