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A dinuclear ruthenium complex completely inhibits the invasive properties of malignant melanoma within a living human skin model.

Following an overview of the approaches and techniques used to acheive super-resolution microscopy, this review presents the advantages supplied by nanoparticle based probes for these applications. The various clases of nanoparticles that have been developed toward these goals are then critically described and these discussions are illustrated with a variety of examples from the recent literature.

In previous studies we have described the therapeutic action of luminescent dinuclear ruthenium(II) complexes based on the tetrapyridylphenazine, tpphz, bridging ligand on pathogenic strains of Escherichia coli and Enterococcus faecalis. Herein, the antimicrobial activity of the complex against pernicious Gram-negative ESKAPE pathogenic strains of Acinetobacter baumannii (AB12, AB16, AB184 and AB210) and Pseudomonas aeruginosa (PA2017, PA_007_IMP and PA_004_CRCN) are reported. Estimated minimum inhibitory concentrations and minimum bactericidal concentrations for the complexes revealed the complex shows potent activity against all A. baumannii strains, in both glucose defined minimal media and standard nutrient rich Mueller-Hinton-II. Although the activity was lower in P. aureginosa, a moderately high potency was observed and retained in carbapenem-resistant strains. Optical microscopy showed that the compound is rapidly internalized by A. baumannii. As previous reports had revealed the complex exhibited no toxicity in Galleria Mellonella up to concentrations of 80 mg/kg, the ability to clear pathogenic infection within this model was explored. The pathogenic concentrations to the larvae for each bacterium were determined to be ≥ 105 for AB184 and ≥ 103 CFU/mL for PA2017. It was found a single dose of the compound totally cleared a pathogenic A. baumannii infection from all treated G. mellonella within 96 hours. Uniquely, in these conditions thanks to the imaging properties of the complex the clearance of the bacteria within the hemolymph of G. mellonella could be directly visualized through both optical and transmission electron microscopy.

Threading intercalators bind DNA with high affinities. Here, we describe single‐molecule studies on a cell‐permeant luminescent dinuclear ruthenium(II) complex that has been previously shown to thread only into short, unstable duplex structures. Using optical tweezers and confocal microscopy, we show that this complex threads and locks into force‐extended duplex DNA in a two‐step mechanism. Detailed kinetic studies reveal that an individual stereoisomer of the complex exhibits the highest binding affinity reported for such a mono‐intercalator. This stereoisomer better preserves the biophysical properties of DNA than the widely used SYTOX Orange. Interestingly, threading into torsionally constrained DNA decreases dramatically, but is rescued on negatively supercoiled DNA. Given the “light‐switch” properties of this complex on binding DNA, it can be readily used as a long‐lived luminescent label for duplex or negatively supercoiled DNA through a unique “load‐and‐lock” protocol., Using single‐molecule optical tweezers and confocal microscopy we perform kinetic analysis of a novel dinuclear ruthenium(II) DNA imaging probe. Reversable binding is observed on relaxed DNA, however on stretching DNA, threading can occur, resulting in high affinity stable binding. Threading is highly stereoisomer specific and dependent on DNA supercoiling state. This DNA probe has exciting potential for DNA structure specific imaging.

The dinuclear Ru

The dental restorative material mercury amalgam has been used for centuries and during this time waste disposal methods have developed considerably. This review provides an overview of how mercury is managed in dental clinics, as well as outlining some possible environmental implications that could occur with poor waste management.

Super-resolution microscopy (SRM) has revolutionized the study of cell biology, enabling researchers to visualize cellular structures with nanometric resolution, single-molecule sensitivity, and with multiple colors using conventional fluorophores. SRM is well-suited for volumetric live-cell imaging and helps in extracting quantitative information on spatial distributions. It can be used to estimate the absolute numbers of proteins or other macromolecules or nanostructured material within subcellular compartments, characterize their structures, and their nano/bio interactions. Although a number of recent general reviews on SRM have elaborated its role in chemical and clinical biology, as well as nanomedicine, herein, we provide an overview of the use of luminescent nanocarriers (LNC) in SRM imaging and single-molecule tracking. The role of LNCs in controlling the brightness and stability of emissive states is discussed with a special focus on organelle-specific delivery and how this approach can be utilized to produce novel optical-switched systems. We also discuss the challenges related to the molecular targeting of such material in biological systems. In doing so, we will provide practical guidance for super-resolution imaging in nanomedicine research, its technical challenges, and the opportunities for future advancement.

Amyloid β oligomers (Aβo) are the main toxic species in Alzheimer's disease, which have been targeted for single drug treatment with very little success. In this work we report a new approach for identifying functional Aβo binding compounds. A tailored library of 971 fluorine containing compounds was selected by a computational method, developed to generate molecular diversity. These compounds were screened for Aβo binding by a combined 19F and STD NMR technique. Six hits were evaluated in three parallel biochemical and functional assays. Two compounds disrupted Aβo binding to its receptor PrPC in HEK293 cells. They reduced the pFyn levels triggered by Aβo treatment in neuroprogenitor cells derived from human induced pluripotent stem cells (hiPSC). Inhibitory effects on pTau production in cortical neurons derived from hiPSC were also observed. These drug-like compounds connect three of the pillars in Alzheimer's disease pathology, i.e. prion, Aβ and Tau, affecting three different pathways through specific binding to Aβo and are, indeed, promising candidates for further development.

The dinuclear photo-oxidizing RuII complex [{Ru(TAP2)}2(tpphz)]4+ (TAP = 1,4,5,8- tetraazaphenanthrene, tpphz = tetrapyrido[3,2-a:2',3'-c:3″,2''-h:2‴,3'''-j]phenazine), 14+, is readily taken up by live cells localizing in mitochondria and nuclei. In this study, the two-photon absorption cross section of 14+ is quantified and its use as a two-photon absorbing phototherapeutic is reported. It was confirmed that the complex is readily photoexcited using near-infrared, NIR, and light through two-photon absorption, TPA. In 2-D cell cultures, irradiation with NIR light at low power results in precisely focused phototoxicity effects in which human melanoma cells were killed after 5 min of light exposure. Similar experiments were then carried out in human cancer spheroids that provide a realistic tumor model for the development of therapeutics and phototherapeutics. Using the characteristic emission of the complex as a probe, its uptake into 280 μm spheroids was investigated and confirmed that the spheroid takes up the complex. Notably TPA excitation results in more intense luminescence being observed throughout the depth of the spheroids, although emission intensity still drops off toward the necrotic core. As 14+ can directly photo-oxidize DNA without the mediation of singlet oxygen or other reactive oxygen species, phototoxicity within the deeper, hypoxic layers of the spheroids was also investigated. To quantify the penetration of these phototoxic effects, 14+ was photoexcited through TPA at a power of 60 mW, which was progressively focused in 10 μm steps throughout the entire z-axis of individual spheroids. These experiments revealed that, in irradiated spheroids treated with 14+, acute and rapid photoinduced cell death was observed throughout their depth, including the hypoxic region.

Cellular uptake, luminescence imaging and antimicrobial activity against clinically relevant methicillin-resistant S. aureus (MRSA) bacteria are reported. The osmium(ii) complexes [Os(N^N)3]2+ (N^N = 1-benzyl-4-(pyrid-2-yl)-1,2,3-triazole (12+); 1-benzyl-4-(pyrimidin-2-yl)-1,2,3-triazole (22+); 1-benzyl-4-(pyrazin-2-yl)-1,2,3-triazole (32+)) were prepared and isolated as the chloride salts of their meridional and facial isomers. The complexes display prominent spin-forbidden ground state to triplet metal-to-ligand charge transfer (3MLCT) state absorption bands enabling excitation as low as 600 nm for fac/mer-32+ and observation of emission in aqueous solution in the deep-red/near-IR regions of the spectrum. Cellular uptake studies within MRSA cells show antimicrobial activity for 12+ and 22+ with greater toxicity for the meridional isomers in each case and mer-12+ showing the greatest potency (32 μg mL−1 in defined minimal media). Super-resolution imaging experiments demonstrate binding of mer- and fac-12+ to bacterial DNA with high Pearson's colocalisation coefficients (up to 0.95 using DAPI). Phototoxicity studies showed the complexes exhibited a higher antimicrobial activity upon irradiation with light., Cellular uptake, luminescence imaging and antimicrobial activity of facial and meridional isomers of Os(ii) triazole-based complexes against methicillin-resistant S. aureus, MRSA.

The synthesis of new dinuclear complexes containing linked RuII(dppz) and ReI(dppz) moieties is reported. The photophysical and biological properties of the new complex, which incorporates a N,N'-bis(4-pyridylmethyl)-1,6-hexanediamine tether ligand, are compared to a previously reported RuII/ReI complex linked by a simple dipyridyl alkane ligand. Although both complexes bind to DNA with similar affinities, steady-state and time-resolved photophysical studies reveal that the nature of the linker affects the excited state dynamics of the complexes and their DNA photocleavage properties. Quantum-based DFT calculations on these systems offer insights into these effects. While both complexes are live cells permeant, their intracellular localizations are significantly affected by the nature of the linker. Notably, one of the complexes displayed concentration-dependent localization and possesses photophysical properties that are compatible with SIM and STED nanoscopy. This allowed the dynamics of its intracellular localization to be tracked at super resolutions.

With the aim of developing photostable near-infrared cell imaging probes, a convenient route to the synthesis of heteroleptic OsII complexes containing the Os(TAP)2 fragment is reported. This method was used to synthesize the dinuclear OsII complex, [{Os(TAP)2}2tpphz]4+ (where tpphz = tetrapyrido[3,2-a:2',3'-c:3″,2''-h:2‴,3'''-j]phenazine and TAP = 1,4,5,8- tetraazaphenanthrene). Using a combination of resonance Raman and time-resolved absorption spectroscopy, as well as computational studies, the excited state dynamics of the new complex were dissected. These studies revealed that, although the complex has several close lying excited states, its near-infrared, NIR, emission (λmax = 780 nm) is due to a low-lying Os → TAP based 3MCLT state. Cell-based studies revealed that unlike its RuII analogue, the new complex is neither cytotoxic nor photocytotoxic. However, as it is highly photostable as well as live-cell permeant and displays NIR luminescence within the biological optical window, its properties make it an ideal probe for optical microscopy, demonstrated by its use as a super-resolution NIR STED probe for nuclear DNA.

With the aim of developing a sensitizer for photodynamic therapy, a previously reported luminescent dinuclear complex that functions as a DNA probe in live cells was modified to produce a new iso-structural derivative containing RuII(TAP)2 fragments (TAP = 1,4,5,8-tetraazaphenanthrene). The structure of the new complex has been confirmed by a variety of techniques including single crystal X-ray analysis. Unlike its parent, the new complex displays Ru → L-based 3MLCT emission in both MeCN and water. Results from electrochemical studies and emission quenching experiments involving guanosine monophosphate are consistent with an excited state located on a TAP moiety. This hypothesis is further supported by detailed DFT calculations, which take into account solvent effects on excited state dynamics. Cell-free steady-state and time-resolved optical studies on the interaction of the new complex with duplex and quadruplex DNA show that the complex binds with high affinity to both structures and indicate that its photoexcited state is also quenched by DNA, a process that is accompanied by the generation of the guanine radical cation sites as photo-oxidization products. Like the parent complex, this new compound is taken up by live cells where it primarily localizes within the nucleus and displays low cytotoxicity in the absence of light. However, in complete contrast to [{RuII(phen)2}2(tpphz)]4+, the new complex is therapeutically activated by light to become highly phototoxic toward malignant human melanoma cell lines showing that it is a promising lead for the treatment of this recalcitrant cancer.

Nucleoside polyphosphates (NPPs) are mainly produced in mitochondria and used as a universal energy source for various cellular events. Although numerous fluorescent probes for adenosine triphosphate (ATP) have been reported, they are not ideally suited for live monitoring of the subtle variation of the mitochondrial ATP level. A new coumarin-based fluorescent probe is synthesized, and this reagent is utilized for specific recognition of NPPs in mitochondria by super-resolution microscopy in physiological condition. Detailed 31P NMR studies reveal that the probe, L·Zn(II), binds to NPPs through their pendent phosphate functionalities. Such binding leads to a substantial enhancement in the luminescence intensity of L·Zn(II) + ADP or L·Zn(II) + ATP as compared to L·Zn(II). This—as well as 1H NMR spectroscopy—has enabled us to evaluate the probe’s binding affinities to these NPPs. Structured illumination and wide field fluorescence microscopy confirmed that this physiologically benign reagent is localized wi...

[Image: see text] Increasing concern over rising levels of antibiotic resistance among pathogenic bacteria has prompted significant research into developing efficacious alternatives to antibiotic treatment. Previously, we have reported on the therapeutic activity of a dinuclear ruthenium(II) complex against pathogenic, multi-drug-resistant bacterial pathogens. Herein, we report that the solubility properties of this lead are comparable to those exhibited by orally available therapeutics that in comparison to clinically relevant antibiotics it induces very slow evolution of resistance in the uropathogenic, therapeutically resistant, E. coli strain EC958, and this resistance was lost when exposure to the compound was temporarily removed. With the aim of further investigating the mechanism of action of this compound, the regulation of nine target genes relating to the membrane, DNA damage, and other stress responses provoked by exposure to the compound was also studied. This analysis confirmed that the compound causes a significant transcriptional downregulation of genes involved in membrane transport and the tricarboxylic acid cycle. By contrast, expression of the chaperone protein-coding gene, spy, was significantly increased suggesting a requirement for repair of damaged proteins in the region of the outer membrane. The complex was also found to display activity comparable to that in E. coli in a range of other therapeutically relevant Gram-negative pathogens.

Six luminescent, mononuclear ruthenium(ii) complexes based on the tetrapyridophenazine (tpphz) and dipyridophenazine (dppz) ligands are reported. The therapeutic activities of the complexes against Gram-negative bacteria (E. coli, A. baumannii, P. aeruginosa) and Gram-positive bacteria (E. faecalis and S. aureus) including pathogenic multi- and pan-drug resistant strains were assessed. Estimated minimum inhibitory and bactericidal concentrations show the activity of the lead compound is comparable to ampicillin and oxacillin in therapeutically sensitive strains and this activity was retained in resistant strains. Unlike related dinuclear analogues the lead compound does not damage bacterial membranes but is still rapidly taken up by both Gram-positive and Gram-negative bacteria in a glucose independent manner. Direct imaging of the complexes through super-resolution nanoscopy and transmission electron microscopy reveals that once internalized the complexes' intracellular target for both Gram-negative and Gram-positive strains is bacterial DNA. Model toxicity screens showed the compound is non-toxic to Galleria mellonella even at exposure concentrations that are orders of magnitude higher than the bacterial MIC., A mononuclear ruthenium(ii) complex based of the tpphz ligand is shown to be a broad-band antimicrobial theranostic active against a range of AMR pathogens.

Theranostic radionuclides that emit Auger electrons (AE) can generate highly localised DNA damage and the accompanying gamma ray emission can be used for single-photon emission computed tomography (SPECT) imaging. Mismatched DNA base pairs (mismatches) are DNA lesions that are abundant in cells deficient in MMR (mismatch mediated repair) proteins. This form of genetic instability is prevalent in the MMR-deficient subset of colorectal cancers and is a potential target for AE radiotherapeutics. Herein we report the synthesis of a mismatch DNA binding bis-ruthenium(II) dipyridophenazine (dppz) complex that can be radiolabelled with the Auger electron emitting radionuclide indium-111 (111In). Greater stabilisation accompanied by enhanced MLCT (metal to ligand charge-transfer) luminescence of both the bis-Ru(dppz) chelator and non-radioactive indium-loaded complex was observed in the presence of a TT mismatch-containing duplex compared to matched DNA. The radioactive construct [111In]In-bisRu(dppz) ([111In][In-2]4+) targets cell nuclei and is radiotoxic towards MMR-deficient human colorectal cancer cells showing substantially less detrimental effects in a paired cell line with restored MMR function. Additional cell line studies revealed that [111In][In-2]4+ is preferentially radiotoxic towards MMR-deficient colorectal cancer cells accompanied by increased DNA damage due to 111In decay. The biodistribution of [111In][In-2]4+ in live mice was demonstrated using SPECT. These results illustrate how a Ru(II) polypyridyl complex can incorporate mismatch DNA binding and radiometal chelation in a single molecule, generating a DNA-targeting AE radiopharmaceutical that displays selective radiotoxicity towards MMR-deficient cancer cells and is compatible with whole organism SPECT imaging.

Mitochondria act as a prime therapeutic target for the treatment of various diseases and dysfunctional states. In this study, we have demonstrated the proof of concept of surface functionalization for achieving two-photon active mitochondriotropic lanthanide nanorods for higher resolution fluorescence imaging, and thereby, the spectral profile of mitochondria. The presence of Gd in the nanorods also enabled us to utilize this material as a T(1)-T(2) dual-mode contrast reagent for recording magnetic resonance images of the mouse brain.

Auger electron emitter indium-111 demonstrates cancer-selective radiotoxicity and SPECT imaging compatibility when conjugated to a ruthenium(ii) polypyridyl complex., Theranostic radionuclides that emit Auger electrons (AE) can generate highly localised DNA damage and the accompanying gamma ray emission can be used for single-photon emission computed tomography (SPECT) imaging. Mismatched DNA base pairs (mismatches) are DNA lesions that are abundant in cells deficient in MMR (mismatch mediated repair) proteins. This form of genetic instability is prevalent in the MMR-deficient subset of colorectal cancers and is a potential target for AE radiotherapeutics. Herein we report the synthesis of a mismatch DNA binding bis-ruthenium(ii) dipyridophenazine (dppz) complex that can be radiolabelled with the Auger electron emitting radionuclide indium-111 (111In). Greater stabilisation accompanied by enhanced MLCT (metal to ligand charge-transfer) luminescence of both the bis-Ru(dppz) chelator and non-radioactive indium-loaded complex was observed in the presence of a TT mismatch-containing duplex compared to matched DNA. The radioactive construct [111In]In-bisRu(dppz) ([111In][In-2]4+) targets cell nuclei and is radiotoxic towards MMR-deficient human colorectal cancer cells showing substantially less detrimental effects in a paired cell line with restored MMR function. Additional cell line studies revealed that [111In][In-2]4+ is preferentially radiotoxic towards MMR-deficient colorectal cancer cells accompanied by increased DNA damage due to 111In decay. The biodistribution of [111In][In-2]4+ in live mice was demonstrated using SPECT. These results illustrate how a Ru(ii) polypyridyl complex can incorporate mismatch DNA binding and radiometal chelation in a single molecule, generating a DNA-targeting AE radiopharmaceutical that displays selective radiotoxicity towards MMR-deficient cancer cells and is compatible with whole organism SPECT imaging.

The synthesis of new dinuclear complexes containing linked Ru

In previous studies we reported that specific dinuclear RuII complexes are particularly active against pathogenic Gram-negative bacteria and, unusually for this class of compounds, appeared to display lowered activity against Gram-positive bacteria. With the aim of identifying resistance mechanisms specific to Gram-positive bacteria, the uptake and antimicrobial activity of the lead complex against Staphylococcus aureus SH1000 and other isolates, including MRSA was investigated. This revealed differential, strain specific, sensitivity to the complex. Exploiting the inherent luminescent properties of the RuII complex, super-resolution STED nanoscopy was used to image its initial interaction with S. aureus and confirm its cellular internalization. Membrane damage assays and transmission electron microscopy confirm that the complex disrupts the bacterial membrane structure before internalization, which ultimately results in a small amount of DNA damage. A known resistance mechanism against cationic antimicrobials in Gram-positive bacteria involves increased expression of the mprF gene as this results in an accumulation of positively charged lysyl-phosphatidylglycerol on the outer leaflet of the cytoplasmic membrane that electrostatically repel cationic species. Consistent with this model, it was found that an mprF deficient strain was particularly susceptible to treatment with the lead complex. More detailed co-staining studies also revealed that the complex was more active in S. aureus strains missing, or with altered, wall teichoic acids.

BODIPY derivative, SF-1, exclusively shows a fluorescence ON response to HOCl and images endogenously generated HOCl in RAW 264.7 macrophages. Widefield and super resolution structured illumination microscopy images confirm localization in the Golgi complex and lysosomes, and hence specifically detects HOCl generated in these organelles. SF-1 is compatible with 3D-SIM imaging of individual cells.

Selective detection of nitroxyl (HNO), which has recently been identified as a reactive nitrogen species, is a challenging task. We report a BODIPY-based luminescence ON reagent for detection of HNO in aqueous solution and in live RAW 264.7 cells, based on the soft nucleophilicity of the phosphine oxide functionality toward HNO. The probe shows high selectivity to HNO over other reactive oxygen/nitrogen and sulfur species. Luminescence properties of the BODIPY-based chemodosimetric reagent make it an ideal candidate for use as a reagent for super-resolution structured illumination microscopy. The viability of the reagent for biological in vivo imaging application was also confirmed using Artemia as a model.

With the long-term aim of enhancing the binding properties of dinuclear RuII-based DNA light-switch complexes, a series of eight structurally related mono- and dinuclear systems are reported in which the linker of the bridging ligand has been modulated. These tethered systems have been designed to explore issues of steric demand at the binding site and the thermodynamic cost of entropy loss upon binding. An analysis of detailed spectroscopic and isothermal titration calorimetry (ITC) studies on the new complexes reveal that one of the linkers produces a dinuclear systems that binds to duplex DNA with an affinity (Kb > 107 M-1) that is higher than its corresponding monometallic complex and is the highest affinity for a non-threading bis-intercalating metal complex. These studies confirm that the tether has a major effect on the binding properties of dinuclear complexes containing nintercalating units and establishes key design rules for the construction of dinuclear complexes with enhanced

The preparation, spectroscopic characterization, structural study and magnetic investigation of three new complexes of formula [Co(bpcam)2]ClO4·dmso·H2O (1), [Co(bpcam)2]2[Co(NCS)4]·dmso·H2O (2) and [Ni(bpcam)2]·H2O (3) [Hbpcam = bis(2-pyrimidylcarbonyl)amide] are reported. Each bpcam group in 1–3 acts as a tridentate ligand being coordinated to the cobalt(III) (1 and 2)/nickel(II) (3) ions through three nitrogen atoms in a mer-arrangement. Six-coordinate cobalt(III) and nickel(II) occur in 1 and 3 respectively, whereas six-coordinate cobalt(III) and four-coordinate cobalt(II) coexist in 2. Cyclic voltammetry of 1 in acetonitrile shows the occurrence of one quasi reversible CoIII ↔ CoII process. Direct current (dc) variable-temperature magnetic susceptibility measurements on polycrystalline samples of 2 and 3 were carried out in the temperature range 1.9–295 K. The magnetic behaviour of 2 obeys to the zero-field splitting effects (DCo) of the 4A2 ground term of the pseudotetrahedral [Co(NCS)4]2− complex anion. A Curie law for a magnetically isolated nickel(II) ion in the high temperatures domain occurs for 3, the small decrease of χMT below 10 K being due zero-field splitting effects. The analysis of the magnetic susceptibility data of 2 and 3 through the spin Hamiltonian H = DM[Sz2 − S(S + 1)/3] + gMβHS [M = Co(2) and Ni(3)] led to the following set of values: gM = 2.37 (2) and 2.12 (3) and |DM| = 34.7 (2) and 1.72 cm−1 (3).

β-NaYF4:Yb,Gd up-conversion nanoparticles, UCNPs, surface functionalized with suitable targetting peptides function as nontoxic lysosome-specific imaging probes.\ud \ud

Medicinal leads that are also compatible with imaging technologies are attractive, as they facilitate the development of therapeutics through direct mechanistic observations at the molecular level. In this context, the uptake and antimicrobial activities of several luminescent dinuclear RuII complexes against E. coli were assessed and compared to results obtained for another ESKAPE pathogen, the Gram-positive major opportunistic pathogen Enterococcus faecalis, V583. The most promising lead displays potent activity, particularly against the Gram-negative bacteria, and potency is retained in the uropathogenic multidrug resistant EC958 ST131 strain. Exploiting the inherent luminescent properties of this complex, super-resolution STED nanoscopy was used to image its initial localization at/in cellular membranes and its subsequent transfer to the cell poles. Membrane damage assays confirm that the complex disrupts the bacterial membrane structure before internalization. Mammalian cell culture and animal model studies indicate that the complex is not toxic to eukaryotes, even at concentrations that are several orders of magnitude higher than its minimum inhibitory concentration (MIC). Taken together, these results have identified a lead molecular architecture for hard-to-treat, multiresistant, Gram-negative bacteria, which displays activities that are already comparable to optimized natural product-based leads.

Herein we report the separation of the three stereoisomers of the DNA light-switch compound [{Ru(bpy)2}2(tpphz)]4+ (tpphz = tetrapyrido[3,2-a:2',3'-c:3″,2″-h:2‴,3‴-j]phenazine) by column chromatography and the characterization of each stereoisomer by X-ray crystallography. The interaction of these compounds with a DNA octanucleotide d(GCATATCG).d(CGATATGC) has been studied using NMR techniques. Selective deuteration of the bipyridyl rings was needed to provide sufficient spectral resolution to characterize structures. NMR-derived structures for these complexes show a threading intercalation binding mode with slow and chirality-dependent rates. This represents the first solution structure of an intercalated bis-ruthenium ligand. Intriguingly, we find that the binding site selectivity is dependent on the nature of the stereoisomer employed, with Λ RuII centers showing a better intercalation fit.

Drug resistance to platinum chemotherapeutics targeting DNA often involves abrogation of apoptosis and has emerged as a significant challenge in modern, non-targeted chemotherapy. Consequently, there is great interest in the anti-cancer properties of metal complexes-particularly those that interact with DNA-and mechanisms of consequent cell death. Herein we compare a parent cytotoxic complex, [Ru(phen)2(tpphz)]2+ [phen = 1,10-phenanthroline, tpphz = tetrapyridyl[3,2- a:2',3'- c:3″,2″- h:2‴,3‴- j]phenazine], with a mononuclear analogue with a modified intercalating ligand, [Ru(phen)2(taptp)]2+ [taptp = 4,5,9,18-tetraazaphenanthreno[9,10- b] triphenylene], and two structurally related dinuclear, tpphz-bridged, heterometallic complexes, RuRe and RuPt. All three of these structural changes result in a switch from intercalation to groove-binding DNA interaction and concomitant reduction in cytotoxic potency, but no significant change in relative cytotoxicity toward platinum-resistant A2780CIS cancer cells, indicating that the DNA interaction mode is not critical for the mechanism of platinum resistance. All variants exhibited a light-switch effect, which for the first time was exploited to investigate timing of cell death by live-cell microscopy. Surprisingly, cell death occurred rapidly as a consequence of oncosis, characterized by loss of cytoplasmic volume control, absence of significant mitochondrial membrane potential loss, and lack of activation of apoptotic cell death markers. Importantly, a novel, quantitative proteomic analysis of the A2780 cell genome following exposure of the cells to either mononuclear complex reveals changes in protein expression associated with global cell responses to oxidative stress and DNA replication/repair cellular pathways. This combination of multiple targeting modalities and induction of a non-apoptotic death mechanism makes these complexes highly promising chemotherapeutic cytotoxicity leads.

Two-photon active Graphene Quantum Dots (GQDs) are obtained from extracts of the neem root. These biocompatible GQDs are found to be suitable for structured illumination microscopy. Two-photon microscopy ensured lysosome specificity of GQDs in live cells and confocal luminescence microscopic studies showed tissue-dependent localization of GQDs in Zebrafish

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.

With the aim of developing a sensitizer for photodynamic therapy, a previously reported luminescent dinuclear complex that functions as a DNA probe in live cells was modified to produce a new iso-structural derivative containing Ru

The synthesis of two new tetracationic mononuclear RuII complexes containing the tetrapyridyl [3,2-a:2',3'-c:3'',2''-h:2''',3'''-j] phenazine ligand in which the uncoordinated site has been converted into a dicationic ethylene-bipyridyldiylium unit is reported. The structure of the complexes is fully assigned through detailed NMR studies and, in one case, through an X-ray crystallography study. Voltammetry, optical spectroscopy and computational studies confirm that the bipyridyldiylium moiety has a low-lying reduction that quenches the 3MLCT-based emission usually observed in such systems. The new complexes interact with DNA in a quite different manner to their dicationic analogues: they both bind to duplex DNA with micromolar affinity through groove binding. These observations are rationalized through a consideration of their structural and electronic properties.

Surface engineering of nanocarriers allows fine-tuning of their interactions with biological organisms, potentially forming the basis of devices for the monitoring of intracellular events or for intracellular drug delivery. In this context, biodegradable nanocarriers or nanocapsules capable of carrying bioactive molecules or drugs into the mitochondrial matrix could offer new capabilities in treating mitochondrial diseases. Nanocapsules with a polymeric backbone that undergoes programmed rupture in response to a specific chemical or enzymatic stimulus with subsequent release of the bioactive molecule or drug at mitochondria would be particularly attractive for this function. With this goal in mind, we have developed biologically benign nanocapsules using polyurethane-based, polymeric backbone that incorporates repetitive ester functionalities. The resulting nanocapsules are found to be highly stable and monodispersed in size. Importantly, a new non-isocyanate route is adapted for the synthesis of these non-isocyanate polyurethane nanocapsules (NIPU). The embedded ester linkages of these capsules' shells have facilitated complete degradation of the polymeric backbone in response to a stimulus provided by an esterase enzyme. Hydrophilic payloads like rhodamine or doxorubicin can be loaded inside these nanocarriers during their synthesis by an interfacial polymerization reaction. The postgrafting of the nanocapsules with phosphonium ion, a mitochondria-targeting receptor functionality, has helped us achieve the site-specific release of the drug. Co-localization experiments with commercial mitotracker green as well as mitotracker deep red confirmed localization of the cargo in mitochondria. Our in vitro studies confirm that specific release of doxorubicin within mitochondria causes higher cytotoxicity and cell death compared to free doxorubicin. Endogenous enzyme triggered nanocapsule rupture and release of the encapsulated dye is also demonstrated in a zebrafish model. The results of this proof-of-concept study illustrate that NIPU nanocarriers can provide a site-specific delivery vehicle and improve the therapeutic efficacy of a drug or be used to produce organelle-specific imaging studies.

A new physiologically benign and cell membrane permeable BODIPY based molecular probe, MB-Sn, specifically senses intracellular hydrogen polysulfides (H2Sn, n > 1) localized in the endoplasmic reticulum. This reagent is suitable for mapping the intracellular distribution of H2Sn by wide-field as well as super-resolution Structured Illumination Microscopy (SIM).

[Ru(phen)2(tpphz)]2+ simultaneously inhibits DNA replication, blocks mitosis and enhances DNA-damaging ionising radiation in oesophageal cancer cells., Substitutionally inert ruthenium(ii) polypyridyl complexes have been developed as DNA intercalating agents yet cellular DNA damage responses to this binding modality are largely unexplored. Here, we show the nuclear-targeting complex [Ru(phen)2(tpphz)]2+ (phen = 1,10-phenanthroline, tpphz = tetrapyridophenazine) generates rapid and pronounced stalling of replication fork progression in p53-deficient human oesophageal cancer cells. In response, replication stress and double-strand break (DSB) DNA damage response (DDR) pathways are activated and cell proliferation is inhibited by growth arrest. Moreover, mitotic progression is compromised by [Ru(phen)2(tpphz)]2+, where the generation of metaphase chromosome spindle attachment failure results in spindle assembly checkpoint (SAC) activation. This dual mechanism of action results in preferential growth inhibition of rapidly-proliferating oesophageal cancer cells with elevated mitotic indices. In addition to these single-agent effects, [Ru(phen)2(tpphz)]2+ functions as a radiosensitizer with efficiency comparable to cisplatin, which occurs through a synergistic enhancement of DNA damage. These results establish that DNA replication is the target for [Ru(phen)2(tpphz)]2+ and provide the first experimental evidence that ruthenium-based intercalation targets multiple genome integrity pathways in cancer cells, thereby achieving enhanced selectivity compared to existing DNA-damaging agents such as cisplatin.

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 Sudlow's 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.

The synthesis of two new Ir(III) complexes which are effectively isostructural with well-established [Ru(NN)2(dppz)](2+) systems is reported (dppz=dipyridophenazine; NN=2,2'-bipyridyl, or 1,10-phenanthroline). One of these Ir(III) complexes is tricationic and has a conventional N6 coordination sphere. The second dicationic complex has a N5C coordination sphere, incorporating a cyclometalated analogue of the dppz ligand. Both complexes show good water solubility. Experimental and computational studies show that the photoexcited states of the two complexes are very different from each other and also differ from their Ru(II) analogues. Both of the complexes bind to duplex DNA with affinities that are two orders of magnitude higher than previously reported Ir(dppz)-based systems and are comparable with Ru(II)(dppz) analogues.

Using a new mononuclear “building block,” for the first time, a dinuclear RuII(dppn) complex and a heteroleptic system containing both RuII(dppz) and RuII(dppn) moieties are reported. The complexes, including the mixed dppz/dppn system, are 1O2 sensitizers. However, unlike the homoleptic dppn systems, the mixed dppz/dppn complex also displays a luminescence “switch on” DNA light-switch effect. In both cisplatin sensitive and resistant human ovarian carcinoma lines the dinuclear complexes show enhanced uptake compared to their mononuclear analogue. Thanks to a favorable combination of singlet oxygen generation and cellular uptake properties all three of the new complexes are phototoxic and display potent activity against chemotherapeutically resistant cells.

Using a new mononuclear "building block," for the first time, a dinuclear Ru

The synergistic effect of oxygen, light, and photosensitizer (PS) has found applications in medicine for the treatment of cancer through photodynamic therapy (PDT). Induction of apoptosis to cancerous cells will prevent tumor metastasis that spreads cancer cells to the neighboring organs/tissues. Herein, we report the two apoptotic Ru(II)–polypyridyl complexes that are functionalized with pendant amino acid moieties tyrosine (1) and tryptophan (2), respectively. These two water soluble complexes were found to interact strongly (K1a = (1.18 ± 0.28) × 105 M−1 and K2a = (1.57 ± 0.77) × 105 M−1) with CT-DNA. Isothermal titration calorimetry (ITC) studies revealed that these complexes bind to CT-DNA through an entropically driven process. Both the complexes showed photo-induced cytotoxicity and exhibit apoptotic activity under photo-irradiation conditions. The comet assay indicated that these complexes can damage cellular DNA, which is attributed to the significant build-up of 1O2 level even on irradiation with low intensity light (10 J cm−2, λRange 450–480 nm). This photoinduced DNA damage and apoptosis in A549 cells was induced by reactive oxygen species (ROS) and occurred through up-regulation of apoptotic marker caspase-3. Control experiments under dark conditions revealed an insignificant cytotoxicity towards these cells for two photosensitive molecules.

Herein we describe the DNA binding properties of two new water-soluble ruthenium complexes; experimental and computational data reveal that both complexes display dual emission from MLCT and LLCT excited states. The interaction of the new complexes with DNA was also investigated. Although one of the complexes only binds DNA though groove binding, the second complex has separate ligands capable of groove binding and intercalation. Nevertheless, it was found that both complexes interact with duplex DNA with high affinity. DNA induced distinctive changes in the emission of the complexes; although the groove binding complex only displays a modest increase in emission on binding, the complex that contains the intercalating RuII(dppz) moiety displays a large increase in MLCT-based emission on DNA binding while emission from the LLCT excited state is unaffected. This means that the complex functions as the first ratiometric sensor for DNA.

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.

Techniques concerning postdisaster assessment from remotely sensed images have been studied by different research communities in the past decade. Such an assessment benefits a range of stakeholders, e.g., government organizations, insurance industry, local communities, and individual homeowners. This work explores detailed damage assessment on an individual building basis by utilizing supervised classification. In contrast with previous research efforts in the field, this work attempts at predicting the type of damages such as missing tiles, collapsed rooftop, and presence of holes, gaps, or cavities. Various existing and novel intensity-, edge-, and color-based features are evaluated. Additionally, preprocessing steps that automatically correct photometric and geometric differences are proposed. Furthermore, a study on the reliability of high-resolution aerial imagery in damage interpretation is conducted by comparing results with the assessment of expert volunteers. Results show that the proposed damage detection framework is very effective and performs at a level similar to that of the experts. This paper concludes that the type and extent of damage to individual rooftops can be identified with good accuracy from high-resolution aerial images. It is envisaged that the automated tools presented in this paper would play a significant role in rapid posthurricane damage estimation and in helping to better manage rescue and recovery missions.

The interaction with duplex DNA of a small library of structurally related complexes that all contain a d6-metal ion coordinated to either the 2,2′:4,4′′:4′,4′′′-quaterpyridyl ligand or its methylated derivative are reported. This library is made up of a mixture of newly synthesised and previously reported systems. Despite their structural similarities the complexes display an almost 20-fold variation in binding affinities. Although effects due to the overall charge of the complexes are apparent, the differences in binding characteristics are deeper than this; indeed, in a number of cases, changes in overall charge have little effect on binding affinity. Intriguingly, despite interacting with DNA through unfused ring systems, although two of the complexes studied are groove binders, the majority are non-classical intercalators. A rationale for these effects has been obtained through a combination of experimental and computational studies.

This review outlines the synthesis and properties of mono and oligonuclear polypyridyl ruthenium complexes incorporating a range of S 3 and S 4 thiacrown ligands, focusing on the mixed valence complexes that have been produced in these studies. This work has revealed that the chemical, electrochemical, and electronic properties of the metal centers are modulated by the nature of the coordinated thiacrown ligand. In particular the back-bonding properties of the ligands mean that they strongly stabilize the ruthenium(II) state and also produce metal centers that are relatively kinetically labile. These distinctive electronic properties have been exploited in the construction of mixed valence systems with unusual electrochemical properties and also facilitated the self-assembly of oligonuclear molecular architectures with multiple accessible oxidation states. Computational studies on the isovalent and mixed valence complexes reflect the experimental data by indicating that, within a series of S 4 coordinated dinuclear complexes, the properties of the mixed valence state are subtly dependent on the cavity size of the coordinated thiacrown.

Metal-ion-directed self-assembly has been used to construct kinetically inert, water-soluble heterometallic Ru2Re2 hosts that are potential sensors for bioanions. A previously reported metallomacrocycle and a new derivative synthesised by this approach are found to be general sensors for bioanions in water, showing an "off-on" luminescent change that is selective for nucleotides over uncharged nucleobases. Through a change in the ancillary ligands coordinated to the ruthenium centres of the host, an "off-on" sensor has been produced. Whilst this host only shows a modest enhancement in binding affinities for nucleotides relative to the other two host systems, its sensing response is much more specific. Although a distinctive "off-on" luminescence response is observed for the addition of adenosine triphosphosphate (ATP), related structures such as adenine and guanosine triphosphate (GTP) do not induce any emission change in the host. Detailed and demanding DFT studies on the ATP- and GTP-bound host-guest complexes reveal subtle differences in their geometries that modulate the stacking interactions between the nucleotide guests and the ancillary ligands of the host. It is suggested that this change in stacking geometries affects solvent accessibility to the binding pocket of the host and thus leads to observed difference in the host luminescence response to the guests.