Magazine Articles |
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Bourg N., S. S., Dupuis G., Lévêque-Fort S. (2015). De la microscopie à la nanoscopie de fluorescence. L'actualité Chimique, (397-398), 35–40.
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Farcy, R., Benoit, C., Moustie, R., Eyrignoux, T., Beaumel, P., Toullec, A., & Fontaine-Aupart, M. P. (2015). Aiguille fibrée pour l’aide au diagnostic du cancer du sein par fluorescence endogène. Photoniques, 79, 22–25.
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Peer-reviewed Publications |
Abed, N., Said-Hassane, F., Zouhiri, F., Mougin, J., Nicolas, V., Desmaele, D., Gref, R., & Couvreur, P. (2015). An efficient system for intracellular delivery of beta-lactam antibiotics to overcome bacterial resistance. Scientific Reports, 5.
Résumé: The “Golden era” of antibiotics is definitely an old story and this is especially true for intracellular bacterial infections. The poor intracellular bioavailability of antibiotics reduces the efficency of many treatments and thereby promotes resistances. Therefore, the development of nanodevices coupled with antibiotics that are capable of targeting and releasing the drug into the infected-cells appears to be a promising solution to circumvent these complications. Here, we took advantage of two natural terpenes (farnesyl and geranyl) to design nanodevices for an efficient intracellular delivery of penicillin G. The covalent linkage between the terpene moieties and the antibiotic leads to formation of prodrugs that self-assemble to form nanoparticles with a high drug payload between 55-63%. Futhermore, the addition of an environmentally-sensitive bond between the antibiotic and the terpene led to an efficient antibacterial activity against the intracellular pathogen Staphylococcus aureus with reduced intracellular replication of about 99.9% compared to untreated infected cells. Using HPLC analysis, we demonstrated and quantified the intracellular release of PenG when this sensitive-bond (SB) was present on the prodrug, showing the success of this technology to deliver antibiotics directly into cells.
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Agostoni, V., Horcajada, P., Noiray, M., Malanga, M., Aykac, A., Jicsinszky, L., Vargas-Berenguel, A., Semiramoth, N., Daoud-Mahammed, S., Nicolas, V., Martineau, C., Taulelle, F., Vigneron, J., Etcheberry, A., Serre, C., & Gref, R. (2015). A “green” strategy to construct non-covalent, stable and bioactive coatings on porous MOF nanoparticles. Scientific Reports, 5, 7925.
Résumé: Nanoparticles made of metal-organic frameworks (nanoMOFs) attract a growing interest in gas storage, separation, catalysis, sensing and more recently, biomedicine. Achieving stable, versatile coatings on highly porous nanoMOFs without altering their ability to adsorb molecules of interest represents today a major challenge. Here we bring the proof of concept that the outer surface of porous nanoMOFs can be specifically functionalized in a rapid, biofriendly and non-covalent manner, leading to stable and versatile coatings. Cyclodextrin molecules bearing strong iron complexing groups (phosphates) were firmly anchored to the nanoMOFs' surface, within only a few minutes, simply by incubation with aqueous nanoMOF suspensions. The coating procedure did not affect the nanoMOF porosity, crystallinity, adsorption and release abilities. The stable cyclodextrin-based coating was further functionalized with: i) targeting moieties to increase the nanoMOF interaction with specific receptors and ii) poly(ethylene glycol) chains to escape the immune system. These results pave the way towards the design of surface-engineered nanoMOFs of interest for applications in the field of targeted drug delivery, catalysis, separation and sensing.
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Bon, P., Bourg, N., Lecart, S., Monneret, S., Fort, E., Wenger, J., & Leveque-Fort, S. (2015). Three-dimensional nanometre localization of nanoparticles to enhance super-resolution microscopy. Nat Commun, 6, 7764.
Résumé: Meeting the nanometre resolution promised by super-resolution microscopy techniques (pointillist: PALM, STORM, scanning: STED) requires stabilizing the sample drifts in real time during the whole acquisition process. Metal nanoparticles are excellent probes to track the lateral drifts as they provide crisp and photostable information. However, achieving nanometre axial super-localization is still a major challenge, as diffraction imposes large depths-of-fields. Here we demonstrate fast full three-dimensional nanometre super-localization of gold nanoparticles through simultaneous intensity and phase imaging with a wavefront-sensing camera based on quadriwave lateral shearing interferometry. We show how to combine the intensity and phase information to provide the key to the third axial dimension. Presently, we demonstrate even in the occurrence of large three-dimensional fluctuations of several microns, unprecedented sub-nanometre localization accuracies down to 0.7 nm in lateral and 2.7 nm in axial directions at 50 frames per second. We demonstrate that nanoscale stabilization greatly enhances the image quality and resolution in direct stochastic optical reconstruction microscopy imaging.
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Bourg, N., Mayet, C., Dupuis, G., Barroca, T., Bon, P., Lecart, S., Fort, E., & Leveque-Fort, S. (2015). Direct optical nanoscopy with axially localized detection. Nature Photonics, 9(9), 587–+.
Résumé: Evanescent light excitation is widely used in super-resolution fluorescence microscopy to confine light and reduce background noise. Here, we propose a method of exploiting evanescent light in the context of emission. When a fluorophore is located in close proximity to a medium with a higher refractive index, its near-field component is converted into light that propagates beyond the critical angle. This so-called supercritical-angle fluorescence can be captured using a high-numerical-aperture objective and used to determine the axial position of the fluorophore with nanometre precision. We introduce a new technique for three-dimensional nanoscopy that combines direct stochastic optical reconstruction microscopy (dSTORM) with dedicated detection of supercritical-angle fluorescence emission. We demonstrate that our approach of direct optical nanoscopy with axially localized detection (DONALD) typically yields an isotropic three-dimensional localization precision of 20 nm within an axial range of similar to 150 nm above the coverslip.
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Chan, A. J., Steenkeste, K., Canette, A., Eloy, M., Brosson, D., Gaboriaud, F., & Fontaine-Aupart, M. - P. (2015). Natural Rubber-Filler Interactions: What Are the Parameters? Langmuir, 31(45), 12437–12446.
Résumé: Reinforcement of a polymer matrix through the incorporation of nanoparticles (fillers) is a common industrial practice that greatly enhances the mechanical properties of the composite material. The origin of such mechanical reinforcement has been linked to the interaction between the polymer and filler as well as the homogeneous dispersion of the filler within the polymer matrix. In natural rubber (NR) technology, knowledge of the conditions necessary to achieve more efficient NR-filler interactions is improving continuously. This study explores the important physicochemical parameters required to achieve NR-filler interactions under dilute aqueous conditions by varying both the properties of the filler (size, composition, surface activity, concentration) and the aqueous solution (ionic strength, ion valency). By combining fluorescence and electron microscopy methods, we show that NR and silica interact only in the presence of ions and that heteroaggregation is favored more than homoaggregation of silica-silica or NR-NR. The interaction kinetics increases with the ion valence, whereas the morphology of the heteroaggregates depends on the size of silica and the volume percent ratio (dry silica/dry NR). We observe dendritic structures using silica with a diameter (d) of 100 nm at a similar to 20-50 vol % ratio, whereas we obtain raspberry-like structures using silica with d = 30 nm particles. We observe that in liquid the interaction is controlled by the hydrophilic bioshell, in contrast to dried conditions, where hydrophobic polymer dominates the interaction of NR with the fillers. A good correlation between the nanoscopic aggregation behavior and the macroscopic aggregation dynamics of the particles was observed. These results provide insight into improving the reinforcement of a polymer matrix using NR-filler films.
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Chan, A. J., Steenkeste, K., Ely, M., Brssn, D., Gaboriaud, F., & Fontaine-Aupart, M. - P. (2015). Lipid Content In Small And Large Natural Rubber Particles. Rubber Chemistry And Technology, 88(2), 248–257.
Résumé: Knowledge of the surface composition of natural rubber (NR) latex is essential to manufacturers of latex goods. Films made from only small rubber particles (SRPs) and a mix of SRPs and large rubber particles (LRPs) differ in mechanical properties. The reason for this difference, which is still under debate, is hypothesized to be linked with biomolecules (proteins and lipids) present in the NR particle surface. In this study, we characterize the surface chemistry, particularly lipid content of the SRP and LRP, by performing investigations directly on these particles in aqueous conditions. Fluorescent probes were used to display protein and lipid affinity and analyze them in situ with steady-state fluorescence spectroscopy, fluorescence correlation spectroscopy, and fluorescence lifetime measurements. Results are atypical in showing that lipids are more abundant in LRPs than in SRPs, suggesting thicker and/or denser membranes in LRPs. The degree of membrane compacity affects rigidity, influences biomolecular interactions, and might impact natural rubber coagulation. These results provide additional insights into colloidal behavior of NR for more efficient industrial applications.
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De Wit, R., Gautret, P., Bettarel, Y., Roques, C., Marliere, C., Ramonda, M., Thanh, T. N., Quang, H. T., & Bouvier, T. (2015). Viruses Occur Incorporated in Biogenic High-Mg Calcite from Hypersaline Microbial Mats. Plos One, 10(6), e0130552.
Résumé: Using three different microscopy techniques (epifluorescence, electronic and atomic force microscopy), we showed that high-Mg calcite grains in calcifying microbial mats from the hypersaline lake “La Salada de Chiprana”, Spain, contain viruses with a diameter of 50-80 nm. Energy-dispersive X-ray spectrometer analysis revealed that they contain nitrogen and phosphorus in a molar ratio of similar to 9, which is typical for viruses. Nucleic acid staining revealed that they contain DNA or RNA. As characteristic for hypersaline environments, the concentrations of free and attached viruses were high (>10(10) viruses per g of mat). In addition, we showed that acid treatment (dissolution of calcite) resulted in release of viruses into suspension and estimated that there were similar to 15 x 109 viruses per g of calcite. We suggest that virus-mineral interactions are one of the possible ways for the formation of nano-sized structures often described as “nanobacteria” and that viruses may play a role in initiating calcification.
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El-Daher, M. T., Hangen, E., Bruyere, J., Poizat, G., Al-Ramahi, I., Pardo, R., Bourg, N., Souquere, S., Mayet, C., Pierron, G., Leveque-Fort, S., Botas, J., Humbert, S., & Saudou, F. (2015). Huntingtin proteolysis releases non-polyQ fragments that cause toxicity through dynamin 1 dysregulation. Embo Journal, 34(17), 2255–2271.
Résumé: Cleavage of mutant huntingtin (HTT) is an essential process in Huntington's disease (HD), an inherited neurodegenerative disorder. Cleavage generates N-ter fragments that contain the polyQ stretch and whose nuclear toxicity is well established. However, the functional defects induced by cleavage of full-length HTT remain elusive. Moreover, the contribution of non-polyQ C-terminal fragments is unknown. Using time- and site-specific control of full-length HTT proteolysis, we show that specific cleavages are required to disrupt intramolecular interactions within HTT and to cause toxicity in cells and flies. Surprisingly, in addition to the canonical pathogenic N-ter fragments, the C-ter fragments generated, that do not contain the polyQ stretch, induced toxicity via dilation of the endoplasmic reticulum (ER) and increased ER stress. C-ter HTT bound to dynamin 1 and subsequently impaired its activity at ER membranes. Our findings support a role for HTT on dynamin 1 function and ER homoeostasis. Proteolysis-induced alteration of this function may be relevant to disease.
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Fraix, A., Kandoth, N., Gref, R., & Sortino, S. (2015). A Multicomponent Gel for Nitric Oxide Photorelease with Fluorescence Reporting. Asian Journal Of Organic Chemistry, 4(3), 256–261.
Résumé: An engineered hydrogel platform has been developed, in the absence of any toxic solvents or reagents, by the supramolecular self-assembly of three different components: a poly--cyclodextrin polymer, a hydrophobically modified dextran, and a photoactivatable bichromophoric molecular conjugate designed to photorelease nitric oxide (NO) with a fluorescent reporting function. The multivalent character of the interactions between the all components and the poor solubility of the conjugate in aqueous medium ensure the stability of the hydrogel and the lack of leaching of the photoactive cargo from the gel network under physiological conditions, even in the absence of protective coating agents. The photochemical properties of the molecular conjugate are retained in the supramolecular matrix, as demonstrated by the remote-controlled release of NO upon visible light excitation simultaneously to the activation of a fluorescent reporting function, which allows easy monitoring of the photoreleased NO.
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Ladaviere, C., & Gref, R. (2015). Toward an optimized treatment of intracellular bacterial infections: input of nanoparticulate drug delivery systems. Nanomedicine, 10(19), 3033–3055.
Résumé: Intracellular pathogenic bacteria can lead to some of the most life-threatening infections. By evolving a number of ingenious mechanisms, these bacteria have the ability to invade, colonize and survive in the host cells in active or latent forms over prolonged period of time. A variety of nanoparticulate systems have been developed to optimize the delivery of antibiotics. Main advantages of nanoparticulate systems as compared with free drugs are an efficient drug encapsulation, protection from inactivation, targeting infection sites and the possibility to deliver drugs by overcoming cellular barriers. Nevertheless, despite the great progresses in treating intracellular infections using nanoparticulate carriers, some challenges still remain, such as targeting cellular subcompartments with bacteria and delivering synergistic drug combinations. Engineered nanoparticles should allow controlling drug release both inside cells and within the extracellular space before reaching the target cells.
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Marliere, C., & Dhahri, S. (2015). An in vivo study of electrical charge distribution on the bacterial cell wall by atomic force microscopy in vibrating force mode. Nanoscale, 7(19), 8843–8857.
Résumé: We report an in vivo electromechanical atomic force microscopy (AFM) study of charge distribution on the cell wall of Gram+ Rhodococcus wratislaviensis bacteria, naturally adherent to a glass substrate, under physiological conditions. The method presented in this paper relies on a detailed study of AFM approach/retract curves giving the variation of the interaction force versus distance between the tip and the sample. In addition to classical height and mechanical (as stiffness) data, mapping of local electrical properties, such as bacterial surface charge, was proved to be feasible at a spatial resolution better than a few tens of nanometers. This innovative method relies on the measurement of the cantilever's surface stress through its deflection far from (>10 nm) the repulsive contact zone: the variations of surface stress come from the modification of electrical surface charge of the cantilever (as in classical electrocapillary measurements) likely stemming from its charging during contact of both the tip and the sample electrical double layers. This method offers an important improvement in local electrical and electrochemical measurements at the solid/liquid interface, particularly in high-molarity electrolytes when compared to techniques focused on the direct use of electrostatic force. It thus opens a new way to directly investigate in situ biological electrical surface processes involved in numerous practical applications and fundamental problems such as bacterial adhesion, biofilm formation, microbial fuel cells, etc.
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Nie, S. Y., Zhang, X. F., Gref, R., Couvreur, P., Qian, Y., & Zhang, L. J. (2015). Multilamellar Nanoparticles Self-Assembled from Opposite Charged Blends: Insights from Mesoscopic Simulation. Journal Of Physical Chemistry C, 119(35), 20649–20661.
Résumé: Multi lamellar nanoparticles (NPs) are spontaneously formed when mixing two components with opposite charges, meaningful for drug delivery. However, details of NPs association and mechanisms of this process remain largely unknown, due to the limitation of experimental technique. In this work, we use dissipative particle dynamics (DPD) simulation for the first time to determine the structure property relationships of multilamellar NPs formed by charged blends. As a case study, a system with polyanionic fondparinux (Fpx) and cationic derivatives squalenoyl (CSq, including Sql(+) and Sq(++)) in aqueous media is investigated, with a focus on the optimized formation condition and mechanism of regular spherical multilamellar NPs. In particular, we find that highly ordered multilamellar structures tend to form when the nonbonded interaction between Fpx CSq and hydrophobic interaction contributed by CSq are well-balanced. The DPD results strongly agree with corresponding experimental results of this novel nanoparticulate drug carrier. This study could help develop promising multilamellar NPs formed by charged blends through self-assembly for drug delivery.
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Ralay-Ranaivo, B., Borgel, D., Couvreur, P., & Gref, R. (2015). Trends in the development of oral anticoagulants. Therapeutic Delivery, 6(6), 685–703.
Résumé: Anticoagulation remains the therapy of choice for the prevention and treatment of venous and arterial thromboembolic disorders which can cause major organ damage or death. Heparins represent the antithrombotic drugs of choice in short and medium-term prophylaxis and therapy of thromboembolic diseases. Fondaparinux, a synthetic and structural analog of the antithrombin-binding pentasaccharide domain of heparin, has selective anti-Xa activity and longer half-life. However, anticoagulants are poorly absorbed by oral route because of their high molecular weight, hydrophilicity and negative charges. Long-term anticoagulation therapy is problematic because of side effects and frequent monitoring. Formulation approaches are particularly promising.
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Rodriguez-Ruiz, V., Maksimenko, A., Anand, R., Monti, S., Agostoni, V., Couvreur, P., Lampropoulou, M., Yannakopoulou, K., & Gref, R. (2015). Efficient “green” encapsulation of a highly hydrophilic anticancer drug in metal-organic framework nanoparticles. Journal Of Drug Targeting, 23(7-8), 759–767.
Résumé: Metal-organic frameworks (MOFs) are coordination polymers of interest for biomedical applications. Of particular importance, nanoparticles made of iron(III) trimesate (MIL-100, MIL standing for Material Institut Lavoisier) (nanoMOFs) can be conveniently synthesised under mild and green conditions. They were shown to be biodegradable, biocompatible and efficient to encapsulate a variety of active molecules. We have addressed here the challenges to encapsulate a highly hydrophilic anticancer prodrug, phosphated gemcitabin (Gem-MP) known for its instability and inability to bypass cell membranes. MIL-100 nanoMOFs acted as efficient “nanosponges”, soaking Gem-MP from its aqueous solution with almost perfect efficiency (>98%). Maximal loadings reached similar to 30 wt% reflecting the strong interaction between the drug and the iron trimesate matrices. Neither degradation nor loss of crystalline structure was observed after the loading process. Storage of the loaded nanoMOFs in water did not result in drug release over three days. However, Gem-MP was released in media containing phosphates, as a consequence to particle degradation. Drug-loaded nanoMOFs were effective against pancreatic PANC-1 cells, in contrast to free drug and empty nanoMOFs. However, an efflux phenomenon could contribute to reduce the efficacy of the nanocarriers. Size optimization and surface modification of the nanoMOFs are expected to further improve these findings.
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Sarsa, A., Alcaraz-Pelegrina, J. M., & Le Sech, C. (2015). Isotopic Effects on Covalent Bond Confined in a Penetrable Sphere. Journal Of Physical Chemistry B, 119(45), 14364–14372.
Résumé: A model of confinement of the covalent bond by a finite potential beyond the Born-Oppenheimer approximation is presented. A two-electron molecule is located at the center of a penetrable spherical cavity. The Schrodinger equation has been solved by using the diffusion Monte Carlo method. Total energies, internuclear distances, and vibrational frequencies of the confined molecular system have been obtained. Even for confining potentials of a few electronvolts, a noticeable increase in the bond energy and the nuclear vibrational frequency is observed, and the internuclear distance is lowered. The gap between the zero point energy of different molecular isotopes increases with confinement. The confinement of the electron pair might play a role in chemical reactivity, providing an alternative explanation for the tunnel effect, when large values of primary kinetic isotopic effect are observed. The Swain-Schaad relation is still verified when confinement changes the zero point energy. A semiquantitative illustration is proposed using the data relative to an hydrogen transfer involving a C-H cleavage catalyzed by the bovine serum amine oxidase. Changes on the confining conditions, corresponding to a confinement/deconfinement process, result in a significant decrease in the activation energy of the chemical transformation. It is proposed that confinement/deconfinement of the electron-pair bonding by external electrostatic forces inside the active pocket of an enzyme could be one of the basic mechanisms of the enzyme catalysis.
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Saunier, J., Herry, J. M., Marliere, C., Renault, M., Bellon-Fontaine, M. N., & Yagoubi, N. (2015). Modification of the bacterial adhesion of Staphylococcus aureus by antioxidant blooming on polyurethane films. Materials Science & Engineering C-Materials For Biological Applications, 56, 522–531.
Résumé: Medical device-related infections are a major problem in hospital. The risk of developing an infection is linked to the bacterial adhesion ability of pathogen strains on the device and their ability to form a biofilm. Here we focused on polymer surfaces exhibiting a blooming of antioxidant (Irganox 3114 (R)) and Irganox 1076 (R)) on their surface. We tried to put into evidence the effect of such a phenomenon on the bacterial adhesion in terms of number of viable cultivable bacteria and bacteria localization on the surface. We showed that the blooming has a tendency to increase the Staphylococcus aureus adhesion phenomenon in part for topographic reasons. (C) 2015 Elsevier B.V. All rights reserved.
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