Efficient extracellular vesicles freeze-dry method for direct formulations preparation and use

Despite the great knowledge achieved in the field of extracellular vesicles (EVs), the short lifetime of EVs liquid formulation still hampers the transfer of EVs technology to clinical applications. In this context, freeze-dried EVs would be advantageous thanks to the enhanced stability of solid formulations. Although some previous attempts have already been reported, the efficiency of EVs lyophilization methodologies used remains insufficient, and the characterization of the resulting EVs is still incomplete. The current work aims to describe an alternative and easy-to-be-applied methodology for EVs lyophilization. The use of sucrose as lyoprotectant at 8.5%wt improved the cryopreservation efficiency. After the subsequent cycles of freeze-drying, properties such as size, morphology, purity, EVs specific markers, biocompatibility and the maintenance of their functionality were confirmed in freeze-dried EVs samples. To sum up, we have designed a methodology for the lyophilization of extracellular vesicles that enables the preservation of the physicochemical properties and functionality of EVs.