(356c) Protein Corona Formation on Lipid Nanoparticles Influences Delivery Efficiency of mRNA Cargo in Cells

Lipid nanoparticles (LNPs) are the most clinically advanced nonviral RNA delivery systems, exemplified by their success in the mRNA-based SARS-CoV-2 vaccines. Although formulation-based modifications have begun to improve tissue-specific delivery beyond the liver, the field still lacks a mechanistic understanding of how LNPs interact with biological environments to influence organ and cell-type targeting, uptake, and expression. In vivo, LNPs rapidly acquire a protein corona, a dynamic layer of adsorbed biomolecules that can redefine their biological identity and fate. However, systematic characterization of this corona has been hindered by technical challenges in selectively isolating soft nanoparticles from biological samples.

To address this challenge, we developed a label-free, quantitative proteomics workflow to study the protein corona formed on LNPs using continuous density gradient ultracentrifugation followed by mass spectrometry. This approach enables reproducible isolation and profiling of the LNP-associated protein corona while controlling for the presence of endogenous nanoparticles found in biological fluids, especially lipoprotein particles in blood. We applied this methodology to quantify proteins consistently enriched in the LNP corona including vitronectin, C-reactive protein, and alpha-2-macroglobulin. We next explored the impact of these corona proteins on cell uptake and mRNA expression in HepG2 human liver cells with a combination of confocal microscopy and flow cytometry. Notably, high levels of uptake did not correlate with increased mRNA expression, likely due to protein corona-mediated alterations in lysosomal trafficking.

In sum, we establish a framework for characterizing the protein corona on LNPs and other soft nanoparticles used in delivery applications. Our findings contribute to the growing evidence that biomolecular interactions greatly influence LNP delivery outcomes and underscore the need to consider the protein corona in the design of LNP-based therapeutics. By understanding these protein-nanoparticle interactions, we can tune the design of future mRNA-based biotechnologies for improved translation to clinical practice.