Bioengineered milk-derived extracellular vesicles implementing high drug loading and membrane integrity for efficient oral drug delivery

Milk-derived extracellular vesicles (EVs) are promising for oral drug delivery, yet different loading methods exhibit distinct impacts on drug encapsulation and membrane integrity. This study demonstrated that sonication method achieved high drug encapsulation in commercial milk-derived EVs (S-CM EVs), but impaired EV structure, compromising transcytosis. Incubation method (I-CM EVs) preserved EVs delivery ability, but had low drug loading. Further proteomic and transmembrane studies showed that sonication greatly damaged membrane proteins involved in trans-epithelial transportation, especially endoplasmic reticulum-Golgi pathway. To overcome this dilemma, we generated a hybrid CM EVs (H-CM EVs) by fusing I-CM EVs and S-CM EVs. H-CM EVs demonstrated comparable drug encapsulation to S-CM EVs (56.14%), significantly higher than I-CM EVs (11.92%). Importantly, H-CM EVs could maintain efficient drug delivery capability by restoring membrane fluidity, repairing damaged proteins, and enhancing enzyme resistance of S-CM EVs. H-CM EVs exhibited excellent absorption characteristics with 1.85-fold higher of area under the curve and 2.50-fold higher of max plasma concentration than those of S-CM EVs. On type Ⅰ diabetic mice, orally delivery of insulin loaded H-CM EVs and I-CM EVs showed improved hypoglycemic effects with pharmacological availabilities of 5.15% and 5.31%, which was 1.7-fold higher than that of S-CM EVs (3.00%). This H-CM EVs platform not only achieved high drug loading and maintained functionality for effective oral delivery but also highlighted the significant translational potential for improved clinical outcomes.