Lipidation is widely used to improve peptide pharmacokinetics; however, its influence on peptide–membrane interactions relevant to oral delivery requires further investigation. Here, we investigated how lipidation pattern, amino acid sequence, and peptide oligomeric state influence the interactions of four GLP-1 analogues with a model membrane composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC). The analogues comprised non-lipidated J211; mono-lipidated, acid-terminated J229 and semaglutide; and bis-lipidated, methyl-terminated MEDI7219. Quartz crystal microbalance with dissipation monitoring (QCM-D) was combined with molecular dynamics (MD) simulations to characterise peptide–membrane interactions.

QCM-D revealed distinct analogue-dependent interfacial responses, indicating that lipidation alone did not determine peptide–bilayer interactions. The non-lipidated analogue J211 produced the largest transient decrease in frequency and the greatest increase in dissipation, consistent with formation of a hydrated, mechanically soft, and largely reversible membrane-associated state. Interestingly, the mono-lipidated J229 produced a weaker and reversible response. In contrast, semaglutide and bis-lipidated MEDI7219 induced only small changes in frequency and dissipation, consistent with weak interactions but the membrane perturbation was retained under the studied conditions. In the presence of 2.5 mM sodium caprate (C10), peptide-specific responses were generally attenuated and shifted towards a C10-dominated membrane-perturbation profile, although analogue-dependent differences still remained.

MD simulations showed analogue-dependent membrane engagement, where interaction depth and interaction stability were influenced by lipidation and peptide sequence. MEDI7219 was found to insert itself deepest into the membrane leaflet with stable interactions between the peptide and the lipid bilayer. The lipid tail of semaglutide remained persistently inserted although not as deep as MEDI7219, whereas (the similarly monolipidated) J229 engaged more transiently and reversibly. The non-lipidated J211 only associated with the bilayer surface through aromatic and charged residues. Overall, the lipidated peptides perturbed the local membrane structure the most, providing a molecular-level context for the distinct experimental QCM-D responses.

Together, these findings demonstrate that peptide–membrane interactions are governed not by lipidation alone, but by the combined effects of lipidation pattern, backbone sequence, peptide association state, and formulation conditions. This work provides mechanistic insight relevant to the developability of orally delivered peptides.