TY - JOUR

T1 - 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

AU - Arnau del Valle, Carla

AU - Williams, Lewis

AU - Thomas, Paul

AU - Johnson, Robert

AU - Raveenthiraraj, Sathuwarman

AU - Warren, Derek

AU - Sobolewski, Anastasia

AU - Muñoz, María Paz

AU - Galindo, Francisco

AU - Marín, María J.

PY - 2022/9/30

Y1 - 2022/9/30

N2 - 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.

AB - 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.

KW - Endothelial cells

KW - Macrophages cells

KW - Near-infrared

KW - Nitric oxide detection

KW - Two-photon microscopy

U2 - 10.1016/j.jphotobiol.2022.112512

DO - 10.1016/j.jphotobiol.2022.112512

M3 - Journal article

VL - 234

JO - Journal of Photochemistry and Photobiology B: Biology

JF - Journal of Photochemistry and Photobiology B: Biology

SN - 1011-1344

M1 - 112512

ER -