Lyophilization preserves the intrinsic cardioprotective activity of bioinspired cell-derived nanovesicles

Recently, bioinspired cell-derived nanovesicles (CDNs) have gained much interest in the field of nanomedicine due to the preservation of biomolecular structure characteristics derived from their parent cells, which impart CDNs with unique properties in terms of binding and uptake by target cells and intrinsic biological activities. Although the production of CDNs can be easily and reproducibly achieved with any kind of cell culture, application of CDNs for therapeutic purposes has been greatly hampered by their physical and chemical instability during long-term storage in aqueous dispersion. In the present study, we conceived a lyophilization approach that would preserve critical characteristics regarding stability (vesicles’ size and protein content), structural integrity, and biological activity of CDNs for enabling long-term storage in freeze-dried form. Compared to the lyoprotectant sucrose, trehalose-lyoprotected CDNs showed significantly higher glass transition temperature and lower residual moisture content. As assessed by ATR-FTIR and far-UV circular dichroism, lyophilization in the presence of the lyoprotectant effectively maintained the secondary structure of cellular proteins. After reconstitution, lyoprotected CDNs were efficiently associated with HeLa cells, CT26 cells, and bone marrow-derived macrophages at a rate comparable to the freshly prepared CDNs. In vivo, both lyoprotected and freshly prepared CDNs, for the first time ever reported, targeted the injured heart, and exerted intrinsic cardioprotective effects within 24 h, attributable to the antioxidant capacity of CDNs in a myocardial ischemia/reperfusion injury animal model. Taken together, these results pave the way for further development of CDNs as cell-based therapeutics stabilized by lyophilization that enabled long-term storage while preserving their activity.