New Biocompatible ß-Phosphorylated Linear Nitrones Targeting Mitochondria: Protective Effect in Apoptotic Cells.

The mitochondrion, an essential organelle involved in cellular respiration, energy production, and cell death, is the main cellular source of reactive oxygen species (ROS), including superoxide. Mitochondrial diseases resulting from uncontrolled/excess ROS generation are an emerging public health concern and there is current interest in specific mitochondriotropic probes to get information on in-situ ROS production. As such, nitrones vectorized by the triphenylphosphonium (TPP) cation have recently drawn attention despite reported cytotoxicity. Herein, we describe the synthesis of 13 low-toxic derivatives of N-benzylidene-1-diethoxyphosphoryl-1-methylethylamine N-oxide (PPN) alkyl chain-grafted to a pyridinium, triethylammonium or berberinium lipophilic cation. These nitrones showed in-vitro superoxide quenching activity and EPR/spin-trapping efficiency towards biologically relevant free radicals, including superoxide and hydroxyl radicals. Their mitochondrial penetration was confirmed by 31 P NMR spectroscopy, and their anti-apoptotic properties were assessed in Schwann cells treated with hydrogen peroxide. Two pyridinium-substituted PPNs were identified as potentially better alternatives to TPP nitrones conjugates for studying mitochondrial oxidative damage.

Highly bioresistant, hydrophilic and rigidly linked trityl-nitroxide biradicals for cellular high-field dynamic nuclear polarization.

Cellular dynamic nuclear polarization (DNP) has been an effective means of overcoming the intrinsic sensitivity limitations of solid-state nuclear magnetic resonance (ssNMR) spectroscopy, thus enabling atomic-level biomolecular characterization in native environments. Achieving DNP signal enhancement relies on doping biological preparations with biradical polarizing agents (PAs). Unfortunately, PA performance within cells is often limited by their sensitivity to the reductive nature of the cellular lumen. Herein, we report the synthesis and characterization of a highly bioresistant and hydrophilic PA (StaPol-1) comprising the trityl radical OX063 ligated to a gem-diethyl pyrroline nitroxide via a rigid piperazine linker. EPR experiments in the presence of reducing agents such as ascorbate and in HeLa cell lysates demonstrate the reduction resistance of StaPol-1. High DNP enhancements seen in small molecules, proteins and cell lysates at 18.8 T confirm that StaPol-1 is an excellent PA for DNP ssNMR investigations of biomolecular systems at high magnetic fields in reductive environments.

EPR spectroscopic evidence of iron-catalysed free radical formation in chronic mountain sickness: Dietary causes and vascular consequences.

Chronic mountain sickness (CMS) is a high-altitude (HA) maladaptation syndrome characterised by elevated systemic oxidative-nitrosative stress (OXNOS) due to a free radical-mediated reduction in vascular nitric oxide (NO) bioavailability. To better define underlying mechanisms and vascular consequences, this study compared healthy male lowlanders (80 m, n = 10) against age/sex-matched highlanders born and bred in La Paz, Bolivia (3600 m) with (CMS+, n = 10) and without (CMS-, n = 10) CMS. Cephalic venous blood was assayed using electron paramagnetic resonance spectroscopy and reductive ozone-based chemiluminescence. Nutritional intake was assessed via dietary recall. Systemic vascular function and structure were assessed via flow-mediated dilatation, aortic pulse wave velocity and carotid intima-media thickness using duplex ultrasound and applanation tonometry. Basal systemic OXNOS was permanently elevated in highlanders (P = <0.001 vs. lowlanders) and further exaggerated in CMS+, reflected by increased hydroxyl radical spin adduct formation (P = <0.001 vs. CMS-) subsequent to liberation of free 'catalytic' iron consistent with a Fenton and/or nucleophilic addition mechanism(s). This was accompanied by elevated global protein carbonylation (P = 0.046 vs. CMS-) and corresponding reduction in plasma nitrite (P = <0.001 vs. lowlanders). Dietary intake of vitamins C and E, carotene, magnesium and retinol were lower in highlanders and especially deficient in CMS + due to reduced consumption of fruit and vegetables (P = <0.001 to 0.028 vs. lowlanders/CMS-). Systemic vascular function and structure were also impaired in highlanders (P = <0.001 to 0.040 vs. lowlanders) with more marked dysfunction observed in CMS+ (P = 0.035 to 0.043 vs. CMS-) in direct proportion to systemic OXNOS (r = -0.692 to 0.595, P = <0.001 to 0.045). Collectively, these findings suggest that lifelong exposure to iron-catalysed systemic OXNOS, compounded by a dietary deficiency of antioxidant micronutrients, likely contributes to the systemic vascular complications and increased morbidity/mortality in CMS+. TRIAL REGISTRY: ClinicalTrials.gov; No: NCT01182792; URL: www.clinicaltrials.gov.

Highly Efficient Trityl-Nitroxide Biradicals for Biomolecular High-Field Dynamic Nuclear Polarization.

Dynamic nuclear polarization (DNP) is a powerful method to enhance the sensitivity of solid-state magnetic nuclear resonance (ssNMR) spectroscopy. However, its biomolecular applications at high magnetic fields (preferably>14 T) have so far been limited by the intrinsically low efficiency of polarizing agents and sample preparation aspects. Herein, we report a new class of trityl-nitroxide biradicals, dubbed SNAPols that combine high DNP efficiency with greatly enhanced hydrophilicity. SNAPol-1, the best compound in the series, shows DNP enhancement factors at 18.8 T of more than 100 in small molecules and globular proteins and also exhibits strong DNP enhancements in membrane proteins and cellular preparations. By integrating optimal sensitivity and high resolution, we expect widespread applications of this new polarizing agent in high-field DNP/ssNMR spectroscopy, especially for complex biomolecules.

Synthesis and Redox Properties of Water-Soluble Asymmetric Trityl Radicals.

Tetrathiatriarylmethyl (trityl) radicals have been recently shown to react with biological oxidoreductants including glutathione (GSH), ascorbic acid (Asc), and superoxide anion radical (O2•-). However, how the substituents affect the reactivity of trityl radicals is still unknown. In this work, five asymmetric trityl radicals were synthesized and their reactivities with GSH, Asc, and O2•- investigated. Under aerobic conditions, GSH induces fast decays for the thioether- (TSA) and N-methyleneglycine-substituted (TGA) derivatives and slow decay for the 4-carboxyphenyl-containing one (TPA). Under anaerobic conditions, the direct reduction of these radicals by GSH also occurs with rate constants (kGSH) from 1.8 × 10-4 M-1 s-1 for TPA to 1.0 × 10-2 M-1 s-1 for TGA. Moreover, these radicals can also react with O2•- with rate constants (kSO) from 1.2 × 103 M-1 s-1 for ET-01 to 1.6 × 104 M-1 s-1 for TGA. Surprisingly, these radicals are completely inert to Asc in both aerobic and anaerobic conditions. Additionally, the substituents exert an important effect on redox potentials of these trityl radicals. This work demonstrates that the redox properties of the trityl radicals strongly depend on their substituents, and TPA with high stability toward GSH shows great potential for intracellular applications.

In situ simultaneous electrochemical ESR study of radicals generated from 2,2-dinitroethene-1,1-diamine (FOX-7). Intramolecular chemical exchange resulting in an alternation line-width effect.

The molecule of 2,2-dinitroethene-1,1-diamine (FOX-7) is one of the most interesting molecules with multiple redox centres stabilized by push-pull effect. To reveal the detailed mechanism of its electrochemical process radical intermediates formed in the course of its electrochemical reduction in organic aprotic media have been studied by in situ simultaneous electrochemical ESR measurements (SEESR). The radical generated on the second reduction step possesses an alternating line-width (AL) effect in the ESR spectra as a result of intramolecular dynamic processes in the timescale of ESR splitting constants. The spectra measured at different temperatures (230-335 K) were analysed with the help of a fitting program which includes a molecular dynamics. Observed dynamics describes well an asymmetric 2-site exchange model for the whole temperature range. With help of the optimized parameters and quantum chemical calculations this radical has been identified as 2,2-dinitroethane-1-amine-1-imine radical dianion, [(H2N)(HN)C=C(NO2)2]2-. The dynamic process responsible for the AL effect consists of mutual turning (changing of dihedral angle) of the both nitro groups, resulting in an intramolecular spin-density (electron) transfer. The dynamic parameters of the process have been established.

Postmodification via Thiol-Click Chemistry Yields Hydrophilic Trityl-Nitroxide Biradicals for Biomolecular High-Field Dynamic Nuclear Polarization.

Dynamic nuclear polarization (DNP) is a powerful method to enhance nuclear magnetic resonance (NMR) signal intensities, enabling unprecedented applications in life and material science. An ultimate goal is to expand the use of DNP-enhanced solid-state NMR to ultrahigh magnetic fields where optimal spectral resolution and sensitivity are integrated. Trityl-nitroxide (TN) biradicals have attracted significant interest in high-field DNP, but their application to complex (bio)molecules has so far been limited. Here we report a novel postmodification strategy for synthesis of hydrophilic TN biradicals in order to improve their use in biomolecular applications. Initially, three TN biradicals (referred to as NATriPols 1-3) with amino-acid linkers were synthesized. EPR studies showed that the α-position of the amino-acid linkers is an ideal modification site for these biradicals since their electron-electron magnetic interactions are marginally affected by the substituents at this position. On the basis of this finding, we synthesized NATriPol-4 with pyridine disulfide appended at the α-position. Postmodification of NATriPol-4 via thiol-click chemistry resulted in various TN biradicals including hydrophilic NATriPol-5 in a quantitative manner. Interestingly, DNP enhancements at 18.8 T of NATriPols for 13C,15N-proline in a glycerol/water matrix are inversely correlated with their hydrophobicity. Importantly, applications of hydrophilic NATriPol-5 and NATriPol-3 to biomolecules including a globular soluble protein and a membrane targeting peptide reveal significantly improved performance compared to TEMTriPol-1 and AMUPol. Our work provides an efficient approach for one-step synthesis of new polarizing agents with tunable physicochemical properties, thus expediting optimization of new biradicals for biomolecular applications at ultrahigh magnetic fields.

Host-guest interaction of nitroxide radicals with water-soluble pillar[6]arenes.

The host-guest interaction of nitroxide radicals with water-soluble pillar[n]arenes was studied for the first time by electron paramagnetic resonance spectroscopy and NMR spectroscopy. Our results showed that this interaction strongly depended on the 4-substituents of nitroxides and the cavity size of pillar[n]arenes. The host-guest interaction with water-soluble pillar[6]arene WP6 effectively increased the thermodynamic and kinetic stability of nitroxide radical 4-AT toward ascorbic acid, thus expanding its potential biomedical applications.

EPR Spectroscopy: A Powerful Tool to Analyze Supramolecular Host•Guest Complexes of Stable Radicals with Cucurbiturils.

Stable organic free radicals are increasingly studied compounds due to the multiple and unusual properties imparted by the single electron(s). However, being paramagnetic, classical methods such as NMR spectroscopy can hardly be used due to relaxation and line broadening effects. EPR spectroscopy is thus better suited to get information about the immediate surroundings of the single electrons. EPR has enabled obtaining useful data in the context of host•guest chemistry, and a classical example is reported here for the stable (2,2,6,6-tetramethyl-4-oxo-piperidin-1-yl)oxyl or 4-oxo-TEMPO nitroxide (TEMPONE) inside the macrocycle host cucurbit[7]uril (CB[7]). Generally and also observed here, a contraction of the spectrum is observed as a result of the reduced nitrogen coupling constant due to inclusion complexation in the hydrophobic cavity of the host. Simulations of EPR spectra allowed determining the corresponding binding constant pointing to a weaker affinity for CB[7], compared to TEMPO with CB[7]. We complement this work by the results of EPR spectroscopy of a biradical: bis-TEMPO-bis-ketal (bTbk) with cucurbit[8]uril (CB[8]). Initial investigations pointed to very weak effects on the spectrum of the guest and incorrectly led us to conclude an absence of binding. However, simulations of EPR spectra combined with NMR data of reduced bTbk allowed showing inclusion complexation. EPR titrations were performed, and the corresponding binding constant was determined. 1H NMR spectra with reduced bTbk suggested a shuttle mechanism, at nearly one equivalent of CB[8], for which the host moves rapidly between two stations.

Discriminative Detection of Biothiols by Electron Paramagnetic Resonance Spectroscopy using a Methanethiosulfonate Trityl Probe.

Biothiols, such as glutathione (GSH), homocysteine (Hcy), and cysteine (Cys), coexist in biological systems with diverse biological roles. Thus, analytical techniques that can detect, quantify, and distinguish between multiple biothiols are desirable but challenging. Herein, we demonstrate the simultaneous detection and quantitation of multiple biothiols, including up to three different biothiols in a single sample, using electron paramagnetic resonance (EPR) spectroscopy and a trityl-radical-based probe (MTST). We term this technique EPR thiol-trapping. MTST could trap thiols through its methanethiosulfonate group to form the corresponding disulfide conjugate with an EPR spectrum characteristic of the trapped thiol. MTST was used to investigate effects of l-buthionine sulfoximine (BSO) and pyrrolidine dithiocarbamate (PDTC) on the efflux of GSH and Cys from HepG2 cells.

Membrane-specific spin trap, 5-dodecylcarbamoyl-5-N-dodecylacetamide-1-pyroline-N-oxide (diC12PO): theoretical, bioorthogonal fluorescence imaging and EPR studies.

Membranous organelles are major endogenous sources of reactive oxygen and nitrogen species. When present at high levels, these species can cause macromolecular damage and disease. To better detect and scavenge free radical forms of the reactive species at their sources, we investigated whether nitrone spin traps could be selectively targeted to intracellular membranes using a bioorthogonal imaging approach. Electron paramagnetic resonance imaging demonstrated that the novel cyclic nitrone 5-dodecylcarbamoyl-5-N-dodecylacetamide-1-pyroline-N-oxide (diC12PO) could be used to target the nitrone moiety to liposomes composed of phosphatidyl choline. To test localization with authentic membranes in living cells, fluorophores were introduced via strain-promoted alkyne-nitrone cycloaddition (SPANC). Two fluorophore-conjugated alkynes were investigated: hexynamide-fluoresceine (HYA-FL) and dibenzylcyclooctyne-PEG4-5/6-sulforhodamine B (DBCO-Rhod). Computational and mass spectrometry experiments confirmed the cycloadduct formation of DBCO-Rhod (but not HYA-FL) with diC12PO in cell-free solution. Confocal microscopy of bovine aortic endothelial cells treated sequentially with diC12PO and DBCO-Rhod demonstrated clear localization of fluorescence with intracellular membranes. These results indicate that targeting of nitrone spin traps to cellular membranes is feasible, and that a bioorthogonal approach can aid the interrogation of their intracellular compartmentalization properties.

Embedding cyclic nitrone in mesoporous silica particles for EPR spin trapping of superoxide and other radicals.

The generation of superoxide radical anion in biological systems is one of the major initiating events in the redox biology of NADPH oxidases and mitochondrial redox signalling. However, the pallette of chemical tools for superoxide detection is very limited, hampering progress in understanding the chemical biology of superoxide. Although EPR spin trapping is regarded as the most rigorous technique for superoxide detection, rapid reduction of the EPR-active superoxide spin adducts to EPR-silent hydroxylamines, or to hydroxyl radical adducts by bioreductants, significantly limits the applicability of this technique in biological systems. To overcome these limitations, in this work, we report the synthesis and characterization of a new mesoporous silica functionalized with a phosphorylated cyclic spin trap (DIPPMPO nitrone). The DIPPMPO-grafted silica is a versatile spin-trap agent enabling the identification of a wide range of carbon or oxygen-centered transient radicals in organic and in aqueous media. Moreover, superoxide was efficiently trapped under in vitro conditions in both cell-free and cellular systems. The generated superoxide adduct exhibited an exceptional half-life of 3.5 h and a resistance toward bioreductant agents such as glutathione for several hours.

Triangular Regulation of Cucurbit[8]uril 1:1 Complexes.

Triangular shapes have inspired scientists over time and are common in nature, such as the flower petals of oxalis triangularis, the triangular faces of tetrahedrite crystals, and the icosahedron faces of virus capsids. Supramolecular chemistry has enabled the construction of triangular assemblies, many of which possess functional features. Among these structures, cucurbiturils have been used to build supramolecular triangles, and we recently reported paramagnetic cucurbit[8]uril (CB[8]) triangles, but the reasons for their formation remain unclear. Several parameters have now been identified to explain their formation. At first sight, the radical nature of the guest was of prime importance in obtaining the triangles, and we focused on extending this concept to biradicals to get supramolecular hexaradicals. Two sodium ions were systematically observed by ESI-MS in trimer structures, and the presence of Na+ triggered or strengthened the triangulation of CB[8]/guest 1:1 complexes in solution. X-ray crystallography and molecular modeling have allowed the proposal of two plausible sites of residence for the two sodium cations. We then found that a diamagnetic guest with an H-bond acceptor function is equally good at forming CB[8] triangles. Hence, a guest molecule containing a ketone function has been precisely triangulated thanks to CB[8] and sodium cations as determined by DOSY-NMR and DLS. A binding constant for the triangulation of 1:1 to 3:3 complexes is proposed. This concept has finally been extended to the triangulation of ditopic guests toward network formation by the reticulation of CB[8] triangles using dinitroxide biradicals.

Diastereoisomers of l-proline-linked trityl-nitroxide biradicals: synthesis and effect of chiral configurations on exchange interactions.

The exchange (J) interaction of organic biradicals is a crucial factor controlling their physiochemical properties and potential applications and can be modulated by changing the nature of the linker. In the present work, we for the first time demonstrate the effect of chiral configurations of radical parts on the J values of trityl-nitroxide (TN) biradicals. Four diastereoisomers (TNT1, TNT2, TNL1 and TNL2) of TN biradicals were synthesized and purified by the conjugation of a racemic (R/S) nitroxide with the racemic (M/P) trityl radical vial-proline. The absolute configurations of these diastereoisomers were assigned by comparing experimental and calculated electronic circular dichroism (ECD) spectra as (M, S, S) for TNT1, (P, S, S) for TNT2, (M, S, R) for TNL1 and (P, S, R) for TNL2. Electron paramagnetic resonance (EPR) results showed that the configuration of the nitroxide part instead of the trityl part is dominant in controlling the exchange interactions and the order of the J values at room temperature is TNT1 (252 G) > TNT2 (127 G) ≫ TNL2 (33 G) > TNL1 (14 G). Moreover, the J values of TNL1/TNL2 with the S configuration in the nitroxide part vary with temperature and the polarity of solvents due to their flexible linker, whereas the J values of TNT1/TNT2 are almost insensitive to these two factors due to the rigidity of their linkers. The distinct exchange interactions between TNT1,2 and TNL1,2 in the frozen state led to strongly different high-field dynamic nuclear polarization (DNP) enhancements with ε = 7 for TNT1,2 and 40 for TNL1,2 under 800 MHz DNP conditions.

Synthesis and Characterization of the Perthiatriarylmethyl Radical and Its Dendritic Derivatives with High Sensitivity and Selectivity to Superoxide Radical.

EPR spectroscopy, coupled with the use of tetrathiatriarylmethyl (TAM) radicals, has been a reliable method to detect the superoxide radical (O2.- ). However, the specificity and biocompatibility of TAM radicals need to be further improved. Although derivatization may overcome the drawbacks of current TAM radicals, esterification or amidation through the carboxylic groups greatly changes their redox properties and makes them inert to O2.- . Herein, the synthesis of a perthiatriarylmethyl (PST) radical and its dendritic derivatives, PST-TA and PST-NA, in which PST is covalently linked with dendrons containing three (TA) and nine (NA) carboxylic acids, respectively. The results show that PST rapidly reacts with O2.- to yield a unique quinone methide product. Dendritic modification of PST slightly decreases the reactivities of PST-TA and PST-NA, but notably increases their biostability toward various oxidoreductants. The detection limit of PST-NA to O2.- was estimated to be 2.1 nm min-1 over 60 min of detection. Importantly, PST-NA shows threefold higher sensitivity to O2.- in the presence and absence of ascorbic acid than that of the classic spin-trapping technique. In addition, the application of PST-NA to detect extracellular O2.- generation in stimulated RAW 264.7 macrophages was also explored. This study demonstrates that PST-NA has great potential for specific detection and quantitation of O2.- in extracellular sites.

Thiol-Dependent Reduction of the Triester and Triamide Derivatives of Finland Trityl Radical Triggers O2-Dependent Superoxide Production.

Tetrathiatriaylmethyl (trityl) radicals have found wide biomedical applications as magnetic resonance probes. Trityl radicals and their derivatives are generally stable toward biological reducing agents such as glutathione (GSH) and ascorbate. We demonstrate that the triester (ET-03) and triamide (AT-03) derivatives of the Finland trityl radical exhibit unique reduction by thiols such as GSH and cysteine (Cys) to generate the corresponding trityl carbanions as evidenced by the loss of EPR signal and appearance of characteristic UV-vis absorbance at 644 nm under anaerobic conditions. The trityl carbanions can be quickly converted back to the original trityl radicals by oxygen (O2) in air, thus rendering the reaction between the trityl derivative and biothiol undetectable under aerobic conditions. The reduction product of O2 by the trityl carbanions was shown to be superoxide radical (O2•-) by EPR spin-trapping. Kinetic studies showed that the reaction rate constants (k) depend on the types of both trityl radicals and thiols with the order of kET-03/Cys (0.336 M-1 s-1) > kET-03/GSH (0.070 M-1 s-1) > kAT-03/Cys (0.032 M-1 s-1) > kAT-03/GSH (0.027 M-1 s-1). The reactivity of trityl radicals with thiols is closely related to the para-substituents of trityl radicals as well as the pKa of the thiols and is further reflected by the rate of O2•- production and consumptions of O2 and thiols. This novel reaction represents a new metabolic process of trityl derivatives and should be considered in the design and application of new trityl radical probes.

Synthesis and properties of a series of ß-cyclodextrin/nitrone spin traps for improved superoxide detection.

Three new DEPMPO-based spin traps have been designed and synthesized for improved superoxide detection, each carrying a cyclodextrin (CD) moiety but with a different alkyl chain on the phosphorus atom or with a long spacer arm. EPR spectroscopy allowed us to estimate the half-life of the superoxide spin adducts which is close to the value previously reported for CD-DEPMPO (t1/2 ≈ 50-55 min under the conditions investigated). The spectra are typical of superoxide adducts (almost no features of the HO˙ adduct that usually forms with time for other nitrone spin traps such as DMPO) and we show that at 250 µM, the new spin trap enables the reliable detection of superoxide by 1 scan at the position opposite to the corresponding spin trap without the CD moiety. The resistance of the spin adducts to a reduction process has been evaluated, and the superoxide spin adducts are sensitive to ascorbate and glutathione (GSH), but not to glutathione peroxidase/GSH, reflecting the exposed nature of the nitroxide moiety to the bulk solvent. To understand these results, 2D-ROESY NMR studies and molecular dynamics pointed to a shallow or surface self-inclusion of the nitrone spin traps and of nitroxide spin adducts presumably due to the high flexibility of the permethyl-ß-CD rim.

Exploring the boundaries of direct detection and characterization of labile isomers - a case study of copper(ii)-dipeptide systems.

The investigation of the linkage isomers of biologically essential and kinetically labile metal complexes in aqueous solutions poses a challenge, as these microspecies cannot be separately studied. Therefore, derivatives are commonly used to initially determine the stability or spectral characteristics of at least one of the isomers. Here we directly detect the isomers, describe the metal ion coordination sphere, speciation and thermodynamic parameters by a synergistic application of temperature dependent EPR and CD spectroscopic measurements in copper(ii)-dipeptide systems including His-Gly and His-Ala ligands. The ΔH = (-23 ± 4) kJ mol-1 value of the standard enthalpy change corresponding to the peptide-type to histamine-type isomerisation equilibrium of the [CuL]+ complex was corroborated by several techniques. The preferential coordination of the side-chains was observed at lower temperatures, whereas, metal-binding of the backbone atoms became favourable upon increasing temperature. This study exemplifies the necessity of using temperature dependent multiple methodologies for a reliable description of similar systems for upstream applications.

New Amino-Acid-Based ß-Phosphorylated Nitroxides for Probing Acidic pH in Biological Systems by EPR Spectroscopy.

There is increasing interest in measuring pH in biological samples by using nitroxides with pH-dependent electron paramagnetic resonance (EPR) spectra. Aiming to improve the spectral sensitivity (ΔaX ) of these probes (i.e., the difference between the EPR hyperfine splitting (hfs) in their protonated and unprotonated forms), we characterized a series of novel linear α-carboxy, α'-diethoxyphosphoryl nitroxides constructed on an amino acid core and featuring an (α or α')-C-H bond. In buffer, the three main hfs (aN , aH , and aP ) of their EPR spectra vary reversibly with pH and, from aP or aH titration curves, a two- to fourfold increase in sensitivity was achieved compared to reference imidazoline or imidazolidine nitroxides. The crystallized carboxylate 10 b (pKa ≈3.6), which demonstrated low cytotoxicity and good resistance to bioreduction, was applied to probe stomach acidity in rats. The results pave the way to a novel generation of highly sensitive EPR pH markers.

Biocompatibility and antibacterial activity of nitrogen-doped titanium dioxide nanoparticles for use in dental resin formulations.

The addition of antibacterial functionality to dental resins presents an opportunity to extend their useful lifetime by reducing secondary caries caused by bacterial recolonization. In this study, the potential efficacy of nitrogen-doped titanium dioxide nanoparticles for this purpose was determined. Nitrogen doping was carried out to extend the ultraviolet absorbance into longer wavelength blue light for increased biocompatibility. Titanium dioxide nanoparticles (approximately 20-30 nm) were synthesized with and without nitrogen doping using a sol-gel method. Ultraviolet-Visible spectroscopy indicated a band of trap states, with increasing blue light absorbance as the concentration of the nitrogen dopant increased. Electron paramagnetic resonance measurements indicated the formation of superoxide and hydroxyl radicals upon particle exposure to visible light and oxygen. The particles were significantly toxic to Escherichia coli in a dose-dependent manner after a 1-hour exposure to a blue light source (480 nm). Intracellular reactive oxygen species assay demonstrated that the particles caused a stress response in human gingival epithelial cells when exposed to 1 hour of blue light, though this did not result in detectable release of cytokines. No decrease in cell viability was observed by water-soluble tetrazolium dye assay. The results show that nitrogen-doped titanium dioxide nanoparticles have antibacterial activity when exposed to blue light, and are biocompatible at these concentrations.