Nanobubble based sonobiopsy reveals circulating protein signatures of BBB opening in healthy and glioblastoma-bearing mice.

Focused ultrasound (FUS) and microbubbles can transiently increase blood brain barrier (BBB) permeability, yet BBB opening (BBBO) verification relies mainly on contrast-enhanced MRI, offering limited insight into the molecular consequences of barrier modulation. Sonobiopsy, which uses FUS induced BBBO to release brain derived molecules into the bloodstream, provides a molecular readout from blood samples. Nanobubbles (NBs) are smaller agents that circulate more effectively in the brain microvasculature and have shown enhanced BBBO in capillaries. Here, we establish NB-based sonobiopsy as a platform for enhanced biomarker efflux and molecular sensitivity, and apply it to define the proteomic signature of BBBO in healthy and glioblastoma (GBM) models. Plasma collected before and after NB-mediated FUS underwent data-independent acquisition-based mass spectrometry, revealing post-BBBO changes in healthy and tumor bearing mice. In healthy cohorts, 77 proteins were reproducibly altered after BBBO. Six proteins (Dpysl3, Myl1, Mybpc1, Vsig4, Krt33a, Krtap6-5) were detectable only after BBBO in healthy mice. In 005 glioma bearing mice, the BBBO signature identified in healthy animals was preserved with clear pre- vs post-BBBO separation. Candidate BBBO-associated proteins identified in the NB cohort were subsequently evaluated in an MB cohort, where the pre-to-post BBBO changes observed with NBs were not reproduced. These findings establish NB-mediated proteomic sonobiopsy as a promising method for BBBO verification across healthy and disease-relevant conditions and support the development of scalable molecular readouts for BBBO confirmation. This platform may also complement disease-focused sonobiopsy approaches, including protein-based biomarker studies in neuro-oncology.