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International audience; We present an angle-resolved photoelectron spectroscopy study on isolated gold nanoparticles in the photon energy range between 6 and 12 eV performed at the DESIRS beamline of Synchrotron SOLEIL with a velocity map imaging spectrometer. The data are reported for polydisperse gold aerosols formed from hydrocolloids of bare and citrate-capped primary nanoparticles. The aerosols were brought to interact with the synchrotron radiation via a high-performance aerodynamic lens system developed for this study. The electronic structure of the aerosols was elucidated by threshold photoemission spectroscopy, which showed excellent agreement with the theoretical density of states. The photoionization cross sections of isolated gold particles were derived in the ∼104 to 106 Mb range between 8 and 12 eV. We introduce a new methodology to extract all of the photoemission observables from the electron images as a function of electron energy, including photoelectron spectrum, anisotropy parameter b2, and asymmetry parameter, α(E), which describes the shadowing asymmetry along the radiation propagation axis. The results show that electron energy distribution depends on the procedure used in the synthesis of the nanoparticles and on the ligand concentration. The dependence of the asymmetry parameter on electron energy along the valence band is rationalized through theoretical modeling in terms of the electric field distribution inside the particle and the losses due to inelastic scattering.

Fluorescent nanocomposite films were prepared by solution mixing of polyvinyl alcohol (PVA) and tryptophan functionalized silver (AgTrp) nanoparticles. Synchrotron excitation deep-ultraviolet (DUV) fluorescence imaging was used to follow the distribution of AgTrp nanoparticles within the polymer matrix. Pronounced growth of dendrite-like structures in the presence of AgTrp nanoparticles was confirmed by bright field and fluorescence imaging as well as with atomic force microscopies. Both tryptophan and AgTrp nanoparticles influence thermal properties of the matrix. Differential scanning calorimetry measurements showed that they reduce crystallinity and significantly increase the glass transition temperature of PVA (in the case of PVA-AgTrp film the Tg increases by ~20 °C). Dielectric properties of the films were studied under two different conditions (dark and illumination). The illumination with 250 nm light induces a significant increase in conductance of the PVA-AgTrp nanocomposite. The relative changes, with respect to the conductance measured in the dark, were almost ~200%.

A maltotriose-conjugate can deliver molecules into the cytoplasmic space of Gram-negative bacteria by parasitizing the maltose uptake pathway., Small molecule accumulation in Gram-negative bacteria is a key challenge to discover novel antibiotics, because of their two membranes and efflux pumps expelling toxic molecules. An approach to overcome this challenge is to hijack uptake pathways so that bacterial transporters shuttle the antibiotic to the cytoplasm. Here, we have characterized maltodextrin–fluorophore conjugates that can pass through both the outer and inner membranes mediated by components of the Escherichia coli maltose regulon. Single-channel electrophysiology recording demonstrated that the compounds permeate across the LamB channel leading to accumulation in the periplasm. We have also demonstrated that a maltotriose conjugate distributes into both the periplasm and cytoplasm. In the cytoplasm, the molecule activates the maltose regulon and triggers the expression of maltose binding protein in the periplasmic space indicating that the complete maltose entry pathway is induced. This maltotriose conjugate can (i) reach the periplasmic and cytoplasmic compartments to significant internal concentrations and (ii) auto-induce its own entry pathway via the activation of the maltose regulon, representing an interesting prototype to deliver molecules to the cytoplasm of Gram-negative bacteria.

Background In Gram-negative bacteria, passing through the double membrane barrier to reach the inhibitory concentration inside the bacterium is a pivotal step for antibiotic activity. Spectrofluorimetry has been developed to follow fluoroquinolone accumulation inside bacteria using intrinsic bacterial fluorescence as an internal standard. However, adaptation for non-fluorescent antibiotics is needed; quantitative methods based on MS offer the possibility of expanding the detection range obtained by spectrofluorimetry. Objectives To validate, with spectrofluorimetry, the use of MS to measure antibiotic accumulation in cells and to determine the relationship between antibiotic concentrations and the amount of intrabacterial accumulation in different efflux backgrounds on the same batch of molecules. Methods Spectrofluorimetry was performed in parallel with MS on the same samples to measure the ciprofloxacin and fleroxacin accumulation in cells expressing various efflux pump levels. A microplate protocol was set up to determine the antibiotic accumulation as a function of external antibiotic concentrations. Results A correlation existed between the data obtained with spectrofluorimetry and MS, whatever the efflux pump or tested antibiotic. The results highlighted different dynamics of uptake between ciprofloxacin and fleroxacin as well as the relationship between the level of efflux activity and antibiotic accumulation. Conclusions We have developed a microplate protocol and cross-validated two complementary methods: spectrofluorimetry, which contains a reliable internal standard; and MS, which allows detection of low antibiotic amounts. These assays allow study of the dose effect and the efflux impact on the intrabacterial accumulation of antibiotics.

International audience; The efficacy of antibacterial molecules depends on their capacity to reach inhibitory concentrations in the vicinity of their target. This is particularly challenging for drugs directed against Gram-negative bacteria, which have a complex envelope comprising two membranes and efflux pumps. Precise determination of the bacterial drug content is an essential prerequisite for drug development. Here we describe three approaches that have been developed in our laboratories to quantify drugs accumulated in intact cells by spectrofluorimetry, microspectrofluorimetry, and kinetics microspectrofluorimetry (KMSF). These different procedures provide complementary results that highlight the contribution of membrane-associated mechanisms, including influx through the outer membrane (OM) and efflux, and the importance of the physicochemical properties of the transported drugs for the intracellular concentration of a given antibiotic in a given bacterial population. The three key stages of this protocol are preparation of the bacterial strains in the presence of the antibiotic; preparation of the whole-cell lysates (WCLs) and fluorescence readings; and data analysis, including normalization and quantitation of the intracellular antibiotic fluorescence relative to the internal standard and the antibiotic standard curve, respectively. Fluorimetry is limited to naturally fluorescent or labeled compounds, but in contrast to existing alternative methods such as mass spectrometry, it uniquely allows single-cell analysis. From culture growth to data analysis, the protocol described here takes 5 d.

The investigation of the interaction of silver nanoparticles and live bacteria cells is of particular importance for understanding and controlling their bactericidal properties. In this study, the process of internalization of silver nanoparticles in Escherichia coli cells was followed by means of synchrotron excitation deep ultraviolet (DUV) fluorescence imaging. Antimicrobial nanostructures that can absorb and emit light in the UV region were prepared by functionalization of silver nanoparticles with tryptophan amino acid and used as environmentally sensitive fluorescent probes. The nanostructures were characterized by morphological (TEM) and spectroscopic methods (UV-vis, FTIR, XPS, and photoluminescence). The TEM images and the analyses of the UV-vis spectra suggested that the addition of tryptophan led to the formation of hybrid nanostructures with pronounced eccentricity and larger sizes with respect to that of the initial silver nanoparticles. The DUV imaging showed that it was possible to distinguish the fluorescent signal pertaining to silver-tryptophan nanostructures from the autofluorescence of the bacteria. The spatial resolution of the fluorescence images was 154 nm which was sufficient to perform analyses of the accumulation of the nanostructures within a single bacterium. The DUV imaging results imply that the tryptophan-functionalized silver nanoparticles interact with cell membranes via insertion of the amino acid into the phospholipid bilayer and enter the cells.

The interaction of partially reduced graphene oxide (prGO) and Huh7.5.1 liver cancer cells was investigated by means of DUV fluorescence bioimaging. The prGO sample was obtained by the reduction (to a certain extent) of the initially prepared graphene oxide (GO) nanosheets with hydrazine. The fluorescence of the GO nanosheets increases with time of the reduction due to a change in ratio of the sp2 and sp3 carbon sites and the prGO sample was extracted from the dispersion after 6 min, when the intensity of the fluorescence reached its maximum. The reduction process was left to proceed further to saturation until highly reduced graphene oxide (denoted here as rGO) was obtained. GO, prGO and rGO samples were investigated by structural (scanning electron microscopy (SEM), scanning transmission electron microscopy coupled with energy dispersive spectrometry (STEM-EDS)) and spectroscopic (UV-vis, photoluminescence (PL), Raman) methods. After that, Huh7.5.1 cells were incubated with GO, prGO and rGO nanosheets and used in bioimaging studies, which were performed on DISCO beamline of synchrotron SOLEIL. It was found that the prGO significantly enhanced the fluorescence of the cells and increased the intensity of the signal by ~2.5 times. Time-lapse fluorescence microscopy experiments showed that fluorescence dynamics strongly depends on the type of nanosheets used. The obtained prGO nanostructure can be easily conjugated with aromatic ring containing drugs, which opens a possibility for its applications in fluorescence microscopy monitored drug delivery. © 2018 IOP Publishing Ltd. This is the peer-reviewed version of the article: Dojčilović, R., Pajović, J.D., Božanić, D.K., Jović, N., Pavlović, V.P., Pavlović, V.B., Acković, L.L., Zeković, I., Dramićanin, M., Kaščaková, S., Réfrégiers, M., Rašić, G., Vlahović, B., Djoković, V., 2018. DUV fluorescence bioimaging study of the interaction of partially reduced graphene oxide and liver cancer cells. 2D Materials 5. [https://doi.org/10.1088/2053-1583/aad72b]

International audience; To understand the antibiotic resistance in Gram-negative bacteria, a key point is to investigate antibiotic accumulation which is defined by influx and efflux. Several methods exist to evaluate the membrane permeability and efflux pump activity but they present some disadvantages and limitations. An optimized spectrofluorimetric method using the intrinsic tryptophan fluorescence as internal standard as well as a complementary microfluorimetric assay following the time-course accumulation in intact individual cells have been developed. Comparing the latter population and single cell approaches can lead to the understanding of the phenotypic heterogeneity within a population. The two methodologies lead to the determination of parameters, concentration, accumulation rates, localization that contribute to emerging concepts (RTC2T, SICAR) with the aim to identify and detail the antibiotic chemotypes that are involved in influx/efflux.

International audience; A main challenge in chemotherapy is to determine the in cellulo parameters modulating the drug concentration required for therapeutic action. It is absolutely urgent to understand membrane permeation and intracellular concentration of antibiotics in clinical isolates: passing the membrane barrier to reach the threshold concentration inside the bacterial periplasm or cytoplasm is the pivotal step of antibacterial activity. Ceftazidime (CAZ) is a key molecule of the combination therapy for treating resistant bacteria. We designed and synthesized different fluorescent CAZ derivatives (CAZ*, CAZ**) to dissect the early step of translocation-accumulation across bacterial membrane. Their activities were determined on E. coli strains and on selected clinical isolates overexpressing ß-lactamases. The accumulation of CAZ* and CAZ** were determined by microspectrofluorimetry and epifluorimetry. The derivatives were properly translocated to the periplasmic space when we permeabilize the outer membrane barrier. The periplasmic location of CAZ** was related to a significant antibacterial activity and with the outer membrane permeability. This study demonstrated the correlation between periplasmic accumulation and antibiotic activity. We also validated the method for approaching ß-lactam permeation relative to membrane permeability and paved the way for an original matrix for determining " Structure Intracellular Accumulation Activity Relationship " for the development of new therapeutic candidates. With the misuse and overuse of the various antibiotic families and the scarcity of new drugs available, multid-rug resistance is now a major bacterial threat to healthcare worldwide 1–4. A major concern discussed in various studies is the intracellular concentration of the antibiotic molecule 5–7. In Gram-negative bacteria, the challenge for a drug is to permeate the outer membrane (OM) that protects the cell against external attacks 8, 9. This membrane plays a key role in controlling the diffusion of external toxic molecules and the antibiotics must translocate through the outer membrane to reach the periplasmic target or additionally pass the inner membrane to reach the cytoplasmic target 10, 11. During their translocation, the molecules can use various hydrophilic channels such as porins that form trimeric hydrophilic pores present in the OM 7, 8. Today a key challenge for hospital microbiologists is to bypass the resistance in a Gram-negative strain that can produce carbapenemase and/or ß-lactamase 12, 13. Recent studies proposed an alternative way by using a combination of ceftazidime (CAZ) + ß-lactamase inhibitor 14–16. Regarding this strategy, it is urgently required to dissect the relationships between the role of membrane and enzymatic barrier and the antibacterial activity of CAZ as recently reported 17. Presently, various groups have studied the binding of ß-lactams to penicillin binding proteins (PBP) using fluorophore-conjugated-penicillin, termed Bocillin 18, 19 , fluorescent cephalosporin C 20 or fluorescent meropenem 21. Despite these various efforts to develop labeled antibiotic probes, no key information has been obtained regarding the role of porins, penetration rate, and the effect of ß-lactamase on periplasmic concentrations when addressing the choice of combination of clinically-used cephalosporin and ß-lactamase

Bacterial multidrug resistance is a worrying health issue. In Gram-negative antibacterial research, the challenge is to define the antibiotic permeation across the membranes. Passing through the membrane barrier to reach the inhibitory concentration inside the bacterium is a pivotal step for antibacterial molecules. A spectrofluorimetric methodology has been developed to detect fluoroquinolones in bacterial population and inside individual Gram-negative bacterial cells. In this work, we studied the antibiotic accumulation in cells expressing various levels of efflux pumps. The assays allow us to determine the intracellular concentration of the fluoroquinolones to study the relationships between the level of efflux activity and the antibiotic accumulation, and finally to evaluate the impact of fluoroquinolone structures in this process. This represents the first protocol to identify some structural parameters involved in antibiotic translocation and accumulation, and to illustrate the recently proposed “Structure Intracellular Concentration Activity Relationship” (SICAR) concept.

The interaction of the tryptophan functionalized Ag nanoparticles and live Candida albicans cells was studied by synchrotron excitation deep-ultraviolet (DUV) fluorescence imaging at the DISCO beamline of Synchrotron SOLEIL. DUV imaging showed that incubation of the fungus with functionalized nanoparticles results in significant increase in the fluorescence signal. The analysis of the images revealed that the interaction of the nanoparticles with (pseudo)hyphae polymorphs of the diploid fungus was less pronounced than in the case of yeast cells or budding spores. The changes in the intensity of the fluorescence signals of the cells after incubation were followed in [327-353 nm] and [370-410 nm] spectral ranges that correspond to the fluorescence of tryptophan in non-polar and polar environment, respectively. As a consequence of the environmental sensitivity of the silver-tryptophan fluorescent nanoprobe, we were able to determine the possible accumulation sites of the nanoparticles. The analysis of the intensity decay kinetics showed that the photobleaching effects were more pronounced in the case of the functionalized nanoparticle treated cells. The results of time-integrated emission in the mentioned spectral ranges suggested that the nanoparticles penetrate the cells, but that the majority of the nanoparticles attach to the cells surfaces. (C) 2017 Elsevier B.V. All rights reserved.

Fluorescent nanostructures were prepared by functionalization of gold nanoparticles with riboflavin molecules and used as probes for synchrotron radiation deep ultraviolet (SR-DUV) fluorescence imaging of gluteraldehyde-fixed Candida albicans cells. The nanoparticles were characterized by transmission electron microscopy (TEM) and optical spectroscopy techniques. The TEM analysis showed that the nanostructures were 6 nm in diameter, while the results of the fluorescence spectroscopies confirmed the photoluminescence of the nanoparticles. The SR-DUV imaging showed that it was possible to distinguish the fluorescence of the nanoparticles from the autofluorescence of the cells, as well as that the local maxima of the signal pertaining to the fluorescence of gold-riboflavin nanostructures were mostly positioned on the surfaces of the C. albicans cells of spherical morphology.

Biocompatible fluorescent nanostructures were prepared by a functionalization of gold nanoparticles with the amino acid tryptophan. The gold-tryptophan bioconjugates were investigated by TEM and HRTEM and various spectroscopy methods (XPS, FTIR, UV-vis and photoluminescence). It was found that the gold nanoparticles, initially 8. nm in diameter, aggregate in the presence of the amino acid. From the XPS and FTIR spectroscopy results, it was concluded that the tryptophan gold interactions mainly take place via indole and carboxyl groups. Although the indole group is involved in the interaction with the gold surfaces, the tryptophan-gold hybrids showed strong fluorescence due to the presence of multilayers of tryptophan. Deep ultra violet (DUV) imaging performed at the SOLEIL synchrotron showed that it is possible to detect these hybrid nanostructures within Escherichia coli cells. Ministry of Education and Science of the Republic of Serbia (Project Nos. 172056, 45020, and 171020). Scopus

Nanocomposites of electroactive ceramics and ferroelectric polymers exploit favorable features of the matrix polymer and the nanostructured filler to produce new functional materials for pressure and IR sensors. In this study, the influence of mechanical activation of barium titanate (BaTiO3) particles on the structural properties of BaTiO3/polyvinylidene fluoride (PVDF) nanocomposites was investigated. Nanocomposite films were prepared by the solution casting method and characterized by scanning electron microscopy, x-ray diffraction and Raman spectroscopy. It was found that mechanically activated fillers promote the formation of a ferroelectric beta-phase during crystallization of PVDF. 3rd International Conference on the Physics of Optical Materials and Devices, Sep 02-06, 2012, Belgrade, Serbia