Synergy of Bacterial Lipid Liposomes and Monoterpene‐Mediated Membrane Perturbation for Enhanced Intracellular Mycobacteria Eradication and Biofilm Disruption

In this work, we created liposomes using lipids from the outer membrane layer of Mycobacterial smegmatis (Msm) and doped them with rifampicin and lipophilic terpene, limonene, resulting in a dual-loaded liposomal formulation. Limonene incorporation resulted in a prolonged release of encapsulated rifampicin over time and reduced minimum inhibitory concentration in comparison to the free drug or limonene-free liposomes loaded solely with the drug. Limonene showed a prolonged release over several days and could potentially find applications in fragrant antibacterial products in the future. The bacterial count evaluated by colony-forming units was found to be reduced with limonene-doped drug-loaded mycobacterial liposomes in comparison to those without limonene. Mechanistically, it was shown that limonene enhances membrane fluidity and influences permeability, interacting with intact bacteria and thereby improving drug delivery, which promotes greater bacterial destruction. Limonene in the antibacterial liposomal formulation was also found to enhance the release of intracellular material because of compromised membranes aiding in bacterial destruction. Finally, mycobacterial liposome formulation loaded with drugs and doped with limonene successfully eliminated Msm biofilms more effectively than either the free drug or liposomes without limonene. This clearly demonstrates the collaboration between limonene and rifampicin in combination antimycobacterial treatment.