Uboldi et al. have recently demonstrated that AuNPs induced cytotoxicity in the AT-II-like human epithelial cells, A549 and H441, and the effects exerted by gold nanoparticles (AuNPs) correlated with the presence of sodium citrate on the particle surface . In this study we wished to determine if the same effect would be observed in human endothelial cells after exposure to the same gold nanoparticles. AuS0302-RIT, AuS0302-RIS02 and AuS0302-RIS04, which have sizes of 10 nm, 11 nm and 25 nm respectively, as well as different amounts of sodium citrate on their surface, were studied with respect to their potential cytotoxicity and their uptake behavior.
We found that only high concentrations of AuNPs (500 μM and 1000 μM) negatively influence the cell viability in both endothelial cell types. The significant increase in cell viability after the treatment with 500 μM and 1000 μM AuS0302-RIT is not fully understood but may be explained by a higher mitochondrial activity of cells incubated with up to 1000 μM gold nanoparticles or a very slight cross-reaction of gold nanoparticles with the substrate of the MTS assay. In addition, a small but not significant increase in cell viability of hCMEC and NCIH441 could also be observed . Thus, to exclude misinterpretation of the data three different assays (MTS, LDH, and proliferation experiments) were performed to determine the effects on the cell viability. In summary, a positive effect on cell viability after treatment with 500–1000 μM AuS0302-RIT cannot be determined. Furthermore a decrease in proliferation rate as measured by an assay for the proliferation factor Ki-67 [19–21], was higher in primary human dermal microvascular endothelial cells (HDMEC) compared to the human cerebral microvascular endothelial cell line, (hCMEC/D3). Even 50 μM AuS0302-RIS04 decreased the proliferation rate in HDMEC while the proliferation in hCMEC exposed to this concentration was not affected. The higher amount of internalized AuS0302-RIS04 might be the reason for the decrease of cell proliferation of HDMEC compared to hCMEC after exposure to 50 μM gold nanoparticles. Dyer and Patterson showed that numerous properties and characteristics of endothelial cells from various locations of the body differ . These variations (i.e. permeability, surface receptors) might generally explain the reduced proliferation rate (here determined by Ki67 expression) in HDMEC after exposure to 50 μM AuS0302-RIS04. In addition to that the increased particle concentration within the cells might decrease the motility of the cells and thus might impair the cell growth and the proliferation. Mironava et al. has previously shown that increased uptake of nanoparticles in human dermal fibroblast was accompanied by a higher amount of vesicles within the cells which impaired the cytoskeleton and influenced cell division . However an overload of the endothelial cells with gold nanoparticles could not be observed using transmission electron microscopy (TEM). In addition, cell cycle arrest might also be an explanation for decreased proliferation. It has previously shown that human prostate cancer cells (DU-145) arrested in G2/M phase of the cell cycle after exposure to AuNPs . In general, this arrest was shown to be accompanied by increased apoptosis [25, 26]. However, after treatment with 50 μM of gold nanoparticles no increase of cell death (measured by LDH-assay) was determined in our investigations. Only much higher concentrations of gold nanoparticles (>500 μM) resulted in an increased cell death in both endothelial cell types. These observations might be explained by the arrest in G2/M phase. Since AuS0302-RIS02 (11 nm) and AuS0302–RIS04 (25 nm) but not AuS0302-RIT (10 nm) induced these effects, it could be concluded that the size of the AuNPs was not a significant factor, but that the higher amount of sodium citrate on the particle surface was the cause for the increase of cytotoxicity. Certainly, the higher concentrations of gold nanoparticles (≥500 μM) resulted in a decreased proliferation rate of both endothelial cells even after exposure to AuS0302-RIT. However the effects on proliferation by particles with a high amount of sodium citrate were higher compared to the effects caused by AuS0302-RIT. In general, citrate-stabilized gold nanoparticles may build agglomerates in cell culture media with serum proteins. So, not only single gold nanoparticles but potential agglomerates come into contact with cells. Due to the different citrate concentrations of the particles different amounts of agglomerates may be built and various impacts on cells may be the result. This may explain the effect of the gold nanoparticles with higher citrate concentration on all cell types. However, the group around Gosens has previously shown that the effects of single and agglomerated citrate-stabilized gold nanoparticles on lung cells were not altered . Thus, the effects of agglomerates can be neglected. Beside this, other factors may be responsible and a detailed clarification of subcellular events regarding the exact nature of the toxic effects (apoptosis, necrosis) and the impairment of the cell proliferation needs to be done. In general, the physico-chemical properties of nanoparticles (i.e. surface modifications) influence the interaction of particles with cells and the effects related to these interactions .
In addition to the surface modification, it has been demonstrated by different groups that the size of gold nanoparticles has an influence on their internalization and the subsequent cellular reaction, such as cytotoxicity . Although only two different-sized gold nanoparticles (10-11 nm and 25 nm) were examined in the present studies, an obvious size effect was not correlated to cytotoxic effect when endothelial cells were exposed to these nanoparticles. Furthermore, differences with two nanoparticles of the same size could also be observed. Both AuS0302-RIT and AuS0302RIS02 were the same size, however, a decrease in the expression of the proliferation factor Ki-67 as well as the increased toxicity was observed after exposure of cells to 1000 μM AuS0302-RIS02, indicating that size was not- but that the concentration of citrate was the relevant factor for causing these effects. In addition, the same effects were detected after the exposure of the endothelial cells to AuS0302-RIS04 which contained the same amount of citrate but were a different size. These results were also observed by Uboldi et al. who demonstrated that it was not the size but the amount of sodium citrate present in the gold nanoparticles that were responsible for the cytotoxic effects on the lung epithelial cell lines, A549 and NCIH441 .
Interestingly, the effects exerted on the proliferation rate of A549 and NCIH441 after exposure to 500 μM to 1000 μM AuNPs were more severe compared to those observed on the endothelial cells examined in the present study with the same nanoparticles . The proliferation rate of epithelial cells was decreased compared to endothelial cells after exposure to high concentrations of gold nanoparticles with a higher concentration of sodium citrate on their surface. Moreover, the cytotoxic effects and the effects on cell viability were milder in endothelial than in epithelial cells as measured by the LDH and MTS assays. These studies indicate that specific cell types respond differently to a particular nanoparticle.
Further studies were carried out to determine whether a correlation existed between the amount of internalized particles and cytotoxicity. Cells examined by transmission electron microscopy showed that the AuNPs were internalized by both epithelial and endothelial cells. In previous studies, it was shown that in the epithelial cells A549 and NCIH441 the internalized AuNPs were located in vesicles enclosed in the perinuclear region . The same results were observed in the human endothelial cells examined in these studies. Others, in contrast to these findings, demonstrated by TEM analyses that 25 nm-sized gold nanoparticles could be detected in a freely dispersed form in the cytoplasm of airway epithelial cells A549 . In addition to the intracellular localization of the AuNPs the TEM analyses showed that the exposure to gold nanoparticles did not alter the cell morphology.
A quantification of internalized AuNPs was carried out by the more sensitive atomic emission spectroscopy (ICP-AES). These analytical results obtained after analysis of cell populations exposed to nanoparticles corresponded with the TEM images of single cells, namely the presence of sodium citrate did not influence the uptake behavior of the gold nanoparticles in endothelial and epithelial cells. Similar amounts of gold nanoparticles were taken up regardless of the presence or absence of citrate. In addition, ICP-AES analysis showed that epithelial cells internalized more gold nanoparticles than endothelial cells. Similar results were reported by Xia et al. who also showed that different uptake mechanisms of cationic polystyrene nanospheres were observed in human endothelial cells (HMEC) compared to epithelial cells (BEAS-2B). However, these results were not correlated to any quantitative parameter of internalization of the nanospheres. Nevertheless, BEAS-2B cells reacted in a more sensitive manner compared to endothelial cells and exhibited necrotic cell death after exposure. These findings were also observed in RAW 264.7 murine macrophages, with the authors concluding that this difference was due to the more sensitive character of epithelial cells as the first-line-of-defense-features in pulmonary cell types . In our study, the more sensitive character of the epithelial cells after exposure to AuS0302-nanoparticles was reflected by a decreased proliferation rate demonstrated by the Ki-67 assay, and not increased cytotoxicity after exposure to the AuNPs as reported by the group of Xia. The higher sensitivity could also not be explained in detail by Xia et al. One possible explanation for the difference in the uptake between epithelial and endothelial cells might be a different interaction of nanoparticles with specific domains of the plasma membrane. According to Nel et al. different forces are present between the particle and the cell surface which resist or promote nanoparticle wrapping . Both promotive and resistive forces, which might differ from one cell type to another, could influence the uptake of nanoparticles. This hypothesis needs to be investigated further but could be an explanation for the different quantities of gold nanoparticles measured within the various cell types.
The more severe effect on the proliferation of epithelial cells compared to endothelial cells could also be explained by the higher number of gold nanoparticles internalized, in combination with the presence of sodium citrate on the particle surface. AuS0302-RIT nanoparticles were internalized in high amounts after 24 hours exposure, while the proliferation rate in epithelial cells was only slightly decreased compared to AuS0302-RIS02 and AuS0302-RIS04 even after 48 hours. Nevertheless, the influence of sodium citrate on the surface of particles not internalized could also play a role in inducing toxic effects on the cells.