(141f) Influence of Protein Corona on Lipid Nanoparticle Uptake

Lipid nanoparticles (LNPs) are the most clinically advanced nonviral RNA-delivery vehicles, demonstrating successful delivery of mRNA-based vaccines such as the Moderna and Pfizer/BioNTech SARS-CoV-2 vaccines. Previous work to improve LNP potency and targeting capabilities has primarily focused on variations in LNP composition. Although this approach has led to some efficient LNP-mediated gene expression and delivery to tissues outside of the liver, we still do not understand the fundamental mechanisms that drive increased potency or organ- and cell-type-specific biodistribution. As these LNPs travel through biological tissues and compartments, biomolecules such as proteins spontaneously interact with the nanoparticles, forming an associated protein corona. Protein recruitment to the LNPs has been shown to play a role in enhanced delivery, yet protein corona formation on such soft, biomimetic nanoparticles remains poorly understood and is largely unexplored due to difficulties associated with isolating the adsorbed protein fraction. In this work, we have developed a quantitative, label-free mass spectrometry-based proteomics approach to probe the nano-bio interface of a novel class of LNPs for mRNA delivery. We have optimized a separation method that, for the first time, limits the contamination of endogenous biofluid nanoparticles in protein corona isolates from LNPs. With this method, we discovered proteins with a high affinity for the LNPs such as ApoE and C-reactive protein, in agreement with the minimal previous literature on such lipid-based carriers. Our data suggests that this method is a promising avenue for corona characterization. We apply our workflow to investigate how altering the LNP formulation – variations in helper lipid within the lipid nanoparticle – impacts the formation of the protein corona in various relevant delivery bioenvironments and explore the impact of these protein targets on expression of mRNA in HepG2 human liver cells. By understanding these fundamental protein-nanoparticle interactions, we aim to rationally tune the design of these mRNA-based biotechnologies for improved translation to clinical practice.