From the Cover: Disease-Induced Disparities in Formation of the Nanoparticle-Biocorona and the Toxicological Consequences

Abstract

Nanoparticle (NP) association with macromolecules in a physiological environment forms a biocorona (BC), which alters NP distribution, activity, and toxicity. While BC formation is dependent on NP physicochemical properties, little information exists on the influence of the physiological environment. Obese individuals and those with cardiovascular disease exist with altered serum chemistry, which is expected to influence BC formation and NP toxicity. We hypothesize that a BC formed on NPs following incubation in hyperlipidemic serum will result in altered NP-BC protein content, cellular association, and toxicity compared to normal serum conditions. We utilized Fe3O4 NPs, which are being developed as MRI contrast and tumor targeting agents to test our hypothesis. We used rat aortic endothelial cells (RAECs) within a dynamic flow in vitro exposure system to more accurately depict the in vivo environment. A BC was formed on 20nm PVP-suspended Fe3O4 NPs following incubation in water, 10% normal or hyperlipidemic rat serum. Addition of BCs resulted in increased hydrodynamic size and decreased surface charge. More cholesterol associated with Fe3O4 NPs after incubation in hyperlipidemic as compared with normal serum. Using quantitative proteomics, we identified unique differences in BC protein components between the 2 serum types. Under flow conditions, formation of a BC from both serum types reduced RAECs association of Fe3O4 NPs. Addition of BCs was found to exacerbate RAECs inflammatory gene responses to Fe3O4 NPs (Fe3O4-hyperlipidemic > Fe3O4-normal > Fe3O4) including increased expression of IL-6, TNF-α, Cxcl-2, VCAM-1, and ICAM-1. Overall, these findings demonstrate that disease-induced variations in physiological environments have a significant impact NP-BC formation, cellular association, and cell response.

Keywords: darkfield microscopy; endothelial cell; hyperlipidemia; iron oxide nanoparticles; nanotoxicology; protein corona.; proteomics.

FIG. 1

False-colored transmission electron micrographs for (a) Fe₃O₄ NPs without a BC, (b) high-resolution micrograph of Fe₃O₄ NPs without a BC showing interlayer spacing of ∼0.49 nm matching [111] plane, (c) Fe₃O₄ – normal BC, and (d) Fe₃O₄ – lipid BC. Biocorona is shown in blue (c) and green colors (d). The NPs were observed to be embedded in the BCs. Please see online version to view figure in color.

FIG. 2

Venn diagram representing the distribution of protein components found to associate with 20 nm Fe₃O₄ NPs following incubation in 10% normal (blue) or hyperlipidemic (red) serum. Please see online version to view figure in color.

FIG. 3

Ratios of common protein components found to associate with 20 nm Fe₃O₄ NPs following incubation in both 10% normal or hyperlipidemic serum. Data are expressed as ratio of protein abundance in normal serum BC compared with hyperlidemic BC.

FIG. 4

Measurement of 20 nm Fe₃O₄ NP association by ICP-MS and darkfield microscopy. Rat aortic endothelial cells (RAEC) were exposed to serum-free media (control), Fe₃O₄ NPs without a biocorona (BC), or Fe₃O₄ NPs with either a normal serum or lipid serum BC. Cells were exposed to a concentration of 20 μg/ml for 2 h under flow conditions of 1 ml/min. (A) Following exposure cells were assessed by ICP-MS for Fe₃O₄ NP association. Values are expressed as mean ± SEM (n = 3/group). *Significant difference from controls (P < .05). (B) Alterations in RAEC association of Fe₃O₄ NPs due to addition of BCs were qualitatively evaluated via darkfield microscopy. Fe₃O₄ NPs without a BC are identified with red arrows, Fe₃O₄ NP-normal BC with blue arrows, and Fe₃O₄ NP-lipid BC with green arrows. Please see online version to view figure in color.

FIG. 5

Heat map of selected endothelial cell cytokine and inflammatory genes following exposure to Fe₃O₄ NPs. Rat aortic endothelial cells (RAECs) were exposed to serum-free media (control), Fe₃O₄ NPs without a BC or Fe₃O₄ NPs with either a normal serum or lipid serum BC. Cells were exposed to a concentration of 20 μg/ml for 1 h under flow conditions of 1 ml/min. Red indicates genes that have high expression values, green indicates genes that have low expression values across all groups, and black indicates median expression. Fold change values and P values are included for all 85 assessed genes in Supplementary Table 2. Please see online version to view figure in color.

FIG. 6

Assessment of Fe₃O₄ NP-BC alterations in cell surface expression of VCAM-1 by immunofluorescent staining. Rat aortic endothelial cells (RAECs) were exposed to serum-free media (control), Fe₃O₄ NPs without a BC or Fe₃O₄ NPs with either a normal serum or lipid serum BC. Cells were exposed to a concentration of 20 μg/ml for 1 h under flow conditions of 1 ml/min. Cells were then immunofluorescently stained with DAPI to identify the nucleus (Blue) and with a VCAM-1 antibody to identify endothelial cell surface expression of VCAM-1 (Green). Please see online version to view figure in color.