1. +7 831 465 5672+7 831 465 4223;2. Nizhny Novgorod State Medical Academy, Nizhny Novgorod, Russia;3. Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russia;4. Institute of Applied Physics RAS, Nizhny Novgorod, Russia;5. Shemyakin‐Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russia;6. Pirogov Russian National Research Medical University, Moscow, Russia
Abstract:
The purpose of this study was to evaluate photobleaching of the genetically encoded photosensitizer KillerRed in tumor spheroids upon pulsed and continuous wave (CW) laser irradiation and to analyze the mechanisms of cancer cell death after the treatment. We observed the light‐dose dependent mechanism of KillerRed photobleaching over a wide range of fluence rates. Loss of fluorescence was limited to 80% at light doses of 150 J/cm2 and more. Based on the bleaching curves, six PDT regimes were applied for irradiation using CW and pulsed regimes at a power density of 160 mW/cm2 and light doses of 140 J/cm2, 170 J/cm2 and 200 J/cm2. Irradiation of KillerRed‐expressing spheroids in the pulsed mode (pulse duration 15 ns, pulse repetition rate 10 Hz) induced predominantly apoptotic cell death, while in the case of CW mode the cancer cells underwent necrosis. In general, these results improve our understanding of photobleaching mechanisms in GFP‐like proteins and show the importance of appropriate selection of treatment mode for PDT with KillerRed.
Representative fluorescence image of two KillerRed‐expressing spheroids before and immediately after CW irradiation.
