The relevance of extracellular vesicles (EVs) has grown exponentially, together with innovative basic research branches that feed medical and bioengineering applications. Such attraction has been fostered by the biological roles of EVs, as they carry biomolecules from any cell type to trigger systemic paracrine signaling or to dispose metabolism products. To fulfill their roles, EVs are transported through circulating biofluids, which can be exploited for the administration of therapeutic nanostructures or collected to intercept relevant EV-contained biomarkers. Despite their potential, EVs are ubiquitous and considerably heterogeneous. Therefore, it is fundamental to profile and identify subpopulations of interest. In this study, we optimized EV-labeling protocols on two different high-resolution single-particle platforms, the NanoFCM NanoAnalyzer (nFCM) and Particle Metrix ZetaView Fluorescence Nanoparticle Tracking Analyzer (F-NTA). In addition to the information obtained by particles’ scattered light, purified and non-purified EVs from different cell sources were fluorescently stained with combinations of specific dyes and antibodies to facilitate their identification and characterization. Despite the validity and compatibility of EV-labeling strategies, they should be optimized for each platform. Since EVs can be easily confounded with similar-sized nanoparticles, it is imperative to control instrument settings and the specificity of staining protocols in order to conduct a rigorous and informative analysis.

Due to their intercellular communication properties and involvement in a wide range of biological processes, extracellular vesicles (EVs) are increasingly being studied and exploited for different applications. Nevertheless, their complex nature and heterogeneity, as well as the challenges related to their purification and characterization procedures, require a cautious assessment of the qualitative and quantitative parameters that need to be monitored. This translates into a multitude of choices and putative solutions that any EV researcher must confront in both research and translational environments. In this respect, decision‐making tools may help assess various options, weigh pros and cons, and ultimately arrive at a thought‐out decision that considers both the best fit‐to‐source and fit‐to‐scope EV application(s). Here, we present a multi‐criteria EV decision‐making grid (EV‐DMG) as a novel, efficient, customizable, and easy‐to‐use tool to support EV research and innovation. By identifying and weighing key assessment criteria for comparing distinct EV‐based preparations and related processes, our EV‐DMG may assist any EV community member in making informed, traceable, and reproducible decisions regarding the management of EV sources or samples. Ultimately, this EV‐DMG may guide the adoption of the most suitable EV production and analytical pipelines for targeting a defined aim or application.

Extracellular Vesicles (EVs) from seminal plasma have achieved attention due to their potential physiopathological role in male reproductive systems. This study employed a comprehensive proteomic and transcriptomic approach to investigate the composition and molecular signatures of EVs isolated from human seminal plasma. EVs from Normozoospermic (NORMO), OligoAsthenoTeratozoospermic (OAT), and Azoospermic (AZO) subjects were isolated using a modified polymer precipitation-based protocol and characterized for size and morphology. Comprehensive proteomic analysis, using both gel-free and gel-based approaches, revealed distinct protein profiles in each group (p<0.01), highlighting potential molecules and pathways involved in sperm function and fertility. The data are available via ProteomeXchange with identifiers PXD051361 and PXD051390, respectively. Transcriptomic analysis confirmed the trend of a general downregulation of AZO and OAT compared to NORMO shedding light on regulatory mechanisms of sperm development. Bioinformatic tools were applied for functional omics analysis; the integration of proteomic and transcriptomic data provided a comprehensive understanding of the cargo content and regulatory networks present in EVs. This study contributes to elucidating the key role of EVs in the paracrine communication regulating spermatogenesis. A full understanding of these pathways not only suggests potential mechanisms regulating male fertility but also offers new insights into the development of diagnostic tools targeting male reproductive disorders.

Due to their intercellular communication properties and their involvement in a wide range of biological processes, extracellular vesicles (EVs) are increasingly being studied and exploited for different applications. Nevertheless, their complex nature and heterogeneity, as well as the challenges related to their isolation, purification and characterization procedures, require cautious assessment of the quality and quantity parameters to monitor. This translates into a multitude of choices and putative solutions that lie in front of any EV researcher, in both research and translational environments, resembling a labyrinth with multiple paths to cross and, possibly, more than one exit. In this respect, decision-making tools might represent our modern Ariadne’s string to follow not to get lost or distracted along the journey, to choose the shorter and best-fit-to-source EV application(s) and vice versa . Here, we present the implementation of a multi-criteria EV decision-making grid (EV-DMG) as a novel, customizable, efficient and easy-to-use tool to support responsible EV research and innovation. By identifying and weighting key assessment criteria for comparing distinct EV-based preparations and/or processes, our EV-DMG may assist any EV community member in making informed, transparent and reproducible decisions regarding the EV sources and/or samples to be managed, as well as the most suitable production and/or analytical pipelines to be adopted for targeting a defined aim or application.

Plant‐derived extracellular vesicles (EVs) have shown numerous health benefits, including modulation of the human gut microbiota. Herein, we employed isothermal microcalorimetry (IMC) to explore the effects of ginger‐derived nanovesicles (G‐NVs) on the growth and metabolic activity of Bacteroides thetaiotaomicron (Bt) , a dominant saccharolytic commensal with promising next‐generation probiotic potential. Bt was exposed to either G‐NVs or the ginger extract separated from G‐NVs (G‐CTL) in defined media under anaerobic conditions. Both ginger samples enhanced bacterial specific growth rate and maximum metabolic activity, inducing the latter earlier. However, higher biomass production and greater secretion of acetic, succinic and propionic acids occurred only in response to the G‐CTL. Complete sugar depletion and unchanged free amino acid levels indicated preferential carbohydrate utilisation by Bt . Overall, these findings revealed that Bt ’s metabolic state is shaped by both G‐NVs and G‐CTL, yet through distinct mechanisms, with G‐NVs inducing rapid stimulation without increasing total metabolic output and G‐CTL providing a sustained, dose‐dependent effect. To our knowledge, this is the first study applying IMC to monitor in real‐time the impact of EVs on microbial growth and metabolism, underscoring IMC's utility for mechanistic studies of EV‐microbe interactions. Furthermore, this research sets the ground for innovative strategies in nutraceutical and microbiome‐targeted therapy development.