Cyclic-Linear Hybrid Peptides as Molecular Transporters of Chemotherapeutic Agents

Arginine/tryptophan-rich peptides are amphipathic, enabling membrane interaction, cargo transport, and antimicrobial activity. To enhance these properties, we synthesized and evaluated hybrid peptide [RW]₄K-AH135 (1), combining the antimicrobial α-helical peptide AH135 (AMP) with the cell-penetrating peptide (CPP) [RW]₄K, and [R₅W₄]K-AH135 (2), combining AH135 with another AMP [R₅W₄]K. Peptides were synthesized by Fmoc solid-phase peptide synthesis and cyclization in solution phase, purified by HPLC, and characterized 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). In contrast, the hybrids functioned as molecular transporters in cancer cells. [R₅W₄]-AH135 enhanced the activity of multiple chemotherapeutics in MDA-MB-231 and SK-OV-3 cells, improving growth inhibition by approximately 15-47% at 72 h, and improved doxorubicin efficacy by approximately 37% in resistant MES-SA cells. Both hybrids promoted the intracellular delivery of green fluorescent protein (50 nM), as confirmed by flow cytometry and confocal microscopy (p < 0.001). [RW]₄K-AH135 showed higher uptake in both lines in a dose- and time-dependent manner. [R₅W₄]K-AH135 acted as a chemotherapeutic agent sensitizer, whereas [RW]₄K-AH135 was a superior carrier. Thus, these hybrids act as molecular transporters and represent promising platforms for drug conjugation and intracellular delivery.