Assessment of the In Vivo Toxicity of Gold Nanoparticles

Abstract

The environmental impact of nanoparticles is evident; however, their toxicity due to their nanosize is rarely discussed. Gold nanoparticles (GNPs) may serve as a promising model to address the size-dependent biological response to nanoparticles because they show good biocompatibility and their size can be controlled with great precision during their chemical synthesis. Naked GNPs ranging from 3 to 100 nm were injected intraperitoneally into BALB/C mice at a dose of 8 mg/kg/week. GNPs of 3, 5, 50, and 100 nm did not show harmful effects; however, GNPs ranging from 8 to 37 nm induced severe sickness in mice. Mice injected with GNPs in this range showed fatigue, loss of appetite, change of fur color, and weight loss. Starting from day 14, mice in this group exhibited a camel-like back and crooked spine. The majority of mice in these groups died within 21 days. Injection of 5 and 3 nm GNPs, however, did not induce sickness or lethality in mice. Pathological examination of the major organs of the mice in the diseased groups indicated an increase of Kupffer cells in the liver, loss of structural integrity in the lungs, and diffusion of white pulp in the spleen. The pathological abnormality was associated with the presence of gold particles at the diseased sites, which were verified by ex vivo Coherent anti-Stoke Raman scattering microscopy. Modifying the surface of the GNPs by incorporating immunogenic peptides ameliorated their toxicity. This reduction in the toxicity is associated with an increase in the ability to induce antibody response. The toxicity of GNPs may be a fundamental determinant of the environmental toxicity of nanoparticles.

Figure 1

TEM images for the GNPs synthesized in the current study. GNPs with diameters of 3, 5, 8, 12, 17, 37, 50, and 100 nm were examined under an electron microscope. Scale bars are 20 nm for images of 3, 5, 8, 12, and 17 nm GNPs. Scale bars are 50 nm for images of 37, 50, and 100 nm GNPs

Figure 2

Average lifespan of mice receiving GNPs with diameters between 8 and 37 nm was shortened to different extents. The average lifespan (L₅₀) was defined as the time beyond which half of the mice died. Mice injected with GNPs outside the lethal range behaved normally. The break marks on the top of bars indicate no death observed during the experimental period

Figure 3

H&E staining showed GNP-induced abnormality in major organs. (Top to bottom) HE staining for liver, lung, and spleen. Theleft columnshows tissues from 5 nm GNP-treated animals. Theright columnshows tissues from 17 nm GNP-treated mice

Figure 4

ELISA of GNPs using anti-endotoxin IgG. ELISA was performed by using anti-endotoxin IgG against various sizes of GNPs synthesized in the lab. Lipopolysaccharide (LPS) served as a positive control, while BSA served as a negative control

Figure 5

CARS microscopy of livers isolated from GNP-treated and control mice. The wavelengths of the pump and the Stokes lasers (Pump = 870 nm and Stokes = 1064 nm) were tuned to match a Raman shift (~2100 cm⁻¹), falling in the so-called “silent region” of the vibrational spectra of cells and tissues. As expected, the CARS images of the “control” did not show appreciable contrast under the non-resonant condition whereas the CARS signals were dramatically enhanced and appeared as scattered bright spots on the images taken from the specimens treated with GNPs. The enhancement presumably resulted from strong scattering from the GNPs and the large third-order polarizability of the GNPs. Enhanced bright spots were observed in neither the control group (a) nor the mice injected with 5 nm GNP (b). Livers obtained from 17 nm GNP-treated mice showed intense bright spots (c). Livers obtained from 50 nm GNP-treated mice showed only a moderate number of spots (d)

Figure 6

MTT assay to obtain LC₅₀for different sizes of GNPs using Hela cells as a model system. After seeding and proper attachment, the Hela cells were treated with 5, 8, 12, and 17 nm GNPs at the concentrations, indicated on the horizontal axis. The percentage of survival was plotted against GNPs concentration

Figure 7

The lethality and immunogenicity of surface-modified 17 nm GNPs. Average lifespans of mice injected with modified 17 nm GNPs are shown in empty columns. Each experimental group received GNP conjugated with BSA, lysozyme, pFMDV, or pH5N1. Unmodified GNPs served as a positive control (17 nm GNP). Titers of antiserum withdrawn from GNP-injected mice against corresponding antigens are shown in filled columns. pH5N1- and pFMDV-coated GNPs induced the highest titer in mouse serum; BSA- and lysozyme-coated GNPs induced a moderate titer; and unmodified GNPs did not induce an antibody response in mice (*)