Galactosyl, alkyl, and acidic groups modify uptake and subcellular deposition of pyropheophorbide-a by epithelial tumor cells and determine photosensitizing efficacy

Photosensitizers currently used for photodynamic therapy of cancer show enhanced accumulation in tumor tissue but lack cancer cell specificity. To augment cellular uptake, the targeting of pyropheophorbide-a to carbohydrate-binding components of cancer cells was explored. Galactose was attached to pyropheophorbide-a at positions 17(2) and 20. Since the modification at position 17(2) removed a carboxylic group, which is relevant for cell specificity, this study evaluated the relative contribution of galactosyl and carboxyl groups at either position 17(2) or 20, with or without a (hexyloxy)ethyl at position 3, to cellular uptake by human epithelial cancer cells. The subcellular deposition was monitored using fluorescence microscopy and the photoreaction was quantified using biomarkers. The results demonstrated that any galactose addition suppresses transmembrane diffusion and promotes endocytosis and lysosomal accumulation. An anionic group at position 17(2) or 20 enhances lysosomal retention. Neutralization of the carboxylic group at position 17(2) facilitates transfer to mitochondria/endoplasmatic reticulum and promotes tumor cell-specific retention. Replacing (hexyloxy)ethyl with an ethyl group at position 3 increased both cellular uptake and egress but did not alter subcellular localization. These findings suggest that specific neutral galactosylated pheophorbides may provide an enhanced therapeutic effect for those tumor types that do not retain unmodified pyropheophorbide. However, the galactose conjugates also serve as substrates for preferential uptake by liver cells resulting in hepatic sequestration, reduced systemic distribution, and lower accumulation in tumor tissue.