The internalization mechanism and subcellular distribution of hypericin (Hyp), hypericin tetrasulfonic acid (HypS4) and 1,3,4,6-tetrahydroxyhelianthrone (Hel) were studied in murine colon carcinoma CT26 cells, in protein-free medium or in the presence of serum proteins. The correlation between the extent of uptake of the sensitizers by cells that were incubated in the presence of different serum components, and the internalization mechanisms, was studied. The results indicate that sensitizer internalization may be a result of three mechanisms: partitioning, pinocytosis and endocytosis, and as a direct consequence is targeted to specific subcellular sites. While Hyp and Hel, the two lipophilic sensitizers, were localized in the endoplasmic reticulum after protein-free internalization, the hydrophilic HypS4 was localized in the cytoplasmic membrane and in lysosomes. An endolysosomal internalization route was revealed for Hyp and Hel under serum-enriched conditions showing lysosomal localization, as for HypS4. The lysosomal accumulation of Hyp-serum and specifically Hyp-LDL points to an endocytotic mechanism which is supported by its higher uptake parameter in an LDL-enriched medium, compared to the medium with 10% serum. The different uptake parameters of Hyp to cells, with or without serum, reflect the different mechanisms. Smaller differences in the uptake parameter for HypS4 reflect the distinction between partitioning and endocytosis, which, in this case, are both targeted to the lysosomes. The same uptake parameter of Hel to cells incubated in media with or without serum indicates the absence of the endocytotic mechanism. The interrelationship between subcellular targeting and photodynamic treatment was shown for the three sensitizers Hyp was found to be the most efficient sensitizer for PDT under our illumination protocol and it was dependent on internalization and localization sites.

Photodynamic therapy (PDT) involves a two-stage process. A light-absorbing photosensitiser (Ps) is endocytosed and then stimulated by light, inducing transfer of energy to a cytoplasmic acceptor molecule and the generation of reactive oxygen species that initiate damage to cellular membrane components and cytolysis. The expanded use of PDT in the clinic is hindered by the lack of Ps targetcell specificity and the limited tissue penetration by external light radiation. This study demonstrates that bioconjugates composed of transferrin and haematoporphyrin (Tf -Hp), significantly improve the specificity and efficiency of PDT for erythroleukemic cells by a factor of almost seven-fold. Fluorescence microscopy showed that the conjugates accumulate in intracellular vesicles whereas free Hp was mostly membrane bound. Experiments with cells deliberately exposed to Tf -Hp at oLD 100 doses showed that surviving cells did not develop resistance to subsequent treatments with the conjugate. Furthermore, we show that the compound luminol induces intracellular chemiluminescence. This strategy was then used to obviate the use of external radiation for Ps activation by incubating the cells with luminol either before or together with Tf -Hp. This novel chemical means of PDT activation induced cytotoxicity in 95% of cells. These combined approaches provide an opportunity to develop broader and more effective applications of PDT.

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S U M M A R YWe studied the staining pattern of merocyanine 540 (MC540) by spectral imaging of murine CT26 and human HT29 colon carcinoma cells incubated with the dye MC540. This dye, usually considered a potential membrane probe, localized mainly in the cytoplasmic vesicles of the colon carcinoma cells. However, in cells incubated in an environment similar to that of a tumor (pH 6.7), high fluorescence was detected in the nuclear membrane and nucleoli. Under these acidic conditions, resembling the Krebs effect, a population of CT26 cells displayed fluorescent plasma membranes. In differentiating cells, exhibiting cell cycle arrest at G 1 -phase and an elevated level of alkaline phosphatase, MC540 fluorescence was confined to cytoplasmic vesicles and was not detected in the plasma membrane or in the nucleoli. Cell sorting analysis of both cell types at pH 5.