Mechanism of Photosensitization by Microsphere-bound Chlorin e6 in Human Bladder Carcinoma Cells1

Photodynamic therapy is an experimental method of cancer treatment in which a photosensitizer is administered and subsequently the tumor is irradiated with light. Due to problems of prolonged skin phototoxicity with hematoporphyrin derivative, new photosensitizers and methods of localization are being sought. The goal of this study was to compare the photosensitizer chlorin e«(C c,,) free and bound to 1-^m-diameter microspheres (MS) for phototoxicity, uptake and efflux characteristics, phago cytosis rates in malignant and benign cells, and effects of NuN,. D.O. and buthionine sulfoximine on phototoxic efficacy. Incubation of MGHII human bladder carcinoma cells with Ce«-MS(0.43 IHMCe6-equivalent; 18 h) and subsequent irradiation using an argon laser-pumped dye laser at a radiant exposure of 20 J/cm2 caused 100% cell death 24 h after irradiation. In contrast, MGH-U1 cells incubated with free Ce6 (0.43 JIM; 18 h) remained 100% viable 24 h after irradiation at a radiant exposure of up to 50 J/cm2. The presence of !).•() during irradiation increased the phototoxicity to MGH-U1 cells, whereas the presence of NaNj decreased it; these data support an important role for '();. Irradiation of MGH-U1 cells in the presence of the glutathione depleter buthionine sulfoximine also increased the phototoxicity, demonstrating a role for intracellular glutathione and possibly free radical intermediates. The cellular uptake of Ce6 w-as approximately 50 times lower than that of Ce6-MS at equivalent incubation concentrations. Efflux experiments showed that the phototoxicity of ( V,,-MS was reduced by 40% for a 5-h washout time as compared to no washout time. In contrast, for free Ce«, the decrease was 95.3% under identical conditions. Because the total intracellular concen tration of Ce6-MS after an efflux time of 5 h was only slightly changed, the decreased phototoxicity is attributed to an altered intracellular local ization. Confocal laser scanning fluorescence microscopy data appear consistent with this hypothesis although they are not conclusive. The observed patterns were different at 0 and 5 h. Comparison of the phago cytosis rates of <i-,,-MS by carcinoma and benign cells showed that on average 20 MS/cell were phagocytosed by MGH-U1 compared with 2.5 and 8.3 in the benign human fibroblasts and keratinocytes, respectively. After incubation with Ce«-MS(0.1 ¿IM; 18 h) and irradiation at 10 J/cm2 the surviving fraction of MGH-U1 cells was 76.3 ±0.95% (mean ±SE) and 40 ±3.49% for fibroblasts. In contrast, for keratinocytes the surviv ing fraction was 93.5 ± 0.83%. The data demonstrate that Ce6-MS conjugates efficiently photosensitize carcinoma cells by multiple mecha nisms and have different cellular pharmacodynamics compared to free