Ultrasound-Controlled Blood-Brain Barrier (BBB) Opening in a BBB-Glioblastoma Microfluidic Chip for Drug Delivery Studies

Yihong Li; Liyang Wu; Huadong Fan

doi:10.3791/72041

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Method Article

Ultrasound-Controlled Blood-Brain Barrier (BBB) Opening in a BBB-Glioblastoma Microfluidic Chip for Drug Delivery Studies

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DOI:

10.3791/72041

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August 18th, 2026

Yihong Li^*¹^,²^,³ , Liyang Wu^*¹^,²^,³ , Huadong Fan¹^,²^,⁴

¹Ningbo No.2 Hospital, ²Innovation Center for Diagnosis and Treatment of Neurological Diseases, Ningbo Institute of Life and Health Industry, University of Chinese Academy of Sciences, ³Lab of Nanopharmacology Research for Neurodegeneration, Department of Research and Development of Science and Technology, Ningbo Institute of Life and Health Industry, University of Chinese Academy of Sciences, ⁴Lab of Dementia and Neurorehabilitation Research, Department of Research and Development of Science and Technology, Ningbo Institute of Life and Health Industry, University of Chinese Academy of Sciences

^* These authors contributed equally

Summary

This protocol describes the construction of a microfluidic BBB‑glioblastoma (BBB‑GBM) chip model. The model is validated for BBB integrity and enables investigation of low‑intensity ultrasound‑induced transient and largely reversible BBB opening, thereby allowing quantitative evaluation of ultrasound‑mediated nanomicelle transport across the BBB and tumor-targeting efficiency.

Abstract

The blood-brain barrier (BBB) is a major obstacle to treating glioblastoma (GBM) by restricting the entry of most therapeutic molecules into the brain. Interspecies differences in BBB structure and function complicate the clinical translation of animal models, highlighting the need for human-relevant in vitro BBB platforms for assessing drug permeability in GBM therapy. Here, a protocol is described for constructing a microfluidic BBB-GBM chip model. This model establishes a tri-culture system comprising human cerebral microvascular endothelial cells (HCMECs), primary astrocytes (ACs), and U87-MG cells within a Matrigel-embedded microfluidic platform. BBB integrity is verified through continuous zonula occludens-1 (ZO-1) immunostaining and FITC-dextran permeability assay. Low-intensity ultrasound (US) (1 MHz, 1 W/cm², 30 s) is then employed to induce transient and largely reversible BBB opening, allowing tumor-targeting nanomicelles (SFN@RB@SPMs) to efficiently traverse the barrier and accumulate in GBM cells. A key feature of this protocol is the integration of real-time BBB permeability and drug delivery measurements within a single ultrasound-responsive chip, providing a powerful platform to dissect the mechanisms of ultrasound-augmented drug delivery in GBM.

Introduction

The treatment of glioblastoma (GBM) is strongly limited by the BBB, which severely limits the entry of nearly all therapeutic agents into the brain¹^,². The BBB is formed by non-fenestrated endothelial cells joined by tight junction proteins (e.g., occludin, ZO-1), with additional support from pericytes and astrocytic end-feet³. Although animal models have provided valuable insights into BBB function, species-specific variations in physiology and metabolism limit their ability to accurately predict human drug responses⁴^,⁵. Conseq....