Size-dependent internalization of exomemes

Exosomes are biologically active extracellular vesicles, whose size range from 30 to 100nm, which are released by many cell types in various body fluids. These particles have been proved to be strong players in cell-cell communication, thus reaching interest especially about their involvement in cancer progression, invasion and metastasis. For example, our group has already investigated the tumor supporting function of exosomes released by Glioma-Associated Stem Cells (GASC). Considering the mechanism of action of exosomes, besides the undeniable role exerted by the message they deliver, much interest is now focused on to their physical properties, since they could also influence the biological function observed, as shown for bioengineered nanoparticles, and consequently their therapeutic potential. As exosomal size is nanoscopic, the estimation of particle size and density has been elusive, and the use of several techniques has been applied, such as Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM), Nanoparticle Tracking Analysis (NTA) and Dynamic Light Scattering (DLS), each displaying strength and weaknesses. Moreover, another extremely relevant matter, for which it has not yet reached a consensus, regards the ideal method to isolate exosomes, as it could lead to different exosomal populations with different features. In this thesis, we compared the exosomes preparations obtained by two different enrichment methods, wondering if this could affect the exosome uptake by cells and their ability to alter the biological functions of target cells. Therefore, we isolated exosomes released by patient-derived high-grade GASC and human glioblastoma cell line A172 in cell supernatants using both ultracentrifugation (UC) and ExoQuick (EQ) precipitation methods. Then we assessed the purified particles both for their physical properties (characterizing particle size and particle density) using AFM and NTA, and molecular properties (evaluating total protein content and a specific exosomal marker) using Bradford, BiCinchoninic Acid Assay (BCA) and CD9 ELISA assay. Then we performed cell uptake assays to observe the differential internalization of the preparations, and finally we evaluated whether this differential internalization could influence glioma cell motility. Our results demonstrate that polymer-based precipitation results in particles that have a size distribution smaller than that of ultracentrifuge-isolated ones. Moreover, we further established that smaller exosomes are better uptaken by the receiving cells and, furthermore, this affects cell motility. These data suggest that the isolation method could profoundly affect the size distribution of the obtained exosomal preparation and this is associated with differences in their physical and biological properties, thus improving or decreasing their potential therapeutic capability