Large uptake of titania and iron oxide nanoparticles in the nucleus of lung epithelial cells as measured by Raman imaging and multivariate classification

Abstract

It is a challenging task to characterize the biodistribution of nanoparticles in cells and tissue on a subcellular level. Conventional methods to study the interaction of nanoparticles with living cells rely on labeling techniques that either selectively stain the particles or selectively tag them with tracer molecules. In this work, Raman imaging, a label-free technique that requires no extensive sample preparation, was combined with multivariate classification to quantify the spatial distribution of oxide nanoparticles inside living lung epithelial cells (A549). Cells were exposed to TiO2 (titania) and/or α-FeO(OH) (goethite) nanoparticles at various incubation times (4 or 48 h). Using multivariate classification of hyperspectral Raman data with partial least-squares discriminant analysis, we show that a surprisingly large fraction of spectra, classified as belonging to the cell nucleus, show Raman bands associated with nanoparticles. Up to 40% of spectra from the cell nucleus show Raman bands associated with nanoparticles. Complementary transmission electron microscopy data for thin cell sections qualitatively support the conclusions.

Figure 1

Example of an optical micrograph of an A549 cell, with the measurement grid overlaid. Spots marked by O (outside cell nucleus) or N (nucleus) are included in the PLS-DA model.

Figure 2

(a) Score plot for the first two PLS components in the PLS-DA model. Grey dots: observations from the cell nucleus. Black dots: observations from outside the cell nucleus. (b) PLS weights for the first PLS component. The positions of Raman bands assigned to DNA are marked by dots.

Figure 3

(a) Mean spectrum and SD (upper and lower curves) for all measurements predicted to belong to the cell nucleus. (b) Mean spectrum and SD (upper and lower curves) for all measurements predicted to belong to the membrane and cytoplasmic regions. (c) 95% CI, as calculated from Student’s t-test, for the mean difference of the plots in a and b. The positions of Raman bands assigned to DNA are indicated by dots.

Figure 4

(a) Optical micrograph of A459 cell exposed to α-FeO(OH). (b) Pseudo-color map of the classification of each pixel in panel a. (c) Intensity map showing the Raman intensity of the band at 388 cm⁻¹ corresponding to the E_g mode in α-FeO(OH) for each voxel indicated in a. (d) Optical micrograph of A459 cell exposed to TiO₂. (e) Pseudo-color map of the classification of each pixel in d. (f) Intensity map showing the Raman intensity of the band at 638 cm⁻¹ corresponding to the E_g mode in anatase TiO₂ for each pixel indicated in d. The crosses depicted in a and d show the measurement spots where the laser beam was focused.

Figure 5

Raman spectra of TiO₂ and α-FeO(OH) nanoparticles. The intense band at 322 cm⁻¹ is from the CaF₂ substrate.

Figure 6

Bar charts showing the percentage of measurements from outside the nucleus, inside the nucleus, and unknown locations, as deduced from the PLS-DA model, which shows characteristic Raman bands due to (a) anatase TiO₂, (b) α-FeO(OH), or (c) mixtures of α-FeO(OH) and TiO₂. The asterisk (^∗) in panel a shows the result from single-point measurements after 4 h of exposure.

Figure 7

TEM images of sections of A549 cells exposed to (a) 10 μg ml⁻¹α-FeO(OH) nanoparticles, (b) 10 μg ml⁻¹ TiO₂ nanoparticles, or (c) 10 μg ml⁻¹ of α-FeO(OH) and 10 μg ml⁻¹ TiO₂ nanoparticles.