Thermodynamic Delivery of Trehalose into Extracellular Vesicles for Enhanced Lyophilization

Abstract Extracellular vesicles (EVs) hold great potential as therapeutic agents and drug carriers. Conventionally, EVs are cryopreserved at ultra‐low temperatures, with substantial cryoinjury associated with the freeze/thaw cycle. Lyophilization has emerged as a promising alternative approach; however, suboptimal outcomes remain owing to the challenge of lyoprotectant delivery into EVs. Here, the atypical transport property of the EV membrane is unraveled, and lyoprotective trehalose is delivered into EVs by combining substantial hypotonicity (below intravesicular colloid osmolality) and mild heat shock (42 °C). Consequently, the lyophilization of trehalose‐laden EVs is notably enhanced, with less EV loss, more RNA and protein retention, and superior therapeutic efficacy. Moreover, the EVs can be co‐reconstituted and co‐lyophilized with hyaluronic and methylcellulose (HAMC) hydrogel carriers, yielding excellent preservation outcomes and controlled EV release. Lyophilized EVs or HAMC‐EVs maintain Treg cell regulation in vitro and experimental autoimmune encephalomyelitis disease treatment in vivo. Overall, not only are EVs and EV–hydrogel constructs efficiently lyophilized for widespread application, but a thermodynamic approach is also developed to safely and uniformly deliver various hydrophilic molecules into EVs.