Visualizing the Invisible: Dual Click Imaging of Ruthenium-Based Photoactivated Chemotherapy Agents and Their DNA Synthesis Inhibition in Fixed Cancer Cells

Like many drugs, ruthenium-based photoactivated chemotherapy
(PACT)
complexes are hard to follow in cells due to their absence of emissive
properties. Here, two alkyne-functionalized Ru-based PACT compounds
with the formula [Ru(HCC-tpy)(N̂N)(Hmte)](PF6)2 were synthesized, where HCC-tpy = 4′-ethynyl-2,2′:6′,2″-terpyridine,
N̂N = 3,3′-biisoquinoline (i-biq, [2](PF6)2) or di(isoquinolin-3-yl)amine (i-Hdiqa,
[4](PF6)2), and Hmte = 2-(methylthio)ethanol.
Their challenging synthesis involved a protection–deprotection
strategy to avoid the reaction of the free alkyne group with the coordinatively
unsaturated ruthenium center. The thermal stability and photosubstitution
quantum yield (Φ[2] = 0.022 and Φ[4] = 0.080) of the PACT complexes were essentially
preserved upon alkyne functionalization. Interestingly, however, cellular
uptake was doubled after alkyne functionalization, resulting in increased
cytotoxicity against A549 cancer cells for both complexes in the dark
and after green light activation (EC50,light = 5 and 7
μM, respectively). To follow the complexes and see the effect
of light activation, post-treatment fluorophore labeling via copper-catalyzed
azide–alkyne cycloaddition was realized in fixed cells at 2
different time points, which allowed for imaging the otherwise invisible
molecules. The images showed that the Ru complexes accumulated in
the cytoplasm only after light irradiation and that they colocalized
with the lysosomes and the Golgi apparatus. Moreover, we combined
this approach with metabolic labeling of DNA, and showed by dual click
imaging that DNA replication was inhibited by complex 4. The strategy described herein, pioneered for nonemissive, photosubstitutionally
active ruthenium complexes, opens a new avenue for investigating the
selective attack of lung cancer cells by PACT.