Structural and Functional Diversity in Rigid Thiosemicarbazones with Extended Aromatic Frameworks: Microwave-Assisted Synthesis and Structural Investigations.

The long-standing interest in thiosemicarbazones (TSCs) has been largely driven by their potential toward theranostic applications including cellular imaging assays and multimodality imaging. We focus herein on the results of our new investigations into: (a) the structural chemistry of a family of rigid mono(thiosemicarbazone) ligands characterized by extended and aromatic backbones and (b) the formation of their corresponding thiosemicarbazonato Zn(II) and Cu(II) metal complexes. The synthesis of new ligands and their Zn(II) complexes was performed using a rapid, efficient and straightforward microwave-assisted method which superseded their preparation by conventional heating. We describe hereby new microwave irradiation protocols that are suitable for both imine bond formation reactions in the thiosemicabazone ligand synthesis and for Zn(II) metalation reactions. The new thiosemicarbazone ligands, denoted HL, mono(4-R-3-thiosemicarbazone)quinone, and their corresponding Zn(II) complexes, denoted ZnL2, mono(4-R-3-thiosemicarbazone)quinone, where R = H, Me, Ethyl, Allyl, and Phenyl, quinone = acenapthnenequinone (AN), aceanthrenequinone (AA), phenanthrenequinone (PH), and pyrene-4,5-dione (PY) were isolated and fully characterized spectroscopically and by mass spectrometry. A plethora of single crystal X-ray diffraction structures were obtained and analyzed and the geometries were also validated by DFT calculations. The Zn(II) complexes presented either distorted octahedral geometry or tetrahedral arrangements of the O/N/S donors around the metal center. The modification of the thiosemicarbazide moiety at the exocyclic N atoms with a range of organic linkers was also explored, opening the way to bioconjugation protocols for these compounds. The radiolabeling of these thiosemicarbazones with 64Cu was achieved under mild conditions for the first time: this cyclotron-available radioisotope of copper (t1/2 = 12.7 h; ß+ 17.8%; ß- 38.4%) is well-known for its proficiency in positron emission tomography (PET) imaging and for its theranostic potential, on the basis of the preclinical and clinical cancer research of established bis(thiosemicarbazones), such as the hypoxia tracer 64Cu-labeled copper(diacetyl-bis(N4-methylthiosemicarbazone)], [64Cu]Cu(ATSM). Our labeling reactions proceeded in high radiochemical incorporation (>80% for the most sterically unencumbered ligands) showing promise of these species as building blocks for theranostics and synthetic scaffolds for multimodality imaging probes. The corresponding "cold" Cu(II) metalations were also performed under the mild conditions mimicking the radiolabeling protocols. Interestingly, room temperature or mild heating led to Cu(II) incorporation in the 1:1, as well as 1:2 metal: ligand ratios in the new complexes, as evident from extensive mass spectrometry investigations backed by EPR measurements, and the formation of Cu(L)2-type species prevails, especially for the AN-Ph thiosemicarbazone ligand (L-). The cytotoxicity levels of a selection of ligands and Zn(II) complexes in this class were further tested in commonly used human cancer cell lines (HeLa, human cervical cancer cells, and PC-3, human prostate cancer cells). Tests showed that their IC50 levels are comparable to that of the clinical drug cis-platin, evaluated under similar conditions. The cellular internalizations of the selected ZnL2-type compounds Zn(AN-Allyl)2, Zn(AA-Allyl)2, Zn(PH-Allyl)2, and Zn(PY-Allyl)2 were evaluated in living PC-3 cells using laser confocal fluorescent spectroscopy and these experiments showed exclusively cytoplasmic distributions.

Hybrid Hierarchical Heterostructures of Nanoceramic Phosphors as Imaging Agents for Multiplexing and Living Cancer Cells Translocation.

Existing fluorescent labels used in life sciences are based on organic compounds with limited lifetime or on quantum dots which are either expensive or toxic and have low kinetic stability in biological environments. To address these challenges, luminescent nanomaterials have been conceived as hierarchical, core-shell structures with spherical morphology and highly controlled dimensions. These tailor-made nanophosphors incorporate Ln:YVO4 nanoparticles (Ln = Eu(III) and Er(III)) as 50 nm cores and display intense and narrow emission maxima centered at ∼565 nm. These cores can be encapsulated in silica shells with highly controlled dimensions as well as functionalized with chitosan or PEG5000 to reduce nonspecific interactions with biomolecules in living cells. Confocal fluorescence microscopy in living prostate cancer cells confirmed the potential of these platforms to overcome the disadvantages of commercial fluorophores and their feasibility as labels for multiplexing, biosensing, and imaging in life science assays.

Multiphoton fluorescence lifetime imaging microscopy (FLIM) and super-resolution fluorescence imaging with a supramolecular biopolymer for the controlled tagging of polysaccharides.

A new supramolecular polysaccharide complex, comprising a functionalised coumarin tag featuring a boronic acid and ß-d-glucan (a natural product extract from barley, Hordeum Vulgare) was assembled based on the ability of the boronate motif to specifically recognise and bind to 1,2- or 1,3-diols in water. The complexation ratio of the fluorophore : biopolymer strand was determined from fluorescence titration experiments in aqueous environments and binding isotherms best described this interaction using a 2 : 1 model with estimated association constants of K2:1a1 = 5.0 × 104 M-1 and K2:1a2 = 3.3 × 1011 M-1. The resulting hybrid (denoted 5@ß-d-glucan) was evaluated for its cellular uptake as an intact functional biopolymer and its distribution compared to that of the pinacol-protected coumarin boronic acid derivative using two-photon fluorescence lifetime imaging microscopy (FLIM) in living cells. The new fluorescent ß-d-glucan conjugate has a high kinetic stability in aqueous environments with respect to the formation of the free boronic acid derivative compound 5 and retains fluorescence emissive properties both in solution and in living cells, as shown by two-photon fluorescence spectroscopy coupled with time-correlated single photon counting (TCSPC). Super-resolution fluorescence imaging using Airyscan detection as well as TM AFM and Raman spectroscopy investigations confirmed the formation of fluorescent and nano-dimensional aggregates of up to 20 nm dimensions which self-assemble on several different inert surfaces, such as borosilicate glass and mica surfaces, and these aggregates can also be observed within living cells with optical imaging techniques. The cytoplasmic distribution of the 5@ß-d-glucan complex was demonstrated in several different cancer cell lines (HeLa and PC-3) as well as in healthy cells (J774.2 macrophages and FEK-4). Both new compounds (pinacol protected boronated coumarin) 5-P and its complex hybrid 5@ß-d-glucan successfully penetrate cellular membranes with the minimum morphological alterations to cells and distribute evenly in the cytoplasm. The glucan biopolymer retains its activity towards macrophages in the presence of the coumarin tag functionality, demonstrating the potential of this natural ß-d-glucan to act as a functional self-assembled theranostic scaffold capable of mediating the delivery of anchored small organic molecules with imaging and drug delivery applications.

Lysosomal tracking with a cationic naphthalimide using multiphoton fluorescence lifetime imaging microscopy.

A naphthalimide-based chemosensing motif turns ON the fluorescence emission in solution in the presence of aqueous iron(iii) chloride, and maintains this property in living cancer cells. The emission response to Fe(iii) ions occurs simultaneously with a change in pH. The protonation of methyl piperazine-conjugated naphthalimide promotes its lysosomal localisation as assessed by co-localisation tests and fluorescence lifetime imaging microscopy (FLIM) in vitro.

Alkaline-Earth Derivatives of the Reactive [HB(C6F5)3]- Anion.

The ß-diketiminato magnesium amidoboranes [HC{(Me)CNDipp}2Mg(NMe2BH2NMe2·BH3)] and [HC{(t-Bu)CNDipp}2Mg(NMe2·BH3)] are readily converted to the corresponding derivatives of the [HB(C6F5)3]- anion by treatment with B(C6F5)3. The bis(borohydride) derivatives of the heaviest alkaline-earth elements, strontium and barium, may be similarly synthesized by reaction of strontium or barium dimethylamidoboranes and B(C6F5)3 and by metathesis reactions of either SrI2 or BaI2 and 2 molar equiv of K(HB(C6F5)3). The strontium and barium compounds have been fully characterized in solution and in the solid state as the respective tris(diethyl ether) and tetrakis(tetrahydrofuran) adducts. The magnesium compound [HC{(Me)CNDipp}2Mg(HB(C6F5)3)] has been applied to the catalytic hydroboration of i-PrN═C═N-i-Pr with HBpin. In contrast to carbodiimide hydroboration catalyzed by the corresponding ß-diketiminato magnesium hydride, which results in the exclusive production of the monoborylated amidine, use of the [HB(C6F5)3]- derivative provides the product of bis-borylation, the aminal H2C(N{Bpin}i-Pr)2, under mild conditions. A series of stoichiometric reactions highlight that, while this reactivity is likely to be primarily magnesium mediated, B(C6F5)3 plays a vital role both in the delivery of reactive hydride and through the Lewis acid activation of the heteroallene substrate and various reactive intermediates.

Microwave gallium-68 radiochemistry for kinetically stable bis(thiosemicarbazone) complexes: structural investigations and cellular uptake under hypoxia.

We report the microwave synthesis of several bis(thiosemicarbazones) and the rapid gallium-68 incorporation to give the corresponding metal complexes. These proved kinetically stable under 'cold' and 'hot' biological assays and were investigated using laser scanning confocal microscopy, flow cytometry and radioactive cell retention studies under normoxia and hypoxia. (68)Ga complex retention was found to be 34% higher in hypoxic cells than in normoxic cells over 30 min, further increasing to 53% at 120 min. Our data suggests that this class of gallium complexes show hypoxia selectivity suitable for imaging in living cells and in vivo tests by microPET in nude athymic mice showed that they are excreted within 1 h of their administration.

A fluorescent Arg-Gly-Asp (RGD) peptide-naphthalenediimide (NDI) conjugate for imaging integrin α(v)ß(3) in vitro.

We have developed a fluorescent peptide conjugate (TrpNDIRGDfK) based on the coupling of cyclo(RGDfK) to a new tryptophan-tagged amino acid naphthalenediimide (TrpNDI). Confocal fluorescence microscopy coupled with fluorescence lifetime imaging (FLIM) mapping, single and two-photon fluorescence excitation, lifetime components and corresponding decay profiles were used as parameters able to investigate qualitatively the cellular behavior regarding the molecular environment and biolocalisation of TrpNDI and TrpNDI-RGDfK in cancer cells.

Confocal and fluorescence lifetime imaging sheds light on the fate of a pyrene-tagged carbon monoxide-releasing Fischer carbene chromium complex.

The synthesis of a new pyrene-containing Fischer carbene complex is described. The complex has a broad absorbance spectrum between 300 and 400 nm and, on excitation at 345 nm in CH2Cl2 solution, emission is observed at 395 and 415 nm. Emission is also observed in PBS buffer, but in this case the resulting spectra are much broader. Confocal and fluorescence lifetime imaging indicate that emission occurs on treating HeLa cells with the complex and co-localisation studies demonstrate that this is from the mitochondria and lipid-rich regions of the cell.

Metallic nanoparticles as synthetic building blocks for cancer diagnostics: from materials design to molecular imaging applications.

Metallic nanoparticles have been a matter of intense exploration within the last decade due to their potential to change the face of the medical world through their role as 'nanotheranostics'. Their envisaged capacity to act as synthetic platforms for a multimodal imaging approach to diagnosis and treatment of degenerative diseases, including cancer, remains a matter of lively debate. Certain synthetic metal-based nanomaterials, e.g. gold and iron oxide nanoparticles, are already in clinical use or under advanced preclinical investigations following in vitro and in vivo preclinical imaging success. We surveyed the recent publications landscape including those reported metallic nanoparticles having established applications in vivo, as well as some of the new metallic nanoparticles which, despite their potential as cancer nanodiagnostics, are currently awaiting in vivo evaluation. The objective of this review is to highlight the current metallic nanoparticles and/or alloys as well as their derivatives with multimodal imaging potential, focusing on their chemistry as a springboard to discussing their role in the future of nanomedicines design. We also highlight here some of the fundamentals of molecular and nano-imaging techniques of relevance to the metal-based colloids, alloys and metallic nanoparticles discerning their future prospects as cancer nanodiagnostics. The current approaches for metallic and alloy surface derivatisation, aiming to achieve functional and biocompatible materials for multimodal bioimaging applications, are discussed in order to bring about some new perspectives on the efficiency of metallic nanoparticles as synthetic scaffolds for imaging probe design and forecast their future use in medical imaging techniques (optical, CT, PET, SPECT and MRI).

Synthesis and evaluation of a boronate-tagged 1,8-naphthalimide probe for fluoride recognition.

A biocompatible fluoride receptor has been developed where the interaction between the boronic acid ester and amine (NH) results in fluoride ion selectivity and enhanced fluorescence quenching.

Re and (99m)Tc complexes of BodP3--multi-modality imaging probes.

A fluorescent tridentate phosphine, BodP3 (2), forms rhenium complexes which effectively image cancer cells. Related technetium analogues are also readily prepared and have potential as dual SPECT/fluorescent biological probes.

Ditopic boronic acid and imine-based naphthalimide fluorescence sensor for copper(II).

Copper ions are essential for many biological processes. However, high concentrations of copper can be detrimental to the cell or organism. A novel naphthalimide derivative bearing a monoboronic acid group (BNP) was investigated as a Cu(2+) selective fluorescent sensor in living cells. This derivative is one of the rare examples of reversible fluorescent chemosensors for Cu(2+) which uses a boronic acid group for a binding site. Moreover, the adduct BNP-Cu(2+) displays a fluorescence enhancement with fructose. The uptake of this novel compound in HeLa cancer cells was imaged using confocal fluorescence microscopy techniques including two-photon fluorescence lifetime imaging microscopy.

Hydrothermal conversion of one-photon-fluorescent poly(4-vinylpyridine) into two-photon-fluorescent carbon nanodots.

A novel two-photon-fluorescent N,O-heteroatom-rich carbon nanomaterial has been synthesized and characterized. The new carbon nanoparticles were produced by hydrothermal conversion from a one-photon-fluorescent poly(4-vinylpyridine) precursor (P4VP). The carbonized particles (cP4VP dots) with nonuniform particle diameter (ranging from sub-6 to 20 nm with some aggregates up to 200 nm) exhibit strong fluorescence properties in different solvents and have also been investigated for applications in cell culture media. The cP4VP dots retain their intrinsic fluorescence in a cellular environment and exhibit an average excited-state lifetime of 2.0 ± 0.9 ns in the cell. The cP4VP dots enter HeLa cells and do not cause significant damage to outer cell membranes. They provide one-photon or two-photon fluorescent synthetic scaffolds for imaging applications and/or drug delivery.

Fluorescent gallium and indium bis(thiosemicarbazonates) and their radiolabelled analogues: synthesis, structures and cellular confocal fluorescence imaging investigations.

New fluorescent and biocompatible aromatic Ga(III)- and In(III)-bis(thiosemicarbazonato) complexes for dual mode optical and PET or SPECT molecular imaging have been synthesised via a synthetic method based on transmetallation reactions from Zn(II) precursors. Complexes have been fully characterised in the solid state by single crystal X-ray diffraction and in solution by spectroscopic methods (UV/Vis, fluorescence, (1)H and (13)C{(1)H} NMR). The bis(thiosemicarbazones) radiolabelled rapidly in high yields under mild conditions with (111)In (a gamma and Auger emitter for SPECT imaging and radiotherapy with t(1/2) = 2.8 d) and (68)Ga (a generator-available positron emitter for PET imaging with t(1/2) = 68 min). Cytotoxicity and biolocalisation studies using confocal fluorescence imaging and fluorescence lifetime imaging (FLIM) techniques have been used to study their in vitro activities and stabilities in HeLa and PC-3 cells to ascertain their suitability as synthetic scaffolds for future multimodality molecular imaging in cancer diagnosis and therapy. The observation that the indium complexes show certain nuclear uptake could be of relevance towards developing (111)In therapeutic agents based on Auger electron emission to induce DNA damage.

Towards nanomedicines: design protocols to assemble, visualize and test carbon nanotube probes for multi-modality biomedical imaging.

Nanomedicine is an interdisciplinary field, still in its infancy, where an accurate scientific assessment of potential risks and benefits is urgently needed, as is the engagement of end users and the public in this facet of the nanotechnology debate. There is increasing interest in improving our understanding of the interactions between nanomaterials and living systems, with regard to both the underlying chemistry and the physics of effects on the nanoscale. Ultimately, such knowledge promises new vistas for designing the 'smart' medicines of the future, of which targeted personalized drugs are the holy grail. Imaging and therapeutic components, including metallic radioisotopes, semiconductor quantum dots and magnetic materials, may be used to construct 'nanocarriers' (by encapsulation or conjugation) by rapid and simple (covalent and supramolecular) chemistry. The biomedical functions of the resulting materials are as yet largely unexplored. Encapsulation in nanocarriers could achieve delivery of the reagents (imaging and therapeutic drugs) to the sites of action in the body, while minimizing systemic toxicity and enzymatic degradation. These functional systems have the potential to become a general solution in drug delivery. Here we review recent developments concerning the applications of nanoparticles, including carbon nanotubes, as synthetic scaffolds for designing nanomedicines. This article will also focus on how understanding and design at the molecular level could help interdisciplinary teams develop research towards new diagnostics and therapeutics both in the short and the long term.

Fluorescent copper(II) bis(thiosemicarbazonates): synthesis, structures, electron paramagnetic resonance, radiolabeling, in vitro cytotoxicity and confocal fluorescence microscopy studies.

Copper bis(4-ethyl-3-thiosemicarbazonato) acenaphthenequinone (1) and copper bis(4-methyl-3-thiosemicarbazonato) acenaphthenequinone (2) are synthesized and characterized in solution, in the solid state, and radiolabeled. Serum-protein binding radioassays show good stability in solution and about 25 % binding to protein over 1 h, which is comparable with the hypoxia selective tracer [(64)Cu(ATSM)]. Cyclic voltammetry shows fast and reversible reduction at redox potentials similar to the values known for hypoxia-selective copper compounds. However, despite this, complex 1 does not show any hypoxic-selective uptake in HeLa cells over 1-h standard assays. Possible reasons for this are studied by using the intrinsic fluorescence of the Cu(II) complexes to determine the cellular distributions and uptake mechanism by confocal microscopy. The complexes are found to bind to the external cell membrane and disperse evenly in the cytoplasm only after a very slow cell internalization (>1 h). No significant changes in distribution are observed by fluorescence imaging under hypoxic conditions. The rate of localization in the cytoplasm contrasts with their Zn(II) analogues, which are known to have fast cell uptake (up to 20 min) and a clear localization in lysosomes and mitochondria. The cytotoxicity mechanism of 1 over 24 h against a number of adherent cell lines is seen to be by membrane disruption and is of a comparable magnitude to that of [Cu(ATSM)], as demonstrated by methyl tetrazolium (MTT) and lactate dehydrogenase (LDH) assays.