Phototoxicity of Tridentate Ru(II) Polypyridyl Complex with Expanded Bite Angles toward Mammalian Cells and Multicellular Tumor Spheroids.

Tridentate ligand-coordinated ruthenium (II) polypyridyl complexes with large N-Ru-N bite angles have been shown to promote ligand field splitting and reduce singlet-triplet state mixing leading to dramatically extended emission quantum yields and lifetimes under ambient conditions. These effects are anticipated to enhance their photoinduced singlet oxygen production, promoting prospects for such complexes as type II phototherapeutics. In this contribution, we examined this putative effect for [Ru(bqp)(bqpCOOEt)]2+, Ru-bqp-ester, a heteroleptic complex containing bqp = [2,6-bi(quinolin-8-yl)pyridine], a well-established large bite angle tridentate ligand, as well as its peptide conjugates [Ru(bqp)(bqpCONH-ahx-FrFKFrFK(Ac)-CONH2)]5+ (Ru-bqp-MPP) and [Ru(bqp) (bqp)(CONH-ahx-RRRRRRRR-CONH2)]10+ (Ru-bqp-R8) that were prepared in an effort to promote live cell/tissue permeability and targeting of the parent. Membrane permeability of both parent and peptide conjugates were compared across 2D cell monolayers; A549, Chinese hamster ovary, human pancreatic cancer (HPAC), and 3D HPAC multicellular tumor spheroids (MCTS) using confocal microscopy. Both the parent complex and peptide conjugates showed exceptional permeability with rapid uptake in both 2D and 3D cell models but with little distinction in permeability or distribution in cells between the parent or peptide conjugates. Unexpectedly, the uptake was temperature independent and so attributed to passive permeation. Both dark and photo-toxicity of the Ru(II) complexes were assessed across cell types, and the parent showed notably low dark toxicity. In contrast, the parent and conjugates were found to be highly phototoxic, with impressive phototoxic indices (PIs) toward HPAC cell monolayers in particular, with PI values ranging from ∼580 to 760. Overall, our data indicate that the Ru(II) parent complex and its peptide conjugates show promise at both cell monolayers and 3D MCTS as photosensitizers for photodynamic therapy.

Selective, Disruptive Luminescent Ru(II) Polypyridyl Probes of G-Quadruplex.

Sensors capable of transducing G-quadruplex DNA binding are important both in solution and for imaging and interrogation in cellulo. Ru(II)-based light switches incorporating dipyridylphenazine (dppz) ligands are effective probes for recognition and imaging of DNA and its polymorphs including G-quadruplex, although selectivity is a limitation. While the majority of Ru(II)-based light switches reported to date, stabilize the quadruplex, imaging/theranostic probes that can disrupt G4s are of potentially enormous value in study and therapy for a range of disease states. We report here, on a Ru(II) complex (Ru-PDC3) that assembles the light switch capability of a Ru(II) dipyridylphenazine complex with the well-known G4-selective ligand Phen-DC3, into a single structure. The complex shows the anticipated light switch effect and strong affinity for G4 structures. Affinity depended on the G4 topology and sequence, but across all structures bar one, it was roughly an order of magnitude greater than for duplex or single-stranded DNA. Moreover, photophysical and Raman spectral data showed clear discrimination between duplex DNA and G4-bound structures offering the prospect of discrimination in imaging as well as in solution. Crucially, unlike the constituent components of the probe, Ru-PDC3 is a powerful G4 disrupter. From circular dichroism (CD), a reduction of ellipticity of the G4 between 70 and 95% was observed depending on topology and in many cases was accompanied by an induced CD signal for the metal complex. The extent of change in ellipticity is amongst the largest reported for small-molecule ligand G4 binding. While a promising G4 probe, without modification, the complex is fully water-soluble and readily permeable to live cells.

A phototoxic thulium complex exhibiting intracellular ROS production upon 630 nm excitation in cancer cells.

A lanthanide(III) complex with a thulium metal centre connected via a terpyridine unit to a light harvesting antenna with strong absorption in the therapeutic window [>590 nm] was synthesised and tested as a possible photosensitiser (PS) in photodynamic therapy (PDT). The complex exhibited significant phototoxic activity on cancer cells upon irradiation in the therapeutic window and from intracellular and solution studies ROS production was identified as the compound's phototoxic mode of action. In cell viability assays, a 10-fold lowered IC50 value was obtained upon irradiation compared to the dark control.

Metal Peptide Conjugates in Cell and Tissue Imaging and Biosensing.

Metal complex luminophores have seen dramatic expansion in application as imaging probes over the past decade. This has been enabled by growing understanding of methods to promote their cell permeation and intracellular targeting. Amongst the successful approaches that have been applied in this regard is peptide-facilitated delivery. Cell-permeating or signal peptides can be readily conjugated to metal complex luminophores and have shown excellent response in carrying such cargo through the cell membrane. In this article, we describe the rationale behind applying metal complexes as probes and sensors in cell imaging and outline the advantages to be gained by applying peptides as the carrier for complex luminophores. We describe some of the progress that has been made in applying peptides in metal complex peptide-driven conjugates as a strategy for cell permeation and targeting of transition metal luminophores. Finally, we provide key examples of their application and outline areas for future progress.

Ru(ii)/BODIPY core co-encapsulated ratiometric nanotools for intracellular O2 sensing in live cancer cells.

Oxygen is a crucial reagent in many biochemical processes within living cells and its concentration can be an effective marker in disease, particularly in cancer where tissue hypoxia has been shown to indicate tumour growth. Probes that can reflect the oxygen concentration and distribution using ratiometric signals can be applied to a range of conventional methods without the need for specialised equipment and are particularly useful. The preparation and in cellulo study of luminescent ratiometric core-shell nanoparticles are presented. Here, a new lipophilic and oxygen-responsive Ru(ii) tris-heteroleptic polypyridyl complex is co-encapsulated with a reference BODIPY dye into the core of poly-l-lysine-coated polystyrene particles. The co-core encapsulation ensures oxygen response but reduces the impact of the environment on both probes. Single wavelength excitation of the particles, suspended in aqueous buffer, at 480 nm, triggers well-resolved dual emission from both dyes with peak maxima at 515 nm and 618 nm. A robust ratiometric oxygen response is observed from water, with a linear dynamic range of 3.6-262 µM which matches well with typical biological ranges. The uptake of RuBDP NPs was found to be cell-line dependent, but in cancerous cell lines, the particles were strongly permeable with late endosomal and partial lysosomal co-staining observed within 3 to 4 hours, eventually leading to extensive staining of the cytoplasm. The co-localisation of the ruthenium and BODIPY emission confirms that the particles remain intact in cellulo with no indication of dye leaching. The ratiometric O2 sensing response of the particles in cellulo was demonstrated using a plate-based assay and by confocal xyλ scanning of cells exposed to hypoxic conditions.

Os(II)-Bridged Polyarginine Conjugates: The Additive Effects of Peptides in Promoting or Preventing Permeation in Cells and Multicellular Tumor Spheroids.

The preparation of two polyarginine conjugates of the complex Os(II) [bis-(4'-(4-carboxyphenyl)-2,2':6',2″-terpyridine)] [Os-(Rn)2]x+ (n = 4 and 8; x = 10 and 18) is reported, to explore whether the R8 peptide sequence that promotes cell uptake requires a contiguous amino acid sequence for membrane permeation or if this can be accomplished in a linearly bridged structure with the additive effect of shorter peptide sequences. The conjugates exhibit NIR emission centered at 754 nm and essentially oxygen-insensitive emission with a lifetime of 89 ns in phosphate-buffered saline. The uptake, distribution, and cytotoxicity of the parent complex and peptide derivatives were compared in 2D cell monolayers and a three-dimensional (3D) multicellular tumor spheroid (MCTS) model. Whereas, the bis-octaarginine sequences were impermeable to cells and spheroids, and the bis-tetraarginine conjugate showed excellent cellular uptake and accumulation in two 2D monolayer cell lines and remarkable in-depth penetration of 3D MCTSs of pancreatic cancer cells. Overall, the data indicates that cell permeability can be promoted via non-contiguous sequences of arginine residues bridged across the metal centre.

Photophysics and Cell Uptake of Self-Assembled Ru(II)Polypyridyl Vesicles.

Effective delivery of luminescent probes for cell imaging requires both cell membrane permeation and directing to discrete target organelles. Combined, these requirements can present a significant challenge for metal complex luminophores, that have excellent properties as imaging probes but typically show poor membrane permeability. Here, we report on highly luminescent Ruthenium polypyridyl complexes based on the parent; [Ru(dpp)2(x-ATAP)](PF6)2 structure, where dpp is 4,7-diphenyl-1,10-phenanthroline and x-ATAP is 5-amino-1,10-phenanthroline with pendant alkyl-acetylthio chains of varying length; where x is 6; 5-Amido-1,10-phenanthroline-(6-acetylthio-hexanyl). 8; 5-Amido-1,10-phenanthroline-(8-acetylthio-octanyl). 11; 5-Amido-1,10-phenanthroline-(11-acetylthio-undecanyl); and 16; 5-Amido-1,10-phenanthroline-(16-acetylthio-hexadecanyl). Soluble in organic media, the alkyl-acetylthiolated complexes form nanoaggregates of low polydispersity in aqueous solution. From dynamic light scattering the nanoaggregate diameter was measured as 189 nm and 135 nm for 5 × 10-6 M aqueous solutions of [Ru(dpp)2(N∧N)](PF6)2 with the hexadecanoyl and hexanyl tails respectivly. The nanoaggregate exhibited dual exponential emission decays with kinetics that matched closely those of the [Ru(dpp)2(16-ATAP)]2+ incorporated into the membrane of a DPPC liposome. Cell permeability and distribution of [Ru(dpp)2(11-ATAP)]2+ or [Ru(dpp)2(16-ATAP)]2+ were evaluated in detail in live HeLa and CHO cell lines and it was found from aqueous media, that the nanoaggregate complexes spontaneously cross the membrane of mammalian cells. This process seems, on the basis of temperature dependent studies to be activated. Fluorescence imaging of live cells reveal that the complexes localize highly specifically within organelles and that organelle localization changes dramatically in switching the pendent alkyl chains from C16 to C11 as well as on cell line identity. Our data suggests that building metal complexes capable of self-assembling into nano-dimensional vesicles in this way may be a useful means of promoting cell membrane permeability and driving selective targeting that is facile and relatively low cost compared to use of biomolecular vectors.

Metabolites of Tobacco- and E-Cigarette-Related Nitrosamines Can Drive Cu2+-Mediated DNA Oxidation.

Nitrosamine metabolites resulting from cigarette smoking and E-cigarette (E-cig) vaping cause DNA damage that can lead to genotoxicity. While DNA adducts of metabolites of nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) are well-known tobacco-related cancer biomarkers, only a few studies implicate NNN and NNK in DNA oxidation in humans. NNK and NNN were found in the urine of E-cigarette users who never smoked cigarettes. This paper proposes the first chemical pathways of DNA oxidation driven by NNK and NNN metabolites in redox reactions with Cu2+ and NADPH leading to reactive oxygen species (ROS). A microfluidic array with thin films of DNA and metabolic enzymes that make metabolites of NNN and NNK in the presence of Cu2+ and NADPH was used to estimate relative rates of DNA oxidation. Detection by electrochemiluminescence (ECL) employed a new ECL dye [Os(tpy-benz-COOH)2]2+ that is selective for and sensitive to the primary DNA oxidation product 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) in DNA. Enzyme-DNA films on magnetic beads were used to produce nitrosamine metabolites that enter ROS-forming redox cycles with Cu2+ and NADPH, and liquid chromatography-mass spectrometry (LC-MS) was used to quantify 8-oxodG and identify metabolites. ROS were detected by optical sensors. Metabolites of NNK and NNN + Cu2+ + NADPH generated relatively high rates of DNA oxidation. Lung is the exposure route in smoking and vaping, human lung tissue contains Cu2+ and NADPH, and lung microsomal enzymes gave the highest rates of DNA oxidation in this study. Also, E-cigarette vapor contains 6-fold more copper than that in cigarette smoke, which could exacerbate DNA oxidation.

Photostable NIR emitting ruthenium(II) conjugates; uptake and biological activity in live cells.

A photostable Ru(2,2-biquinoline)2(3-(2-pyridyl)-5-(4-carboxyphenyl)-1,2,4-triazolate) (Ru(biq)2(trzbenzCOOH)) complex that exhibits near-infrared (NIR) emission centred at 786 nm is reported. The parent complex was conjugated via amide coupling to a cell-penetrating peptide sequence octa-arginine (R8), and two signal peptide sequences; the nuclear localizing sequence (NLS) VQRKRQKLMP and the mitochondria penetrating peptide (MPP) FrFKFrFK(Ac) (r = D isomer of arginine, Ac = terminal lysine amine acetyl blocked). Notably, none of the peptide conjugates were cell-permeable as chloride salts but efficient and rapid membrane permeation was observed post ion exchange with perchlorate counterion. Also, surprisingly, all three peptide conjugates exhibited potent dark cytotoxicity in both CHO and HeLa cell lines. The peptide conjugates induce cell death through a caspase dependent apoptotic pathway. At the minimum concentration of dye (approx. 15 µM) required for cell imaging, only 20% of the cells were viable after a 24 h incubation period. To overcome cytotoxicity, the parent complex was PEGylated; this dramatically decreased cytotoxicity, where 50% of cells were viable even at 150 µM concentration after 24 h. Confocal luminescence microscopy indicated that all four bioconjugates, peptides in perchlorate form and polyethylene glycol (PEG) in chloride form, were rapidly internalized within the cell. However, interestingly the precise localisation by the signal peptides observed in related complexes was not observed here and the peptide conjugates were unsuitable as luminescent probes for cell microscopy due to their high cell toxicity. The poor targeting of signal peptides in this instance is attributed to the high lipophilicity of the metal centre.

Mitochondrial targeted osmium polypyridyl probe shows concentration dependent uptake, localisation and mechanism of cell death.

A symmetric osmium(ii) [bis-(4'-(4-carboxyphenyl)-2,2':6',2''-terpyridine)] was prepared and conjugated to two mitochondrial-targeting peptide sequences; FrFKFrFK (r = d-arginine). The parent and conjugate complexes showed strong near infra-red emission centred at λmax 745 nm that was modestly oxygen dependent in the case of the parent and oxygen independent in the case of the conjugate, attributed in the latter case, surprisingly, to a shorter emission lifetime of the conjugate compared to the parent. Confocal fluorescence imaging of sub-live HeLa and MCF 7 cells showed the parent complex was cell impermeable whereas the conjugate was rapidly internalised into the cell and distributed in a concentration dependent manner. At concentrations below approximately 30 µmol, the conjugate localised to the mitochondria of both cell types where it was observed to trigger apoptosis induced by the collapse of the mitochondrial membrane potential (MPP). At concentrations exceeding 30 µmol the conjugate was similarly internalised rapidly but distributed throughout the cell, including to the nucleus and nucleolus. At these concentrations, it was observed to precipitate a caspase-dependent apoptotic pathway. The combination of concentration dependent organelle targeting, NIR emission coincident with the biological window, and distribution dependent cytotoxicity offers an interesting approach to theranostics with the possibility of eliciting site dependent therapeutic effect whilst monitoring the therapeutic effect with luminescence imaging.