N-rich carbon nanosphere as fluorescent nanoprobe for intracellular iron.

Nitrogen rich carbon nanoparticles are known to provide higher fluorescence stokes shift, and thereby are potential candidates for fluorescent sensors. Herein, a facile one-step hydrothermal synthesis is reported for N-rich carbon nanospheres (G-CNS) from caffeine and o-phenylenediamine as precursors. The as-synthesized G-CNS showed high fluorescence with λem at 509 nm, with a highly selective fluorescence turn-off response towards Fe2+/Fe3+, rendering these carbon nanospheres as potential candidates to detect intracellular labile iron pool in live cells. The intracellular labile iron pool in iron-overloaded cells was sensed using the synthesized G-CNS. Mechanistically, the fluorescence quenching via dynamic pathway involves the formation of an excited state charge transfer process, which undergoes non-radiative decay.

Synthesis, Quality Control, and Bench-to-Bed Translation of a New [68Ga]Ga-Labeled NOTA-Conjugated Bisphosphonate for Imaging Skeletal Metastases by Positron Emission Tomography.

Background: Early detection of skeletal metastasis is of great interest to determine the prognosis of cancer. Positron emission tomography-computed tomography (PET-CT) imaging provides a better temporal and spectral resolution than single photon emission computed tomography-computed tomography (SPECT-CT) imaging, and hence is more suitable to detect small metastatic lesions. Although [18F]NaF has been approved by U.S. FDA for a similar purpose, requirement of a medical cyclotron for its regular formulation restricts its extensive utilization. Efforts have been made to find suitable alternative molecules that can be labeled with 68Ga and used in PET-CT imaging. Objective: The main objective of this study is to synthesize and evaluate a new [68Ga]Ga-labeled NOTA-conjugated geminal bisphosphonate for its potential use in early detection of skeletal metastases using PET-CT. Methods: The authors performed a multistep synthesis of a new NOTA-conjugated bisphosphonic acid using thiourea linker and radiolabeled the molecule with 68Ga. The radiolabeled formulation was evaluated for its in vitro stability, affinity for hydroxyapatite (HA) particles, preclinical biodistribution in animal models, and PET-CT imaging in patients. Results: The bifunctional chelator (NOTA)-conjugated bisphosphonate was synthesized with 97.8% purity and radiolabeled with 68Ga in high yield (>98%). The radiolabeled formulation was found to retain its stability in vitro to the extent of >95% up to 4 h in physiological saline and human serum. The formulation also showed high affinity for HA particles in vitro with Kd = 907 ± 14 mL/g. Preclinical biodistribution studies in normal Wistar rats demonstrated rapid and almost exclusive skeletal accumulation of the complex. PET-CT imaging in a patient confirmed its ability to detect small metastatic skeletal lesions. Conclusions: The newly synthesized [68Ga]Ga-labeled NOTA-conjugated bisphosphonate is a promising radiotracer for PET-CT imaging for skeletal metastases.

Deep Eutectic Solvent-Based Highly Sensitive Turn-On Fluorescent Probe for D2O.

Owing to the importance of heavy water in spectroscopy, nuclear energy generation, chemical characterization, and biological industry, a design of a robust, cheap, nontoxic, and sensitive D2O sensor is very important. In this work, taking advantage of the singular emission fluorescence of the deep eutectic solvent prepared in our laboratory, we propose a first of its kind highly sensitive turn-on fluorescent sensor to effectively sense D2O at an ultratrace level based on rapid exchange of the labile DES proton with deuterium. This method can be used as a full-range heavy water detection strategy with a limit of detection of 0.079% (v/v) or 870 ppm. The isotopic purity (IP) obtained from DES fluorescence measurements is also in close agreement with that of the conventional FT-IR method. The current DES-based sensor thus allows both sensing and isotopic purity of D2O and can serve as one of the most sensitive monitoring strategies for heavy water analysis.

Mechanistic Approach to Reveal Interaction of Uranyl Ions in Alkyltriphenylphosphonium Bromide-Based Deep Eutectic Solvent.

Speciation is known to control fundamental aspects of metal processing and electrochemical behavior such as solubility and redox potentials. Deep eutectic solvents (DESs) are an emerging class of green, low-cost and designer solvents and are being explored as alternatives for recycling nuclear fuel and critical materials. However, there is a lack of knowledge about the behavior of metals in them. Here, for the first time, we synthesized three new DESs based on alkyltriphenylphosphonium bromide (CnPPh3Br), with varied alkyl chain lengths (n), as the hydrogen-bond acceptor along with decanoic acid (DA) as the hydrogen-bond donor and explored the redox speciation of uranyl nitrate. The changes in the Fourier transform infrared and NMR spectra helped elucidate the formation of hydrogen bonds in DES. The absorption maxima of uranyl in DES was red-shifted by 10 nm compared to the free uranyl, with concomitant increase in intensity and luminescence lifetime, which suggested a strong interaction of uranyl nitrate with DES. Cyclic voltammetry was probed to understand the redox thermodynamics, transport properties, and heterogeneous electron transfer kinetics of the irreversible electron transfer of uranyl ions in the three DESs. Electrochemical and spectroscopic techniques together with density functional theory calculations unlocked microscopic insights into the solvation and speciation of UO22+ ions in three DESs and also the associated unusual trends observed in the physical properties of the DESs. The hydrogen-bonded structure of DES plays a crucial role in the redox behavior of the UO22+ ion due to its strong potent complexation with its components. The basic findings of the present work can have far-reaching consequences for the extraction, electrochemical separation, and future development of redox-based separation processes in the nuclear fuel cycle.

Multimodal Applications of Zinc Gallate-Based Persistent Luminescent Nanoparticles in Cancer Treatment: Tumor Margining, Diagnosis, and Boron Neutron Capture Therapy.

On the basis of the boron neutron capture therapy (BNCT) modality, we have designed and synthesized a zinc gallate (ZnGa2O4)-based nanoformulation for developing an innovative theranostic approach for cancer treatment. Initially, the (ZnGa1.995Cr0.005O4 or ZnGa2O4:(0.5%)Cr persistent luminescence nanoparticles (PLNPs) embedded on silica matrix were synthesized. Their surface functionalization was performed using organic synthesis strategies to attach the amine functional moieties which were further coupled with poly(vicinal diol). These diols were helpful for conjugation with 10B(OH)3, which subsequently served to couple with an in-house-synthesized variant of pH-(low)-insertion peptide (pHLIP) finally giving a tumor-targeting nanoformulation. Most importantly, the polymeric diols helped in conjugation of a substantial number of 10B to provide the therapeutic dose required for effective BNCT. This nanoformulation internalized substantially (∼80%) to WEHI-164 cancer cells within 6 h. Tumor homing studies indicated that the accumulation of this formulation at the acidic tumor site was within 2 h. The in vitro evaluation of the formulation against WEHI-164 cancer cells followed by neutron irradiation revealed its potent cytotoxicity with IC50 ∼ 25 µM. In the case of studies on animal models, the melanoma-induced C57BL/6 and fibrosarcoma-induced BALB/c mice were treated with formulations through intratumoral and intravenous injections, respectively, followed by neutron irradiation, leading to a significant killing of the cancer cells, which was evidenced by a reduction in tumor volume (75-80%) as compared with a control tumor. Furthermore, the histopathological studies confirmed a damaging effect only on tumor cells, while there was no sign of damage to the vital organs in treated mice as well as in controls.

Synthesis of Bioactive Diarylheptanoids from Alpinia officinarum and Their Mechanism of Action for Anticancer Properties in Breast Cancer Cells.

An efficient synthesis of the Alpinia officinarum-derived diarylheptanoids, viz., enantiomers of a ß-hydroxyketone (1) and an α,ß-unsaturated ketone (2) was developed starting from commercially available eugenol. Among these, compound 2 showed a superior antiproliferative effect against human breast adenocarcinoma MCF-7 cells. Besides reducing clonogenic cell survival, compound 2 dose-dependently increased the sub G1 cell population and arrested the G2-phase of the cell cycle, as revealed by flow cytometry. Mechanistically, compound 2 acts as an intracellular pro-oxidant by generating copious amounts of reactive oxygen species. Compound 2 also induced both loss of mitochondrial membrane potential (MMP) as well as lysosomal membrane permeabilization (LMP) in the MCF-7 cells. The impaired mitochondrial and lysosomal functions due to reactive oxygen species (ROS)-generation by compound 2 may contribute to its apoptotic property.

Binding Constant Determined from the Angstrom-Scale Change in Hydrodynamic Radius of Transferrin upon Binding with Europium Using Dual-Focus Fluorescence Correlation Spectroscopy.

Monitoring the binding of a large fluorescently tagged molecule to a small solute by fluorescence correlation spectroscopy (FCS) is rather uncommon because the binding-related change in diffusion coefficient is very small. Here, we use a high-precision variant of FCS, namely, dual-focus FCS (2fFCS), for measuring the angstrom-scale change of the hydrodynamic radius of the bilobal metal transport protein transferrin (Tf) upon binding europium ions. Applying a sequential 1:2 complexation model, we use these measurements for determining the binding constants (K). Our results show a 0.7 Å change of the protein's hydrodynamic radius upon 1:1 Tf-Eu complex formation and a second change of 1.8 Å upon subsequent binding of a second europium ion. More than one unit variation in logK indicates an intrinsic dissimilarity in metal affinity of the C- and N-lobes of Tf, which agrees well with earlier reported ensemble spectroscopy results.

Determining Metal Ion Complexation Kinetics with Fluorescent Ligands by Using Fluorescence Correlation Spectroscopy.

Fluorescence correlation spectroscopy (FCS) has been extensively used to measure equilibrium binding constants (K) or association and dissociation rates in many reversible chemical reactions across chemistry and biology. For the majority of investigated reactions, the binding constant was on the order of ∼100 M-1 , with dissociation constants faster or equal to 103  s-1 , which ensured that enough association/dissociation events occur during the typical diffusion-determined transition time of molecules through the FCS detection volume. However, complexation reactions involving metal ions and chelating ligands exhibit equilibrium constants exceeding 104  M-1 . In the present paper, we explore the applicability of FCS for measuring reaction rates of such complexation reactions, and apply it to binding of iron, europium and uranyl ions to a fluorescent chelating ligand, calcein. For this purpose, we exploit the fact that the ligand fluorescence becomes strongly quenched after binding a metal ion, which results in strong intensity fluctuations that lead to a partial correlation decay in FCS. We also present measurements for the strongly radioactive ions of 241 Am3+ , where the extreme sensitivity of FCS allows us to work with sample concentrations and volumes that exhibit close to negligible radioactivity levels. A general discussion of the applicability of FCS to the investigation of metal-ligand binding reactions concludes our paper.

A chemoenzymatic synthesis of ceramide trafficking inhibitor HPA-12.

A chemoenzymatic synthesis of the title compound has been developed using an efficient and highly enantioselective lipase-catalyzed acylation in a hydrophobic ionic liquid, [bmim][PF6], followed by a diastereoselective asymmetric dihydroxylation as the key steps for incorporating the stereogenic centers. The further conversion to the appropriate intermediates and subsequent acylation with lauric acid furnished the target compound.

Cell Permeable Imidazole-Desferrioxamine Conjugates: Synthesis and In Vitro Evaluation.

Desferrioxamine (DFO), a clinically approved iron chelator used for iron overload, is unable to chelate labile plasma iron (LPI) because of its limited cell permeability. Herein, alkyl chain modified imidazolium cations with varied hydrophobicities have been conjugated with DFO. The iron binding abilities and the antioxidant properties of the conjugates were found to be similar to DFO. The degree of cellular internalization was much higher in the octyl-imidazolium-DFO conjugate (IV) compared with DFO, and IV was able to chelate LPI in vitro. This opens up a new avenue in using N-alkyl imidazolium salts as a delivery vector for hydrophilic cell-impermeable drugs.

Remarkably enhanced direct dissolution of plutonium oxide in task-specific ionic liquid: insights from electrochemical and theoretical investigations.

The present work envisages an approach for direct dissolution of PuO2 in a task-specific ionic liquid (TSIL). An attractive possibility to electrodeposit plutonium from the mixture of TSIL and PuO2 has been explored further. The carboxyl functional group attached to the TSIL plays a key role in facilitating the dissolution of plutonium ions.

Bi-mediated allylation of aldehydes in [bmim][Br]: a mechanistic investigation.

The inexpensive room temperature ionic liquid (RTIL), [bmim][Br] has been found to be a superior medium for the Bi-mediated Barbier-type allylation of aldehydes compared to other conventional solvents. It plays the dual role of a solvent and a metal activator enabling higher yields of the products in a shorter reaction time using stoichiometric/near-stoichiometric amounts of reagents. Plausibly, [bmim][Br] activates Bi metal by a charge transfer mechanism. The 1H VT-NMR studies suggested that both the allylating species, allylbismuth dibromide and diallylbismuth bromide, are generated in situ.

Syntheses and evaluation of 68 Ga- and 153 Sm-labeled DOTA-conjugated bisphosphonate ligand for potential use in detection of skeletal metastases and management of pain arising from skeletal metastases.

This article reports the syntheses and evaluation of 68 Ga- and 153 Sm-complexes of a new DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)-conjugated geminal bisphosphonate, DOTA-Bn-SCN-BP, for their potential uses in the early detection of skeletal metastases by imaging and palliation of pain arising from skeletal metastases, respectively. The conjugate was synthesized in high purity following an easily adaptable three-step reaction scheme. Gallium-68- and 153 Sm-complexes were prepared in high yield (>98%) and showed excellent in vitro stability in phosphate-buffered saline (PBS) and human serum. Both the complexes showed high affinity for hydroxyapatite particles in in vitro binding study. In biodistribution studies carried out in normal Wistar rats, both the complexes exhibited rapid skeletal accumulation with almost no retention in any other major organ. The newly synthesized molecule DOTA-Bn-SCN-BP would therefore be a promising targeting ligand for the development of radiopharmaceuticals for both imaging skeletal metastases and palliation of pain arising out of it in patients with cancer when radiolabeled with 68 Ga and 153 Sm, respectively. A systematic comparative evaluation, however, showed that there was no significant improvement of skeletal accumulation of the 153 Sm-DOTA-Bn-SCN-BP complex over 153 Sm-DOTMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethylenephosphonic acid) as the later itself demonstrated optimal properties required for an agent for bone pain palliation.

Triphenylphosphonium-desferrioxamine as a candidate mitochondrial iron chelator.

Cell-impermeant iron chelator desferrioxamine (DFO) can have access to organelles if appended to suitable vectors. Mitochondria are important targets for the treatment of iron overload-related neurodegenerative diseases. Triphenylphosphonium (TPP) is a delocalized lipophilic cation used to ferry molecules to mitochondria. Here we report the synthesis and characterization of the conjugate TPP-DFO as a mitochondrial iron chelator. TPP-DFO maintained both a high affinity for iron and the antioxidant activity when compared to parent DFO. TPP-DFO was less toxic than TPP alone to A2780 cells (IC50 = 135.60 ± 1.08 and 4.34 ± 1.06 µmol L-1, respectively) and its native fluorescence was used to assess its mitochondrial localization (Rr = +0.56). These results suggest that TPP-DFO could be an interesting alternative for the treatment of mitochondrial iron overload e.g. in Friedreich's ataxia.

syn-Selective crotylation of aldehydes using bismuth-crotyl bromide-(1-butyl-3-methylimidazolium bromide) combination: some synthetic applications.

The Bi-[bmim][Br] combination has been found to offer high syn-selectivity in the crotylation of aldehydes with crotyl bromide using practically stoichiometric amounts of the reagents. The room temperature ionic liquid (RTIL), [bmim][Br], activated Bi metal in the presence of oxygen to produce crotylbismuthdibromide, which reacted with the aldehydes at room temperature. The major anti-syn diastereomeric product obtained from the crotylation of (R)-cyclohexylideneglyceraldehyde was utilized for the synthesis of dictyostatin and cryptophycin segments, and (+)-cis-aerangis lactone, using standard synthetic protocols.

Mitochondria-penetrating peptides conjugated to desferrioxamine as chelators for mitochondrial labile iron.

Desferrioxamine (DFO) is a bacterial siderophore with a high affinity for iron, but low cell penetration. As part of our ongoing project focused on DFO-conjugates, we synthesized, purified, characterized and studied new mtDFOs (DFO conjugated to the Mitochondria Penetrating Peptides TAT49-57, 1A, SS02 and SS20) using a succinic linker. These new conjugates retained their strong iron binding ability and antioxidant capacity. They were relatively non toxic to A2780 cells (IC50 40-100 µM) and had good mitochondrial localization (Rr +0.45 -+0.68) as observed when labeled with carboxy-tetramethylrhodamine (TAMRA) In general, mtDFO caused only modest levels of mitochondrial DNA (mtDNA) damage. DFO-SS02 retained the antioxidant ability of the parent peptide, shown by the inhibition of mitochondrial superoxide formation. None of the compounds displayed cell cycle arrest or enhanced apoptosis. Taken together, these results indicate that mtDFO could be promising compounds for amelioration of the disease symptoms of iron overload in mitochondria.

Deferasirox-TAT(47-57) peptide conjugate as a water soluble, bifunctional iron chelator with potential use in neuromedicine.

Deferasirox (DFX), an orally active and clinically approved iron chelator, is being used extensively for the treatment of iron overload. However, its water insolubility makes it cumbersome for practical use. In addition to this, the low efficacy of DFX to remove brain iron prompted us to synthesize and evaluate a DFX-TAT(47-57) peptide conjugate for its iron chelation properties and permeability across RBE4 cell line, an in vitro model of the blood-brain barrier. The water-soluble conjugate was able to remove labile iron from buffered solution as well as from iron overloaded sera, and the permeability of DFX-TAT(47-57) conjugate into RBE4 cells was not affected compared to parent deferasirox. The iron bound conjugate was also able to translocate through the cell membrane.

Self-assembled penetratin-deferasirox micelles as potential carriers for hydrophobic drug delivery.

There has been a growing interest in the use of micelles with nanofiber geometry as nanocarriers for hydrophobic drugs. Here we show that the conjugate of penetratin, a cell-penetrating peptide (CPP) with blood-brain barrier (BBB) permeability, and deferasirox (DFX), a hydrophobic iron chelator, self-assembles to form micelles at a very low concentration (∼15 mg/L). The critical micelle concentration (CMC) was determined, and the micelles were used for solubilizing curcumin, a hydrophobic anti-neurodegenerative drug, for successful delivery across RBE4 cells, a BBB model. Transmission Electron Microscope images of the curcumin-loaded micelles confirmed the formation of nanofibers. These results indicate the potential of CPP-drug conjugates for use as nanocarriers.

Cell penetrating peptide (CPP)-conjugated desferrioxamine for enhanced neuroprotection: synthesis and in vitro evaluation.

Iron overload causes progressive and sometimes irreversible damage due to accelerated production of reactive oxygen species. Desferrioxamine (DFO), a siderophore, has been used clinically to remove excess iron. However, the applications of DFO are limited because of its inability to access intracellular labile iron. Cell penetrating peptides (CPPs) have become an efficient delivery vector for the enhanced internalization of drugs into the cytosol. We describe, herein, an efficient method for covalently conjugating DFO to the CPPs TAT(47-57) and Penetratin. Both conjugates suppressed the redox activity of labile plasma iron in buffered solutions and in iron-overloaded sera. Enhanced access to intracellular labile iron compared to the parent siderophore was achieved in HeLa and RBE4 (a model of blood-brain-barrier) cell lines. Iron complexes of both conjugates also had better permeability in both cell models. DFO antioxidant and iron binding properties were preserved and its bioavailability was increased upon CPP conjugation, which opens new therapeutic possibilities for neurodegenerative processes associated with brain iron overload.

Desferrioxamine-caffeine (DFCAF) as a cell permeant moderator of the oxidative stress caused by iron overload.

Desferrioxamine (DFO) is a potent iron chelator used in the treatment of iron overload (IO) disorders. However, due to its low cell permeability and fast clearance, DFO administration is usually prolonged and of limited use for the treatment of IO in tissues such as the brain. Caffeine is a safe, rapidly absorbable molecule that can be linked to other compounds to improve their cell permeability. In this work, we successfully prepared and described DFO-caffeine, a conjugate with iron scavenging ability, antioxidant properties and enhanced permeation in the HeLa cell model.