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Localized delivery of non-viral gene-bearing nanoparticles into the rat brain following focused ultrasound-mediated BBB opening
© Mead et al; licensee BioMed Central Ltd. 2015
Published: 30 June 2015
By preventing more than 98% of currently used pharamaceutical agents from entering the brain, the blood-brain barrier critically reduces the ability of therapeutics to treat a variety of central nervous system (CNS) disorders including glioblastoma and neurodegenerative diseases. Focused ultrasound (FUS) mediated microbubble oscillation and subsequent blood brain barrier (BBB) permeabilization has been explored as a powerful non-invasive strategy for the delivery of circulating therapeutic agents into the CNS. FUS in conjunction with microbubbles has been shown to facilitate non-damaging, reversible and localized disruption of the BBB, leading to substantial increases in nanoparticle (NP) concentrations in ultrasound-treated tissue. Once beyond the BBB, the extracellular matrix acts as a steric and adhesive barrier and limits NP distribution. Coating sub-100 nm nanoparticles with a dense brush layer of polyethylene glycol (PEG) to limit the interactions with the ECM leads to a significant improvement of their diffusivity in brain tissue. The current study investigates the ability of FUS to deliver densely PEGylated brain penetrating cationic polymer-based gene vectors across the BBB to mediate robust and long-term transgene expression.
Anesthetized 200g Sprague-Dawley rats were secured in a stereotaxic frame and their heads were depilated. A 1.5-inch single element 1MHz focused ultrasound transducer was ultrasonically coupled. The tail vein was cannulated, and a coinjection of microbubbles (105/g) and 50 nm PEGylated polyethylenimine (PEG-PEI) nanoparticles (50 ug, 100 ug, 200 ug or 350 ug doses) immediately preceded ultrasound treatment. We delivered luciferase and mCherry transgenes under control of a beta-actin promoter. All sonications were performed with a 0.5% duty cycle, a total time of two minutes, and a peak negative pressure of 0.6 MPa. Luciferase expression was assessed through bioluminescent imaging following a 150 mg/kg injection of luciferin in an In Vivo Imaging System. Following euthanasia, animals were perfused with saline, and brains were dehydrated and cryosectioned. Mounted sections were stained with mCherry, Draq5 or H&E.
Results and conclusions
This work was funded through grant support from the National Institutes of Health (R01 CA164789). B.P.M. was funded by NHLBI-sponsored Basic Cardiovascular Research Training Grant (5 T32 HL007284).
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