Long-Term Stabilization of Non-Bovine Milk Exosomes Using Lyophilization: Toward Oral Delivery of PROTAC Therapeutics.

The clinical translation of exosome-based therapeutics is limited by challenges in long-term storage, stability, and scalability. We developed and optimized a lyophilization protocol to stabilize camel and donkey milk-derived exosomes for oral delivery of ARV-110, a proteolysis-targeting chimera (PROTAC). Using 5% trehalose, 5% mannitol, and 2.5% glycine as cryoprotectants, lyophilization preserved exosome integrity, protein content, and expression of key markers (CD63, CD81, TSG101, Alix) during storage at -80°C. Reconstituted exosomes, maintained particle size and zeta potential comparable to fresh preparations. In vitro, ARV-110-loaded lyophilized exosomes showed enhanced cytotoxicity in LLC cells (p < 0.001) and increased transepithelial transport in MDCK assays (p < 0.05). In rats, ARV-110 delivered via lyophilized exosomes achieved a 1.77-fold higher Cmax and 1.73-fold higher AUC0-24 compared to free drug, with no change in Tmax. Lyophilized exosomes retained over 90% of protein content after 8 weeks at -80°C, whereas non-lyophilized controls retained <25%. These findings demonstrate that lyophilized milk exosomes are a scalable, biocompatible platform for oral delivery of poorly bioavailable therapeutics like PROTACs, addressing major formulation and stability challenges and supporting their translational potential in drug delivery.