Abstract 13414: Studying Sonoporation-Induced Drug Payload Penetration Beyond the Blood-Brain Barrier Using Multi-Cellular Human Brain Spheroids

Abstract

Introduction: Ultrasound-targeted microbubble cavitation ( UTMC ) facilitates delivery of cell-impermeant drugs across the endothelial barrier, such as the blood brain barrier ( BBB ) . Using cultured endothelial cell ( EC ) monolayers, we reported that UTMC causes sonoporation and inter-endothelial gaps, which may mediate vascular hyperpermeability, although mechanisms are incompletely understood. Further, the effects of UTMC on extravascular cells, and extent of payload penetration are unknown, as the monolayer does not recapitulate the 3D multicellular environment. Here, we established a 3D multicellular human brain spheroid model to study UTMC bioeffects and underlying mechanisms. Methods: Spheroids were generated using neurons and astrocytes derived from human iPSCs, primary human brain- microglia, ECs and pericytes, and placed in a water tank with lipid microbubbles ( MBs ). Ultrasound (1 MHz frequency, 250-500 kPa peak negative pressures, 10 μs pulse duration, 10 ms pulse interval) was delivered for 20 s and spheroids were immediately collected. Immunostaining was used to localize cell types; propidium iodide (PI) uptake to mark sonoporation; calcein-AM and sytox to detect cell viability; and Texas red dextran (TRD) (10 kDa) to quantify permeability, using confocal microscopy. Results: Immunostaining showed ECs and pericytes at the spheroid periphery, with high expression of membranous ZO-1. BBB functionality was confirmed with histamine treatment, which increased permeability to TRD (28.8% increase in TRD mean intensity, p =0.0015). Interestingly, PI uptake (sonoporation) occurred in cells beyond the BBB. TRD penetrated up to ~100 μm beyond the BBB upon UTMC (12.2% increase). Inhibition of endothelial nitric oxide synthase with L-NAME reduced UTMC-induced TRD penetration (19% decrease, p =0.0458). Conclusions: In this novel 3D human brain spheroid model with intact BBB, UTMC caused nitric-oxide dependent BBB breach. Moreover, this is the first report of UTMC causing “remote” sonoporation of cells not in direct contact with MBs, which bears further study. Our model allows the study of mechanisms mediating UTMC efficacy for targeted delivery of drugs across the endothelial barrier, which should facilitate its clinical translation.