Intracellular Trafficking of Cationic Bicelles and siRNA Cargo in an <i>In Vitro</i> Blood–Brain Barrier Model

Blood-brain barrier dysfunction (BBB), triggered by inflammatory changes in the periphery and the brain, is an early event in the pathogenesis of Alzheimer's disease (AD). Therapeutic strategies that restore BBB integrity and function by targeting inflammatory signaling hold great promise for halting the progression of AD. siRNA-based therapeutics offer a precise means of silencing proinflammatory targets, but the efficient and targeted delivery of siRNA to the brain endothelium remains a significant challenge. To address this, we formulated cationic bicelles (DPPC/DC₇PC/DOTAP, molar ratio 63.8/25.0/11.2) to deliver siRNA to the BBB. In this study, we investigated the pathways of endocytic uptake and intracellular trafficking for siRNA-loaded bicelles in a human <i>in vitro</i> BBB model. Using pharmacological inhibitors and targeted siRNA knockdowns, we demonstrated that bicelles are internalized via multiple endocytic mechanisms, including clathrin-mediated, caveolin-mediated, dynamin-independent, and lipid raft-associated mechanisms. Using fluorescence microscopy, we showed that the bicelles and siRNA are internalized together and then trafficked into distinct intracellular compartments. Bicelles accumulated in the early and late endosomes while siRNA accumulated outside of the endolysosomal system. A three-compartmental model was used to quantitatively describe bicelle uptake and trafficking over the course of 12 h. These findings significantly advance our understanding of how the cationic bicelles deliver siRNA to the BBB endothelium and provide a mechanistic foundation for developing next-generation siRNA therapeutics aimed at restoring neurovascular health in Alzheimer's patients.