Gold nanoparticles induce cytotoxicity in the alveolar type-II cell lines A549 and NCIH441
© Uboldi et al; licensee BioMed Central Ltd. 2009
Received: 26 January 2009
Accepted: 22 June 2009
Published: 22 June 2009
During the last years engineered nanoparticles (NPs) have been extensively used in different technologies and consequently many questions have arisen about the risk and the impact on human health following exposure to nanoparticles. Nevertheless, at present knowledge about the cytotoxicity induced by NPs is still largely incomplete. In this context, we have investigated the cytotoxicity induced by gold nanoparticles (AuNPs), which differed in size and purification grade (presence or absence of sodium citrate residues on the particle surface) in vitro, in the human alveolar type-II (ATII)-like cell lines A549 and NCIH441.
We found that the presence of sodium citrate residues on AuNPs impaired the viability of the ATII-like cell lines A549 and NCIH441. Interestingly, the presence of an excess of sodium citrate on the surface of NPs not only reduced the in vitro viability of the cell lines A549 and NCIH441, as shown by MTT assay, but also affected cellular proliferation and increased the release of lactate dehydrogenase (LDH), as demonstrated by Ki-67 and LDH-release assays respectively. Furthermore, we investigated the internalization of AuNPs by transmission electron microscopy (TEM) and we observed that particles were internalized by active endocytosis in the cell lines A549 and NCIH441 within 3 hr. In addition, gold particles accumulated in membrane-bound vesicles and were not found freely dispersed in the cytoplasm.
Our data suggest that the presence of contaminants, such as sodium citrate, on the surface of gold nanoparticles might play a pivotal role in inducing cytotoxicity in vitro, but does not influence the uptake of the particles in human ATII-like cell lines.
Nanoparticles (NPs) are generally defined as having a diameter below 100 nm and they are believed to display different properties compared to their bulk material [1–4]. NPs are currently being used extensively in different industrial technologies, in biomedical applications [5–7] and in cosmetics . The main route of exposure to NPs is the respiratory tract, but the human organism can come into contact with particles via ingestion, deposition and injection. The epithelial surface area of a human lung, which is approximately 140 m2 , interacts with an enormous number of inhaled nanosized materials with each breath. Inhaled nanoparticles are then deposited along the respiratory tract depending on their size: discrete inhaled nanoparticles preferentially accumulate in the nasal region, while smaller NPs are able to reach the deep lung, inducing inflammation and the formation of reactive oxidative species in the alveolar region [10–12]. In addition, the alveolar region, which is composed of many different cell types in close association to the endothelium and therefore connected to the blood circulation, is considered to be a privileged site for NPs deposition and translocation. It has been shown that after being internalized NPs are able to escape their site of deposition, entering the blood or the lymphatic circulation and redistributing to other organs, such as the liver and the central nervous system [13–16]. Thus, for example, in rats inhaled ultrafine titanium dioxide NPs have been shown to be taken up and rapidly redistributed from the lung compartment to the circulation [17–19]. In humans, inhaled carbon nanoparticles were reported to translocate from the lung tissue into the blood stream and become distributed in extrapulmonary sites . However, due to the complexity of the pulmonary system, as well as the increasing number of NPs released in the environment, a systematic evaluation of nanoparticles-mediated cytotoxicity has not as yet been possible.
Currently gold nanoparticles (AuNPs) are used in different biomedical applications, such as intracellular gene regulation , chemotherapy  and drug delivery [5, 6], as well as in optical and electronic applications . Investigations on the in vitro cytotoxicity of gold nanoparticles have already been performed, but so far results were inconclusive. AuNPs were shown to induce cytotoxicity in Cos-1 cells  and in human dermal fibroblasts , but not in human leukemic cells  or murine macrophages , where particles were taken up and stored in intracellular perinuclear vesicles. In addition, it is still unknown whether the size or the surface coating of the gold nanosized particles induces cellular damage and cytotoxicity in vitro. In this context, Pan et al.  have shown that the size of the particles plays a pivotal role in inducing cytotoxicity in HeLa cells. They have demonstrated that small AuNPs (1–2 nm size), but not the larger (15 nm size), could induce toxicity in HeLa cells. On the other hand, Niidome et al.  have shown that the toxic potential is triggered by the surface modification of the gold nanoparticles. In fact, bromide-stabilized gold nanorods induced severe cytotoxicity in HeLa cells, whereas PEG-modified gold particles, which displayed a neutral surface, could only induce moderate toxicity. Nevertheless, Connor et al.  demonstrated that neither the surface characteristics nor the size of gold nanoparticles seemed to play a role in inducing cytotoxicity in the human leukemic cell line K562. In fact, they indicated that gold nanoparticles with different size and surface modifications could be internalized by the cells but did not exert cytotoxicity. On the basis of this the authors proposed that the shape of the NPs, and eventually intracellular modifications of the nanoparticles determined by the cellular environment, could be responsible for causing toxicity.
Due to the contradictory results of the previous investigations, the present study was aimed at examining the effect of gold nanoparticles in the deep respiratory tract. To this end, the human alveolar type-II (ATII)-like cell lines A549 and NCIH441 were exposed to AuNPs with different diameter (9.5 – 25 nm) and purification grade (presence or absence of an excess of sodium citrate), and the toxicity induced was evaluated by classical cytotoxicity assays. Briefly, we demonstrated that gold nanoparticles with an excess of sodium citrate impaired the viability, the membrane integrity and the proliferation of the human ATII-like cell lines A549 and NCIH441 in vitro. As a result, we then examined whether the non-toxic potential of the gold nanoparticles without sodium citrate was due to an impaired uptake of the particles. By transmission electron microscopy (TEM) we observed that AuNPs were indeed internalized by the ATII-like cell lines A549 and NCIH441, independent of the presence or absence of contaminants on the particle surface, and independent of the size and the shape of the particles.
Results and Discussion
Synthesis and characterization of gold nanoparticles
Physicochemical characterization of the gold nanoparticles.
dV mean (nm)
D STEM (nm)
To remove the excess of sodium citrate retained during the synthesis, the sample AuS0302 underwent filtration and dialysis, and the purified product was identified as AuS0302-RIT. The physicochemical properties of the particles were preserved during tangential flow filtration (Table 1), and this procedure did not induce nanoparticle agglomeration, as was shown by STEM (Figure 1). Analysis of the dried permeate by differential scanning calorimetry – thermogravimetry (DSC-TG) revealed that by filtration nearly 92% of sodium citrate was removed from the final filtrate solution AuS0302-RIT (data not shown). Nevertheless, the presence of PVP residues on the surface of the gold nanoparticles cannot be excluded. In addition, possible effects on the particles surface following dialysis were investigated by measuring the Zeta potential of the particles, revealing that the Zeta potential before (-13 ± 2 mV) and after (-12 ± 2 mV) dialysis did not change.
Cytotoxicity of gold nanoparticles
Lactate dehydrogenase (LDH) release assay
To summarize, the presence of sodium citrate on the surface of gold nanoparticles reduced the viability (as shown by MTT assay) and impaired the proliferation (as demonstrated by Ki-67 assay) in both the human ATII-like cell lines A549 and NCIH441. In addition, the results obtained by MTT and Ki-67 assay suggest that the cell line NCIH441 is slightly more resistant to the AuNPs-induced cytotoxicity compared to the cell line A549. However, our data indicate that it is the presence of contaminants, such as sodium citrate, and not the size of the particles that impairs the viability and the proliferation of the cell lines A549 and NCIH441. These results are consistent with previous investigations performed with dermal fibroblasts  and leukemic cells . Pernodet et al.  demonstrated that gold/citrate nanoparticles impaired the proliferation of dermal fibroblasts and induced an abnormal formation of actin filaments, causing therefore a reduced cellular motility and influencing the cell morphology. On the contrary, Connor et al.  reported that citrated and biotinylated 18 nm AuNPs did not induce toxicity in leukemic cells (cell line K562), whereas smaller particles were much more toxic. In addition, gold nanoparticles did not exert any toxic effect, as shown by the MTT assay, in the human gastrointestinal cancer cells Panc-1 and HepB3 , nor in HeLa cells [27, 31]. Hauck et al.  have demonstrated that the use of different surface coatings does not influence the toxicity induced by gold nanoparticles in HeLa cells, but it was of extreme importance to tune the uptake of AuNPs. In addition, Hauck et al.  suggest that the lack of toxicity was due to the fact that AuNPs were stored intracellularly in membrane-bound vesicles and therefore particles could not directly interfere with the nuclei or with other cytoplasmic organelles and induce toxic events. Nevertheless, there is evidence that the size of the gold nanoparticles induces in vitro cytotoxicity in HeLa cells. In fact, Pan et al.  have shown that the size, and not the particle chemistry, is responsible for determining the toxicity of the gold particles. After exposing for 48 hr the cells to increasing concentrations of nanosized gold particles, by MTT assay Pan et al. observed that Au clusters of different sizes had a different toxic potential. In addition, they demonstrated that on using different particles stabilizers, the toxicity observed was almost indistinguishable, leading the authors to conclude that it is the size of the particles to play a pivotal role in inducing in vitro cytotoxicity.
Citrate is commonly used as reducing agent and its presence on the surface of gold nanoparticles might interfere with the MTT assay, thus leading to an overestimation of the viability data and to the conclusion that AuNPs exert only a mild toxicity or, in some cases, no toxicity at all in the cell lines A549 and NCIH441. Consequently, the cytotoxicity induced by AuNPs was further investigated by Ki-67 and LDH release assay. By Ki-67 assay it has been shown that the sodium citrate-nanoparticles AuS0302-RIS02 and AuS0302-RIS04 impaired (up to 50–70% reduction) the proliferation of the cell lines A549 and NCIH441. A slightly reduced proliferation was also measured following exposure to the sample AuS0302-RIT, which presents a significantly reduced amount of sodium citrate as surface contaminant. Investigating the amount of lactate dehydrogenase (LDH) released upon exposure to AuNPs we have shown that the major effects are detectable after 72 hr treatment. In addition, in the cell lines A549 and NCIH441 a dose-dependent LDH release was observed. Summarizing, we demonstrated the cytotoxic potential of gold nanoparticles in alveolar type-II-like cell lines using different assays (MTT, Ki-67 and LDH release) and different end-points (cell viability, cell proliferation and membrane integrity).
Internalization of gold nanoparticles
In conclusion, our results suggest that the presence of contaminants, such as sodium citrate, affects the viability and the proliferation of the human alveolar type-II-like cell lines A549 and NCIH441 in vitro, rather than the size of the particles. Dialyzed and purified gold nanoparticles could induce a milder cytotoxicity in A549 and NCIH441 cells compared to the particles with an excess of sodium citrate. In addition, the absence of contaminants does not influence the uptake of gold particles. Our data support the fact that the human ATII-like cell lines A549 and NCIH441 internalize gold NPs by active endocytosis independent of the size and of the presence or absence of contaminants in solution. Moreover, the uptake can be inhibited by exposing the cells to low temperature. Further studies are underway to elucidate the uptake mechanism and the intracellular fate of gold nanoparticles.
Synthesis and characterization of gold nanoparticles
Gold nanoparticles (AuNPs) were synthesized in water by reducing tetrachloroauric acid (HAuCl4, purchased by Colorobbia S.p.A., Italy) in a sodium citrate solution containing polyvinylpirrolidone (PVP; (C6H9NO)n) as stabilizing agent [23, 36]. To produce nanoparticles which retain their physicochemical properties over time and do not aggregate, the wet chemical synthesis of AuNPs via reduction of gold salts was employed . A water solution of 0.5% (w/w) PVP, purchased by Toscochimica (Prato, Italy), was heated to 60°C and HAuCl4, characterized by a gold content of 30%, as determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES), was added to obtain a final gold concentration of 0.2% (w/w). To this intermediate compound, an aqueous solution of sodium citrate (16.5% w/w) was added and the resulting product was kept at 60°C for 1 hr and afterwards air-cooled to room temperature. The resulting sample was named AuS0302 and was characterized by the presence of an excess of sodium citrate and a particle diameter of 9.5 nm as shown by STEM images. A second series of gold nanoparticles with larger average size (samples AuS0302-RIS02 and AuS0302-RIS04) were prepared as follows: to synthesize AuS0302-RIS02, AuS0302 was used as initial seed (25% of the total gold content) and mixed with PVP. The solution was heated to 60°C and suitable amounts of tetrachloroauric acid were added to maintain the final gold concentration at 0.2% (w/w). As final step, sodium citrate was added and the solution kept at 60°C, prior to cooling to RT. The batch AuS0302-RIS04 was prepared using a similar procedure, but the compound AuS0302-RIS02 was used as seed for this synthesis. To remove excess of sodium citrate from the sample AuS0302, tangential flow dialysis was performed using an Amicon system (Millipore) equipped with Biomax membrane (cut-off 10.000 KDa) (Millipore). The total volume dialysed was 36 times the initial volume and the resulting samples, which had only about 8% sodium citrate on the particle surface, was termed AuS0302-RIT.
Cell culture and Cytotoxicity assays
The human alveolar type-II (ATII)-like cell lines A549 (ATCC number CCL-185,) and NCIH441 (ATCC number HTB-174) were purchased by LGC Promochem (Wesel, Germany). Cells were cultured in complete cell culture medium composed of RPMI 1640 with L-Glutamine (Invitrogen Corporation, Germany) supplemented with 10% (v/v) Fetal Bovine Serum (Sigma Aldrich, Germany) and 1% (v/v) 10000 U/ml Penicillin and 10000 U/ml Streptomycin (Invitrogen Corporation, Germany). Cells were maintained under standard cell culture conditions (5% CO2, 95% humidity and 37°C in HERAEUS incubators, Germany) and passaged weekly.
The cytotoxicity induced by gold nanoparticles was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Ki-67 assay and lactate dehydrogenase (LDH) release assay. All tests were performed using sub-confluent cells in the logarithmic growth phase. Cells (4.0 × 104 A549 cells/ml and 1.5 × 105 NCIH441 cells/ml; 100 μl/well in complete cell culture medium) were seeded in 96 well plates (TPP, Switzerland). Cells were then exposed for 24, 48 and 72 hr to concentrations of gold nanoparticles ranging from 0 mM to 0.7 mM. Nanoparticles were dispersed in complete cell culture medium RPMI 1640 with L-Glutamine (Invitrogen Corporation, Germany), with 5% Fetal Bovine Serum (Sigma Aldrich, Germany) and 1% (v/v) 10000 U/ml Penicillin and 10000 U/ml Streptomycin (Invitrogen Corporation, Germany). For each gold nanoparticle a blank solution was tested and no cytotoxicity could be observed. A 10% DMSO solution was used as positive control. Three independent experiments and 3 replicates for each experiment were performed.
In the MTT assay, after 60 hr incubation cell culture medium was replaced with fresh medium containing AuNPs at concentrations ranging from 0 mM to 0.7 mM. After 24, 48 and 72 hr continuous exposure to AuNPs, medium was removed, cells were washed with PBS and then incubated with cell culture medium containing 20% MTT solution (stock solution 5 mg MTT/ml PBS; Sigma Aldrich, Germany). After 3 hr incubation at 37°C and 5% CO2, 100 μl lysis buffer (20 g SDS dissolved in 50 ml ddH2O and supplemented with 50 ml N, N-Dimethylformamide. pH 4.7) per well was added. Cells were further incubated overnight and the absorbance was measured by spectrophotometry at λ1 = 620 nm and λ2 = 750 nm. Results were analyzed as the average of viability (% of the untreated control) ± Standard Deviation (SD).
For the Ki-67 assay, sub-confluent cells were exposed to gold nanoparticles for 24–72 hr, fixed using 1% (v/v) methanol/ethanol solution and washed five times in PBS. Fixed cells were permeabilized with 0.1% Triton-x 100 (v/v) in PBS, and after 10 minutes of permeabilization at room temperature, cells were washed three times in a solution containing PBS/0.05% Tween-20 (v/v) and incubated (45 minutes at 37°C on a rotating plate) with 1 μg/ml mouse anti-human Ki-67 antibody (clone MIB-1; DAKO, Germany). Cells were then incubated for 45 minutes at 37°C with anti-mouse IgG1 peroxidase (DAKO, Germany), and washed with PBS/0.05% Tween-20. Afterwards cells were incubated 10–20 minutes at 37°C with a solution (170 μl/well) composed by 5 mL 10× citric buffer mixed with 45 mL water, 20 μL 30% oxygen peroxide and o-Phenylenediamine. The solution was pipetted in a new 96 well plate containing 3 M chloridric acid (HCl) (50 μl/well), and the fluorescence was detected by spectrophotometry at 492 nm. Results are expressed as the mean value of cellular proliferation (% of the untreated control) ± Standard Deviation (SD).
In the LDH assay (Promega Corporation, Germany) 50 μl/well supernatant, collected after exposing the cell lines A549 and NCIH441 to AuNPs, were incubated with an equivalent volume of substrate solution. After incubating the plate for 30 minutes at RT, 50 μl/well stop solution was pipetted and data were acquired by spectrophotometry at 490 nm. Results are expressed as mean LDH release (expressed as experimental LDH release at 490 nm/maximum LDH release at 490 nm ± Standard Deviation (SD).
Uptake and transmission electron microscopy studies
The human ATII-like cell lines A549 and NCIH441 were seeded on fibronectin-coated Thermanox cover slips (NUNC – Thermo Fisher Scientific, Germany) and cultivated until confluent. For the incubation at 37°C, cells were exposed to 0.3 mM and 1 mM gold nanoparticles AuS0302-RIT, AuS0302-RIS02 and AuS0302-RIS04. For the incubation at 4°C, cells were first pre-cooled for 30 minutes at 4°C, and then exposed to ice-cold AuNPs in the same concentration as for the 37°C test. For both cell incubation conditions, after 3 hr exposure to AuNPs cells were fixed in 3.7% (v/v) paraformaldehyde in PBS (20 min at RT). Afterwards cells were prepared for transmission electron microscopy analysis as follows: cells were fixed in 1% (w/v) osmium tetroxide for 2 hr and dehydrated in ethanol. Cells were passaged through propylene oxide, then samples were embedded in agar-100 resin (PLANO, Germany) and polymerized at 60°C for 48 hr. Ultrathin sections were cut with an ultramicrotome (Leica Microsystems, Germany), placed onto copper grids and stained with 1% (w/v) uranyl acetate in alcoholic solution and lead citrate. Ultrastructural analysis and photomicroscopy were performed with a transmission electron microscope EM 410 (Philips; Eindhoven, Netherlands).
This work has been supported by the European Commission, STREP project FP6 – 2004 – NMP – TI4 – 032731. The authors are grateful to Mrs Susanne Barth for the excellent assistance in cell culture. The authors acknowledge Mrs Marianne Müller, Mrs Karin Molter and Mrs Luise Meyer for their technical assistance during TEM analysis.
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