Biofilm penetration, triggered release and in vivo activity of inhaled liposomal amikacin in chronic Pseudomonas aeruginosa lung infections

Abstract

Chronic infections of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients are intractable antibiotic targets because of their biofilm mode of growth. We have investigated the biofilm penetration, mechanism of drug release and in vivo antimicrobial activity of a unique nanoscale liposomal formulation of amikacin designed specifically for nebulization and inhaled delivery. Penetration of fluorescently labelled liposomes into sputum or P. aeruginosa (PA3064) biofilms was monitored by a filter assay and by epifluorescence or confocal scanning laser microscopy. Amikacin release in vitro and rat lung levels after inhalation of nebulized material were measured by fluorescence polarization immunoassay. A 14 day agar bead model of chronic Pseudomonas lung infection in rats was used to assess the efficacy of liposomal amikacin versus free aminoglycosides in the reduction of bacterial count. Fluorescent liposomes penetrated readily into biofilms and infected mucus, whereas larger (1 microm) fluorescent beads did not. Amikacin release from liposomes was mediated by sputum or Pseudomonas biofilm supernatants. Rhamnolipids were implicated as the major releasing factors in these supernatants, active at one rhamnolipid per several hundred lipids within the liposomes. Inhaled liposomal amikacin was released in a slow, sustained manner in normal rat lungs and was orders of magnitude more efficacious than inhaled free amikacin in infected lungs. Penetration of biofilm and targeted, sustained release from liposomes can explain the superior in vivo efficacy of inhaled liposomal amikacin versus free drug observed in a 14 day infection model. Inhaled liposomal amikacin may represent an important therapy for chronic lung infections.