A Protein Corona Adsorbed to a Bacterial Magnetosome Affects Its Cellular Uptake

Abstract

Purpose: It is well known that when exposed to human blood plasma, nanoparticles are predominantly coated by a layer of proteins, forming a corona that will mediate the subsequent cell interactions. Magnetosomes are protein-rich membrane nanoparticles which are synthesized by magnetic bacteria; these have gained a lot of attention owing to their unique magnetic and biochemical characteristics. Nevertheless, whether bacterial magnetosomes have a corona after interacting with the plasma, and how such a corona affects nanoparticle-cell interactions is yet to be elucidated. The aim of this study was to characterize corona formation around a bacterial magnetosome and to assess the functional consequences.

Methods: Magnetosomes were isolated from the magnetotactic bacteria, M. gryphiswaldense (MSR-1). Size, morphology, and zeta potential were measured by transmission electron microscopy and dynamic light scattering. A quantitative characterization of plasma corona proteins was performed using LC-MS/MS. Protein absorption was further examined by circular dichroism and the effect of the corona on cellular uptake was investigated by microscopy and spectroscopy.

Results: Various serum proteins were found to be selectively adsorbed on the surface of the bacterial magnetosomes following plasma exposure, forming a corona. Compared to the pristine magnetosomes, the acquired corona promoted efficient cellular uptake by human vascular endothelial cells. Using a protein-interaction prediction method, we identified cell surface receptors that could potentially associate with abundant corona components. Of these, one abundant corona protein, ApoE, may be responsible for internalization of the magnetosome-corona complex through LDL receptor-mediated internalization.

Conclusion: Our findings provide clues as to the physiological response to magnetosomes and also reveal the corona composition of this membrane-coated nanomaterial after exposure to blood plasma.

Keywords: LC-MS/MS; biogenic magnetic nanoparticle; cellular interaction; cellular uptake.

Figure 1

Characterization of magnetosome and PEG-Fe₃O₄. (A) TEM images of typical magnetosome and PEG-Fe₃O₄ particles. Scale bars, 200 nm. (B) SDS-PAGE image of plasma corona proteins on particles.

Figure 2

Overview of identified corona proteins postprocessing.

Figure 3

Protein plasma concentrations as well as iBAQ intensities of corona proteins identified by LC-MS/MS. Identified magnetosome corona proteins (A) and the most 20 abundant corona proteins (B) iBAQ intensities and relative concentrations in plasma. (C) Plasma protein concentrations from Plasma Proteome Database (PPD). Y-axis represents different proteins and x-axis is different concentrations observed for each protein. As in PPD, one protein usually has several concentrations. Intensities in log 2 scales are indicated by different colors. (D) Overlaps of proteins identifications from magnetosome or PEG-Fe₃O₄ incubated with plasma by LC-MS/MS. Venn plot shows the most 20 abundant corona proteins and top 20 abundant plasma proteins. (E) Comparison of the top 20 abundant proteins (in gene names) relative abundance in the magnetosome and PEG-Fe₃O₄ corona.

Figure 4

Characterization of the top 20 abundant plasma proteins identified on magnetosomes by LC-MS/MS. Classifications according to their isoelectric point (pI) (A), molecular weight (MW) (B) or GRAVY values (C). Amino acid compositions are shown in (D). (E) CD spectra of top three magnetosome corona proteins alone (0.02 mg/mL, black line) or in the presence of magnetosome (MAG) or PEG-Fe₃O₄ (Fe₃O₄) with different concentrations (0.02, 0.01 and 0.0033 mg/mL Fe concentration, represented by yellow, blue and green dashed lines, respectively). Spectra are shown in mean residue molar ellipticity (Mol.Ellip.). Black dashed lines correspond to spectral peaks at 208 and 222 nm (principle α-helix peak).

Figure 5

Bioinformatics analysis of magnetosome-corona protein. (A) GO molecular function enrichment of the top 20 abundant proteins. (p < 0.05) Each protein was represented by a certain gene name; surrounding color shows the relative abundance. Details are given in Table S4. (B) Corona protein-interaction network. Interacted receptors with membrane location were obtained from BioGRID, IMEx, IntAct, MINT and UniProt databases. Abundant corona proteins are colored by their relative abundance.

Figure 6

Internalization of magnetosome-corona complexes by EC cells. (A) Enhancement of magnetosome cell uptake in the presence of protein corona. Prussian blue staining and intracellular iron concentrations of EC cells (control) incubated with magnetosomes (10 μg/mL Fe concentration) with or without corona for 3 h. Scale bars 20 μm. **p<0.01 (B) Confocal images of EC cells treated with magnetosomes (20 μg/mL Fe concentration) with or without corona for 5 h. Large colocalization of LDL receptors (green) and magnetosomes (red) signals were found in the presence of corona. Scale bars 10 μm.