Applications of nanoparticles for chemistry, biochemistry and biomedicine require a detailed understanding of the nature of the nanoparticles and of their surface interactions with both functionalizing organic groups and biological environments. Here we present ongoing studies of the nature and surface interactions of iron oxide nanoparticles (IONPs) intended for magnetic resonance imaging detection and hyperthermia treatment.

The IONPs are synthesized by a novel low temperature aqueous route devoid of surfactant chemistry or capping agents. In addition to routine characterization of the IONPs by TEM, XRD, FTIR, XPS, DLS, and magnetic characterization, we use aberration-corrected high resolution TEM to monitor their structural quality. Because of an observed sensitivity to the electron beam at a 200 kV high tension (HT), we use a HT of 80 kV, with effects of chromatic aberration reduced by implementing a monochromatic “rainbow” illumination in the incident beam [1]. This imaging allows observations of minute changes in surface structural quality. Initially formed particles are rounded, showing signs of slight structural disorder in the first atomic layers at the surface (Fig. 1). In contrast, aged particles have atomically sharp structural ordering at surfaces which are markedly more faceted (Fig. 2). No beam damage effects are observed other than the hopping of atoms on surface ledge sites.

For bioengineering, it is critical to understand how molecules and proteins interact with these IONPs. Beginning with the former, the particles are functionalized with folic acid, the molecule most often used for “nonspecific” targeting. Using the same monochromated, Cs-corrected imaging conditions as above, in Figure 3 we demonstrate the ability to image ligand attachment and surface coverage, similarly to Lee et al. [2]. The folic acid molecules show phase contrast with definition down to about 2 Å; this phase contrast will be compared to simulations for better interpretability. Ligands are observed to form two or three strand thick shells around the surfaces of the IONPs. Using fast frame acquisition, very specific effects of electron irradiation are recorded: the molecules wriggle under the beam, often with one end appearing to detach and reattach to the surface of the IONP. This behavior is explained in terms of relative bonding potentials for either the amino acid or the carboxylic ends of the molecules absorbed to the IONP surfaces.

Once the IONPs are injected into a biological environment it is known that any such functionalization is rapidly replaced by a surface coverage of proteins – the protein corona – which then dictates how the IONPs interact with this environment [3]. To understand this biomedically-important process, for the first time, we obtain in vivo conditions which create a protein corona whose nature mimics that observed for in vitro studies. As well as again using phase contrast imaging of dry samples, we also study the morphology of this corona by simple negative-staining to create contrast between the protein and the carbon support film. Contrary to the commonly held view that the proteins make a uniform encapsulating layer around nanoparticles, their coverage is distinctly patchy (Fig. 4); consistent with the attaching proteins having widely varying sizes. As a next step HAADF STEM tomography will be used to map the 3D morphology of this non-uniform coronal coverage.

With research ongoing on these IONPs for “magnetotheranostics”, the other main perspective of this work is to pursue imaging of such interactions in representative liquid environments, particularly using high resolution cryo-TEM imaging to monitor better the nature of organic functionalization in an aqueous environment.

References and Acknowledgements

[1] P.C. Tiemeijer et al., Ultramicrosc. 114 (2012) 72–81.

[2] Z. Lee et al., Nano Lett. 9 (2009) 3365–3369.

[3] M.P. Monopoli et al., Nature Nano. 7 (2012) 779–786.

The authors acknowledge funding from Nano-Tera.ch, project Magnetotheranostics number 530 627.

Figures:

Figure 1. Aberration-corrected phase contrast image of first-forming IONPs by novel low temperature aqueous route. Slight disorder is observed in the surface crystalline structure of the central particle. Background image intensity varies because of the non-uniform nature of the highly monochromatic "rainbow" illumination. Scale bar 5 nm.

Figure 2. Aberration-corrected phase contrast image of more aged IONP, showing a tendency to form a cubic morphology with a highly ordered crystalline atomic lattice at the surface. Scale bar 5 nm.

Figure 3. Aberration-corrected phase contrast image of IONPs functionalized with folic acid molecules. The carbon backbones of the folic acid molecules are clearly seen as a few strands of "ribbons" extending around the nanoparticle surfaces. The molecules show phase contrast definition down to ~ 2 Å in length. Scale bar 5 nm.

Figure 4. Example of bright field TEM image of negatively-stained IONPs after exposure to biological plasma and removal of soft protein to leave hard protein corona. This hard protein corona is observed as patchy regions of bright contrast around the darker IONPs, as for example indicated by the two arrows. Scale bar 50 nm.

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EMC Abstracts - https://emc-proceedings.com/abstract/imaging-interactions-of-iron-oxide-nanoparticles-with-organic-ligands-and-the-biological-environment/