Spatio-temporal nano-biomineralization and pro-antibiotic release for synergistically combatting multidrug-resistant bacteria

With the emergence and prevalence of drug resistance in microorganisms, bacterial infections and related diseases are major causes of global health concerns. A host of factors, including regulatory restrictions hindering the production of new antibiotics by the pharmaceutical industries, makes it essential to develop a platform for combatting multidrug-resistant bacteria with known drugs and druggable alternatives. Herein, we present a combinatorial approach that harnesses the bactericidal properties of silver nanoparticles along with a levofloxacin prodrug embedded within the lipid nanoparticle. The spatio-temporal synergistic effect of combined prodrug technology and biomineralization enables targeted, controlled drug release within bacterial cells, enhancing bioavailability, overcoming drug resistance, and minimizing systemic toxicity while leveraging in-situ silver nanoparticle formation for amplified antimicrobial efficacy. Our studies report the formation of homogeneous nanoparticles of ∼ 100 ± 20 nm in diameter within bacteria resulting in synergistic enhancement of bactericidal effect. The in-vitro studies with Gram-positive Bacillus subtilis resulted in important insight into the mechanism of uptake and reduction of silver ion incorporated within the lipid nanoparticles inside the bacterial cell, which have been extrapolated to in-vivo studies. This concept was further consolidated with in-vitro studies on multidrug-resistant Gram-negative bacteria Klebsiella pneumoniae. We further showed time-dependent intracellular uptake of the nanocarriers and cleavage of the prodrug in the presence of bacterial esterase enzyme using hyperspectral imaging, confocal microscopy, and mass spectrometry. In vivo studies carried out in a rodent model also revealed significant enhancement of the nanocarriers’ bactericidal properties compared to individual components and demonstrated their biocompatibility.