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Nature CommunicationsBioengineeringPeer-reviewed journal article

Protein corona formed on lipid nanoparticles compromises delivery efficiency of mRNA cargo

Elizabeth VokeiD, Mariah Lynn ArraliD, Henry John SquireiD, Teng-Jui LiniD, Lining ZhengiD, Roxana CoreasiD, Alison LuiiD, Anthony T. IavaroneiD, Rebecca L. PinalsiD, Kathryn A. WhiteheadiD, Markita Patricia LandryiD

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Abstract

Lipid nanoparticles (LNPs) are the most clinically advanced nonviral RNA-delivery vehicles, though challenges remain in fully understanding how LNPs interact with biological systems. In vivo, proteins form an associated corona on LNPs that redefines their physicochemical properties and influences delivery outcomes. Despite its importance, the LNP protein corona is challenging to study owing to the technical difficulty of selectively recovering soft nanoparticles from biological samples. Herein, we developed a quantitative, label-free mass spectrometry-based proteomics approach to characterize the protein corona on LNPs. Critically, this protein corona isolation workflow avoids artifacts introduced by the presence of endogenous nanoparticles in human biofluids. We applied continuous density gradient ultracentrifugation for protein-LNP complex isolation, with mass spectrometry for protein identification normalized to protein composition in the biofluid alone. With this approach, we quantify proteins consistently enriched in the LNP corona including vitronectin, C-reactive protein, and alpha-2-macroglobulin. We explore the impact of these corona proteins on cell uptake and mRNA expression in HepG2 human liver cells, and find that, surprisingly, increased levels of cell uptake do not correlate with increased mRNA expression in part likely due to protein corona-induced lysosomal trafficking of LNPs. Our results underscore the need to consider the protein corona in the design of LNP-based therapeutics. Abstract Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=58 SRC="FIGDIR/small/633942v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@2fef29org.highwire.dtl.DTLVardef@fe7613org.highwire.dtl.DTLVardef@1f9de01org.highwire.dtl.DTLVardef@15e5237_HPS_FORMAT_FIGEXP M_FIG C_FIG

In-depth summary

Claude Opus 5.5 · xhigh effort · 5 Nov 2026 · from the full text of version 1 (24 Jan 2025)

A 16-hour continuous iodixanol density gradient workflow isolates lipid nanoparticle protein coronas from human plasma and shows that vitronectin and C-reactive protein coronas impair mRNA delivery in liver cells.

Existing lipid nanoparticle (LNP) corona isolation methods either require surface modifications or co-isolate endogenous plasma lipoproteins and exosomes, obscuring which adsorbed proteins govern delivery. Separating native biofluid particles via continuous density gradients with plasma-control normalization reveals how specific hard-corona proteins alter cellular uptake, lysosomal trafficking, and mRNA translation.

Key results

  1. ShownA 16-hour continuous iodixanol density gradient ultracentrifugation workflow isolates colloidally stable protein-LNP complexes from pooled human plasma with minimal endogenous lipoprotein overlap.

    Approximately 68% of fluorescently tagged 306O10 LNPs localized in gradient fractions 2-6, whereas endogenous lipoprotein cholesterol localized primarily in fractions 5-10, yielding low peptide coefficients of variation (median CV% 11.8 for LNP and 19.0 for plasma control across 56 identified proteins). · Improved workflow for protein corona isolation…; Proteomic characterization of the protein corona…; Figure legends

  2. ShownNormalizing protein-LNP fractions against matched plasma-alone gradient fractions identifies 39 significantly enriched and 14 depleted corona proteins while avoiding bias toward high-abundance plasma proteins.

    Out of 56 quantified proteins, 53 differed significantly (FDR q < 0.05) between LNP and plasma-alone fractions; fold-change enrichment showed near-zero or negative correlation with unnormalized percent relative abundance, excluding abundant ApoA-I and ApoA-II while consistently enriching alpha-2-macroglobulin (A2M), C-reactive protein (CR), and vitronectin (VTN) across 3 independent batches. · Proteomic characterization of the protein corona…; Effect of proteins enriched in the LNP corona on…; Figure legends

  3. ShownPre-formed coronas of vitronectin (VTN) or C-reactive protein (CR) substantially reduce LNP-mediated luciferase mRNA expression in HepG2 liver cells without affecting cell viability.

    Pre-incubating LNPs with VTN or CR (0.05 ng mRNA:1 ng protein) reduced luminescence by approximately 50% and 90%, respectively, compared to no-corona LNPs after 24 hours (p <= 0.05 and p <= 0.01, n=3 biological replicates), with VTN showing dose-dependent inhibition above 0.005 mg/mL, while ApoE, A2M, or a four-protein mixture caused no significant change. · Protein-nanoparticle interactions impact…; Figure legends

  4. ShownPre-formed ApoE and VTN coronas increase cellular Cy5-mRNA association and outer membrane adhesion, while ApoE also elevates lysosomal co-localization, revealing a mismatch between cellular uptake and mRNA translation.

    Confocal microscopy showed 5-fold and 4-fold higher cellular Cy5-mRNA signal for ApoE-LNPs and VTN-LNPs vs bare LNPs, with 3.5% and 8.5% more signal retained in the eroded outer membrane region, respectively; ApoE-LNPs also exhibited a 5.8-fold increase in LysoTracker lysosomal co-localization per cell (n=4 biological replicates). · Protein-nanoparticle interactions impact…; Discussion; Figure legends

Methods, data and reproducibility

Incubated 306O10 lipid nanoparticles (LNPs) with pooled human plasma, separated protein-LNP complexes from endogenous plasma particles via 16-h, six-layer continuous iodixanol density gradient ultracentrifugation, and quantified corona proteins against plasma-alone fractions by label-free LC-MS/MS. Pre-formed coronas with top enriched proteins (VTN, CR, A2M, ApoE) on mRNA LNPs to assay expression and uptake in HepG2.

  • 306O10 lipid nanoparticles (LNPs) loaded with Cre recombinase, firefly luciferase, or Cy5-labeled luciferase mRNA incubated with pooled human blood plasma · human · n = 3 independent experimental batches with 3 technical replicates each
  • HepG2 human liver cells treated with LNPs carrying pre-formed single-protein or mixed-protein coronas · human · n = 15,000 cells/well; n=3-4 biological replicates and n=4 technical replicates
  • Escherichia coli chaperone protein ClpB internal standard for label-free LC-MS/MS quantification · E. coli · n = 50 fmol per 5 uL sample · P63284
Methods & tools
MassLynx, Progenesis QI for Proteomics, DAVID, KEGG, GraphPad Prism, Python
Software & environment
MassLynx (version 4.1), Progenesis QI for Proteomics (version 4.2), GraphPad Prism (v.10.2.3), Python (v3)
Reproducibility gaps
Raw LC-MS/MS proteomics datasets and public data repository accessions are not reported in the text.
Limitations (3)
  • The continuous density gradient ultracentrifugation workflow isolates high-affinity hard corona proteins that remain bound over 16 hours of centrifugation, leaving the transient and dynamic soft corona uncharacterized. · stated by the authors
  • Pooled gradient fractions 5-6 retain marginal overlap between LNPs and endogenous plasma lipoproteins, requiring normalization against a matched plasma-alone gradient control. · stated by the authors
  • Fluorescent lipid tags (lissamine rhodamine PE) used to track LNP fractions exhibited a minor secondary peak at higher gradient fractions, likely due to partial fluorophore dissociation from the nanoparticles. · stated by the authors

Read this if you design mRNA lipid nanoparticles or use mass spectrometry proteomics to characterize nanoparticle protein coronas and their impact on cellular delivery.

Citations
129
FWCI
38.23
Field percentile
top 1%

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