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Hypocrellin-B acetate as a fluorogenic substrate for enzyme-assisted cell photosensitization

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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.

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Authors and Affiliations

  1. IGM-CNR, Sezione Istochimica e Citometria, Università di Pavia, Via A. Ferrata 9, 27100, Pavia, Italy

    A. C. Croce, M. G. Bottone, U. De Simone & G. Bottiroli

  2. Dipartimento di Chimica, Sezione Chimica Organica, Università di Pavia, Italy

    E. Fasani

  3. Dipartimento di Biologia Animale, Università di Pavia, Italy

    M. G. Bottone, G. Santin & C. Pellicciari

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  1. A. C. Croce
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  2. E. Fasani
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  3. M. G. Bottone
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  4. U. De Simone
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  5. G. Santin
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  6. C. Pellicciari
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  7. G. Bottiroli
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Correspondence to M. G. Bottone.

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Croce, A.C., Fasani, E., Bottone, M.G. et al. Hypocrellin-B acetate as a fluorogenic substrate for enzyme-assisted cell photosensitization. Photochem Photobiol Sci 10, 1783–1790 (2011). https://doi.org/10.1039/c1pp05136a

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  • DOI: https://doi.org/10.1039/c1pp05136a

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