title: Hypocrellin-B acetate as a fluorogenic substrate for enzyme-assisted cell photosensitization.
abstract: Photosensitizing molecules (PSs) undergo chemico-physical changes upon addition of suitable substituents, influencing both their photophysical properties and their ability to accumulate into cells. Once inside the cells, the modified PS acts as a fluorogenic substrate: the added substituent is removed by a specific enzyme, restoring the native PS in subcellular sensitive sites. We investigated the photophysical properties and interaction with HeLa cells of Hypocrellin-B (HypB), as native molecule and upon acetate-group addition (HypB-Ac). Chemical modification alters both absorption and fluorescence features of HypB; consequently, the dynamics of the enzyme hydrolysis of HypB-Ac can be monitored through restoring the native HypB spectral properties. At the cellular level, only the HypB emission signal was detected within 5 min of incubation with either HypB or HypB-Ac, allowing a direct comparison of the time courses of their intracellular accumulation. Plateau values were reached within 15 min of incubation with both compounds, the emission signals being significantly higher in HypB-Ac than in HypB treated cells. Consistently, imaging showed a rapid appearance of red fluorescence in the cytoplasm, with more abundant bright spots in HypB-Ac treated cells. Both compounds did not induce dark toxicity at concentrations up to 1 × 10(-6) M, while upon irradiation at 480 nm phototoxicity was significantly higher for cells exposed to HypB-Ac than for HypB-loaded cells. These findings suggest an improved efficacy of acetylated HypB to be internalized by cells through membrane trafficking, with a preferential interaction of the photoactive molecules on sensitive intracellular sites. After irradiation, in HypB-Ac treated cells, prominent disorganization of several cytoplasmic organelles such as the endoplasmic reticulum, Golgi apparatus, lysosomes, microfilaments and microtubules were observed.
authors: IGM-CNR, Sezione Istochimica e Citometria, Università di Pavia, Italy.
authors: A C
authors: AC
authors: Croce
authors: E
authors: E
authors: Fasani
authors: M G
authors: MG
authors: Bottone
authors: U
authors: U
authors: De Simone
authors: G
authors: G
authors: Santin
authors: C
authors: C
authors: Pellicciari
authors: G
authors: G
authors: Bottiroli
mesh_terms: D004798:Enzymes / Q000378:metabolism*; D004950:Esterases / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008027:Light; D008856:Microscopy, Fluorescence; D010569:Perylene / Q000031:analogs & derivatives* / Q000737:chemistry / Q000633:toxicity; D017319:Photosensitizing Agents / Q000737:chemistry / Q000633:toxicity*; D011809:Quinones / Q000737:chemistry / Q000633:toxicity*; D026082:Singlet Oxygen / Q000378:metabolism; D013997:Time Factors
chemical_list: D004798:Enzymes; D005456:Fluorescent Dyes; D017319:Photosensitizing Agents; D011809:Quinones; C570352:hypocrellin B acetate; D026082:Singlet Oxygen; D010569:Perylene; D004950:Esterases; C064522:hypocrellin B