We herein describe an Atomic Force Microscopy (AFM)-based experimental procedure which allows the simultaneous mechanical and morphological characterization of several hundred individual nanosized vesicles within the hour timescale. When deposited on a flat rigid surface from aqueous solution, vesicles are deformed by adhesion forces into oblate spheroids whose geometry is a direct consequence of their mechanical stiffness. AFM image analysis can be used to quantitatively measure the contact angle of individual vesicles, which is a sizeindependent descriptor of their deformation and, consequently, of their stiffness. The same geometrical measurements can be used to infer vesicle diameter in its original, spherical shape. We demonstrate the applicability of the proposed approach to natural vesicles obtained from different sources, recovering their size and stiffness distributions by simple AFM imaging in liquid. We show how the combined EV stiffness/size readout is able to discriminate between subpopulations of vesicular and nonvesicular objects in the same sample, and between populations of vesicles with similar sizes but different .

We herein describe an Atomic Force Microscopy (AFM)-based experimental procedure which allows the simultaneous mechanical and morphological characterization of several hundred individual nanosized vesicles within the hour timescale.When deposited on a flat rigid surface from aqueous solution, vesicles are deformed by adhesion forces into oblate spheroids whose geometry is a direct consequence of their mechanical stiffness. AFM image analysis can be used to quantitatively measure the contact angle of individual vesicles, which is a sizeindependent descriptor of their deformation and, consequently, of their stiffness. The same geometrical measurements can be used to infer vesicle diameter in its original, spherical shape.We demonstrate the applicability of the proposed approach to natural vesicles obtained from different sources, recovering their size and stiffness distributions by simple AFM imaging in liquid. We show how the combined EV stiffness/size readout is able to discriminate between subpopulations of vesicular and nonvesicular objects in the same sample, and between populations of vesicles with similar sizes but different mechanical characteristics. We also discuss a force spectroscopy calibration procedure to quantitatively link the stiffness of EVs to their average contact angle.Finally, we discuss expected extensions and applications of the methodology.

Despite the significant advances in the last decades, low implantation rate per transferred embryo still remains a major concern in assisted reproductive techniques, highlighting a need to better characterize endometrial receptivity also by mean of specific biomarkers. Based on physiology and on the intimate contact with endometrium as the tissue of interest, in this study we developed and validated an optimized protocol that uses extracellular vesicles (EVs) recovered from uterine flushings and from a cervical brush, the latter never used until now as an EVs source, as surrogates for endometrial biopsies. This method combines the safety of sampling with the ability to study the expression profile across the uterine cycle. We have compared the yield and composition of EVs recovered from different biofluids samples and fractions thereof, opting for chemical precipitation as the EV isolation procedure, assuring the highest yield without introducing any bias in specific EV recovery. Moreover, collected EVs, in particular exosome-like vesicles, express putative endometrial markers, such as glycodelin A and receptors for estrogen and progesterone, thus confirming their endometrial origin. We also identified uterine flushing EVs, in particular those recovered from its mucous fraction, as the richest source of endometrial transcripts, likely correlated to cellular (epithelial) origin of these vesicles. Finally, our pilot quantitative assessment of three endometrial gene profiles, in samples collected at different time points along the luteal phase, revealed the fluctuations apparently recapitulating gene expression variability prior reported during the menstrual cycle. Unlike tissue biopsy that is subjected to inter- and intra-sample differences, our data suggest that EVs from liquid biopsies (from uterine flushings and a cervical brush) obtained through less-invasive procedures, can be substrate to detect and track the tissue representative expression profiles, better depicting the total endometrium complexity.

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.

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