Oral Administration Properties Evaluation of Three Milk‐derived Extracellular Vesicles Based on Ultracentrifugation Extraction Methods

Abstract Milk‐derived extracellular vesicles (M‐EVs) are low‐cost, can be prepared in large quantities, and can cross the gastrointestinal barrier for oral administration. However, the composition of milk is complex, and M‐EVs obtained by different extraction methods may affect their oral delivery. Based on this, a new method for extracting M‐EVs based on cryogenic freezing treatment (Cryo‐M‐EVs) is proposed and compared with the previously reported acetic acid treatment (Acid‐M‐EVs) method and the conventional ultracentrifugation method (Ulltr‐M‐EVs). The new method simplifies the pretreatment step and achieves 25‐fold and twofold higher yields than Acid‐M‐EVs and Ulltr‐M‐EVs. And it is interesting to note that Cryo‐M‐EVs and Acid‐M‐EVs have higher cellular uptake efficiency, and Cryo‐M‐EVs present the best transepithelial transport effect. After oral administration of the three M‐EVs extracted by three methods in mice, Cryo‐M‐EVs effectively successfully cross the gastrointestinal barrier and achieve hepatic accumulation, whereas Acid‐M‐EVs and Ultr‐M‐EVs mostly reside in the intestine. The M‐EVs obtained by the three extraction methods show a favorable safety profile at the cellular as well as animal level. Therefore, when M‐EVs obtained by different extraction methods are used for oral drug delivery, their accumulation properties at different sites can be utilized to better deal with different diseases.