Double Emulsions Enable In Situ Generation of Permeation Enhancers for Oral Delivery of Peptides

Double Emulsions Enable In Situ Generation of Permeation Enhancers for Oral Delivery of Peptides Hannah Pohlit¹,², Lingxiao Li², Estela Isabel Bini³, Dario Colucci³, Cristhian Fernando Salas Cotaquispe¹, Maja Sikström¹, Per Larsson²,⁴, Christel A.S. Bergström²,⁴, Shakhawath Hossain²,⁴, David J. Brayden³, Alexandra Teleki¹,²* ¹Department of Pharmacy, Science for Life Laboratory, Uppsala University, Sweden ²The Swedish Drug Delivery Center, Department of Pharmacy, Uppsala University, Sweden ³School of Veterinary Sciences, University College Dublin, Ireland ⁴Department of Pharmacy, Uppsala Biomedical Center, Uppsala University, Sweden * Corresponding author (alexandra.teleki@scilifelab.uu.se) Abstract Oral delivery of peptide therapeutics remains limited by gastrointestinal degradation and poor epithelial permeability. Here, water-in-oil-in-water (W/O/W) double emulsions produced by microfluidics were designed to co-encapsulate octreotide and medium-chain triglycerides, enabling digestion-triggered generation of permeation-enhancing fatty acids. Production parameters were systematically optimized to obtain stable, monodisperse droplets with defined core-shell morphology. The emulsions comprised an inner aqueous phase containing the payload, encapsulated within a Miglyol 812N oil phase stabilized by polyglycerol polyricinoleate (PGPR), and dispersed in an external aqueous phase stabilized by Tween 80, yielding droplets of ~190 μm with a single inner aqueous core (~78 μm). Lipolysis studies confirmed minimal fatty acid release under gastric conditions and substantial release of caprylic (C8) and capric (C10) acids during intestinal digestion, accompanied by release of encapsulated cargo. In differentiated Caco-2 monolayers, digested emulsions increased apparent permeability (Pₐₚₚ) of fluorescein isothiocyanate-dextran (FD-4) and octreotide in a fatty acid concentration-dependent manner. Immunostaining showed occludin redistribution under permeation‑enhancing conditions. Ex vivo studies in rat colonic mucosa using Ussing chambers demonstrated a ~4-fold increase in FD-4 permeability for the digested emulsions, comparable to matched concentrations of free fatty acids, while octreotide permeability remained unchanged. Coarse-grained molecular dynamics simulations revealed strong association of octreotide with mixed bile salt–fatty acid micelles, limiting its freely dissolved fraction, whereas FD-4 remained predominantly solvated, consistent with the experimental findings. This study demonstrates that digestion of structured double emulsions enables in situ generation of permeation enhancers while simultaneously releasing hydrophilic cargo, providing a formulation strategy for oral delivery of peptide therapeutics. Keywords Lipid-based formulation, Lipolysis, Permeation enhancers, Apparent permeability, Molecular dynamics simulations, Intestinal permeability