Acid-Promoted D-A-D Type Far-Red Fluorescent Probe with High Photostability for Lysosomal Nitric Oxide Imaging

Abstract

To accurately monitor the variations of lysosomal nitric oxide (NO) under physiological condition remains a great challenge for understanding the biological function of NO. Herein, we developed a new chemotype probe, namely, MBTD, for acid-promoted and far-red fluorescence imaging of lysosomal NO in vitro and ex vivo. MBTD was rationally designed by incorporating o-phenylenediamino (OPD) moiety into the donor-acceptor-donor (D-A-D) type fluorophore based on a dual intramolecular charge transfer (ICT) mechanism. Compared to previously reported OPD-based NO probes, MBTD displays several distinct advantages including large stroke shift, huge on-off ratio with minimal autofluorescence, and high NO specificity. Particularly, MBTD exhibits an acid-promoted response to NO with high acid tolerance, which greatly improves the spatial resolution to lysosomal NO by excluding the background noise from other nonacidic organelles. Furthermore, MBTD displayed much longer-lived and more stable fluorescence emission in comparison with the commercialized NO probe. MBTD was employed for ratiometric examination of the exogenous or endogenous NO of macrophages. More importantly, MBTD was able to detect the variation of lysosomal NO level in an acute liver injury mouse model ex vivo, implying the potential of MBTD for real-time monitoring the therapeutic efficacy of drug candidates for the treatment of acute liver injury. MBTD as a novel type of NO probe might open a new avenue for precisely sensing lysosomal NO-related pathological and therapeutic process.

Biomarker‐Responsive Fluorescent Probes for In‐Vivo Imaging of Liver Injury

“…To fully reveal the role of NO in acute liver injury, an OPD-based donor-acceptor-donor (D-A-D) type ratiometric NO probe with far-red emission, namely MBTD, was designed based on the dual intramolecular charge transfer (ICT) mechanism (Figure 3A). [14] Upon the addition of NO, the thiadiazolefused OPD was transferred to tricyclic thiadiazole-fused benzotriazole with stronger electron withdrawing capability. Thus the dual ICT effect between the donor and acceptor was strengthened and the emission of the product was red-shifted from Reproduced with permission.…”

Cardiovascular bioimaging of nitric oxide: Achievements, challenges, and the future

“…72,74,79 Luciferase expression bioluminescence was used to demonstrate changes in iNOS gene expression in macrophages infiltrated to ligated carotid arteries in a murine model of vascular inflammation. 43 Many studies have reported the use of RAW 264.7 cells (murine macrophages) to detect NO in cell culture, 59,78,81,82 with some two-photon fluorescent probes applied in ex vivo tissue sections from the rat/mouse liver in the presence of exogenous NO. 60,81,82 Some of these sensors have been applied in vivo for in situ imaging of NO in kidneys subjected to ischemia-reperfusion injury 112 and in superficial tumors in mice.…”

“…The first approach involves sensing and imaging modalities that directly quench/trap NO before it is metabolized to other species, which can then be detected and quantified by measuring a current or light-emission. Such NO specific sensors have been shown to detect NO in solution, extracellular, and intracellular components and even at a subcellular level in mitochondria, 57 lysosomes, 58,59 endoplasmic reticulum, 60 and endosomes. 61 The second approach uses sensors that quench and detect by-products of NO metabolism or changes in NOS enzyme activity.…”

Novel Pyrazine-Bridged D-A-D Type Charge Neutral Probe for Membrane Permeable Long-Term Live Cell Imaging

“…On the other hand, D-A-D type chromophores have currently attracted much research attention for showing great potentials as a fine-tuning platform for opto-electronic applications ( Yang et al, 2020 ; Liu et al, 2021 ). In recent reports, D-A-D fluorescent probes have been used in imaging of lysosomal nitric oxide ( Wang et al, 2018 ) and biothiol ( Chen et al, 2018 ). With enriched variety of structure–activity relationships and a larger degree of conjugation that generates a longer wavelength emission, the D-A-D type molecular systems can reach desired performance indicators as probes through a rational design.…”