In Vitro Studies Regarding the Safety of Chitosan and Hyaluronic Acid-Based Nanohydrogels Containing Contrast Agents for Magnetic Resonance Imaging.

The aim of this study was to investigate the biocompatibility of contrast agents, such as gadolinium 1, 4, 7, 10 tetraazacyclo-dodecane tetraacetic acid (GdDOTA) and gadolinium dioctyl terephthalate (GdDOTP), encapsulated in a polymeric matrix containing chitosan and hyaluronic acid using RAW264.7 murine macrophages and human blood samples. The cell viability and cytotoxicity were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, while cell cycle analysis was determined in RAW264.7 cells using flow cytometry. The mitochondrial membrane potential (MMP), hemolytic index, complement activation, and thrombogenic potential of gadolinium (Gd) containing nanohydrogels were measured by fluorometric and spectrophotometric methods. Taken together, our results demonstrate the good bio- and hemocompatibility of chitosan-based nanohydrogels with the RAW264.7 cell line and human blood cells, suggesting that these could be used as injectable formulations for the magnetic resonance imaging diagnostic of lymph nodes.

Comparison of MRI Properties between Multimeric DOTAGA and DO3A Gadolinium-Dendron Conjugates.

The inherent lack of sensitivity of MRI needs the development of new Gd contrast agents in order to extend the application of this technique to cellular imaging. For this purpose, two multimeric MR contrast agents obtained by peptidic coupling between an amido amine dendron and GdDOTAGA chelates (premetalation strategy, G1-4GdDOTAGA) or DO3A derivatives which then were postmetalated (G1-4GdDO3A) have been prepared. By comparison to the monomers, an increase of longitudinal relaxivity has been observed for both structures. Especially for G1-4GdDO3A, a marked increase is observed between 20 and 60 MHz. This structure differs from G1-4GdDOTAGA by an increased rigidity due to the aromatic linker between each chelate and the organic framework. This has the effect of limiting local rotational movements, which has a positive impact on relaxivity.

Silicon Wafer Functionalization with a Luminescent Tb(III) Coordination Complex: Synthesis, Characterization, and Application to the Optical Detection of NO in the Gas Phase.

A new luminescent Tb-DOTAGA (1,4,7,10-tetraazacyclododecane-1-glutaric-4,7,10- triacetic acid) complex (TbL) was synthesized and covalently immobilized on a silicon wafer. The grafting process was monitored by means of IR and XPS spectroscopies and the optical properties of the functionalized silicon wafer (TbL@Si) were investigated by fluorescence experiments. A homemade setup was then implemented in order to follow TbL@Si optical properties in the presence of gaseous nitric oxide (NO). The prima facie results indicated that in the presence of NO, the wafer fluorescence was partially quenched. This quenching was reversible as soon as NO was pumped outside the fluorescence cell, which could be interesting for the further development of lanthanide labelled silicon wafers as gas phase sensors.

Synthesis and Characterization of PEGylated and Fluorinated Chitosans: Application to the Synthesis of Targeted Nanoparticles for Drug Delivery.

To synthesize chitosan nanoparticles (CS NPs), ionic gelation is a very attractive method. It relies on the spontaneous supramolecular assembly of cationic CS with anionic compounds, which leads to nanohydrogels. To extend ionic gelation to functionalized CS, the assessment of CS degree of substitution (DSCS) is a key step. In this paper, we have developed a hyphenated strategy for functionalized CS characterization, based upon 1H, DOSY and, when relevant, 1D diffusion-filtered 19F NMR spectroscopies. For that, we have synthesized two series of water-soluble CS via amidation of CS amino groups with mPEG2000-COOH or fluorinated synthons (TFB-COOH). The aforementioned NMR techniques helped to discriminate between ungrafted and grafted synthons and finally to determine DSCS. According to DSCS values, the selection of CS-mPEG2000 or CS-TFB copolymers can be made to obtain, in the presence of hyaluronic acid (HA) and tripolyphosphate (TPP), CS-mPEG2000-TPP/HA or CS-TFB-TPP/HA nanohydrogels suitable for drug delivery.

The internal structure of gadolinium and perfluorocarbon-loaded polymer nanoparticles affects 19F MRI relaxation times.

19F magnetic resonance imaging (19F MRI) is an emerging technique for quantitative imaging in novel therapies, such as cellular therapies and theranostic nanocarriers. Nanocarriers loaded with liquid perfluorocarbon (PFC) typically have a (single) core-shell structure with PFC in the core due to the poor miscibility of PFC with organic and inorganic solvents. Paramagnetic relaxation enhancement acts only at a distance of a few angstroms. Thus, efficient modulation of the 19F signal is possible only with fluorophilic PFC-soluble chelates. However, these chelates cannot interact with the surrounding environment and they might result in image artifacts. Conversely, chelates bound to the nanoparticle shell typically have a minimal effect on the 19F signal and a strong impact on the aqueous environment. We show that the confinement of PFC in biodegradable polymeric nanoparticles (NPs) with a multicore structure enables the modulation of longitudinal (T1) and transverse (T2) 19F relaxation, as well as proton (1H) signals, using non-fluorophilic paramagnetic chelates. We compared multicore NPs versus a conventional single core structure, where the PFC is encapsulated in the core(s) and the chelate in the surrounding polymeric matrix. This modulated relaxation also makes multicore NPs sensitive to various acidic pH environments, while preserving their stability. This effect was not observed with single core nanocapsules (NCs). Importantly, paramagnetic chelates affected both T1 and T219F relaxation in multicore NPs, but not in single core NCs. Both relaxation times of the 19F nucleus were enhanced with an increasing concentration of the paramagnetic chelate. Moreover, as the polymeric matrix remained water permeable, proton enhancement additionally was observed in MRI.

Hydrogels incorporating GdDOTA: towards highly efficient dual T1/T2 MRI contrast agents.

Do not tumble dry: Gadolinium-DOTA encapsulated into polysaccharide nanoparticles (GdDOTA NPs) exhibited high relaxivity (r(1) =101.7 s(-1) mM(-1) per Gd(3+) ion at 37 °C and 20 MHz). This high relaxation rate is due to efficient Gd loading, reduced tumbling of the Gd complex, and the hydrogel nature of the nanoparticles. The efficacy of the nanoparticles as a T(1)/T(2) dual-mode contrast agent was studied in C6 cells.

Synthesis and Characterization of Conjugated Hyaluronic Acids. Application to Stability Studies of Chitosan-Hyaluronic Acid Nanogels Based on Fluorescence Resonance Energy Transfer.

Hyaluronic acid (HA) was functionalized with a series of amino synthons (octylamine, polyethylene glycol amine, trifluoropropyl amine, rhodamine). Sodium hyaluronate (HAs) was first converted into its protonated form (HAp) and the reaction was conducted in DMSO by varying the initial ratio (-NH2 (synthon)/COOH (HAp)). HA derivatives were characterized by a combination of techniques (FTIR, 1H NMR, 1D diffusion-filtered 19F NMR, DOSY experiments), and degrees of substitution (DSHA) varying from 0.3% to 47% were determined, according to the grafted synthon. Nanohydrogels were then obtained by ionic gelation between functionalized hyaluronic acids and chitosan (CS) and tripolyphosphate (TPP) as a cross-linker. Nanohydrogels for which HA and CS were respectively labeled by rhodamine and fluorescein which are a fluorescent donor-acceptor pair were subjected to FRET experiments to evaluate the stability of these nano-assemblies.

Photochemical electron transfer mediated addition of naphthylamine derivatives to electron-deficient alkenes.

Using photochemical electron transfer, N,N-dimethylnaphthylamine derivatives are added to α,ß-unsaturated carboxylates. The addition takes place exclusively in the α-position of electron-deficient alkenes and mainly in the 4-position of N,N-dimethylnaphthalen-1-amine. A minor regioisomer results from the addition in the 5-position of this naphthylamine. A physicochemical study reveals that the fluorescence quenching of N,N-dimethylnaphthalen-1-amine is diffusion-controlled and that the back electron transfer is highly efficient. Therefore no transformation is observed at lower concentrations. To overcome this limitation and to induce an efficient transformation, minor amounts of water or another proton donor as well as an excess of the naphthylamine derivative are necessary. A mechanism involving a contact radical ion pair is discussed. Isotopic labeling experiments reveal that no hydrogen is directly transferred between the substrates. The hydrogen transfer to the furanone moiety observed in the overall reaction therefore results from an exchange with the reaction medium. An electrophilic oxoallyl radical generated from the furanone reacts with the naphthylamine used in excess. Concerning some mechanistic details, the reaction is compared with radical and electrophilic aromatic substitutions. The transformation was carried out with a variety of electron-deficient alkenes. Sterically hindered furanone derivatives are less reactive under standard conditions. In a first experiment, such a compound was transformed using heterogeneous electron transfer photocatalysis with TiO(2).

Cyclam-methylbenzimidazole: a selective OFF-ON fluorescent sensor for zinc.

The coordination properties and the photophysical response of a new cyclam fluorescent probe for Zn(II), [L1H: 1-(benzimidazol-2-ylmethyl]-1,4, 8,11-tetraazacyclotetradecane] toward Cu(II), Zn(II), and Cd(II) are reported. The stability constants of the corresponding complexes were determined by means of potentiometric measurements in aqueous solution. The fluorescence of L1H was quenched by the presence of Cu(II), and L1H behaves as an OFF-ON sensor for Zn(II) even in the presence of a wide range of biological divalent cations. Furthermore, on addition of successive amounts of Zn(II), the fluorescence emission of L1H increases linearly by a factor of 12. This can be correlated to the efficient Zn(II) binding of L1H and to the participation of all the amine functions in the metal coordination which prevents the photoinduced electron transfer (PET) effect and promotes a good chelation-enhanced fluorescence (CHEF) effect; this confers to the cyclam probe better sensing properties than the cyclen ionophore.

Synthetic and magnetic studies of a dodecanuclear cobalt wheel.

The synthesis, structural characterisation and preliminary magnetic studies of a Co12 wheel are reported; the magnetic investigations reveal that the electronic ground state has a spin S = 6, which corresponds to ferromagnetic interactions between the twelve Co(II) ions.

Octanuclear cobalt and nickel cages featuring formate ligands.

Two new octanuclear cages are reported which feature formate as a bridging carboxylate which has been formed in situ from decomposition of triphenylacetate used as a ligand.

Tuning the composition of biocompatible Gd nanohydrogels to achieve hypersensitive dual T1/T2 MRI contrast agents.

A series of hydrogel nanoparticles incorporating MRI contrast agents (GdDOTP and MS325) as potential cross-linkers were elaborated by an easy and robust ionotropic gelation process. By this process, high Gd loadings were obtained (between 1.8 and 14.5 × 104 Gd centres per NP). By tuning the cross-linker ionization degree and the nature of the polymer matrix it was possible to boost the r1 relaxivity per Gd centre up to 22-fold. The greatest gains in relaxivity were observed for nanogels for which the polymer matrix was constituted of chitosan and hyaluronan. Relaxivities per Gd centre as high as 100 s-1 mM-1 at 30 MHz can be reached, which highlighted the fact that molecular motion of the Gd chelate was effectively restricted and water access to the inner core of these nanogels was not limited.

A benzimidazole functionalised DO3A chelator showing pH switchable coordination modes with lanthanide ions.

The synthesis of a new macrocyclic chelator incorporating a benzimidazole heterocycle is reported. Lanthanide complexes with macrocyclic chelators based on 1,4,7,10-tetra(carboxymethyl)-1,4,7,10-tetraazacyclododecane (DOTA) and 1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane (DO3A) are of interest in luminescent, radiopharmaceutical and magnetic resonance (MR) biomedical imaging applications. The benzimidazole DO3A chelator allows for sensitisation of europium(iii), terbium(iii) and ytterbium(iii) luminescence by the heterocycle and also shows a pH dependent coordination change due to protonation of the chelator (pKa = 4.1 for the europium(iii) complex). The thermodynamic stability of the complexes has been investigated by potentiometric titration with the gadolinium(iii) complex showing significantly higher stability than the zinc(ii) complex, where log ßZnLH = 28.1 and log ßGdLH = 32.1.

A modified cyclen azaxanthone ligand as a new fluorescent probe for Zn2+.

A new cyclen derivative L, bearing a methyl-chromeno-pyridinylidene hydrazone moiety, was synthesized and studied in MeOH, as potential fluorescent "OFF-on-ON" sensors for Zn(ii). Photophysical properties of this ligand being PET regulated, L was only weakly emissive in the absence of metal ions (OFF). L fluorescence was increased modestly upon addition of one equivalent of Zn(II), and further increased upon addition of a second equivalent. Therefore, Zn : L behaved as a highly sensitive ON sensor for zinc. This efficiency was correlated to Zn(II) coordination via the hydrazone moiety of the fluorophore, producing an efficient CHelation-Enhanced Fluorescence (CHEF) effect. A complementary theoretical study carried out with DFT calculations further elucidated the optical properties.

Enhancing U(eff) in oxime-bridged [Mn(III)6Ln(III)2] hexagonal prisms.

The first 3d-4f clusters built using derivatised salicylaldoximes (R-saoH(2)) describe unusual hexagonal prisms. Replacement of the paramagnetic Gd(III) ions with diamagnetic Ln(III) ions allows for a more thorough understanding of the magnetic properties, whilst replacement with Tb(III) doubles U(eff).

Modification of the Stöber process by a polyazamacrocycle leading to unusual core-shell silica nanoparticles.

In the view of designing functional nanoparticles, the encapsulation of 1,4,7,10-tetraazacyclododecane (cyclen) within silica nanoparticles using the Stöber process was studied. In the presence of cyclen and tetraethoxysilane (TEOS), silica particles exhibiting an unusual core-shell structure were obtained. On then basis of TEM, DLS, and NMR data, we suggest that the particle core is constituted of hybrid primary nanoparticles resulting from cyclen-silica interactions, whereas the shell formation results from further condensation of unreacted silica precursors. Control experiments performed with the zinc-cyclen complex and ammonia addition suggest that cyclen-TEOS interactions arise from the activation of the silicon alkoxide hydrolysis with the polyazamacrocycle amine groups. These data are discussed in the context of silica biomineralization mechanisms, where polyamine/silica interactions have been shown to play a major role. Moreover, the possibility to control the size and the structure of these nanoparticles makes them promising materials for pharmaceutical applications.

Studies of a nickel-based single-molecule magnet.

A cyclic complex [Ni(12)(chp)(12)(O(2)CMe)(12)(thf)(6)(H(2)O)(6)] (1) has been synthesised and studied (chp=6-chloro-2-pyridonate). Complex 1 exhibits ferromagnetic exchange between the S=1 centres, giving an S=12 spin ground state. Detailed studies demonstrate that it is a single-molecule magnet with an energy barrier of approximately 10 K for reorientation of magnetisation. Resonant quantum tunnelling is also observed. The field between resonances allows accurate measurement of D, which is 0.067 K. Inelastic neutron scattering studies have allowed exchange parameters to be derived accurately, which was impossible from susceptibility data alone. Three exchange interactions are required: two ferromagnetic nearest neighbour interactions of approximately 11 and 2 cm(-1) and an anti-ferromagnetic next nearest neighbour interaction of -0.9 cm(-1).