ABSTRACT: Multidrug membrane transporters can extrude a wide range of substrates, which cause multidrug resistance and ineffective treatment of diseases. In this study, we used three different sized antibiotic drug nanocarriers to study their size-dependent inhibitory effects against Bacillus subtilis. We functionalized 2.4 ± 0.7, 13.0 ± 3.1, and 92.6 ± 4.4 nm silver nanoparticles (Ag NPs) with a monolayer of 11-amino-1-undecanethiol and covalently linked them with antibiotics (ofloxacin, Oflx). The labeling ratios of antibiotics with NPs are 8.6 × 102, 9.4 × 103, and 6.5 × 105 Oflx molecules per NP, respectively. We designed cell culture medium in which both BmrA and ?BmrA cells grew and functioned normally while ensuring the stabilities of nanocarriers (nonaggregation). These approaches allow us to quantitatively study the dependence of their inhibitory effect against two isogenic strains of B. subtilis, WT (normal expression of BmrA) and ?BmrA (deletion of bmrA), upon the NP size, antibiotic dose, and BmrA expression. Our results show that the inhibitory effects of nanocarriers highly depend on NP size and antibiotic dose. The same amount of Oflx on 2.4 ± 0.7, 13.0 ± 3.1, and 92.6 ± 4.4 nm nanocarriers shows the 3× lower, nearly the same, and 10× higher inhibitory effects than that of free Oflx, against both WT and ?BmrA, respectively. Control experiments of the respective sized AgMUNH2 NPs (absence of Oflx) show insignificant inhibitory effects toward both strains. Taken together, the results show multiple factors, such as labeling ratios, multivalent effects, and pharmacodynamics (Oflx localization and distribution), which might play the roles in the size-dependent inhibitory effects on the growth of both WT and ?BmrA strains. Interestingly, the inhibitory effects of nanocarriers are independent of the expression of BmrA, which could be attributed to the higher efflux of nanocarriers by other membrane transporters in both strains.
