Pharmacokinetics and
phototoxicity of
purpurin-18 (Pp18) in human colon
carcinoma cells (Colo-205) was studied using
liposomes as delivery vehicles. Cytotoxicity was measured using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and
neutral red uptake assay, and mode of cell death was assessed by the study of cell morphology and nuclear staining with Hoechst 33342-propidium
iodide. Pp18 solubilized in
dimethyl sulfoxide saline solution was observed to aggregate (Q-band absorption 740 nm), resulting in very poor cellular uptake. Pp18 incorporated in
liposome remained in monomeric form (Q-band absorption 695 nm), but due to the presence of an
anhydride ring in the molecule it readily yielded another
photosensitizer,
chlorin p6 (Q-band absorption 662 nm). Measurements at various pH showed that Pp18 in
liposome was stable at acidic pH (6.5). Incubation of cells with 6.0 microM Pp18 in
liposome at pH 6.5 showed a rapid cellular uptake. Spectrofluorometric measurements showed the presence of both Pp18 and
chlorin p6, indicating conversion of some amount of Pp18 into
chlorin p6 in the cells. Fluorescence microscopy revealed that the fluorescence was localized mainly in the cytoplasm, sparing the nucleus. Illumination of cells to white light after 4-h incubation with Pp18
liposome preparation was observed to lead to dose-dependent decrease in cell viability. At low irradiation time, cells displayed formation of plasma membrane
blebs and micronuclei typical of apoptotic cell death. In contrast, at higher irradiation time, cell swelling and vacuolization in nucleus was observed, suggesting cell death due to
necrosis. Irradiation with narrow bandwidth light showed that at low pH, the relative
phototoxicity due to pp18 was higher than that due to
chlorin p6. It is suggested that the pH-dependent conversion of pp18 to
chlorin p6 can be exploited to increase
PDT selectivity.