Far-Red Fluorescence Probe for Monitoring Singlet Oxygen during Photodynamic Therapy

S, Kim.;T, Tachikawa.; M, Fujitsuka.; T, Majima. J. Am. Chem. Soc. 2014. 136, 11707.

DOI: 10.1021/ja504279r

Singlet oxygen (¹O₂), molecular oxygen in thelowest excited state, has a critical role in the cell-killing mechanismof photodynamic therapy (PDT). Although ¹O₂ phosphorescencemeasurement has been mainly used to monitor 1O2 formationduring PDT, its intensity is far insufficient to obtain two-dimensional images of intracellular ¹O₂ with the subcellular spatialresolution using the currently available near-IR detector. Here, wepropose a new far-red fluorescence probe of ¹O₂, namely, Si-DMA,composed of silicon-containing rhodamine and anthracenemoieties as a chromophore and a ¹O₂ reactive site, respectively. In the presence of ¹O₂, fluorescence of Si-DMA increases 17 times due to endoperoxide formation at the anthracene moiety. With the advantage of negligible self-oxidation by photoirradiation (ΦΔ < 0.02) and selective mitochondrial localization, Si-DMA is particularly suitable for imaging ¹O₂ during PDT. Among three different intracellular photosensitizers (Sens), Si-DMA could selectively detect the ¹O₂ that is generated by 5- aminolevulinic acid-derived protoporphyrin IX, colocalized with Si-DMA in mitochondria. On the other hand, mitochondria- targeted KillerRed and lysosomal porphyrins could not induce fluorescence change of Si-DMA. This surprising selectivity of Si- DMA response depending on the Sens localization and photosensitization mechanism is caused by a limited intracellular ¹O₂ diffusion distance (∼300 nm) and negligible generation of ¹O₂ by type-I Sens, respectively. For the first time, we successfully visualized ¹O₂ generated during PDT with a spatial resolution of a single mitochondrial tubule.

Introduction

PDS and ROS

PDS photodynamic therapy is widely practiced medical treatment. Typical PDS takes advantage of ROS (reactive oxygen species) that are spontaneously generated from organic probe used during the treatment. To be specific, ¹O₂ is recognized as the main cause of cell death because other ROS like HO^• and O₂^•- are generated via ¹O₂.^[1] Hence, detection and visualization of ¹O₂ molecule location within living cell are very important for further its development and understanding. Here, Majima group at Osaka university realized the live-visualization of ¹O₂ using silicon-containing rhodamine.

Molecular design

In this paper, high photo-stable, mitochondria selective, red emissive and ¹O₂ selective dye was reported. The new dye was designed as shown below.
(1) Considering its photostability and photo-physical properties, silicon based fluorescein was adopted in core fluorophore.
(2) The often used phenyl group in the head part was replaced with 9,10-dimethylanthracene (DMA) because it react with 1O2 with high selectivity.

In vitro characterization

As shown below, anthracene’s band in short wavelength region dramatically ceased when 1O2 reagent was added. Simultaneously, increase in fluorescence intensity was also observed, meaning that the oxygen bridgedstructure was formed on anthracene, which hampered ¹O₂ transfer from the main fluorophore. Absorption spectrum change suggested that the dye formed H-aggregation in PBS buffer. Last date showed that the dye has the highest selectivity to ¹O₂among other ROS species.

In vivo experiment

Comparison with Mitotracker green revealed that the Si-DMA had specific localization in mitochondria. Next, Si-DMA was stained together with PpIX and TMPyP4 and KillerRed for its further characterization. After co-stained followed by appropriate wavelengths photoirradiation, only PpIX stained with Si-DMA showed dramatic change in fluorescence intensity. These results revealed that Si-DMA can selectively detect ¹O₂ in mitochondria.

PpIX is pervasively used PDS that which localizes in mitochondria. TMPyP4 is also known to generate1O2but it selectively localize in lysosome not mitochondria. KillerRed is PDS protein that unlike other dyes generates superoxide anion rather than ¹O₂.^[2]