Inter-nanocarrier and nanocarrier-to-cell transfer assays demonstrate the risk of an immediate unloading of dye from labeled lipid nanocapsules

Release studies constitute a fundamental part of the nanovector characterization. However, it can be difficult to correctly assess the release of lipophilic compounds from lipid nanocarriers using conventional assays. Previously, we proposed a method including an extraction with oil to measure the loading stability of lipophilic dyes in lipid nanocapsules (LNCs). The method indicated a rapid release of Nile Red from LNCs, while the loading of lipophilic carbocyanine dyes remained stable. This method, although interesting for a rapid screening of the fluorescence labeling stability of nanocarriers, is far from what happens in vivo, where lipid acceptor phases are nanostructured. Here, lipophilic dye loading stability has been assessed, by monitoring dye transfer from LNCs toward stable colloidal lipid nanocompartments, i.e. non-loaded LNCs, using new methodology based on size exclusion chromatography (SEC) and Förster Resonance Energy Transfer (FRET). Dye transfer between LNCs and THP-1 cells (as model for circulating cells) has also been studied by FACS. The assays reveal an almost instantaneous transfer of Nile Red between LNCs, from LNCs to THP-1 cells, between THP-1 cells, and a reversal transfer from THP-1 cells to LNCs. On the contrary, there was no detectable transfer of the lipophilic carbocyanine dyes. Dye release was also analyzed using dialyses, which only revealed a very slow release of Nile Red from LNCs, demonstrating the weakness of membrane based assays for investigations of the lipophilic compound loading stability in lipid nanocarriers. These results highlight the importance of using relevant release assays, and the potential risk of an immediate unloading of lipophilic fluorescent dyes from lipid nanocarriers, in the presence of a lipid acceptor nanocompartment. Some misinterpretations of cellular trafficking and in vivo biodistribution of fluorescent nanoparticles should be avoided

Probing dynamics of HIV-1 nucleocapsid protein/target hexanucleotide complexes by 2-aminopurine

The nucleocapsid protein (NC) plays an important role in HIV-1, mainly through interactions with the genomic RNA and its DNA copies. Though the structures of several complexes of NC with oligonucleotides (ODNs) are known, detailed information on the ODN dynamics in the complexes is missing. To address this, we investigated the steady state and time-resolved fluorescence properties of 2-aminopurine (2Ap), a fluorescent adenine analog introduced at positions 2 and 5 of AACGCC and AATGCC sequences. In the absence of NC, 2Ap fluorescence was strongly quenched in the flexible ODNs, mainly through picosecond to nanosecond dynamic quenching by its neighboring bases. NC strongly restricted the ODN flexibility and 2Ap local mobility, impeding the collisions of 2Ap with its neighbors and thus, reducing its dynamic quenching. Phe16→Ala and Trp37→Leu mutations largely decreased the ability of NC to affect the local dynamics of 2Ap at positions 2 and 5, respectively, while a fingerless NC was totally ineffective. The restriction of 2Ap local mobility was thus associated with the NC hydrophobic platform at the top of the folded fingers. Since this platform supports the NC chaperone properties, the restriction of the local mobility of the bases is likely a mechanistic component of these properties

Deciphering the pH-dependence of ground- and excited-state equilibria of thienoguanine

The thienoguanine nucleobase (thGb) is an isomorphic fluorescent analogue of guanine. In aqueous buffer at neutral pH, thGb exists as a mixture of two ground-state H1 and H3 keto-amino tautomers with distinct absorption and emission spectra and high quantum yield. In this work, we performed the first systematic photophysical characterization of thGb as a function of pH (2 to 12). Steady-state and time-resolved fluorescence spectroscopies, supplemented with theoretical calculations, enabled us to identify three additional thGb forms, resulting from pH-dependent ground-state and excited-state reactions. Moreover, a thorough analysis allowed us to retrieve their individual absorption and emission spectra as well as the equilibrium constants which govern their interconversion. From these data, the complete photoluminescence pathway of thGb in aqueous solution and its dependence as a function of pH was deduced. As the identified forms differ by their spectra and fluorescence lifetime, thGb could be used as a probe for sensing local pH changes under acidic conditions

Highly lipophilic fluorescent dyes in nano-emulsions: towards bright non-leaking nano-droplets

Dye-loaded lipid nano-droplets present an attractive alternative to inorganic nanoparticles, as they are composed of non-toxic biodegradable materials and are easy to prepare. However, to achieve high fluorescence brightness, the nano-droplets have to be heavily loaded with the dyes avoiding fluorescence self-quenching and release (leakage) of the encapsulated dyes from the nano-droplets in biological media. In the present work, we have designed highly lipophilic fluorescent derivatives of 3-alkoxyflavone (F888) and Nile Red (NR668) that can be encapsulated in the lipophilic core of stable nano-emulsion droplets at exceptionally high concentrations in the oil core, i.e. up to 170 mM and 17 mM, respectively, corresponding to ∼830 and 80 dyes per 40 nm droplet. Despite this high loading, these dyes keep high fluorescence quantum yields and thus, provide high nano-droplet brightness, probably due to their bulky structure preventing self-quenching. Moreover, simultaneous encapsulation of both dyes at high concentrations in single nano-droplets allows the observation of FRET. FRET and fluorescence correlation spectroscopy (FCS) studies showed that NR668 release in the serum-containing medium is very slow, while the reference hydrophobic dye Nile Red leaks immediately. This drastic difference in the leakage profile between NR668 and Nile Red was confirmed by in vitro cellular studies as well as by in vivo angiography imaging on zebrafish models, where the NR668-loaded nano-droplets remained in the blood, while the parent Nile Red leaked rapidly from the droplets distributing all over the animal body. This study suggests new molecular design strategies for obtaining bright nano-droplets without dye leakage and their use as efficient and stable optical contrast agents in vitro and in vivo

Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West Africa

West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.status: publishe

Imaging lipid order changes in endosome membranes of live cells by using a Nile Red-based membrane probe

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Mechanism of zinc coordination by point-mutated structures of the distal CCHC binding motif of the HIV-1 NCp₇ protein

The kinetics of Zn2+ binding by two point-mutated forms of the HIV-1 NCp7 C-terminal zinc finger, each containing tridentate binding motif HCC [Ser49(35−50)NCp7] or CCC [Ala44(35−50)NCp7], has been studied by stopped-flow spectrofluorimetry. Both the formation and dissociation rate constants of the complexes between Zn2+ and the two model peptides depend on pH. The results are interpreted on the basis of a multistep reaction model involving three Zn2+ binding paths due to three deprotonated states of the coordinating motif, acting as monodentate, bidentate, and tridentate ligands. For Ser49(35−50)NCp7 around neutral pH, binding preferentially occurs via the deprotonated Cys36 in the bidentate state also involving His44. The binding rate constants for the monodentate and bidentate states are 1 × 106 and 3.9 × 107 M-1 s-1, respectively. For Ala44(35−50)NCp7, intermolecular Zn2+ binding predominantly occurs via the deprotonated Cys36 in the monodentate state with a rate constant of 3.6 × 107 M-1 s-1. In both mutants, the final state of the Zn2+ complex is reached by subsequent stepwise ligand deprotonation and intramolecular substitution of coordinated water molecules. The rate constants for the intermolecular binding paths of the bidentate and tridentate states of Ala44(35−50)NCp7 and of the tridentate state of Ser49(35−50)NCp7 are much smaller than expected according to electrostatic considerations. This is attributed to conformational constraints required to achieve proper metal coordination during folding. The dissociation of Zn2+ from both peptides is again characterized by a multistep process and takes place fastest via the protonated Zn2+-bound bidentate and monodentate states, with rate constants of ∼0.3 and ∼103 s-1, respectively, for Ser49(35−50)NCp7 and ∼4 × 10-3 and ∼500 s-1, respectively, for Ala44(35−50)NCp7

Comparison of the Oilseed rape mosaic virus and Tobacco mosaic virus movement proteins (MP) reveal common and dissimilar MP functions for tobamovirus spread

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