Quantification of the Uptake of Silver Nanoparticles and Ions to HepG2

Mar 4, 2013 - ABSTRACT: The toxic mechanism of silver nanoparticles. (AgNPs) is still debating, partially because of the common co- occurrence and the...
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Quantification of the Uptake of Silver Nanoparticles and Ions to HepG2 Cells Su-juan Yu, Jing-bo Chao, Jia Sun, Yong-guang Yin, Jing-fu Liu,* and Gui-bin Jiang State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, Beijing 100085, China ABSTRACT: The toxic mechanism of silver nanoparticles (AgNPs) is still debating, partially because of the common cooccurrence and the lack of methods for separation of AgNPs and Ag+ in biological matrices. For the first time, Triton-X 114-based cloud point extraction (CPE) was proposed to separate AgNPs and Ag+ in the cell lysates of exposed HepG2 cells. Cell lysates were subjected to CPE after adding Na2S2O3, which facilitated the transfer of AgNPs into the nether Triton X114-rich phase by salt effect and the preserve of Ag+ in the upper aqueous phase through the formation of hydrophilic complex. Then the AgNP and Ag+ contents in the exposed cells were determined by ICP-MS after microwave digestion of the two phases, respectively. Under the optimized conditions, over 67% of AgNPs in cell lysates were extracted into the Triton X-114-rich phase while 94% of Ag+ remained in the aqueous phase, and the limits of detection for AgNPs and Ag+ were 2.94 μg/L and 2.40 μg/L, respectively. This developed analytical method was applied to quantify the uptake of AgNPs to the HepG2 cells. After exposure to 10 mg/L AgNPs for 24 h, about 67.8 ng Ag were assimilated per 104 cells, in which about 10.3% silver existed as Ag+. Compared to the pristine AgNPs (with 5.2% Ag+) for exposure, the higher ratio of Ag+ to AgNPs in the exposed cells (10.3% Ag+) suggests the transformation of AgNPs into Ag+ in the cells and/or the higher uptake rate of Ag+ than that of AgNPs. Given that the toxicity of Ag+ is much higher than that of AgNPs, the substantial content of Ag+ in the exposed cells suggests that the contribution of Ag+ should be taken into account in evaluating the toxicity of AgNPs to organisms, and previous results obtained by regarding the total Ag content in organisms as AgNPs should be reconsidered.

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AgNPs may lead to the slowly release of Ag+,13,14 which was known as one of the most toxic heavy metals. Therefore, a central question is whether the toxicity is owing to nanoparticle itself or is related to Ag+ stemmed from the AgNP dissolution. Navarro et al.15 studied the toxicity of AgNPs to a freshwater algae Chlamydomonas reinhardtii by eliminating the effects of Ag+ with cysteine. They found that the toxicity was mainly due to the ions and AgNPs probably acted as sources of Ag+. Also, AgNPs exhibited higher toxicity to human mesenchymal stem cells (hMSCs) with the prolonged store time,16 indicating that the toxicity is mostly related to Ag+ that slowly released during storage. However, Kawata et al. observed that Ag+ could not fully elucidate the toxicity to HepG2 cells,17 suggesting both nanoparticles and ions devoted to the toxicity. It was also observed that the toxic effects of AgNPs on Lolium multif lorum were more than the silver ions.18 Recently, Xiu et al.19,20 provided compelling evidence that Ag+ exhibited much more antimicrobial activity than AgNPs, and AgNPs themselves were not toxic to bacteria. To achieve detailed insights into the observed toxicity of AgNPs and distinguish the toxicity of AgNPs and Ag+, it is in urgent need to develop analytical methods for selective determination of AgNPs and Ag+ in different matrices.

INTRODUCTION Due to their excellent antibacterial activity, silver nanoparticles (AgNPs) have been used in a great number of consume products and medical devices, such as textiles,1 food packaging,2 room sprays,3 wound dressings and implantable catheters.4 It is estimated that AgNPs are among one of the fastest growing product categories in nanotechnology industry, and have the highest degree of commercialization.5 An inevitable consequence of the widespread use of AgNPs is their release into the environment both deliberately and accidentally. Studies have shown that silver can easily release into wastewater from AgNPs containing textiles1 and sock fabrics6 during washing, and the release of AgNPs from exterior paints was also observed.7 This may pose a threat to the environmental organisms and the human body. In an in vivo study it was found that AgNPs deposited in embryos at each developmental stage of zebrafish, and high dose of AgNPs could give rise to obvious developmental abnormality.8 Moreover, cytotoxicity and genotoxicity tests with human lung fibroblast cells (IMR-90) and glioblastoma cells (U251) showed that AgNPs could disrupt the mitochondrial respiratory chain, cause the accumulation of ROS, and even induce DNA damage.9 Although there are many studies on the toxicity of AgNPs, the mechanisms of AgNP toxicity remain unclear. Because of the nanoscale dimension, AgNPs may directly pass through cell membrane and interact with DNA or important enzymes to cause damage.10−12 On the other hand, partial oxidation of © 2013 American Chemical Society

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October 24, 2012 February 22, 2013 March 4, 2013 March 4, 2013 dx.doi.org/10.1021/es304346p | Environ. Sci. Technol. 2013, 47, 3268−3274

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(EDS) analysis were carried out with an H-7500 (Hitachi, Japan) at 80 kV or a high resolution TEM coupled with an EDS (TECANI G20, FEI, Hillsboro OR) at 200 kV. TEM samples were prepared by loading 5 μL aliquots of the aqueous sample or the TX-114-rich phase (at 30 times diluted with 1:1 methanol and water) onto carbon-coated copper grid and drying at room temperature. The Zeta potential in the range of pH 2.5−10 and the hydrodynamic diameter of AgNPs were measured by dynamic light scattering (DLS) with a Malvern Nano ZS (Malvern Instruments, UK). Measurement of Ag+. The total Ag+ concentration in the stock AgNP solution was analyzed by our previous reported CPE method for speciation analysis of AgNPs and Ag+.27 Briefly, into the 10 mL long tapered glass centrifuge tube was added ∼9.5 mL of AgNPs solution that was adjusted to pH 3.0 with diluted HNO3, and then 0.1 mL of 1 M Na2S2O3, and 0.2 mL of 10% (w/v) TX-114 were added. After incubation in a 40 °C water bath for 30 min, the mixture was centrifuged at 2000 rpm (∼640g) at room temperature for 5 min to facilitate the phase separation. After CPE, the AgNPs were selectively extracted into the TX-114-rich phase. By respective determining the total Ag concentration (CTAg) without CPE and the concentration of the AgNPs (CNAg) extracted in TX-114-rich phase with ICP-MS after microwave digestion, the total Ag+ concentration (CTAg+) was calculated as CTAg+ = CTAg - CNAg. To test whether the centrifugal filter units are suitable for separating Ag+ from AgNPs in cell lysates, 10 mL cell lysates spiked with 1 mg/L Ag+ were added into Amicon centrifugal ultrafilter units (Ultra-15 3K, Millipore, cellulose membranes with 1−2 nm pore size), and subjected to centrifugation for 30 min at 8000 rpm. Then the filtrates were collected for further analysis by ICP-MS. Cell Culture and Treatments. Human hepatoma HepG2 cells, which have been wildly used in toxicological studies, were adopted in this present study. The cells were cultured in cell culture medium (DMEM/High Glucose-FCS) consisting of DMEM/High Glucose-FCS (GIBCO, Invitrogen GmbH) supplemented with 10% fetal calf serum (FCS, GIBCO, Invitrogen GmbH) using 100 mm cell culture plates. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2. When the cells were grown to 70% confluency in 100 mm culture dishes, they were incubated with the cell culture medium spiked with 10 mg/L of AgNPs for 24 h. Following the incubation period, medium was removed and the adherent cells were washed several times with phosphate buffered saline (PBS) solution, and then detached from the culture plates by adding 1 mL of 0.25% enzyme trypsin for 3 min at 37 °C. Subsequently, the cells were washed with PBS and collected. Then the mixture was centrifuged at 1500 rpm for 5 min at 4 °C, and cell number was counted. For optimization of the CPE parameters, the cells were cultured in the same way but without the AgNPs exposure procedure. The isoelectric point of cell lysates was also determined by measuring the Zeta potential of cell lysates in the range of pH 3−10 with a Malvern Nano ZS (Malvern Instruments, UK), which was conducted after the collected cells were disrupted by sonication and adjusted to 10 mL with ultrapure water. CPE of AgNPs in HepG2 Cells. For optimizing the CPE parameters, HepG2 cells cultured without exposure to AgNPs were used as matrix. Briefly, the obtained cells in a 10 mL long tapered glass centrifuge tube were disrupted by sonication, and adjusted to 9 mL by adding ultrapure water. After adding 100

Currently available methods for quantification of AgNPs are mainly based on the difference between the total contents of Ag and the amounts of Ag+. The total Ag amounts were often measured by ICP-MS,15 ICP-OES21 or atomic absorption spectroscopy13 after digestion, while Ag+ were quantified by ion-selective electrode,15 or by ICP-MS after separation with centrifugal ultrafiltration,13,15,21 diffusive gradients in thin films15 or field flow fractionation.22 Several toxicology studies also reported the uptake and intracellular distribution of AgNPs in different cell lines,11,12,23 in which the uptake amount of AgNPs was determined by measuring the total silver content after digesting the exposed cells. In this way, the exact amounts of AgNPs in cells would be overestimated, as it had been proven that besides AgNPs could enter into cells by endocytosis,9−12 Ag+ can also be uptake into the cells.12,23 In addition, the AgNPs in cells can be metabolized to release Ag+. For example, reactive oxygen species (ROS) are natural byproducts of respiring organisms and generated during the metabolism of oxygen by the electron transport chain. Previous study has revealed that ROS were more powerful oxidants than O2 and could significantly enhance the release of Ag+.13 Thus, there has been a strong request for the development of approaches to selectively extract AgNPs or Ag+ from complex biological media. Surfactant Triton X-114 (TX-114)-based cloud point extraction (CPE) is a cost-effective approach to concentrate trace nanomaterials in environmental waters, and effectively preserve the size and shape of nanomaterials during extraction.24−26 Recently, the CPE method was also applied to speciation analysis of AgNPs and Ag+ in several commercial available antibacterial products.27 After the CPE procedure, AgNPs were enriched in the TX-114-rich phase, whereas Ag+ remained in the aqueous phase. By measuring the total Ag contents and the AgNPs in the TX-114 phase, the Ag+ concentration could be calculated. Herein, we report for the first time the selective extraction of AgNPs from exposed cells by CPE, aiming to develop an efficient method for the quantification of AgNPs in biological matrices. Parameters that influence the extraction efficiency of AgNPs from exposed cells like salt concentration, surfactant content, pH, and incubation time were valued. The extracted AgNPs were identified by transmission electron microscope (TEM) and energy dispersive X-ray spectroscopy (EDS), and quantified by ICP-MS after microwave digestion.

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MATERIALS AND METHODS Materials. Water suspension of PVP-coated AgNPs was purchased from Shanghai Huzheng Nanotechnology CO., Ltd. (Shanghai, China). The stock AgNPs solution was prepared by centrifugation at 17 000 rpm (∼19 000g) for 30 min, then redispersed in water, and stored at 4 °C in the dark for further use. TX-114 was obtained from Acros Organics (Geel, Belgium). Nitric acid (65%) was purchased from Merck (Darmstadt, Germany). Ag+ stock solution (1000 mg/L) prepared in 5% (v/v) HNO3 was purchased from National Institute of Metrology (Beijing, China). All the other chemicals were obtained from Sinopharm Chemical Reagent Co. (Beijing, China) with grades of analytical or better, and were used without further purification. Ultrapure water (18.3 MΩ) produced with a Milli-Q Gradient system (Millipore, Bedford) was used throughout the experiments. AgNP Particle Characterization. Transmission electron microscope (TEM) and energy dispersive X-ray spectroscopy 3269

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Figure 1. Characterization of pristine AgNPs and identification of AgNPs enriched in TX-114 rich phase after CPE. Up, TEM image (A) and particle size distribution (B) of the stock AgNP solution; down, TEM image (C) and EDS image (D) of AgNPs in the TX-114 rich phase after CPE of the exposed cells. The scalar bar is 50 nm.

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μL of 100 mg/L AgNPs and mixed thoroughly, the mixture was adjusted to pH∼3.5 with diluted HNO3. Then, into the mixture was added 0.2 mL each of 1 mol/L Na2S2O3 and 10% (w/v) TX-114 in sequence. The mixture was mixed and incubated at 40 °C in a water bath for 30 min, and centrifuged at 3000 rpm at room temperature for 5 min to facilitate the phase separation. For the exposure experiment, the extraction was directly conducted immediately after cells were disrupted. The obtained TX-114-rich phase (∼100 μL) with the concentrated AgNPs and the upper aqueous phase were collected, respectively, for quantification and characterization. Microwave Digestion and ICP-MS Detection. AgNPs were quantified by measuring the Ag contents in the TX-114rich phase by ICP-MS (Agilent 7500ce) with Ag standard solutions prepared by diluting a certified reference material. Before ICP-MS determination, the TX-114-rich phase was digested by microwave-assisted digestion (CEM Mars 5, Xpress, Matthews, NC) with a procedure modified from the US EPA method 3052. Briefly, 5 mL concentrated HNO3 and 1 mL concentrated H2O2 were added into the obtained TX-114rich phase and the mixture was irradiated at 120 °C (800 W) for 10 min, followed by 180 °C (1600 W) for 30 min. After digestion, samples were diluted with water to 50−100 mL and stored at 4 °C for further analysis. Determination of the Uptake of Ag by Cells. The total concentration of Ag uptake by cells was calculated as the sum of the Ag contents of the upper aqueous phase and the TX-114rich phase. And the uptake amount of Ag was also recalculated as ng Ag per 104 cells (cell number after AgNPs exposure) and ng cm−2 of growth area of cells (100 mm cell culture plate) to compare with previous studies.

RESULTS AND DISCUSSION Characterization of AgNPs. Figure 1 shows the TEM image and size distribution of the PVP-coated AgNPs used in this study. These nanoparticles are relatively monodispersed in size with an average diameter of 12.4 ± 0.3 nm by counting more than 200 particles. DLS analysis showed that the hydrodynamic diameter of AgNPs is 31.4 nm and the polydispersivity index (PDI) is 0.164, indicating the AgNP solution is well dispersed and without aggregation. Optimization of the CPE Parameters. In our previous study for the extraction of AgNPs in waters,24 parameters influencing the extraction efficiency had been optimized. Given the biological matrix, containing large numbers of carboxyl and amino groups that can associate with AgNPs, is much more complex than water, the critical CPE parameters were optimized using cell lysates spiked with 1 mg/L AgNPs or Ag+. Our previous study22,24 demonstrated that Na2S2O3 played a vital role in the CPE of AgNPs, as it served not only as a salt to reduce the Coulomb repulsion between charged AgNPs and facilitate the separation, but also as a chelate reagent to form water-soluble complex with Ag+ to avoid its coextraction with AgNPs. The role of Na2S2O3 was valued in CPE of AgNPs from cell lysates. Results shown in Figure 2 indicate that the extraction efficiency increased with Na2S2O3 concentration from 2.5 to 10 mM, and then leveled off. Thus, 20 mM Na2S2O3 was adapted as a conservative result in the following experiments to fully eliminate the interference of Ag+. As the solution pH governs the surface charge of AgNPs, which has a strong impact on the extraction efficiency, it is of importance to optimize the extraction pH. Figure 3A shows that the extraction efficiency reached maximum at pH∼3.5, and decreased with pH in the range of pH 4−8. This result is in accordance with the zero point charge pH (pHpzc) which was 3270

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extraction of cell lysates, which are mainly proteins and can binding Ag+. This was evidenced by the appearance of abundant floccules at pH 4−5. To verify this, we measured the isoelectric points of the cell lysates. Figure 3B shows that the isoelectric point of the cell lysates was around pH 4.8. Therefore, at pH 4−5 the cell lysates with associated Ag+ were extracted, which resulted in the high extraction efficiency of Ag+. Taking all of these into consideration, pH∼3.5 was adopted to achieve efficient separation of AgNPs and Ag+. The influence of TX-114 on the extraction efficiency of AgNPs was studied in the range of 0.05%-0.4% (w/v) TX-114, which has a critical micellar concentration ∼0.01% (w/v).28 Results illustrated in Figure 4 revealed that the extraction Figure 2. Effect of Na2S2O3 concentration on the extraction efficiency of AgNPs in cell lysates. To 10 mL cell lysis solution containing 0.97 mg/L AgNPs was added 0.2% (m/v) TX-114 and different amounts of Na2S2O3. The resulting solution was then incubated at 40 °C for 30 min.

Figure 4. Effect of TX-114 concentration on the extraction efficiency of AgNPs in cell lysates. To 10 mL cell lysis solution containing 0.97 mg/L AgNPs was added 20 mM Na2S2O3 and different contents of TX-114. The resulting solution was adjusted to pH 3.5 and incubated at 40 °C for 30 min.

efficiency of AgNPs increased with TX-114 concentration from 0.05% to 0.1%, and then leveled off. In the subsequent studies, 0.2% TX-114 was adopted to ensure the fully extraction of AgNPs. It has been reported that temperature and incubation time also affect the extraction efficiency;28−30 therefore, in this present study the impact of extraction temperature and time was evaluated. As the reported cloud point of TX-114 was 23− 25 °C,28 and our previous work in the concentration of AgNPs in waters had proved that the highest extraction efficiency was obtained in the range of 35−50 °C,24 40 °C was used in this study. The incubation time was evaluated in the range of 10−50 min at 40 °C, and results in Figure 5 revealed that the extraction efficiency increased from 10 to 20 min, then kept constant between 20 and 50 min, suggesting the extraction equilibration was reached within 20 min. In the subsequent studies, incubation at 40 °C for 30 min was employed. Effects of Sonication. To test if the sonication process causes the dissolution of AgNPs into Ag+ and therefore analysis errors, control experiment was conducted by adding AgNPs before the cell disruption and then followed the regular extraction. Results showed that there was no significant difference in the extraction efficiency between the two procedures (67.1 ± 4.6% for the regular versus 65.4 ± 6.1% for the control), indicating the influence of sonication is negligible. Separation Efficiency between AgNPs and Ag+. In the proposed CPE procedure, AgNPs were extracted into the surfactant-rich phase, while Ag+ remained in the aqueous phase. Therefore, the quantified Ag content in the nether TX-114-rich

Figure 3. (A) Effect of sample pH on the extraction efficiency of AgNPs and Ag+ in cell lysates. To 10 mL cell lysis solution containing 0.97 mg/L AgNPs or 0.89 mg/L Ag+ was added 0.2% (m/v) TX-114 and 20 mM Na2S2O3. The resulting solution was then incubated at 40 °C for 30 min. (B) Zeta potential of AgNPs and cell lysate at different pH.

measured to be about 2.5 (Figure 3B). Higher solution alkalinity induced the formation of Ag2O, which explained the slight fluctuation at pH∼9. Although higher extraction efficiency is expected at the pHpzc (2.5),23 there is a risk that Na2S2O3 may decompose into sulfur precipitation at such acidity, which interferes the CPE of target AgNPs. The added Na2S2O3 was expected to complex with Ag+ and therefore prohibit the coextraction of Ag+. However, as shown in Figure 3A, while the extraction efficiency is negligible at pH∼3.5 and pH 6−9, comparable extraction efficiencies for Ag+ were obtained at pH 4−5. We speculated that the high extraction efficiency of Ag+ at pH 4−5 was attributed to the 3271

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revealing that about 8% Ag was lost possibly due to the adsorption of AgNPs onto the vessels. Analytical Performance. The method precision and detection limits (LODs) for AgNPs and Ag+ were determined by analysis of cell lysates spiked with AgNPs and Ag+ at various levels. The LOD, defined as 3 times of baseline noise (S/N = 3), was 2.94 μg/L for AgNPs and 2.40 μg/L for Ag+, respectively. The relative standard deviation (RSD) was 8.9% for AgNPs at a spiked level of 0.97 mg/L (n = 4), and 9.7% for Ag+ at a spiked level of 0.71 mg/L (n = 4), respectively. The spiked recovery was tested to further understand the extent to which biological media may affect the extraction of AgNPs. Experiments showed that the recoveries of AgNPs were between 50 and 82% for samples with spiked levels in the range of 0.6−3 mg/L, but reduced to only 25% when the spiked AgNPs decreased to 0.06 mg/L. This result suggests that a small part of the spiked AgNPs were associated with the cell lysates and remained in the aqueous phase after the CPE procedure. An efficient approach to eliminate the matrix effects is to prepare the AgNP standards in the sample matrixes. In this study, five standards were prepared by spiking 0.125, 0.623, 1.25, 1.87, and 3.11 mg/L AgNPs into the cell lysates, respectively, and after CPE the amount of AgNPs in the TX114 phase was determined by ICP-MS after digestion. The ICP-MS response showed a good linear correlation with the spiked AgNPs concentration with a correlation coefficient of 0.9953. With this calibration curve, the spiked recoveries of AgNPs in cell lysates were 93.1%, 81.5%, and 122.5% at spiking levels of 2.49, 0.934, and 0.249 mg/L, respectively. These results demonstrated that preparing AgNP standards in cell lysates could significantly improve the analysis accuracy of AgNPs in samples and was adopted in the following studies. The above results also indicate that higher spiked level of AgNPs gave rise to more satisfactory recovery and therefore more believable quantification results. In view of the relatively high AgNP concentration in cell lysates of the exposed cells (3.04 mg/L, see the next section), the determined uptake amount of AgNPs by HepG2 cells in the following study is reliable. Uptake of AgNPs and Ag+ to HepG2 Cells. Due to the small dimension of AgNPs that in the range of 1−100 nm, one major toxicological concern is their possible penetration into organisms and accumulation in the cells. Generally, it is regarded that AgNPs may enter cells by endocytosis that the membranes engulfs the particles and deliver them across the cell membrane and into the interior.36 Several studies have reported the uptake of AgNPs in different cell lines, and TEM images clearly showed the AgNPs located in the vesicles, around the cell nucleus and distributed in the cytoplasm.11,23 In the present study, we use the above developed extraction procedure to separate the AgNPs in exposed cells and thus

Figure 5. Effect of extraction time on the extraction efficiency of AgNPs in cell lysates. To 10 mL cell lysis solution containing 0.97 mg/ L AgNPs was added 0.2% (m/v) TX-114 and 20 mM Na2S2O3.The mixture was adjusted to pH 3.5 and incubated at 40 °C for different time.

phase and the upper aqueous phase accounted for the AgNPs and Ag+ content in the samples, respectively. To evaluate the separation of AgNPs and Ag+ under the optimized conditions, three respective cell lysate samples spiked with AgNPs, Ag+, and a mixture of Ag+ and AgNPs were extracted with the proposed procedure. The Ag contents in both aqueous phases and TX-114-rich phases were quantified, respectively, by ICPMS after microwave digestion. As shown in Table 1, only a very small amount of Ag+ (6.1%) was extracted into the TX-114-rich phases, and the respective apparent extraction efficiency for AgNPs were 67.5 ± 3.3% and 70.4 ± 2.1% for the mixture of Ag+ and AgNPs, indicating that the proposed method could successfully isolate AgNPs from Ag+. Comparison with Other Methods. Previous studies have reported the use of centrifugal ultrafilter devices to separate Ag+ from AgNPs.13,31,32 However, most of these reports were just for simple AgNP solutions; in our case, the complex composition in cell lysates may associate with Ag+ and make false results. To verify this, 10 mL cell lysates spiked with 1 mg/ L Ag+ were transferred into Amicon centrifugal ultrafilter units and subjected to centrifugation. Results showed that only less than 10% Ag+ presented in the filtrate, revealing this method is incapable of separating Ag+ from AgNPs in biological matrices as more than 90% Ag+ remained in the filter residue, where AgNPs would present. This result suggests that conventional filtration-based techniques are invalid to separate Ag+ from AgNPs in cell lysates. Adsorption of AgNPs on the Vessels. Given that the nanomaterials were reported to easily adsorb onto test vessels,33−35 it is of interest to evaluate the total recovery, which was defined as the sum of the Ag contents in aqueous and TX-114-rich phases relative to the total spiked Ag amount. Table 1 shows that the average total recovery was around 92%,

Table 1. Detected Ag Contents in both TX-114 Phase and Aqueous Phase, And the Recovery of Ag after CPE in cell Lysates Spiked with Individual and Mixed Ag+ and AgNPs detected (mg/L Ag)

a

sample

spiked (mg/L Ag)

TX-114 phase

aqueous phase

extraction efficiency (%)

recovery (%)

Ag+ AgNPs AgNPs + Ag+

0.889 0.966 0.966 (AgNPs) + 0.889 (Ag+)

0.054 ± 0.003 0.652 ± 0.032 0.680 ± 0.020

0.760 ± 0.014 0.234 ± 0.168 1.036 ± 0.066

6.1 ± 0.33 67.5 ± 3.3 70.4 ± 2.1a

91.7 ± 1.4 91.7 ± 3.3 94.1 ± 4.0

Apparent extraction efficiency calculated relative to 0.966 mg/L AgNPs and assuming that Ag+ was not extracted. 3272

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anaerobic conditions, however, their toxicity greatly enhanced after exposure to air for 6 h, and the toxicity still increased with prolonged air exposure, indicating the aerobic toxicity of AgNPs may account for the release of Ag+. Given the toxicity of Ag+ is much higher than that of AgNPs, the presence of 10.3% silver as Ag+ in cells may contribute substantially to the apparent toxicity of AgNPs. Therefore, our findings imply that some of the reported toxic results should be reconsidered as they did not take into account of the presence of substantial Ag+. Further experiment should be conducted to understand the exact role of Ag+ played in the AgNPs toxicity and to what extent that Ag+ contributed to the toxicity. In summary, this study demonstrated that conventional methods for separation of AgNPs and Ag+ in AgNP suspensions such as centrifugal ultrafiltration are not suitable for samples with complex matrix like cell lysates, whereas CPE showed excellent performance for this purpose. For the first time, we quantified the contents of AgNPs and Ag+ in exposed organism, and found that the ratio of Ag+ in relation to the AgNPs in the exposed HepG2 cells is substantially higher than that of the pristine AgNPs. Our results suggest the occurrence of transformation of AgNPs into Ag+ in the cells and/or the higher uptake rate of Ag+ than that of AgNPs, and the contribution of Ag+ should be taken into account in evaluating the toxicity of AgNPs to organism.

quantify the uptake of AgNPs. To this end, HepG2 cells were directly incubated with the exposure medium, and the cells were collected and subjected to CPE to separate AgNPs from Ag+ in the cells. Identification of AgNPs. Figure 1 shows the TEM image of TX-114-rich phase after CPE of AgNP exposed cells, which indicates the existence of nanoparticles. EDS analysis showed the presence of strong signal of Ag and the absence of signals of chloride, excluding the formation of AgCl and supporting the presence of AgNPs. The high contents of phosphorus and sulfur may ascribe to the cell debris and the S2O32‑ used in the extraction, respectively. Quantification of AgNPs. After exposure to 10 mg/L AgNPs for 24 h and subjected to CPE, the detected Ag contents in the TX-114-rich phase and the aqueous phase were 3.04 and 0.35 mg/L, respectively. This corresponds to about 67.8 ng Ag was detected per 104 cells. Very limited studies11,12,23 reported the uptake amounts of AgNPs after exposure. In a previous study for evaluating the effect of particle size on the toxicity of AgNPs to HepG2 cells,12 about 4 ng and 16 ng Ag/104 cells was detected after cell exposure to 1 mg/L of 20 and 5 nm AgNPs for 6 h, respectively. When recalculating the uptake of Ag into the amount of silver taken up in ng cm−2 of growth area of cells, our result (431.8 ± 112.1 ng cm−2) is higher than another study (307.9 ± 169.6 ng cm−2) which test the uptake and toxicity of AgNPs in rainbow trout gill cells.23 The varied exposure conditions such as exposure time and concentration, cell line, as well as type and size of AgNPs may account for the different uptake amounts of AgNPs reported in different studies. The quantified Ag content in the nether TX-114-rich phase (30.4 ± 7.3 μg) and the upper aqueous phase (3.5 ± 1.5 μg) accounted for the AgNPs and Ag+ content in the exposed cells, respectively. This result suggests that about 10.3% of the total amount of silver was present in ionic form, indicating that the strategy of accounting total silver content as AgNPs in previous studies could result in errors. As the AgNPs contain only 5.2% Ag+ before exposure, the significantly higher content of Ag+ in the exposed cells (10.3%) may stem from the transformation of AgNPs into Ag+ in the cells and/or the higher transferring rate of Ag+ into the cells than that of AgNPs. Previous studies have shown that Ag+ exposed cells contain larger amount of silver in comparison to cells exposed to AgNPs at the same exposure doses.12,23 The toxic effects of AgNPs on organisms, in their own right or as the carrier of Ag+, have been reported a lot. Some early studies suggested that AgNPs would be oxidized and release Ag+ in cells, and caused toxicity by a Trojan-horse type mechanism.37 A study38 in evaluating pH and organic matter effect on AgNPs toxicity to bacterial growth found that the intrinsic nanoparticle effect could not be explained by the dissolution Ag+. However, another article39 demonstrated that the toxicity of AgNPs is dependent on the dissolved silver and the capping agents. They found that the amount of Ag+ contributed to the toxicity for all the tested AgNPs, and there was a linear correlation between the dissolved silver and AgNPs toxicity. Xiu et al.19 tried to discern the different antimicrobial activity of AgNPs and Ag+ under anaerobic conditions. They found that Ag+ was 20 times more toxic to E. coli than AgNPs (EC50: 0.10 ± 0.01 vs 2.04 ± 0.07 mg/L), suggesting that Ag+ may be the major drive for the AgNPs toxicity. Their further study20 revealed that PEG coated 5 and 11 nm AgNPs showed no measurable inhibitory effect on the growth of E. coli under

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-10-62849192; fax: +86-10-62849192; e-mail: jfl[email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (2011CB936001), the National Science Fund for Distinguished Young Scholars of China (21025729), and the National Natural Science Foundation of China (21227012).

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REFERENCES

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dx.doi.org/10.1021/es304346p | Environ. Sci. Technol. 2013, 47, 3268−3274