Gene therapy is a promising approach for correcting acquired or inherited brain diseases, nevertheless, faces a challenge in effectively delivering nucleic acids to the brain. Ionizable lipid nanoparticles (LNPs) are commonly used as delivery systems, however they are often screened in in vitro settings which poorly replicates in vivo biological barriers. Here, we used a high-throughput in vivo and in vitro screening methods to assess a library of LNPs for nucleic acid delivery to neurons and brain tissue. LNPs were formulated via microfluidic mixing with different helper lipid and molar ratios, each containing a unique barcode DNA (b-DNA). LNPs were characterized and pooled for intravenous injection in C57BL/6 mice, and their biodistribution was assessed via next-generation sequencing. The top-performing LNPs, which exhibited higher b-DNA delivery to the brain, were further assessed for transfection efficiency in primary neurons using pDNA and mRNA. We established concentration-response curves, monitored protein expression overtime, and performed cell viability assays. Our results showed that DOPE-based LNPs outperformed other formulations in brain delivery and neuronal transfection. Additionally, altering the ionizable lipid in our top formulation to FDA-approved options did not improve neuronal transfection efficiency. Finally, our identified top performing LNP4 was able to induce luciferase expression in brain after intravenous administration. No signs of neurological damage, inflammation or behavioral impairments were observed. Our results demonstrate that our LNPs efficiently deliver nucleic acids to neurons and the brain, while our screening strategy accelerates the design of LNPs for brain gene therapy.
