Core material and surface chemistry of Layer-by-Layer (LbL) nanoparticles independently direct uptake, transport, and trafficking in preclinical blood-brain barrier (BBB) models

Development of new treatments for neurological disorders, especially brain tumors and neurodegenerative diseases, is hampered by poor accumulation of new therapeutic candidates in the brain. Drug carrying nanoparticles are a promising strategy to deliver therapeutics, but there is a major need to understand interactions between nanomaterials and the cells of the blood-brain barrier (BBB), and to what degree these interactions can be predicted by preclinical models. Here, we use a library of eighteen layer-by-layer electrostatically assembled nanoparticles (LbL-NPs) to independently assess the impact of nanoparticle core stiffness and surface chemistry on in vitro uptake and transport in three common assays, as well as intracellular trafficking in hCMEC/D3 endothelial cells. We demonstrate that nanoparticle core stiffness impacts the magnitude of material transported, while surface chemistry influences how the nanoparticles are trafficked within the cell. Finally, we demonstrate that these factors similarly dictate in vivo BBB transport using intravital imaging through cranial windows in mice, and we discover that a hyaluronic acid surface chemistry provides an unpredicted boost to transport. Taken together, these findings highlight the importance of considering factors such as assay geometry, nanomaterial labelling strategies, and fluid flow in designing preclinical assays to improve nanoparticle screening throughput for drug delivery to the brain. ### Competing Interest Statement P.T.H. is a co-founder and member of the board of LayerBio, a member of the Scientific Advisory Board of Moderna, and a member of the Board of Alector, Advanced Chemotherapy Technologies, and Burroughs-Wellcome Fund. All other authors report no competing interests.