Abstract

A series of gold nanoparticles were examined for uptake and acute toxicity in human leukemia cells. The nanoparticles (average diameter=18 nm), which possessed various surface modifiers, were not toxic to cells during continuous exposure for three days. Citrate-capped nanoparticles were further examined for their cellular uptake by absorbance and transmission electron microscopy (see image). Results indicate that although some precursors of nanoparticles may be toxic, the nanoparticles themselves are not necessarily detrimental to cellular function. A series of gold nanoparticles were examined for uptake and acute toxicity in human leukemia cells. The results indicate that although some precursors of nanoparticles may be toxic, the nanoparticles themselves are not necessarily detrimental to cellular function. Nanoscience and nanotechnology hold great promise for many applications, including biomedical uses. Yet despite the huge potential benefit of nanomaterials in the realm of biomedical and industrial applications, very little is known about potential short- and long-term deleterious effects of such nanomaterials on human and environmental health.1–3 Specifically, there is very little information on the effect of size, shape, and surface functional groups on the bioavailability, uptake, subcellular distribution, metabolism, and degradation of many of the nanomaterials being explored. Recent reports have begun to examine these issues for carbon nanotubes,4–6 CdSe nanoparticles,7–10 and gold nanoparticles.11–14 Here we report an investigation of the cellular uptake and cytotoxicity of gold nanoparticles with human cells. The present studies were undertaken in order to determine the interactions of a series of defined nanoparticles containing a variety of surface modifiers and stabilizers with an established human cancer cell line. The nanoparticle library consisted of gold spheres with average diameters of ≈4, 12, or 18 nm, and containing a variety of surface modifiers. Cysteine and citrate-capped 4-nm nanoparticles and glucose-reduced 12-nm nanoparticles were synthesized as previously described (the synthesis of the various nanoparticles employed in this study, along with the materials used, are given in the Supporting Information).15–17 For the 18-nm nanoparticles, the surface modifiers were citrate, biotin, and cetyltrimethylammonium bromide (CTAB).18 The nanoparticle library was tested for cytotoxicity using the K562 leukemia cell line.19 Following three days of continuous exposure to the nanoparticles, cell viability was determined using the MTT assay.20 In this assay, cells that properly metabolize a dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) undergo visible color changes that are monitored spectrophotometrically; cells that are incapable of metabolizing the dye remain colorless. The 18-nm nanoparticle preparations with citrate and biotin surface modifiers did not appear to be toxic at concentrations up to 250 μM (gold atoms) under these conditions (Figure 1 A1). In contrast, the gold-salt (AuCl4) precursor solution was over 90 % toxic at a concentration of 200 μM (Figure 1 A1). Note that the gold-salt precursor solution was adjusted to a pH value of 7 prior to the cytotoxicity experiment. The nanoparticle preparations with glucose or cysteine surface modifiers, or with a reduced gold surface, were not toxic at concentrations up to 25 μM. To further confirm the lack of toxicity, cell numbers were counted on days 2–5 during continuous exposure to a 25 μM concentration of 18-nm citrate-capped nanoparticles. No difference was seen in either the growth rate of the untreated control cells or the cells exposed to the nanoparticles (see Supporting Information, Figure S1). Survival curves for human K562 cells exposed to nanoparticles. Cells were continuously exposed to nanoparticles for 3 days. Cell viability was measured by the MTT assay. The data are plotted as the percentage of surviving cells compared to untreated controls. a) Plot showing the survival of cells exposed to the AuCl4 precursor solution (▴) or to 18-nm nanoparticles containing citrate (⧫) or biotin (▪); b) plot showing the survival of cells exposed to CTAB alone (▪), 18-nm nanoparticles with CTAB (⧫), or 18 nm nanoparticles with CTAB that were washed three times prior to incubation with the cells (▴). The preparation of 18-nm nanoparticles that contained CTAB displayed significant toxicity (Figure 1 B1). CTAB alone showed a similar toxicity (Figure 1 B1). It was thus necessary to determine whether unbound CTAB or the CTAB-modified nanoparticles caused the observed cytotoxicity. Therefore, CTAB-modified nanoparticles were centrifuged and washed with deionized water three times to remove unbound CTAB. The washed CTAB-modified nanoparticles were found to be not toxic under the conditions examined, which suggests that CTAB bound to the gold nanoparticles does not cause toxicity (Figure 1 B1). NMR studies of the washed CTAB-modified nanoparticles indicated that all of the remaining CTAB was associated with the nanoparticles (data not shown). The lack of detectable cytotoxicity raised the question of whether the nanoparticles were capable of being taken up into the cells. In order to assess the extent of the uptake of gold nanoparticles into cells, the nanoparticle concentration in the cell culture media was monitored by visible spectroscopy at time points from 1 to 24 h post-exposure (Figure 2 C2). The cells were exposed to 18-nm citrate-capped nanoparticles at a concentration of 25 μM for time points from 15 min to 24 h. The concentration of the gold nanoparticles in the media dropped to a plateau within 1 h of the initial exposure, which suggests that the nanoparticles were rapidly taken up into cells (Figure 2 C2). Control experiments with a media that lacked cells suggested against adsorption of the gold nanoparticles onto serum proteins or the cell culture plates (data not shown). The presence in cells of the 18-nm citrate-capped gold nanoparticles was confirmed by transmission electron microscopy (TEM) of the cells following exposure. Figure 2 A and B2 shows electron micrographs at different magnifications of a cell that contains gold nanoparticles following exposure (to 18-nm citrate-capped nanoparticles) for 1 h. The nanoparticles are clustered in a subcellular location that we speculate are endocytic vesicles, although further experiments would be necessary to conclusively demonstrate this. Interestingly, the images taken at higher magnifications show that the gross morphology of the nanoparticles has not changed dramatically, that is, the nanoparticles appear as ≈18-nm spheres even after being taken up by the cells (Figure 2 B2). Further experiments will be required to determine what, if any, changes to the surface groups on the nanoparticles have occurred after being exposed to the cellular environment. Electron micrographs at different magnifications of a cell containing nanoparticles. Cells were exposed to nanoparticles for 24 h, fixed with osmium tetroxide, sectioned, and visualized with a Hitachi H-8000 electron microscope. a) Image at 8000× magnification of a representative cell with nanoparticles subcellularly localized. The small box represents the area magnified in (b); b) image at 60 000× magnification of gold nanoparticles within cells. The inset is a 150 000× magnification of the gold nanoparticles. c) visible spectroscopy plot (measured at 526 nm) of the concentration of gold nanoparticles in cell culture media following incubation with the cells (data is compared to the initial concentration). The media was exposed to 18-nm citrate-capped nanoparticles for the times shown. Following exposure, the cells were removed from the media by centrifugation at 300 g. Cells were grown in cell culture media lacking phenol red for the absorbance experiments. Taken together, the data suggest that spherical gold nanoparticles with a variety of surface modifiers are not inherently toxic to human cells, despite being taken up into cells. The results with the CTAB-capped nanoparticles and the gold-salt solution indicate that although some precursors of nanoparticles might cause toxicity, the nanoparticles themselves are not necessarily detrimental to cellular function. Many more variables require further testing, including shapes other than spheres, and different functional groups on the surfaces of the nanoparticles. The long-term effects of the presence of nanoparticles would also have to be examined. Lastly, it will be important to determine whether nanoparticles are themselves modified by the cellular environment, thus potentially altering the properties of the nanoparticles for biosensing, imaging, or delivery applications. Supporting information for this article is available on the WWW under http://www.small-journal.com or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.