Indirect detection of photosensitizer ex vivo.
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 mm3 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 microg x 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 x 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 x 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.
Bourré
Ludovic
L
Laboratoire de Photobiologie des Cancers, Département Laser, Hôpital Laënnec, 44093 cedex 01, Nantes, France.
Thibaut
Sonia
S
Briffaud
Amélie
A
Rousset
Nathalie
N
Eléouet
Sabine
S
Lajat
Youenn
Y
Patrice
Thierry
T
D000818:Animals; D001429:Bacteriochlorophylls / Q000493:pharmacokinetics*; D004305:Dose-Response Relationship, Drug; D005452:Fluoresceins; D019073:HT29 Cells; D006801:Humans; D008297:Male; D008652:Mesoporphyrins / Q000493:pharmacokinetics*; D051379:Mice; D017319:Photosensitizing Agents / Q000493:pharmacokinetics*; D017382:Reactive Oxygen Species; D013050:Spectrometry, Fluorescence / Q000379:methods
D001429:Bacteriochlorophylls; D005452:Fluoresceins; D008652:Mesoporphyrins; D017319:Photosensitizing Agents; D017382:Reactive Oxygen Species; C037631:2',7'-dichlorofluorescein; C072269:temoporfin