The Biomolecular Corona of Lipid Nanoparticles for Gene Therapy

Purpose: LNPs are clinically advanced vectors for siRNA, mRNA, and DNA delivery, but their in vivo behavior is strongly influenced by the biological environment. Upon administration, biomolecules adsorb to the LNP surface and form a “biomolecular corona,” which defines the particle’s biological identity, affects stability, clearance, biodistribution, and targeting, and remains poorly understood for LNPs.

Hypothesis: If LNPs contact biological fluids—blood, interstitial fluid, or lung surfactants—then a biomolecular corona forms on their surface, and this corona determines LNP stability, immune recognition, biodistribution, and cell targeting. Modulating the corona, or exploiting specific corona proteins such as ApoE, can enable tissue-specific gene delivery, including beyond the liver.

Aims: Summarize recent discoveries about the biomolecular corona of nanomedicines and translate them to LNPs for nucleic acid delivery. - Describe how LNP stability, integrity, systemic circulation, immune recognition, biodistribution, and targeting are influenced by the biological environment. - Use Onpattro as a case study of successful LNP development and the key role of ApoE in liver tropism. - Outline techniques to isolate and analyze the LNP corona, with advantages and drawbacks. - Discuss clinical implications and the potential of exploiting the corona as a targeting strategy for next-generation gene therapies.

Delivery system: Platform: lipid nanoparticles (LNPs) for gene therapy, typically composed of an ionizable cationic lipid, a phospholipid, cholesterol, and a PEG-lipid. - Payloads: siRNA, mRNA, DNA. - Key example: Onpattro (patisiran), an LNP-siRNA therapeutic for hereditary transthyretin-mediated amyloidosis. - Corona-forming biomolecules: serum albumin, complement C3, IgM, apolipoprotein E (ApoE), serotransferrin, alpha-2-macroglobulin, IgHA1, ApoA-IV, ApoA-I, clusterin, and others. - Targeting/functionalization: endogenous targeting via corona proteins, especially ApoE for hepatocytes; PEG-lipid chain length (C14 vs C18) and density; detachable PEG; SORT lipids for organ tropism; ligand grafting is discussed but can be masked by the corona.

Approach: Review and synthesis of nanomedicine and LNP literature. Cited models include in vitro cell studies, murine and non-human primate studies, and clinical data for Onpattro. The review discusses corona formation in blood, serum, plasma, and other biological fluids; disease- and species-specific corona effects; and techniques for corona isolation and analysis. As a review, it reports no primary experimental groups, n values, doses, or controls.

Key methods: No primary methods. The review discusses data generated by cited studies using: - Liquid chromatography–mass spectrometry (LC-MS) for corona protein identification. - Size exclusion chromatography (SEC), (ultra)centrifugation, density gradient ultracentrifugation, and magnetic separation for corona isolation. - Cross-linking and photoaffinity labeling for hard-corona capture. - Dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), electrophoretic light scattering (ELS/zeta potential), and (cryo-)TEM for physicochemical characterization. - NMR, fluorescence correlation spectroscopy (FCS), and surface plasmon resonance (SPR) for protein interactions. - FRET and radiolabeled lipids (³H-CHE, ¹⁴C-DSPC) for LNP integrity and lipid dissociation.

Key results: Top 10 corona proteins of AmBisome after incubation in 100% human serum: serum albumin 166,624 ppm, complement C3 125,283 ppm, IgM 42,156 ppm, ApoE 38,054 ppm, serotransferrin 20,767 ppm, alpha-2-macroglobulin 18,900 ppm, IgHA1 11,451 ppm, ApoA-IV 10,705 ppm, ApoA-I 9,582 ppm, clusterin 6,738 ppm. - Onpattro liver tropism depends on ApoE adsorption to the LNP corona, enabling recognition by hepatocyte LDL receptors. Without ApoE, transfection potency is lost. - PEG-lipid chain length strongly affects circulation and biodistribution: increasing PEG-C18 from 1.5 to 5 mol% improved circulation times up to ~10 h, whereas shorter PEG-C14 did not show the same effect. The lowest PEG concentration influencing pharmacokinetics was 1.5 mol%. - Detachable PEG-DMG (C14) exchanges with blood proteins such as ApoE, promoting hepatocyte uptake and gene silencing. - Corona composition can mask targeting ligands; the presence of a ligand does not guarantee targeting because corona proteins may interfere. - Corona “fingerprints” can predict nanoparticle–cell interactions better than size, charge, or aggregation state alone in some studies. - SORT lipids shift LNP tropism from liver to spleen and then lung, possibly via changes in apparent pKa and corona composition. - Only a small fraction of corona proteins may enhance LNP–cell association; many corona proteins are not in native form and cannot mediate targeting.

Interpretation: The authors conclude that the biomolecular corona defines the biological identity of LNPs and is a major determinant of their stability, clearance, biodistribution, and targeting. Onpattro’s success demonstrates that corona proteins—especially ApoE—can be exploited for endogenous targeting. They argue that LNP design, characterization, and testing must account for corona formation, and that understanding and modulating the corona will be key to developing next-generation gene therapies beyond the liver.

Limitations: Review article; no primary data, effect sizes, n values, doses, or controls. - Only a limited number of studies have specifically analyzed the corona of LNPs for nucleic acid delivery. - Corona composition is highly dependent on biological fluid type, concentration, temperature, species, disease state, and individual patient, complicating generalization. - In vitro LNP characterization is often performed in non-physiological conditions (e.g., 10% FBS, bovine serum), poorly predicting in vivo behavior. - Corona isolation techniques have major limitations: SEC and centrifugation cannot easily separate LNPs from lipoproteins or extracellular vesicles; magnetic separation requires modified LNPs; cross-linking/photoaffinity labeling may alter the corona. - Soft corona is often lost during washing and is poorly characterized. - Corona can mask targeting ligands, and ligand-mediated targeting often fails to improve efficacy. - PEGylation has drawbacks, including reduced cellular uptake, complement activation, hypersensitivity, and anti-PEG antibodies. - A “one size fits all” nanomedicine approach is inadequate; personalized corona considerations are needed.