Production and Characterization of Yeast Extracellular Vesicles: A Comparison of Three Extraction Protocols

In recent years, extracellular vesicles (EVs) produced by organisms from all three domains of life have increasingly attracted the interest of the scientific community. Their favourable characteristics - small size, ability to circulate in biological fluids, and the presence of a variety of bioactive molecules within their cargo - make them promising candidates for drug delivery and immune intervention strategies. This work arises from the need to explore the potential of yeast EVs as novel nanocarriers for drug delivery. In particular, EVs produced by Saccharomyces cerevisiae were studied. S. cerevisiae is a well-established model organism that shares several features with more complex eukaryotes, making it particularly suitable for preliminary studies on EVs. The main objective of this thesis is to compare three different extraction protocols to determine the most effective one in terms of yield, purity, and homogeneity of the EV content. Specifically, S. cerevisiae cells were cultivated in YM medium. Once they reached the stationary phase, EVs were extracted from the culture broth. A series of three centrifugation steps was performed to remove cell debris and residues. After each step, the pellet was collected to isolate the biomass. Following the final centrifugation, the supernatant was filtered using a 0.45 µm membrane and subsequently sterilized with a 0.22 µm membrane. The resulting supernatant was then divided into three separate batches, each processed using a different extraction protocol: one with a 100 kDa cut-off membrane, one with a 30 kDa membrane, and one combining both 100 kDa and 30 kDa membranes. Each batch also underwent diafiltration to remove residues from the supernatant. The EV-containing supernatants were then characterized based on their protein composition - via Bradford assay and SDS-PAGE - and their physical properties - using Nanoparticle Tracking Analysis (NTA) to assess vesicle size distribution. The results of these analyses were compared to evaluate which protocol provided the most suitable extraction method, focusing on two key aspects: a monodisperse size distribution (i.e., as close as possible to a single population) and the presence of proteins typically associated with EVs.