Response of Serum-Isolated Extracellular Vesicles to Focused Ultrasound-Mediated Blood-Brain Barrier Opening.

To characterize the response of extracellular vesicles (EV) in the serum of mice and Alzheimer's disease (AD) patients following focused ultrasound (FUS)-mediated blood-brain barrier (BBB) opening (FUS-BBBO) as a means to improve liquid biopsy. Blood was collected from C57BL/6 mice before, and one hour after FUS-BBBO, and from AD patients before, one hour after, and three days after FUS-BBBO. EVs were isolated from serum using the Exoquick precipitation solution and their concentration was quantified using nanoparticle tracking analysis. The transcriptomic and proteomic content of EVs from mice was assessed using RNA sequencing and mass spectrometry protein analysis respectively. Additionally, the release of EVs in mice was inhibited using the GW4869 drug to assess the role of EVs in the restoration of the BBB. Finally, the biomarker content of EVs in AD patients was detected using a Luminex multiplex assay. We observed a 164±85% (95% confidence interval: 78.998 - 249.202) increase in murine EV concentration one hour after treatment, as well as an increase in EV RNA associated with FUS-BBBO neuroimmunotherapy. Inhibition of EVs reduced the inflammatory response and BBBO volume in mice. Patient EV concentration also increased one hour after treatment and was dependent on the volume of BBB opening three days post-treatment. Furthermore, EV isolation was found to significantly enhance (p<0.05) the detection of FUS-BBBO-induced amplification of AD and CNS biomarkers such as GFAP, beta-amyloid 42 and phosphorylated tau 181, exhibiting on average a 1.2 times higher log-fold change in biomarker levels in isolated EVs compared to total serum. Overall, we hereby present the first evidence of altered murine and AD patient EV concentration and content in response to FUS-BBBO, providing evidence of EVs' role within FUS-BBBO neuroimmunotherapy as well as their utility in improving FUS-BBBO biomarker amplification. Our results pave the way for clinical applications of EV-based liquid biopsy in patients with neurodegenerative diseases following FUS-BBBO, as a way of noninvasively monitoring disease progression.