Abstract
Photodynamic therapy (PDT) leads to cell death by using a combination of a photosensitizer and an external light source for the production of lethal doses of reactive oxygen species (ROS). Since a major limitation of PDT is the poor penetration of UV-visible light in tissues, there is a strong need for organic compounds whose activation is compatible with near-infrared excitation. Triphenylamines (TPAs) are fluorescent compounds, recently shown to efficiently trigger cell death upon visible light irradiation (458 nm), however outside the so-called optical/therapeutic window. Here, we report that TPAs target cytosolic organelles of living cells, mainly mitochondria, triggering a fast apoptosis upon two-photon excitation, thanks to their large two-photon absorption cross-sections in the 760–860 nm range. Direct ROS imaging in the cell context upon multiphoton excitation of TPA and three-color flow cytometric analysis showing phosphatidylserine externalization indicate that TPA photoactivation is primarily related to the mitochondrial apoptotic pathway via ROS production, although significant differences in the time courses of cell death-related events were observed, depending on the compound. TPAs represent a new class of water-soluble organic photosensitizers compatible with direct two-photon excitation, enabling simultaneous multiphoton fluorescence imaging of cell death since a concomitant subcellular TPA re-distribution occurs in apoptotic cells.
Highlights
Significant enhancements of Photodynamic therapy (PDT) efficacy can be achieved using combination treatments in which the effect of a photosensitizer can be improved by another chemical compound targeting and inhibiting cellular pathways, such as for instance the survivin pathway[3] or the proteasome-dependent protein degradation[4], or selectively enhancing accumulation of the PDT compound within tumor cells[5]
An alternative approach was developed based on up-converting (NIR-to-visible) nanoparticles loaded with standard PDT compounds activatable by visible light leading to reactive oxygen species (ROS) formation under NIR irradiation at 980 nm[8]
We have previously demonstrated that triphenylamine compounds (TPA) are suitable for two-photon imaging of fixed cell nuclei[18,19], the effect of their two-photon excitation using NIR light was never addressed in the context of living cells and is of particular interest for therapeutic applications
Summary
Significant enhancements of PDT efficacy can be achieved using combination treatments in which the effect of a photosensitizer can be improved by another chemical compound targeting and inhibiting cellular pathways, such as for instance the survivin pathway[3] or the proteasome-dependent protein degradation[4], or selectively enhancing accumulation of the PDT compound within tumor cells[5]. Most of the standard PDT compounds developed so far are characterized by one-photon activation wavelengths in the red part of the visible spectrum, typically in the 630–690 nm range in the so-called therapeutic window, to optimize light penetration by minimizing both light scattering and absorption at the tissue level[1]. There is a need for compounds whose activation occurs directly at wavelengths with optimal tissue penetration, typically in the NIR region of the spectrum (750–950 nm) This two-photon therapeutic window ensures a deeper penetration of light in tissue. Outside this range, visible light is strongly scattered or absorbed by redox co-factors or endogenous proteins, mainly hemoproteins, while NIR light absorption by water is high above 950 nm. An indirect approach involves either energy transfer-mediated activation of a standard (one-photon) PDT compound (acceptor) by a two-photon absorbing dye acting as a donor within the same nanoparticle[14] or a plasmon-mediated enhancement of two-photon excitation of the photosensitizer (e.g. the case of porphyrin in gold nanoparticles)[15]
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