Ultrasound-responsive multifunctional nanodroplets for enhanced biofilm penetration and synergistic sonodynamic/gas therapy of bacterial implant infections

Conventional antibiotic treatment of bacterial infections associated with biofilms usually suffers from poor penetration and drug resistance. Ultrasound (US)-responsive antibacterial systems have shown great promise in the elimination of bacterial biofilms, benefiting from their unique sonophysical and sonochemical effects. In this study, PFP@Lip-BNN6/Ce6 nanodroplets (PLBC NDs) were prepared by using perfluoropentane (PFP) to load chlorin e6 (Ce6) and a nitric oxide (NO) precursor (BNN6) for treating <em>Staphylococcus aureus</em> (<em>S. aureus</em>) implant infection. PLBC NDs physically disrupt the biofilm structure by US-triggered PFP phase transition and cavitation to enhance the permeation of Ce6 and BNN6. Under US irradiation, Ce6 generates various reactive oxygen species (ROS), such as singlet oxygen (¹O₂) and superoxide anion (O₂^.−); BNN6 releases NO and then reacts with O₂^.− to form peroxynitrite anion (ONOO⁻), one of the long-lived reactive nitrogen species (RNS), thus realizing synergistic ROS/RNS antibacterial activity. <em>In vitro </em>experiments showed that PLBC NDs reduced the biofilm biomass of <em>S. aureus</em> in 96-well plates by 65.9%, with a bacterial inactivation rate of 4.4 Log (99.995%), significantly surpassing single treatments. Transcriptomic analysis indicated that PLBC NDs can interfere with key pathways of <em>S. aureus</em> biosynthesis, metabolism, and oxidative stress. In a mouse titanium implant infection model, PLBC NDs reduced the number of viable bacteria in infected tissues by 3.5 Log (99.97%) and promoted macrophage polarization towards an anti-inflammatory phenotype (M2). Toxicity assessments demonstrated the favorable safety profile of PLBC NDs. This study presents a multifunctional US-responsive nanoplatform integrating sonophysical disruption and sonochemical killing for effective biofilm infection treatment.