Eradication of Intracellular <i>Staphylococcus aureus</i> Persisters via On-Site Antibiotic Delivery by Poly(amino acid) Nanoparticles

Intracellular <i>Staphylococcus aureus</i> persisters are a dormant bacterial subpopulation responsible for chronic and recurrent infections due to their ability to evade antibiotic treatment within host cells. However, effective strategies for eliminating these intracellular pathogens remain limited. Herein, we proposed a versatile poly(amino acid)-based platform, F(AM), for the effective eradication of intracellular <i>Staphylococcus aureus</i> persisters via on-site antibiotic delivery. The F(AM) platform exhibited dual-targeting capability toward macrophages and <i>Staphylococcus aureus</i> persisters, efficiently penetrating cellular barriers and achieving precise antibiotic delivery at intracellular bacterial niches. Sitafloxacin, rifampicin, and polymyxin B were identified from a panel of 11 antibiotic candidates and individually loaded into the F(AM) platform. The resulting nanoparticles markedly improved intracellular drug accumulation, protected antibiotics from degradation within the adverse intracellular environment, and overcame microenvironment-induced bacterial metabolic shifts. Compared with free antibiotics, the drug-loaded F(AM) nanoparticles notably improved their intracellular bactericidal activity. Collectively, this study highlights F(AM) as a robust and versatile platform for overcoming intracellular barriers and restoring antibiotic efficacy, offering a valuable tool for antipersister strategies and intracellular pharmacokinetic investigations.