Exosomes are gaining a prominent role in research due to their intriguing biology and several therapeutic opportunities. However, their accurate purification from body fluids and detailed physicochemical characterization remain open issues. We isolated exosomes from serum of patients with Multiple Myeloma by four of the most popular purification methods and assessed the presence of residual contaminants in the preparations through an ad hoc combination of biochemical and biophysical techniques - including Western Blot, colloidal nanoplasmonics, atomic force microscopy (AFM) and scanning helium ion microscopy (HIM). The preparations obtained by iodixanol and sucrose gradients were highly pure. To the contrary, those achieved with limited processing (serial centrifugation or one step precipitation kit) resulted contaminated by a residual matrix, embedding the exosomes. The contaminated preparations showed lower ability to induce NfkB nuclear translocation in endothelial cells with respect to the pure ones, probably because the matrix prevents the interaction and fusion of the exosomes with the cell membrane. These findings suggest that exosome preparation purity must be carefully assessed since it may interfere with exosome biological activity. Contaminants can be reliably probed only by an integrated characterization approach aimed at both the molecular and the colloidal length scales.

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 .

Extracellular Vesicles (EVs) - cell secreted vesicles that carry rich molecular information of the parental cell and constitute an important mode of intercellular communication - are becoming a primary topic in translational medicine. EVs (that comprise exosomes and microvesicles/microparticles) have a size ranging from 40 nm to 1 μm and share several physicochemical proprieties, including size, density, surface charge, and light interaction, with other nano-objects present in body fluids, such as single and aggregated proteins. This makes separation, titration, and characterization of EVs challenging and time-consuming. Here we present a cost-effective and fast colorimetric assay for probing by eye protein contaminants and determine the concentration of EV preparations, which exploits the synergy between colloidal gold nanoplasmonics, nanoparticle-protein corona, and nanoparticle-membrane interaction. The assay hits a limit of detection of protein contaminants of 5 ng/μL and has a dynamic range of EV concentration ranging from 35 fM to 35 pM, which matches the typical range of EV concentration in body fluids. This work provides the first example of the exploitation of the nanoparticle-protein corona in analytical chemistry.

Parkinson's disease (Pd) is an important disabling age-related disorder and is the second most common neurodegenerative disease. currently, no established molecular biomarkers exist for the early diagnosis of Pd. circulating microRNAs (miRNAs), either vesicle-free or encapsulated in extracellular vesicles (EVs), have emerged as potential blood-based biomarkers also for neurodegenerative diseases. In this exploratory study, we focused on miR-34a-5p because of its well-documented involvement in neurobiology. To explore a differential profile of circulating miR-34a-5p in PD, Pd patients and age-matched control subjects were enrolled. Serial ultracentrifugation steps and density gradient were used to separate EV subpopulations from plasma according to their different sedimentation properties (Large, Medium, Small EVs). characterization of EV types was performed using western blotting and atomic force microscopy (AFM); purity from protein contaminants was checked with the colorimetric nanoplasmonic assay. circulating miR-34a-5p levels were evaluated using qPcR in plasma and in each EV type. miR-34a-5p was significantly up-regulated in small EVs devoid of exogenous protein contaminants (pure SEVs) from Pd patients and ROc analysis indicated a good diagnostic performance in discriminating patients from controls (AUc=0.74, P<0.05). Moreover, miR-34a-5p levels in pure SEVs were associated with disease duration, Hoehn and Yahr and Beck depression Inventory scores. These results underline the necessity to examine the miRNA content of each EV subpopulation to identify miRNA candidates with potential diagnostic value and lay the basis for future studies to validate the overexpression of circulating miR-34a-5p in Pd via the use of pure SEVs.