Toxicity of gold nanoparticles in a commercial dietary supplement drink on connective tissue fibroblast cells
Due to an increased use of gold nanoparticles (AuNPs) in various consumer products we aimed to investigate the cytotoxic impact of AuNPs found in a commercial dietary supplement drink (SD-AuNPs). The cytotoxic impact of SD-AuNPs was determined in mouse connective tissue fibroblasts (L929). The cells were treated with different concentrations of SD-AuNPs (0.2, 0.5, 10, 20, 30, 40, and 50 µg/mL). The results indicated that the toxicological effect of SD-AuNPs on L929 cells occurred in a dose-dependent manner. Significant reduction of cell viability was found in L929 cells treated with 10, 20, 30, 40, and 50 µg/mL SD-AuNPs for 24 h. L929 cells treated with 40 and 50 µg/mL SD-AuNPs produced significant intracellular reactive oxygen species and TNF-α. High levels of intracellular uptake of these compounds were also observed in L929 cells treated with 50 µg/mL SD-AuNPs.
KeywordsGold nanoparticles Commercial dietary supplement ROS generation Inflammation Connective tissue fibroblasts
Gold nanoparticles (AuNPs) have been of interest in biomedical applications [1, 2, 3, 4]. Furthermore, they have been included in consumer food and drink products . Accordingly, direct exposure of humans to AuNPs through ingestion, inhalation, and skin contact has increased. Although many reports have shown that AuNPs have less toxicity than other types of metal-based nanoparticles, their prevalent use makes it still of importance to evaluate the effect of AuNPs contained in consumer products. The intensive use of AuNPs in daily life can lead to more chances of exposure of the human body to these nanoparticles. Several investigations on the toxic effects of synthesized AuNPs, both from academic research laboratories and commercial sources, have been reported [5, 6, 7]. Recently, there have been many commercial dietary supplement drinks on the market that contain AuNPs (SD-AuNPs). These companies claim that addition of AuNPs into dietary supplement drinks provides health benefits, including acting as an anti-inflammatory agent, and can lead to mood enhancement, and improvements in cognitive function . Therefore, these products can be officially labeled as dietary supplement drinks. However, the addition of AuNPs into these dietary supplement drinks increases the direct exposure of the human body to AuNPs. Most products that contain nanoparticles do not provide detailed information on the prepared nanoparticles used in the products. This issue has led to a growing concern for human health because any risk posed by those nanoparticles is unknown. Especially based on our available knowledge, the information of cytotoxic investigation of AuNPs contained in dietary supplement drinks is very rare. Due to this reason, we were interested in testing whether SD-AuNPs could have an impact on cellular activities. To perform these studies, we used connective tissue fibroblast cells (L929) as test cells in our study. The reason that we focused on this type of fibroblast cells is because they are the predominantly common cells in connective tissue, which is the most abundant tissue in the body. Fibroblast cells play a major role in building to the extracellular matrix to support and maintain tissue structures including oral cavity and intestine. When nanomaterials containing in the drink are consumed, both oral and intestinal absorption of nanomaterials by fibroblasts can occur. Furthermore, L929 cells have been widely used in a standardized nanotoxicity test [9, 10, 11, 12]. Thus, we used L929 cells to explore the toxicity of SD-AuNPs. It should be emphasized that we separated SD-AuNPs out from a dietary supplement drinks through centrifugation, characterized their properties, and then used them for cytotoxicity assessment. A commercial dietary supplement drink with a claim of containing AuNPs was used in this work. We investigated how SD-AuNPs impact the cell viability, reactive oxygen species (ROS) generation, and the inflammatory responses of L929 fibroblasts. Cellular uptake of SD-AuNPs was also investigated to provide more information on the relationship between the amount of AuNPs taken up by fibroblasts and cytotoxicity. A study of cellular response to SD-AuNPs was also included.
2 Materials and methods
2.1 Characterization of SD-AuNPs from a representative dietary supplement drink
The size of SD-AuNPs contained in a representative dietary supplement drink was characterized by using transmission electron microscopy (TEM; TECNAI 20 TWIN, USA). To separate SD-AuNPs from the drink, 1 mL of drink was centrifuged at 9,391 g for 15 min to pellet SD-AuNPs. The SD-GNP pellet was then dispersed in Milli-Q water and the suspension was applied to a copper grid coated with formvar film and dried for TEM analysis. Light absorption of SD-AuNPs was also measured using UV-vis spectroscopy (Shimadzu UV-2550, Japan). The zeta potential and hydrodynamic diameter of the SD-AuNPs were measured by using dynamic light scattering (DLS, Zetasizer, Malvern, UK). The SD-AuNPs were dispersed in Milli-Q water or in cell culture media to check whether the cell culture media has any impact on light absorption, zeta potential, and hydrodynamic size of SD-AuNPs. The SD-AuNPs used in this work is a commercial product purchased from Japan. Based on product information, the surface of SD-AuNPs in the test drink was stabilized with carboxymethyl cellulose (CMC). Other ingredients are water and minerals (sodium, potassium, calcium, and magnesium).
2.2 Cell culture
L929 cells (Riken Cell Bank) were cultured in RPM 1640 medium supplemented with 10% FBS plus 1% penicillin–streptomycin. Cells were maintained in a 5% CO2 humidified incubator at 37 °C.
2.3 Measurement of SD-AuNPs gold concentration by inductively coupled plasma mass spectrometry (ICP-MS)
The gold concentration in the SD-AuNPs was investigated using ICP-MS (Agilent Technologies, USA). A 4.5 mL of SD-AuNPs diluted in Milli-Q water (100 ×) was mixed with a 250 µL of 65% HNO3 and a 250 µL of 37% HCl. The mixed solution was heated using a block heater at 100 °C for 5 h, and ICP-MS measurements were performed. A solution of Au(III) at concentrations of 0, 5, 10, 20, 50, 100, and 200 ppb was prepared for making a linear calibration curve. Optimization of rf power, nebulizer gas flow, auxiliary gas flow, and plasma gas flow was performed before the measurement.
2.4 Cell viability assay
A cell viability measurement was performed by seeding L929 cells at a density of 1 × 104 cells/well into a 96-well plate and culturing in RPMI 1640 supplemented with 10% FBS and 1% antibiotic solution for 24 h at 37 °C. After culturing, various concentrations of SD-AuNPs (0.2, 0.5, 10, 20, 30, 40, and 50 µg/mL) in fresh cell culture media were applied to the cells for 24 h. L929 cells in media were used as control. L929 cells exposed to 1 µg/mL of lipopolysaccharide (LPS from Escherichia coli; O111:B4, Sigma) were under the same conditions as the positive control. After treatment, cells were washed twice with a serum-free medium, and the viability was measured using a CellTiter-Glo assay (Promega) following the protocol provided by the manufacturer. Luminescence signals were collected using a microplate reader (SpectraMax® M3, USA). The relative cell viability was calculated by comparing the luminescent signal of the treated cells with those of the untreated cells.
2.5 ROS generation detection
L929 cells (1 × 104 cells/well) were seeded into a 96-well plate. After incubation for 24 h the cells were exposed to various concentrations of SD-AuNPs (from 0.2 to 50 µg/mL) for 3 h at 37 °C. Cells treated with H2O2 were also prepared as a positive control. The treated condition was performed in RPMI 1640 supplemented with 10% FBS and 1% antibiotic solution. The cells were washed twice with a serum-free medium and treated with 10 µM dichloro-dihydro-fluorescein diacetate (DCFH-DA, Sigma) for 30 min. After treatment, the DCFH-DA was removed and the cells were washed twice with cold PBS before lysing with 20% Tween-20 (in PBS w/o Ca2+ and Mg2+). During the lysing process, cells were shaken for 30 min. The fluorescence signals were measured at 492 nm and 595 nm using a microplate reader.
2.6 Pro-inflammatory cytokine measurement
L929 cells (1 × 104 cells/well) were exposed to SD-AuNPs at concentrations of 0.2, 0.5, 10, 20, 30, 40, and 50 µg/mL for 24 h. After exposure, the cell culture media were collected and centrifuged at 9,391 g for 15 min to remove SD-AuNPs remaining in the cell culture media (RPMI 1640 supplemented with 10% FBS and 1% antibiotic solution). Thereafter, the supernatant of each treatment was stored at − 80 °C and used for determining inflammatory cytokine production (Interleukin-1beta (IL-1β); Interleukin-6 (IL-6); and Tumor necrosis factor (TNF-α)) using an enzyme-linked immunosorbent assay (ELISA) kit (Biolegend). This assay was performed by following the instructions from the manufacturer.
2.7 Cellular uptake of SD-AuNPs and localization of SD-AuNPs in L929 cells
The cellular uptake of SD-AuNPs by L929 cells was investigated using ICP-MS (PerkinElmer, USA). The measurement was done by following the protocol published by Kim et al. . L929 cells were seeded onto a 24-well plate at a density of 2 × 104 cells/well and incubated for 24 h at 37° C in a 5% CO2 incubator. Cells were then cultured with RPMI-1640 medium plus 10% FBS containing SD-AuNPs at 0.2 and 50 µg/mL for 24 h. After the incubation, cells were washed three times with PBS and then lysed by addition of 20% Tween-20 in PBS and sonicated for 30 min. The cell lysate was digested overnight by addition of 250 µL of digestion buffer (3 mL of HNO3 and 1 mL of H2O2). Next, a 3 mL of aqua regia (HCl: HNO3 = 3:1) was added into the samples and incubated for 2 h. After incubation, the samples were diluted, and the final samples contained 5% aqua regia. A gold standard solution (Au(III)) at concentrations of 0, 0.2, 0.5, 1.0, 2.0, 5.0, 10, and 20 ppb was prepared to make a calibration curve used for determining the amount of gold taken up by the cells. The operating conditions of the ICP-MS were also set before measuring the gold content.
The localization of SD-AuNPs inside cells was investigated by using TEM. L929 cells at a density of 1 × 105 cells/mL were cultured in a cell culture dish containing a glass cover slip for 24 h. Following incubation, cells were treated with 0.2 and 50 µg/mL SD-AuNPs for 24 h. Cells were then washed with 0.1 M sodium cacodylate buffer (SCB). After washing, cells were pre-fixed with 2.5% glutaraldehyde dissolved in 0.1 M SCB. The pre-fixed cell samples were stored at 4 °C overnight. Next, cells were washed with 0.1 M SCB three times and then the cells were post-fixed with 1% osmium tetroxide (OsO4) dissolved in 0.2 M SCB at 4 °C for 1 h. Cells were washed again after fixing by using the same washing process mentioned previously. Following this, cells were dehydrated by performing a sequential treatment with serial dilutions of ethanol at different concentrations (30%, 50%, 70%, 80%, 90%, and 95%) for 15 min (at each concentration) at 4 °C. 100% ethanol was added to the cells and soaked for 15 min at 4 °C. This was repeated 4 times in total. Following this soaking step, the embedding process was done. With this process, a mixture of propylene oxide and 100% ethanol at a ratio of 1:1 was added to cells, incubated for 15 min at room temperature, and then decanted. Mixtures of propylene oxide and araldite resin at ratios of 3:1, 1:1, and 1:3 were then prepared at room temperature. A 3:1 propylene oxide/araldite resin mixture was added to the cells and incubated for 1 h and then the mixture was decanted. The process was repeated using a 1:1 propylene oxide/araldite resin mixture. Finally, the 3:1 propylene oxide/araldite resin mixture was added to the cells and the sample was polymerized in a hot air oven at 45 °C for 2 days, and then at 60 °C for another 2 days. The embedding process was prepared by transferring the sample into a mold and pure araldite resin was added. The embedded sample was further incubated in a hot air oven at 60 °C for 2 days. Finally, ultrathin sections were cut from the cell samples at a thickness of 60–90 nm and observed by TEM (268 Morgagni, Thermofisher, The Netherlands) at 80 kV.
2.8 Statistical analysis
All experimental data are expressed as a mean ± standard error (SE). The data were analyzed by using one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test in GraphPad Prism® version 6. The significance of statistical data was calculated at p ˂ 0.01.
3 Results and discussion
3.1 Characterization of SD-AuNPs
3.2 Cell viability and cellular uptake of SD-AuNPs
3.3 Reactive oxygen species (ROS) generation
3.4 Inflammatory cytokine response
We have demonstrated the impact of SD-AuNPs in a commercial supplement drink on L929 cells. The cytotoxic impact of SD-AuNPs on L929 cells is highly dependent on concentration. Although AuNPs have been applied in many biological applications, it is important to be aware of their toxicity. Our results provide information that although the product is sold in the form of a supplement drink, consumers still need to pay strong attention to the consumption dose (the suggested dose of this SD-AuNPs is ~ 0.2 µg/mL) to avoid any adverse effects that may occur with ingestion.
This work received major funding support from the Capability Building Unit for Nanoscience and Nanotechnology, Faculty of Science, Mahidol University. The authors thank the Center of Excellence on Environmental Health and Toxicology (EHT), Toxicology and Materials Science and Engineering Programs, Center of Nanoimaging (CNI), and Prof. Piyarat Govitrapong at Faculty of Science, Mahidol University for supporting some facilities used in this work. The authors also thank minor funding support from the Development and Promotion for Science and Technology talents project (DPST). Furthermore, the authors are thankful for the extra funding from the Faculty of Science to support this work. Finally, the authors thank Dr. Sujin Jiracheewanun and Mr. Preecha Sowanthip for their technical support.
Compliance with ethical standards
Conflict of interest
On behalf of all authors, the corresponding author states that there is no conflict of interest.
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