Cyclic-Linear Hybrid Peptides as Molecular Transporters of Chemotherapeutic agents

Arginine/tryptophan-rich peptides are amphipathic and cationic, enabling membrane interaction, cargo transport, and antimicrobial activity. To enhance these properties, we synthesized and evaluated hybrid peptide [RW]4K–AH135 (1), combining antimicrobial α-helical peptide (AMP) AH135 with cell-penetrating peptide (CPP) [RW]4K, and [R5W4]K–AH135 (2), combining AH135 with another AMP [R5W4]K. The peptides were synthesized by Fmoc solid-phase peptide synthesis and cyclization in solution phase, purified by reversed-phase HPLC, and verified by MALDI-TOF. While AH135 and [R₅W₄] showed antimicrobial activity (MICs 3.1–6.2 µg/mL), both hybrids were inactive (MIC ≥ 50 µg/mL). However, in cancer cell lines, the hybrids functioned as molecular transporters. [R₅W₄]K–AH135 enhanced the activity of multiple chemotherapeutics in MDA-MB-231 and SK-OV-3 cells, improving growth inhibition by ~15–47% at 72 h, and improved doxorubicin efficacy by ~37% in resistant MES-SA cells. Both hybrid peptides promoted the intracellular delivery of green fluorescent protein (GFP) (50 nM) as determined by flow cytometry and confocal microscopy (p < 0.001); [RW]4K–AH135 provided higher uptake in both lines in a dose- and time-dependent manner, with predominantly cytosolic localization. [R5W4]K–AH135 acted as a chemotherapeutic agent sensitizer, whereas [RW]4K–AH135 was a superior macromolecule carrier. Thus, these cyclic–linear constructs function as molecular transporters, supporting their development as platforms for drug conjugation and intracellular delivery.