Abstract
Triphenylamines (TPAs) were previously shown to trigger cell death under prolonged one- or two-photon illumination. Their initial subcellular localization, before prolonged illumination, is exclusively cytoplasmic and they translocate to the nucleus upon photoactivation. However, depending on their structure, they display significant differences in terms of precise initial localization and subsequent photoinduced cell death mechanism. Here, we investigated the structural features of TPAs that influence cell death by studying a series of molecules differing by the number and chemical nature of vinyl branches. All compounds triggered cell death upon one-photon excitation, however to different extents, the nature of the electron acceptor group being determinant for the overall cell death efficiency. Photobleaching susceptibility was also an important parameter for discriminating efficient/inefficient compounds in two-photon experiments. Furthermore, the number of branches, but not their chemical nature, was crucial for determining the cellular uptake mechanism of TPAs and their intracellular fate. The uptake of all TPAs is an active endocytic process but two- and three-branch compounds are taken up via distinct endocytosis pathways, clathrin-dependent or -independent (predominantly caveolae-dependent), respectively. Two-branch TPAs preferentially target mitochondria and photoinduce both apoptosis and a proper necrotic process, whereas three-branch TPAs preferentially target late endosomes and photoinduce apoptosis only.
Highlights
Over the past decade, there has been a growing interest in developing and studying two-photon absorption probes for cell imaging and biomedical applications
Vinyl-triphenylamine (TPA) compounds correspond to a family of fluorescent molecules exhibiting high two-photon absorption cross-sections (σ2)[13,14,15,16]. Their scaffold is comprised of a triphenylamine core bearing two (TP2) or three (TP3) vinyl branches terminated by cationic electron acceptor groups, e.g. N-methyl benzimidazolium (Bzim) or pyridinium branched in position para (Py) or ortho (Pyo) with respect to the vinyl bond (Fig. 1)
Despite poor reactive oxygen species (ROS) quantum yields (1O2, O2, OH) as measured in vitro, the apparent photodynamic therapy (PDT) effect of photoactivated TPAs in the cell context mainly relies on high amplification of endogenous ROS production, due at least in part to their mitochondrial targeting properties
Summary
There has been a growing interest in developing and studying two-photon absorption probes for cell imaging and biomedical applications. Near infrared (NIR) and infrared (IR) wavelengths used for two-photon excitation minimize autofluorescence and maximize tissue penetration due to smaller light scattering/absorption by the tissue and endogenous biomolecules[1] Such probes were used for several types of applications including fluorescent markers such as nucleus and organelle markers or chemical probes[2,3,4,5]. Upon one- (visible light) or two-photon (NIR) excitation, these two compounds triggered cell death, a phenomenon mediated by ROS production and strictly dependent on TPA photoactivation[16,17] This cell death process is accompanied by a cytoplasm-nucleus relocalization of the TPA fluorescence signal and concomitant morphological hallmarks of apoptosis such as plasma membrane blebbing and cell shrinkage[18], making them good candidates for application in PDT. We found that the chemical nature of the electron acceptor group is critical for the overall cell death efficiency, with TP-Pyo derivatives being much less efficient than TP-Bzim or TP-Py derivatives, regardless of the excitation mode (one- or two-photon), whereas the number of branches primarily determines the cell uptake pathway and subsequent intracellular properties, i.e. subcellular localization, ROS generation efficiency and cell death mechanism
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