Exhaled nitric oxide amplification via functionalized vesicles enables non-invasive diagnosis of pulmonary infection

Pulmonary infections remain difficult to diagnose rapidly and non-invasively, as existing biomarkers lack both sensitivity and specificity. Here we present functionalized biomarker vesicles (FBVs), an inhalable nanotechnology platform that amplifies fractional exhaled nitric oxide (FeNO) in an infection-specific manner. FBVs are engineered from inactivated bacterial membranes and encapsulate L-arginine and NADPH, enabling selective uptake by M1-polarized macrophages within infected lung tissue. Upon phagocytosis or membrane fusion with iNOS-containing vesicles, FBVs release their payload to drive localized nitric oxide production, directly linking FeNO amplification to pathogen-induced immune activation. Optimized FBVs (800 nm) demonstrated efficient pulmonary delivery, robust macrophage uptake, and NO output tightly correlated with iNOS expression. In mouse infection models, FBV-induced FeNO distinguished infected from healthy with high accuracy (AUC = 0.94). In clinical bronchoalveolar lavage samples, FBV-based detection outperformed extracellular vesicle quantification, achieving superior diagnostic performance (AUC = 0.93). This strategy establishes a non-invasive diagnostic modality that couples host immune activation to measurable breath biomarkers, providing a direct path toward clinical translation for respiratory infection management.