Martin R. Gill

Dinuclear Ruthenium(II) Complexes as Two‐Photon, Time‐Resolved Emission Microscopy Probes for Cellular DNA

The first transition-metal complex-based two-photon absorbing luminescence lifetime probes for cellular DNA are presented. This allows cell imaging of DNA free from endogenous fluorophores and potentially facilitates deep tissue imaging. In this initial study, ruthenium(II) luminophores are used as phosphorescent lifetime imaging microscopy (PLIM) probes for nuclear DNA in both live and fixed cells. The DNA-bound probes display characteristic emission lifetimes of more than 160 ns, while shorter-lived cytoplasmic emission is also observed. These timescales are orders of magnitude longer than conventional FLIM, leading to previously unattainable levels of sensitivity, and autofluorescence-free imaging.

Ruthenium(II) Metallo-intercalators: DNA Imaging and Cytotoxicity

Cisplatin is currently employed as the first step in treating a variety of cancers, including metastatic breast and ovarian tumours, and it achieves its toxicity by irreversibly creating intrastrand DNA cross-links, provoking a cellular DNA damage response and ultimately leading to programmed cell death or cell cycle arrest. However, acquired drug resistance represents a key challenge in platinum-based chemotherapy and there remains a significant need for therapeutic strategies that address the emergence of cisplatin resistance in tumours. Since the observation that [Ru(bpy)2(dppz)] 2+ (bpy: 2,2’-bipyridine; dppz: dipyrido[3,2-a:2’,3’-c]phenazine) and [Ru(phen)2(dppz)] 2+ (phen: 1,10-phenanthroline) function as “light switches” upon reversibly binding to DNA—they display quenched luminescence in aqueous media but emission from a MLCT (metal-to-ligand charge transfer) excited state as a result of intercalation—there has been a burgeoning interest in d transition metal polypyridyl coordination complexes that reversibly bind to DNA. Such work has particularly centred on structureand site-specific DNA probes but more recently there has been growing attention on further developing these complexes for application as imaging agents in living biological systems while cytotoxicity studies on these systems have shown the importance of ancillary and intercalating ligand to be apparent in the overall toxicity. In this context, the biological activity of Ru “light switch” systems is of great interest and, conceptually, the therapeutic application is particularly attractive, as light switch complexes bind to DNA by an entirely different mechanism to cisplatin (through reversible interactions—usually intercalation—rather than irreversible coordination), and so cytotoxic metallo-intercalators are predicted to remain active against cells with acquired cisplatin resistance. Here, we report the successful cellular uptake of two [Ru(L)2(tpphz)] intercalating systems into live cancer cells (tpphz: tetrapyrido[3,2-a:2’,3’-c:3’’,2’’-h:2’’’,3’’’-j]phenazine; 1 L: bpy; 2 L: phen; Scheme 1). These complexes are multifunctional in cellulo imaging probes and also display cytotoxicity. Using their properties as imaging agents, their ability to bind to cellular DNA in live and fixed cells was confirmed by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM), and we found their cytotoxic potency towards cancer cells to be similar in magnitude to cisplatin. Strikingly, this toxicity is retained even with cisplatin resistant cancer cells. Complexes 1 and 2 were prepared as described previously, and although the in vitro DNA binding properties of 1 have been previously reported, corresponding data for 2 have not been reported. Optical absorption and emission response of 2 to the addition of calf thymus DNA (CT-DNA) in addition to viscosity studies showed that this complex binds to DNA through intercalation and displays an in vitro DNA light-switch effect (Figure 1 and Figure S1 in the Supporting Information). By fitting the emission data to the McGhee–von Hippel model for binding to an isotropic lattice, the equilibrium binding constant, Kb, was estimated to be 3.0 10 5 m , a similar order of

Photoactive RuII–Polypyridyl Complexes that Display Sequence Selectivity and High-Affinity Binding to Duplex DNA through Groove Binding

The duplex-DNA binding properties of a nonintercalating polypyridyl ruthenium(II) complex that incorporates a linear extended ligand with a catechol moiety has been probed with a variety of photo- and biophysical techniques. These studies reveal that the complex groove binds to DNA sequences biphasically, and displays binding constants equivalent to those of high-affinity metallointercalators. The complex also displays preferential binding to AT-rich sequences. Changes in the structure of the coordinated catechol ligand and the incorporation of intercalating ancillary ligands into the complex were found to modulate both the optical-binding response and binding parameters of the system, which indicates that the catechol moiety plays a crucial role in the observed enhancement to binding affinities.

Live cell luminescence imaging as a function of delivery mechanism.

Mitochondria in live cells can be imaged with a ruthenium(II) complex that usually binds and images nuclear DNA. The cellular uptake mechanism of this probe was changed by using a biocompatible pH-sensitive polymersome vector. This change in delivery route, determines the final cellular location of the probe and thus modulates its imaging properties.

Serum Albumin Binding Inhibits Nuclear Uptake of Luminescent Metal-Complex-Based DNA Imaging Probes.

The DNA binding and cellular localization properties of a new luminescent heterobimetallic Ir(III) Ru(II) tetrapyridophenazine complex are reported. Surprisingly, in standard cell media, in which its tetracationic, isostructural Ru(II) Ru(II) analogue is localized in the nucleus, the new tricationic complex is poorly taken up by live cells and demonstrates no nuclear staining. Consequent cell-free studies reveal that the Ir(III) Ru(II) complex binds bovine serum albumin, BSA, in Sudlows Site I with a similar increase in emission and binding affinity to that observed with DNA. Contrastingly, in serum-free conditions the complex is rapidly internalized by live cells, where it localizes in cell nuclei and functions as a DNA imaging agent. The absence of serum proteins also greatly alters the cytotoxicity of the complex, where high levels of oncosis/necrosis are observed due to this enhanced uptake. This suggests that simply increasing the lipophilicity of a DNA imaging probe to enhance cellular uptake can be counterproductive as, due to increased binding to serum albumin protein, this strategy can actually disrupt nuclear targeting.

A ruthenium polypyridyl intercalator stalls DNA replication forks, radiosensitizes human cancer cells and is enhanced by Chk1 inhibition.

Ruthenium(II) polypyridyl complexes can intercalate DNA with high affinity and prevent cell proliferation; however, the direct impact of ruthenium-based intercalation on cellular DNA replication remains unknown. Here we show the multi-intercalator [Ru(dppz)2(PIP)]2+ (dppz = dipyridophenazine, PIP = 2-(phenyl)imidazo[4,5-f][1,10]phenanthroline) immediately stalls replication fork progression in HeLa human cervical cancer cells. In response to this replication blockade, the DNA damage response (DDR) cell signalling network is activated, with checkpoint kinase 1 (Chk1) activation indicating prolonged replication-associated DNA damage, and cell proliferation is inhibited by G1-S cell-cycle arrest. Co-incubation with a Chk1 inhibitor achieves synergistic apoptosis in cancer cells, with a significant increase in phospho(Ser139) histone H2AX (γ-H2AX) levels and foci indicating increased conversion of stalled replication forks to double-strand breaks (DSBs). Normal human epithelial cells remain unaffected by this concurrent treatment. Furthermore, pre-treatment of HeLa cells with [Ru(dppz)2(PIP)]2+ before external beam ionising radiation results in a supra-additive decrease in cell survival accompanied by increased γ-H2AX expression, indicating the compound functions as a radiosensitizer. Together, these results indicate ruthenium-based intercalation can block replication fork progression and demonstrate how these DNA-binding agents may be combined with DDR inhibitors or ionising radiation to achieve more efficient cancer cell killing.

A Self‐Assembled Metallomacrocycle Singlet Oxygen Sensitizer for Photodynamic Therapy

Although metal-ion-directed self-assembly has been widely used to construct a vast number of macrocycles and cages, it is only recently that the biological properties of these systems have begun to be explored. However, up until now, none of these studies have involved intrinsically photoexcitable self-assembled structures. Herein we report the first metallomacrocycle that functions as an intracellular singlet oxygen sensitizer. Not only does this Ru2 Re2 system possess potent photocytotoxicity at light fluences below those used for current medically employed systems, it offers an entirely new paradigm for the construction of sensitizers for photodynamic therapy.

Multimodal Super-resolution Optical Microscopy Using a Transition-Metal-Based Probe Provides Unprecedented Capabilities for Imaging Both Nuclear Chromatin and Mitochondria

Detailed studies on the live cell uptake properties of a dinuclear membrane-permeable RuII cell probe show that, at low concentrations, the complex localizes and images mitochondria. At concentrations above ∼20 μM, the complex images nuclear DNA. Because the complex is extremely photostable, has a large Stokes shift, and displays intrinsic subcellular targeting, its compatibility with super-resolution techniques was investigated. It was found to be very well suited to image mitochondria and nuclear chromatin in two color, 2C-SIM, and STED and 3D-STED, both in fixed and live cells. In particular, due to its vastly improved photostability compared to that of conventional SR probes, it can provide images of nuclear DNA at unprecedented resolution.