Solvatochromic and Fluorogenic Dyes as Environment-Sensitive Probes: Design and Biological Applications.

Fluorescent environment-sensitive probes are specially designed dyes that change their fluorescence intensity (fluorogenic dyes) or color (e.g., solvatochromic dyes) in response to change in their microenvironment polarity, viscosity, and molecular order. The studies of the past decade, including those of our group, have shown that these molecules become universal tools in fluorescence sensing and imaging. In fact, any biomolecular interaction or change in biomolecular organization results in modification of the local microenvironment, which can be directly monitored by these types of probes. In this Account, the main examples of environment-sensitive probes are summarized according to their design concepts. Solvatochromic dyes constitute a large class of environment-sensitive probes which change their color in response to polarity. Generally, they are push-pull dyes undergoing intramolecular charge transfer. Emission of their highly polarized excited state shifts to the red in more polar solvents. Excited-state intramolecular proton transfer is the second key concept to design efficient solvatochromic dyes, which respond to the microenvironment by changing relative intensity of the two emissive tautomeric forms. Due to their sensitivity to polarity and hydration, solvatochromic dyes have been successfully applied to biological membranes for studying lipid domains (rafts), apoptosis and endocytosis. As fluorescent labels, solvatochromic dyes can detect practically any type of biomolecular interactions, involving proteins, nucleic acids and biomembranes, because the binding event excludes local water molecules from the interaction site. On the other hand, fluorogenic probes usually exploit intramolecular rotation (conformation change) as a design concept, with molecular rotors being main representatives. These probes were particularly efficient for imaging viscosity and lipid order in biomembranes as well as to light up biomolecular targets, such as antibodies, aptamers and receptors. The emerging concepts to achieve fluorogenic response to the microenvironment include ground-state isomerization, aggregation-caused quenching, and aggregation-induced emission. The ground-state isomerization exploits, for instance, polarity-dependent spiro-lactone formation in silica-rhodamines. The aggregation-caused quenching uses disruption of the self-quenched dimers and nanoassemblies of dyes in less polar environments of lipid membranes and biomolecules. The aggregation-induced emission couples target recognition with formation of highly fluorescent dye aggregates. Overall, solvatochromic and fluorogenic probes enable background-free bioimaging in wash-free conditions as well as quantitative analysis when combined with advanced microscopy, such as fluorescence lifetime (FLIM) and ratiometric imaging. Further development of fluorescent environment-sensitive probes should address some remaining problems: (i) improving their optical properties, especially brightness, photostability, and far-red to near-infrared operating range; (ii) minimizing nonspecific interactions of the probes in biological systems; (iii) their adaptation for advanced microscopies, notably for superresolution and in vivo imaging.journal article2017 Feb 212017 01 09importe

Ca-NIR: a ratiometric near-infrared calcium probe based on a dihydroxanthene-hemicyanine fluorophore.

Fluorescent calcium probes are essential tools for studying the fluctuation of calcium ions in cells. Herein, we developed Ca-NIR, the first ratiometric calcium probe emitting in the near infrared region. This probe arose from the fusion of a BAPTA chelator and a dihydroxanthene-hemicyanine fluorophore. It is efficiently excited with common 630-640 nm lasers and displays two distinct emission bands depending on the calcium concentration (Kd = ∼8 μM). The physicochemical and spectroscopic properties of Ca-NIR allowed for ratiometric imaging of calcium distribution in live cells.journal article2017 Jun 01imported"Supporting information" disponible sur le site de l'éditeu

Non-coordinating anions assemble cyanine amphiphiles into ultra-small fluorescent nanoparticles

A non-coordinating anion, fluorinated tetraphenylborate, assembles specially designed cationic cyanine amphiphiles into 7–8 nm fluorescent nanoparticles that are >40-fold brighter than a single cyanine dye. This kind of anion, combining hydrophobic and electrostatic forces in aqueous media, constitutes promising building blocks in the self-assembly of functional nanomaterials

PEGylated Red-Emitting Calcium Probe with Improved Sensing Properties for Neuroscience.

Monitoring calcium concentration in the cytosol is of main importance as this ion drives many biological cascades within the cell. To this end, molecular calcium probes are widely used. Most of them, especially the red emitting probes, suffer from nonspecific interactions with inner membranes due to the hydrophobic nature of their fluorophore. To circumvent this issue, calcium probes conjugated to dextran can be used to enhance the hydrophilicity and reduce the nonspecific interaction and compartmentalization. However, dextran conjugates also feature important drawbacks including lower affinity, lower dynamic range, and slow diffusion. Herein, we combined the advantage of molecular probes and dextran conjugate without their drawbacks by designing a new red emitting turn-on calcium probe based on PET quenching, Rhod-PEG, in which the rhodamine fluorophore bears four PEG4 units. This modification led to a high affinity calcium probe (Kd = 748 nM) with reduced nonspecific interactions, enhanced photostability, two-photon absorbance, and brightness compared to the commercially available Rhod-2. After spectral characterizations, we showed that Rhod-PEG quickly and efficiently diffused through the dendrites of pyramidal neurons with an enhanced sensitivity (ΔF/F0) at shorter time after patching compared to Rhod-2.journal article2017 Nov 222017 10 24imported"Supporting information" disponible sur le site de l'éditeur à l'adresse suivante : http://pubs.acs.org/doi/suppl/10.1021/acssensors.7b0066

Photopolymerized micelles of diacetylene amphiphile: physical characterization and cell delivery properties:

A series of polydiacetylene (PDA) - based micelles were prepared from diacetylenic surfactant bearing polyethylene glycol, by increasing UV-irradiation times. These polymeric lipid micelles were analyzed by physicochemical methods, electron microscopy and NMR analysis. Cellular delivery of fluorescent dye suggests that adjusting the polymerization state is vital to reach the full in vitro potential of PDA-based delivery system

Rsc Adv

A Forster resonance energy transfer (FRET) system of semiconductor quantum dots and porphyrins represents a new promising photosensitizing tool for the photodynamic therapy of cancer. In this work, we demonstrate the ability of a non-covalent complex formed between commercial lipid-coated CdSe/ ZnS quantum dots (QD) bearing different terminal groups (carboxyl, amine or non-functionalized) and a second-generation photosensitizer, chlorin e(6) (Ce-6) to enter living HeLa cells with maintained integrity and perform FRET from two-photon excited QD to bound Ce-6 molecules. Spectroscopic changes, the highly efficient FRET, observed upon Ce-6 binding to QD, and remarkable stability of the QD-Ce-6 complex in different media suggest that Ce-6 penetrates inside the lipid coating close to the inorganic core of QD. Two-photon fluorescence lifetime imaging microscopy (FLIM) on living HeLa cells revealed that QD-Ce-6 complexes localize within the plasma membrane and intracellular compartments and preserve high FRET efficiency (similar to 50%). The latter was confirmed by recovery of QD emission lifetime after photobleaching of Ce-6. The intracellular distribution pattern and FRET efficiency of QD-Ce-6 complexes did not depend on the charge of QD terminal groups. Given the non-covalent nature of the complex, its exceptional stability in cellulo can be explained by a combination of hydrophobic interactions and coordination of carboxyl groups of Ce6 with the ZnS shell of QD. These findings suggest a simple route to the preparation of QD-photosensitizer complexes featuring efficient FRET and high stability in cellulo without using time-consuming conjugation protocols

In Vivo FRET Imaging to Predict the Risk Associated with Hepatic Accumulation of Squalene-Based Prodrug Nanoparticles.

Förster resonance energy transfer (FRET) is used here for the first time to monitor the in vivo fate of nanoparticles made of the squalene-gemcitabine prodrug and two novel derivatives of squalene with the cyanine dyes 5.5 and 7.5, which behave as efficient FRET pair in the NIR region. Following intravenous administration, nanoparticles initially accumulate in the liver, then they show loss of their integrity within 2 h and clearance of the squalene bioconjugates is observed within 24 h. Such awareness is a key prerequisite before introduction into clinical settings.journal article2018 Feb2017 11 30importedSupporting information : librement accessible sur le site de l'éditeur

Pathogenic variants of sphingomyelin synthase SMS2 disrupt lipid landscapes in the secretory pathway

Sphingomyelin is a dominant sphingolipid in mammalian cells. Its production in the trans-Golgi traps cholesterol synthesized in the ER to promote formation of a sphingomyelin/sterol gradient along the secretory pathway. This gradient marks a fundamental transition in physical membrane properties that help specify organelle identify and function. We previously identified mutations in sphingomyelin synthase SMS2 that cause osteoporosis and skeletal dysplasia. Here, we show that SMS2 variants linked to the most severe bone phenotypes retain full enzymatic activity but fail to leave the ER owing to a defective autonomous ER export signal. Cells harboring pathogenic SMS2 variants accumulate sphingomyelin in the ER and display a disrupted transbilayer sphingomyelin asymmetry. These aberrant sphingomyelin distributions also occur in patient-derived fibroblasts and are accompanied by imbalances in cholesterol organization, glycerophospholipid profiles, and lipid order in the secretory pathway. We postulate that pathogenic SMS2 variants undermine the capacity of osteogenic cells to uphold nonrandom lipid distributions that are critical for their bone forming activity.Peer reviewe

Org Biomol Chem

A new fluorescent label N-[4'-(dimethylamino)-3-hydroxyflavone-7-yl]-N-methyl-beta-alanine () was synthesized. Due to two electron donor groups at the opposite ends of the chromophore, an excited state intramolecular proton transfer (ESIPT) resulting in a dual emission was observed even in highly polar media and its fluorescence quantum yield was found to be remarkably high in a broad range of solvents including water. As a consequence, this label exhibits a remarkable sensitivity to the hydration of its environment, which is observed as a color switch between the emission of the ESIPT product (T* form) and that of the normal N* form. The label was coupled to the N-terminus of penetratin, a cell penetrating peptide, in order to study its interactions with lipid membranes and internalization inside the cells. As expected, the binding of penetratin to lipid membranes resulted in a dramatic switch in the relative intensity of its two emission bands as compared to its emission in buffer. Our studies with different lipid compositions confirmed the preference of penetratin to lipid membranes of the liquid disordered phase. After incubation of low concentrations of labeled penetratin with living cells, ratiometric imaging revealed, in addition to membrane-bound species, a significant fraction of free peptide in cytosol showing the characteristic emission from aqueous medium. At higher concentrations of penetratin, mainly peptides bound to cell membrane structures were observed. These observations confirmed the ability of penetratin to enter the cytosol by direct translocation through the cell plasma membrane, in addition to the classical entry by endocytosis. The present probe constitutes thus a powerful tool to study the interaction of peptides with living cells and their internalization mechanisms