Colloidal Structure Dictates Antimicrobial Efficacy in LL‐37 Self‐Assemblies With Glycerol Monooleate

Abstract The antimicrobial peptide LL‐37 is a promising alternative to conventional antibiotics to combat bacteria in suspension and biofilms. Its self‐assembly with polar lipids is suggested to improve its potential for therapeutic applications with higher stability against degradation and bioavailability. This study investigates the self‐assembly of LL‐37 with glyceryl monooleate (GMO), establishing the link between colloidal structure and antimicrobial activity. Small‐angle X‐ray scattering, dynamic light scattering and cryogenic transmission electron microscopy show structural transformation from dispersions of inverse bicontinuous structure (cubosomes) to multilamellar vesicles and direct rod‐like mixed‐micelles upon increasing the content of LL‐37 in GMO. In vitro assays against planktonic and biofilm cells demonstrate that 128 µg mL ⁻¹ of GMO cubosomes have no impact on Pseudomonas aeruginosa . Still, the cubosomes reduce the Staphylococcus aureus planktonic population by ≈ 1‐log after 24 h. Cylindrical micelles formed at LL‐37/GMO 9/1 and 8/2 with 128 µg mL ⁻¹ LL‐37 decrease the Pseudomonas aeruginosa population by 6‐log. This activity is gradually abolished when LL‐37 is encapsulated in vesicles or cubosomes. They also demonstrate low antibiofilm efficacy and promote the biomass of Staphylococcus aureus biofilms. These results highlight the importance of colloidal structure for therapeutic outcomes, providing insights for advanced lipid nanocarrier designs.