This study aimed to investigate the permeation behaviour and intracellular fate of hydrophobic ion pairs (HIP).

HIP were formed by combining a daptomycin-derived model peptide (DD) with ethyl lauroyl arginate (ELA) and lipophilic fluorescent dye 4-(4-dihexadecylaminostyryl)-N-methylpyridinium iodide (DiA). A representative HIP (DD: ELA: DiA, molar ratio 1:4:0.5) was incorporated into self-emulsifying drug delivery systems (SEDDS) and characterized for size, zeta potential, stability, hemolytic activity, cytotoxicity, and cellular uptake. Permeability was assessed using the Parallel Artificial Membrane Permeability Assay (PAMPA) model and Caco-2 monolayers.

SEDDS exhibited droplet sizes below 200 nm, a polydispersity index (PDI) < 0.4, positive surface charges, and high stability. Hemolysis studies indicated potential for endosomal escape, while dose-dependent toxicity became apparent after 4 and 24 h of incubation. Flow cytometry revealed enhanced cellular uptake: HIP and SEDDS increased internalization of DD by 12- and 32-fold, compared to free peptide. Permeation studies demonstrated marked improvements in DD transport. In the PAMPA assay, HIP and SEDDS increased passive diffusion by 2.8- and 6.5-fold. Similarly, in the Caco-2 model, HIP and SEDDS enhanced permeation by 17- and 57-fold, compared to free DD. DiA permeation remained minimal, suggesting that HIP disassociates intracellularly, allowing selective release of the peptide.

These findings confirm that HIP enhances membrane permeation of DD and dissociates after uptake. The combination of HIP and SEDDS presents a robust strategy for improving the oral bioavailability of peptide therapeutics.