Focused-Ultrasound Mediated Gene Delivery to Brain Endothelium Without Blood-Brain Barrier Opening
Introduction
The blood-brain barrier (BBB) prevents the brain’s uptake of therapeutic agents from the blood. To overcome this problem, BBB opening by focused ultrasound (FUS) associated microbubbles (MBs) has become an effective method to deliver various therapeutic agents to the brain. However, there are limitations of BBB opening such as possibly inflammatoray response in the brain tissue and applicability in a large brain region and in a repetitive way. Sonoporation, based on the microbubble cavitation induced by FUS, is a promising approach of gene delivery to the BBB, specifically the endothelial cells, without opening the BBB. In particular, gene delivery to the brain endothelial cells may open new revenues to manipulate specific barrier functions such as drug efflux to enhance therapeutic delivery. Our study focuses on the feasibility of selective endothelial gene delivery without opening the BBB.
Methods
Mice were anesthetized with isoflurane. A tail-vein catheter was inserted to permit intravenous (i.v.) injections of MBs, plasmids (pcDNA3-luciferase) and fluorescent dye (FITC). The heads of the mice were shaved and depilated, and the animals were then placed in a supine position over a degassed water bath coupled to an MR-compatible small animal FUS system (RK-50; FUS Instruments). Mice received an injection of the conjugated MBs and luciferase plasmid (500 µg per mouse). Sonication applied to the right hippocampus region began immediately after the injection (1.46 MHz, 100 ms burst duration, 1000 ms burst period, 300 bursts).
Results
To study how sonoporation parameters affect transfection, we investigated FUS acoustic pressure ranging from 0.1 to 0.6 MPa and the number of MBs ranging from 1×108 to 5×108. With extremely high acoustic pressure (0.6 MPa) and a large number of MBs (5×108), clear brain injury was found at the sonication region. With low acoustic pressure (0.1 MPa) and a small number of MBs (1×108), there is no luciferase expression in the brain. Data points with parameter values between these two cases provide results of seeing expression. To test how sonoporation parameters affect BBB integrity, we inject FITC (330 Da) as a tracer to examine BBB disruption. With 0.2 MPa of acoustic pressure and 5×108 of MBs, there is an obvious dye leakage in the right hippocampus region with sonication. However, when the acoustic pressure or the number of MBs is reduced, we observed no dye leakage, indicating no BBB disruption. In sum, in the cases of 0.2 MPa - 1×108 and 0.1 MPa - 5×108, the luciferase plasmid is delivered to the brain without opening the BBB. Our ongoing work is to examine whether the plasmid is delivered to brain endothelial cells.
Conclusion
Here, we demonstrate that FUS sonoporation can deliver gene plasmid to the brain endothelial cells, and the transfection efficacy depends on sonoporation parameters, such as acoustic pressure and MB concentration. The development of novel non-invasive gene therapy to brain endothelial cells represents a new method to manipulate the BBB and has the potential to enhance therapeutic delivery into the brain.