Focused ultrasound (FUS) can be combined with intravenously injected microbubbles (MBs) to transiently and noninvasively increase the permeability of the blood-brain barrier (BBB) to enable the targeted delivery of stem cells to the brain. Although this process has been demonstrated in animal models, several key issues remain unresolved: (1) whether vascular disruption enhances the delivery of stem cells beyond using BBB opening alone, (2) how the temporal sequence of stem-cell administration and BBB opening affects efficiency, (3) the relative contributions of BBB permeability and inflammation to stem-cell accumulation, and (4) the temporal distribution and persistence of stem cells after BBB opening. To address these questions, we first used an in vitro HUVEC (human umbilical vein endothelial cell) monolayer barrier model and red-fluorescent-protein-labeled cord-blood mesenchymal stem cells (MSCs) to evaluate the efficacy of delivering MSCs using 1-MHz FUS at 300-600 kPa with MBs. The optimal condition for increasing the endothelial barrier permeability was using FUS at 300 kPa, which induced a 1.3-fold reduction in the fluorescence intensity of the ZO-1 tight-junction protein at the cell borders (203 ± 4 μm gaps), while cell viability remained high (93.5 %) and the migration of MSCs increased 1.8-2.6-fold. We then established two in vivo BBB-opening conditions by fine-tuning the acoustic pressure: (1) safe opening with minimal inflammation and (2) opening with severe vascular disruption. We found that safe BBB opening achieved the efficient delivery of MSCs, and this was not increased by vascular disruption, which could even reduce the penetration of MSCs. Furthermore, FUS + MBs-mediated delivery preserved the intrinsic properties of MSCs and was safer than intracranial injection. The number of MSCs accumulating in the brain progressively increased to peak at 24 h after FUS sonication. These findings suggest that both BBB opening and inflammation contribute to MSC migration, with transient BBB opening enabling the rapid and efficient delivery of MSCs, whereas inflammation supports prolonged recruitment, maintaining MSC accumulation for up to 7 days.
