Non-viral targeted inducible nitric oxide synthase (iNOS) delivery to the brain for modulating sterile inflammation induced by ultrasound-mediated BBB opening
Focused ultrasound (FUS) combined with circulating microbubbles (MBs) enables noninvasive, spatially targeted opening of the blood–brain barrier (BBB) for drug and gene delivery. However, accumulating evidence indicates that even mild BBB opening parameters can induce sterile neuroinflammation, characterized by microglial activation and astrocytosis, which could pose a significant barrier to clinical translation. In this study, we explored whether localized NO production in the brain, derived by inducible nitric oxide synthase (iNOS) transfection, could modulate inflammatory responses associated with FUS-mediated BBB opening. iNOS plasmid DNA was delivered to the mouse hippocampus using cationic MBs and low-pressure FUS. BBB opening alone elicited a robust sterile inflammatory response without overt tissue damage. High-dose iNOS transfection further exacerbated inflammation and induced apoptosis, indicating dose-dependent neurotoxicity. In contrast, low-dose iNOS delivery resulted in minimal to no microglial accumulation, reduced astrocyte activation, and no detectable apoptosis or structural injury, despite successful opening of the BBB. These findings suggest that modest, localized NO production can suppress ultrasound-induced sterile inflammation in a dose-dependent manner. This work identifies a previously unrecognized anti-inflammatory role for low-level iNOS expression and suggests a straightforward strategy to improve the safety and therapeutic window of FUS-mediated BBB opening for neurotherapeutic applications.