To directly observe the mechanism of peptide permeation in the presence of SNAC computationally, we performed unbiased CpHMD simulations with a system containing 400 SNAC molecules (periodic box size: 11 nm x 11 nm x 15 nm), semaglutide, and a model POPC/cholesterol epithelial membrane (see Fig. ). Semaglutide and SNAC were randomly placed around the membrane, and the system was run for 100 ns to equilibrate. The membrane was constructed using Packmol, with its composition based on estimates of the cholesterol content in epithelial cells and previously reported simulations of SNAC. During the first 5 ns, semaglutide once again rapidly aggregated with neighboring SNAC molecules in the aqueous layer. Subsequently, semaglutide clusters in the water layer spontaneously fused with a membrane-bound SNAC aggregate, positioning the peptide flat on the membrane surface after ~50 ns. Similar synergistic aggregation mechanisms have been described previously for antimicrobial peptides. Next, given its hydrophobic nature, we hypothesized that the lipid tail of semaglutide (which includes the fatty acid chain and γGlu-2xOEG linker ligated to K26 as shown in Supplementary Figs. and) likely inserts first into the membrane, serving as a membrane anchor for the peptide. To confirm this hypothesis, we calculated the free energy profile for membrane insertion of the semaglutide lipid tail with the CpHMD method, using umbrella sampling followed by WHAM analysis 45 (Supplementary Figs. 16-18). Our results showed that incorporating the lipid tail anchor into the membrane center is favorable overall (ΔG = -4.8 kcal mol -1 ). However, pulling the acid end of the lipid tail to the opposite leaflet imposed an 8.2 kcal mol -1 free energy barrier due to the γGlu-2xOEG linker being pulled past the phosphate headgroups. Based on these results, we constructed an initial structure with the lipid tail of semaglutide anchored inside the membrane for extended, 1-μs unbiased CpHMD simulations of this system as shown in Fig. . As this simulation progressed (see Supplementary Movie 8 for a visual depiction of this process), the initial, smaller SNAC aggregates gradually expanded within the membrane, forming larger SNAC-filled membrane defects spanning the phospholipid head groups (Supplementary Fig. ).