Shedding Light on the Antibacterial Mechanisms of Silver Nanoparticles with Correlative Microscopy

Abstract

The antimicrobial activity of silver nanoparticles (AgNPs) has been widely studied over the past two decades, with particular emphasis on their potential applications in oral care. Our group pioneered the development of an AgNP-based formulation capable of arresting tooth decay, and clinical trials demonstrated its efficacy in halting the progression of dental caries after a single application. In vitro assays further confirmed that AgNPs exert bactericidal and bacteriostatic effects against Streptococcus mutans, the primary cariogenic pathogen, as well as other oral species. Nevertheless, the mechanisms underlying both the caries-arresting effect and the antibacterial action of AgNPs at the cellular level remain incompletely understood. In this thesis, we employed a multimodal microscopy approach to address these knowledge gaps. By combining focused ion beam (FIB), scanning electron microscopy (SEM), energydispersive X-ray spectroscopy (EDS), and X-ray microtomography, we investigated the interactions between AgNPs and human dentin. The analysis revealed that nanoparticles penetrate dentinal tubules and adhere to their walls, with estimated local silver concentrations sufficient to inhibit S. mutans growth, thus suggesting the formation of a long-lasting antimicrobial reservoir at the lesion site. Additionally, we investigated AgNP–bacteria interactions at the single-cell level, using bio-atomic force microscopy (Bio-AFM) and AFM-based infrared spectroscopy (AFM-IR). These experiments demonstrated rapid biomechanical and morphological alterations in S. mutans upon AgNP exposure, including cell stiffening, enlargement, and deformation, alongside evidence of membrane disruption driven by locally released Ag+ ions. Taken together, the results deepen our understanding of how AgNPs arrest caries progression and disrupt bacterial physiology shortly after contact. Beyond their relevance for AgNPs applications in dentistry, the findings contribute to the broader debate on the antibacterial mechanisms of AgNPs and highlight the value of correlative microscopy in investigating complex nano–bio interactions.