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Neoplastic cell response to tiopronin-coated gold nanoparticles
BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 9 (2013) 264 – 273
Research Article
nanomedjournal.com
Neoplastic cell response to tiopronin-coated gold nanoparticles Lei Cui, BSc a , Payam Zahedi, PhD a , Justin Saraceno, BA a , Robert Bristow, PhD, MD b, c, e , David Jaffray, PhD b, d, e , Christine Allen, PhD a, e,⁎ a Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada Departments of Radiation Oncology and Medical Biophysics, University of Toronto, Toronto, Canada c Ontario Cancer Institute/Princess Margaret Hospital, University Health Network, Toronto, Ontario, Canada d Department of Radiation Physics, Princess Margaret Hospital, University Health Network, Toronto, Ontario, Canada e STTARR Innovation Centre, Radiation Medicine Program, Princess Margaret Hospital, University Health Network, Toronto, Ontario, Canada Received 3 November 2011; accepted 25 May 2012 b
Abstract The present study characterized the in vitro biological response of a comprehensive set of cancer cell lines to gold nanoparticles (2.7 nm) coated with tiopronin (AuNPs-TP). Our findings suggest that upon entering cells, the AuNPs-TP are sequestered in vacuoles such as endosomes and lysosomes, and mostly localize in perinuclear areas. Peak cell accumulation was achieved at 8 hours after incubation. L929 and H520 cells showed more than 75% surviving fraction when treated with 0.5 mg/mL of AuNPs-TP for 24 hours, whereas the surviving fractions were 60% in MCF-7 and 20% in HeLa cells. Reactive oxygen species (ROS) production by the AuNPs-TP was dependent on cell line and exposure time. Antioxidants inhibited ROS generation to various extents, with glutathione and tiopronin being most effective. Overall, exposure time, concentration of the AuNPs-TP, and cell line influenced neoplastic cell response. Furthermore, the mechanism of cytotoxicity of the AuNPs-TP was found to be ROS generation. From the Clinical Editor: This study describes the basic intracellular characteristics of Tiopronin-Au nanoparticles from the standpoint of their anti-cancer activity in different cancer cell cultures. © 2013 Elsevier Inc. All rights reserved. Key words: Gold nanoparticles; Cancer; Cellular accumulation; Cytotoxicity; Reactive oxygen species
The interest in nanotechnology for medical applications such as drug delivery, imaging, and tissue engineering has grown significantly. 1 In particular, gold nanoparticles (AuNPs) have been heavily explored for their use in cancer diagnosis and therapy. 2,3 Au is attractive for such applications because of its inert nature (i.e., stable and nonmetabolizable), biocompatibility, ease of NP size control, and well-developed surface chemistry for functionalization. 4 Varying the size, shape, and surface properties of AuNPs allows for customized optical characteristics, 5 pharmacokinetics and biodistribution, 6–8 and biocompatibility. 9,10 The size of NPs has been shown to influence their biodistribution in vivo. 11 For instance, smaller NPs (b5 nm) penetrate tumors more deeply than do larger particles (N10 nm). 12 In vitro as well, cellular uptake of NPs has been
This research was funded by an operating grant from the Canadian Institutes of Health Research to D.A. Jaffray, R. Bristow, and C. Allen. ⁎Corresponding author: Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario M5S 3M2, Canada. E-mail address: [email protected] (C. Allen).
shown to be dependent on size. 8 The surface properties of NPs, which are largely determined by the surfactants used during preparation and for coating, is another crucial factor that can determine their in vitro and in vivo performance. 13,14 To date, one of the most commonly used surfactants for AuNPs has been thiol-terminated poly(ethylene glycol) (PEG-SH). 15 Two limitations associated with PEG-SH are its relatively large molecular weight and limitations with respect to further functionalization. The method of synthesizing small AuNPs coated with the hydrophilic molecule tiopronin (AuNPs-TP) was first developed by Templeton et al., 16 and there are several advantages associated with the use of this surfactant. First, the core size of the AuNPs-TP can be controlled by changing the molar ratio of Au to tiopronin. As well, the hydrophilicity of tiopronin makes the AuNPs-TP water soluble, enabling administration in vivo. Furthermore, the thiol group in tiopronin makes it capable of tight conjugation to Au atoms through the strong sulfur-gold (S-Au) bond. 17 Also, the small size of the tiopronin molecule means that there is less steric repulsion between the stabilizing molecules, leading to greater coverage of the surface of AuNPs. 16 The strong S-Au bond and high surface coverage
1549-9634/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nano.2012.05.016 Please cite this article as: Cui L., Zahedi P., Saraceno J., Bristow R., Jaffray D., Allen C., Neoplastic cell response to tiopronin-coated gold nanoparticles. Nanomedicine: NBM 2013;9:264-273, http://dx.doi.org/10.1016/j.nano.2012.05.016
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Figure 1. Preparation of AuNPs coated with a monolayer of tiopronin.
make the AuNPs-TP resistant to ligand exchange reactions, reduce the possibility of absorption of other molecules such as proteins onto their surface, and minimize aggregation. In addition, functional groups such as fluorescent probes and active targeting peptides can be easily added to the AuNPs-TP through conjugation with the carboxyl group found on tiopronin. 18,19 Although there are numerous advantages associated with AuNPs-TP, their biological properties have only been characterized to a limited extent. 19,20 Herein, in vitro studies were performed to evaluate and gain an improved understanding of cellular response to AuNPs-TP. Given that AuNPs have been mostly investigated for applications in cancer diagnosis and therapy, three human cancer cell lines (MCF-7 breast cancer cells, HeLa cervical cancer cells, and H520 lung cancer cells) were selected for these studies; a murine fibroblast cell line (L929) was used as a control. Cellular accumulation, intracellular distribution, cytotoxicity, and reactive oxygen species (ROS) production of the AuNPs-TP were assessed. In addition, a series of antioxidants [N-acetylcysteine (NAC), reduced L-cysteine, glutathione (GSH), and tiopronin] were employed to determine their influence on cellular ROS levels following co-treatment with the AuNPs-TP. A concentration range of 0.01–0.5 mg/mL AuNPs-TP was used for these studies. Similar concentration ranges have been investigated in vitro with AuNP systems. 8,10,19 Methods Detailed information on the materials and methods for preparation and characterization of AuNPs-TP, cell culture, qualitative assessment of cellular accumulation of AuNPs-TP, and statistical analysis is available in the Supplementary Methods online at http://www.nanomedjournal.com. Quantitative assessment of cellular accumulation of AuNPs-TP For quantitative analysis of cellular accumulation, cells were seeded in six-well plates at a density of approximately 1 × 10 6 cells per well. Cells were treated with two different concentrations of AuNPs-TP (0.05 and 0.25 mg/mL) for 1, 4, 8, 24, 48, or 72 hours. At each time point cell media were removed, cells were washed three times with phosphate buffered saline (PBS) and then harvested with 0.25% Trypsin with EDTA (Gibco BRL, Grand Island, New York). Cells were counted using a hemocytometer, centrifuged to a pellet, digested with HNO3 at
90°C for 60 minutes, and diluted with double-distilled water (ddH2O). The amount of Au was measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES) and normalized to the number of cells in each sample. The results were reported as the amount of Au (in picograms) per cell. The number of AuNPs-TP in cells was calculated as described in the Supplementary Methods. Evaluation of cytotoxicity of AuNPs-TP Clonogenic assays were performed as described previously. 21,22 Cells were seeded in six-well plates at a density of 1 × 10 6 cells per well, and treated with various concentrations of AuNPs-TP (0.01–0.5 mg/mL). For the HeLa cells, cotreatment with 0.5 mg/mL AuNPs-TP and 3 mM GSH was also tested. After 24 hours of incubation, cells were washed twice with PBS and trypsinized. For each treatment, cells were counted and added into six-well plates at different cell densities (i.e., 300–1800 cells per well). After 7–10 days the colonies were washed with PBS, fixed with methanol, and stained with 1% crystal violet. The number of colonies that consisted of at least 50 cells was counted on each plate. Cell surviving fraction (SF) was expressed as plating efficiency compared to that of nontreated cells. Measurement of ROS production The amount of ROS was measured using the 2′,7′dichlorofluorescein diacetate (DCFH-DA) assay as previously described. 23 Cells were seeded into 96-well plates at a density of 20,000 cells per well. After recovery, cells were washed twice with Hank's Balanced Salt Solution and incubated with 100 μM DCFH-DA for 60 minutes. Cells were then washed with Hank's Balanced Salt Solution and exposed to different treatments, with nontreated cells used as negative controls. Cells treated with 0.3% H2O2 and 10 μM 3-morpholinosydnonimine hydrochloride (SIN) were employed as ROSgenerating positive controls. The treatment groups included 0.5 mg/mL AuNPs-TP; 0.5 mg/mL AuNPs-TP + 3 mM NAC; AuNPs-TP + reduced L-cysteine; AuNPs-TP + GSH; AuNPsTP + TP; and 0.5 mg/mL AuNPs-TP + 50 μM Z-VAD-fmk (Bachem Americas, Torrance, California). At different time points (1, 4, 8, and 24 hours) after treatment, fluorescent signal was measured with a SpectraMax Gemini Plus (Molecular Devices, Sunnyvale, California) microplate reader (λex = 480 nm, λem = 520
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Figure 2. Characterization of the AuNPs-TP. (A) A representative TEM image of the AuNPs-TP. Scale bar, 20 nm. (B) Core size distribution histogram calculated from over 1000 AuNPs-TP. (C) Proton nuclear magnetic resonance spectrum of 0.5 mg/mL AuNPs-TP suspension in D2O. (D) UV-vis spectrum of 1 mg/mL AuNPs-TP in dd-H2O. (E) Percentage of Au that remains in the supernatant following incubation in cell culture medium at 37°C. Data represent mean ± SD (n = 3). (F–H) TEM images of AuNPs-TP in cell culture media following 24, 48, and 72 hours of incubation at 37°C. Scale bar, 20 nm.
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Figure 3. TEM images of AuNPs-TP accumulation in four different cell types. (A, B) MCF-7; (C, D) HeLa; (E, F) H520; (G, H) L929. As highlighted by the arrows in images A and B, once the AuNPs-TP enter cells they appear to sequester in large vacuoles such as endosomes and lysosomes, and mostly localize in the perinuclear region of cells. A similar trend was observed for all cell lines evaluated. Scale bars: 2 μm (A, C, E, G); 100 nm (B, D, F, H).
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Figure 4. In vitro cellular accumulation of AuNPs-TP in four different cell types. (A) MCF-7; (B) HeLa; (C) H520; (D) L929. Accumulation was quantified by inductively coupled plasma atomic emission spectroscopy, with incubation at two different concentrations (0.05 and 0.25 mg/mL) of AuNPs-TP. *Statistically significant difference between the two concentrations (P b 0.05); #statistically significant difference in cell accumulation at different time points in comparison to that at the 8-hour time point (P b 0.05). Data represent mean ± SD (n = 3).
nm). The results were reported as relative ROS production compared to nontreated cells.
Results Synthesis and characterization of AuNPs-TP A representative transmission electron microscopy (TEM) image of the AuNPs-TP suspended in dd-H2O is shown in Figure 2, A. The AuNPs-TP had a mean core diameter of 2.7 nm as obtained from the core size distribution histogram (Figure 2, B). It was determined that each 2.7-nm AuNP-TP was composed of approximately 600 Au atoms. Au atoms composed 76.5% ± 0.3% of the mass of the AuNPs-TP, and the coating efficiency of TP (i.e., the percentage of Au atoms at the surface of the NPs that are coated with TP) was calculated to be 87.3% ± 0.3%. As seen from the proton nuclear magnetic resonance spectrum (Figure 2, C), the final product was spectroscopically pure, with the absence of signals due to unreacted thiol or disulfide byproduct. 16 As evidenced from the ultraviolet-visible (UV-vis) spectrum (Figure 2, D), an absence of absorbance at 520 nm demonstrated that the AuNPs-TP were stable in dd-H2O
with no aggregation. However, it was found that upon incubation of AuNPs-TP in cell culture medium a fraction of the NPs aggregates and precipitates out of solution. Following 24, 48, and 72 hours of incubation in cell culture medium, 82% ± 2%, 78% ± 2%, and 76% ± 2% of the AuNPs-TP, respectively, were found to remain in the supernatant. TEM analysis revealed that the AuNPs-TP that remain in the supernatant retain their size and are well dispersed in the medium (Figure 2, F–H). Cellular accumulation of AuNPs-TP To study the intracellular fate of the AuNPs-TP, cells were observed by TEM. TEM images of four cell lines pretreated with AuNPs-TP are presented in Figure 3. From these images it can be seen that AuNPs-TP mostly localized in the perinuclear areas of cells. AuNPs-TP in cytoplasm were sequestered in large clusters in vacuoles such as the endosomes and lysosomes. The AuNPs-TP did not localize in organelles such as the nucleus or mitochondria. Figure 4 summarizes the quantitative evaluation of cellular accumulation of AuNPs-TP. The total intracellular level of Au is presented in Supplementary Figure S1, A–D. To assess the effect of AuNPs-TP concentration on cellular accumulation,
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cells were incubated with two different concentrations (0.05 mg/mL and 0.25 mg/mL). For all cell lines used, the amount of cellular accumulation was dependent on AuNPs-TP concentration. Statistically significant differences (P b 0.05) in cell accumulation were observed between the two concentrations at all time points. Similar accumulation profiles were observed in MCF-7 and HeLa cells. In the first 8 hours of incubation there was an increase in Au accumulation; the levels then decreased in the following 40 hours and reached a constant level at 48 hours. A similar profile was also observed in the L929 cells incubated with 0.25 mg/mL AuNPs-TP. However, for the H520 cells the cellular accumulation profiles (for both concentrations of AuNP-TP) reached a maximum at 8 hours, and this level remained relatively constant up to 72 hours. For the L929 cells incubated with 0.05 mg/mL AuNPs-TP a significant decrease was observed at 24 hours compared to 8 hours, after which the level increased to a value similar to that achieved at the 8-hour time point. Statistically significant differences (P b 0.05) in cell accumulation at different time points in comparison to the uptake at the 8-hour time point are shown in Figure 4. At the peak time point for accumulation (i.e., 8 hours), HeLa and L929 cells incubated with 0.25 mg/mL of AuNPs-TP showed higher accumulation of AuNPs-TP (HeLa, 42 pg/ cell, 2.1 × 10 8 AuNPs-TP per cell, and L929, 48 pg/cell, 2.4 × 10 8 AuNPs-TP/cell) compared to MCF-7 (26 pg/cell, 1.3 × 10 8 AuNPs-TP per cell), and H520 (15 pg/cell, 0.78 × 10 8 AuNPs-TP per cell) cells. Interestingly, despite the large differences seen in accumulation at the 8-hour incubation period, all cell lines showed a similar concentration of Au accumulation at 72 hours. These concentrations were approximately 15 pg/cell (0.78 × 10 8 AuNPs-TP per cell) and 5 pg/cell (0.26 × 10 8 AuNPs-TP per cell) when incubated with 0.25 mg/mL and 0.05 mg/mL of the AuNPs-TP, respectively. Cytotoxicity of AuNPs-TP The biocompatibility of the AuNPs-TP was assessed by clonogenic assay. Incubation of AuNPs-TP with L929 and H520 cells resulted in more than 75% surviving fraction (SF) even at the highest AuNPs-TP concentration (i.e., 0.5 mg/mL). For HeLa cells, the SF decreased significantly (P b 0.05) when treated with 0.25 and 0.5 mg/mL of the AuNPs-TP. Specifically, HeLa cells showed only 20% SF at the highest concentration tested. For MCF-7 cells, a significant decrease (P b 0.05) in SF was observed at 0.5 mg/mL. (See Figure 5.) Measurement of ROS production To determine if the cytotoxic effect of the AuNPs-TP in HeLa and L929 cell lines could be attributed to oxidative stress the ROS generated by the AuNPs-TP was measured using the DCFH-DA assay (Figure 6). Incubation of cells with 0.3% H2O2 or 10 μM SIN for 1 hour, as positive controls, resulted in high ROS levels compared to nontreated cells. The amount of ROS produced following treatment with the AuNPs-TP increased with incubation time yet was lower than the positive controls. ROS production following treatment with the AuNPs-TP in combination with various antioxidants (i.e., NAC, reduced L-cysteine, GSH, or tiopronin) was also evaluated. The degree of ROS
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Figure 5. Cell surviving fraction (SF) after 24 hours of treatment with different concentrations of AuNPs-TP. SF as determined by clonogenic assays is reported as plating efficiency compared to nontreated cells. *Statistically significant difference between various concentrations for HeLa cells; #statistically significant difference between various concentrations for MCF-7 cells (P b 0.05). Data represent mean ± SD (n = 3).
inhibition depended on the antioxidant employed (Figure 6). GSH and tiopronin were more effective in decreasing ROS compared to NAC and reduced L-cysteine. Co-treatment of cells with Z-VAD-fmk and AuNPs-TP resulted in ROS levels that were similar to those achieved following treatment with AuNPs-TP alone. The ability of GSH to rescue cells from oxidative stress induced by AuNPs-TP was evaluated. Cell clonogenicity was compared between HeLa cells treated with AuNPs-TP alone and in combination with GSH. It was found that the cell SF was increased by fourfold following combination treatment [i.e., SF = 19% ± 0.45% (only AuNPs-TP) vs. 91% ± 4.4% (AuNPs-TP + GSH)].
Discussion With its unique physical and chemical characteristics, gold in the form of AuNPs has been widely explored for biomedical applications such as drug delivery, imaging, disease diagnosis, and therapy. 2,3 The size, shape, and surface coating of AuNPs can be modified to alter in vitro cellular response. 7,24 Tiopronin has several advantages over conventionally used PEG-based surfactants; however, little is known about the biological performance of AuNPs-TP. In this study, AuNPs-TP were synthesized and assessed in terms of size, purity, in vitro cell accumulation, and cytotoxicity. The mechanism of cytotoxicity was also investigated. A significant challenge in the chemical synthesis of AuNPs is achieving monodispersity in size. 25 In this study the mean core diameter of AuNPs-TP suspended in dd-H2O was 2.7 nm, and the size distribution histogram showed a relatively narrow size distribution (1.5–3.9 nm). This relatively high monodispersity of the AuNPs-TP was achieved using the method described by Templeton et al., in which synchronized growth and coating of the NPs are achieved with the thiol-containing reducing
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Figure 6. Amount of ROS produced relative to nontreated cells following treatment with AuNPs-TP (0.5 mg/mL) in combination with antioxidants including NAC, reduced L-cysteine, GSH, or tiopronin (3 mM) and the apoptotic inhibitor Z-VAD-fmk (50 μM) in (A) HeLa cells and (B) L929 cells. The insets show relative ROS produced in cells following treatment with 0.3% H2O2 or 10 μM SIN for 1 hour compared to nontreated cells. Data represent mean ± SD (n = 4).
reaction. 16 The stability of the AuNPs-TP in dd-H2O was assessed based on their optical properties (i.e., surface plasmon resonance). The UV spectrum of AuNPs with diameters greater than 3 nm is known to include absorption at a wavelength of approximately 520 nm. As well, any change in the surface of the AuNPs-TP such as absorption of macromolecules or aggregation can lead to changes in the UV-vis spectrum. 26,27 The absence of peaks in the UV-vis spectra (i.e., in the 520-nm range) of the AuNPs-TP confirmed their stability and lack of aggregation in dd-H2O. However, aggregation of AuNPs was observed upon incubation in cell culture medium. Aggregation occurs when NPs interact with cell culture medium, given that ions such as Na + and Cl − in the medium neutralize the surface charge of the NPs. 28 This aggregation is said to be an immediate and irreversible process. It is believed that absorption of proteins onto the surface of some AuNPs can assist in stabilizing the particles and preventing aggregation. However, absorption of proteins onto AuNPs-TP is unlikely given the high coating efficiency of TP (87.3% ± 0.3%) and the strong S-Au bond, which is resistant to ligand exchange reactions. Cellular accumulation of the AuNPs-TP was visualized in four different cell lines by TEM. The AuNPs-TP that entered the cells were sequestered in large clusters in vacuoles in the perinuclear areas of the cytoplasm. Previous studies have made similar observations. 20,29 For applications such as drug delivery or radiosensitization it may be important to target the AuNPs to specific subcellular organelles such as the nucleus. This delivery to specific intracellular compartments may be achieved by conjugating targeting moieties to the surface of the AuNPsTP. De la Fuente et al. conjugated the TAT peptide onto the surface of AuNPs-TP and demonstrated successful transport to the cell nucleus. 19 Quantitative assessment of cellular accumulation of the AuNPs-TP is of relevance given that the cellular concentration
determines potential toxicity and therapeutic effect. Many variables contribute to the accumulation profile of NPs in cells, including the physicochemical properties of the NPs (e.g., size, morphology, surface properties), cell type, concentration of the NPs, and incubation conditions. These variables, in turn, determine the rate of cell proliferation (i.e., doubling time), extent and rate of endocytosis of NPs, as well as extent and rate of exocytosis. For instance, previous studies have shown that the size of AuNPs is an important factor that influences the rate of endocytosis and exocytosis, and thus the level of cellular accumulation. 8,29 Chithrani et al. demonstrated that incubation of HeLa cells with AuNPs of 50 nm diameter resulted in the highest level of cell accumulation following a 10-hour incubation period, in comparison to AuNPs with diameters of 14 nm and 74 nm. 29 Despite their small size, the AuNPs-TP evaluated in the current study showed a higher level of cellular accumulation (in HeLa cells) at 8 hours (42 pg/cell) than the 50-nm AuNPs (8 pg/cell). 8 The time-dependent accumulation profiles of AuNPs-TP revealed a peak at 8 hours for all cell lines incubated with AuNPs-TP at a concentration of 0.25 mg/mL. A similar trend was observed in previous studies, wherein peak AuNP accumulation was observed following 6–10 hours of incubation. 8,30 The initial increase in the cellular level of AuNPs-TP that occurs at the early time point is largely attributed to endocytosis. Several factors may contribute to the decrease in and constant cellular levels of AuNPs-TP that were achieved at later time points, including endocytosis and exocytosis of the AuNPs-TP that occur simultaneously, cell proliferation, saturation of uptake, decrease in AuNPs-TP dose due to cell uptake and NP aggregation, and state of AuNPs-TP in the cell culture medium. Each of these factors has been considered below. As shown in Supplementary Figure S1, A–D, the total amount of AuNPs-TP that is internalized into cells increases over the 72-
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hour time period. This indicates that cellular uptake of AuNPsTP occurs throughout the incubation period. In the current study quantitative analysis of exocytosis was not conducted, and therefore the contribution of exocytosis to the cellular levels of AuNPs-TP remains unknown. A previous study by Chithrani et al. reported that following a 6-hour incubation period and a subsequent 8-hour washout period, approximately 8%, 20%, and 40% of the AuNPs that had been endocytosed were exocytosed for AuNPs with diameters of 74, 50, and 14 nm, respectively. 29 One drawback of this study is that exocytosis was only observed under washout conditions, wherein cells are exposed to particlefree medium. A recent study by Kim et al. indicates that cell proliferation is the major factor that leads to dose reduction of NPs in cells. 31,32 The authors demonstrated constant levels of NPs throughout the cell cycle until cell division in A549 cells. Indeed, in the current study a comparison between the decrease in the number of AuNPs-TP per cell following the 8-hour time point and the respective doubling time for each cell line indicates that cell proliferation plays a significant role. The magnitude of the decrease in number of AuNPs-TP per cell decreased in the following order: L929 N HeLa N MCF-7 N H520. The doubling times for the cell lines are as follows: L929, 14 hours; HeLa, 24 hours; MCF-7, 29 hours; H520, 61 hours. Therefore, it can be seen that the decrease in number of AuNPs per cell is related to the concomitant increase in cell number. Given that the peak in the accumulation profile at 8 hours is also observed, although less prominently, for HeLa, MCF-7, and L929 cells incubated with the lower concentration (0.05 mg/mL) of AuNPs-TP, this peak cannot be attributed to saturation of uptake. In addition, at all time points it is only a fraction of the AuNPs-TP present in solution that are taken up into cells. Specifically, for L929 cells, which showed the highest level of cell uptake following the 8-hour incubation period, the amount of intracellular Au was 54.8 ± 2.7 μg, which accounts for only 5.7 wt% of the total Au in medium containing 0.25 mg/mL AuNPs-TP. This result suggests that there is always a signficant excess of AuNPs-TP available in the medium. Furthermore, as shown in Figure 2, E, there was no significant difference between the percentage of AuNPs-TP that remain dispersed in the supernatant following 8 and 24 hours of incubation. Therefore, the decrease in the cellular level of AuNPs-TP that occurs beyond the 8-hour time point cannot be attributed to a decrease in the total number of AuNPs available in solution due to aggregation. The state of AuNPs-TP in the cell culture medium could also influence the degree of cellular uptake. TEM analysis of AuNPsTP following incubation in cell culture medium for 24, 48, and 72 hours revealed the AuNPs-TP that remain in the supernatant retain their size and are well dispersed in the medium. However, it is recognized that the size of particles incubated in cell culture medium alone (i.e., in the absence of cells) may not be representative of their size in all regions of the cell culture wells in the presence of cells. Based on the literature, the relationship between the state of AuNPs in solution and cell uptake is complex. For example, Cho et al. recently investigated the effects of aggregation and consequent sedimentation of AuNPs on cell uptake. They demonstrated that sedimentation
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leads to a higher concentration of AuNPs in the cell uptake zone; as a result, a higher uptake was observed in cells in an upright configuration compared to inverted cells. 33 This observation suggests that the actual dose of AuNPs in the cell uptake zone is higher than that measured in the supernatant. Furthermore, a study by Albanese et al. demonstrated differential uptake patterns for single and aggregated NPs in different cell lines. For example, HeLa and A549 cells showed preferential uptake of single NPs, whereas uptake of aggregated NPs was favored in MDA-MB-435 cells. The authors ascribed this phenomenon to different mechanisms of cellular uptake being operative in different cell lines. In addition to receptor-mediated endocytosis, which is a major cell uptake pathway in cell lines such as A549, receptor-independent uptake also plays a significant role in cell lines such as MDA-MB-435. 28 Oxidative stress, which includes ROS generation, is currently the most established theory for NP toxicity. 34 Oxidative stress has been shown to occur in cells following exposure to NPs of single composition such as silica, 35 silver, 36 polystyrene, 37 and gold. 38 The highly curved surface of nanosized particles is said to result in greater defects in crystal structure, therefore disrupting the electronic configuration in the bulk material. 34 These surface properties create reactive electron donor and acceptor groups, which can interact with molecules such as oxygen (O2). For example, as reviewed by Nel et al., the transfer of an electron from a reactive donor group at the surface of a NP to O2 results in the creation of superoxide radicals. 34 Therefore, even though bulk Au is considered to be chemically inert and nontoxic, AuNPs behave very differently than their bulk counterpart. 39 In this study the clonogenic assay was used to characterize the effect of the AuNPs-TP on cell proliferation. The results showed that cytotoxicity depended on both the concentration of AuNPsTP and the type of cell line used. Using the MTT assay (a colorimetric assay used primarily to determine viability and proliferation of cells), tiopronin was found to be nontoxic in HeLa and MCF-7 cells at a concentration equivalent to that present on the surface of 0.5 mg/mL AuNPs-TP (data not shown). As well, as shown in Figure 6, the presence of free tiopronin inhibited ROS generation by AuNPs. This result confirms that the toxicity of the AuNPs-TP cannot be attributed to tiopronin. The effect of various thiol-containing antioxidants (i.e., NAC, reduced L-cysteine, GSH, tiopronin) on reducing ROS production due to AuNPs-TP exposure was evaluated. The intracellular levels of ROS were measured using the DCFH-DA assay. 23 Within cells, DCFH-DA is hydrolyzed to DCFH by esterase; DCFH is then oxidized to fluorescent DCF in the presence of ROS, and the fluorescence intensity produced is proportional to the ROS concentration. 40 Results from the DCFH-DA assay demonstrated that AuNPs-TP induced high levels of ROS following a 24-hour exposure, in comparison to nontreated cells. The increase in ROS following AuNPs-TP treatment explains the toxicities observed for the AuNPs-TP at high concentration. A comparison of relative ROS levels (3.69 ± 0.30 for HeLa and 5.38 ± 0.57 for L929) and SF (19.0% ± 0.45% for HeLa and 76.7% ± 5.08% for L929) between the HeLa and L929 cells reveals that the relative ROS level for a specific cell line cannot
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be used to predict SF (i.e., a higher relative ROS level does not imply a lower SF). Cellular response to ROS depends on the cell's redox potential, 41 which is mainly determined by the intracellular amount of GSH. 42 A previous study has shown that the amount of intracellular GSH varies between cell lines, 43 and therefore the cellular response to ROS can also vary. The addition of antioxidants significantly decreased ROS production. Two mechanisms have been proposed for the inhibitory effects of thiol-containing antioxidants on AuNPinduced ROS production. 38,44 First, thiol-containing antioxidants are able to directly neutralize ROS as a result of their reducing nature. Second, these agents bind to the AuNPs through Au-S bonds, shielding otherwise exposed reactive sites and thus lowering the catalytic activity of the AuNPs. Findings in a study by Pan et al. demonstrate that this second mechanism is probably more important given that a non-thiol-containing antioxidant, ascorbic acid, was unable to reduce the cytotoxicity of AuNPs but several thiol-containing antioxidants were found to significantly reduce their toxic effects. 38 Importantly, the amount of ROS produced in cells may be overestimated using the DCFH-DA assay if apoptosis is induced. During apoptosis cytochrome c, a potent catalyst for oxidation of DCFH, 45 is released from mitochondria into the cytoplasm. 38 In the current study, Z-VAD-fmk, a caspase inhibitor, was employed to determine if the observed ROS levels could be in part attributed to apoptosis. 46 The inability of Z-VAD-fmk to reduce the level of DCF demonstrated that ROS were mainly responsible for the oxidation of DCFH instead of cytochrome c. This also provides indirect evidence that the main mechanism of cell death induced by the AuNPs-TP is necrosis and not apoptosis. Other research groups compared the SF of cells treated with Z-VAD-fmk combined with AuNPs to that of AuNPs alone and showed that Z-VAD-fmk did not increase the SF 38; this further supports the concept that cell death caused by AuNP exposure occurs by necrosis and not apoptosis. In this study GSH was found to be one of the most potent antioxidants, resulting in a significant decrease in ROS generation at all time points. GSH is an endogenous antioxidant that protects cells from oxidative stress by lowering membrane lipid peroxidation. 47 GSH depletion has been observed in cells exposed to NPs. 38,48–50 Co-treatment of HeLa cells with GSH and AuNPs-TP resulted in a fourfold increase in the SF, in comparison to cells treated with AuNPsTP alone. These results confirm that oxidative stress is the key cause of AuNPs-TP cytotoxicity. To sum up, Au in the form of NPs can be customized for use in a variety of applications by altering size, morphology, and surface properties. In this study AuNPs-TP of 2.7 nm diameter were synthesized and characterized. To our knowledge, this is the first time that the neoplastic cell response to AuNPs-TP has been evaluated. The mechanism of cytotoxicity of the AuNPs-TP was found to be ROS generation. Furthermore, antioxidants effectively inhibited ROS and reduced the cytotoxicity of the AuNPs-TP. Overall, the cellular accumulation, cytotoxicity, and ROS production of the AuNPs-TP were shown to be dependent on time, concentration, and cell line. Future studies will focus on evaluating the in vivo distribution of the AuNPs-TP at the wholebody, tissue, and cellular levels.
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nano.2012.05.016.
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Research Article
nanomedjournal.com
Neoplastic cell response to tiopronin-coated gold nanoparticles Lei Cui, BSc a , Payam Zahedi, PhD a , Justin Saraceno, BA a , Robert Bristow, PhD, MD b, c, e , David Jaffray, PhD b, d, e , Christine Allen, PhD a, e,⁎ a Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada Departments of Radiation Oncology and Medical Biophysics, University of Toronto, Toronto, Canada c Ontario Cancer Institute/Princess Margaret Hospital, University Health Network, Toronto, Ontario, Canada d Department of Radiation Physics, Princess Margaret Hospital, University Health Network, Toronto, Ontario, Canada e STTARR Innovation Centre, Radiation Medicine Program, Princess Margaret Hospital, University Health Network, Toronto, Ontario, Canada Received 3 November 2011; accepted 25 May 2012 b
Abstract The present study characterized the in vitro biological response of a comprehensive set of cancer cell lines to gold nanoparticles (2.7 nm) coated with tiopronin (AuNPs-TP). Our findings suggest that upon entering cells, the AuNPs-TP are sequestered in vacuoles such as endosomes and lysosomes, and mostly localize in perinuclear areas. Peak cell accumulation was achieved at 8 hours after incubation. L929 and H520 cells showed more than 75% surviving fraction when treated with 0.5 mg/mL of AuNPs-TP for 24 hours, whereas the surviving fractions were 60% in MCF-7 and 20% in HeLa cells. Reactive oxygen species (ROS) production by the AuNPs-TP was dependent on cell line and exposure time. Antioxidants inhibited ROS generation to various extents, with glutathione and tiopronin being most effective. Overall, exposure time, concentration of the AuNPs-TP, and cell line influenced neoplastic cell response. Furthermore, the mechanism of cytotoxicity of the AuNPs-TP was found to be ROS generation. From the Clinical Editor: This study describes the basic intracellular characteristics of Tiopronin-Au nanoparticles from the standpoint of their anti-cancer activity in different cancer cell cultures. © 2013 Elsevier Inc. All rights reserved. Key words: Gold nanoparticles; Cancer; Cellular accumulation; Cytotoxicity; Reactive oxygen species
The interest in nanotechnology for medical applications such as drug delivery, imaging, and tissue engineering has grown significantly. 1 In particular, gold nanoparticles (AuNPs) have been heavily explored for their use in cancer diagnosis and therapy. 2,3 Au is attractive for such applications because of its inert nature (i.e., stable and nonmetabolizable), biocompatibility, ease of NP size control, and well-developed surface chemistry for functionalization. 4 Varying the size, shape, and surface properties of AuNPs allows for customized optical characteristics, 5 pharmacokinetics and biodistribution, 6–8 and biocompatibility. 9,10 The size of NPs has been shown to influence their biodistribution in vivo. 11 For instance, smaller NPs (b5 nm) penetrate tumors more deeply than do larger particles (N10 nm). 12 In vitro as well, cellular uptake of NPs has been
This research was funded by an operating grant from the Canadian Institutes of Health Research to D.A. Jaffray, R. Bristow, and C. Allen. ⁎Corresponding author: Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario M5S 3M2, Canada. E-mail address: [email protected] (C. Allen).
shown to be dependent on size. 8 The surface properties of NPs, which are largely determined by the surfactants used during preparation and for coating, is another crucial factor that can determine their in vitro and in vivo performance. 13,14 To date, one of the most commonly used surfactants for AuNPs has been thiol-terminated poly(ethylene glycol) (PEG-SH). 15 Two limitations associated with PEG-SH are its relatively large molecular weight and limitations with respect to further functionalization. The method of synthesizing small AuNPs coated with the hydrophilic molecule tiopronin (AuNPs-TP) was first developed by Templeton et al., 16 and there are several advantages associated with the use of this surfactant. First, the core size of the AuNPs-TP can be controlled by changing the molar ratio of Au to tiopronin. As well, the hydrophilicity of tiopronin makes the AuNPs-TP water soluble, enabling administration in vivo. Furthermore, the thiol group in tiopronin makes it capable of tight conjugation to Au atoms through the strong sulfur-gold (S-Au) bond. 17 Also, the small size of the tiopronin molecule means that there is less steric repulsion between the stabilizing molecules, leading to greater coverage of the surface of AuNPs. 16 The strong S-Au bond and high surface coverage
1549-9634/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nano.2012.05.016 Please cite this article as: Cui L., Zahedi P., Saraceno J., Bristow R., Jaffray D., Allen C., Neoplastic cell response to tiopronin-coated gold nanoparticles. Nanomedicine: NBM 2013;9:264-273, http://dx.doi.org/10.1016/j.nano.2012.05.016
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Figure 1. Preparation of AuNPs coated with a monolayer of tiopronin.
make the AuNPs-TP resistant to ligand exchange reactions, reduce the possibility of absorption of other molecules such as proteins onto their surface, and minimize aggregation. In addition, functional groups such as fluorescent probes and active targeting peptides can be easily added to the AuNPs-TP through conjugation with the carboxyl group found on tiopronin. 18,19 Although there are numerous advantages associated with AuNPs-TP, their biological properties have only been characterized to a limited extent. 19,20 Herein, in vitro studies were performed to evaluate and gain an improved understanding of cellular response to AuNPs-TP. Given that AuNPs have been mostly investigated for applications in cancer diagnosis and therapy, three human cancer cell lines (MCF-7 breast cancer cells, HeLa cervical cancer cells, and H520 lung cancer cells) were selected for these studies; a murine fibroblast cell line (L929) was used as a control. Cellular accumulation, intracellular distribution, cytotoxicity, and reactive oxygen species (ROS) production of the AuNPs-TP were assessed. In addition, a series of antioxidants [N-acetylcysteine (NAC), reduced L-cysteine, glutathione (GSH), and tiopronin] were employed to determine their influence on cellular ROS levels following co-treatment with the AuNPs-TP. A concentration range of 0.01–0.5 mg/mL AuNPs-TP was used for these studies. Similar concentration ranges have been investigated in vitro with AuNP systems. 8,10,19 Methods Detailed information on the materials and methods for preparation and characterization of AuNPs-TP, cell culture, qualitative assessment of cellular accumulation of AuNPs-TP, and statistical analysis is available in the Supplementary Methods online at http://www.nanomedjournal.com. Quantitative assessment of cellular accumulation of AuNPs-TP For quantitative analysis of cellular accumulation, cells were seeded in six-well plates at a density of approximately 1 × 10 6 cells per well. Cells were treated with two different concentrations of AuNPs-TP (0.05 and 0.25 mg/mL) for 1, 4, 8, 24, 48, or 72 hours. At each time point cell media were removed, cells were washed three times with phosphate buffered saline (PBS) and then harvested with 0.25% Trypsin with EDTA (Gibco BRL, Grand Island, New York). Cells were counted using a hemocytometer, centrifuged to a pellet, digested with HNO3 at
90°C for 60 minutes, and diluted with double-distilled water (ddH2O). The amount of Au was measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES) and normalized to the number of cells in each sample. The results were reported as the amount of Au (in picograms) per cell. The number of AuNPs-TP in cells was calculated as described in the Supplementary Methods. Evaluation of cytotoxicity of AuNPs-TP Clonogenic assays were performed as described previously. 21,22 Cells were seeded in six-well plates at a density of 1 × 10 6 cells per well, and treated with various concentrations of AuNPs-TP (0.01–0.5 mg/mL). For the HeLa cells, cotreatment with 0.5 mg/mL AuNPs-TP and 3 mM GSH was also tested. After 24 hours of incubation, cells were washed twice with PBS and trypsinized. For each treatment, cells were counted and added into six-well plates at different cell densities (i.e., 300–1800 cells per well). After 7–10 days the colonies were washed with PBS, fixed with methanol, and stained with 1% crystal violet. The number of colonies that consisted of at least 50 cells was counted on each plate. Cell surviving fraction (SF) was expressed as plating efficiency compared to that of nontreated cells. Measurement of ROS production The amount of ROS was measured using the 2′,7′dichlorofluorescein diacetate (DCFH-DA) assay as previously described. 23 Cells were seeded into 96-well plates at a density of 20,000 cells per well. After recovery, cells were washed twice with Hank's Balanced Salt Solution and incubated with 100 μM DCFH-DA for 60 minutes. Cells were then washed with Hank's Balanced Salt Solution and exposed to different treatments, with nontreated cells used as negative controls. Cells treated with 0.3% H2O2 and 10 μM 3-morpholinosydnonimine hydrochloride (SIN) were employed as ROSgenerating positive controls. The treatment groups included 0.5 mg/mL AuNPs-TP; 0.5 mg/mL AuNPs-TP + 3 mM NAC; AuNPs-TP + reduced L-cysteine; AuNPs-TP + GSH; AuNPsTP + TP; and 0.5 mg/mL AuNPs-TP + 50 μM Z-VAD-fmk (Bachem Americas, Torrance, California). At different time points (1, 4, 8, and 24 hours) after treatment, fluorescent signal was measured with a SpectraMax Gemini Plus (Molecular Devices, Sunnyvale, California) microplate reader (λex = 480 nm, λem = 520
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Figure 2. Characterization of the AuNPs-TP. (A) A representative TEM image of the AuNPs-TP. Scale bar, 20 nm. (B) Core size distribution histogram calculated from over 1000 AuNPs-TP. (C) Proton nuclear magnetic resonance spectrum of 0.5 mg/mL AuNPs-TP suspension in D2O. (D) UV-vis spectrum of 1 mg/mL AuNPs-TP in dd-H2O. (E) Percentage of Au that remains in the supernatant following incubation in cell culture medium at 37°C. Data represent mean ± SD (n = 3). (F–H) TEM images of AuNPs-TP in cell culture media following 24, 48, and 72 hours of incubation at 37°C. Scale bar, 20 nm.
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Figure 3. TEM images of AuNPs-TP accumulation in four different cell types. (A, B) MCF-7; (C, D) HeLa; (E, F) H520; (G, H) L929. As highlighted by the arrows in images A and B, once the AuNPs-TP enter cells they appear to sequester in large vacuoles such as endosomes and lysosomes, and mostly localize in the perinuclear region of cells. A similar trend was observed for all cell lines evaluated. Scale bars: 2 μm (A, C, E, G); 100 nm (B, D, F, H).
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Figure 4. In vitro cellular accumulation of AuNPs-TP in four different cell types. (A) MCF-7; (B) HeLa; (C) H520; (D) L929. Accumulation was quantified by inductively coupled plasma atomic emission spectroscopy, with incubation at two different concentrations (0.05 and 0.25 mg/mL) of AuNPs-TP. *Statistically significant difference between the two concentrations (P b 0.05); #statistically significant difference in cell accumulation at different time points in comparison to that at the 8-hour time point (P b 0.05). Data represent mean ± SD (n = 3).
nm). The results were reported as relative ROS production compared to nontreated cells.
Results Synthesis and characterization of AuNPs-TP A representative transmission electron microscopy (TEM) image of the AuNPs-TP suspended in dd-H2O is shown in Figure 2, A. The AuNPs-TP had a mean core diameter of 2.7 nm as obtained from the core size distribution histogram (Figure 2, B). It was determined that each 2.7-nm AuNP-TP was composed of approximately 600 Au atoms. Au atoms composed 76.5% ± 0.3% of the mass of the AuNPs-TP, and the coating efficiency of TP (i.e., the percentage of Au atoms at the surface of the NPs that are coated with TP) was calculated to be 87.3% ± 0.3%. As seen from the proton nuclear magnetic resonance spectrum (Figure 2, C), the final product was spectroscopically pure, with the absence of signals due to unreacted thiol or disulfide byproduct. 16 As evidenced from the ultraviolet-visible (UV-vis) spectrum (Figure 2, D), an absence of absorbance at 520 nm demonstrated that the AuNPs-TP were stable in dd-H2O
with no aggregation. However, it was found that upon incubation of AuNPs-TP in cell culture medium a fraction of the NPs aggregates and precipitates out of solution. Following 24, 48, and 72 hours of incubation in cell culture medium, 82% ± 2%, 78% ± 2%, and 76% ± 2% of the AuNPs-TP, respectively, were found to remain in the supernatant. TEM analysis revealed that the AuNPs-TP that remain in the supernatant retain their size and are well dispersed in the medium (Figure 2, F–H). Cellular accumulation of AuNPs-TP To study the intracellular fate of the AuNPs-TP, cells were observed by TEM. TEM images of four cell lines pretreated with AuNPs-TP are presented in Figure 3. From these images it can be seen that AuNPs-TP mostly localized in the perinuclear areas of cells. AuNPs-TP in cytoplasm were sequestered in large clusters in vacuoles such as the endosomes and lysosomes. The AuNPs-TP did not localize in organelles such as the nucleus or mitochondria. Figure 4 summarizes the quantitative evaluation of cellular accumulation of AuNPs-TP. The total intracellular level of Au is presented in Supplementary Figure S1, A–D. To assess the effect of AuNPs-TP concentration on cellular accumulation,
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cells were incubated with two different concentrations (0.05 mg/mL and 0.25 mg/mL). For all cell lines used, the amount of cellular accumulation was dependent on AuNPs-TP concentration. Statistically significant differences (P b 0.05) in cell accumulation were observed between the two concentrations at all time points. Similar accumulation profiles were observed in MCF-7 and HeLa cells. In the first 8 hours of incubation there was an increase in Au accumulation; the levels then decreased in the following 40 hours and reached a constant level at 48 hours. A similar profile was also observed in the L929 cells incubated with 0.25 mg/mL AuNPs-TP. However, for the H520 cells the cellular accumulation profiles (for both concentrations of AuNP-TP) reached a maximum at 8 hours, and this level remained relatively constant up to 72 hours. For the L929 cells incubated with 0.05 mg/mL AuNPs-TP a significant decrease was observed at 24 hours compared to 8 hours, after which the level increased to a value similar to that achieved at the 8-hour time point. Statistically significant differences (P b 0.05) in cell accumulation at different time points in comparison to the uptake at the 8-hour time point are shown in Figure 4. At the peak time point for accumulation (i.e., 8 hours), HeLa and L929 cells incubated with 0.25 mg/mL of AuNPs-TP showed higher accumulation of AuNPs-TP (HeLa, 42 pg/ cell, 2.1 × 10 8 AuNPs-TP per cell, and L929, 48 pg/cell, 2.4 × 10 8 AuNPs-TP/cell) compared to MCF-7 (26 pg/cell, 1.3 × 10 8 AuNPs-TP per cell), and H520 (15 pg/cell, 0.78 × 10 8 AuNPs-TP per cell) cells. Interestingly, despite the large differences seen in accumulation at the 8-hour incubation period, all cell lines showed a similar concentration of Au accumulation at 72 hours. These concentrations were approximately 15 pg/cell (0.78 × 10 8 AuNPs-TP per cell) and 5 pg/cell (0.26 × 10 8 AuNPs-TP per cell) when incubated with 0.25 mg/mL and 0.05 mg/mL of the AuNPs-TP, respectively. Cytotoxicity of AuNPs-TP The biocompatibility of the AuNPs-TP was assessed by clonogenic assay. Incubation of AuNPs-TP with L929 and H520 cells resulted in more than 75% surviving fraction (SF) even at the highest AuNPs-TP concentration (i.e., 0.5 mg/mL). For HeLa cells, the SF decreased significantly (P b 0.05) when treated with 0.25 and 0.5 mg/mL of the AuNPs-TP. Specifically, HeLa cells showed only 20% SF at the highest concentration tested. For MCF-7 cells, a significant decrease (P b 0.05) in SF was observed at 0.5 mg/mL. (See Figure 5.) Measurement of ROS production To determine if the cytotoxic effect of the AuNPs-TP in HeLa and L929 cell lines could be attributed to oxidative stress the ROS generated by the AuNPs-TP was measured using the DCFH-DA assay (Figure 6). Incubation of cells with 0.3% H2O2 or 10 μM SIN for 1 hour, as positive controls, resulted in high ROS levels compared to nontreated cells. The amount of ROS produced following treatment with the AuNPs-TP increased with incubation time yet was lower than the positive controls. ROS production following treatment with the AuNPs-TP in combination with various antioxidants (i.e., NAC, reduced L-cysteine, GSH, or tiopronin) was also evaluated. The degree of ROS
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Figure 5. Cell surviving fraction (SF) after 24 hours of treatment with different concentrations of AuNPs-TP. SF as determined by clonogenic assays is reported as plating efficiency compared to nontreated cells. *Statistically significant difference between various concentrations for HeLa cells; #statistically significant difference between various concentrations for MCF-7 cells (P b 0.05). Data represent mean ± SD (n = 3).
inhibition depended on the antioxidant employed (Figure 6). GSH and tiopronin were more effective in decreasing ROS compared to NAC and reduced L-cysteine. Co-treatment of cells with Z-VAD-fmk and AuNPs-TP resulted in ROS levels that were similar to those achieved following treatment with AuNPs-TP alone. The ability of GSH to rescue cells from oxidative stress induced by AuNPs-TP was evaluated. Cell clonogenicity was compared between HeLa cells treated with AuNPs-TP alone and in combination with GSH. It was found that the cell SF was increased by fourfold following combination treatment [i.e., SF = 19% ± 0.45% (only AuNPs-TP) vs. 91% ± 4.4% (AuNPs-TP + GSH)].
Discussion With its unique physical and chemical characteristics, gold in the form of AuNPs has been widely explored for biomedical applications such as drug delivery, imaging, disease diagnosis, and therapy. 2,3 The size, shape, and surface coating of AuNPs can be modified to alter in vitro cellular response. 7,24 Tiopronin has several advantages over conventionally used PEG-based surfactants; however, little is known about the biological performance of AuNPs-TP. In this study, AuNPs-TP were synthesized and assessed in terms of size, purity, in vitro cell accumulation, and cytotoxicity. The mechanism of cytotoxicity was also investigated. A significant challenge in the chemical synthesis of AuNPs is achieving monodispersity in size. 25 In this study the mean core diameter of AuNPs-TP suspended in dd-H2O was 2.7 nm, and the size distribution histogram showed a relatively narrow size distribution (1.5–3.9 nm). This relatively high monodispersity of the AuNPs-TP was achieved using the method described by Templeton et al., in which synchronized growth and coating of the NPs are achieved with the thiol-containing reducing
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Figure 6. Amount of ROS produced relative to nontreated cells following treatment with AuNPs-TP (0.5 mg/mL) in combination with antioxidants including NAC, reduced L-cysteine, GSH, or tiopronin (3 mM) and the apoptotic inhibitor Z-VAD-fmk (50 μM) in (A) HeLa cells and (B) L929 cells. The insets show relative ROS produced in cells following treatment with 0.3% H2O2 or 10 μM SIN for 1 hour compared to nontreated cells. Data represent mean ± SD (n = 4).
reaction. 16 The stability of the AuNPs-TP in dd-H2O was assessed based on their optical properties (i.e., surface plasmon resonance). The UV spectrum of AuNPs with diameters greater than 3 nm is known to include absorption at a wavelength of approximately 520 nm. As well, any change in the surface of the AuNPs-TP such as absorption of macromolecules or aggregation can lead to changes in the UV-vis spectrum. 26,27 The absence of peaks in the UV-vis spectra (i.e., in the 520-nm range) of the AuNPs-TP confirmed their stability and lack of aggregation in dd-H2O. However, aggregation of AuNPs was observed upon incubation in cell culture medium. Aggregation occurs when NPs interact with cell culture medium, given that ions such as Na + and Cl − in the medium neutralize the surface charge of the NPs. 28 This aggregation is said to be an immediate and irreversible process. It is believed that absorption of proteins onto the surface of some AuNPs can assist in stabilizing the particles and preventing aggregation. However, absorption of proteins onto AuNPs-TP is unlikely given the high coating efficiency of TP (87.3% ± 0.3%) and the strong S-Au bond, which is resistant to ligand exchange reactions. Cellular accumulation of the AuNPs-TP was visualized in four different cell lines by TEM. The AuNPs-TP that entered the cells were sequestered in large clusters in vacuoles in the perinuclear areas of the cytoplasm. Previous studies have made similar observations. 20,29 For applications such as drug delivery or radiosensitization it may be important to target the AuNPs to specific subcellular organelles such as the nucleus. This delivery to specific intracellular compartments may be achieved by conjugating targeting moieties to the surface of the AuNPsTP. De la Fuente et al. conjugated the TAT peptide onto the surface of AuNPs-TP and demonstrated successful transport to the cell nucleus. 19 Quantitative assessment of cellular accumulation of the AuNPs-TP is of relevance given that the cellular concentration
determines potential toxicity and therapeutic effect. Many variables contribute to the accumulation profile of NPs in cells, including the physicochemical properties of the NPs (e.g., size, morphology, surface properties), cell type, concentration of the NPs, and incubation conditions. These variables, in turn, determine the rate of cell proliferation (i.e., doubling time), extent and rate of endocytosis of NPs, as well as extent and rate of exocytosis. For instance, previous studies have shown that the size of AuNPs is an important factor that influences the rate of endocytosis and exocytosis, and thus the level of cellular accumulation. 8,29 Chithrani et al. demonstrated that incubation of HeLa cells with AuNPs of 50 nm diameter resulted in the highest level of cell accumulation following a 10-hour incubation period, in comparison to AuNPs with diameters of 14 nm and 74 nm. 29 Despite their small size, the AuNPs-TP evaluated in the current study showed a higher level of cellular accumulation (in HeLa cells) at 8 hours (42 pg/cell) than the 50-nm AuNPs (8 pg/cell). 8 The time-dependent accumulation profiles of AuNPs-TP revealed a peak at 8 hours for all cell lines incubated with AuNPs-TP at a concentration of 0.25 mg/mL. A similar trend was observed in previous studies, wherein peak AuNP accumulation was observed following 6–10 hours of incubation. 8,30 The initial increase in the cellular level of AuNPs-TP that occurs at the early time point is largely attributed to endocytosis. Several factors may contribute to the decrease in and constant cellular levels of AuNPs-TP that were achieved at later time points, including endocytosis and exocytosis of the AuNPs-TP that occur simultaneously, cell proliferation, saturation of uptake, decrease in AuNPs-TP dose due to cell uptake and NP aggregation, and state of AuNPs-TP in the cell culture medium. Each of these factors has been considered below. As shown in Supplementary Figure S1, A–D, the total amount of AuNPs-TP that is internalized into cells increases over the 72-
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hour time period. This indicates that cellular uptake of AuNPsTP occurs throughout the incubation period. In the current study quantitative analysis of exocytosis was not conducted, and therefore the contribution of exocytosis to the cellular levels of AuNPs-TP remains unknown. A previous study by Chithrani et al. reported that following a 6-hour incubation period and a subsequent 8-hour washout period, approximately 8%, 20%, and 40% of the AuNPs that had been endocytosed were exocytosed for AuNPs with diameters of 74, 50, and 14 nm, respectively. 29 One drawback of this study is that exocytosis was only observed under washout conditions, wherein cells are exposed to particlefree medium. A recent study by Kim et al. indicates that cell proliferation is the major factor that leads to dose reduction of NPs in cells. 31,32 The authors demonstrated constant levels of NPs throughout the cell cycle until cell division in A549 cells. Indeed, in the current study a comparison between the decrease in the number of AuNPs-TP per cell following the 8-hour time point and the respective doubling time for each cell line indicates that cell proliferation plays a significant role. The magnitude of the decrease in number of AuNPs-TP per cell decreased in the following order: L929 N HeLa N MCF-7 N H520. The doubling times for the cell lines are as follows: L929, 14 hours; HeLa, 24 hours; MCF-7, 29 hours; H520, 61 hours. Therefore, it can be seen that the decrease in number of AuNPs per cell is related to the concomitant increase in cell number. Given that the peak in the accumulation profile at 8 hours is also observed, although less prominently, for HeLa, MCF-7, and L929 cells incubated with the lower concentration (0.05 mg/mL) of AuNPs-TP, this peak cannot be attributed to saturation of uptake. In addition, at all time points it is only a fraction of the AuNPs-TP present in solution that are taken up into cells. Specifically, for L929 cells, which showed the highest level of cell uptake following the 8-hour incubation period, the amount of intracellular Au was 54.8 ± 2.7 μg, which accounts for only 5.7 wt% of the total Au in medium containing 0.25 mg/mL AuNPs-TP. This result suggests that there is always a signficant excess of AuNPs-TP available in the medium. Furthermore, as shown in Figure 2, E, there was no significant difference between the percentage of AuNPs-TP that remain dispersed in the supernatant following 8 and 24 hours of incubation. Therefore, the decrease in the cellular level of AuNPs-TP that occurs beyond the 8-hour time point cannot be attributed to a decrease in the total number of AuNPs available in solution due to aggregation. The state of AuNPs-TP in the cell culture medium could also influence the degree of cellular uptake. TEM analysis of AuNPsTP following incubation in cell culture medium for 24, 48, and 72 hours revealed the AuNPs-TP that remain in the supernatant retain their size and are well dispersed in the medium. However, it is recognized that the size of particles incubated in cell culture medium alone (i.e., in the absence of cells) may not be representative of their size in all regions of the cell culture wells in the presence of cells. Based on the literature, the relationship between the state of AuNPs in solution and cell uptake is complex. For example, Cho et al. recently investigated the effects of aggregation and consequent sedimentation of AuNPs on cell uptake. They demonstrated that sedimentation
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leads to a higher concentration of AuNPs in the cell uptake zone; as a result, a higher uptake was observed in cells in an upright configuration compared to inverted cells. 33 This observation suggests that the actual dose of AuNPs in the cell uptake zone is higher than that measured in the supernatant. Furthermore, a study by Albanese et al. demonstrated differential uptake patterns for single and aggregated NPs in different cell lines. For example, HeLa and A549 cells showed preferential uptake of single NPs, whereas uptake of aggregated NPs was favored in MDA-MB-435 cells. The authors ascribed this phenomenon to different mechanisms of cellular uptake being operative in different cell lines. In addition to receptor-mediated endocytosis, which is a major cell uptake pathway in cell lines such as A549, receptor-independent uptake also plays a significant role in cell lines such as MDA-MB-435. 28 Oxidative stress, which includes ROS generation, is currently the most established theory for NP toxicity. 34 Oxidative stress has been shown to occur in cells following exposure to NPs of single composition such as silica, 35 silver, 36 polystyrene, 37 and gold. 38 The highly curved surface of nanosized particles is said to result in greater defects in crystal structure, therefore disrupting the electronic configuration in the bulk material. 34 These surface properties create reactive electron donor and acceptor groups, which can interact with molecules such as oxygen (O2). For example, as reviewed by Nel et al., the transfer of an electron from a reactive donor group at the surface of a NP to O2 results in the creation of superoxide radicals. 34 Therefore, even though bulk Au is considered to be chemically inert and nontoxic, AuNPs behave very differently than their bulk counterpart. 39 In this study the clonogenic assay was used to characterize the effect of the AuNPs-TP on cell proliferation. The results showed that cytotoxicity depended on both the concentration of AuNPsTP and the type of cell line used. Using the MTT assay (a colorimetric assay used primarily to determine viability and proliferation of cells), tiopronin was found to be nontoxic in HeLa and MCF-7 cells at a concentration equivalent to that present on the surface of 0.5 mg/mL AuNPs-TP (data not shown). As well, as shown in Figure 6, the presence of free tiopronin inhibited ROS generation by AuNPs. This result confirms that the toxicity of the AuNPs-TP cannot be attributed to tiopronin. The effect of various thiol-containing antioxidants (i.e., NAC, reduced L-cysteine, GSH, tiopronin) on reducing ROS production due to AuNPs-TP exposure was evaluated. The intracellular levels of ROS were measured using the DCFH-DA assay. 23 Within cells, DCFH-DA is hydrolyzed to DCFH by esterase; DCFH is then oxidized to fluorescent DCF in the presence of ROS, and the fluorescence intensity produced is proportional to the ROS concentration. 40 Results from the DCFH-DA assay demonstrated that AuNPs-TP induced high levels of ROS following a 24-hour exposure, in comparison to nontreated cells. The increase in ROS following AuNPs-TP treatment explains the toxicities observed for the AuNPs-TP at high concentration. A comparison of relative ROS levels (3.69 ± 0.30 for HeLa and 5.38 ± 0.57 for L929) and SF (19.0% ± 0.45% for HeLa and 76.7% ± 5.08% for L929) between the HeLa and L929 cells reveals that the relative ROS level for a specific cell line cannot
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be used to predict SF (i.e., a higher relative ROS level does not imply a lower SF). Cellular response to ROS depends on the cell's redox potential, 41 which is mainly determined by the intracellular amount of GSH. 42 A previous study has shown that the amount of intracellular GSH varies between cell lines, 43 and therefore the cellular response to ROS can also vary. The addition of antioxidants significantly decreased ROS production. Two mechanisms have been proposed for the inhibitory effects of thiol-containing antioxidants on AuNPinduced ROS production. 38,44 First, thiol-containing antioxidants are able to directly neutralize ROS as a result of their reducing nature. Second, these agents bind to the AuNPs through Au-S bonds, shielding otherwise exposed reactive sites and thus lowering the catalytic activity of the AuNPs. Findings in a study by Pan et al. demonstrate that this second mechanism is probably more important given that a non-thiol-containing antioxidant, ascorbic acid, was unable to reduce the cytotoxicity of AuNPs but several thiol-containing antioxidants were found to significantly reduce their toxic effects. 38 Importantly, the amount of ROS produced in cells may be overestimated using the DCFH-DA assay if apoptosis is induced. During apoptosis cytochrome c, a potent catalyst for oxidation of DCFH, 45 is released from mitochondria into the cytoplasm. 38 In the current study, Z-VAD-fmk, a caspase inhibitor, was employed to determine if the observed ROS levels could be in part attributed to apoptosis. 46 The inability of Z-VAD-fmk to reduce the level of DCF demonstrated that ROS were mainly responsible for the oxidation of DCFH instead of cytochrome c. This also provides indirect evidence that the main mechanism of cell death induced by the AuNPs-TP is necrosis and not apoptosis. Other research groups compared the SF of cells treated with Z-VAD-fmk combined with AuNPs to that of AuNPs alone and showed that Z-VAD-fmk did not increase the SF 38; this further supports the concept that cell death caused by AuNP exposure occurs by necrosis and not apoptosis. In this study GSH was found to be one of the most potent antioxidants, resulting in a significant decrease in ROS generation at all time points. GSH is an endogenous antioxidant that protects cells from oxidative stress by lowering membrane lipid peroxidation. 47 GSH depletion has been observed in cells exposed to NPs. 38,48–50 Co-treatment of HeLa cells with GSH and AuNPs-TP resulted in a fourfold increase in the SF, in comparison to cells treated with AuNPsTP alone. These results confirm that oxidative stress is the key cause of AuNPs-TP cytotoxicity. To sum up, Au in the form of NPs can be customized for use in a variety of applications by altering size, morphology, and surface properties. In this study AuNPs-TP of 2.7 nm diameter were synthesized and characterized. To our knowledge, this is the first time that the neoplastic cell response to AuNPs-TP has been evaluated. The mechanism of cytotoxicity of the AuNPs-TP was found to be ROS generation. Furthermore, antioxidants effectively inhibited ROS and reduced the cytotoxicity of the AuNPs-TP. Overall, the cellular accumulation, cytotoxicity, and ROS production of the AuNPs-TP were shown to be dependent on time, concentration, and cell line. Future studies will focus on evaluating the in vivo distribution of the AuNPs-TP at the wholebody, tissue, and cellular levels.
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.nano.2012.05.016.
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