Indirect detection of photosensitizer ex vivo

• Published in: Journal of photochemistry and photobiology. B, Biology Vol. 67; no. 1; pp. 23 - 31
• Main Authors: Bourré, Ludovic, Thibaut, Sonia, Briffaud, Amélie, Rousset, Nathalie, Eléouet, Sabine, Lajat, Youenn, Patrice, Thierry
• Format: Journal Article
• Language: English
• Published: Switzerland Elsevier B.V 01-05-2002
• Subjects: 2′-7′ dichlorofluorescin diacetate; Animals; Bacteriochlorophylls - pharmacokinetics; Biopsy; Cancer; Dose-Response Relationship, Drug; Fluoresceins; Fluorescence; HT29 Cells; Humans; Male; Mesoporphyrins - pharmacokinetics; Mice; Photodynamic therapy (PDT); Photosensitizing Agents - pharmacokinetics; Reactive Oxygen Species; Reactive oxygen species (ROS); Spectrometry, Fluorescence - methods; Fluorescence; 2′-7′ dichlorofluorescin diacetate; Photodynamic therapy (PDT); Biopsy; Reactive oxygen species (ROS); Cancer
• ISSN: 1011-1344; 1873-2682
• DOI: 10.1016/S1011-1344(02)00279-8

Photodynamic therapy induces the production of reactive oxygen species (ROS) within tissues exposed to laser light after administration of a sensitizer. In the context of continuing clinical and commercial development of chemicals with sensitizing properties, a minimally invasive assay is needed to determine the tissue kinetics of fluorescent or non-fluorescent photoreactive drugs. The level of ROS was determined ex vivo from 1 mm
3 biopsy samples using 2′-7′ dichlorofluorescin diacetate (DCFH-DA), a fluorescent probe which was converted into highly fluorescent dichlorofluorescein (DCF) in the presence of ROS. This assay was tested on meta(tetrahydroxyphenyl)chlorin (
m-THPC, FOSCAN
®), a powerful and fluorescent sensitizer, and bacteriochlorophyll derivative WST09 (TOOKAD
®), a near-infrared absorbing sensitizer that is only slightly fluorescent. In conjunction with the ROS assay, the tissue accumulation of
m-THPC was determined on biopsy samples using an optic fibre spectrofluorometer (OFS). DCF fluorescence was proportional to the level of oxidation induced by horseradish peroxidase used as a control and to the concentration (range: 0–5 μg ml
−1) of both selected photosensitizers irradiated in a tube together with DCFH. Regardless of the organ studied, an excellent correlation was found between fluorescence measurement by OFS and ROS determination for
m-THPC.
m-THPC (2 mg kg
−1 iv) accumulation in tumour tissues was best after 48 h, and the best signal was obtained in liver. With non-fluorescent WST09 (2 mg kg
−1), ROS determination showed the best tumour uptake 48 h after injection, with a tumour/muscle ratio of 5.4. The ROS assay appears to be feasible for determining sensitizer concentration in regular grip biopsy tissue samples.