AN AUTOMATED AND BARCODED PLATFORM FOR SCALABLE IN VIVO SCREENING OF BRAIN-TARGETED LIPID NANOPARTICLES

The blood–brain barrier (BBB) is a major obstacle to delivering biologics and nucleic acid therapeutics to the central nervous system. Although lipid nanoparticles (LNPs) are a clinically validated non‑viral delivery platform, their intrinsic BBB permeability is limited. Functionalizing LNPs with targeting peptides that engage receptor‑mediated transcytosis pathways, such as the transferrin receptor (TfR), offers a path to enhanced brain delivery. However, systematic in vivo evaluation of peptide‑conjugated LNPs (pLNPs) is restricted by low‑throughput formulation and screening approaches.
To address this, we developed an integrated high‑throughput platform for large‑scale production and in vivo screening of pLNP libraries. Automated workflows using Biomek i7, Biomek FX Span‑8, and BioTek MultiFlo FX enabled reproducible assembly across diverse formulation parameters, including lipid composition, PEG‑lipid chemistry, mixing speeds, and phase volumes. Plate‑based dynamic light scattering and automated Ribogreen assays provided rapid characterization of particle size, polydispersity, and encapsulation efficiency. A DNA barcoding strategy enabled multiplexed in vivo assessment of BBB penetration and biodistribution.
This workflow produced up to 1,000 formulations per hour while minimizing material use and operator variability and LNPs exhibited consistent physicochemical properties. For in vivo validation, a TfR‑targeted pLNP formulation carrying 96 barcodes was systemically administered, and barcode recovery from brain and liver revealed distinct tissue‑specific delivery profiles. This scalable platform accelerates the discovery of LNP design features and peptide ligands that enhance BBB transport for next‑generation CNS drug delivery.