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.

Extracellular vesicles (EVs) large‐scale production is a crucial point for the translation of EVs from discovery to application of EV‐based products. In October 2021, the International Society for Extracellular Vesicles (ISEV), along with support by the FET‐OPEN projects, “The Extracellular Vesicle Foundry” (evFOUNDRY) and “Extracellular vesicles from a natural source for tailor‐made nanomaterials” (VES4US), organized a workshop entitled “massivEVs” to discuss the potential challenges for translation of EV‐based products. This report gives an overview of the topics discussed during “massivEVs”, the most important points raised, and the points of consensus reached after discussion among academia and industry representatives. Overall, the review of the existing EV manufacturing, upscaling challenges and directions for their resolution highlighted in the workshop painted an optimistic future for the expanding EV field.

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.

Identification of extracellular vesicle (EV) subpopulations remains an open challenge. To date, the common strategy is based on searching and probing set of molecular components and physical properties intended to be univocally characteristics of the target subpopulation. Pitfalls include the risk to opt for an unsuitable marker set – which may either not represent the subpopulation or also cover other unintended subpopulations – and the need to use different characterization techniques and equipment. This approach focused on specific markers may result inadequate to routinely deal with EV subpopulations that have an intrinsic high level of heterogeneity. In this paper, we show that Fourier‐transform Infrared (FT‐IR) spectroscopy can provide a collective fingerprint of EV subpopulations in one single experiment. FT‐IR measurements were performed on large (LEVs, ~600 nm), medium (MEVs, ~200 nm) and small (SEVs ~60 nm) EVs enriched from two different cell lines medium: murine prostate cancer (TRAMP‐C2) and skin melanoma (B16). Spectral regions between 3100–2800 cm ⁻¹ and 1880–900 cm ⁻¹ , corresponding to functional groups mainly ascribed to lipid and protein contributions, were acquired and processed by Principal Component Analysis (PCA). LEVs, MEVs and SEVs were separately grouped for both the considered cell lines. Moreover, subpopulations of the same size but from different sources were assigned (with different degrees of accuracy) to two different groups. These findings demonstrate that FT‐IR has the potential to quickly fingerprint EV subpopulations as a whole, suggesting an appealing complement/alternative for their characterization and grading, extendable to healthy and pathological EVs and fully artificial nanovesicles.