Low-frequency ultrasound-mediated blood-brain barrier opening enables non-invasive lipid nanoparticle RNA delivery to glioblastoma

ABSTRACT Ionizable Lipid Nanoparticles (LNPs) are an FDA-approved non-viral RNA delivery system, though their use for brain therapy is restricted by the blood-brain barrier (BBB). Focused ultrasound combined with microbubbles can disrupt the BBB, but delivering large particles requires balancing increased peak negative pressures while maintaining microvascular integrity. Herein, we optimized low-frequency ultrasound parameters to induce high-amplitude microbubble oscillations, enabling the safe delivery of LNPs across the BBB. First, BBB opening was assessed at different frequencies (850, 250, and 80 kHz) and pressures by monitoring the extravasation of Evans blue (~1 kDa). Next, the delivery of 4, 70, and 150 kDa Dextrans, LNPs entrapping Cy5-siRNAs (~70 nm in diameter), and LNPs entrapping mRNA (~100 nm in diameter) was evaluated via microscopy and bioluminescence. In a glioblastoma syngeneic mouse model, siRNA-Cy5-LNP was successfully delivered. A frequency of 850 kHz and 125 kPa pressure induced safe BBB opening, enabling delivery of both small molecules and LNPs. In healthy brains, LNP entrapping siRNAs delivery increased 10-fold compared to controls, and LNPs with mRNAs showed a 12-fold increase in bioluminescence after 24 hours. In glioblastoma tumors, LNPs with siRNAs delivery resulted in a 6.7-fold increase in fluorescence. This study paves the way for non-invasive LNP delivery to the brain, offering a versatile platform for brain therapies. Competing Interest Statement D.P. receives licensing fees (to patents on which he was an inventor) from, invested in, consults (or on scientific advisory boards or boards of directors) for, lectured (and received a fee), or conducts sponsored research at TAU for the following entities: ART Biosciences, BioNtech SE, Earli Inc., Geneditor Biologics Inc., Kernal Biologics, Newphase Ltd., NeoVac Ltd., RiboX Therapeutics, SirTLabs Corporation, Teva Pharmaceuticals Inc. All other authors declare no competing financial interests.