Detection of subcellular nitric oxide in mitochondria using a pyrylium probe: assays in cell cultures and peripheral blood

Fluorescent probes for the detection of intracellular nitric oxide (NO) are abundant, but those targeted to the mitochondria are scarce. Among those molecules targeting mitochondrial NO (mNO), the majority use a triphenylphosphonium (TPP) cation as a vector to reach such organelles. Here we describe a simple molecule (mtNOpy) based on the pyrylium structure, made in a few synthetic steps, capable of detecting selectively NO (aerated medium) over other reactive species. The calculated detection limit for mtNOpy is 88 nM. The main novelty of this probe is that it has a simple molecular architecture and can act both as a fluorogenic and as a mitochondriotropic agent, without using TPP. mtNOpy has been tested in two different scenarios: (a) in a controlled environment of cell line cultures (human colon carcinoma HT-29 cells and mouse macrophage RAW 264.7 cells), using confocal laser scanning microscopy, and (b) on a much more complex sample of peripheral blood, using flow cytometry. In the first context, mtNOpy has been found to be responsive (turn-on fluorescence) to exogenous and endogenous NO stimuli (via SNAP donor and LPS stimulation, respectively). In the second area, mtNOpy has been able to discriminate between NO-generating phagocytes (neutrophils and monocytes) from other leukocytes (NK, B and T cells)

Structure-performance relationships of four lysosomal markers used for the imaging of HT-29 cancer cells and a cellular model of lysosomal storage disease (Niemann-Pick C)

Four new BODIPY derivatives with a potential tendency to aggregation have been synthesized and characterized by means of NMR techniques, mass spectrometry and UV–Vis/fluorescence spectroscopies. The objective of this study is to determine which structural factors of the new molecules influence most notably the cellular uptake, intracellular location and fluorescence imaging abilities. The behaviour of the compounds in organic solvent and aqueous solution has been studied. In organic solvents (DMSO, ethanol and toluene), the photophysical properties of the new molecules are almost independent of the building blocks used to synthesize the pendant moieties (non-fluorogenic parts). In an aqueous environment (HEPES Buffer Solution, pH 7), at 10 μM, three of the compounds (1, 2 and 4) tend to form weakly emissive nanoparticles (DLS determination) whereas one of them (3) remains soluble and highly fluorescent. In the nanomolar range of concentration, all the compounds are aqueous soluble. The cellular internalisation of the compounds (10 nM) has been studied in human colon adenocarcinoma HT-29 cells by means of flow cytometry and confocal laser scanning microscopy. All the compounds were uptaken by HT-29 cells, but notably molecule 3 (made with lysine as a building block) was the one displaying a higher loading and a more clear lysosomal location (0.88 Pearson's correlation coefficient in colocalization assays using lysosomal fluorescent probe LysoTracker DND-99). Molecule 3 also performed better than the valine derivative 1 as a lysosomal marker in a cellular model (human adrenal carcinoma SW13/cl.2 cells) of lysosomal storage disease (Niemann-Pick type C).Funding for open access charge: CRUE-Universitat Jaume

Glutathione-responsive molecular nanoparticles from a dianionic bolaamphiphile and their use as carriers for targeted delivery

The formation in aqueous media of molecular nanoparticles from a bolaamphiphile (SucIleCsa) incorporating a disulfide moiety is described. The particles can be loaded efficiently with the lipophilic mitochondrial marker DiOC6(3), quenching its fluorescence, which is recovered upon reductive particle disassembly. DiOC6(3) transport into human colorectal adenocarcinoma cells (HT-29) is demonstrated using flow cytometry and confocal scanning fluorescence microscopy. A significant increase in intracellular fluorescence is observed when the cells are stimulated to produce glutathione (GSH). These new molecular nanoparticles can be considered a theranostic tool that simultaneously achieves targeted delivery of lipophilic substances and signals high levels of GSH