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

A novel leaflet-selective fluorescence labeling technique reveals differences between inner and outer leaflets at high bilayer curvature

AbstractUnderstanding the differences in the physical properties of the inner and outer leaflet of membranes and how the leaflets are coupled to each other requires methods that can selectively label both the outer and inner leaflets. In this report we introduce a combined chromatography/cyclodextrin method for selective labeling of the inner leaflet. Combining this method with selective labeling of the outer leaflet, we are able to show that there is a distinct difference in polar headgroup physical properties of the inner and outer leaflet headgroups in small unilamellar vesicles composed of a wide variety of phosphatidylcholines and a phosphaticylcholine/sphingomyelin mixture. It appears that the inner leaflet headgroups are more tightly packed than those of the outer leaflet. This differential packing disappears when vesicle size increases, showing that it is a consequence of membrane curvature. Differential packing is also reduced as acyl chain length is decreased. In the future, selective leaflet labeling is likely to be a powerful tool for investigating the properties of asymmetric lipid vesicles

Fighting Aggregation‐Caused Quenching and Leakage of Dyes in Fluorescent Polymer Nanoparticles: Universal Role of Counterion

Dye‐loaded polymer nanoparticles (NPs) emerge as a powerful tool for bioimaging applications, owing to their exceptional brightness and controlled small size. However, aggregation‐caused quenching (ACQ) and leakage of dyes at high loading remain important challenges of these nanomaterials. The use of bulky hydrophobic counterions has been recently proposed as an effective approach to minimize ACQ and dye leakage, but the role of counterion structure is still poorly understood. Here, a systematic study based on ten counterions, ranging from small hydrophilic perchlorate up to large hydrophobic tetraphenylborate derivatives, reveals how counterion nature can control encapsulation and emission of a cationic dye (rhodamine B octadecyl ester) in NPs prepared by nanoprecipitation of a biodegradable polymer, poly‐lactide‐co‐glycolide (PLGA). We found that increase in counterion hydrophobicity enhances dye encapsulation efficiency and prevents dye adsorption at the particle surface. Cellular imaging studies revealed that ≥95 % encapsulation efficiency, achieved with most hydrophobic counterions (fluorinated tetraphenylborates), is absolutely required because non‐encapsulated dye species at the surface of NPs are the origin of dye leakage and strong fluorescence background in cells. The size of counterions is found to be essential to prevent ACQ, where the largest species, serving as effective spacer between dyes, provide the highest fluorescence quantum yield. Moreover, we found that the most hydrophobic counterions favor dye–dye coupling inside NPs, leading to ON/OFF fluorescence switching of single particles. By contrast, less hydrophobic counterions tend to disperse dyes in the polymer matrix favoring stable emission of NPs. The obtained structure‐property relationships validate the counterion‐based approach as a mature concept to fight ACQ and dye leakage in the development of advanced polymeric nanomaterials with controlled optical properties

Simultaneous probing of hydration and polarity of lipid bilayers with 3-hydroxyflavone fluorescent dyes

AbstractThe penetration of water into the hydrophobic interior leads to polarity and hydration profiles across lipid membranes which are fundamental in the maintenance of membrane architecture as well as in transport and insertion processes into the membrane. The present paper is an original attempt to evaluate simultaneously polarity and hydration properties of lipid bilayers by a fluorescence approach. We applied two 3-hydroxyflavone probes anchored in lipid bilayers at a relatively precise depth through their attached ammonium groups. They are present in two forms: either in H-bond-free form displaying a two-band emission due to an excited state intramolecular proton transfer reaction (ESIPT), or in H-bonded form displaying a single-band emission with no ESIPT. The individual emission profiles of these forms were obtained by deconvolution of the probes' fluorescence spectra. The polarity of the probe surrounding the bilayer was estimated from the two-band spectra of the H-bond-free form, while the local hydration was estimated from the relative contribution of the two forms. Our results confirm that by increasing the lipid order (phase transition from fluid to gel phase, addition of cholesterol or decrease in the lipid unsaturation), the polarity and to a lesser extent, the hydration of the bilayers decrease simultaneously. In contrast, when fluidity (i.e. lipid order) is kept invariant, increase of temperature and of bilayer curvature leads to a higher bilayer hydration with no effect on the polarity. Furthermore, no correlation was found between dipole potential and the hydration of the bilayers

BODIPY-loaded polymer nanoparticles: chemical structure of cargo defines leakage from nanocarrier in living cells

Uncontrolled release of encapsulated drugs and contrast agents from biodegradable polymer nanoparticles (NPs) is a central problem in drug delivery and bioimaging. In particular, it concerns polymeric NPs prepared by nanoprecipitation, where this release (so-called burst release) can be very significant, leading to side effects and/or bioimaging artifacts. Here, we made a systematic study on the effect of chemical structure of cargo molecules, BODIPY dye derivatives, on their capacity to be loaded into ~50 nm PLGA NPs without leakage in biological media. Absorption and fluorescence spectroscopy suggested that most of dyes, except the most polar BODIPY derivative, formed blended structures with polymer NPs. Fluorescence correlation spectroscopy of dye-loaded NPs in the presence of serum proteins revealed that only the most hydrophobic BODIPY dyes, bearing one octadecyl chain or two octyl chains, remain inside NPs, while all other derivatives are released into serum medium. The time-laps absorption and fluorescence studies confirmed this result, suggesting the release kinetics for the leaky NPs on the time scale of hours. Fluorescence microscopy of living cells incubated with BODIPY-loaded NPs showed that most of them exhibit strong dye leakage observed as homogeneous distribution of fluorescence all over the cytoplasm. Importantly, NPs loaded with the most hydrophobic dyes, exhibited high stability showing a dotted pattern in the perinuclear region, typical for endosomes and lysosomes. Our results highlight significance of the cargo hydrophobicity for efficient encapsulation inside polymeric NPs prepared by nanoprecipitation, which enables designing stable cargo-loaded nanomaterials for bioimaging and drug delivery.

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

Optimizing the Fluorescence Properties of Nanoemulsions for Single Particle Tracking in Live Cells

Nanoemulsions (NEs) are biocompatible lipid nanoparticles composed of an oily core stabilized by a surfactant shell. It is acknowledged that the surface decoration with poly­(ethylene glycol), through the use of nonionic surfactants, confers high stealth in biological medium with reduced nonspecific interactions. Tracking individual NE by fluorescence microscopy techniques would lead to a better understanding of their behavior in cells and thus require the development of bright single particles with enhanced photostability. However, the understanding of the relationship between the physicochemical properties and chemical composition of the NEs, on the one hand, and its fluorescence properties of encapsulated dyes, on the other hand, remains limited. Herein, we synthesized three new dioxaborine barbituryl styryl (DBS) dyes that displayed high molar extinction coefficients (up to 120 000 M–1 cm–1) with relatively low quantum yields in solvents and impressive fluorescence enhancement when dissolved in viscous oils (up to 0.98). The reported screening of nine different oils allowed disclosing a range of efficient “oil/dye” couples and understanding the main parameters that lead to the brightest NEs. We determine vitamin E acetate/DBS-C8 as the representative most efficient couple, combining high dye loading capabilities and low aggregation-induced quenching, leading to <50 nm ultrabright NEs (with brightness as high as 30 × 106 M–1 cm–1) with negligible dye leakage in biological media. Beyond a comprehensive optical and physicochemical characterization of fluorescent NEs, cellular two-photon excitation imaging was performed with polymer-coated cell penetrating NEs. Thanks to their impressive brightness and photostability, NEs displaying different charge surfaces were microinjected in HeLa cells and were individually tracked in the cytosol to study their relative velocity

Lanthanide-based bulky counterions against aggregation-caused quenching of dyes in fluorescent polymeric nanoparticles

Dye-loaded polymeric nanoparticles (NPs) are promising bioimaging agents because of their available surface chemistry, high brightness, and tunable optical properties. However, high dye loadings can cause the aggregation-caused quenching (ACQ) of the encapsulated fluorophores. Previously, we proposed to mitigate the ACQ inside polymeric NPs by insulating cationic dyes with bulky hydrophobic counterions. In order to implement new functionalities into dye-loaded NPs, here, we extend the concept of bulky counterions to anionic lanthanide-based complexes. We show that by employing Gd-based counterions with octadecyl rhodamine B loaded NPs at 30 wt% versus polymer, the fluorescence quantum yield can be increased to 10-fold (to 0.34). Moreover, Gd-anion provides NPs with enhanced contrast in electron microscopy. A combination of a luminescent Eu-based counterion with a far-red to near-infrared cyanine 5 dye (DiD) yields Forster resonance energy transfer NPs, where the UV-excited Eu-based counterion transfers energy to DiD, generating delayed fluorescence and large stokes shift of similar to 340 nm. Cellular studies reveal low cytotoxicity of NPs and their capacity to internalize without detectable dye leakage, in contrast to leaky NPs with small counterions. Our findings show that the aggregation behavior of cationic dyes in the polymeric NPs can be controlled by bulky lanthanide anions, which will help in developing bright luminescent multifunctional nanomaterials