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Photochemical internalization (PCI) is a method for intracellular delivery of hydrophilic macromolecular drugs with intracellular targets as well as other drugs with limited ability to penetrate cellular membranes. Such drugs enter cells by means of endocytosis and are to a large extent degraded by hydrolytic enzymes in the lysosomes unless they possess a mechanism for cytosolic translocation. PCI is based on photodynamic therapy (PDT) specifically targeting the endosomes and lysosomes of the cells, so that the drugs in these vesicles can escape into the cytosol from where they can reach their targets. The preferential retention of the photosensitizer (PS) in tumor tissue in combination with controlled light delivery makes PCI relatively selective for cancer tissue. The tumor specificity of PCI can be further increased by delivery of drugs that selectively target the tumors. Indeed, this has been shown by PCI delivery of several targeted protein toxins. Targeted protein toxins may be regarded as ideal drugs for PCI delivery, and may represent the clinical future for the PCI technology.

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We have used the site specific and light-depended drug delivery method photochemical internalization (PCI) to release an immunotoxin (IT), targeting the CD44 receptor, into the cytosol of target cells. The IT consisted of a pan CD44 mAb (clone IM7) bound to the ribosome inactivating protein (RIP) saporin by a biotin-streptavidin linker named IM7-saporin. PCI is based upon photosensitizing compounds localized in the membrane of endosomes and lysosomes causing membrane rupture upon illumination followed by release of the IT into the cytosol. In this in vitro study, we have used 7 different human cancer cell lines of various origins to investigate the cytotoxic effect of PCI-based targeting of the cancer stem cell (CSC) marker CD44. Epi-fluorescence microscopy shows both specific binding and uptake of the IM7-Alexa488, after 30 min and 18 h of incubation, and colocalization with the PCI-photosensitizer TPCS2a prior to light-triggered cytosolic release of the CD44-targeting IT. PCI of IM7-saporin resulted in efficient and specific cytotoxicity in CD44-expressing but not in CD44-negative cancer cells. A higher level of reactive oxygen species (ROS) was found in untreated and photodynamic therapy (PDT)-treated LNCaP (CD44(neg)) compared to that of DU145 (CD44(pos)) prostate cancer (PC) cells. This may explain the PDT-resistance observed in the DU145 cells. PCI-based targeting of CD44-expressing cancer cells gives very potent and specific cytotoxic effects and may represent a rational strategy for achieving site-selective elimination of CSCs in aggressive androgen-independent and treatment-resistant PC cells preventing cytotoxic effects on distant normal stem cells.