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.

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.

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.

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.

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.

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.