A highly photostable and versatile two-photon fluorescent probe for the detection of a wide range of intracellular nitric oxide concentrations in macrophages and endothelial cells

Arnau del Valle, Carla, Williams, Lewis, Thomas, Paul, Johnson, Robert ORCID: https://orcid.org/0000-0003-3618-238X, Raveenthiraraj, Sathuwarman, Warren, Derek, Sobolewski, Anastasia, Muñoz, María Paz ORCID: https://orcid.org/0000-0001-9037-349X, Galindo, Francisco and Marín, María J. ORCID: https://orcid.org/0000-0001-8021-5498 (2022) A highly photostable and versatile two-photon fluorescent probe for the detection of a wide range of intracellular nitric oxide concentrations in macrophages and endothelial cells. Journal of Photochemistry and Photobiology B: Biology, 234. ISSN 1011-1344

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Nitric oxide (NO) is involved in many biological processes affecting the cardiovascular, nervous and immune systems. Intracellular NO can be monitored using fluorescent probes in combination with fluorescence imaging techniques. Most of the currently available NO fluorescent molecular probes are excited via one-photon excitation using UV or Vis light, which results in poor penetration and high photodamage to living tissues. Here, we report a two-photon fluorescent molecular probe, DANPY-NO, able to detect NO in live cells. The probe consists of an o-phenylenediamine linked to a naphthalimide core; and operates via photoinduced electron transfer. DANPY-NO exhibits good sensitivity (LOD of 77.8 nM) and high selectivity towards NO, and is stable over a broad range of pHs. The probe targeted acidic organelles within macrophages and endothelial cells, and demonstrated enhanced photostability over a commercially available NO probe. DANPY-NO was used to selectively detect endogenous NO in RAW264.7ϒ NO − macrophages, THP-1 human leukemic cells, primary mouse (bone marrow-derived) macrophages and endothelial cells. The probe was also able to detect exogenous NO in endothelial cells and distinguish between increasing concentrations of NO. The NO detection was evidenced using confocal laser scanning and two-photon microscopies, and flow cytometry. Further evidence was obtained by recording the changes in the intracellular fluorescence emission spectrum of the probe. Importantly, the probe displayed negligible toxicity to the analysed biological samples. The excellent sensitivity, selectivity, stability and versatility of DANPY-NO confirm its potential for in vitro and in vivo imaging of NO.

Item Type: Article
Additional Information: Acknowledgment: The authors acknowledge Dr. A. Goldson for training and guidance on the flow cytometer; Dr. P. Wilson for training and guidance on the multiphoton microscope. The authors would like to thank the Faculty of Sciences and School of Chemistry at the University of East Anglia and Mr. and Mrs. Whittaker oncology fellowship for financial support, and the EPSRC (Grant EP/S017909/1) that supported the purchase of the Edinburgh Instrument FS5 fluorescence spectrometer used in this work.
Uncontrolled Keywords: endothelial cells,macrophages cells,near-infrared,nitric oxide detection,two-photon microscopy,radiation,radiological and ultrasound technology,biophysics,radiology nuclear medicine and imaging ,/dk/atira/pure/subjectarea/asjc/3100/3108
Faculty \ School: Faculty of Science > School of Pharmacy
Faculty of Science > School of Chemistry
Faculty of Science
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Depositing User: LivePure Connector
Date Deposited: 12 Jul 2022 16:30
Last Modified: 01 Jan 2023 01:53
URI: https://ueaeprints.uea.ac.uk/id/eprint/86073
DOI: 10.1016/j.jphotobiol.2022.112512

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