The blood-brain barrier (BBB) remains a major challenge in the treatment of central nervous system (CNS) diseases. Lipid nanoparticles (LNPs) have emerged as promising carriers for small interfering RNA (siRNA) delivery to the brain. However, their ability to penetrate the BBB remains limited, with typically <1 % of the injected dose reaching the brain. Among various design strategies, PEGylation of lipids has been widely used to improve nanoparticle stability, prolong circulation time, and modulate tissue distribution. Notably, the length of the Polyethylene Glycol-lipid (PEG-lipid) anchor has been shown to significantly affect LNP pharmacokinetics and tissue distribution, particularly in hepatic and tumor tissues. However, its impact on brain-targeted delivery remains poorly understood. Here, we systematically investigated the impact of different PEG-lipid anchor lengths on the brain delivery performance of LNPs. Our results demonstrate that LNPs with longer PEG-lipid anchors exhibit enhanced BBB penetration, increased brain accumulation, and reduced hepatic uptake following intravenous administration. Further investigation using a BBB model revealed that LNPs with longer anchors exhibited enhanced exocytosis in brain endothelial cells, resulting in superior transcytosis and the highest BBB crossing efficiency. To validate these findings under pathophysiological conditions, we evaluated the formulations in an orthotopic GBM mouse model. Notably, LNPs with longer PEG-lipid anchors achieved the most effective siRNA-mediated knockdown in vivo. These findings identify PEG-lipid anchor length as a critical and tunable design parameter for brain-targeted siRNA delivery, offering new insights for optimizing LNP performance in CNS therapeutics. STATEMENT OF SIGNIFICANCE: 1. Tuning the Polyethylene Glycol (PEG)-lipid anchor length of Lipid Nanoparticles enhances the efficiency of brain-targeted siRNA delivery. 2. Longer PEG-lipid anchors enhance BBB penetration and brain accumulation. 3. Enhanced BBB transport by longer anchors is mediated through increased transcytosis. 4. Longer PEG-lipid anchors enable stronger gene silencing in glioblastoma models.
