The potential of exosomes as biomarker resources for diagnostics, prognostics and even for therapeutics is an area of intense research. Despite the various approaches available, there is no consensus with respect to the best methodology for isolating exosomes and to provide substantial yields with reliable quality. Differential centrifugation is the most commonly used method but it is time-consuming and requires large sample volumes, thus alternative methods are urgently needed. In this study two precipitation-based methods and one column-based approach were compared for exosome isolation from distinct biofluids (serum, plasma and cerebrospinal fluid). Exosome characterization included morphological analyses, determination of particle concentration, stability and exosome preparations’ purity, using different complementary approaches such as Nanoparticle Tracking Analysis, Electrophoretic Light Scattering, Transmission Electron Microscopy, EXOCET colorimetric assay, protein quantification methods and western blotting. The three commercial kits tested successfully isolated exosomes from the biofluids under study, although ExoS showed the best performance in terms of exosome yield and purity. Data shows that methods other than differential centrifugation can be applied to quickly and efficiently isolate exosomes from reduced biofluid volumes. The possibility to use small volumes is fundamental in the context of translational and clinical research, thus the results here presented contribute significantly in this respect.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with a very poor prognosis. Its treatment is hindered by a lack of new therapeutic alternatives and the existence of the blood–brain barrier (BBB), which restricts the access of drugs commonly used in ALS, such as riluzole, to the brain. To overcome these limitations and increase brain targeting, riluzole-loaded nanostructured lipid carriers (NLC) were prepared and functionalized with lactoferrin (Lf), facilitating transport across the BBB by interacting with Lf receptors expressed in the brain endothelium. NLC were characterized with respect to their physicochemical properties (size, zeta potential, polydispersity index) as well as their stability, encapsulation efficiency, morphology, in vitro release profile, and biocompatibility. Moreover, crystallinity and melting behavior were assessed by DSC and PXRD. Nanoparticles exhibited initial mean diameters between 180 and 220 nm and a polydispersity index below 0.3, indicating a narrow size distribution. NLC remained stable over at least 3 months. Riluzole encapsulation efficiency was very high, around 94–98%. FTIR and protein quantification studies confirmed the conjugation of Lf on the surface of the nanocarriers, with TEM images showing that the functionalized NLC presented a smooth surface and uniform spherical shape. An MTT assay revealed that the nanocarriers developed in this study did not cause a substantial reduction in the viability of NSC-34 and hCMEC/D3 cells at a riluzole concentration up to 10 μM, being therefore biocompatible. The results suggest that Lf-functionalized NLC are a suitable and promising delivery system to target riluzole to the brain.

Exosomes are small extracellular vesicles (EVs) present in human biofluids that can transport specific disease-associated molecules. Consequently blood-derived exosomes have emerged as important peripheral biomarker sources for a wide range of diseases, among them Alzheimer’s disease (AD). Although there is no effective cure for AD, an accurate diagnosis, relying on easily accessible peripheral biofluids, is still necessary to discriminate this disease from other dementias, test potential therapies and even monitor rate of disease progression. The ultimate goal is to produce a cost-effective and widely available alternative, which can also be employed as a first clinical screen. In this study, EVs with exosome-like characteristics were isolated from serum of Controls and AD cases through precipitation- and column-based methods, followed by mass spectrometry analysis. The resulting proteomes were characterized by Gene Ontology (GO) and multivariate analyses. Although GO terms were similar for exosomes’ proteomes of Controls and ADs, using both methodologies, a clear segregation of disease cases was obtained when using the precipitation-based method. Nine significantly different abundant proteins were identified between Controls and AD cases, representing putative biomarker candidate targets. Among them are AACT and C4BPα, two Aβ-binding proteins, whose exosome levels were further validated in individuals from independent cohorts using antibody-based approaches. The findings discussed represent an important contribution to the identification of novel exosomal biomarker candidates useful as potential blood-based tools for AD diagnosis.

Alzheimer’s disease (AD) diagnosis is based on psychological and imaging tests but can also include monitoring cerebrospinal fluid (CSF) biomarkers. However, CSF based-neurochemical approaches are expensive and invasive, limiting their use to well-eq

The use of secretion pathways for effector molecule delivery by microorganisms is a trademark of pathogenesis. Leishmania extracellular vesicles (EVs) were shown to have significant immunomodulatory potential. Still, they will act in conjunction with other released parasite‐derived products that might modify the EVs effects. Notwithstanding, the immunomodulatory properties of these non‐vesicular components and their influence in the infectious process remains unknown. To address this, we explored both in vitro and in vivo the immunomodulatory potential of promastigotes extracellular material (EXO), obtained as a whole or separated in two different fractions: EVs or vesicle depleted EXO (VDE). Using an air pouch model, we observed that EVs and VDE induced a dose‐dependent cell recruitment profile different from the one obtained with parasites, attracting significantly fewer neutrophils and more dendritic cells (DCs). Additionally, when we co‐inoculated parasites with extracellular products a drop in cell recruitment was observed. Moreover, in vitro, while VDE (but not EVs) downregulated the expression of DCs and macrophages activation markers, both products were able to diminish the responsiveness of these cells to LPS. Finally, the presence of Leishmania infantum extracellular products in the inoculum promoted a dose‐dependent infection potentiation in vivo, highlighting their relevance for the infectious process. In conclusion, our data demonstrate that EVs are not the only relevant players among the parasite exogenous products. This, together with the dose‐dependency observed, opens new avenues to the comprehension of Leishmania infectious process. The approach presented here should be exploited to revisit existing data and considered for future studies in other infection models.