Milk exosomes anchored with hydrophilic and zwitterionic motifs enhance mucus permeability for applications in oral gene delivery.

Exosomes have emerged as a promising tool for the delivery of drugs and genetic materials, owing to their biocompatibility and non-immunogenic nature. However, challenges persist in achieving successful oral delivery due to their susceptibility to degradation in the harsh gastrointestinal (GI) environment and impeded transport across the mucus-epithelium barrier. To overcome these challenges, we have developed high-purity bovine milk exosomes (mExo) as a scalable and efficient oral drug delivery system, which can be customized by incorporating hydrophilic and zwitterionic motifs on their surface. In our study, we observed significantly improved transport rates by 2.5-4.5-fold in native porcine intestinal mucus after the introduction of hydrophilic and zwitterionic surface modifications, as demonstrated by transwell setup and fluorescence recovery after photobleaching (FRAP) analysis. Remarkably, mExo functionalized by a block peptide (BP), consisting of cationic and anionic amino acids arranged in blocks at the two ends, demonstrated superior tolerability in the acidic gastric environment (with a protein recovery rate of 84.8 ± 7.7%) and exhibited a 2.5-fold increase in uptake by intestinal epithelial cells. Furthermore, both mExo and mExo-BP demonstrated successful intracellular delivery of functional siRNA, resulting in up to 65% suppression of the target green fluorescence protein (GFP) gene expression at a low dose of siRNA (5 pmol) without causing significant toxicity. These findings highlight the immense potential of modifying mExo with hydrophilic and zwitterionic motifs for effective oral delivery of siRNA therapies.