Comprehensive design and characterization of pH-gradient-loaded donkey-milk exosomes for oral octreotide delivery: A bench-to-in silico roadmap

While an oral octreotide formulation (Mycapssa®) utilizing transient permeability enhancement has recently been FDA-approved, its clinical utility is often constrained by strict fasting requirements and reliance on the transient disruption of intestinal tight junctions. To develop a more biomimetic and efficient alternative, we developed the first oral formulation of Octreotide (OCT) using donkey-milk exosomes (DME). These naturally derived vesicles are enriched with membrane-fusion and transcytosis proteins that protect biologics from gastric degradation and facilitate endogenous epithelial transport. Exosomes were isolated from donkey milk powder, characterized by nanoparticle tracking analysis, western blotting, and proteomics, and found to possess a mean size of 138.4 ± 4.37 nm, and zeta potential of -42.07 ± 0.99 mV, and abundant transport-associated proteins including PIGR, MFGE8, AnxA2, CD9, CD63, and CD81. OCT was encapsulated using a pH-gradient method, achieving 7.25% entrapment efficiency. Analytical techniques including ATR-FTIR and DSC reveals the characteristic functional groups and thermal transitions of the components, confirming that octreotide was effectively integrated into the DME formulation. In-vitro dissolution studies demonstrated protective release behavior, while MDCK monolayer assays revealed a 37-fold enhancement in permeability compared to free OCT. Pharmacokinetic performance was predicted by GastroPlus™, which projected a rise in fraction absorbed from 8.5% to 88.2%, a reduction in Tmax from 5.5 to 2.2 h, and a four-fold increase in AUC. These predictions were confirmed in-vivo, where oral administration of OCT-exosomes (2 mg/kg) in BALB/c mice achieved a 16-fold increase in systemic exposure (AUC_0-24: 435,200 pg·h/mL) compared with free OCT, alongside targeted biodistribution to the liver, spleen, kidney, and intestine. This study provides the first proof-of-concept that donkey-milk exosomes can enable effective oral delivery of octreotide. By combining stable encapsulation, epithelial transport facilitation, predictive modeling, and in-vivo validation, this platform offers a scalable and broadly applicable strategy for transforming injectable peptides and biologics into oral therapeutics.

Bioavailability; Exosomes; Octreotide; pH gradient method.