M. Francesca Ottaviani

Redox-responsive branched-bottlebrush polymers for in vivo MRI and fluorescence imaging

Stimuli-responsive multimodality imaging agents have broad potential in medical diagnostics. Herein, we report the development of a new class of branched-bottlebrush polymer dual-modality organic radical contrast agents--ORCAFluors--for combined magnetic resonance and near-infrared fluorescence imaging in vivo. These nitroxide radical-based nanostructures have longitudinal and transverse relaxation times that are on par with commonly used heavy-metal-based magnetic resonance imaging (MRI) contrast agents. Furthermore, these materials display a unique compensatory redox response: fluorescence is partially quenched by surrounding nitroxides in the native state; exposure to ascorbate or ascorbate/glutathione leads to nitroxide reduction and a concomitant 2- to 3.5-fold increase in fluorescence emission. This behaviour enables correlation of MRI contrast, fluorescence intensity and spin concentration with tissues known to possess high concentrations of ascorbate in mice. Our in vitro and in vivo results, along with our modular synthetic approach, make ORCAFluors a promising new platform for multimodality molecular imaging.

An EPR and NMR study of supramolecular effects on paramagnetic interaction between a nitroxide incarcerated within a nanocapsule with a nitroxide in bulk aqueous media.

A 15N-labeled nitroxide was incarcerated into an octa acid nanocapsule, which was confirmed by 1H NMR and EPR spectroscopy. Electron paramagnetic interaction between the 15N-labeled incarcerated nitroxide and a 14N-labeled free nitroxide in the external aqueous solution was observed by EPR spectroscopy. The observation of spin-spin interaction, through the walls of the cancer and is reflected in the simultaneous line-broadening of both the 15N-labeled and 14N-labeled nitroxides. The computer-assisted analysis of the EPR data further provides direct information on the motion and the polarity of both the incarcerated paramagnetic nitroxide and the nitroxides in the external bulk aqueous phase. We also show how communication between an incarcerated guest and molecules in the bulk solvent can be enhanced or inhibited by supramolecular factors such as Coulombic attraction or repulsion between a charged guest@host complex (incarcerated 15N nitroxide) and charged molecules in the aqueous phase.

EPR study of spin labeled brush polymers in organic solvents.

Spin-labeled polylactide brush polymers were synthesized via ring-opening metathesis polymerization (ROMP), and nitroxide radicals were incorporated at three different locations of brush polymers: the end and the middle of the backbone, and the end of the side chains (periphery). Electron paramagnetic resonance (EPR) was used to quantitatively probe the macromolecular structure of brush polymers in dilute solutions. The peripheral spin-labels showed significantly higher mobility than the backbone labels, and in dimethylsulfoxide (DMSO), the backbone end labels were shown to be more mobile than the middle labels. Reduction of the nitroxide labels by a polymeric reductant revealed location-dependent reactivity of the nitroxide labels: peripheral nitroxides were much more reactive than the backbone nitroxides. In contrast, almost no difference was observed when a small molecule reductant was used. These results reveal that the dense side chains of brush polymers significantly reduce the interaction of the backbone region with external macromolecules, but allow free diffusion of small molecules.

EPR investigations on the formation of micelle-templated silica

Abstract The mechanism of formation of aluminium-containing micelle-templated silicas (MTS) was investigated by in situ EPR combined with XRD. Analysis of the spectra of EPR probes, 4-alkyldimethylammonium-2,2,6,6-tetramethyl-piperidine-1-oxyl (CATm), allowed the monitoring of the synthesis of MTS at 343 K in the presence of alkyltrimethylammonium bromide (C n H 2 n +1 N + (CH 3 ) 3 Br − = n TAB) surfactants with hydrocarbon chains from 8 to 18 carbon atoms. The kinetics of MTS formation is strongly dependent on the surfactant chain length, larger micelles inducing a faster MTS formation. Furthermore, the fraction of CATm probes strongly interacting with polar sites on the silica surface increases by increasing the chain length. The hydrophilic–hydrophobic balance of the surface was correlated with the structure of the hexagonal pores, allowing the characterization of hydrophobic corners and hydrophilic sides.

The dynamics of a nitroxide radical in water adsorbed on porous supports studied by ESR

Abstract The ESR of the neutral spin probe 2,2,6,6-tetramethyl-4-hydroxypiperidine-1-oxyl in the study of its dynamics of water adsorbed onto silica gels with pore diameters in the range 4 to 100 nm is described. The correlation times for the motion and their activation energies were calculated from the ESR linewidth at different temperatures. The mobility of the spin probe decreases with decreasing pore size, thus reflecting an increase in the average viscosity of the adsorbed water. Different behaviors are shown by water in silica gels with pore diameters 10–100 nm and in silica gels with p.d. 4 nm. The results obtained with the nitroxide are compared with those obtained with Cu(II) and Mn(II) on the same supports, and discussed in terms of the nature of the adsorbed water.

Aggregational process of the positively charged surfactants CTAC and CAT16 in the presence of starburst dendrimers: an electron paramagnetic resonance spectroscopic study

Abstract The aggregation process of positively charged surfactants (CAT16 alone and mixed CAT16-CTAB) in the presence of half generation polyamidoamine starburst dendrimers ( n .5-SBDs) was analyzed by means of a computer-aided analysis of the electron paramagnetic resonance (EPR) spectra. The partition of the probes in different environments was evaluated from the relative intensities of the corresponding EPR signals, which superimposed on each other to form the overall EPR line shape. Mobility and polarity parameters were evaluated for CAT16 monomers under the following conditions: (i) free in solution below the critical micellar concentration (cmc) of the surfactants; (ii) free in solution in equilibrium with surfactant aggregates (above their cmc); (iii) interacting as monomers with the n .5-SBDs (primary interactions). A parameter related to the local concentration (or the structural packing) of CAT16 was evaluated under the following conditions: (i) CAT16 aggregates; (ii) mixed CTAB-CAT16 aggregates. It was hypothesized that the formation of different supramolecular structures — SBDs/surfactants — varied as a function of the dendrimer size (generation) and the concentrations of both the dendrimer and the surfactant.

Interaction between encapsulated excited organic molecules and free nitroxides: communication across a molecular wall

Communication between two molecules, one confined and excited (triplet or singlet) and one free and paramagnetic, has been explored through quenching of fluorescence and/or phosphorescence by nitroxides as paramagnetic radical species. Quenching of excited states by nitroxides has been investigated in solution, and the mechanism is speculated to involve charge transfer and/or exchange processes, both of which require close orbital interaction between excited molecule and quencher. We show in this report that such a quenching, which involves electron-electron spin communication, can occur even when there is a molecular wall between the two. The excited state molecule is confined within an organic capsule made up of two molecules of a deep cavity cavitand, octa acid, that exists in the anionic form in basic aqueous solution. The nitroxide is kept free in aqueous solution. (1)H NMR and EPR experiments were carried out to ascertain the location of the two molecules. The distance between the excited molecule and the paramagnetic quencher was manipulated by the use of cationic, anionic, and neutral nitroxide and also by selectively including the cationic nitroxide within the cavity of cucurbituril. Results presented here highlight the role of the lifetime of the encounter complex in electron-electron spin communication when the direct orbital overlap between the two molecules is prevented by the intermediary wall.

Guest rotations within a capsuleplex probed by NMR and EPR techniques.

With the help of (1)H NMR and EPR techniques, we have probed the dynamics of guest molecules included within a water-soluble deep cavity cavitand known by the trivial name octa acid. All guest molecules investigated here form 2:1 (host/guest) complexes in water, and two host molecules encapsulate the guest molecule by forming a closed capsule. We have probed the dynamics of the guest molecule within this closed container through (1)H NMR and EPR techniques. The timescales offered by these two techniques are quite different, millisecond and nanosecond, respectively. For EPR studies, paramagnetic nitroxide guest molecules and for (1)H NMR studies, a wide variety of structurally diverse neutral organic guest molecules were employed. The guest molecules freely rotate along their x axis (long molecular axis and magnetic axis) on the NMR timescale; however, their rotation is slowed with respect to that in water on the EPR timescale. Rotation along the x axis is dependent on the length of the alkyl chain attached to the nitroxide probe. Overall rotation along the y or z axis was very much dependent on the structure of the guest molecule. The guests investigated could be classified into three groups: (a) those that do not rotate along the y or z axis both at room and elevated (55 degrees C) temperatures, (b) those that rotate freely at room temperature, and (c) those that do not rotate at room temperature but do so at higher temperatures. One should note that rotation here refers to the NMR timescale and it is quite possible that all molecules may rotate at much longer timescales than the one probed here. A slight variation in structure alters the rotational mobility of the guest molecules.

Self aggregation of supramolecules of nitroxides@cucurbit[8]uril revealed by EPR spectra.

Supramolecular complexation behavior of cucurbiturils with paramagnetic nitroxide spin probes was examined by (1)H NMR, X-ray diffraction studies of crystals, computation, and EPR. Both cucurbit[7]uril (CB7) and cucurbit[8]uril (CB8) form a 1:1 complex with 4-(N,N,N-trimethylammonium)-2,2,6,6-tetramethylpiperidinyl-N-oxy bromide (CAT1). The structure of the complex in the solid state was inferred by X-ray diffraction studies and in the gas phase by computation (B3LYP/6-31G(d)). Whereas ESI-MS data provided evidence for the existence of the complex in solution, indirect evidence was obtained through (1)H NMR studies with a structural diamagnetic analogue, 4-(N,N,N-trimethylammonium)-2,2,6,6-tetramethyl-N-methylpiperidine iodide (DCAT1). The EPR spectrum of the CAT1@CB7 complex consisting of three lines suggested that probe CAT1 is associated with host CB7 such that the nitroxide part is exposed to water. The spectral pattern was independent of the concentration of the complex and the presence of salt such as NaCl. The most interesting observation was made with CB8 as the host. In this case, in addition to the expected three-line spectrum, an additional spectrum consisting of seven lines was recorded. The contribution of the seven-line spectrum to the total spectrum was dependent on the concentration of the complex and added salt (NaCl) to the aqueous solution. The coupling constant for the seven-line spectrum for (14)N-substituted CAT1 is 5 G, and that for the four-line spectrum for (15)N-substituted CAT1 is 7.15 G. The only manner by which we could reproduce the observed spectra by simulation for both (14)N- and (15)N-substituted CAT1@CB8 was by assuming a spin exchange among three nitroxide radicals. To account for this observation, we hypothesize that three CAT1 molecules included within CB8 interact in such a way that there is an association of three supramolecules of CAT1@CB8 (i.e., [CAT1@CB8](3)) in a triangular geometry that leads to spin exchange between the three radical centers. We have established, with the help of 13 additional examples, that this is a general phenomenon. We are in the process of understanding this unusual phenomenon.

Time Evolution of the Aggregation Process of Peptides Involved in Neurodegenerative Diseases and Preventing Aggregation Effect of Phosphorus Dendrimers Studied by EPR

A key pathological event of prion and Alzheimer diseases is the formation of prion and amyloid plaques generated by peptide aggregation in the form of fibrils. Dendrimers have revealed their ability to prevent fibril formation and therefore cure neurodegenerative diseases. To provide information about the kinetics and the mechanism of peptide fibril formation and about the ability of the dendrimers to prevent peptide aggregation, we performed a computer-aided EPR analysis of the selected nitroxide spin probe 4-octyl-dimethylammonium,2,2,6,6-tetramethyl-piperidine-1-oxyl bromide (CAT8) in water solutions of the β-amyloid peptide Aβ 1-28 and the prion peptide PrP 185-208, which contain the fibril nucleation sites, in the absence and in the presence of phosphorus dendrimers. After a careful selection of the experimental conditions that allow aggregation to occur and to be monitored by EPR analysis over time, it was found that the Aβ 1-28 fibrils formed in 220 min at 0.5 mM peptide, 0.05 mM CAT8, 0.04 mg/mL heparin, and pH = 5. As a consequence, the interacting sites available for cooperative interactions with CAT8 were engaged in the peptide-peptide interactions and a fraction of the probe was extracted in the fluid fibril/water interphase, while another fraction was trapped at the peptide/peptide interphase, showing a decrease in mobility. Conversely, in the presence of the dendrimer (at the selected, after several trials, peptide/dendrimer molar ratio = 50), due to dipole-dipole interactions with peptide monomers, the probe remained at the dendrimer/peptide interphase and the spectral parameters negligibly changed over time. A fraction of probes inserted in PrP 185-208 low-packed aggregates and monitored their fast formation after 90 min. However, the binding organization of the prion peptide negligibly changed upon aggregation in comparison to Aβ 1-28. It is proposed that dendrimers mainly interfere in the lag (nucleation) phase of the prion peptide.