Star-shaped PCL nanoparticles combining quercetin release and α-amylase surface functionalization enable AFM-quantified removal of biofilms of clinical Staphylococcus aureus isolates.

Healthcare-associated infections (HAIs) driven by multi-drug-resistant biofilms remain a critical challenge in clinical settings. The emergence of antibiotic-resistant strains has intensified the need for innovative antimicrobial strategies, particularly for effective surface treatments in clinical environments. This study presents a novel bifunctional nanoplatform designed to eradicate Staphylococcus aureus biofilms through a dual-action mechanism. We synthesized a custom star-shaped polycaprolactone (PCL-COOH) architecture to serve as a robust nanocarrier, enabling high quercetin loading (PCL-Q NPs) and effective surface conjugation of α-amylase (PCL-E NPs). The nanoparticles demonstrated excellent physical stability over a 120-day storage period and a sustained quercetin release profile up to 90 days. Unlike standard studies using laboratory strains, our platform was rigorously validated against four distinct clinical isolates of S. aureus collected from hospital surfaces, accounting for the natural heterogeneity of environmental biofilms. To ensure objective evaluation, we developed a high-resolution AFM-based quantification method utilizing systematic binarization and topographical analysis, moving beyond traditional bulk assays. Results showed enhanced combined activity for most isolates: the enzymatic component disrupts the extracellular polymeric substance (EPS) matrix, while the released quercetin neutralizes the sequestered bacterial cells, thus supporting the potential of the proposed approach as a dual-action strategy for healthcare-surface biofilm removal.