Fast Biofilm Penetration and Anti-PAO1 Activity of Nebulized Azithromycin in Nanoarchaeosomes Maria Julia Altube Melina M. B. Martínez Barbara Malheiros Paulo C. Maffía Leandro R. S. Barbosa Maria Jose Morilla Eder Lilia Romero 10.1021/acs.molpharmaceut.9b00721.s001 https://acs.figshare.com/articles/journal_contribution/Fast_Biofilm_Penetration_and_Anti-PAO1_Activity_of_Nebulized_Azithromycin_in_Nanoarchaeosomes/11310494 Azithromycin (AZ) is a broad-spectrum antibiotic with anti-inflammatory and antiquorum sensing activity against biofilm forming bacteria such as Pseudomonas aeruginosa. AZ administered by oral or parenteral routes, however, neither efficiently accesses nor remains 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 (TPAs), rich in 2,3-di-<i>O</i>-phytanyl-<i>sn</i>-glycero-1-phospho-(3′-<i>sn</i>-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 minimal inhibitory concentration and minimal bactericidal concentration, higher preformed biofilm disruptive, and anti-PAO1 activity in biofilms than AZ. NanoARC penetrated and disrupted the structure of the PAO1 biofilm within only 1 h. Two milliliters of 15 μg/mL AZ nanoARC-AZ nebulized for 5 min rendered AZ doses compatible with in vitro antibacterial activity. The strong association between AZ and the nanoARC bilayer, combined with electrostatic attraction and trapping into perpendicular methyl groups of archaeolipids, as determined by Laurdan fluorescence anisotropy, generalized polarization, and small-angle X-ray scattering, was critical to stabilize during storage and endure shear forces of nebulization. NanoARC-AZ was noncytotoxic on A549 cells and human THP-1-derived macrophages, deserving further preclinical exploration as enhancers of AZ anti-PAO1 activity. 2019-12-03 15:23:30 biofilm depths AZ anti-PAO 1 activity Nebulized Azithromycin Fast Biofilm Penetration TPA THP -1-derived macrophages nanoARC bilayer bactericidal concentration AZ doses anti-PAO 1 activity Nanoarchaeosomes Azithromycin shear forces inhalable nanovesicle formulation parenteral routes inhaled nanocarriers antimicrobial activity Anti-PAO 1 Activity Laurdan fluorescence anisotropy small-angle X-ray 5 min methyl groups 549 cells 1 h PAO 1 biofilm broad-spectrum antibiotic Halorubrum tebenquichense archaebacteria Pseudomonas aeruginosa glycero -1-phospho sn preformed biofilm