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Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

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Visualizing the Invisible: Dual Click Imaging of Ruthenium-Based Photoactivated Chemotherapy Agents and Their DNA

Anja Busemann¹, Lisa Rieger², Rachael M Cunningham³

¹Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, Netherlands.

Journal of the American Chemical Society

|November 5, 2025

Summary

Researchers developed new ruthenium-based photoactivated chemotherapy (PACT) drugs that can be tracked in cells. These alkyne-functionalized PACT complexes show improved cellular uptake and cytotoxicity against lung cancer cells.

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Single Molecule Analysis of Laser Localized Psoralen Adducts

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Area of Science:

Inorganic Chemistry
Medicinal Chemistry
Chemical Biology

Background:

Ruthenium-based photoactivated chemotherapy (PACT) complexes are difficult to visualize within cells due to their lack of inherent fluorescence.
Tracking drug localization and action is crucial for understanding their efficacy and optimizing cancer treatment strategies.

Purpose of the Study:

To synthesize and characterize novel alkyne-functionalized ruthenium PACT complexes.
To investigate the cellular uptake, cytotoxicity, and intracellular localization of these complexes upon light activation.
To develop a method for visualizing the otherwise non-emissive ruthenium complexes within cells.

Main Methods:

Synthesis of two alkyne-functionalized ruthenium complexes using a protection-deprotection strategy.
Assessment of thermal stability and photosubstitution quantum yields.
Evaluation of cellular uptake and cytotoxicity against A549 lung cancer cells in dark and light conditions.
Post-treatment fluorophore labeling via copper-catalyzed azide-alkyne cycloaddition for cellular imaging.
Dual click imaging combined with metabolic DNA labeling to assess DNA replication inhibition.

Main Results:

Alkyne functionalization preserved the stability and photosubstitution properties of the PACT complexes.
Cellular uptake and cytotoxicity were significantly enhanced after alkyne functionalization.
Light activation led to cytoplasmic accumulation of ruthenium complexes, co-localizing with lysosomes and the Golgi apparatus.
Complex 4 demonstrated inhibition of DNA replication in lung cancer cells.

Conclusions:

Alkyne functionalization is a viable strategy to enhance the cellular activity of ruthenium PACT complexes.
The developed imaging technique allows visualization of non-emissive ruthenium complexes, enabling studies on their intracellular fate and mechanism of action.
This approach provides a new platform for studying the selective targeting of lung cancer cells by PACT agents.