Semaglutide is a glucagon-like peptide-1 receptor agonist widely used for the treatment of type 2 diabetes and obesity. Despite its clinical efficacy, oral administration remains challenging because of its limited gastrointestinal stability, poor epithelial permeability, and low affinity for lipid-based delivery systems. In the present study, a combined hydrophobic ion pairing (HIP) and solid lipid nanoparticle (SLN) approach was explored to improve semaglutide incorporation and delivery-related properties. Semaglutide was complexed with the cationic lipid DOTAP at different molar ratios (1:0-1:18) and subsequently incorporated into cetyl palmitate-based SLNs produced by microfluidic mixing using a herringbone device. The resulting formulations were characterized in terms of particle size, ζ-potential, encapsulation efficiency, morphology, solid-state organization, colloidal stability, release behavior, mucus interaction, cytocompatibility, and epithelial permeability. Among the various formulations prepared, the one prepared with a molar ratio semaglutide: DOTAP of 1:18 and a peptide concentration of 10% (w/w) (F10) showed the best results, combining particle sizes of less than 300 nm with almost complete encapsulation efficiency and a highly positive ζ-potential. FTIR, DSC, TGA and SAXS analyses confirmed the correct formation of the complex and its incorporation into the lipid matrix. The F10 formulation demonstrated good stability under simulated gastrointestinal conditions and a sustained-release profile. The formulation also exhibited strong interactions with mucus, whilst retaining the ability to diffuse through the mucin network. Cytocompatibility studies demonstrated acceptable cell viability at relevant concentrations, whilst permeability experiments through Caco-2 monolayers revealed an approximately 6-fold increase in apparent permeability compared to free semaglutide. Therefore, these findings indicate that the combination of DOTAP-mediated hydrophobic ion pairing and microfluidic-assisted SLN production represents a potentially promising strategy for improving semaglutide encapsulation, gastrointestinal stability, and epithelial transport, while maintaining a favorable balance between mucus interaction and mucodiffusion.
