Enzyme‐assisted Cell Photosensitization: A Proposal for an Efficient Approach to Tumor Therapy and Diagnosis. The Rose Bengal Fluorogenic Substrate

Abstract

Rose bengal, a xanthene derivative among the most efficient producer of singlet oxygen, was submitted to a chemical modification consisting in the introduction of an acetate group into the aromatic ring fluorophore structure. The acetate group acts as a quencher, thus inactivating both fluorescence and photosensitization properties of the molecule. In the modified structure, rose bengal acts as a fluorogenic substrate giving rise to the cellular reaction termed fluorochromasia. The acetate group is recognized by a carboxylic esterase activity that splits it. Removal of the quencher group results in restoring the native structure of photosensitizer inside the cells. The intracellular turnover of rose bengal acetate was studied in rat glioma-derived cultures cells, in terms of the balance of the processes of influx and enzyme hydrolysis of the fluorogenic substrate, and of the efflux of the fluorescent product. A large intracellular accumulation of photosensitizer is obtained when treatments are performed with the fluorogenic substrate, even at the drug concentration at which rose bengal does not enter the cells. The intracellular localization allows rose bengal to exert a more effective photosensitization effect. Provided that the quencher group is selected according to the metabolic properties of the tumor cells, the use of fluorogenic substrates as photosensitizer precursors could improve fluorescence diagnosis and the photodynamic therapy of tumors, exploiting the biological properties that distinguish pathological from normal conditions.

Apoptosis in tumour cells photosensitized with Rose Bengal acetate is induced by multiple organelle photodamage

“…The addition of acetate groups to the xanthene ring makes RB more hydrophobic, thus favouring its intracellular accumulation, and aVects the RB photophysical properties, quenching both Xuorescence emission and the photosensitizing properties (Bottiroli et al 1997). On this basis, RB acetate (RB-Ac) has been proposed as a suitable compound for a new strategy in photodynamic therapy since it may act as a Xuorogenic substrate: once inside the cell, the acetate groups are removed by esterases with restoration of both Xuorescence and the photosensitizing properties of the native RB (Bottiroli et al 1997).…”

Photodynamic and Tissue Tolerable Plasma Therapies as Alternatives to Antimicrobials to Control Pathogenic Biofilms

“…Most of these dyes can be excited with wavelengths between 400 and 500 nm, corresponding to light sources in the blue region [26,27].…”

Rose Bengal Acetate photodynamic therapy-induced autophagy

“…Indeed, upon RBAc administration, the photoactive molecules of RB accumulate in perinuclear region and than diffuse dynamically throughout the cytoplasm. 20,[37][38][39][40] Endosomes, Golgi apparatus and the ER are the precocious intracellular sites of accumulation of RB. 37,40,41 The photodamage to ER exerted by the RBAc-PDT, as demonstrated by the drastic reduced incorporation of DiOC 6 and by the increased level of cleaved caspase-12, promotes autophagy.…”

“…20 A stock solution (10 -2 M) was obtained by diluting RBAc in dimethyl sulfoxide: final concentration (10 -5 M) was obtained directly in the culture medium at the time of incubation. All other chemicals used were of the commercially available highest purity.…”