Automated high-throughput preparation and characterization of oligonucleotide-loaded lipid nanoparticles

[Display omitted] Lipid nanoparticles (LNPs) are increasingly employed to improve delivery efficiency and therapeutic efficacy of nucleic acids. Various formulation parameters can affect the quality attributes of these nanoparticle formulations, but currently there is a lack of systemic screening approaches to address this challenge. Here, we developed an automated high-throughput screening (HTS) workflow for streamline preparation and analytical characterization of LNPs loaded with antisense oligonucleotides (ASOs) in a full 96-well plate within 3 hrs. ASO-loaded LNPs were formulated by an automated solvent-injection method using a robotic liquid handler, and assessed for particle size distribution, encapsulation efficiency, and stability with different formulation compositions and ASO loadings. Results indicated that the PEGylated lipid content significantly affected the particle size distribution, while the ionizable lipid / ASO charge ratio impacted the encapsulation efficiency of ASOs. Furthermore, results from our HTS approach correlated with those from the state-of-the-art scale-up method using a microfluidic formulator, therefore opening up a new avenue for robust formulation development and design of experiment methods, while reducing material usage by 10 folds, improving analytical outputs and accumulation of information by 100 folds.