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

Wellman Laboratories of Photomedicine, Departments of Urology [R. B.] and Dermatology [M. S., C. R. S., T. H.], Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114

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 e₆ (Ce₆) free and bound to 1-µm-diameter micro-spheres (MS) for phototoxicity, uptake and efflux characteristics, phagocytosis rates in malignant and benign cells, and effects of NaN₃, D₂O, and buthionine sulfoximine on phototoxic efficacy. Incubation of MGH-U1 human bladder carcinoma cells with Ce₆-MS (0.43 µM Ce₆-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 µM; 18 h) remained 100% viable 24 h after irradiation at a radiant exposure of up to 50 J/cm². The presence of D₂O during irradiation increased the phototoxicity to MGH-U1 cells, whereas the presence of NaN₃ decreased it; these data support an important role for ¹O₂. 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 was approximately 50 times lower than that of Ce₆-MS at equivalent incubation concentrations. Efflux experiments showed that the phototoxicity of Ce₆-MS was reduced by 40% for a 5-h washout time as compared to no washout time. In contrast, for free Ce₆, the decrease was 95.3% under identical conditions. Because the total intracellular concentration of Ce₆-MS after an efflux time of 5 h was only slightly changed, the decreased phototoxicity is attributed to an altered intracellular localization. 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 phagocytosis rates of Ce₆-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 Ce₆-MS (0.1 µM; 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 surviving fraction was 93.5 ± 0.83%. The data demonstrate that Ce₆-MS conjugates efficiently photosensitize carcinoma cells by multiple mechanisms and have different cellular pharmacodynamics compared to free Ce₆.

¹ This work was sponsored by Office of Naval Research Contract N00014-86-K-0117, NIH Grant GMA-1,2R01-AR23595, and a Whittaker Foundation grant. R. B. and M. S. are recipients of research fellowships by the Deutsche Forschungsgemeinschaft.

² Present address: Division of Dermatopathology, New York Hospital, Cornell University Medical College, 525 E. 68th Street, New York, NY 10021.

³ To whom requests for reprints should be addressed, at Department of Dermatology, Massachusetts General Hospital, Wellman 2, Boston, MA 02114.

Received 12/26/90. Accepted 6/ 5/91.