29th Annual Meeting of the American Society of Photobiology

Downtown Marriot

Chicago, Il.

Intracellular Localization and Photobleaching of Hypericin

Abstract-
Hypericin is known as a powerful photosensitizer. The intracellular localization of hypericin fluorescence in three cultured cell lines (adenocarcinoma WiDr, carcinoma NHIK 3025 and glioblastoma D54Mg) was compared with the distribution of the fluorescent probes specific to lysosomes (LysoTracker Blue DND-22), mitochondria (MitoTracker Green FM) and ER (ERTracker Blue-White DPX). The staining pattern of hypericin was different as compare to intracellular distribution of mitochondria or lysosomes. It was diffusely concentrated in the perinucleolar cytoplasmic area rich in ER and Golgi. Sometimes hypericin was associated with the nuclear envelope. The plasma membrane was not stained but often the dye was accumulated between the tightly contacting WiDr cells in colonies. Hypericin concentrations less than 10 M were not toxic for WiDr cells in the dark. Orange light (600 nm; 6 mW/cm²) killed WiDr cells stained with 1 M hypericin with LD₅₀ near 1 J/cm². The spectral properties and the photobleaching characteristics of hypericin in HSA solutions, bound to cells and in mouse skin were also studied. Photobleaching was not oxygen dependent and singlet oxygen probably played no significant role in this process. During light exposure, the optical absorbance decayed slower than the fluorescence. This migth be due to the facts that drug aggregates are nonfluorescent and more stable than monomers. The emission bands in the spectral regions 420 - 560 nm and above 600 nm characterized the formed photoproducts. The excitation spectra of these products resembled that of hypericin, indicating that only minor chemical changes took place on peripheral groups of the molecule. Hypericin appeared to be more photostable than most sensitizers used in PDT, such as mesotetrahydroxy phenyl chlorine and Photofrin.

Keywords: PDT, hypericin, localization, photobleaching