A novel spray-dried milk extracellular vesicles formulation with long-term immunomodulatory activity and functional stability

Milk is a rich source of bioactive extracellular vesicles (EVs), with potential therapeutic functions for intestinal inflammation. A major limitation for the clinical use is their preservation, as they typically require storage at − 80 °C. To address this, our study aimed to develop a stable, room-temperature dry powder formulation of milk EVs (mEVs) that preserves their bioactivity. The mEVs were isolated via differential centrifugation with an EDTA cleaning step and subsequently spray-dried into microparticles using mannitol and leucine as excipients. The resulting microparticles were morphologically characterized as collapsed structures with a volume-weighted mean diameter of 11–20 µm and low aggregation. We found that the drying process did not cause molecular alterations, as the dried mEVs preserved protein and nucleic acid content comparable to that of unprocessed mEVs. Furthermore, the formulation showed minimal cytotoxicity on THP-1 cells (up to 1011 particles/mL), as measured by MTT assay. In a cellular inflammatory model, spray-dried mEVs stored for up to 18 months significantly reduced the gene expression of key pro-inflammatory cytokines (IL1B, IL6, CXCL8, and TNFA) to a degree comparable to unprocessed mEVs. This confirms the long-term functional stability of the dry mEV formulation. Our findings demonstrate that proper spray-drying approaches are a flexible and effective strategies for preserving mEVs, paving the way to the development of EVs loaded microparticles for a broad range of functional and therapeutic applications.