Targetable, two-photon fluorescent probes for local nitric oxide capture in the plasma membranes of live cells and brain tissues

Abstract

Although the plasma membrane is a major site for nitric oxide (NO) generation and action, few targetable probes that specifically sense and image NO in the plasma membrane have been reported. In this study, a membrane targetable, two-photon nitric oxide probe, Mem-NO, was developed and evaluated for bio-imaging of both exogenous and endogenous NO. By installing a quaternary ammonium compound as the hydrophilic head and a long alkyl chain as the hydrophobic tail on 4-amino-1,8-naphthalimide, we designed Mem-NO into a bi-polar structure. Due to the interaction with the phospholipid bilayer of plasma membrane, Mem-NO could specifically and stably localize in the plasma membrane. Mem-NO is almost nonfluorescent, but it displayed substantial fluorescence enhancement (16-fold) upon NO capture with sensitive (74 nM limit of detection) and fast response (within 1 min). Moreover, Mem-NO displayed strong two-photon excitation fluorescence activity (δ = 177 GM at 810 nm) and low cytotoxicity. It was found that Mem-NO is capable of two-photon imaging of endogenous NO in live neurons and human umbilical vein endothelial cells (HUVECs) and exogenous NO in mouse brain tissues. Therefore, Mem-NO qualifies as an essential and unique analytical tool for monitoring NO for future physiological, pathological, and pharmacological studies.

A Novel Ruthenium-based Molecular Sensor to Detect Endothelial Nitric Oxide

“…We tested the capacity of [Ru(bpy) 2 (dabpy)] 2+ to detect NO in cultured, attached endothelial cells in the presence of both endogenous (acetylcholine and H 2 O 2 ) and exogenous (NOC13) sources of NO. While 10 µM acetylcholine and 150 µM H 2 O 2 have been specifically used in previous studies to demonstrate the function of other NO sensors 13,29,30 , both agents have been shown to produce endogenous NO in endothelial cells at different concentrations and incubation periods. We therefore performed Western blot protein quantifications of phosphorylated-endothelial nitric oxide synthase (p-eNOS), the activated eNOS enzyme, with 5–100 µM acetylcholine stimulation in HUVECs to confirm the endogenous activity under our experimental conditions (Fig.…”

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

“…Fluorescent intensity decreased on the addition of Nω‐nitro‐ l ‐arginine methyl ester ( l ‐NAME), a well‐studied inhibitor of NO production in endothelial cells 77 . Similar studies have been conducted using bovine endothelial cells and HUVECs with different stimuli 29,67,68,83,84,93 . These studies include, extracellular, intracellular, and membrane targeted 83 fluorophores.…”

Nanoparticles based on the zwitterionic pillar[5]arene and Ag+: synthesis, self-assembly and cytotoxicity in the human lung cancer cell line A549

“…Diamines containing both primary and tertiary amino groups were used as aminating agents [30]. In this work, we used commercially available N,N-dimethylethylenediamine, because there are no side reactions due to the presence of two methyl groups, and the process of further quaternization proceeds under mild conditions [31]. The macrocycle 2 with 89% yield was obtained by aminolysis of decaether 1 by N,N-dimethylethanediamine ( Figure 1, Supporting Information File 1, Figures S1-S12).…”