A three-dimensional human brain spheroid model for studying sonoporation-induced drug penetration beyond the blood-brain barrier

Abstract

Ultrasound (US)-targeted microbubble (MB) cavitation (UTMC) facilitates delivery of cell-impermeant drugs across the blood brain barrier (BBB). The effects of UTMC on extravascular cells, and the extent of payload penetration, are unknown. Spheroids were generated using neurons and astrocytes derived from human iPSCs, primary human brain- microglia, endothelial cells (ECs) and pericytes, and placed in a water tank with lipid MBs. US (1 MHz frequency, 250 kPa peak negative pressure, 10 μs pulse duration, 10 ms pulse interval) was delivered for 10 s. 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 Texas red dextran (TRD) (28.8% increase, p = 0.0015). TRD penetrated up to 100 μm beyond the BBB upon UTMC (12% increase compared to 0 kPa control). Inhibition of endothelial nitric oxide synthase with L-NAME reduced UTMC-induced TRD penetration (19% decrease, p < 0.05). Sonoporation, as shown by uptake of propidium iodide, occurred in cells beyond the BBB. In our novel 3D spheroid model with intact BBB, UTMC caused nitric-oxide dependent BBB breach. This is the first report of UTMC causing “remote” sonoporation of cells not in direct contact with MBs and bears further study.