Diversity of Extracellular Vesicles in Human Follicular Fluid: Morphological Analysis and Quantification.

The oocyte microenvironment constituted by the follicular fluid (FF) is a key for the optimal development of female gametes. Its composition reflects the physiological state of the ovarian follicle. The particularity of FF is to contain a huge diversity of extracellular vesicles specific to women, in the same way as seminal plasma in men. Here, we described and compared morphological aspects of broad subcategories of human FF-related Extracellular Vesicles (EVs). EVs participate in physiological and pathological processes and have potential applications in diagnostics or therapeutics. EVs isolated from FF are involved in different biological functions related to follicular growth, oocyte maturation, and embryo development. However, knowledge on the morphology of FF-derived EVs is limited, mainly due to their sub-micrometer size and to intrinsic limitations in methods applied for their characterization. The aim of this study was to provide a comprehensive morphological description of EVs from FF of healthy subjects and quantification. EVs separation was realized by centrifugation, with comparison of the EV yield obtained from differential centrifugation and one-step ultracentrifugation. Cryo-Transmission Electron Microscopy was used to reveal the morphology, size, and phenotype of EVs. Dynamic Light Scattering (DLS) and Nanoparticle Tracking Analysis (NTA) were used to quantify and analyze the size distribution for each centrifugation step. We performed a comprehensive inventory of human follicular fluid EVs. We show that human FF contains a huge diversity of EVs. This study brings novel insights on EVs from normal FF and provides a reference for further studies of EVs in ovarian diseases.

Photo-electrochemical properties of quantum rods studied by scanning electrochemical microscopy.

Scanning electrochemical microscopy (SECM) is used for studying the intrinsic photo-electrochemical properties of CdSe/CdS quantum rods. They are deposited on a transparent and non-conductive glass plate and investigated by SECM in feedback and generator-collector modes using a series of redox mediators. The method allows the interrogation of the quantum rods under illumination without the interference of the substrate, notably that due to the electron photo-ejection from the substrate, a process that is inherent to any polarized electrode material. Beside the methodological demonstration that could easily be extended to the investigations of the photo-redox properties of nanoparticles, studies highlight the strong reductive properties of quantum rods under illumination.

Self-Organization of Quantum Rods Induced by Lipid Membrane Corrugations.

Self-organization of fluorescent nanoparticles, using biological molecules such as phospholipids to control assembly distances, is a promising method for creating hybrid nanostructures. We report here the formation of hybrid condensed phases made of anisotropic nanoparticles and phospholipids. Such structure formation is driven by electrostatic interaction between the nanoparticles and the phospholipids, and results in the formation of a 2D rectangular liquid crystal, as confirmed by high-resolution Small-Angle X-ray Scattering (SAXS). Moreover, we show that the fluorescent properties of the NPs are not modified by the self-assembly process.

Peptidic ligands to control the three-dimensional self-assembly of quantum rods in aqueous media.

The use of peptidic ligands is validated as a generic chemical platform allowing one to finely control the organization in solid phase of semiconductor nanorods originally dispersed in an aqueous media. An original method to generate, on a macroscopic scale and with the desired geometry, three-dimensional supracrystals composed of quantum rods is introduced. In a first step, nanorods are transferred in an aqueous phase thanks to the substitution of the original capping layer by peptidic ligands. Infrared and nuclear magnetic resonance spectroscopy data prove that the exchange is complete; fluorescence spectroscopy demonstrates that the emitter optical properties are not significantly altered; electrophoresis and dynamic light scattering experiments assess the good colloidal stability of the resulting aqueous suspension. In a second step, water evaporation in a microstructured environment yields superstructures with a chosen geometry and in which nanorods obey a smectic B arrangement, as shown by electron microscopy. Incidentally, bulk drying in a capillary tube generates a similar local order, as evidenced by small angle X-ray scattering.

In Situ Labeling of the Aqueous Compartment of Extracellular Vesicles with Luminescent Gold Nanoclusters.

Extracellular vesicles (EVs) are well-known membrane-limited particles secreted by both healthy and cancerous cells. They are considered as biomarkers for early cancer diagnosis and are involved in many pathologies and physiological pathways. They could serve as diagnostic tools in liquid biopsies, as therapeutics in regenerative medicine, or as drug delivery vehicles. Our aim is here to encapsulate luminescent nanoprobes in the aqueous compartment of human EVs extracted from reproductive fluids. The analysis and labeling of the EVs content with easily detectable luminescent nanoparticles could enable a powerful tool for early diagnosis of specific diseases and also for the design of new therapeutics. In this view, gold nanoclusters (AuNCs) appear as an attractive alternative as nontoxic fluorophore probes because of their luminescence properties, large window of fluorescence lifetimes (1 ns-1 µs), ultrasmall size (<2 nm), good biocompatibility, and specific ability as X-ray photosensitizers. Here, we investigated an attractive method that uses fusogenic liposomes to deliver gold nanoclusters into EVs. This approach guarantees the preservation of the EVs membrane without any breakage, thus maintaining compartmental integrity. Different lipid compositions of liposomes preloaded with AuNCs were selected to interact electrostatically with human EVs and compared in terms of fusion efficiency. The mixture of liposomes and EVs results in membrane mixing as demonstrated by FRET experiments and fusion revealed by flux cytometry and cryo-TEM. The resulting fused EVs exhibit typical fluorescence of the AuNCs together with an increased size in agreement with fusion. Moreover, the fusion events in mixtures of EVs and AuNCs preloaded liposomes were analyzed by using cryo-electron microscopy. Finally, the ratio of released AuNCs during the fusion between the fusogenic liposomes and the EVs was estimated to be less than 20 mol % by Au titration using ICP spectroscopy.

Light-induced reactivation of O2-tolerant membrane-bound [Ni-Fe] hydrogenase from the hyperthermophilic bacterium Aquifex aeolicus under turnover conditions.

We report the effect of UV-Vis light on the membrane-bound [Ni-Fe] hydrogenase from Aquifex aeolicus under turnover conditions. Using electrochemistry, we show a potential dependent light sensitivity and propose that a light-induced structural change of the [Ni-Fe] active site is related to an enhanced reactivation of the hydrogenase under illumination at high potentials.

Internalization of Pegylated Er:Y2O3 Nanoparticles inside HCT-116 Cancer Cells: Implications for Imaging and Drug Delivery.

Lanthanide-doped nanoparticles, featuring sharp emission peaks with narrow bandwidth, exhibit high downconversion luminescence intensity, making them highly valuable in the fields of bioimaging and drug delivery. High-crystallinity Y2O3 nanoparticles (NPs) doped with Er3+ ions were functionalized by using a pegylation procedure to confer water solubility and biocompatibility. The NPs were thoroughly characterized using transmission electron microscopy (TEM), inductively coupled plasma mass spectrometry (ICP-MS), and photoluminescence measurements. The pegylated nanoparticles were studied both from a toxicological perspective and to demonstrate their internalization within HCT-116 cancer cells. Cell viability tests allowed for the identification of the "optimal" concentration, which yields a detectable fluorescence signal without being toxic to the cells. The internalization process was investigated using a combined approach involving confocal microscopy and ICP-MS. The obtained data clearly indicate the efficient internalization of NPs into the cells with emission intensity showing a strong correlation with the concentrations of nanoparticles delivered to the cells. Overall, this research contributes significantly to the fields of nanotechnology and biomedical research, with noteworthy implications for imaging and drug delivery applications.

Encapsulation of Luminescent Gold Nanoclusters into Synthetic Vesicles.

Gold nanoclusters (Au NCs) are attractive luminescent nanoprobes for biomedical applications. In vivo biosensing and bioimaging requires the delivery of the Au NCs into subcellular compartments. In this view, we explore here the possible encapsulation of ultra-small-sized red and blue emitting Au NCs into liposomes of various sizes and chemical compositions. Different methods were investigated to prepare vesicles containing Au NCs in their lumen. The efficiency of the process was correlated to the structural and morphological aspect of the Au NCs' encapsulating vesicles thanks to complementary analyses by SAXS, cryo-TEM, and confocal microscopy techniques. Cell-like-sized vesicles (GUVs) encapsulating red or blue Au NCs were successfully obtained by an innovative method using emulsion phase transfer. Furthermore, exosome-like-sized vesicles (LUVs) containing Au NCs were obtained with an encapsulation yield of 40%, as estimated from ICP-MS.

Luminescent Gold Nanoclusters Interacting with Synthetic and Biological Vesicles.

According to their high electron density and ultrasmall size, gold nanoclusters (AuNCs) have unique luminescence and photoelectrochemical properties that make them very attractive for various biomedical fields. These applications require a clear understanding of their interaction with biological membranes. Here we demonstrate the ability of the AuNCs as markers for lipidic bilayer structures such as synthetic liposomes and biological extracellular vesicles (EVs). The AuNCs can selectively interact with liposomes or EVs through an attractive electrostatic interaction as demonstrated by zetametry and fluorescence microscopy. According to the ratio of nanoclusters to vesicles, the lipidic membranes can be fluorescently labeled without altering their thickness until charge reversion, the AuNCs being located at the level of the phosphate headgroups. In presence of an excess of AuNCs, the vesicles tend to adhere and aggregate. The strong adsorption of AuNCs results in the formation of a lamellar phase as demonstrated by cryo-transmission electron microscopy and small-angle X-ray scattering techniques.

Extra-cellular vesicles of the male genital tract: new actors in male fertility?

Extracellular Vesicles (EVs) are membrane-limited particles containing proteins, lipids, metabolites and nucleic acids that are secreted by healthy and cancerous cells. These vesicles are very heterogeneous in size and content and mediate a variety of biological functions. Three subtypes of EV have been described in the male genital tract: microvesicles, myelinosomes and exosomes. Each type of EVs depends on the location of secretion such as the testis, prostate or epididymis. It has been shown that EVs can fuse together and deliver information to recipient cells, for example spermatozoa in the male genital tract. Cryo-electron microscopy remains the reference technique for determining EV morphology, but quantifying the absolute concentration of these EVs in biological fluids remains a challenge from a clinical point of view. The field of bio detection has considerably increased with the introduction of nanomaterials in biosensors and will provide a better understanding of the impact of these EVs. However, functional modifications of male gametes result from interactions with the components of the intraluminal fluid all along the genital tract and depend on the secretion and absorption of proteins and lipids from the local microenvironment. We cannot therefore exclude the possibility of epigenetic modulation of the information that will be transmitted to the embryo and therefore to the next generation via EVs.

RéSUMé: Les Vésicules Extracellulaires (VE) sont des constituants d'origine membranaire contenant des protéines solubles ou membranaires, des lipides, des métabolites ou des acides nucléiques. Ces vésicules sont très hétérogènes en taille et en contenu. Trois catégories de VE ont été décrites dans le tractus génital mâle: les microvésicules, les myélinosomes et les exosomes. Les types de VE sont différents selon le lieu de production testiculaire, prostatique ou encore épididymaire. Il a été montré que les VE peuvent fusionner et délivrer une information à la cellule réceptrice, en l'occurrence le spermatozoïde dans le tractus génital mâle. La cryo-microscopie électronique reste la technique de référence pour déterminer la morphologie des VE mais la quantification de la concentration absolue de ces VE dans les liquides biologiques reste un challenge dans le cadre d'une approche clinique. Le domaine de la biodétection s'est. considérablement développé avec l'introduction des nanomatériaux dans les biocapteurs et va permettre de mieux comprendre l'impact de ces VE. Or les modifications fonctionnelles des gamètes mâles résultent d'interactions avec les composants du liquide intraluminal tout le long du tractus génital et dépendent de la sécrétion et de l'absorption de protéines et de lipides du microenvironnement local. On ne peut donc pas exclure la possibilité d'une modulation épigénétique des informations qui seront transmises à l'embryon donc à la génération suivante via les VE.

Hybrid gold nanoparticle-quantum dot self-assembled nanostructures driven by complementary artificial proteins.

Hybrid nanostructures are constructed by the direct coupling of fluorescent quantum dots and plasmonic gold nanoparticles. Self-assembly is directed by the strong affinity between two artificial α-repeat proteins that are introduced in the capping layers of the nanoparticles at a controlled surface density. The proteins have been engineered to exhibit a high mutual affinity, corresponding to a dissociation constant in the nanomolar range, towards the protein-functionalized quantum dots and gold nanoparticles. Protein-mediated self-assembly is evidenced by surface plasmon resonance and gel electrophoresis. The size and the structure of colloidal superstructures of complementary nanoparticles are analyzed by transmission electron microscopy and small angle X-ray scattering. The size of the superstructures is determined by the number of proteins per nanoparticle. The well-defined geometry of the rigid protein complex sets a highly uniform interparticle distance of 8 nm that affects the emission properties of the quantum dots in the hybrid ensembles. Our results open the route to the design of hybrid emitter-plasmon colloidal assemblies with controlled near-field coupling and better optical response.

Nanoparticles Self-Assembly Driven by High Affinity Repeat Protein Pairing.

Proteins are the most specific yet versatile biological self-assembling agents with a rich chemistry. Nevertheless, the design of new proteins with recognition capacities is still in its infancy and has seldom been exploited for the self-assembly of functional inorganic nanoparticles. Here, we report on the protein-directed assembly of gold nanoparticles using purpose-designed artificial repeat proteins having a rigid but modular 3D architecture. αRep protein pairs are selected for their high mutual affinity from a library of 10(9) variants. Their conjugation onto gold nanoparticles drives the massive colloidal assembly of free-standing, one-particle thick films. When the average number of proteins per nanoparticle is lowered, the extent of self-assembly is limited to oligomeric particle clusters. Finally, we demonstrate that the aggregates are reversibly disassembled by an excess of one free protein. Our approach could be optimized for applications in biosensing, cell targeting, or functional nanomaterials engineering.

Hydrogen bioelectrooxidation on gold nanoparticle-based electrodes modified by Aquifex aeolicus hydrogenase: Application to hydrogen/oxygen enzymatic biofuel cells.

For the first time, gold nanoparticle-based electrodes have been used as platforms for efficient immobilization of the [NiFe] hydrogenase from the hyperthermophilic bacterium Aquifex aeolicus. AuNPs were characterized by electronic microscopy, dynamic light scattering and UV-Vis spectroscopy. Two sizes around 20.0±5.3 nm and 37.2±4.3 nm nm were synthesized. After thiol-based functionalization, the AuNPs were proved to allow direct H2 oxidation over a large range of temperatures. A high current density up to 1.85±0.15 mA·cm(-2) was reached at the smallest AuNPs, which is 170 times higher than the one recorded at the bare gold electrode. The catalytic current was especially studied as a function of the AuNP size and amount, and procedure for deposition. A synergetic effect between the AuNP porous deposit and the increase surface area was shown. Compared to previously used nanomaterials such as carbon nanofibers, the covalent grafting of the enzyme on the thiol-modified gold nanoparticles was shown to enhance the stability of the hydrogenase. This bioanode was finally coupled to a biocathode where BOD from Myrothecium verrucaria was immobilized on AuNP-based film. The performance of the so-mounted H2/O2 biofuel cell was evaluated, and a power density of 0.25 mW·cm(-2) was recorded.

MDR1 promoter hypermethylation in MCF-7 human breast cancer cells: changes in chromatin structure induced by treatment with 5-Aza-cytidine.

Resistance to the cytotoxic actions of antineoplastic drugs, whether intrinsic or acquired, remains a barrier to the establishment of curative chemotherapy regimens for advanced breast cancer. Over-expression of P-glycoprotein (P-gp), encoded by the MDR1 gene and known to mediate resistance to many antineoplastic drugs, may contribute to poor breast cancer treatment outcome. Nonetheless, the precise molecular mechanisms responsible for high or low level P-gp expression in breast cancer cells have not been established. We assessed the role of DNA hypermethylation near the MDR1 transcriptional regulatory region in MDR1 expression in MCF-7 breast cancer cells, which fail to express MDR1 mRNA, and MCF-7/ADR cells, known to express high MDR1 mRNA levels. When compared to MCF-7/ADR cells, MCF-7 cells manifested markedly diminished MDR1 transcription rates by nuclear run-off assay, but equivalent MDR1 promoter trans-activation activity in transient transfection experiments, indicating that cis factors were most likely responsible for the differences in MDR1 transcription between MCF-7/ADR cells and MCF-7 cells. Bisulfite genomic sequencing analyses revealed substantially less extensive MDR1 promoter methylation in MCF-7/ADR cells than in MCF-7 cells, suggesting that CpG dinucleotide methylation might contribute to the observed MDR1 transcription differences. Chromatin immunoprecipitation analyses indicated an inactive MDR1 chromatin conformation in MCF-7 cells, with a paucity of acetylated histones and the presence of 5-mC-binding proteins MeCP2 and MBD2, and an active MDR1 chromatin conformation in MCF-7/ADR cells, with an abundance of acetylated histones and the presence of the transcriptional trans-activator YB-1. Stable MCF-7 sublines which had been treated with the DNA methyltransferase inhibitor 5-azacytidine, exhibited a reduction in MDR1 promoter methylation and a complex MDR1 chromatin configuration, characterized by the simultaneous presence of transcriptional activators and repressors. In this state, MDR1 expression was markedly sensitive to treatment with the histone deacetylase inhibitor trichostatin A.

Spatiotemporal control of microtubule nucleation and assembly using magnetic nanoparticles.

Decisions on the fate of cells and their functions are dictated by the spatiotemporal dynamics of molecular signalling networks. However, techniques to examine the dynamics of these intracellular processes remain limited. Here, we show that magnetic nanoparticles conjugated with key regulatory proteins can artificially control, in time and space, the Ran/RCC1 signalling pathway that regulates the cell cytoskeleton. In the presence of a magnetic field, RanGTP proteins conjugated to superparamagnetic nanoparticles can induce microtubule fibres to assemble into asymmetric arrays of polarized fibres in Xenopus laevis egg extracts. The orientation of the fibres is dictated by the direction of the magnetic force. When we locally concentrated nanoparticles conjugated with the upstream guanine nucleotide exchange factor RCC1, the assembly of microtubule fibres could be induced over a greater range of distances than RanGTP particles. The method shows how bioactive nanoparticles can be used to engineer signalling networks and spatial self-organization inside a cell environment.