Raluca M. Fratila

Triazolium cations: from the “click” pool to multipurpose applications

Accessing 1,2,3-triazole compounds through the copper-catalysed azide–alkyne “click” cycloaddition reaction is now a routine synthetic tool which has resulted in a huge amount of novel molecules of varied complexity bearing such heterocycles. 3-N-Alkyl-1,2,3-triazolium salts constitute an example of “post-click” chemistry arising from the “click” pool. This Focus review outlines emerging fields of application for triazolium cations, including their use as functional ionic liquids, as precursors of mesoionic carbenes, or as components of supramolecular assemblies and molecular machines.

Small-Volume Nuclear Magnetic Resonance Spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy is one of the most information-rich analytical techniques available. However, it is also inherently insensitive, and this drawback precludes the application of NMR spectroscopy to mass- and volume-limited samples. We review a particular approach to increase the sensitivity of NMR experiments, namely the use of miniaturized coils. When the size of the coil is reduced, the sample volume can be brought down to the nanoliter range. We compare the main coil geometries (solenoidal, planar, and microslot/stripline) and discuss their applications to the analysis of mass-limited samples. We also provide an overview of the hyphenation of microcoil NMR spectroscopy to separation techniques and of the integration with lab-on-a-chip devices and microreactors.

Multivalent nanoparticle networks as ultrasensitive enzyme sensors

As few as 23 enzyme molecules could be detected on the basis of the dispersion of Au nanoparticles in a model bioassay whose sensitivity was boosted by the interplay between multivalent and monovalent supramolecular interactions: a diferrocenyl ligand (red) caused the assembly of nanoparticle clusters; upon its oxidation, a competing monovalent guest molecule (blue) favored the formation of monovalent interactions for more efficient

Evaluation of superparamagnetic iron oxide nanoparticles (Endorem) as a photoacoustic contrast agent for intra-operative nodal staging

Detection of tumor metastases in the lymphatic system is essential for accurate staging of malignancies. Commercially available superparagmagnetic nanoparticles (SPIOs) accumulate in normal lymph tissue after injection at a tumor site, whereas less or no accumulation takes place in metastatic nodes, thus enabling lymphatic staging using MRI. We verify for the first time the potential of SPIOs, such as Endorem(®) as a novel photoacoustic (PA) contrast agent in biological tissue. We injected five Wistar rats subcutaneously with variable amounts of Endorem(®) and scanned the resected lymph nodes using a tomographic PA setup. Findings were compared using histology, vibrating sample magnetometry (VSM) and 14 T MR-imaging. Our PA setup was able to detect the iron oxide accumulations in all the nodes containing the nanoparticles. The distribution inside the nodes corresponded with both MRI and histological findings. VSM revealed that iron quantities inside the nodes varied between 51 ± 4 and 11 ± 1 µg. Nodes without SPIO enhancement did not show up in any of the PA scans. Iron oxide nanoparticles (Endorem(®)) can be used as a PA contrast agent for lymph node analysis and a distinction can be made between nodes with and nodes without the agent. This opens up possibilities for intra-operative nodal staging for patients undergoing nodal resections for metastatic malignancies.

Multinuclear nanoliter one-dimensional and two-dimensional NMR spectroscopy with a single non-resonant microcoil

Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical technique, but its low sensitivity and highly sophisticated, costly, equipment severely constrain more widespread applications. Here we show that a non-resonant planar transceiver microcoil integrated in a microfluidic chip (detection volume 25 nl) can detect different nuclides in the full broad-band range of Larmor frequencies (at 9.4 T from 61 to 400 MHz). Routine one-dimensional (1D) and two-dimensional (2D), homo- and heteronuclear experiments can be carried out using the broad-band coil set-up. Noteworthy, heteronuclear 2D experiments can be performed in a straightforward manner on virtually any combination of nuclides (from classical ¹H-¹³C to more exotic combinations like ¹⁹F-³¹P) both in coupled and decoupled mode. Importantly, the concept of a non-resonant system provides magnetic field-independent NMR probes; moreover, the small-volume alleviates problems related to field inhomogeneity, making the broad-band coil an attractive option for, for example, portable and table-top NMR systems.

Intra-operative ex vivo photoacoustic nodal staging in a rat model using a clinical superparamagnetic iron oxide nanoparticle dispersion

The ability to accurately detect tumor metastases in lymph nodes is essential for intra-operative staging of various malignancies. Histopathological assessment of nodes has the drawback of a time delay before results are available to the surgeon and a likelihood of missing metastases. Photoacoustic (PA) imaging has been shown to possess the potential to detect melanoma metastases in resected in toto lymph nodes based on intrinsic contrast. To extend application of the method to other malignancies, extrinsic contrast for lymphatic mapping is important. We investigate in a metastatic animal model whether clinically approved superparamagnetic iron oxide (SPIO) nanoparticles, applied for MRI, can help PA imaging for staging in an intra-operative ex vivo setting. Imaging results are compared with 14 Tesla MR images and histology. We observe that irregularities in SPIO distribution in PA images of the nodes and a decrease in contrast correlate with metastatic involvement as seen in MR images and histology. The results show that a PA based imaging technique may be valuable for nodal staging in the field of surgical oncology.

Mechanistic Insights on the Magnesium(II) Ion-Activated Reduction of Methyl Benzoylformate with Chelated NADH Peptide β-Lactam Models

Mechanistic details of the Mg(2+) ion-activated enantioselective reduction of methyl benzoylformate have been investigated at a B3LYP/6-31G* theory level, using peptide NADH models 1 rigidified with a beta-lactam ring. Computation of the reaction pathway revealed important structural differences between the intermediate NADH/Mg(2+)/ArCOCO(2)R ternary complexes 3 and the corresponding transition states leading to enantiomeric methyl mandelates. Thus, ternary complexes showed the dihydronicotinamide moiety placed quasiequatorial to a seven-membered chelation pseudoplane including the two amide carbonyls and the Mg(2+) cation, whereas productive transition states were strongly deformed with the dihydronicotinamide group oriented quasiaxial to the chelation pseudoplane. This chelation model was further applied to acyclic nonrigidified NADH models and, based on the fluxional mobility of the peptide chain bonds, experimental enantioselectivities were correctly predicted. Parallel experiments were also conducted in deuterated acetonitrile, using NMR techniques, to study the structure of the binary complexes 2 (NADH/Mg(2+)) and ternary complexes 3 (NADH/Mg(2+)/PhCOCO(2)Me). Finally, owing to the incorporation of two diastereotopic trimethylsilyl NMR-tags in the beta-lactam-NADH peptidomimetics, a nonproductive ternary complex predicted by calculations could be observed and its structure characterized on the basis of ROESY experiments and molecular modeling.

Determination of Kinetic Parameters within a Single Nonisothermal On-Flow Experiment by Nanoliter NMR Spectroscopy

Conventional methods to determine the kinetic parameters for a certain reaction require multiple, separate isothermal experiments, resulting in time- and material-consuming processes. Here, an approach to determine the kinetic information within a single nonisothermal on-flow experiment is presented, consuming less than 10 μmol of reagents and having a total measuring time of typically 10 min. This approach makes use of a microfluidic NMR chip hyphenated to a continuous-flow microreactor and is based on the capabilities of the NMR chip to analyze subnanomole quantities of material in the 25 nL detection volume. Importantly, useful data are acquired from the microreactor platform in specific isothermal and nonisothermal frames. A model fitting the experimental data enables rapid determination of kinetic parameters, as demonstrated for a library of isoxazole and pyrazole derivatives.

An Open Source Image Processing Method to Quantitatively Assess Tissue Growth after Non-Invasive Magnetic Resonance Imaging in Human Bone Marrow Stromal Cell Seeded 3D Polymeric Scaffolds

Monitoring extracellular matrix (ECM) components is one of the key methods used to determine tissue quality in three-dimensional (3D) scaffolds for regenerative medicine and clinical purposes. This is even more important when multipotent human bone marrow stromal cells (hMSCs) are used, as it could offer a method to understand in real time the dynamics of stromal cell differentiation and eventually steer it into the desired lineage. Magnetic Resonance Imaging (MRI) is a promising tool to overcome the challenge of a limited transparency in opaque 3D scaffolds. Technical limitations of MRI involve non-uniform background intensity leading to fluctuating background signals and therewith complicating quantifications on the retrieved images. We present a post-imaging processing sequence that is able to correct for this non-uniform background intensity. To test the processing sequence we investigated the use of MRI for in vitro monitoring of tissue growth in three-dimensional poly(ethylene oxide terephthalate)–poly(butylene terephthalate) (PEOT/PBT) scaffolds. Results showed that MRI, without the need to use contrast agents, is a promising non-invasive tool to quantitatively monitor ECM production and cell distribution during in vitro culture in 3D porous tissue engineered constructs.

Photoacoustic staging of nodal metastases using SPIOs: Comparison between in vivo, in toto and ex vivo imaging in a rat model

Background and objectives: To determine prognosis and treatment, accurate nodal staging is essential in many tumor types. After injection of clinical grade superparamagnetic iron oxide (SPIO) nanoparticles, it has been shown that metastatic lymph nodes can be distinguished from benign specimens using MR imaging. However, MR does not benefit per-operative nodal staging which requires a non-ionizing, small volume, high resolution, fast imaging technique. In vivo non-invasive photoacoustic (PA) imaging of lymph nodes might facilitate nodal staging during surgery, thereby benefiting both surgeon and patient. Materials and methods: In order to investigate the feasibility of an in vivo nodal staging approach using photoacoustics, six Mat-lylu inoculated Copenhagen rats were photo-acoustically imaged after injection of a new Class IIa medical device SPIO magnetic tracer (Sienna+). Lymph nodes were imaged in vivo, in toto (after euthanization) and ex vivo using multiple wavelength illumination. Results were compared with MRI, immunohistochemistry and photographs of the sectioned nodes. Results: These experiments demonstrate that in an ex vivo setting, the PA contrast of Sienna+ is able to facilitate a distinction between metastatic and benign nodes. A non-invasive distinction between both groups is partially impeded by the low amount of PA contrast generated by the SPIO particles compared to that of endogenous absorbers such as hemoglobins. Conclusions: This comparison between in vivo, in toto and ex vivo PA imaging of lymph nodes after SPIO injection demonstrates that the clinical potential of combined PA/SPIO staging should initially be exploited in an ex vivo setting. Improved distinction between chromophores by for example multi-spectral unmixing might in the near future enable non-invasive assessment of nodal involvement.