037 – Microglial Extracellular Vesicles as Therapeutic Vector for Neuroinflammation
Giulia Marostica (1) – Annamaria Finardi (1) – Roberto Furlan (1)
San Raffaele Scientific Institute, Clinical Neuroimmunology Unit, MIlan, Italy (1)
Microglia is considered an eligible target against the progressive multiple sclerosis, but current available therapies do not allow its efficient targeting. As many cell types, microglia communicates with the neighbouring cells through a complex system of extracellular vesicles exchange. Recently my group described that microglia derived-extracellular vesicles, engineered to encapsulate IL4, are taken up by microglia itself, mediating a phenotype switch to a protective phenotype. In vivo studies suggest that these extracellular vesicles can ameliorate established neuroinflammation, thus making them a promising drug-delivery tool to target CNS in multiple sclerosis. My project focuses on understanding the mechanism of action and the signalling pathway of extracellular vesicles delivery and to exploit this knowledge to specifically deliver different potential therapeutic molecules. For this purpose, we decided to start with the characterization of these extracellular vesicles through TRPS technology. BV2 cells were cultured in complete DMEM and transfected with a plasmid carrying murine IL4. After 48h cells were stimulated with PMA(20mg/mL) for 30 min and cell supernatant was collected and processed to isolate EVs. EVs isolation was carried out by first centrifugation of 300g for 10 min, first supernatant was preserved and the pellet was re-suspended and re-centrifuged at 300g for 10 min. The supernatants were mixed together, filtered(5µm) and ultracentrifuged at 100kg for 2h. The EVs pellet was re-suspended in PBS and analysed with qNano. EVs were analysed for size, concentration and zeta-potential. The mean diameter of the EVs coming from different treatment display a range between 120-140nm, while the concentration was measured around 1010particles/mL. Interestingly we found a small difference in the zeta-potential of around 3mV, between IL4 negative and IL4 positive EVs. Moreover, when the EVs populations are displayed by size and zeta-potential we can notice clearly a difference between the populations. This difference is given by a higher zeta-potential and a smaller size of the IL4 positive EVs. This difference could be due to a different formation pathway, with most probably a different content in the EVs. This preliminary data are few to build a solid hypothesis on the causes of this size and zeta-potential difference, although they look promising. Further experiments are planned in order to analyse the single population by cargo and lipid content.