Exposure to silver nanoparticles induces size- and dose-dependent oxidative stress and cytotoxicity in human colon carcinoma cells

Toxicology in Vitro 28 (2014) 1280–1289

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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit

Exposure to silver nanoparticles induces size- and dose-dependent oxidative stress and cytotoxicity in human colon carcinoma cells Rona Miethling-Graff a,⇑, Rita Rumpker a, Madeleine Richter a, Thiago Verano-Braga b, Frank Kjeldsen b, Jonathan Brewer c, James Hoyland d, Horst-Günter Rubahn d, Helmut Erdmann a a

Biotechnology, University of Applied Science, Flensburg, Germany Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark MEMPHYS Center for Biomembrane Physics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark d Mads Clausen Institute, University of Southern Denmark, Sønderborg, Denmark b c

a r t i c l e

i n f o

Article history: Received 11 November 2013 Accepted 23 June 2014 Available online 2 July 2014 Keywords: Silver nanoparticles Cytotoxicity Cellular uptake LoVo cell line

a b s t r a c t The antimicrobial properties of silver nanoparticles (AgNPs) have made these particles one of the most frequently utilized nanomaterials in consumer products; therefore, a comprehensive understanding of their toxicity is necessary. In particular, information about the cellular uptake and size dependence of AgNPs is insufficient. In this study, we evaluated the size-dependent effects of AgNPs by treating the human LoVo cell line, an intestinal epithelium model, with spherical AgNPs of well-defined sizes (10, 20, 40, 60 and 100 nm). The cellular uptake was visualized by confocal laser scanning microscopy, and various cytotoxicity parameters were analyzed in a size- and dose-dependent manner. In addition, the cellular proteomic response to 20 and 100 nm AgNPs was investigated to increase the understanding of potential mechanisms of action. Our data indicated that cellular uptake and toxicity were regulated by size; smaller particles easily penetrated the cells, and 100 nm particles did not. It was hypothesized that this size-dependent effect resulted from the stimulation of a signaling cascade that generated ROS and inflammatory markers, leading to mitochondrial dysfunction and subsequently inducing apoptosis. By contrast, the cell proliferation, was independent of AgNPs particle size, indicating a differentially regulated, ROS-independent pathway. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction In addition to the increasing number of nanotechnology applications in medical diagnostics and therapeutics (Chen and Schluesener, 2008), the utilization of nanostructures in consumer products has recently become more important. Novel physiochemical properties of materials on the nanoscale have enabled an improvement in the functionality and other properties of these

Abbreviations: AgNP, silver nanoparticle; ROS, reactive oxygen species; IL-8, interleukin 8; ELISA, enzyme-linked immunosorbent assay; CLSM, confocal laser scanning microscopy; BrdU, 5-bromo-20 -deoxyuridine; DCFDA, 20 ,70 -dichlorofluorescein diacetate; FITC, fluorescein isothiocyanate; PI, propidium iodide; TEM, transmission electron microscopy; SEM–EDX, scanning electron microscopy/energy dispersive X-ray spectroscopy; PVDF, polyvinylidene fluoride; GO, gene ontology; SUMO, small ubiquitin-like modifier. ⇑ Corresponding author. Address: Biotechnology, University of Applied Science Flensburg, Kanzleistr. 91–93, D-24943 Flensburg, Germany. Tel.: +49 461 8051247; fax: +49 461 805 1300. E-mail address: rona.graff@fh-flensburg.de (R. Miethling-Graff). http://dx.doi.org/10.1016/j.tiv.2014.06.005 0887-2333/Ó 2014 Elsevier Ltd. All rights reserved.

nanoproducts. However, the use of nanoparticles arise risks for human health, because the small size of nanoparticles enables them to cross natural cell barriers, perhaps causing cytotoxic effects. The chemistry, structure and size of nanoparticles dictate the potential adverse effects (Nel et al., 2006; Oberdorster et al., 2005). In 2011, the Woodrow Wilson Inventory listed 1317 products from 30 different countries that were subdivided into the primary categories of health and fitness, personal care, clothing and cosmetics (http://www.nanotechproject.org). Silver is included in 30% of the materials utilized in nanotechnology-based consumer products, which gives it the highest degree of commercialization, followed by carbon (including fullerenes), titanium, silica, zinc and gold. Nanosilver is utilized in food contact and packaging materials, odor-resistant textiles and personal care and cosmetic products, as well as food additives that exploit its antimicrobial properties (Wijnhoven et al., 2009). Therefore, humans are exposed to AgNPs in an increasing degree via inhalation, dermal contact and digestion.

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Despite several studies that have addressed the toxicology of nanoparticles, a knowledge gap remains in fully understanding how cells interact with nanostructures of well-defined sizes. Across numerous cell lines, the toxicity of silver nanoparticles has been reported to be mediated by the induction of oxidative stress that is associated with decreased viability, the inhibition of mitochondrial activity and the initiation of apoptosis and cell death (AshaRani et al., 2009; Carlson et al., 2008; Foldbjerg et al., 2009, 2011; Hussain et al., 2005; Kim et al., 2009). Whether the toxic effects of silver nanoparticles result from a combination of their specific properties or from their release of ions from them is still not answered sufficiently (Bolt et al., 2013). Some studies have indicated that the ionization of AgNPs causes cytotoxicity by a Trojan horse-type mechanism (Park et al., 2010b; Singh and Ramarao, 2012), whereas there is evidence that AgNP toxicity is primarily the result of oxidative damage and is independent of the toxicity of Ag+ ions (Kim et al., 2009). A recent study demonstrated that AgNPs and Ag+ ions affected different sets of proteins and that more proteins were regulated by the nanoparticles than by the free silver ions released into solution by the nanoparticles (Verano-Braga et al., 2014). Particle size and surface area are important material characteristics from a toxicological point of view because the number of reactive groups increases with decreasing size and increasing surface area. Furthermore, the organ uptake and distribution of colloidal nanoparticles is size-dependent in mice (Hillyer and Albrecht, 2001; Park et al., 2010a). However, particle coating, surface treatments and aggregation can change the physiochemical properties and modify the cellular responses (Nel et al., 2006). For instance, the capping agent N-acetylcysteine significantly reduced the toxic effects of AgNPs (Avalos Funez et al., 2013; Chairuangkitti et al., 2013; Foldbjerg et al., 2011; Kim et al., 2009). A few studies have addressed the question of size dependence for silver nanoparticles, and they reported different outcomes. Hussain and coworkers (Hussain et al., 2005) investigated the in vitro toxicity of 15 and 100 nm AgNPs and observed size-independent cellular responses in rat liver cells, whereas Carlson (Carlson et al., 2008) detected size-dependent, ROS-mediated toxicity of 15, 30 and 55 nm hydrocarbon-coated AgNPs in macrophages. Besides different effects on various cell types, a possible cause for the divergent results might be the use of nanoparticles with a broad size distribution. The present study was designed to address the question of size dependence by analyzing spherical silver nanoparticles of welldefined sizes (10, 20, 40, 60 and 100 nm) exhibiting a very narrow size distribution. With respect to numerous applications of nanosilver in the food production chain, the human LoVo intestinal carcinoma cell line was chosen as a model for evaluating potential toxicity. Cellular uptake was visualized by CLSM, and cytotoxicity parameters, including ROS generation, mitochondrial activity, cytokine release, proliferation and the initiation of apoptosis, were analyzed in cells exposed to 10–100 nm silver nanoparticles. In addition, the cellular proteomic response to 20 and 100 nm AgNPs was investigated to gain insight into potential particle sizedependent mechanisms of action.

2. Materials and methods 2.1. Silver nanoparticles Spherical silver nanoparticles (NanoXact™) sized 10, 20, 40, 60 and 100 nm and stabilized with citrate were purchased from nanoComposix (San Diego, CA). The mass concentration was 0.02 mg/ml in 2 mM citrate buffer. The particles were comprehensively characterized by the manufacturer in terms of diameter and size distribution, spectral properties, TEM analysis, hydrodynamic diameter

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and zeta potential. There was a very narrow size distribution of the diameter of the particles, with less than 10% deviation for each lot. Furthermore, the SEM–EDX analysis revealed no impurities in the elemental composition of the stock solution. The behavior of the particles in RPMI 1640 cell culture medium was determined by dynamic light scattering analysis and UV/Vis spectroscopy, and no agglomeration of the nanoparticles was observed within 48 h. This is in accordance with other studies that analyzed the agglomeration of citrate-stabilized AgNPs in RPMI medium (Greulich et al., 2009; Kittler et al., 2010). Prior to the experiments, the AgNP solution was filtered through a 0.22 lm PVDS syringe filter (Roth, Karlsruhe, Germany). 2.2. Cell culture The human LoVo colon carcinoma cell line (ACC 350, German Collection of Microorganisms and Cell Cultures-DSMZ, Germany) was grown as a monolayer in RPMI 1640 medium with stable glutamine supplemented with 10% FBS Superior (Biochrom, Berlin, Germany) at 37 °C in a 5% CO2 humidified atmosphere. Passages 5–15 were used for the cytotoxicity analyses. 2.3. Exposure protocol Cells were cultured for 24 h prior to the experiments in assayappropriate dishes, and all the assays were performed in RPMI 1640 medium containing 10% FBS. After removing the culture medium, the dose-dependent experiments with AgNPs were initiated by adding fresh culture medium containing AgNPs. Controls contained no AgNPs. The AgNP doses were calculated based on the mass of silver and were adjusted by diluting the stock solution with cell culture medium. The doses ranged from 0 to 10 lg/ml. In preliminary tests, PBS buffer was used as vehicle control to exclude any nutrient limitation in the selected concentration range due to the dilution of the culture medium. 2.4. ROS The intracellular generation of reactive oxygen species (ROS) was evaluated with the fluorescence marker H2DCF-DA (20 ,70 dichlorodihydrofluorescein diacetate) as previously described (AshaRani et al., 2009) with minor modifications. Briefly, 5  105 cells were seeded in 12-well plates and pre-incubated for 24 h. After a 24 h exposure to AgNPs, the cells were washed once with PBS and subsequently stained with 1 ml of 5 lM H2DCF-DA for 30 min in the dark at 37 °C/5% CO2. After removing the staining solution and washing with PBS, the cells were trypsinized, pelleted, resuspended and analyzed by flow cytometry for fluorescent DCF. Hydrogen peroxide was utilized as a positive control. 2.5. IL-8 ELISA assay Interleukin-8 release was analyzed in the culture media of LoVo cells exposed to 1 or 10 lg/ml AgNP for 24 or 48 h. Cell supernatants were collected from 12-well plates initially seeded with 5  105 cells/well, centrifuged at 200g for 5 min and frozen at 20 °C. IL-8 cytokine levels were quantified using an ELISA kit (RayBioÒ Human IL-8, Ray Biotech, Inc.) according to the manufacturer’s protocol. The absorbance was measured at 450 nm in a microplate reader (Discovery HT-R, BioTek Instruments, Vermont, US). 2.6. CellTiter-BlueÒ cell viability assay This assay was used to measure mitochondrial activity and is based on the ability of living cells to reduce resazurin into

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fluorescent resorufin. The assay was performed in 96-well microtiter plates according to the manufacturer’s guidelines (Promega, Madison, US) using the multi-mode microplate reader FLx800 (BioTek Instruments, Vermont, US). The fluorescence was measured following a 3 h incubation with 20 ll resazurin reagent after a 24 or 48 h exposure to AgNPs. Initially, exponentially growing LoVo cells were seeded at 1  104 cells/well. All the samples and controls were assayed in at least triplicate. 2.7. BrdU proliferation assay To quantify the proliferation of LoVo cells, BrdU (Bromodeoxyuridine) incorporation into newly synthesized cellular DNA was determined via a colorimetric method using a cell proliferation ELISA kit (Roche Diagnostics, Mannheim, Germany). The ELISA was performed according to the manufacturer’s guidelines. Briefly, 104 cells/well were seeded in a 96-well plate and pre-incubated for 24 h under cell culture conditions. After 24 h, the dose-dependent exposure to AgNPs was initiated by adding fresh culture medium containing AgNPs. After 24 h, the BrdU labeling solution was added, and the cells were incubated for 4 h in the dark at 37 °C in a humidified atmosphere of 5% CO2 in air. Subsequently, the labeling solution was discarded, and the DNA was denatured. The antibody conjugate was incubated for 90 min at room temperature, followed by three washes with PBS. The reaction of the added TMB (tetramethyl-benzidine) substrate directly correlates with the amount of synthesized DNA and therefore to the number of proliferating cells; this reaction was quantified by measuring the absorbance at 370 nm in a microplate reader (Discovery HT-R, BioTek Instruments, Vermont, US). For this assay, all the samples were analyzed in at least four replicates to minimize the deviation. 2.8. Annexin V/PI assay To quantitate the presence of apoptotic and necrotic cells, morphologic changes in the plasma membrane were detected using a FITC Annexin V/propidium iodide (PI) Apoptosis Detection Kit (BD Biosciences, Heidelberg, Germany) and analyzed by flow cytometry. Phosphatidyl-serine, which is externalized in the early stages of apoptosis, was detected by annexin V conjugated to FITC, and late apoptotic/necrotic membrane damage was detected via the intercalation of PI into nuclear DNA. This assay was performed according to the manufacturer’s guidelines using 1  105 cells for staining. Negative, positive and staining controls were included in each assay. Tamoxifen (50 lM) was utilized as a positive control. 2.9. Proteomics approach The proteomics dataset was obtained in a previous study (Verano-Braga et al., 2014) and re-analyzed using different software – Ingenuity Pathways Analysis (Ingenuity Systems, Redwood City, CA) and Protein Center (Thermo, Waltham, MA) – to provide new information on canonical pathways and gene ontology of the differentially regulated proteins, respectively. A brief description of the proteomics approach is given as follows. LoVo cells were exposed to 20 and 100 nm AgNPs at a concentration of 10 lg/ml for 24 h before lysis, and the proteins were digested with trypsin as previously described (Manza et al., 2005; Wisniewski et al., 2009). Subsequently, 30 lg of peptides was labeled with iTRAQ 8-plex reagent according to the manufacturer’s specifications, combined in equal amounts and then fractionated on a TSKGel Amide 80 HILIC HPLC column (length: 15 cm, diameter: 2 mm, particle size: 3 mm) to reduce the sample complexity. Each HILIC fraction was analyzed by nanoflow liquid chromatography coupled to tandem mass spectrometry (nLC–MS/ MS) using an Easy-LC nanoHPLC (Thermo Fisher, Waltham, MA)

interfaced with a Thermo LTQ Orbitrap Velos MS (Thermo Fisher, Waltham, MA). The tandem mass spectrometry data were searched against the Homo sapiens database download from Swiss-Prot (Sprot 2012_07 version; 536,789 sequences; 190,518,892 residues) using an inhouse Mascot server (version 2.3.0; Matrix Science Ltd., London, UK). The intensities of the peptides identified in the 3 independent biological replicates were log 2-transformed and normalized based on the median. The R Rollup function in the DanteR package (http://www.omics.pnl.com) was utilized to merge the peptides from the same protein using the mean. The protein abundance values were calculated using the ANOVA procedure in the DanteR software and were corrected for multiple testing with the Benjamini–Hochberg method (Benjamini and Hochberg, 1995). Only proteins with a p-value 60.01 were considered to be differentially regulated.

2.10. Confocal laser scanning microscopy (CLSM) – nanoparticle uptake Confocal laser scanning microscopy was performed to elucidate the particle size-dependence of cellular uptake and the distribution pattern of the silver nanoparticles. For imaging, 5  105 LoVo cells were cultured in CELLview dishes (Greiner Bio-One, Frickenhausen, Germany) for 24 h. After the subsequent exposure to 10 lg/ml of 20–100 nm AgNPs, the medium was removed, and the cells were stained with DiI (1,10 -Dioctadecyl-3,3,30 ,30 -Tetramethylindocarbocyanine Perchlorate; DiIC18(3)), a lipophilic membrane dye (Molecular ProbesÒ, Life Technologies, Darmstadt, Germany). Five microliters of DiI (1 mM in ethanol) diluted in 1 ml phenol red-free cell culture medium was used to stain the cells for 20 min at 37 °C. Next, the cells were washed three times with fresh medium and allowed to recover for 10 min before proceeding. Finally, 1 ml phenol red-free medium was added, and the cells were stored at 4° to 10 °C. Confocal imaging was performed on a Zeiss LSM 510 META. The samples were imaged using a 63  1.4 NA oil immersion objective (Carl Zeiss). The nanoparticles were imaged using a femtosecond pulsed laser (Mai Tai broadband, Spectra Physics, Mountain View, CA) operated at 800 nm. The emission was collected through a 685 nm short pass filter. Control measurements were performed to ensure that only the silver nanoparticles were imaged in the channel designated for recording the nanoparticles. The fluorescent emission from the DiI was collected through a 560 nm long pass filter. The included images are representative of more than 30 cells. To enumerate the internalized silver nanoparticles, 30–40 cells exposed to the respective AgNP size were analyzed in confocal zstacks of a distance of 0.5 lm for 20 nm particles and 1 lm for all larger nanoparticles. All particles inside the cells were counted manually in each stack. A total of 3233 nanoparticles were accounted for quantification. Because the resolution limit of the CLSM is approximately 300 nm (Stelzer, 1998), it is not possible to determine if the observed particles are single particles or aggregates. However, considering the brightness of the particles and the TEM images of the 20 nm AgNPs, it is most likely that the bright particles observed in the CLSM of the 20 nm AgNPs were aggregates of single particles (Supplemental Fig. 1). However, in the case of the 100 nm AgNPs, the particles detected by CLSM could have been single nanoparticles in some cases. Consequently, when counting the particles inside the cells, the particles are referred to as ‘‘AgNPs/AgNP aggregates’’ as it is not possible to differentiate between the two cases.

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2.11. Flow cytometry

3.2. Pro-inflammatory response

All the fluorescence was analyzed with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). An argon laser with a wavelength of 488 nm was used for excitation. FITC and DCF were analyzed at an emission wavelength of 530/30 nm, and PI was detected at 585/42 nm. The flow cytometry configuration, standard compensation and data acquisition were performed using the CellQuest™Pro software suite provided by Becton Dickinson. A total of 5000 events were collected for each analysis. The raw data were analyzed using Cyflogic™ software.

To investigate the pro-inflammatory response of LoVo cells to AgNPs, IL-8 release from cells exposed to 1 or 10 lg/ml AgNPs for 24 or 48 h was quantitated. A dose of 1 lg/ml of 10 or 20 nm AgNPs induced the progressive release of IL-8 after 24 or 48 h (Fig. 2). After 24 h, a 4.5-fold increase in IL-8 secretion was observed in the supernatant of cells exposed to 10 nm AgNPs, corresponding to 113 pg/ml IL-8 that further accumulated to 147 pg/ ml within the next 24 h. For 20 nm AgNPs, a dose of 1 lg/ml resulted in 36 and 62 pg/ml IL-8 after 24 and 48 h, respectively; for all the other nanoparticle sizes, there was no significant increase in IL-8 at this concentration. By contrast, 10 lg/ml of all the AgNPs significantly increased IL-8 secretion, with the highest concentration being 145 pg/ml IL-8 in response to 10 nm AgNPs after 48 h (Fig. 2B). This corresponded to the maximum yield achieved when cells were exposed to 1 lg/ml of 10 nm AgNPs. Cells exposed to 10 lg/ml of 20–100 nm AgNPs released an average of 108 pg/ml IL-8 after 48 h, which was significantly less than that released in response to 10 nm AgNPs but significant higher than the IL-8 levels secreted by control cells (55 pg/ml).

2.12. Statistical analysis Data are expressed as the mean ± SD of at least three independent experiments, unless stated otherwise. Statistical analyses were performed using SigmaPlot V. 11.0. Differences between the AgNP-treated samples and the untreated controls were analyzed by one-way analysis of variance (ANOVA) followed by the Holm– Sidak test. p Values of less than 0.05 were considered to be statistically significant.

3.3. Cell viability 3. Results 3.1. ROS

Increase of fluorescence relative to control

H2DCF-DA staining was used to evaluate the intracellular generation of reactive oxygen species (ROS) in LoVo cells exposed to silver nanoparticles. After 24 h of exposure, all AgNPs (ranging from 10 to 100 nm in size) significantly increased ROS levels at concentrations above 5 lg/ml compared with the control (Fig. 1). Nanoparticles 10 and 20 nm in diameter significantly increased intracellular ROS levels at a concentration of 2.5 lg/ml. For all the tested concentrations, the most pronounced effects were seen with 10 and 20 nm AgNPs, with up to 6-fold higher ROS levels at 10 lg/ml of 10 nm AgNPs; all larger nanoparticles (between 40 and 100 nm) elicited a similar ROS response. On average, 1.9-fold and 2.6-fold increases in ROS levels were observed with 5 lg/ml and 10 lg/ml AgNPs, respectively. However, cells treated with 100 nm AgNPs always exhibited the lowest, but not statistically significant, intracellular ROS levels.

7 AgNP 10 nm AgNP 20 nm AgNP 40 nm AgNP 60 nm AgNP 100 nm Control

6 5

* *

3.4. Proliferation

4

*

*

**

3

*

2

*

Cell viability, based on mitochondrial activity and cell membrane damage, was evaluated by measuring dehydrogenase activity via the reduction of resazurin to resorufin in the CellTiter BlueÒ assay. After 24 h of exposure, the mitochondrial activity slightly increased in response to 1, 2.5 and 5 lg/ml AgNPs, resulting in an average of 103%, 110% and 112% activity, respectively (Fig. 3A). At 10 lg/ml AgNPs, the mitochondrial activity significantly decreased to 53% for cells exposed to 10 nm AgNPs and to 85% for those exposed to 20 nm AgNPs. For all the other size AgNPs, the mitochondrial activities were no longer elevated but were in the same range as that for the control. This effect was enhanced after 48 h of exposure (Fig. 3B). At this time point, the mitochondrial activity was significantly reduced for all the AgNPs at 10 lg/ml, but the lower doses did not impair the viability. Again, at 10 lg/ml, the smallest AgNPs exhibited the strongest effects, resulting in an activity of only 8% compared with the control. On average, 20–100 nm AgNPs resulted in a decrease to 40% mitochondrial activity, with no significant differences among these sizes. However, there was a trend toward increasing cytotoxicity with smaller particles; as such, the mitochondrial activity of cells exposed to 100 nm AgNPs was the highest at 51% relative to control.

* *

* *

1

The inhibitory effects of AgNPs on cell proliferation were determined by measuring BrdU incorporation during DNA synthesis. The proliferation rate, relative to non-exposed control cells, was evaluated after a 24 h exposure to various size AgNPs at concentrations ranging from 1 to 10 lg/ml (Fig. 4). For all AgNPs, a decreased proliferation rate was observed with increasing dose. This effect was independent of the particle size. At the maximum concentration of 10 lg/ml, only an average of 13% of the cells were proliferating.

0 0

2,5

5

10

3.5. Apoptosis induction

AgNP concentration [µg/ml] Fig. 1. Effect of AgNPs on intracellular ROS levels. ROS levels were determined after 24 h by H2DCF-DA staining using flow cytometry. The data represent the increase in fluorescence relative to the controls and are expressed as the mean ± SD of three independent experiments. Asterisks (*) denote significant differences with respect to control cells (p < 0.05).

To evaluate whether the increase in intracellular ROS in AgNPexposed cells was associated with the induction of apoptosis, Annexin-V-FITC/PI staining was carried out. LoVo cells were exposed to 10 lg/ml of different size AgNPs. After 24 h, apoptosis was initiated in all cells, resulting in a slight, but significant,

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200

200 10 µg/ml

*

150

IL-8 release [pg/ml]

IL-8 release [pg/ml]

1 µg/ml

*

100 *

50

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0

*

150

*

*

*

*

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100

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*

50

0 Control 10

20

40

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Control 10

AgNP size [nm]

20

40

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AgNP size [nm] 24 h

48 h

Fig. 2. Effect of AgNPs on IL-8 release. IL-8 secretion was quantified by ELISA. The cells were exposed to 10–100 nm AgNPs at a dose of 1 or 10 lg/ml for 24 h or 48 h. The data are presented as the mean ± SD of two independent experiments assayed in duplicate. For significance analysis, three independent experiments with normalized data sets were used. Asterisks (*) denote significant differences with respect to control cells at each treatment time (p < 0.05).

Mitochondrial activity [%]

A 160

24 h

140

*

120

* *

*

B 160

48 h

140 120

100

100

80

80 *

60

60

40

40

20

20

0

0 0

1

2,5

5

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0

AgNP concentration [µg/ml] control

AgNP 10 nm

AgNP 20 nm

1

2,5

5

10

AgNP concentration [µg/ml] AgNP 40 nm

AgNP 60 nm

AgNP 100 nm

Proliferation (BrdU incorporation relative to control)

Fig. 3. Effect of AgNPs on mitochondrial activity. Cell viability was evaluated using the CellTiter Blue assay. Cells were treated with 10–100 nm AgNPs at various concentrations for 24 (A) or 48 h (B). The data are presented as the mean ± SD of three independent experiments. Asterisks (*) denote significant differences with respect to control cells (p < 0.05).

1.4 AgNP 10 nm AgNP 20 nm AgNP 40 nm AgNP 60 nm AgNP 100 nm

1.2 1.0 0.8 0.6 0.4 0.2 0 0

2

4

6

8

10

12

AgNP concentration [µg/ml] Fig. 4. Effect of AgNPs on cell proliferation. LoVo cell proliferation was evaluated via the BrdU assay. Cells were exposed to 10–100 nm AgNPs at a concentration of 1–10 lg/ml for 24 h. The data are presented as the mean ± SD of four independent experiments. Asterisks (*) denote significant differences with respect to control cells (p < 0.05).

decrease in the number of viable cells exposed to 40–100 nm AgNPs compared with the control (Fig. 5A). For smaller nanoparticles (10 and 20 nm), the viable population decreased to 52% and

66%, respectively, corresponding to an increased number of apoptotic and necrotic cells. After 48 h, flow cytometric analysis of the control population revealed 83% viable cells, 6% apoptotic cells and 10% late apoptotic/necrotic cells. In contrast, among cells exposed to AgNPs, there were significantly lower proportions of viable cells, corresponding to increased fractions of apoptotic and necrotic cells (Fig. 5B). This effect strongly correlated with particle size. For 100 nm AgNPs, the viable fraction was 65%, whereas for 10 nm AgNPs, only 1% of the population was viable and 88% exhibited late apoptotic or necrotic membrane damage. For 10 and 20 nm AgNPs, lower doses (2, 5 and 7.5 lg/ml; 48 h exposure; data not shown) resulted in a slight but significant decrease in the fraction of viable cells concurrent with an increase in the number of apoptotic cells. For 20 and 10 nm particles, the viable fraction decreased to 86% and 82% at 2 lg/ml, to 70% and 65% at 5 lg/ml and to 66% and 49% at 7.5 lg/ml, respectively. This effect was not observed with 40–100 nm AgNPs (data not shown).

3.6. Proteomics approach To identify the proteins that were involved in the cytotoxic effects triggered by exposure to AgNPs, a mass spectrometry-based quantitative proteomic study was performed using LoVo cells exposed with 10 lg/ml of 20 or 100 nm AgNPs for 24 h. Table 1 shows the top 10 most up- or down-regulated proteins due to 20

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Population of LoVo cells [%]

A

B

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AgNP size [nm] Viable cells

60

40

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AgNP size [nm] Apoptotic cells

Necrotic cells

Fig. 5. Effect of AgNPs on the induction of apoptosis using flow cytometry analysis of Annexin-V-FITC/PI-labeled cells. Cells were exposed to 10 lg/ml of 10–100 nm AgNPs for 24 h (A) or 48 h (B). The data are presented as the mean ± SD of three independent experiments. Asterisks (*) denote significant differences with respect to control cells (p < 0.05).

Table 1 Top 10 most down- or up-regulated proteins by 20 nm and 100 nm AgNPs.

a

Particle size

Gene

Ratioa

Protein name

20 nm

MRPL50 VIM GOLM1 TGFB2 SPCS3 RAB3D SDF4 OCIAD2 UXS1 SOAT1 TBCB EEF1A1 XPO5 PRDX1 TUBB EEF1B2 GFPT1 LRRC47 EML4 HMMR

1.12 1.00 0.96 0.86 0.84 0.83 0.83 0.82 0.71 0.70 0.72 0.72 0.73 0.74 0.75 0.75 0.75 0.77 0.80 1.24

39S ribosomal protein L50, mitochondrial Vimentin Golgi membrane protein 1 Transforming growth factor beta-2 isoform 2 precursor Signal peptidase complex subunit 3 Ras-related protein Rab-3D 45 kDa calcium-binding protein isoform 2 precursor OCIA domain-containing protein 2 isoform 1 UDP-glucuronic acid decarboxylase 1 isoform 2 Sterol O-acyltransferase 1 isoform 1 Tubulin-folding cofactor B Elongation factor 1-alpha 1 Exportin-5 Peroxiredoxin-1 Tubulin beta chain Elongation factor 1-beta Glucosamine-fructose-6-phosphate aminotransferase [isomerizing] 1 isoform 1 Leucine-rich repeat-containing protein 47 Echinoderm microtubule-associated protein-like 4 Hyaluronan mediated motility receptor isoform b

100 nm

TGFB2 RAB3D GPT2 MRPL44 NDUFA11 MTPAP UTRN ATRX SCARB1 MRPS31 SRI HSP90AA1 YWHAZ HMGB1 SH3GL1 RTF1 XPO5 TBCB TFG WARS

0.87 0.70 0.64 0.64 0.61 0.60 0.57 0.56 0.55 0.51 0.69 0.69 0.70 0.72 0.73 0.77 0.77 0.78 0.82 0.91

Transforming growth factor beta-2 isoform 2 precursor Ras-related protein Rab-3D Alanine aminotransferase 2 isoform 1 39S ribosomal protein L44, mitochondrial NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 11 isoform 1 Poly(A) RNA polymerase, mitochondrial precursor Utrophin Transcriptional regulator ATRX isoform 1 Scavenger receptor class B member 1 28S ribosomal protein S31, mitochondrial Sorcin isoform A Heat shock protein HSP 90-alpha isoform 2 14-3-3 Protein zeta/delta High mobility group protein B1 Endophilin-A2 isoform 1 RNA polymerase-associated protein RTF1 homolog Exportin-5 Tubulin-folding cofactor B Protein TFG isoform 1 Tryptophan-tRNA ligase, cytoplasmic isoform a

log 2-Transformed.

or 100 nm AgNPs exposure compared with untreated control cells. A list of all regulated proteins due to 20 and 100 nm AgNPs exposure can be found in the ‘‘Supporting Information Table 2’’ in Verano-Braga et al. (2014). Interestingly, the most down-regulated protein induced by 20 nm AgNPs was the 39S ribosomal protein L50 while a similar protein (39S ribosomal protein L44) was the fourth most down-regulated following 100 nm AgNPs exposure

(Table 1). Both are mammalian mitochondrial proteins involved in the protein synthesis inside this organelle. Therefore, it is possible to speculate that synthesis of new mitochondrial proteins is affected by AgNPs exposure. Regarding the top 10 most up-regulated proteins following cellular exposure to 20 nm particles, it is worth mentioning the protein peroxiredoxin-1, which is involved in cellular redox regulation. Its up-regulation suggests increasing

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levels of peroxides due to 20 nm AgNPs. Although none of the top 10 most up-regulated proteins following 100 nm AgNPs exposure are involved in the redox system, it is important to mention that this particle size indeed induced up-regulation of this class of proteins – see Supporting Information Table 2 in Verano-Braga et al. (2014). To gain insights into the biological and molecular functions of these proteins, a gene ontology (GO) analysis was performed that focused on the overrepresented GO terms ‘‘cell death’’, ‘‘cell growth’’, ‘‘mitochondrial activity’’ and ‘‘antioxidant activity’’. Fig. 6 reveals that more proteins involved in cell death and mitochondrial activity were affected by 20 nm AgNPs than by 100 nm AgNPs, whereas proteins involved in cell growth were similarly affected by both particle sizes. The cellular pathways affected by 20 and 100 nm AgNPs were assessed by using the software Ingenuity Pathway Analysis. Only 20 nm AgNPs induced statistical changes in the ‘‘mitochondrial dysfunction pathway’’ (Supplementary Fig. 2). Among the 19 proteins differentially regulated due to 20 nm AgNPs that are components of this signal cascade, 18 were down-regulated and one up-regulated. Moreover, among these down-regulated proteins, there are components of the complex I, III, IV and V from the electron transport chain, highlighting the immense impact of 20 nm particles on the mitochondrial activity, possibly leading to a negative effect on the ATP synthesis. 3.7. AgNP uptake – confocal laser scanning microscopy (CLSM) To assess whether and to what degree the silver nanoparticles were taken up by the cells, LoVo cells were analyzed by CLSM after exposure to 10 lg/ml AgNPs. After 24 h, the cells were washed with PBS to remove unbound particles, and the cell membrane was stained with a selective fluorescence dye. Fig. 7 illustrates the exemplarily confocal images of LoVo cells exposed to 20–100 nm AgNPs. To verify that the particles were located inside the cells, confocal z-stacks were applied and all particles in each stack were quantified. The analyses revealed that 20 nm particles penetrated the cells, where they were detected individually in the cytosol or concentrated in clusters, some of which are several micrometers in diameter. However, the size of the AgNPs in the confocal images did not reflect their actual size and number as determined by the TEM analysis (Supplemental Fig. 1).

Fig. 6. Gene ontology (GO) analysis of differentially regulated proteins in LoVo cells in response to 20 and 100 nm AgNPs. The data are presented as the mean ± SD of three independent experiments.

In contrast, 40 and 60 nm AgNPs/AgNP aggregates were detected in significantly lower numbers inside the cells, and 100 nm particles were confined predominantly to the cell surface, with only few particles inside the cells. Fig. 8 summarizes the enumeration of internalized 20, 40, 60 and 100 nm AgNPs/AgNP aggregates. Even if the enumeration did not reflect the absolute number of individual particles, there was a clear size dependence in the internalization of AgNPs. Nanoparticles greater than 20 nm were detected significantly less frequently inside the cells. With increasing particle size, the number of internalized particles/aggregates decreased. For 100 nm AgNPs, only an average of 5 particles per cell was enumerated whereas for 20 nm AgNPs, five times more AgNPs/AgNP aggregates per cell were detected.

4. Discussion In this study, the human LoVo colon carcinoma cell line was used to evaluate the cytotoxic effects of silver nanoparticles. As the size of nanoparticles is considered to be one of the major factors for NP-mediated toxicity, we investigated various cellular responses with respect to particle size. Spherical NanoXact™ silver nanoparticles of 10, 20, 40, 60 and 100 nm stabilized with citrate were chosen because they exhibited a very narrow size distribution (less than 10% deviation) and nearly no agglomeration in cell growth medium. ROS production and subsequent oxidative stress have been reported to be early cellular responses to nanoparticles and play a key role in cytotoxicity (AshaRani et al., 2009; Kim et al., 2009; Xia et al., 2006). This is in accordance with the data in our study. After 24 h of exposure, increased intracellular ROS levels were observed in LoVo cells treated with AgNPs in a dose and sizedependent manner, with 10 nm AgNPs eliciting the highest ROS levels. This corresponded to the proteomic response of proteins involved in oxidative stress, which demonstrated that 20 nm particles induced post-translational modifications, via the SUMO pathway (Verano-Braga et al., 2014). Furthermore, the secretion of the cytokine IL-8 was observed; IL-8 is known to be secreted by epithelial cells, and is involved in the innate immune response and is induced by oxidative stress (Vlahopoulos et al., 1999). For 10 and 20 nm AgNPs, cytokine secretion was already enhanced at a dose of 1 lg/ml. High doses (10 lg/ml) of AgNPs of all sizes induced cytokine release to a similar extent, with the exception of 10 nm AgNPs, which elicited an even higher level of secretion. These initial cellular responses of ROS and IL-8 production were succeeded by decreased cell viability caused by mitochondrial dysfunction due to reduced dehydrogenase activity and the induction of apoptosis. However, after 24 h, the mitochondrial activity was slightly increased for AgNP doses up to 5 lg/ml. This phenomenon might be explained by a cell activation in response to NP exposure and has been reported for human mesenchymal stem cells exposed to nontoxic concentrations of nanosilver (Greulich et al., 2009). After 48 h of exposure to 10 lg/ml AgNPs, size-dependent effects on mitochondrial activity and apoptosis were clearly seen. This cellular response cascade has been demonstrated for AgNPs in various cell types, including human IMR-90 lung fibroblasts and U251 glioblastoma cells (AshaRani et al., 2009), alveolar macrophages (Carlson et al., 2008) and the human A549 lung carcinoma cell line (Foldbjerg et al., 2011). In addition, De Berardis and coworkers (De Berardis et al., 2010) evaluated the effect of 50–70 nm ZnO nanoparticles on LoVo cells and reported a significant decrease in cell viability and an induction in apoptosis linked to IL-8 secretion in a dose- and time-dependent manner. Our cytotoxicity data indicated that the initial cellular responses of LoVo cells to silver nanoparticle exposure were triggered to a greater extent by smaller particles that were able to

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Fig. 7. AgNP uptake in LoVo cells. The green images illustrate the nanoparticles, and the red and green images represent an overlay of the nanoparticles (green) with the fluorescent membrane marker DiI c18 (red). The top row of images is from the bottom of the cell, the middle row of images is 4 lm into the cell and the bottom row shows the top of the cell. The panels illustrate the uptake of 20 nm silver particles (a), 40 nm silver particles (b), 60 nm silver particles (c) and 100 nm silver particles (d). The images demonstrate that the 20 nm particles penetrated the cell, where they were concentrated in clusters, some of which were several micrometers in diameter. A cluster of 1 lm could easily contain several thousand 20 nm particles (see also Supplemental Fig. 1). Larger particles were primarily found on the cell surface. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

penetrate the cells. The quantification of cellular AgNP uptake using CLSM imaging revealed a clear size dependence, in spite of the fact that the enumeration was ineffective at quantitating the absolute number of internalized particles. Prasad and coworkers (Prasad et al., 2013) determined the intracellular silver concentration in HepG2 cells by ICP-MS using citrate-stabilized 10 and 75 nm AgNPs and observed similar percent uptake rates. However, an equal mass of nanoparticles of different sizes does not reflect an equal number of particles. Thus, the authors concluded that the increased toxicity of smaller AgNPs derived from a higher number of intracellular particles, which corresponds to our findings. Furthermore, the CLSM data in our study revealed that the particles were located in the cytoplasm. A similar cytoplasmic localization of AgNPs was demonstrated in murine macrophages (Singh and Ramarao, 2012). Particles exceeding 20 nm in size were predominantly detected outside the cells, at the membrane. The

accumulation of AgNPs in cytoplasmic vesicular structures that occurs in macrophages (Yen et al., 2009) has not been observed for AgNPs in LoVo cells. However, the mode of cellular uptake requires further investigation to elucidate the effects of size and coating, which might be cell type-dependent. The proteomic analysis results were consistent with the results from the cell-based assays. The gene ontology (GO) analysis revealed that proteins involved in cell death and, mitochondrial activity were more affected by 20 nm AgNPs than by 100 nm AgNPs. These proteomic results corresponded with those from a recent study in which an in vitro intestinal epithelium model was exposed to AgNPs of four different particle sizes (20, 34, 61 and 113 nm), and whole-genome gene expression was monitored (Bouwmeester et al., 2011). The authors observed that smaller particles modulated the expression of more genes than 61 and 113 nm particles and that these regulated genes were involved in

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Development Fund (Grant 55-1.2-10). J.R. Brewer would like to acknowledge the support of the Carlsberg Foundation. The authors also acknowledge DaMBIC (SDU, Denmark) for the use of experimental facilities.

Number of AgNPs/AgNP aggregates inside the cells

80

60 R2 = 0.999

Appendix A. Supplementary material 40

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tiv.2014.06.005. 20

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Size of AgNP [nm] Fig. 8. Internalization of AgNPs in LoVo cells. Cells were exposed to 20, 40, 60 and 100 nm AgNPs at a concentration of 10 lg/ml for 24 h. Nanoparticles were enumerated in confocal z-stacks of CLSM micrographs of a distance of 0.5 lm for 20 nm particles and 1 lm for all larger nanoparticles. All the particles inside the cells were counted manually in each stack. Due to the resolution limit of the CLSM of about 300 nm, it was not possible to determine if the observed particles were single particles or aggregates. Therefore, the particles are referred to as ‘‘AgNPs/ AgNP aggregates’’. For each particle size, an average of 40 cells and a total of 3233 AgNPs/AgNP aggregates were accounted for quantification. The data are presented as the mean ± SEM.

apoptosis, the response to oxidative stress and metal ion binding. Interestingly, our data revealed that proteins involved in cell growth were affected similarly by both particle sizes (20 nm and 100 nm, respectively) which corresponded with the measured proliferation rates. Results from Chairuangkitti (Chairuangkitti et al., 2013) indicated that the down-regulation of the cell cycle-associated proliferating cell nuclear antigen protein (PCNA) was not affected by oxidative stress in A549 cells. Therefore, cell cycle arrest was ROS-independent, and all the other toxic effects were mediated via a ROS-dependent signaling pathway. These findings suggested that the size independence of LoVo cell proliferation may be attributed to a differentially regulated, ROS-independent pathway. In summary, silver nanoparticles were cytotoxic in LoVo cells as evidenced by various cell-based assays. Except for the effects on proliferation, the toxicity was dependent on particle size and was suggested to be mediated by the stimulation of a signaling cascade that generated ROS and inflammatory markers, caused mitochondrial dysfunction and subsequently induced apoptosis. The CLSM measurements indicated that silver particles exceeding 20 nm were only rarely internalized, resulting in reduced toxicity, whereas smaller particles that penetrated the cells caused significantly more damage. Whether this damage was caused by the ionization of AgNPs needs to be investigated in the future. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version.

Acknowledgements This work was supported by INTERREG IVa SyddanmarkSchleswig-K.E.R.N. with means from the European Regional

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