Two-photon fluorescent (nano) probes for a versatile intracellular detection and quantification of nitric oxide Carla Arnau del Valle, 1,2 Paul Thomas, 3 Francisco Galindo, 4 M.Paz Muñoz-Herranz, 1,5 María J. Marín* 1 1 School of Chemistry, University of East Anglia, UK, 2 Fischell Department of Bioengineering, University of Maryland, United States, 3 School of Biological Sciences, University of East Anglia, UK, 4 Departamento de Química Inorgánica y Orgánica, Universitat Jaume I, Spain, 5 Department of Chemistry, Lancaster University, UK. Nitric oxide (NO) is involved in numerous biological processes, playing an important role in the regulation of diverse physiological and pathophysiological mechanisms of the cardiovascular, nervous and immune systems; and alterations in the intracellular NO concentrations have been linked to a large number of diseased states. 1,2 For example, NO has been associated positively and negatively with cancer, depending on its concentration and the environment surrounding the tumour; and with diseases and disorders such as Alzheimer’s disease, Parkinson’s disease and lysosomal storage disorders. Considering the significant role that NO plays in important biological functions, the development and improvement of methods to detect and quantify intracellular NO are essential to further our understanding of the biological roles of NO. To this end, we have developed near infrared (NIR) excitable fluorescence (nano)probes for the extra- and intracellular detection of NO that take advantage of the high photostability, high biological tissue penetration and minimal photodamage associated with this long-wavelength excitation (Figure 1).
Figure 1. Schematic representation of the application of two-photon excitable flourescent (nano) probes for a versatile intracellular detection of nitric oxide. This contribution will present NIR excitable (nano)probes for the intracellular detection and quantification of NO. A novel molecular probe, DANPY-NO, will be introduced 3 that showed good
sensitivity and selectivity towards NO, and is stable over a broad range of biologically-relevant pHs. DANPY-NO is a versatile probe that has been used to detect intracellular NO produced by iNOS and eNOS in a wide range of cells (mouse macrophages, human leukemic cells, primary mouse macrophages and endothelial cells) using a range of techniques including confocal laser scanning microscopy (images and intracellular fluorescence emission spectra), multiphoton microscopy and flow cytometry. A derivative of DANPY-NO has been used to develop a broadly applicable two-photon excitable fluorescent nanoprobe (DANPY-NO@AuNPs) 4 able to detect and potentially quantify NO levels in an extensive range of cellular environments. The nanoprobe is able to selectively detect NO in solution, with a dynamic range up to 150 μM; and endogenous NO in RAW264.7γ NO - macrophages, THP-1 human leukemic cells, and endogenous and exogenous NO in endothelial cells. The nanoprobe accumulated in the acidic organelles of the tested cell lines showing negligible toxicity. Importantly, DANPY-NO@AuNPs showed potential to quantify intracellular NO concentrations in MDA-MB-231 breast cancer cells. Based on their excellent sensitivity and stability, and outstanding versatility, DANPY-NO@AuNPs can be applied for the spatiotemporal monitoring of in vitro and in vivo NO levels. References 1. R.M.J. Palmer et al. , Nature, 327, 524–526 (1987). 2. A.R. Butler et al. , Chem. Soc. Rev., 22, 233–241 (1993).
3. Arnau del Valle et al. , J. Photochem. Photobiol., B, 234, 112512 (2022). 4. Arnau del Valle et al. , revisions submitted to J. Mater. Chem. B (2023).
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