Self-defensive antibiotic-loaded layer-by-layer coatings: Imaging of localized bacterial acidification and pH-triggering of antibiotic release.
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UNLABELLED: Self-defensive antibiotic-loaded coatings have shown promise in inhibiting growth of pathogenic bacteria adhering to biomaterial implants and devices, but direct proof that their antibacterial release is triggered by bacterially-induced acidification of the immediate environment under buffered conditions remained elusive. Here, we demonstrate that Staphylococcus aureus and Escherichia coli adhering to such coatings generate highly localized acidification, even in buffered conditions, to activate pH-triggered, self-defensive antibiotic release. To this end, we utilized chemically crosslinked layer-by-layer hydrogel coatings of poly(methacrylic acid) with a covalently attached pH-sensitive SNARF-1 fluorescent label for imaging, and unlabeled-antibiotic (gentamicin or polymyxin B) loaded coatings for antibacterial studies. Local acidification of the coatings induced by S. aureus and E. coli adhering to the coatings was demonstrated by confocal-laser-scanning-microscopy via wavelength-resolved imaging. pH-triggered antibiotic release under static, small volume conditions yielded high bacterial killing efficiencies for S. aureus and E. coli. Gentamicin-loaded films retained their antibacterial activity against S. aureus under fluid flow in buffered conditions. Antibacterial activity increased with the number of polymer layers in the films. Altogether, pH-triggered, self-defensive antibiotic-loaded coatings become activated by highly localized acidification in the immediate environment of an adhering bacterium, offering potential for clinical application with minimized side-effects. STATEMENT OF SIGNIFICANCE: Polymeric coatings were created that are able to uptake and selectively release antibiotics upon stimulus by adhering bacteria in order to understand the fundamental mechanisms behind pH-triggered antibiotic release as a potential way to prevent biomaterial-associated infections. Through fluorescent imaging studies, this work importantly shows that adhering bacteria produce highly localized pH changes even in buffer. Accordingly such coatings only demonstrate antibacterial activity by antibiotic release in the presence of adhering bacteria. This is clinically important, because ad libitum releasing antibiotic coatings usually show a burst release and have often lost their antibiotic content when bacteria adhere.
