Abstract
Lipid nanoparticles (LNPs) are gaining traction in the field of nucleic acid delivery following the success of two mRNA vaccines against COVID-19. As one of the constituent lipids on LNP surfaces, PEGylated lipids (PEG-lipids) play an important role in defining LNP physicochemical properties and biological interactions. Previous studies indicate that LNP performance is modulated by tuning PEG-lipid parameters including PEG size and architecture, carbon tail type and length, as well as the PEG-lipid molar ratio in LNPs. Owing to these numerous degrees of freedom, a high-throughput approach is necessary to fully understand LNP behavioral trends over a broad range of PEG-lipid variables. To this end, we report a low-volume, automated, high-throughput screening (HTS) workflow for the preparation, characterization, and in vitro assessment of LNPs loaded with a therapeutic antisense oligonucleotide (ASO). A library of 54 ASO-LNP formulations with distinct PEG-lipid compositions was prepared using a liquid handling robot and assessed for their physiochemical properties as well as gene silencing efficacy in murine cortical neurons. Our results show that the molar ratio of anionic PEG-lipid in LNPs regulates particle size and PEG-lipid carbon tail length controls ASO-LNP gene silencing activity. ASO-LNPs formulated using PEG-lipids with optimal carbon tail lengths achieved up to 5-fold lower mRNA expression in neurons as compared to naked ASO. Representative ASO-LNP formulations were further characterized using dose–response curves and small-angle X-ray scattering to understand structure–activity relationships. Identified hits were also tested for efficacy in primary murine microglia and were scaled-up using a microfluidic formulation technique, demonstrating a smooth translation of ASO-LNP properties and in vitro efficacy. The reported HTS workflow can be used to screen additional multivariate parameters of LNPs with significant time and material savings, therefore guiding the selection and scale-up of optimal formulations for nucleic acid delivery to a variety of cellular targets.
Highlights
Lipid nanoparticles (LNPs) are gaining traction in the field of nucleic acid delivery following the success of two mRNA vaccines against COVID-19
This study presents the rst systematic high-throughput screening (HTS) approach that correlates the impact of PEGylating agents on LNP structure and subsequent in vitro antisense oligonucleotide (ASO) delivery efficacy in multiple primary brain cell types
In our HTS lipid library design, DLin-MC3-DMA (MC3), DSPC, and cholesterol were chosen as the constituent lipids for all ASO-LNP formulations, while the PEG-lipid type and content
Summary
Lipid nanoparticles (LNPs) are gaining traction in the field of nucleic acid delivery following the success of two mRNA vaccines against COVID-19. Owing to these numerous degrees of freedom, a high-throughput approach is necessary to fully understand LNP behavioral trends over a broad range of PEG-lipid variables To this end, we report a low-volume, automated, high-throughput screening (HTS) workflow for the preparation, characterization, and in vitro assessment of LNPs loaded with a therapeutic antisense oligonucleotide (ASO). PEGlipids control particle size distributions during the selfassembly of LNPs and prevent aggregation.[32,33,34,35] PEG prolongs in vivo LNP circulation time by acting as a steric barrier to the adsorption of plasma proteins.[22,36,37,38] While extending half-life increases therapeutic exposure, the hydrophilic PEG corona may hinder interactions between the particle surface and the lipophilic cell membrane, resulting in poor cellular internalization.[37] PEG can block transport protein binding, which is essential for LNP cellular internalization via receptor-mediated endocytosis.[39] To overcome the PEG dilemma and achieve optimal intracellular delivery, it is necessary to understand how PEG-lipid characteristics regulate LNP size, structure, and subsequent cargo delivery. Due to the wide screening space for PEG-lipids, a comprehensive study encompassing multiple PEG-lipid types and variables is necessary to empirically understand how PEG-lipids impact LNP structure and function
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