Photobleaching and phototoxicity of KillerRed in tumor spheroids induced by continuous wave and pulsed laser illumination

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/cm(2) 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/cm(2) and light doses of 140 J/cm(2) , 170 J/cm(2) and 200 J/cm(2). 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.

Novel uses of fluorescent proteins

“…For live models, miniSOG and GFPlike protein KillerRed [13] (the latter produces radicalbased ROS under green-orange light) can be used as genetically encoded photosensitizers. It is a unique tool that enables very precise (in space and time) light-induced killing of specific cell populations [13][14][15][16][17], inactivation of target proteins [13,18 ,19], and damaging genomic DNA [16,20]. Moreover, a new approach to study protein arrangement in large complexes was developed [21 ].…”

Towards PDT with Genetically Encoded Photosensitizer KillerRed: A Comparison of Continuous and Pulsed Laser Regimens in an Animal Tumor Model

“…It is known that photobleaching of KR accompanies the photosensitization reaction [ 1 , 8 , 11 ], and can potentially predict the efficiency of PDT. Another factor accompanying PDT is the potential change in tumor temperature due to light delivery [ 12 , 13 ].…”

“…Although relations between the extent of photobleaching and the phototoxic effects have, as yet, been poorly studied for KR, it is well established that photobleaching accompanies its photochemical reactions. The decrease of KR fluorescence intensity after exposure to light has been shown in cell cultures [ 1 , 6 , 11 ] and in tumor xenografts expressing this protein [ 8 ]. The most probable mechanisms of KR photobleaching are the direct reaction of molecular oxygen with the excited chromophore or the photoreduction of the chromophore owing to electron transfer from neighboring amino acid residues [ 4 , 5 ].…”

“…Fluence rates of 110, 150, and 260 mW/cm 2 for the CW laser and 225 mW/cm 2 for the pulsed laser were tested using exposure times from 5 to 30 min. Similar treatment regimens had previously been tested on 3D tumor spheroids [ 11 ]. Before and during the irradiation, the skin surface temperature was measured using an IR thermograph (CEM-ThermoDiagnostics, CEMTechnology, Russia).…”

A targeted illumination optical fiber probe for high resolution fluorescence imaging and optical switching

“…Targeted illumination with a temporal precision of the order of milliseconds and the spatial precision of few tens of micrometers is a necessity for certain studies such as neuronal signalling11011. Non-targeted illumination is also known to generate abrupt heat effects in regions of non-interests in many studies, hindering their research studies61213.…”