Determining live-cell integrity of metallodrugs using reversible fluorescent metal ion sensors: intrinsic inertness matters for stability in cells

The therapeutic effectiveness of metallodrugs is heavily influenced by their propensity to release metal ions within biological systems. This is particularly relevant in the case of labile first series transition metal ions, the release leading to loss in complex-based activity. While thermodynamic parameters governing the stability and efficacy of metallodrugs can be analyzed using established analytical techniques, assessing live-cell integrity of a metallodrug remains a significant challenge. Determination of in-cell integrity is essential for optimizing the design and therapeutic potential of metallodrugs. Importantly, the study should be performed in living cells because biological environments can significantly impact the stability and lability. Currently, no experimental approach directly visualizes metal ions released by metallodrugs and determines their in-cell-integrity. To address this gap, we propose a novel assay to monitor the extent of release of metal ions from metallodrugs using binding-based, reversible fluorescent metal ion sensors in live cells. We tested the relevance of the assay on a series of manganese antioxidants (Mn superoxide dismutase (SOD) mimics) utilizing Mn(II) selective reversible fluorescent sensors. The results showed that the SOD mimic Mn1, with the lowest intrinsic Kd from the series of antioxidants tested, exhibited the highest amount of Mn-release, while Mn1CP, with the highest Kd, demonstrated lower Mn-release. Interestingly, the level of metal-release correlates with the intrinsic lability of metallodrugs and not with their intrinsic thermodynamic stability. The study demonstrates the importance of assessing in-cell metal ion release for optimizing the therapeutic outcome of metallodrugs. The novel fluorescence assay developed here offers a valuable tool for the evaluation of metallodrug behavior in cellular environments. Overall, this work shows that in-cell metal ion release from these manganese metallodrugs appears to correlate with intrinsic lability and thus is under kinetic control.