Metallohelices emulate the properties of short cationic α-helical peptides.

Naturally occurring peptides in many living systems perform antimicrobial and anticancer host defence roles, but their potential for clinical application is limited by low metabolic stability and relatively high costs of goods. Self-assembled helical metal complexes provide an attractive synthetic platform for non-peptidic architectures that can emulate some of the properties of short cationic α-helical peptides, with tuneable charge, shape, size and amphipathicity. Correspondingly there is a growing body of evidence demonstrating that these supramolecular architectures exhibit bioactivity that emulates that of the natural systems. We review that evidence in the context of synthetic advances in the area, driven by the potential for biomedical applications. We note some design considerations for new biologically-relevant metallohelices, and give our outlook on the future of these compounds as therapeutic peptidomimetics.

Glycoconjugated Metallohelices have Improved Nuclear Delivery and Suppress Tumour Growth In Vivo.

Monosaccharides are added to the hydrophilic face of a self-assembled asymmetric FeII metallohelix, using CuAAC chemistry. The sixteen resulting architectures are water-stable and optically pure, and exhibit improved antiproliferative selectivity against colon cancer cells (HCT116 p53+/+ ) with respect to the non-cancerous ARPE-19 cell line. While the most selective compound is a glucose-appended enantiomer, its cellular entry is not mainly glucose transporter-mediated. Glucose conjugation nevertheless increases nuclear delivery ca 2.5-fold, and a non-destructive interaction with DNA is indicated. Addition of the glucose units affects the binding orientation of the metallohelix to naked DNA, but does not substantially alter the overall affinity. In a mouse model, the glucose conjugated compound was far better tolerated, and tumour growth delays for the parent compound (2.6 d) were improved to 4.3 d; performance as good as cisplatin but with the advantage of no weight loss in the subjects.

Triazole-based, optically-pure metallosupramolecules; highly potent and selective anticancer compounds.

Functionalised triazole aldehydes are used in the highly selective self-assembly of water-compatible, optically pure, low symmetry Fe(ii)- and Zn(ii)-based metallohelices. Sub-micromolar antiproliferative activity is observed against various cancerous cell lines, accompanied by excellent selectivity versus non-cancerous cells and potential for synergistic combinatorial therapy with cisplatin.

Discovery of selective, antimetastatic and anti-cancer stem cell metallohelices via post-assembly modification.

Helicates and related metallofoldamers, synthesised by dynamic self-assembly, represent an area of chemical space inaccessible by traditional organic synthesis, and yet with potential for discovery of new classes of drug. Here we report that water-soluble, optically pure Fe(ii)- and even Zn(ii)-based triplex metallohelices are an excellent platform for post-assembly click reactions. By these means, the in vitro anticancer activity and most importantly the selectivity of a triplex metallohelix Fe(ii) system are dramatically improved. For one compound, a remarkable array of mechanistic and pharmacological behaviours is discovered: inhibition of Na+/K+ ATPase with potency comparable to the drug ouabain, antimetastatic properties (including inhibition of cell migration, re-adhesion and invasion), cancer stem cell targeting, and finally colonosphere inhibition competitive with the drug salinomycin.

Metallohelices that kill Gram-negative pathogens using intracellular antimicrobial peptide pathways.

A range of new water-compatible optically pure metallohelices - made by self-assembly of simple non-peptidic organic components around Fe ions - exhibit similar architecture to some natural cationic antimicrobial peptides (CAMPs) and are found to have high, structure-dependent activity against bacteria, including clinically problematic Gram-negative pathogens. A key compound is shown to freely enter rapidly dividing E. coli cells without significant membrane disruption, and localise in distinct foci near the poles. Several related observations of CAMP-like mechanisms are made via biophysical measurements, whole genome sequencing of tolerance mutants and transcriptomic analysis. These include: high selectivity for binding of G-quadruplex DNA over double stranded DNA; inhibition of both DNA gyrase and topoisomerase I in vitro; curing of a plasmid that contributes to the very high virulence of the E. coli strain used; activation of various two-component sensor/regulator and acid response pathways; and subsequent attempts by the cell to lower the net negative charge of the surface. This impact of the compound on multiple structures and pathways corresponds with our inability to isolate fully resistant mutant strains, and supports the idea that CAMP-inspired chemical scaffolds are a realistic approach for antimicrobial drug discovery, without the practical barriers to development that are associated with natural CAMPS.

Estrogenic Mechanisms and Cardiac Responses Following Early Life Exposure to Bisphenol A (BPA) and Its Metabolite 4-Methyl-2,4-bis( p-hydroxyphenyl)pent-1-ene (MBP) in Zebrafish.

Environmental exposure to Bisphenol A (BPA) has been associated with a range of adverse health effects, including on the cardiovascular system in humans. Lack of agreement on its mechanism(s) of action likely stem from comparisons between in vivo and in vitro test systems and potential multiple effects pathways. In rodents, in vivo, metabolic activation of BPA produces 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), which is reported to be up to 1000 times more potent as an estrogen than BPA. We investigated the estrogenic effects and estrogen receptor signaling pathway(s) of BPA and MBP following early life exposure using a transgenic, estrogen responsive (ERE-TG) zebrafish and a targeted morpholino approach to knockdown the three fish estrogen receptor (ER) subtypes. The functional consequences of BPA exposure on the cardiovascular system of zebrafish larvae were also examined. The heart atrioventricular valves and the bulbus arteriosus were primary target tissues for both BPA and MBP in the ERE-TG zebrafish, and MBP was approximately 1000-fold more potent than BPA as an estrogen in these tissues. Estrogen receptor knockdown with morpholinos indicated that the estrogenic responses in the heart for both BPA and MBP were mediated via an estrogen receptor 1 (esr1) dependent pathway. At the highest BPA concentration tested (2500 µg/L), alterations in the atrial:ventricular beat ratio indicated a functional impact on the heart of 5 days post fertilization (dpf) larvae, and there was also a significantly reduced heart rate in these larvae at 14 dpf. Our findings indicate that some of the reported adverse effects on heart function associated with BPA exposure (in mammals) may act through an estrogenic mechanism, but that fish are unlikely to be susceptible to adverse effects on heart development for environmentally relevant exposures.

Tailoring iridium luminescence and gold nanoparticle size for imaging of microvascular blood flow.

AIM: Imaging of blood flow in narrow channels and close to vessel walls is important in cardiovascular research for understanding pathogenesis. Our aim was to provide novel nanoprobes with visible emission and long lifetimes as trackers of flow. MATERIALS & METHODS: Gold nanoparticles coated with an iridium complex were prepared. Luminescence imaging was used to monitor their flows in different hematocrit blood and in murine tissues. RESULTS: The velocities are independent of hematocrit level and the nanoparticles entering blood circulation can be clearly detected in vessels in lungs, mesentery and the skeletal muscle. CONCLUSION: The work introduces for the first time iridium-based yellow-green luminescence with nanoparticle size of 100 nm for visualizing and monitoring flows with much higher resolution than conventional alternatives.

Simultaneous Triple Imaging with Two PARASHIFT Probes: Encoding Anatomical, pH and Temperature Information using Magnetic Resonance Shift Imaging.

The chemical shift of paramagnetically shifted resonances in lanthanide(III) complexes encodes information about temperature and has also been made to report pH in parallel, through introduction of a single phosphonate group adjacent to the reporter tert-butyl resonance. The enhanced sensitivity of this new probe has allowed the simultaneous triple imaging of the water signal and the shifted tert-butyl signals of thulium and dysprosium complexes of a common ligand, separated by over 160 ppm. In parallel spectral imaging experiments, the temperature and pH dependence of the frequency of the Tm and Dy signals has been deconvoluted, allowing the pH and temperature in the liver, kidney and bladder to be measured.

PARASHIFT Probes: Solution NMR and X-ray Structural Studies of Macrocyclic Ytterbium and Yttrium Complexes.

Ytterbium and yttrium complexes of octadentate ligands based on 1,4,7,10-tetraazacyclododecane with a coordinated pyridyl group and either tricarboxylate (L1) or triphosphinate (L2) donors form twisted-square-antiprismatic structures. The former crystallizes in the centrosymmetric group P21/c, with the two molecules related by an inversion center, whereas the latter was found as an unusual kryptoracemate in the chiral space group P21. Pure shift NMR and EXSY spectroscopy allowed the dynamic exchange between the (RRR)-Δ-(δδδδ) and (RRR)-Λ-(λλλλ) TSAP diastereomers of the [Y.L2] complex to be detected. The rate-limiting step in the exchange between Δ and Λ isomers involves cooperative ligand arm rotation, which is much faster for [Ln.L1] than for [Ln.L2]. Detailed analysis of NOESY, COSY, HSQC, and HMBC spectra confirms that the major conformer in solution is (RRR)-Λ-(λλλλ), consistent with crystal structure analysis and DFT calculations. The magnetic susceptibility tensors for [Yb.L1] and [Yb.L2], obtained from a full pseudocontact chemical shift analysis, are very different, in agreement with a CASSCF calculation. The remarkably different pseudocontact shift behavior is explained by the change in the orientation of the pseudocontact shift field, as defined by the Euler angles of the susceptibility tensor.

A new paramagnetically shifted imaging probe for MRI.

PURPOSE: To develop and characterize a new paramagnetic contrast agent for molecular imaging by MRI. METHODS: A contrast agent was developed for direct MRI detection through the paramagnetically shifted proton magnetic resonances of two chemically equivalent tert-butyl reporter groups within a dysprosium(III) complex. The complex was characterized in phantoms and imaged in physiologically intact mice at 7 Tesla (T) using three-dimensional (3D) gradient echo and spectroscopic imaging (MRSI) sequences to measure spatial distribution and signal frequency. RESULTS: The reporter protons reside ∼6.5 Å from the paramagnetic center, resulting in fast T1 relaxation (T1 = 8 ms) and a large paramagnetic frequency shift exceeding 60 ppm. Fast relaxation allowed short scan repetition times with high excitation flip angle, resulting in high sensitivity. The large dipolar shift allowed direct frequency selective excitation and acquisition of the dysprosium(III) complex, independent of the tissue water signal. The biokinetics of the complex were followed in vivo with a temporal resolution of 62 s following a single, low-dose intravenous injection. The lower concentration limit for detection was ∼23 µM. Through MRSI, the temperature dependence of the paramagnetic shift (0.28 ppm.K-1 ) was exploited to examine tissue temperature variation. CONCLUSIONS: These data demonstrate a new MRI agent with the potential for physiological monitoring by MRI. Magn Reson Med 77:1307-1317, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Molecular hydrogen and catalytic combustion in the production of hyperpolarized 83Kr and 129Xe MRI contrast agents.

Hyperpolarized (hp) (83)Kr is a promising MRI contrast agent for the diagnosis of pulmonary diseases affecting the surface of the respiratory zone. However, the distinct physical properties of (83)Kr that enable unique MRI contrast also complicate the production of hp (83)Kr. This work presents a previously unexplored approach in the generation of hp (83)Kr that can likewise be used for the production of hp (129)Xe. Molecular nitrogen, typically used as buffer gas in spin-exchange optical pumping (SEOP), was replaced by molecular hydrogen without penalty for the achievable hyperpolarization. In this particular study, the highest obtained nuclear spin polarizations were P =29% for(83)Kr and P= 63% for (129)Xe. The results were reproduced over many SEOP cycles despite the laser-induced on-resonance formation of rubidium hydride (RbH). Following SEOP, the H2 was reactively removed via catalytic combustion without measurable losses in hyperpolarized spin state of either (83)Kr or (129)Xe. Highly spin-polarized (83)Kr can now be purified for the first time, to our knowledge, to provide high signal intensity for the advancement of in vivo hp (83)Kr MRI. More generally, a chemical reaction appears as a viable alternative to the cryogenic separation process, the primary purification method of hp(129)Xe for the past 2 1/2 decades. The inherent simplicity of the combustion process will facilitate hp (129)Xe production and should allow for on-demand continuous flow of purified and highly spin-polarized (129)Xe.

Electromagnetic susceptibility anisotropy and its importance for paramagnetic NMR and optical spectroscopy in lanthanide coordination chemistry.

The importance of the directional dependence of magnetic susceptibility in magnetic resonance and of electric susceptibility in the optical spectroscopy of lanthanide coordination complexes is assessed. A body of more reliable shift, relaxation and optical emission data is emerging for well-defined isostructural series of complexes, allowing detailed comparative analyses to be undertaken. Such work is highlighting the limitations of the current NMR shift and relaxation theories, as well as emphasising the absence of a compelling theoretical framework to explain optical emission phenomena.

Another challenge to paramagnetic relaxation theory: a study of paramagnetic proton NMR relaxation in closely related series of pyridine-derivatised dysprosium complexes.

Measurements of the relaxation rate behaviour of two series of dysprosium complexes have been performed in solution, over the field range 1.0 to 16.5 Tesla. The field dependence has been modelled using Bloch-Redfield-Wangsness theory, allowing estimates of the electronic relaxation time, T1e, and the size of the magnetic susceptibility, µeff, to be made. Changes in relaxation rate of the order of 50% at higher fields were measured, following variation of the para-substituent in the single pyridine donor. The magnetic susceptibilities deviated unexpectedly from the free-ion values for certain derivatives in each series examined, in a manner that was independent of the electron-releasing/withdrawing ability of the pyridine substituent, suggesting that the polarisability of just one pyridine donor in octadenate ligands can play a significant role in defining the magnetic susceptibility anisotropy.

Challenging lanthanide relaxation theory: erbium and thulium complexes that show NMR relaxation rates faster than dysprosium and terbium analogues.

Measurements of the proton NMR paramagnetic relaxation rates for several series of isostructural lanthanide(III) complexes have been performed in aqueous solution over the field range 1.0 to 16.5 Tesla. The field dependence has been modeled using Bloch-Redfield-Wangsness theory, allowing values for the electronic relaxation time, Tle and the magnetic susceptibility, µeff, to be estimated. Anomalous relaxation rate profiles were obtained, notably for erbium and thulium complexes of low symmetry 8-coordinate aza-phosphinate complexes. Such behaviour challenges accepted theory and can be interpreted in terms of changes in Tle values that are a function of the transient ligand field induced by solvent collision and vary considerably between Ln(3+) ions, along with magnetic susceptibilities that deviate significantly from free-ion values.

Molecular Sensing with Hyperpolarized (129) Xe Using Switchable Chemical Exchange Relaxation Transfer.

An approach for hyperpolarized (129) Xe molecular sensors is explored using paramagnetic relaxation agents that can be deactivated upon chemical or enzymatic reaction with an analyte. Cryptophane encapsulated (129) Xe within the vicinity of the paramagnetic center experiences fast relaxation that, through chemical exchange of xenon atoms between cage and solvent pool, causes accelerated hyperpolarized (129) Xe signal decay in the dissolved phase. In this proof-of-concept work, the relaxivity of Gadolinium(III) -DOTA on (129) Xe in the solvent was increased eightfold through tethering of the paramagnetic molecule to a cryptophane cage. This potent relaxation agent can be 'turned off' specifically for (129) Xe through chemical reactions that spatially separate the Gd(III) centre from the attached cryptophane cage. Unlike (129) Xe chemical shift based sensors, the new concept does not require high spectral resolution and may lead to a new generation of responsive contrast agents for molecular MRI.

Critical analysis of the limitations of Bleaney's theory of magnetic anisotropy in paramagnetic lanthanide coordination complexes.

The origins of the breakdown of Bleaney's theory of magnetic anisotropy are described, based on an analysis of eleven different complexes of the second half of the 4f elements that form isostructural series. An examination of the chemical shift and relaxation rate behaviour of resonances located at least four bonds away from the paramagnetic centre was undertaken, and correlated to theoretical predictions. The key limitations relate to comparability of ligand field splitting with spin-orbit coupling, variation in the position of the principal magnetic axis between Ln complexes and the importance of multipolar terms in describing lanthanide ligand field interactions.

High coating of Ru(II) complexes on gold nanoparticles for single particle luminescence imaging in cells.

Gold nanoparticles are efficiently labelled with a luminescent ruthenium complex, producing 13 and 100 nm diameter, monodisperse red-emissive imaging probes with luminescence lifetimes prolonged over the molecular unit. Single, 100 nm particles are observed in whole cell luminescence imaging which reveals their biomolecular association with chromatin in the nucleus of cancer cells.

Silica nanoparticles for micro-particle imaging velocimetry: fluorosurfactant improves nanoparticle stability and brightness of immobilized iridium(III) complexes.

To establish highly luminescent nanoparticles for monitoring fluid flows, we examined the preparation of silica nanoparticles based on immobilization of a cyclometalated iridium(III) complex and an examination of the photophysical studies provided a good insight into the Ir(III) microenvironment in order to reveal the most suitable silica nanoparticles for micro particle imaging velocimetry (µ-PIV) studies. Iridium complexes covalently incorporated at the surface of preformed silica nanoparticles, [Ir-4]@Si500-Z, using a fluorinated polymer during their preparation, demonstrated better stability than those without the polymer, [Ir-4]@Si500, as well as an increase in steady state photoluminescence intensity (and therefore particle brightness) and lifetimes which are increased by 7-fold compared with nanoparticles with the same metal complex attached covalently throughout their core, [Ir-4]⊂Si500. Screening of the nanoparticles in fluid flows using epi-luminescence microscopy also confirm that the brightest, and therefore most suitable particles for microparticle imaging velocimetry (µ-PIV) measurements are those with the Ir(III) complex immobilized at the surface with fluorosurfactant, that is [Ir-4]@Si500-Z. µ-PIV studies demonstrate the suitability of these nanoparticles as nanotracers in microchannels.

The impact of size on the fate and toxicity of nanoparticulate silver in aquatic systems.

The increased use of silver nanomaterials presents a risk to aquatic systems due to the high toxicity of silver. The stability, dissolution rates and toxicity of citrate- and polyvinylpyrrolidone-coated silver nanoparticles (AgNPs) were investigated in synthetic freshwater and natural seawater media, with the effects of natural organic matter investigated in freshwater. When sterically stabilised by the large PVP molecules, AgNPs were more stable than when charge-stabilised using citrate, and were even relatively stable in seawater. In freshwater and seawater, citrate-coated AgNPs (Ag-Cit) had a faster rate of dissolution than PVP-coated AgNPs (Ag-PVP), while micron-sized silver exhibited the slowest dissolution rate. However, similar dissolved silver was measured for both AgNPs after 72h in freshwater (500-600µgL(-1)) and seawater (1300-1500µgL(-1)), with higher concentrations in seawater attributed to chloride complexation. When determined on a mass basis, the 72-h IC50 (inhibitory concentration giving 50% reduction in algal growth rate) for Pseudokirchneriella subcapitata and Phaeodactylum tricornutum and the 48-h LC50 for Ceriodaphnia dubia exposure to Ag(+) (1.1, 400 and 0.11µgL(-1), respectively), Ag-Cit (3.0, 2380 and 0.15µgL(-1), respectively) and Ag-PVP (19.5, 3690 and 2.0µgL(-1), respectively) varied widely, with toxicity in the order Ag(+)>Ag-Cit>Ag-PVP. Micron-sized silver treatments elicited much lower toxicity than ionic Ag(+) or AgNP to P. subcapitata. However, when related to the dissolved silver released from the nanoparticles the toxicities were similar to ionic silver treatments. The presence of natural organic matter stabilised the particles and reduced toxicity in freshwater. These results indicate that dissolved silver was responsible for the toxicity and highlight the need to account for matrix components such as chloride and organic matter in natural waters that influence AgNP fate and mitigate toxicity.

Single particle inductively coupled plasma-mass spectrometry: a performance evaluation and method comparison in the determination of nanoparticle size.

Sizing engineered nanoparticles in simple, laboratory systems is now a robust field of science; however, application of available techniques to more complex, natural systems is hindered by numerous challenges including low nanoparticle number concentrations, polydispersity from aggregation and/or dissolution, and interference from other incidental particulates. A new emerging technique, single particle inductively coupled plasma-mass spectrometry (spICPMS), has the potential to address many of these analytical challenges when sizing inorganic nanoparticles in environmental matrices. However, to date, there is little beyond the initial feasibility studies that investigates the performance characteristics and validation of spICPMS as a nanoparticle sizing technique. This study compares sizing of four silver nanoparticle dispersions (nominal diameters of 40, 60, 80, and 100 nm) by spICPMS to four established sizing techniques: dynamic light scattering, differential centrifugal sedimentation, nanoparticle tracking analysis, and TEM. Results show that spICPMS is able to size silver nanoparticles, across different sizes and particle number concentrations, with accuracy similar to the other commercially available techniques. Furthermore, a novel approach to evaluating particle coincidence is presented. In addition, spICPMS size measurements were successfully performed on nanoparticles suspended in algal growth media at low concentrations. Overall, while further development of the technique is needed, spICPMS yields important advantages over other techniques when sizing nanoparticles in environmentally relevant media.