In the present study, four mitochondria-specific and two-photon phosphorescence iridium(III) complexes, Ir1–Ir4, were developed for mitochondria imaging in hypoxic tumor cells. The iridium(III) complex has two anthraquinone groups that are hypoxia-sensitive moieties. The phosphorescence of the iridium(III) complex was quenched by the functions of the intramolecular quinone unit, and it was restored through two-electron bioreduction under hypoxia. When the probes were reduced by reductase to hydroquinone derivative products under hypoxia, a significant enhancement in phosphorescence intensity was observed under one- (λ=405 nm) and two-photon (λ=720 nm) excitation, with a two-photon absorption cross section of 76–153 GM at λ=720 nm. More importantly, these probes possessed excellent specificity for mitochondria, which allowed imaging and tracking of the mitochondrial morphological changes in a hypoxic environment over a long period of time. Moreover, the probes can visualize hypoxic mitochondria in 3D multicellular spheroids and living zebrafish through two-photon phosphorescence imaging.

Hypoxia is the critical feature of the tumor microenvironment that is known to lead to resistance to many chemotherapeutic drugs. Six novel ruthenium(II) anthraquinone complexes were designed and synthesized; they exhibit similar or superior cytotoxicity compared to cisplatin in hypoxic HeLa, A549, and multidrug-resistant (A549R) tumor cell lines. Their anticancer activities are related to their lipophilicity and cellular uptake; therefore, these physicochemical properties of the complexes can be changed by modifying the ligands to obtain better anticancer candidates. Complex 1, the most potent member of the series, is highly active against hypoxic HeLa cancer cells (IC₅₀=0.53 μM). This complex likely has 46-fold better activity than cisplatin (IC₅₀=24.62 μM) in HeLa cells. This complex tends to accumulate in the mitochondria and the nucleus of hypoxic HeLa cells. Further mechanistic studies show that complex 1 induced cell apoptosis during hypoxia through multiple pathways, including those of DNA damage, mitochondrial dysfunction, and the inhibition of DNA replication and HIF-1α expression, making it an outstanding candidate for further in vivo studies.

A series of dinuclear ruthenium(II) complexes were synthesised, and the complexes were determined to be new highly selective compounds for binding to telomeric G-quadruplex DNA. The interactions of these complexes with telomeric G-quadruplex DNA were studied by using circular dichroism (CD) spectroscopy, fluorescence resonance energy transfer (FRET) melting assays, isothermal titration calorimetry (ITC) and molecular modelling. The results showed that the complexes 1, 2 and 4 induced and stabilised the formation of antiparallel G-quadruplexes of telomeric DNA in the absence of salt or in the presence of 100 mM K⁺-containing buffer. Furthermore, complexes 1 and 2 strongly bind to and effectively stabilise the telomeric G-quadruplex structure and have significant selectivity for G-quadruplex over duplex DNA. In comparison, complex 3 had a much lesser effect on the G-quadruplex, suggesting that possession of a suitably sized plane for good π–π stacking with the G-quadruplets is essential for the interaction of the dinuclear ruthenium(II) complexes with the G-quadruplex. Moreover, telomerase inhibition by the four complexes and their cellular effects were studied, and complex 1 was determined to be the most promising inhibitor of both telomerase and HeLa cell proliferation.

Fluorescence detection is the most effective tool for tracking gene delivery in living cells. To reduce photodamage and autofluorescence and to increase deep penetration into cells, choosing appropriate fluorophores that are capable of two-photon activation under irradiation in the NIR or IR regions is an effective approach. In this work, we have developed six tetranuclear ruthenium(II) complexes, GV1–6, and have studied their one- and two-photon luminescence properties. DNA interaction studies have demonstrated that GV2–6, bearing hydrophobic alkyl ether chains, show more efficient DNA condensing ability but lower DNA binding constants than GV1. However, the hydrophobic alkyl ether chains also enhance the DNA delivery ability of GV2–6 compared with that of GV1. More importantly, we have applied GV1–6 as non-viral gene vectors for tracking DNA delivery in living cells by one- and two-photon fluorescence microscopies. In two-photon microscopy, a high signal-to-noise contrast was achieved by irradiation with an 830 nm laser. This is the first example of the use of transition-metal complexes for two-photon luminescent tracking of the cellular pathways of gene delivery and as DNA carriers. Our work provides new insights into improving real-time tracking during gene delivery and transfection as well as important information for the design of multifunctional non-viral vectors.

The aim of this study was to illustrate the dramatically different anticancer activities between coordinatively saturated polypyridyl (1 a–4 a) and cyclometalated (1 b–4 b) ruthenium(II) complexes. The cyclometalated complexes 1 b–4 b function as DNA transcription inhibitors, exhibiting switch-on cytotoxicity against a 2D cancer cell monolayer, whereas the polypyridyl complexes 1 a–4 a are relatively inactive. Moreover, complexes 1 b–4 b exhibit excellent cytotoxicity against 3D multicellular tumor spheroids (MCTSs), which serve as an intermediate model between in vitro 2D cell monolayers and in vivo 3D solid tumors. The hydrophobicity, efficient cell uptake, and nucleus targeting ability, as well as the high DNA binding affinity of complexes 1 b–4 b, likely contribute to their enhanced anticancer activity. We surmise that cyclometalation could be a universal approach to significantly enhance the anticancer activity of substituted polypyridyl Ru^II complexes. We also suggest that 3D MCTSs may be a more practical platform for anticancer drug screening than 2D cancer monolayer approaches.

Five cyclometalated iridium(III) complexes with 2-phenylimidazo[4,5-f][1,10]phenanthroline derivatives (IrL1–IrL5) were synthesized and developed to image and track mitochondria in living cells under two-photon (750 nm) excitation, with two-photon absorption cross-sections of 48.8–65.5 GM at 750 nm. Confocal microscopy and inductive coupled plasma-mass spectrometry (ICP-MS) demonstrated that these complexes selectively accumulate in mitochondria within 5 min, without needing additional reagents for membrane permeabilization, or replacement of the culture medium. In addition, photobleaching experiments and luminescence measurements confirmed the photostability of these complexes under continuous laser irradiation and physiological pH resistance. Moreover, results using 3D multicellular spheroids demonstrate the proficiency of these two-photon luminescent complexes in deep penetration imaging. Two-photon excitation using such novel complexes of iridium(III) for exclusive visualization of mitochondria in living cells may substantially enhance practical applications of bioimaging and tracking.

A new dinuclear Ru^II polypyridyl complex, [(bpy)₂Ru(H₂bpip)Ru(bpy)₂]⁴⁺ (RuH₂bpip, bpy=2,2-bipyridine, H₂bpip=2,6-pyridyl(imidazo[4,5-f][1,10]phenanthroline), was developed to act as a one- and two-photon luminescent probe for biological Cu²⁺ detection. This Ru^II complex shows a significant two-photon absorption cross section (400 GM) and displays a remarkable one- and two-photon luminescence switch in the presence of Cu²⁺ ions. Importantly, RuH₂bpip can selectively recognise Cu²⁺ in aqueous media in the presence of other abundant cellular cations (such as Na⁺, K⁺, Mg²⁺, and Ca²⁺), trace metal ions in organisms (such as Zn²⁺, Ag⁺, Fe³⁺, Fe²⁺, Ni²⁺, Mn²⁺, and Co²⁺), prevalent toxic metal ions in the environment (such as Cd²⁺, Hg²⁺, and Cr³⁺), and amino acids, with high sensitivity (detection limit≤3.33×10⁻⁸ M) and a rapid response time (≤15 s). The biological applications of RuH₂bpip were also evaluated and it was found to exhibit low cytotoxicity, good water solubility, and membrane permeability; RuH₂bpip was, therefore, employed as a sensing probe for the detection of Cu²⁺ in living cells and zebrafish.

The interaction of ruthenium(II)-polypyridyl complexes with DNA has attracted considerable interests during the past two decades. This paper presents some recent progresses in our laboratory on the interaction of Ru^II-polypyridyl complexes with DNA. The first part describes the effect of modulating the intercalative ligand on the DNA-binding behaviors of the complexes, such as DNA-binding affinity, DNA-binding enantioselectivity, DNA molecular ‘light switch’ effect, and DNA sequence selectivity. The second part focuses on the DNA photocleavage by the complexes and its mechanism. In the final part, we discuss the topoisomerase inhibition and its mechanism, as well as the antitumor activity of the Ru^II-polypyridyl complexes.

Two new complexes, [Ru(phen)₂(ppd)]²⁺ (1) and [Ru(phen)(ppd)₂]²⁺ (2) (ppd=pteridino[6,7-f] [1,10]phenanthroline-11,13(10H,12H)-dione, phen=1,10-phenanthroline) were synthesized and characterized by ES-MS, ¹H-NMR spectroscopy, and elemental analysis. The intercalative DNA-binding properties of 1 and 2 were investigated by absorption-spectroscopy titration, luminescence-spectroscopy studies, thermal denaturation, and viscosity measurements. The theoretical aspects were further discussed by comparative studies of 1 and 2 by means of DFT calculations and molecular-orbital theory. Photoactivated cleavage of pBR322 DNA by the two complexes were also studied, and 2 was found to be a much better photocleavage reagent than 1. The mechanism studies revealed that singlet oxygen and the excited-states redox potentials of the complex may play an important role in the DNA photocleavage.