Neutral Fluorescent Probes Research Articles

Visualizing dynamic interactions between mitochondria and lysosomes in living cells using novel pH-sensitive fluorescent probes in dual-channel

The neutral fluorescent probes 1a-1c based on D-π-A-π-D structure, with benzopyranoquinoline as the fluorophore and the electron-donating groups at both ends, were developed, which emitted dual-channel fluorescence in different pH environments. Among them, probe 1a with both N,N-diethyl group and morpholine ring as electron-donating groups exhibited the best fluorescence performance, showing a red shift of 85 nm in dual emission at 505 nm and 590 nm, and a Stokes shift of 95 nm in both channels. The neutral 1a displayed green fluorescence specifically targeting mitochondria, while the protonated form 1a+H+ resulting in red fluorescence localized in lysosomes. The ability to distinguish healthy and pathological cells such as cancer or inflammation could be achieved by observing the differences in red fluorescence within lysosomes. Probe 1a could migrate and retarget between mitochondria and lysosomes, and it was discovered by the physiological effects of chloral hydrate experiments. In addition, the dynamic changes in morphology and movement of mitochondria and lysosomes during cellular autophagy through the variation of fluorescence intensity in green and red channels induced by rapamycin and starvation conditions had been investigated deeply.

Naphthyridine-based neutral fluorescent probes for dynamic monitoring lipid droplet polarity

In this paper, several novel nitrogen-containing heterocycle dyes 1a-c with large conjugated planar structures based on naphthyridine derivatives were designed and synthesized, which had excellent optical properties and biological applications. Optical test showed the maximum absorption wavelengths of 1a-c were in the range of 443–485 nm, while the maximum emission wavelengths located at 523–610 nm in difference solvents. Furthermore, they exhibited polarity related fluorescence intensity changes with high fluorescence quantum yields, even up to 88% (1a in THF). Moreover, the transient absorption spectra revealed the excited-state lifetime of dye 1a became shorter with the increase of solvent polarity. Density functional theory calculations are also used to help explain the effect of the polarity of solvents on their fluorescence properties. Interestingly, the dyes 1a-c were somewhat toxic to cancer cells, while had an obvious growth-promoting effect on normal cells. Additionally, 1a-c with excellent lipid droplet (LD) targeting could be used for long-term observation of targeted cellular lis) hysiological activity and further applied to dynamic monitoring of changes in LDs polarity stimulated by TLR4 activator (LPS) in living cells. Overall, the excellent optical properties and biocompatibility of 1a-c indicate that they can serve as fluorescent probes for biological applications, and the results have important implications for understanding the physiological functions of LDs, especially in terms of morphological and polar changes.

Development of a Neutral Diketopyrrolopyrrole Phosphine Oxide for the Selective Bioimaging of Mitochondria at the Nanomolar Level

Development of novel bioimaging materials that exhibit organelle specific accumulation continues to be at the forefront of research interests and efforts. Among the various subcellular organelles, mitochondria, which are found in the cytoplasm of eukaryotic cells, are of particular interest in relation to their vital function. To date, most molecular probes that target mitochondria utilise delocalised lipophilic cations such as triphenylphosphonium and pyridinium. However, the use of such charged motifs is known to be detrimental to the working function of the mitochondrial transmembrane potential and there remains a strong case for development of neutral mitochondrial fluorescent probes. Herein, we demonstrate for the first time the exploitation of diketopyrrolopyrrole-based chemistries for the realisation of a neutral fluorescent probe that exhibits organelle specific accumulation within the mitochondria at the nanomolar level. The synthesised probe, which bears a neutral triphenylphosphine oxide moiety, exhibits a large Stokes shift and high fluorescence quantum yield in water, both highly sought-after properties in the development of bioimaging agents. In vitro studies reveal no interference with cell metabolism when tested for the human MCF7 breast cancer cell and nanomolar subcellular organelle colocalisation with commercially available mitochondrial staining agent Mitotracker Red. In light of its novelty, neutral structure and the preferential accumulation at nanomolar concentrations we anticipate this work to be of significant interest for the increasingly larger community devoted to the realisation of neutral mitochondrial selective systems and more widely to those engaged in the rational development of superior organic architectures in the biological field.

Red Emitting Neutral Fluorescent Glycoconjugates for Membrane Optical Imaging

A family of neutral fluorescent probes was developed, mimicking the overall structure of natural glycolipids in order to optimize their membrane affinity. Nonreducing commercially available di- or trisaccharidic structures were connected to a push-pull chromophore based on dicyanoisophorone electron-accepting group, which proved to fluoresce in the red region with a very large Stokes shift. This straightforward synthetic strategy brought structural variations to a series of probes, which were studied for their optical, biophysical, and biological properties. The insertion properties of the different probes into membranes were evaluated on a model system using the Langmuir monolayer balance technique. Confocal fluorescence microscopy performed on muscle cells showed completely different localizations and loading efficiencies depending on the structure of the probes. When compared to the commercially available ANEPPS, a family of commonly used membrane imaging dyes, the most efficient probes showed a similar brightness, but a sharper pattern was observed. According to this study, compounds bearing one chromophore, a limited size of the carbohydrate moiety, and an overall rod-like shape gave the best results.

Influence of the donor substituent and acceptor alkylation on the structure–analytical properties of mono- and bifunctional biphenyl-type fluorescent reporters

A systematic structural investigation of R-phenyl-substituted 2,2′:6′,2″-terpyridines, a family of mono- and bifunctional charge transfer (CT)-operated fluorescent reporters for protons and metal ions, is presented. These molecules are equipped with non-binding and analyte coordinating donor substituents R (R = CF 3, H, OMe, OH, DMA, A15C5 equaling monoaza-15-crown-5) of various donor strength and display CT-controlled spectroscopic properties and communication of analyte–receptor interactions. The crystal structures of the neutral fluorescent probes are compared to the structures of their terpyridine-alkylated or -protonated counterparts that represent model systems for acceptor protonation or cation coordination. The aim is here a better understanding of the complexation-induced structural and spectroscopic changes and the identification of common packing motifs of bpb-R thereby taking into account the importance of terpyridine building blocks for the construction of supramolecular systems and coordination arrays revealing π–π interactions.

The Interaction between Polycationic Poly-Lysine Dendrimers and Charged and Neutral Fluorescent Probes

The interactions between polycationic poly-lysine dendrimers and hydrophobic fluorescent probes (anionic ANS and neutral Prodan) were studied. R121 and R131 dendrimers were not able to interact with anionic and neutral hydrophobic groups. R124 was able to interact with neutral and anionic hydrophobic fluorescent probes, however mainly through hydrophobic forces. Dendrimers R155 and R169 showed the maximal effects. The strongest interactions observed for R169 can be explained by intramolecular folding (stacking) of its two L-proline residues. Using double fluorescence titration technique for ANS probe allowed to receive such constant of binding and the number of binding centers: for R121, 1.8 x 10(3) (mol/l)(-1) and 1.07; for R124, 12.1 x 10(3) (mol/l)(-1) and 0.48; for R131, 4.7 x 10(3) (mol/l)(-1) and 0.48; for R155, 9.2 x 10(3) (mol/l)(-1) and 1.36; for R169, 39.6 x 10(3) (mol/l)(-1) and 0.97. Thus, neutral and anionic hydrophobic probes can be used for the fast preliminary screening of binding properties of newly synthesized polycationic dendrimers.

A new method for measuring fibrillar amyloid β‐protein using diphenylhexatriene, a fluorescent probe

The two current methods for the measurement of fibrillar (f) amyloid beta‐protein (Aβ), namely Thioflavin T (ThT) fluorescence spectroscopy and Congo red (CR) absorbance spectroscopy, are based on the interaction of fAβ with cationic ThT and anionic CR molecules. The presence of cations and colored factors in the reaction often interferes with ThT and CR assays. Recently, we reported that fibrillar Aβ forms a membrane‐like hydrophobic domain (Chauhan et al. 2001; Neuroreport12, 587–590). We report here that diphenylhexatriene (DPH), a neutral fluorescent probe, interacts with fibrillar Aβ 1‐40 and fibrillar Aβ 1‐42, but not with soluble Aβ. As a result, DPH fluorescence increases several fold in the presence of fibrillar Aβ at the excitation and emission wavelength of 360 and 430 nm, respectively. The blank sample (DPH in buffer) had minimal fluorescence, and soluble Aβ had no effect on DPH fluorescence. The interaction of DPH with fibrillar Aβ was dependent on the incubation time of these two constituents. At fixed Aβ concentration and varying DPH concentration, there was a lag phase of DPH fluorescence from 1 to 4 μm DPH, that was followed by a linear increase in fluorescence from 4 to 8 μm DPH which then reached a plateau. Studies at varying pH showed that emission spectra of fAβ/DPH can be studied at a wide range of pH from 4 to 8. Aβ concentration studies showed that the fluorescence of DPH increases linearly with increase in the concentration of fibrillar Aβ 1‐40/Aβ 1‐42. Kinetic studies showed that one mole of fAβ binds to 15.5 mol of DPH. These results suggest that DPH fluorescence can be used as a standard assay method for the measurement of fibrillar Aβ.

Modulation of Guest Partitioning Within Dendrimer–Surfactant Supramolecular Assemblies

ABSTRACT Electrostatic interactions are used to create a template-assisted supramolecular assembly consisting of a polymeric dendrimer at the core and amphiphilic substrates on the periphery. The dendrimer generation and the chemical structure of the amphiphiles are varied to construct multiple and distinct microenvironments within the dendrimer–ligand complex for encapsulation of small guest molecules. In particular, these investigations employ a guest molecule that is a neutral fluorescent probe that exhibits an emission wavelength with an extreme sensitivity to the polarity of its surroundings. Partitioning of the fluorophore within the various microregions of the dendrimer–surfactant supramolecular complex is distinguished by the characteristic emission wavelengths of the overlapping Gaussian functions comprising the overall fluorescence spectrum. The observed variations in the prodan emission spectrum suggest interaction of prodan at protonated amino groups (460-nm emission), within dendritic branches and surfactant tails (490-nm emission), and in interior regions of the dendrimer core (430-nm emission). We demonstrate that the positioning of the guest molecule within the supramolecular complex can be modulated through the selection of dendrimer generation, surfactant chain length, and dendrimer:surfactant concentration ratio.

Membrane potential differences between adriamycin-sensitive and -resistant cells as measured by flow cytometry

Using the fluorescent membrane potential probe, 3,3'-dihexyl-oxacarbocyanine (DiOC6(3)), we found a 4-fold higher uptake in Adriamycin (ADM)-sensitive versus -resistant Friend leukemia cells (FLC). When sensitive cells were treated in the presence of high potassium (120 mM K +), there was a greater than 80% reduction of DiOC6(3) uptake. Using carbonylcyanide 4-trinuoromethoxyphenylhydrazone (FCCP), a specific inhibitor of mitochondrial membrane potential, DiOC6(3) accumulation was reduced by less than 30% in these cells. Both results support the conclusion that a greater uptake of DiOC6(3) in ADM-sensitive than in -resistant cells indicates an increased plasma transmembrane potential. Since electronegative plasma membrane potentials are a driving force for the transport of lipophilic positively-charged compounds, differences in membrane potentials between sensitive and multiple drug resistant (MDR) tumor cells could have an important influence on drug accumulation and cytotoxicity. The drugs which our ADM-resistant FLC display multiple drug resistance to are positively charged. In MDR FLC, the calcium channel antagonist, verapamil, has been shown to block the efflux of Rhodamine 123 (Rho 123) and other positively-charged compounds. Since DiOC6(3) is also positivelycharged, we used verapamil to investigate its effects on drug uptake. In MDR FLC, verapamil increased DiOC6(3) accumulation by 1.9-fold, whereas in sensitive cells it was increased 1.5-fold. In contrast, verapamil increased the levels of Rho 123 in resistant cells 7.8-fold but lowered them in sensitive cells 1.5-fold. The minimal loss of DiOC6(3) from both sensitive and MDR cells and the above results can best be interpreted as indicating that DiOC6(3) is not transported by the efflux “pump” system but that verapamil induces a plasma membrane potential increase in sensitive and resistant cells that DiOC6(3) is sensitive to. On the other hand, since Rho 123 did appear to be actively effluxed from these resistant cells, the enhancement of this compound by verapamil was more likely due to inhibition of the MDR “pump”. How, or whether, plasma membrane potentials and the MDR efflux “pump” are related remains to be investigated. In the resistant cells, verapamil also induced an increase (13-fold) in the accumulation of the electrically neutral fluorescent probe for calcium, INDO-1/AM. However, verapamil had no effect on the efflux of this compound, which was equivalent in both resistant and sensitive cells. Thus, a new effect of verapamil on drug accumulation in MDR cells is identified here.