The oral delivery of peptide therapeutics remains constrained by poor intestinal permeability and the challenge of enhancing epithelial transport without compromising mucosal integrity. Although sodium N-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC) enables gastric absorption of oral semaglutide and has also been explored for small-intestinal absorption of other peptide drugs, its suitability for enteric intestinal delivery of semaglutide remains unexplored. Here, we performed a comparative mechanistic evaluation of three permeation enhancers (SNAC, N-dodecyl-β-D-maltoside, and lauroyl-l-carnitine) for enteric delivery of semaglutide. Mechanisms were screened in epithelial monolayers and in situ intestinal absorption in rats, and the enhancers carried forward were formulated into enteric-coated capsules and evaluated in beagle dogs, with the leading candidate taken in to cynomolgus monkeys. In differentiated Caco-2 monolayers, all enhancers increased semaglutide transport but exhibited distinct transport mechanisms and barrier-response profiles. SNAC increased permeability without increasing mannitol transport or disrupting ZO-1, indicating a largely non-paracellular, cargo-dependent profile. DDM markedly increased paracellular marker transport and disrupted tight-junction organization, consistent with a primarily paracellular mode of action associated with reduced barrier recoverability. LLC showed a more favorable balance between transport enhancement and barrier recovery, while also increasing intracellular semaglutide-associated fluorescence and FD4 permeability. Rab5 immunostaining and dynasore inhibition indicated a dynamin-sensitive uptake component alongside partial tight-junction modulation. In rats, LLC produced the greatest increase in semaglutide absorption, and this effect was substantially attenuated by dynasore co-treatment. Transmission electron microscopy and tissue immunofluorescence further supported the presence of LLC-associated micelle-like structures, Rab5-related intracellular trafficking and junctional remodelling, with no overt tissue injury on local or systemic histology. For large-animal translation, citric acid and L-arginine improved semaglutide stability in intestinal fluids, and the resulting LLC-containing enteric formulation markedly increased systemic exposure in orally dosed beagle dogs and in cynomolgus monkeys following intraduodenal delivery. Collectively, these findings identify LLC as a promising permeation enhancer within the present SNAC/DDM/LLC comparison for enteric intestinal delivery of semaglutide and highlight how coordinated uptake-related and barrier-modulating mechanisms can support intestinal semaglutide absorption.
