Modifying charge and hydrophilicity of simple Ru(II) polypyridyl complexes radically alters biological activities: old complexes, surprising new tricks

Abstract

Compounds capable of light-triggered cytotoxicity are appealing potential therapeutics, because they can provide spatial and temporal control over cell killing to reduce side effects in cancer therapy. Two simple homoleptic Ru(II) polypyridyl complexes with almost-identical photophysical properties but radically different physiochemical properties were investigated as agents for photodynamic therapy (PDT). The two complexes were identical, except for the incorporation of six sulfonic acids into the ligands of one complex, resulting in a compound carrying an overall -4 charge. The negatively charged compound exhibited significant light-mediated cytotoxicity, and, importantly, the negative charges resulted in radical alterations of the biological activity, compared to the positively charged analogue, including complete abrogation of toxicity in the dark. The charges also altered the subcellular localization properties, mechanism of action, and even the mechanism of cell death. The incorporation of negative charged ligands provides a simple chemical approach to modify the biological properties of light-activated Ru(II) cytotoxic agents.

Chart 1. Structures of Compounds 1 and 2

Figure 1

Agarose gel electrophoresis of pUC19 with increasing concentrations of (A) 1 and (B) 2 in the dark or irradiated (λ > 400 nm). Lanes 1 and 12, DNA molecular weight standard; lane 2, linear (reaction with EcoR1); lane 3, relaxed circle (reaction with Cu(phen)₂); lanes 4–11, 0, 8.25, 16.5, 31.3, 62.5, 125, 250, and 500 μM compound. Cytotoxicity dose response of (C) 1 and (D) 2 in the dark (open squares) or irradiated (closed circles). HL60 cells were incubated for 72 h with compound prior to quantification of viability.

Figure 2

ApoTome microscopy showing subcellular localization of 1 and 2 at 8 h. Co-localization of 1 and 2 in mitochondria or lysosomes is indicated by the apparent yellow emission. (A) Mitotracker Green FM was used to image mitochondria. (B) Lysotracker Green DND-26 was used to image lysosomes. Red color denotes intrinsic emission of 1 and 2, whereas blue color denotes Hoechst staining of the nucleus. The yellow color occurs due to overlap of the red emission from the ruthenium complexes and green emission of the organelle-specific dyes, indicating colocalization. Compound 1 localizes in both the mitochondria and the lysosomes while 2 was not predominantly found in either organelle.

Figure 3

(A) Example images and quantification of the emission of tetramethylrhodamine ethyl ester (TMRE) in the presence and absence of 1 and 2. Compound 1 does not show increased TMRE emission over background emission of 1, while compound 2 does not add to the TMRE emission. Mitochondrial potential and cell viability of A549 cells as a function of time for (B) 1, in the dark; (C) 1, irradiated; (D) 2, in the dark; and (E) 2, irradiated. TMRE was used to quantify membrane potential; values are relative to a no-compound control value of 100.

Figure 4

(A) Western blot analysis of cleaved PARP, caspase 3, and pro-caspase 3. GAPDH is used as a loading control. (B) Agarose gel electrophoresis of genomic DNA harvested from HL60 cells after treatment with various agents. Lanes 1 and 9, ladder; lane 2, no compound control; lane 3, 10% EtOH, 24 h (necrosis control); lane 4, cisplatin, 24 h (apoptosis control); lane 5, 1, irradiated, 8 h; lane 6, 1, in the dark, 8 h; lane 7, 2, irradiated, 24 h; and lane 8, 2, in the dark, 24 h.