Proteomics Analysis Reveals Distinct Corona Composition on Magnetic Nanoparticles with Different Surface Coatings: Implications for Interactions with Primary Human Macrophages

<div><p>Superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as promising contrast agents for magnetic resonance imaging. The influence of different surface coatings on the biocompatibility of SPIONs has been addressed, but the potential impact of the so-called corona of adsorbed proteins on the surface of SPIONs on their biological behavior is less well studied. Here, we determined the composition of the plasma protein corona on silica-coated <i>versus</i> dextran-coated SPIONs using mass spectrometry-based proteomics approaches. Notably, gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed distinct protein corona compositions for the two different SPIONs. Relaxivity of silica-coated SPIONs was modulated by the presence of a protein corona. Moreover, the viability of primary human monocyte-derived macrophages was influenced by the protein corona on silica-coated, but not dextran-coated SPIONs, and the protein corona promoted cellular uptake of silica-coated SPIONs, but did not affect internalization of dextran-coated SPIONs.</p></div

Plasma protein corona composition on CSNP.

<p>Classification of identified corona proteins according to differentially enriched Gene Ontology (GO) categories and KEGG pathways. The relative abundances of the proteins, as a percentage of total estimated protein abundance in the sample, are shown and each replicate is plotted separately. A significant enrichment of plasma proteins absorbed on the CSNP surface involved (A) KEGG Complement and coagulation cascades pathway, (B) GO Regulation of coagulation, (C) GO Negative regulation of coagulation, (D) GO Positive regulation of coagulation, (E) GO Regulation of fibrinolysis, (F) GO Fibrinogen complex, (G) GO Heparin binding, (H) GO Regulation of defense response, and (I) GO Lipid biosynthetic process. For further details, refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129008#pone.0129008.s009" target="_blank">S3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129008#pone.0129008.s011" target="_blank">S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129008#pone.0129008.s012" target="_blank">S6</a> Tables.</p

Relaxivity properties of SPIONs with/without plasma corona

<p>Relaxivity values are measured at 20 MHz (0.47 T) and 60 MHz (1.41 T) in PBS (37°C).</p><p>^aResults presented as mean values ± SD, n = 3.</p><p>^bCSNP@protein corona particles are unstable over the measurements time at 60 MHz.</p><p>Relaxivity properties of SPIONs with/without plasma corona</p

Most abundant CSNP nanoparticle enriched proteins

<p>Uniprot = Uniprot accession, AAs = Number of Amino acids in the protein, MW.kDa. = molecular weight of the protein (calculated), calc.pI = protein isoelectric point (calculated), Symbol = Official gene symbol, GeneName = Official gene name, log2FC = log2 transformed fold‐change relative to plasma, CSNP% = relative abundance of the protein in the CSNP corona, Nmag% = NMag abundance, Plasma% = plasma abundance, FDR q‐value = false discovery rate corrected p‐value for the difference relative to plasma, CSNP = core shell nano particles; Nmag = nanomag‐D‐spio. Plasma = crude plasma (control).</p><p>Most abundant CSNP nanoparticle enriched proteins</p

Distinct plasma protein corona composition on SPIONs with different surface coating.

<p>Cluster analysis of SPION-bound plasma proteins <i>versus</i> plasma proteins. Red color denotes counts higher than average when compared to other samples. Blue denotes counts lower than average (refer to legend top right corner). Clusters are numbered 1–5 (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129008#pone.0129008.s010" target="_blank">S4 Table</a>). Clusters 2 and 5 contain proteins that are enriched in the CSNP corona. Cluster 1 proteins are enriched in the nanomag-D-spio corona and clusters 3–4 contain plasma-enriched proteins. For gene ontology (GO) enrichment analysis of the corona-specific clusters, refer to Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129008#pone.0129008.g005" target="_blank">5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129008#pone.0129008.s005" target="_blank">S5</a>.</p

Most abundant nanomag-D-spio enriched proteins

<p>Uniprot = Uniprot accession, AAs = Number of Amino acids in the protein, MW.kDa. = molecular weight of the protein (calculated), calc.pI = protein isoelectric point (calculated), Symbol = Official gene symbol, GeneName = Official gene name, log2FC = log2 transformed fold‐change relative to plasma, CSNP% = relative abundance of the protein in the CSNP corona, Nmag% = NMag abundance, Plasma% = plasma abundance, FDR q‐value = false discovery rate corrected p‐value for the difference relative to plasma, CSNP = core shell nano particles; Nmag = nanomag‐D‐spio. Plasma = crude plasma (control).</p><p>Most abundant nanomag-D-spio enriched proteins</p

Biocompatibility assessment of SPIONs with or without a ‘hard’ corona of plasma proteins.

<p>Human monocyte-derived macrophages were exposed for 2 h (A, D), 6 h (B, E) or 24 h (C, F) to the indicated doses (μL/mL) of CSNP or CSNP + protein corona (A-C), or to nanomag-D-spio or nanomag-D-spio + protein corona (D-F). Macrophage viability was determined using the MTT assay. Cells were cultured in the absence of FBS to exclude any confounding effects of serum proteins. Results are presented as % mitochondrial function (mean values ± S.D.) from four independent experiments using cells isolated from healthy human donors. Statistical analysis was performed using Tukey post-hoc test following one way ANOVA (**p<0.01, ***p<0.001).</p

Acetate-Induced Disassembly of Spherical Iron Oxide Nanoparticle Clusters into Monodispersed Core–Shell Structures upon Nanoemulsion Fusion

It has been long known that the physical encapsulation of oleic acid-capped iron oxide nanoparticles (OA–IONPs) with the cetyltrimethylammonium (CTA⁺) surfactant induces the formation of spherical iron oxide nanoparticle clusters (IONPCs). However, the behavior and functional properties of IONPCs in chemical reactions have been largely neglected and are still not well-understood. Herein, we report an unconventional ligand-exchange function of IONPCs activated when dispersed in an ethyl acetate/acetate buffer system. The ligand exchange can successfully transform hydrophobic OA–IONP building blocks of IONPCs into highly hydrophilic, acetate-capped iron oxide nanoparticles (Ac–IONPs). More importantly, we demonstrate that the addition of silica precursors (tetraethyl orthosilicate and 3-aminopropyltriethoxysilane) to the acetate/oleate ligand-exchange reaction of the IONPs induces the disassembly of the IONPCs into monodispersed iron oxide–acetate–silica core–shell–shell (IONPs@acetate@SiO₂) nanoparticles. Our observations evidence that the formation of IONPs@acetate@SiO₂ nanoparticles is initiated by a unique micellar fusion mechanism between the Pickering-type emulsions of IONPCs and nanoemulsions of silica precursors formed under ethyl acetate buffered conditions. A dynamic rearrangement of the CTA⁺–oleate bilayer on the IONPC surfaces is proposed to be responsible for the templating process of the silica shells around the individual IONPs. In comparison to previously reported methods in the literature, our work provides a much more detailed experimental evidence of the silica-coating mechanism in a nanoemulsion system. Overall, ethyl acetate is proven to be a very efficient agent for an effortless preparation of monodispersed IONPs@acetate@SiO₂ and hydrophilic Ac–IONPs from IONPCs