Characterization of photodynamic actions of rose bengal on cultured cells

The authors have previously reported successful photodynamic occlusion of corneal blood vessels using intravenous rose bengal and argon green laser irradiation. To explore the action mechanism of this novel technique of photothrombosis, they examined the photodynamic effect of rose bengal on cultured fibroblasts, smooth muscle cells, and vascular endothelium--the cellular components of blood vessels.

Five types of cells were exposed to different concentrations of rose bengal and argon green laser irradiation. The irradiated cell areas were analyzed by fluorescence microscopy and fluorometry. Various potential quenchers and proteins were tested for their modulation of the photodynamic action.

Upon irradiation with 16 W/cm2 of argon green laser light in conjunction with rose bengal concentrations extending above 1 x 10(-4) M, all cultured cell types showed a dose-dependent photobiologic effect characterized by constriction and detachment of the laser-irradiated cell region from the rest of the cell monolayer. In addition, there was dye photobleaching and development of a blue shift of the fluorescence excitation and emission maxima in the irradiated cell areas. Binding of rose bengal to intracellular components was demonstrated by fluorescence microscopy and by fluorometry showing a red shift of the excitation maximum compared to the maximum in solution. This binding was a prerequisite for expression of the described photobiologic effect, because polymer-conjugated rose bengal (Sensitox II) failed to reproduce it. The addition of native or heat-inactivated bovine serum albumin or catalase decreased this photobiologic effect also owing to dye binding, as indicated by G-75 Sephadex gel filtration chromatography.

These results indicate that the specific photobiologic effect of monolayer contraction, which simulates the vasoconstriction seen during photothrombosis under argon green irradiation, appears to be caused by the photochemical interaction of rose bengal bound with intracellular components.