2025/11/25 by Lucia Paolini, Miriam Romano, Valentina Mangolini +11 · 1 voice
Biochemistry, Genetics and Molecular Biology · Medicine · Engineering · #Extracellular vesicles in disease #Erythrocyte Function and Pathophysiology #Nanoplatforms for cancer theranostics
paper · pdf · doi:10.1039/d5bm01349f
openalex publication_date 2025/11/25 · openalex created_date 2025/11/25 · openalex updated_date 2026/07/28
Red blood cell-derived extracellular vesicles (RBC-EVs) are emerging as promising biomaterials for next-generation drug delivery, owing to their intrinsic biocompatibility, immune-evasion properties, and minimal oncogenic risk. However, their broader application is currently limited by unresolved challenges related to heterogeneity, reproducibility, and long-term storage stability. By combining discontinuous sucrose density gradient separation with high-resolution interferometric nanoparticle tracking analysis, we identified a sharp bimodal size distribution of vesicles in freshly prepared samples. We then tracked how long-term storage at -80 °C drove their conversion into a monomodal distribution. To reproduce these conditions in a shorter time frame, we developed an "accelerated-ageing" protocol based on freeze-thaw cycles that generates RBC-EV samples with homogeneous density, size distribution, and biological activity, effectively replicating the properties of preparations stored for six months at -80 °C. This new vesicle population remains stable and retains membrane integrity and cellular internalization capacity, as confirmed by surface-associated enzymatic activity assays and uptake tests in cancer cell lines. These results suggest that freezing-induced "accelerated ageing" represents an effective method for the optimization and standardization of RBC-EVs as building blocks for biomaterial and bioengineering applications.