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, 10 (1), 261

ToF-SIMS 3D Imaging Unveils Important Insights on the Cellular Microenvironment During Biomineralization of Gold Nanostructures

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ToF-SIMS 3D Imaging Unveils Important Insights on the Cellular Microenvironment During Biomineralization of Gold Nanostructures

Ajay Vikram Singh et al. Sci Rep.

Abstract

The biomolecular imaging of cell-nanoparticle (NP) interactions using time-of-flight secondary ion mass spectrometry (ToF-SIMS) represents an evolving tool in nanotoxicology. In this study we present the three dimensional (3D) distribution of nanomaterials within biomolecular agglomerates using ToF-SIMS imaging. This novel approach was used to model the resistance of human alveolar A549 cells against gold (Au) ion toxicity through intra- and extracellular biomineralization. At low Au concentrations (≤1 mM HAuCl4) 3D-ToF-SIMS imaging reveals a homogenous intracellular distribution of Au-NPs in combination with polydisperse spherical NPs biomineralized in different layers on the cell surface. However, at higher precursor concentrations (≥2 mM) supplemented with biogenic spherical NPs as seeds, cells start to biosynthesize partially embedded long aspect ratio fiber-like Au nanostructures. Most interestingly, A549 cells seem to be able to sense the enhanced Au concentration. They change the chemical composition of the extracellular NP agglomerates from threonine-O-3-phosphate aureate to an arginine-Au(I)-imine. Furthermore they adopt the extracellular mineralization process from spheres to irregular structures to nanoribbons in a dose-dependent manner with increasing Au concentrations. The results achieved regarding size, shape and chemical specificity were cross checked by SEM-EDX and single particle (sp-)ICP-MS. Our findings demonstrate the potential of ToF-SIMS 3D imaging to better understand cell-NP interactions and their impact in nanotoxicology.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
The schematic overview of the workflow of the 3D biomolecular imaging of de novo biomineralization of ionic gold into anisotropic (0, 1 & 2D) nanostructures. Left panel show the collection of secondary ions by the detector after the primary ion beam impacts freeze dried A549 cells with embedded anisotropic gold nanostructres. The right panel shows that ToF-SIMS images can be reconstructed into 3D space to give molecular distributions of gold and reducing agent in three different culture environments resulting in 3 different nanostructures (spheres, irregular particles and nanoribbons).
Figure 2
Figure 2
High resolution transmission electron microscopy (HR-TEM) and nanoparticle tracking analysis (NTA) of biomineralized AuNP materials. The Brownian motion and light scattering information from NTA gives the size of distribution and HRTEM and shape of nanostructures formed by A549 cells treated with (A) 0.5 mM; (B) 1.0 mM; (C) 2.0 mM; and (D) 2.0 mM HAuCl4 with spherical Au-NPs as seeds. (E,F) NTA plots showing size distribution after 5 rounds of tracking of spherical NPs (E) corresponding to scheme (A) and nanoribbons (F) corresponding to (C) of right panel in Fig. 1.
Figure 3
Figure 3
Scanning electron microscopy and ToF-SIMS molecular 3D imaging of condition (A) in schematic Fig. 1. Scanning electron microscopy (A,B) and X-ray spectroscopic (C,D) analysis of A549 cells treated with 0.5 mM HAuCl4 confirms NP formation (scale bar 5 µm in A,C and 200 nm in B). The ion signals from Au+ (E, untreated A549 cells; G, ionic gold) and threonine-O-3-phosphate aureate (F, untreated A549 cells; H, A549 cells treated with low dose of ionic gold). (I,J) Topography of extracellular spherical Au nanostructures on top of A549 cells (square ROI 130 µ × 130 µ in I and 80 µ × 80 µ in J). (K) Reconstructed 3D depth profile of mineralized Au structures in A549 cell cultures. The Au+ signal (red, translucent) indicates the spherical Au-NP, whilst the signal for threonine-O-3-phosphate aureate is shown in green. (L) Enlarged view from (K, arrow) showing the combined distribution of threonine-O-3-phosphate (green) and Au-NPs signals (orange) with occasional overlap (ring).
Figure 4
Figure 4
The spatial distribution of threonine-O-3-phosphate as agglomerates in a 3D reconstruction of a nanoparticulate cluster. The reconstruction shows the whole NP agglomerate and successive phosphate agglomerate distribution (depicted in green) in and around deeper layers of the spherical Au-NP. (A) Slicing the spherical NPs with different z-micro stacks and 3D reconstruction from top to bottom as shown in top view: Au particle reconstructed from Au+ signals (orange, solid); threonine-O-3-phosphate aureate (green). (B) Zoomed-in view spatially isolated threonine-O-3-phosphate aureate showing m/z 417.06 green signal within a single Au-NP cluster.
Figure 5
Figure 5
The constructed ion image reveals quasi spherical Au-NPs with increasing Au ions to 1 mM Au ions. (A,B) The topography is displayed by total ion reconstruction image of the A549 cell surface. Inset show SEM image of quasi-spherical nanostructure. (C) 3D reconstructed ion image of a single cluster with the Au+ signals (m/z 196.97) picked from biomineralized cell surface. (D) The zoomed in-depth 3D reconstruction overlay of cluster shown in (C) in side view of spatiotemporal Au+ signals (orange) with threonine-O-3-phosphate aureate (green).
Figure 6
Figure 6
SEM and ToF-SIMS analysis of de novo biomineralized nanostructures at 2 mM Au ions: delta and rhombus shaped particles. (AD) SEM and EDX chemical mapping to confirm anisotropic rhombus/delta shape Au nanoplate formation (Scale bar 1 micron). (E,F) The SIMS signals from Au+ (E) and threonine-O-3-phosphate aureate (F). (G) Reconstructed ToF-SIMS ion image from the A549 cell surface demonstrating spherical Au particles. (H) The 3D reconstruction and overlay of Au+ signal (orange) and threonine-O-3-phosphate aureate (green) from red square ROI from (G).
Figure 7
Figure 7
The de novo SEM and ToF-SIMS analysis of biomineralized long aspect ratio “nanoribbons” at 2 mM Au ions along with spherical Au-NP as seeds. (AC) The SEM micrograph and EDX element mapping confirm the nanoribbon-like structure formation (scale bar 1 µm). (DF) A qualitative view showing nanoribbon covered cell surface layers in ToF-SIMS topography (D,E) and overlapped ion signals in top view (F). The (F) is enlarged section from (D) shown with red square ROI. (F) demonstrates reconstruction of total ion count without background to show gold signals from (E).
Figure 8
Figure 8
The ion image 3D reconstruction of Au+ signals (orange), and arginine-Au(I)-imine (blue) in top (A) view of the Au nanoribbon clusters on A549 cell. (B) The SIMS signals in side view from Au+ (orange) and arginine-Au(I)-imine (blue) showing that there is a change in chemical specificity of biocomplexation during Au mineralization supplemented with spherical NPs as ‘seed’ in cellular microenvironment. (C) The secondary ion signals from Au+ (m/z: 196.97) and arginine-Au(I)-imine (m/z: 417.06) (lower panel) and their comparison with untreated A549 cells (upper panel).

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