Esterified Cholesterol Conjugates Enable LNP‐Mediated mRNA Delivery to the Blood–Brain Barrier Following Intravenous Administration

The blood-brain barrier (BBB) plays a critical role in maintaining central nervous system homeostasis by regulating molecular and cellular exchange between the brain and peripheral circulation. While most delivery strategies focus on crossing the BBB, targeting brain endothelial cells directly may offer a complementary and more controlled alternative for therapeutic intervention. This study presents a ligand-free lipid nanoparticle (LNP) platform that enables efficient mRNA delivery to the brain endothelium via systemic administration. A modular synthesis strategy was used to generate a chemically diverse library of cholesterol-based lipids. Screening of 486 LNPs identified an optimized four-component formulation consisting of C6O2B2 (novel cholesterol-based lipid), DODAP, DSPC, and DMG-PEG, which achieves high transfection efficiency in brain endothelial cells without the incorporation of free cholesterol or disruption of BBB integrity and function, such as those utilized in focused ultrasound (FUS) techniques. This BBB-targeting LNP platform avoids neuronal and glial transfection, thereby offering a noninvasive, and scalable approach for cerebrovascular gene delivery. The work establishes a new class of cholesterol-based lipids and provides a foundation for next-generation RNA therapeutics targeting BBB-associated diseases.