Self-assembled cationic lipopeptides modulate membrane interactions, cellular uptake, and mitochondrial dysfunction in HeLa cells.

Self-assembled cationic lipopeptides are membrane-active amphiphilic systems whose biological responses depend on molecular composition and supramolecular organization. Here, we investigated the structure-dependent cytotoxicity, hemocompatibility, cellular association/internalization, and cell-death mechanisms of four cationic lipopeptides with distinct peptide sequences and lipidation patterns: arginine-containing PRWG derivatives and guanidinocarbonylpyrrole (GCP)-containing PK(GCP)WG derivatives. Cytotoxicity assays showed concentration-dependent reductions in cell viability, with cell-line-dependent responses between HeLa and HEK293 cells. The double-lipidated GCP-containing lipopeptide showed the most favorable differential viability trend between these cell lines under the tested conditions, whereas PRWG-based lipopeptides induced more pronounced apoptosis-associated responses in HeLa cells. Hemolysis assays showed low hemolytic activity after 3 h, supporting preliminary short-term hemocompatibility; however, 24 h exposure revealed concentration- and structure-dependent hemolysis, particularly for PRWG-C18H37. Cellular association/internalization studies using coumarin-6 as a physically incorporated hydrophobic fluorescent probe revealed higher cell-associated fluorescence in HeLa cells than in HEK293 cells, suggesting that membrane interactions contribute to the biological response. Because coumarin-6 was physically incorporated rather than covalently attached, these data should be interpreted as cell-associated fluorescence from coumarin-6-loaded assemblies rather than direct quantitative evidence of intact lipopeptide uptake. Annexin V/7-AAD staining and JC-1 analysis indicated that PRWG-based lipopeptides predominantly induced apoptosis-associated cell death and mitochondrial membrane depolarization in HeLa cells, particularly the double-lipidated PRWG derivative. These findings show that peptide sequence, lipidation pattern, apparent surface charge, supramolecular morphology, and membrane-associated fluorescence collectively modulate the biological activity of cationic lipopeptides, highlighting their relevance as supramolecular platforms for investigating tunable lipopeptide-membrane interactions and cell-death responses.