Selective Interaction of the Antimicrobial Peptide RKW with Bacterial Lipid Bilayers: A Biophysical Approach

Antimicrobial peptides (AMPs) have emerged as promising candidates for next-generation antibiotics due to their broad-spectrum activity, including efficacy against multidrug-resistant bacteria. However, their clinical application remains limited, primarily because of cytotoxicity toward host cells. A deeper understanding of AMP-membrane interactions, particularly through biophysical studies using model membrane systems, is essential for developing safe and effective AMP-based therapeutics. In this study, the interaction of a previously designed AMP, named RKW, with model lipid vesicles mimicking the lipid composition of both prokaryotic and eukaryotic cell membranes was investigated. RKW exhibited a strong preference for negatively charged bacterial membrane models, especially those representing Gram-negative bacteria, while showing minimal or no affinity for zwitterionic or eukaryotic-like membranes. These findings imply that electrostatic interactions are the primary driving force behind its membrane selectivity. Fluorescence spectroscopy and quenching experiments with acrylamide and lipophilic probes revealed that RKW localizes mainly at the membrane interface, likely adopting a parallel orientation relative to the bilayer surface. Furthermore, RKW induced substantial leakage of carboxyfluorescein from bacterial model membranes, indicating potent membrane permeabilisation. This mechanism was corroborated by dynamic light scattering (DLS) analyses, which provided additional evidence of peptide-induced membrane disruption. Collectively, this study elucidates the selective mechanism of action of RKW and underlines its potential as a targeted antimicrobial agent with reduced cytotoxicity toward eukaryotic cells. Toxicological assessments using the <i>Caenorhabditis elegans</i> in vivo model further supported its safety, showing no adverse effects on survival, reproduction, locomotion, or growth.