Unexpected intracellular biodegradation and recrystallization of gold nanoparticles

Abstract

Gold nanoparticles are used in an expanding spectrum of biomedical applications. However, little is known about their long-term fate in the organism as it is generally admitted that the inertness of gold nanoparticles prevents their biodegradation. In this work, the biotransformations of gold nanoparticles captured by primary fibroblasts were monitored during up to 6 mo. The combination of electron microscopy imaging and transcriptomics study reveals an unexpected 2-step process of biotransformation. First, there is the degradation of gold nanoparticles, with faster disappearance of the smallest size. This degradation is mediated by NADPH oxidase that produces highly oxidizing reactive oxygen species in the lysosome combined with a cell-protective expression of the nuclear factor, erythroid 2. Second, a gold recrystallization process generates biomineralized nanostructures consisting of 2.5-nm crystalline particles self-assembled into nanoleaves. Metallothioneins are strongly suspected to participate in buildings blocks biomineralization that self-assembles in a process that could be affected by a chelating agent. These degradation products are similar to aurosomes structures revealed 50 y ago in vivo after gold salt therapy. Overall, we bring to light steps in the lifecycle of gold nanoparticles in which cellular pathways are partially shared with ionic gold, revealing a common gold metabolism.

Keywords: biodegradation; biomineralization; gold nanoparticles; nanoparticles fate.

Fig. 1.

TEM observations on microtome sections of human fibroblasts exposed to 4-nm GNPs observed 1 d to 6 mo after GNPs incubation, evidencing the existence of dense and diffuse electron-dense areas. (A–C) Images of 1 cell 2 wk after GNPs exposure presenting 3 electron-dense areas identified as lysosomes constituted of dense areas (dark orange arrows) and diffuse areas (light orange arrow). (D–K) Representative lysosomes observed 1 d (D and H), 2 wk (E and I), 2 mo (F and J), and 6 mo (G and K) after exposure at 2 magnifications. L, Surface proportion of dense and diffuse areas from 1 d to 6 mo after exposure; 5 to 10 images have been analyzed for each condition, with at least 1 electron-dense area per image.

Fig. 2.

STEM observations of human fibroblasts 2 wk after exposure to 4 nm GNPs showing that diffuse structures are 2D nanoleaves composed of self-assembled 2.5-nm nanoparticles. (A–C) STEM images of a cell domain at 3 different magnifications, showing the local structure of the diffuse areas previously described. (D) Diameter distribution of nanoparticles observed in dense or diffuse areas 2 wk after GNPs exposure measured on at least 115 objects. (E–G) STEM-HAADF images extracted from the tilt series acquired during tomography experiments. The tilt angle of the sample holder with respect to the electron beam is indicated in the top right corner of each image. The same 2 leaf-shaped nanostructures are indicated by arrows on each image. The resulting calculated 3D tomogram can be found in Movie S1.

Fig. 3.

High-resolution TEM observations, electron diffraction, and STEM-EDS performed on human fibroblasts 2 wk after exposure to 4-nm GNPs reveal the crystallinity of nanoparticles composing diffuse areas and a specific signal of sulfur. (A–D) TEM imaging of dense (A and B) and diffuse (C and D) area, presenting crystalline lattice highlighted in red. (E and F) The 2D diffraction patterns obtained on representative regions of dense (E) or diffuse (F) areas. (G) Scattered intensity obtained after radial integration of E and F. (H and K) STEM images of noncontrasted (H; red frame), diffuse (H; light orange frame), and dense (K; dark orange frame) areas analyzed by EDS. (I, J, and L) EDS spectra of noncontrasted (I), diffuse (J), and dense (L) areas and (Inset) enlargement of the 2- to 3-keV area, with Gaussian fits for gold, sulfur, and chloride peaks. The slight horizontal contrast modifications seen at the bottom of the STEM-HAADF image are due to sample charging.

Fig. 4.

TEM observations of human fibroblasts exposed to 22- or 15-nm GNPs reveal the presence of diffuse areas associated to degradation. (A–C) TEM images of representative lysosomes observed 1 d (A), 2 wk (B), or 2 mo (C) after 22-nm GNPs exposure, presenting dense areas (dark orange arrows) and diffuse areas (light orange arrow). (D) Surface proportion of dense and diffuse areas from 1 d to 2 mo after exposure to 22-nm GNPs; 5 to 10 images have been analyzed for each condition, with at least 1 electron-dense area per image. (E–H) Representative lysosomes observed 6 mo after 15-nm GNP exposure, presenting dense and diffuse areas observed at 2 magnifications.

Fig. 5.

Transcriptomics analysis reveals a time-dependent answer to 4-nm GNPs and the activation of 3 main pathways at the longer times of study (NADPH production, ROS detoxification, and metal chelation). (A and B) PCA of transcriptomics data performed with the 500 most variable genes. (C) Venn diagram of GNPs samples differential expression compared with unlabeled control at the same time (FDR q value lower than 10⁻³ only). Lists and arrows summarize highest hits of GSEA (FDR q value lower than 10⁻⁴ only). (D) Expression heatmap of up-regulated genes 2 wk after GNPs exposure. Only genes that are attributed to identified pathways are displayed (38 genes on 124). Expression was considered as significantly different for FDR q value below 0.01. CTL, control; TNF, tumor necrosis factor.

Fig. 6.

GNPs degradation is mediated by NOX-created ROS followed by a probable recrystallization inside MTs and a self-assembly process impacted by BAL. (A and B) TEM observations of human fibroblasts 2 wk after exposure to 4-nm GNPs with (A) or without (B) the NOX inhibitor GKT137831. Dense and diffuse areas are indicated by dark orange and light orange arrows, respectively. (C) Proportion of dense and diffuse areas with or without GKT137831; 30 images or more have been analyzed, with at least 1 electron-dense area per image. (D–H) Liquid TEM snapshot performed in aqueous solution ([HCl] = 0.02 mol/L, [NaCl] = 1 mol/L) under constant radiation at 200 keV. Observations were performed on citrate-coated GNPs and gold nanorods (D = 25 nm, l = 11 nm, L = 45 nm). (I) Ultraviolet (UV)-visible spectroscopy performed steadily during MT filling with sodium aurothiomalate. The legend indicates the ratio MT:Au. (J) UV-visible signal evolution at 293 nm during MT filling. (K and L) High-resolution TEM imaging of MT–gold complex at the MT:Au ratio of 1:120 observed at 2 magnifications. (M–O) STEM observation of human fibroblasts 2 wk after exposure to 4-nm GNPs and cultured with BAL. Diffuse areas of 2 types were observed (N): an aurosome-like one (green frame and M) and a new one (blue frame and O).

Fig. 7.

Proposed mechanism of GNPs degradation and recrystallization process. H⁺ and stoichiometric coefficients are not mentioned for clarity.