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

Ionizable Lipid Nanoparticles (LNP) 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 focused ultrasound (FUS) 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. Two types of LNPs containing different ionizable lipids (SM-102 and Lipid-14) were compared and both achieved successful brain delivery following FUS-mediated BBB opening. In a glioblastoma syngeneic mouse model, where the BBB remains largely intact under baseline conditions, siRNA-Cy5-LNP was successfully delivered. A frequency of 850 kHz and 180 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 h. 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.