Rational design of near-infrared fluorescent probes for superoxide anion radical: Enhancement of self-stability and sensitivity by self-immolative linker.

Fluorescent imaging of cellular superoxide anion radical (O2•-) is of great significance to investigate reactive oxygen species-related pathophysiological processes and drug metabolism. However, the application of this technique is far away from maximum partially due to the lack of suitable probes. In this work, we propose a new strategy for design of near-infrared (NIR) O2•- fluorescent probes in which p-cresol is used as a self-immolative linker to conjugate the NIR fluorophore DDAO (9H-1,3-Dichloro-7-hydroxy-9,9-dimethylacridine-2-one) with the O2•--sensing group (i.e., trifluoromethanesulfonate). The introduction of self-immolative linker effectively increases the self-stability of these probes under physiological conditions. Importantly, the electron-withdrawing halogen substituents on the linker greatly enhance the sensitivity of the probes to O2•-. As such, the representative probe DLS4 exhibits high self-stability over a broad range of pHs (5.0-8.5), high selectivity as well as excellent sensitivity to O2•- with a detection limit (LOD) of 7.3 nM and 720-fold fluorescence enhancement upon reaction with O2•-. Moreover, DLS4 enables imaging of O2•- generation in PMA-stimulated RAW 264.7 cells and HeLa cells, and the fluorescence intensities are proportional to the PMA concentrations. In addition, the doxorubicin-induced cytotoxicity of H9c2 cells was also evaluated using DLS4. The present study provides a novel strategy for molecular design of small-molecule O2•- fluorescent probes and the resulting probes show great potential as reliable tools to study the development and progression of O2•--related diseases and drug metabolism in various systems.

Intracellular delivery of liposome-encapsulated Finland trityl radicals for EPR oximetry.

Tetrathiatriarylmethyl (TAM, trityl) radicals have found wide applications in electron paramagnetic resonance (EPR) oximetry. However, the biomedical applications of TAM radicals were exclusively limited to an extracellular region owing to their negatively charged nature. The intracellular delivery of TAM radicals still remains a challenge. In the present work, we report a liposome-based method to encapsulate the water-soluble Finland trityl radical CT-03 for its intracellular delivery. Using the thin lipid film hydration method, CT-03-loaded liposomes were prepared from DSPC/cholesterol/DOTAP with a mean size of 167.5 ± 2.4 nm and a zeta potential of 27.8 ± 0.8 mV. EPR results showed that CT-03 was entrapped into the liposomes and still exhibited good oxygen (O2) sensitivity. Moreover, CT-03 was successfully delivered into HepG2 cells and HUVECs using the CT-03-loaded liposomes. Importantly, the combination of the liposome-encapsulated radical CT-03 and the other TAM radical CT02-H enabled simultaneous measurements of the intracellular and extracellular O2 concentrations and O2 consumption rates in HepG2 cells. Our present study provides a new approach for intracellular delivery of TAM radicals and could significantly expand their biomedical applications.

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.

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.