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

Characterization of the livestock production system and potential for enhancing productivity through improved feeding in Amoni Division, Mweiga District, Central Kenya, May 2010

Lipid droplets (LDs) are intracellular lipid-rich organelles that regulate the storage of neutral lipids and were recently found to be involved in many physiological processes, metabolic disorders, and diseases including obesity, diabetes, and cancers. Herein we present a family of new fluorogenic merocyanine fluorophores based on an indolenine moiety and a dioxaborine barbiturate derivative. These so-called StatoMerocyanines (SMCy) fluoresce from yellow to the near-infrared (NIR) in oil with an impressive fluorescence enhancement compared to aqueous media. Additionally, SMCy display remarkably high molar extinction coefficients (up to 390 000 M^–1 cm^–1) and high quantum yield values (up to 100%). All the members of this new family specifically stain the LDs in live cells with very low background noise. Unlike Nile Red, a well-known lipid droplet marker, SMCy dyes possess narrow absorption and emission bands in the visible, thus allowing multicolor imaging. SMCy proved to be compatible with fixation and led to high-quality 3D images of lipid droplets in cells and tissues. Their high brightness allowed efficient tissue imaging of adipocytes and circulating LDs. Moreover their remarkably high two-photon absorption cross-section, especially SMCy5.5 (up to 13 300 GM), as well as their capacity to efficiently fluoresce in the NIR region led to two-photon multicolor tissue imaging (liver). Taking advantage of the available color palette, lipid droplet exchange between cells was tracked and imaged, thus demonstrating intercellular communication

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

Synthèses et évaluations immunologiques d oligosaccharides biotinylés (application au diagnostic des candidoses et des allergies alimentaires)

Ces travaux portent sur le développement d une nouvelle méthode de biotinylation d oligosaccharides. Cette technique est mise à profit pour la synthèse et l évaluation immunologiques d a et b 1,2 mannosides dans le cadre des candidoses et de fragments de N-glycanes dans celui des allergies alimentaires. En introduction, différentes techniques d immobilisation des hydrates de carbone sont présentées. Dans un premier chapitre, les méthodes de biotinylation d' oligosaccharides émergeant de la littérature sont décrites et référencées. Dans le chapitre 2, une nouvelle méthode de biotinylation faisant intervenir la biotine sulfone est développée ainsi que la synthèse d un modèle pour évaluer et valider cette technique. Dans un troisième chapitre, cette nouvelle méthode de biotinylation est mise à profit pour la synthèse et l immobilisation d a et b 1,2 mannosides biotinylés. La reconnaissance de ces produits par des anticorps monoclonaux spécifiques aux motifs 1,2 mannosides a été démontrée par des tests immunologiques grâce aux techniques de résonance plasmonique de surface et Luminex. Les résultats biologiques effectués sur des séra de patients présentant une candidose montrent que l utilisation de nos produits est un outil diagnostic efficace pour ce type d infection. Dans un dernier chapitre, cette même technique est utilisée pour la synthèse de fragments de N-glycanes dont les évaluations immunologiques sur des séra de patients allergiques peuvent nous renseigner sur le mécanisme des allergies alimentaires et plus particulièrement sur la compréhension les allergies croisées. Deux alternatives à l utilisation de thiols malodorants et toxiques tels que le thiophénol et l éthanethiol sont présentés. La synthèse des mannosides fait intervenir l utilisation du 2-naphtalènethiol et celle des N-glycanes celle du 2-méthyl-5-tert-butylthiophénol. Les nouveaux thioglycosides sont obtenus avec de très bons rendements et leurs utilisations en glycosylation se sont avérées efficaces.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Recent Advances in Fluorescent Probes for Lipid Droplets

Lipid droplets (LDs) are organelles that serve as the storage of intracellular neutral lipids. LDs regulate many physiological processes. They recently attracted attention after extensive studies showed their involvement in metabolic disorders and diseases such as obesity, diabetes, and cancer. Therefore, it is of the highest importance to have reliable imaging tools. In this review, we focus on recent advances in the development of selective fluorescent probes for LDs. Their photophysical properties are described, and their advantages and drawbacks in fluorescence imaging are discussed. At last, we review the reported applications using these probes including two-photon excitation, in vivo and tissue imaging, as well as LDs tracking