Fast biofilm penetration and anti-PAO1 activity of nebulized azythromicin in nanoarchaeosomes.

Abstract

Azithromycin (AZ) is a broad-spectrum antibiotic with anti-inflammatory and anti-quorum sensing activity against biofilm forming bacteria such as Pseudomonas aeruginosa (Pa). AZ administered by oral or parenteral routes however, neither efficiently access nor remain in therapeutic doses inside pulmonary biofilm depths. Instead, inhaled nanocarriers loaded with AZ may revert the problem of low accessibility and permanence of AZ into biofilms, enhancing its antimicrobial activity. The first inhalable nanovesicle formulation of AZ: nanoarchaeosome-AZ (nanoARC-AZ) is here presented. NanoARC prepared with total polar archaeolipids (TPA, rich in 2,3-di-O-phytanyl-sn-glycero-1-phospho-(3 -sn-glycerol-1 -methylphosphate) (PGP-Me) from Halorubrum tebenquichense archaebacteria, consisted of ∽ 180 nm diameter nanovesicles, loaded with 0.28 w:w AZ:TPA. NanoARC-AZ displayed lower MIC and MBC, higher preformed biofilm disruptive and anti-PAO1 activity in biofilm than AZ. NanoARC penetrated and disrupted the structure of PAO1 biofilm within only 1 h. 2 mL of 15 μg/mL AZ nanoARC-AZ nebulized along 5 min rendered AZ doses compatible with in vitro antibacterial activity. The strong association between AZ and nanoARC bilayer, combined electrostatic attraction and trapping into perpendicular methyl groups of archaeolipids, as determined by Laurdan fluorescence anisotropy, generalized polarization and SAXS, was critical to stabilize during storage and endure shear forces of nebulization. NanoARC-AZ was non-cytotoxic on A549 cells and human THP-1 derived macrophages, deserving further preclinical exploration as enhancers of AZ anti-PAO1 activity.