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David.Awad. MSc_Food Safety.Internship_report

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David.Awad. MSc_Food Safety.Internship_report

  1. 1. Evaluation of the effect of silver nanoparticles (NM-300K) on an in vitro intestinal co-culture model before and after in vitro digestion Author: David Awad Education program: MSc. Food Safety Course code: TOX-70424 Thesis-provider: RIKILT- Institute of Food Safety Supervisors: Dr. Meike van der zande (RIKILT) Prof. Ivonne Rietjens (Toxicology department) Date: 22-6-2015
  2. 2. 2 Abstract Application of silver nanoparticles in the food sector is expanding due to their strong anti-microbial effects. Silver nanoparticles are among the most frequently used materials in many household products and medical uses. This subsequently suggests a possible increase in human exposure via all the relevant routes with the gastrointestinal tract (ingestion) being the most prominent. Information about the digestion process effects on the cytotoxicity of these particles is therefore very important, but still lacking. In this study an in vitro co-culture model for the human intestinal epithelium consisting of Caco-2 and HT29-MTX was used to study the effect of silver nanoparticles and their ionic equivalent (AgNO3) before and after in vitro digestion. This in vitro intestine model was exposed to five different concentrations of digested and undigested AgNPs ranging from 5 to 100µg/ml, as well as to silver ions (digested and undigested). AgNO3 exposure was carried out at around 6% of the AgNP concentrations used. The cytotoxicity of the particles before and after in vitro digestion was determined using MTT, WST-1, and ATPlite assays. Silver nanoparticles (NM-300K) were able to induce cytotoxicity starting from the lowest dose used (5µg/ml). These effects were most likely caused by the silver ions that were released from the AgNPs as the toxic effect of the AgNPs was very comparable to that of AgNO3. In vitro digestion of the AgNPs increased the toxicity of these particles, but the observed increase in cytotoxicity was likely caused mainly by the presence of chyme from the in vitro digestion. The oxidative stress was also investigated. Production of reactive oxygen species was noticeable compared to the positive control (hydrogen peroxide); however, the results of this experiment were not conclusive. Abbreviations BSA: Bovine serum albumin DLS: Dynamic light scattering AgNPs: Silver nanoparticles AgNO3: Silver nitrate PDI: Polydispersity indexes Caco-2: Human colonic adenocarcinoma cells (cell line) HT29-MTX: Human colon adenocarcinoma mucus secreting cells (cell line)
  3. 3. 3 Acknowledgment I would like to thank my supervisor Meike van der Zande, for giving me the opportunity of doing my internship at RIKILT and for her patient guidance and support. Also special thanks to Richard Helsdingen for all the help throughout my experiments. Thanks to Hans Bouwmeester for his comments and ideas that helped me to think critically. Last but not least, i want to thank my colleges Chunyang Dong, Yennie Wulansary, Myriem Bouziane, Zhe Qin, Matthew Burlington and Simin Huang for creating a nice work atmosphere and for motivating me.
  4. 4. 4 Contents 1. Introduction ..............................................................................................................................................5 1.1 Silver nanoparticles............................................................................................................... 5 1.2 Mode of action ....................................................................................................................... 5 1.3 State of the art, bottlenecks and knowledge gaps..................................................................... 6 1.3 Aim of the study..................................................................................................................... 8 2. Materials and Methods .............................................................................................................................8 2.1 Cell culture............................................................................................................................. 8 2.2 Nanoparticles and chemicals preparation........................................................................... 8 2.3 Experiment setup and toxicological assays ......................................................................... 9 2.3.1 Dynamic Light Scattering (DLS)...................................................................................... 9 2.3.3 Cell viability..................................................................................................................... 10 2.3.3.1 WST-1 assay................................................................................................................. 10 2.3.3.2 MTT assay.................................................................................................................... 10 2.3.3.3 ATPlite assay.................................................................................................................... 11 2.3.4 Reactive Oxygen Species (ROS)..................................................................................... 11 2.4 Statistical analysis................................................................................................................ 12 3.Results .......................................................................................................................................................12 3.1 Characterization of the AgNPs........................................................................................... 12 3.2 Cell viability results................................................................................................................... 13 3.2.1 Exposure to chyme ................................................................................................................. 13 3.2.2 Exposure to AgNPs................................................................................................................. 17 3.2 ROS results ......................................................................................................................................20 4. Discussion.................................................................................................................................................21 5. Conclusion................................................................................................................................................25 6. Recommendations ...................................................................................................................................26 7. References ................................................................................................................................................27 8. Appendices ...............................................................................................................................................31
  5. 5. 5 1. Introduction 1.1 Silver nanoparticles In the last decade silver nanoparticles (AgNPs) have received much attention owing to their unique anti-microbial properties that fit them for a vast array of applications. AgNPs display novel properties -chemically, physically and optically- compared to their bulk counterparts (Bergin and Witzmann, 2013; Bohmert et al., 2014; Rai et al., 2009). Nowadays, thanks to its biocidal activity (anti-bacterial) silver in the nano scale is extensively used in commercial products such as cosmetics, drug delivery systems, coatings of refrigerators and packaging materials, water treatment, wound dressings and sports equipment (Braydich-Stolle et al., 2005; Morones et al., 2005; van der Zande et al., 2012). However, since very little information is available on the safety, and human health impact of nano- silver applications, the accelerated increase in production and usage of AgNPs has raised serious concerns and debates about their possible undesired side effects (long and short term biological effects) on public health (Bergin and Witzmann, 2013). In general, the growing usage of AgNP applications could be perceived from a toxicological -risk assessment- perspective as a possible increase in human exposure via all the relevant routes with the gastrointestinal tract (ingestion) being the most prominent route (Bohmert et al., 2014; Frohlich and Roblegg, 2012; Gaiser et al., 2009). Ingestion of AgNPs occurs either through intentional ingestion (direct), consumption of particles released from food containers (indirect) or via secondary ingestion of inhaled particles (Bergin and Witzmann, 2013; Wijnhoven et al., 2009). 1.2 Mode of action It is widely known that silver-based compounds and silver ions are extremely toxic to microorganisms and present strong biocidal effects on many species of bacteria including E. coli one of the well- known microorganisms that is sporadically responsible for product recalls due to serious food poisoning after consumption of contaminated food (Black et al., 1982; Spadaro et al., 1974; Zhao and Stevens, 1998). Although several studies on AgNPs antimicrobial activity have been carried out, the exact biochemical and molecular aspects of the mechanism of action of AgNPs are still unclear. Nevertheless, studies indicate that AgNPs can attach (efficiently interact) to the surface of the cell membranes and/or accumulate (enter) inside the cell causing an increase in permeability and death of the cell (Feng et al., 2000; Lok et al., 2006; Sondi and Salopek-Sondi, 2004). Furthermore, it was found that AgNPs in solutions tend to generate Ag+ ions, which are (to some degree) responsible for its biocidal effects (Dibrov et al., 2002; Semeykina and Skulachev, 1990; van der Zande et al., 2012).
  6. 6. 6 1.3 State of the art, bottlenecks and knowledge gaps Up to now, no comprehensive risk assessment on AgNPs is available due to the existing hurdles in identifying the products produced under various brand names, lack of information on the AgNPs concentration in products and their physiochemical properties, and the fact that only a few regulations claim listing of AgNPs on the label (Braydich-Stolle et al., 2005). Several in vivo studies in rodents (Table 1) have been carried out to assess tissue distribution and/or toxicity of these particles in healthy animals. These studies demonstrated the presence of silver in all examined organs i.e. brain, lung, liver, and kidneys after oral exposure to different doses of AgNPs and for different periods of time. Silver concentrations in the organs were correlated with the amount of silver ions in the AgNP suspension, showing that it is most likely that mainly Ag ions passed the intestinal wall. However, the presence of AgNPs has been shown in some tissues indicating that they are also capable of crossing the intestinal wall. Evidence suggests that the possibility of adverse effects on host tissues caused by acute or sub- chronic oral administration of AgNPs is low, as in studies that used doses up to 100–1,000 mg/kg/day, which is 20,000–200,000 higher than the recommended maximal ingestion of 0.005 mg/kg/day, the adverse effects were still low (Cha et al., 2008; Hadrup et al., 2012; Kim et al., 2008; Loeschner et al., 2011; Park et al., 2010a; Park et al., 2011a; van der Zande et al., 2012) . Table 1: In vivo studies of AgNPs exposures by the oral route, modified from (Bergin and Witzmann, 2013). Size (AgNPs) Dose/endpoint Species Effects NR 0.4–4–40 mg/kg, recurring, 5 d Rat High dose: ↓ inflammation in colitis model 13 nm 2.5 g one dose (150 g/kg)/3 d Mouse Localised lymphocyte infiltration; gene alterations in apoptosis, inflammation 14nm 2.25–9 mg/kg recurring, 28 d Rat (Wistar) No toxicological effects 14 nm 12.6 mg/kg recurring; 28 d Rat (Wistar- Hannover-Galas) Not assessed (distribution-focused study) 7.9 nm 1, 10 mg/kg, one dose, 24 hrs Rat Not assessed (distribution and bioavailability-focused study) 22, 42, 71 and 323 nm 0.25, 0.5, 1 mg/kg recurring, 14 and 28 d Mouse Dose-dependent ↑ inflammatory cytokines in blood; elevated B cells and IgE (60 nm) 30–300–1,000 mg/kg recurring; 90 d Rat LOAEL: 300 mg/kg based on ↑ alkphos, cholesterol; no genetic toxity as measured by micronucleus test on bone marrow; ↑RBC, Hgb, Hct, ↓ aPTT
  7. 7. 7 Although the in vivo studies have provided some prospective on the possible target organs and potential effects of ingested silver nanomaterials, the consequences remain uncertain and hard to predict. Therefore, relevant in vitro models can be used to investigate the underlining mechanisms. However, when extrapolating data obtained from an in vitro model, it is crucial to consider the limitations of the in vitro exposure model i.e. survival reduction, metabolic competence disruption, reduction of cell to cell interaction, disrupted organ topology disruption, and absence of tissue communication. Therefore, in vitro models may fail to precisely reflect the in vivo responses (Bergin and Witzmann, 2013; Bohmert et al., 2014; Braydich-Stolle et al., 2005; Eisenbrand et al., 2002). One of the most commonly used models for the intestinal barrier is the Caco-2 cell system, a well characterized intestinal in vitro model that permits evaluation of the ability of chemicals to cross the intestinal barrier, as well as to study their transport mechanisms. Several studies concluded that permeability values assessed using this model correlate well with human in vivo absorption data for many drugs and chemicals. Hence, the Caco-2 cellular model is frequently used as a permeability assay to predict oral absorption in humans (Catalioto et al., 2008; Hill et al., 1993; Laurent et al., 2007; Schaar et al., 2004). However, the various secretory and absorptive functions of the intestinal epithelium are due to a mixed population of absorptive cells and mucus-producing goblet cells which are not represented in the Caco-2 cells model. This limitation may be overcome by using multi-cell cultures (co-culture) that use mucosal cells in addition to Caco-2 cells to improve the physiological relevance. For instance, a co-culture system of absorptive Caco-2 cells and mucus-secreting HT29- MTX cells was developed and used to evaluate the permeability of a wide range of different drugs and chemicals (Walter et al., 1996). Several studies focusing on intestinal absorption and transport function (translocation) of NPs were performed using the co-culture model of Caco-2 and HT29-MTX cells in which also cell viability was monitored, para-cellular permeability, and monolayer integrity as evidence of cytotoxicity (Bouwmeester et al., 2011; Braydich-Stolle et al., 2005; des Rieux et al., 2005). Some of these studies also took the in vitro digestion process, that takes place in the human body before the NPs reach the intestinal barrier, into account in their studies, but none of these studies evaluated the influence of the in vitro digestion process on the effects (cytotoxicity) of the NPs on the cells. In vitro digestion has been shown to significantly increase the translocation of the examined NPs (Walczak et al., 2015). A lot of information about uptake through the intestines and possible toxic effects of AgNPs (after digestion) is still lacking. However, AgNPs under physiological conditions have been described to reach the intestinal wall in their initial size and composition, intestinal digestion of AgNO3 has been shown to give rise to particle formation. These nanoparticles were 20-30 nm in size, composed of silver complexed with sulphur and/or chlorine (Bouwmeester et al., 2011; Braydich-Stolle et al., 2005; des Rieux et al., 2005; Walczak et al., 2013).
  8. 8. 8 1.3 Aim of the study The principal aim of this study is to evaluate the potential toxicity of NM-300K silver nanomaterial on an in vitro intestinal epithelium composed of co-culture Caco-2 & HT29-MTX cells before and after human in vitro digestion. To this end, various methods that indicate cytotoxic effects will be applied such as WST-1, MTT, ATPlite, and ROS production assays. To characterize the AgNPs the hydrodynamic size was investigated using the Dynamic Light Scattering (DLS). 2. Materials and Methods 2.1 Cell culture The cell lines used in this study are: 1- The human colonic adenocarcinoma (Caco-2) cell line which was obtained from the American Type Culture Collection and was used in all experiments at passage numbers 22–34, 2- The HT29-MTX (human colon adenocarcinoma mucus secreting) cell line which was obtained from the European Collection of Cell Cultures and was used at passages 12–29. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Lonza, Verviers, Belgium) with phenol red, supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Bleiswijk, the Netherlands), 1% penicillin-streptomycin (Sigma, Steinbach, Germany), 1% MEM non-essential amino acids (Gibco) and 1% GlutaMAX (Gibco), further referred to as DMEM+. Cells were maintained in 75 cm2 cell culture flasks (430641 - Corning- New York, USA) at 37°C in a humidified atmosphere of 5% CO2 (HERAcell 150 incubator – Heraeus, Germany). Sub-culturing (passaging) was performed twice a week. For all experiments performed in this study cells were seeded at a density of 40 000 cells per cm2 in a 96 well plate, and for the co-culture experiments the cells were seeded in a ratio of 3:1 Caco-2/HT29-MTX. 2.2 Nanoparticles and chemicals preparation The nanomaterial used in this study is an AgNP material (i.e. NM-300K) from the NM-series, which are random samples from one industrial production batch, produced within industrial specifications, specified for OECD testing. NM-300K is a nano-silver colloidal dispersion with particle sizes < 20 nm for >99% of the particles (measured with TEM), which was obtained from the Joint Research Centre (JRC, Ispra). An AgNP dispersion of 19 mg/ml was made from the stock NM-300K dispersion (111.76 mg/ml) according to the NanoGenotox protocol with minor adjustments. Extended details about the NanoGenotox protocol with the adjustments and the dilutions used in this study can be referred to in Appendix 1 and 2 of this report.
  9. 9. 9 The chemicals and reagents used in this study were used with no further purification. Phosphate Buffered Saline 1X (PBS) (Gibco® - Life Technologies, The Netherlands), thiazolyl blue tetrazolium (MTT) bromide (M5655), dimethyl sulfoxide (DMSO) (102952) and Triton-X100 (0.25%) (Sigma). Rotenone (sigma), hydrogen peroxide (H2O2) (Sigma, Aldrich), AgNO3 (Sigma, Aldrich). 2.3 Experiment setup and toxicological assays 2.3.1 Dynamic Light Scattering (DLS) For DLS measurements, 10 ml NP suspensions at concentrations of 10 μg/ml in ultrapure water with 0.05% BSA and DMEM+ media were prepared separately, and analysed within one hour. From this suspension, 1 ml was transferred to a glass tube, and inserted into the ALV-DLS (ALV-Laser vertriebsgesellschaf mbH, Hessen, Germany), consisting of a thorn RFIB263KF photomultiplier detector, an ALV-SP/86 goniometer, an ALV 50/100/200/400/600 µm pinhole detection system, an ALV7002 external correlator, and a Cobalt Samba- 300 DPSS laser, with a wave length of 532 nm and a power of 300 Mw. The samples were run 10 times with duration of 30 seconds at an angle of 90o . The data was processed with AfterALV software (AfterALV1.0d, Dullware, USA) and Microsoft Excel to calculate the polydispersity index (PDI) and the hydrodynamic diameter of the nanoparticles. 2.3.2 In vitro digestion model The in vitro digestion model was used to mimic the in vivo digestion. The protocol of the in vitro digestion was performed according to the RIKILT SOP-A- (version 2, released on 19th Dec 2013). The digestion was started by adding 1 ml of MilliQ-water (blank) or one of the different concentrations of the Ag-NPs to 3  ml of saliva (pH = 6.8 ± 0.1). The mixture was incubated for 5 min at 37 ± 2 °C. Then, 6 ml of gastric juice (pH = 1.3) was added to the mixture and the pH of the sample was tested and, if necessary, adjusted to 2.5 ± 0.5. The sample was further incubated at 37 °C for 30min. Afterwards, 6 ml of duodenal juice (pH = 8.1) and 3 ml of bile (pH = 8.2) were added respectively. This complete mixture of digestive juices is referred to as chyme. The pH of this mixture was set at 6.5 ± 0.5 with NaOH (1 M) or HCl (37%) and it was incubated for another 2 h. All incubations were performed at 37 °C with rotation head-over-heels rotation of the samples to simulate the peristaltic movements (Walczak et al., 2015; Walter et al., 1996). For details on preparation of the digestion juices see Appendix 3.
  10. 10. 10 2.3.3 Cell viability Some of the frequently used assays to measure cytotoxicity are the MTT (3-(4,5-dimethylthiazol-2- yl)- 2,5-diphenyltetrazolium bromide) and the WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)- 2H-5- tetrazolio]-1,3-benzene disulfonate) assay. The principle of these tests lies in the reduction of tetrazolium salts by enzymes in the mitochondria of live cells into a formazan salt, which can be quantified spectroscopically. Absorbance represents both cell number and viability (Stone et al., 2009b). 2.3.3.1 WST-1 assay Cells were seeded in the 96-well plates at a concentration of 40000cells/ml in a total volume of 100 μl DMEM, followed by incubation at 37ºC, 5% CO2 for 24 hours to allow growth and adhesion. On the second day, the medium was aspirated with care to leave the monolayer undamaged, another 90 μl fresh DMEM were added immediately and the cells were exposed to 10 μl of various Ag NP concentrations. Triton-X100 served as positive control (PC), while the DMEM+ was the negative control and AgNO3 served as the ionic control. The exposed plates were incubated for 24 hours and 10 μl of Cell Proliferation Reagent WST-1 (ROCHE GmbH, Germany) were added to each well. The plates were incubated again for 2hours, and then shaken for 1 minute on a shaker. The absorbance was measured using a spectrophotometer (Synergy HT Multi-Mode Microplate Reader of BioTek) at a wavelength of 440 and 690 nm. Data was obtained using the SoftMax Pro 5.2 software (Molecular Devices, Sunnyvale, USA) and processed with Microsoft Excel. The graphs were created using GraphPad Prism 5. 2.3.3.2 MTT assay Cells were seeded in the 96-well plates at a concentration of 40000cells/ml in a total volume of 100 μl DMEM and for co-culture the ratio of cells seeding was 3:1 Caco-2 cells/HT29-MTX. Followed by incubation at 37ºC, 5% CO2, for 24 hours to allow growth and adhesion. On the second day, the medium was aspirated with care to leave the monolayer undamaged, another 90 μl fresh DMEM were added immediately and the cells were exposed to 10 μl of various Ag NPs concentrations. Rotenone (266.2 µg/ml) served as a positive control (PC). While, the DMEM+ was the negative control and AgNO3 served as the ionic control. The exposed plates were incubated for 24 hours and afterwards 10 μl of MTT reagent were added to each well. The plates were incubated again for 2 hours. Then, the medium was removed, 100 μl of DMSO were added to each well and the plates were shaken for 5- 10minutes on a shaker.
  11. 11. 11 The absorbance was measured using a spectrophotometer at wave length of 540nm and 690 nm (background) using of the Synergy HT Multi-Mode Microplate Reader of (BioTek). Data was obtained using the SoftMax Pro software and processed with the GraphPad Prism 5 software. 2.3.3.3 ATPlite assay ATP is a marker for cell viability, as it is present in all metabolically active cells. The intracellular concentration of ATP declines rapidly when the cells are necrotic or apoptotic. The assay system is based on the production of light caused by the reaction of ATP with added luciferase and D-Luciferin. This is illustrated in the following reaction scheme: For the ATPlite assay the protocol of the “ATPlite Luminescence ATP Detection Assay System “of PerkinElmer was followed. Cells were seeded in the 96-well plates at a concentration of 40000cells/ml in a total volume of 100 μl DMEM and the ratio of cells seeding was 3:1 Caco-2 cells/HT29-MTX followed by incubation at 37ºC, 5% CO2, for 24 hours to allow growth and adhesion. On the second day, the medium was aspirated with care to leave the monolayer undamaged, another 90 μl fresh DMEM+ were added immediately and the cells were exposed to 10 μl of various Ag NPs concentrations. Rotenone (266.2 µg/ml) served as a positive control (PC), while the DMEM+ was the negative control and AgNO3 served as the ionic control. After 24 hours of exposure, 50 µl of mammalian cell lysis solution was added to the wells containing 100 µl of DMEM+ (with phenol red) (Lonza, Verviers, Belgium). The plate was shaken for five minutes on an orbital shaker at 700 rpm. After that 50 µl substrate solution was added to the wells containing the 100 µl of DMEM+ and the 50 µl of lysis solution. The plates were shaken again for five minutes on an orbital shaker at 700rpm. Then the plates were wrapped in aluminium foil and allowed to stand for 10 minutes. The luminescence was measured at 950/35, top reading and sensitivity of 200 and 230, using a Synergy HT Multi-Mode Microplate Reader of (BioTek). 2.3.4 Reactive Oxygen Species (ROS) To measure the formation of ROS due to AgNP exposure, a 2’, 7’-dichlorofluorescein diacetate (DCFDA) (Sigma, Aldrich) assay was performed. This fluorogenic dye is de-acetylated to the non- fluorescent DCFH in the cell by cellular esterases. In presence of ROS, DCFH is oxidized into a fluorescent product, 2’, 7’ –dichlorofluorescin (DCF) that can be detected by fluorescence spectroscopy (Stone et al., 2009a).
  12. 12. 12 To perform this assay, the cells were seeded in 96-well plates at a concentration of 40,000 cells/ml in a total volume of 100 μl DMEM without phenol red (DMEM/F-12 (1X)-REF: 21041-025) (Lonza, Verviers, Belgium) per well. Next followed incubation at 37ºC for 24 hours to allow growth and adhesion. On the second day, the medium was aspirated with care to leave the monolayer undamaged and then 100 μl of DCFDA mix were immediately added to stain the cells. After 45 minutes of incubation, the reagent was removed, replaced with 90 μl fresh DMEM and the cells were exposed to 10 μl of various AgNPs concentrations resulting in a final concentration ranging between 0-100 µg/ml. Hydrogen peroxide (H2O2, 10 mΜ) served as a positive control (PC) and DMEM+ as a negative control. The exposed cells were incubated at 37ºC, 5% CO2, for 24 hours and fluorescence was measured using the Synergy HT Multi-Mode Microplate Reader of (BioTek) at wavelengths of 485 nm excitation and 530 nm emission. Data was obtained using SoftMax Pro 5.2 software and processed with Microsoft Excel. The graphs were created using the GraphPad Prism 5 software. 2.4 Statistical analysis WST-1, MTT, and ATPlite assays were performed in at least 6 biological replicates (n=6) for each treatment and each experiment was repeated once. While ROS production assay experiment was carried out once with 3 biological replicates. Each data point expresses the average of these measurements and is presented as the arithmetic mean ± standard error of the mean (SEM). Statistical analysis was performed in Microsoft Excel using the Student’s t-test and considering a value of p<0.05 as significant. An asterisk (*) over the data points marks statistical significance. 3 Results 3.1Characterization of the AgNPs Before exposure to the cells the AgNPs were dispersed in 0.05% BSA water followed by immersion in in vitro digestion juices, followed by immersion in DMEM+, or they were dispersed in 0.05% BSA water followed by immersion in DMEM+. To investigate the quality and stability of the dispersions in 0.05% BSA water and to assess the possible changes in size of the AgNPs in DMEM+, the suspensions were analysed by DLS. DLS results of NM-300K (10 µg/ml) in 0.05% BSA water revealed 3 peaks, the highest peak was at ~34 nm±0.29, the other peaks were at ~6 nm and ~196 nm (Figure 1A). However, the 6 and 196 nm peaks had a relatively low intensity, indicating that the largest fraction of the AgNPs had a hydrodynamic size of ~34 nm. In DMEM+, 3 peaks were observed, the highest peak was at ~28±0.82 nm, and peaks at ~5 nm and ~182 nm were also observed.
  13. 13. 13 Nevertheless, the 5 and 200 nm peaks had a relatively low intensity, indicating that the largest fraction of the AgNPs had a hydrodynamic size of ~28 nm (Figure 1B). The PDI (a measure of the homogeneity of a solution) results showed polydispersity (≥0.1) of the AgNPs dispersions in both DMEM and BSA water. In BSA water the PDI was around 0.5, while in DMEM+ the PDI was ~0.4. This experiment was repeated every week for 8 weeks and only minor variations in results were observed. This indicates that the size distribution and the diameter values obtained were reliable and reproducible. Figure 1. Hydrodynamic particle size of NM-300K measured by DLS in (A) in 0.05% BSA water And in (B) DMEM+ media. 3.2 Cell viability results 3.2.1 Exposure to chyme The potential cytotoxic effects of AgNPs were investigated on Caco-2 cells, HT29-MTX cells and on a co-culture of Caco-2 and HT29-MTX cells. However, before exposure of the AgNPs to the cells the effect of chyme (end product of the in vitro digestion) on cells was investigated first using MTT and WST-1 cell viability assays. For this, Caco-2 cells were used as a cell model. After 24h of exposure to 5, 10, 15, 20, and 25% chyme (the chyme prepared 3 times -chyme 1, 2, and 3- following the same preparation steps-Appendix 3-). Using the WST-1 assay a cytotoxic effect on the cells was observed starting at 15% chyme (Figure 2A and B). At 25% chyme, the reduction of cell viability was about 70- 80% compared to the non-exposed cells. 0 5 10 15 20 25 1 100 10000 1000000 Intensity(A.U.) Diameter (nm) Silver nanoparticles (NM-300K) D= 34 nm±0.29 A 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 1 100 10000 1000000 Intensity(A.I) Diameter (nm) Silver nanoparticles (NM-300K) B D= 28 nm±0.82
  14. 14. 14 However, using the MTT assay the results showed 30-50% cell mortality at 25% chyme and cytotoxicity started at ~20%. Both assays indicated that chyme is capable of reducing the viability of Caco-2 cells. As a non-toxic concentration of chyme was desired for further cytotoxicity testing with AgNPs, a chyme concentration of 10% was chosen for further experiments. Figure 2. Cytotoxicity of different concentrations of chyme on Caco-2 cells after 24hours of exposure measured using A) MTT and B) WST-1 assays. Data are expressed as a percentage of the (-) control and shown as a mean ± SEM (n=3). The * sign signifies p<0.05 compared to the negative control (0 %). At first, for AgNP exposure experiments only the WST-1 assay was selected to evaluate the potential cytotoxic effect of AgNPs (digested and undigested). However, it was found that there was a dose dependent interference of the AgNPs with the spectroscopic measurement (Appendix 4). Using the WST-1 test a viability of 70-75% was obtained for the highest concentration of 100 µg/ml of undigested AgNPs (Appendix 4), while the MTT test showed lower viability values at the same concentration of ~20-25% cell viability (Appendix 4). Therefore, the MTT assay was always applied simultaneously with the WST-1 assay and a modification of the WST-1 protocol was made by discarding the media with the nanoparticles and adding a fresh medium before the absorbance measurement was performed. Later, also an ATPlite assay evaluation was added. A B
  15. 15. 15 To determine if in vitro digestion affects AgNPs toxicity, WST-1, and MTT viability assays were used on Caco-2 and HT29-MTX cells and their co-cultures exposed to (digested) AgNPs. Cytotoxic effects of AgNPs were observed on Caco-2 cells. Using MTT, a cytotoxic effect for the undigested particles starting at 5 µg/ml was observed, and strong cytotoxicity (<80% viability) was observed at concentrations of 50 µg/ml and higher.. While digested particles induced strong cytotoxicity (<80% viability) starting from 5 µg/ml and continued to reach 10% viability at the highest concentration (Figure 3A). WST-1 results also indicated cytotoxicity at all concentrations, with strong toxicity (<80% viability) at concentrations higher than 10 µg/ml for the undigested AgNPs. Digested AgNPs showed strong cytotoxicity (<80% viability) starting at 5 µg/ml and reaching ~20% viability at 50 and 100 µg/ml (Figure 3B). The 10% chyme appeared to cause sub-toxic effects in the WST-1 assay, but not in the MTT assay (≥80% viability). Figure 3. Cytotoxicity of different concentrations of digested and undigested AgNPs on Caco-2 cells after 24hours of exposure measured using A) MTT and B) WST-1 assays. Data are expressed as a percentage of the (-) control and shown as a mean ± SEM (n=3). The * sign signifies p<0.05 compared to the negative control (0 %). Exposing the HT29-MTX cells to AgNPs (undigested) no cytotoxic effect was observed for all the concentrations used (Figure 4A and B). However, the digested particles induced toxicity starting from 5 µg/ml and continued to reach 5-10% viability at the 100 µg/ml (Figure 4A and B). The chyme did not show toxic effects. A B
  16. 16. 16 Figure 4. Cytotoxicity of different concentrations of digested and undigested AgNPs on HT29- MTX cells after 24hours of exposure measured using A) MTT and B) WST-1 assays. Data are expressed as a percentage of the (-) control and shown as a mean ± SEM (n=3). The * sign signifies p<0.05 compared to the negative control (0 %). As the results from the AgNP exposure to the co-culture of Caco-2 and HT29-MTX (which will be discussed later) showed a higher toxicity to the 10% chyme than the Caco-2 and HT29-MTX cells individually, it was decided to perform the chyme exposure experiment on the co-culture as well. Performing the chyme experiment using a co-culture of Caco-2 and HT29-MTX cells, the toxicity of the chyme increased compared with its effect on the caco-2 cells (Figure 2). At 10% the chyme was able to kill 40% of the co-culture cells suggesting a higher sensitivity of the co-cultured Caco-2 and HT29-MTX cells to the chyme. One can see clearly that the chyme reduces cell viability in a dose dependent manner starting from a 10% concentration and at the highest concentration of 25% only 20% of the cells retain mitochondrial activity (Figure 5A and B). It is worth mentioning that the chyme used in these experiments (caco-2 cells and co-culture) showed some variability of cytotoxicity within the same experiment and same cell type (Figure 5A and B) even though it was prepared following the same steps for each experiment and exposed to a fixed cell density of 40000 cells/ml. A B
  17. 17. 17 Figure 5. Cytotoxicity of different concentrations of chyme on a co-culture (Caco-2cells, HT29- MTX) after 24hours of exposure measured using MTT and WST-1 assays. Data are expressed as a percentage of the (-) control and shown as a mean ± SEM (n=3). The * sign signifies p<0.05 compared to the negative control (0 %). 3.2.2 Exposure to AgNPs In the co-culture model, undigested AgNPs appeared sub –toxic (i.e. between 80-100% viability) up to 25 μg/ml. From 50 μg/ml onwards there was a dose-dependent decrease up to 6% as compared with the negative control at the highest concentration (100 µg/ml) in the ATPlite test, and up to 20% in MTT and WST-1 assays. Interestingly, in digested form, the AgNPs showed a significant dose- dependent cytotoxicity, starting at a concentration of 5 µg/ml. Digested AgNPs caused 50%±5% reduction at the lowest concentration and continued up to 20% ±10% at the highest concentration of 100 µg/ml in the MTT and WST-1 assays. Using the ATPlite assay even lower viability values were obtained for both particles either digested or undigested. Clearly, the cell mortality had a dose response pattern for the digested particles reaching 90% at the highest concentration (Figure 6). The 10% chyme control showed a cell viability of ~60% in all three tests indicating clear cytotoxicity of the chyme at this concentration. BA
  18. 18. 18 Figure 6. Cytotoxicity of different concentrations of digested and undigested AgNPs on co- culture (Caco-2cells, HT29-MTX) after 24hours of exposure measured using A) MTT, B) WST-1, and C) ATPlite assays. Data are expressed as a percentage of the (-) control and shown as a mean ± SEM (n=6). The * sign signifies p<0.05 compared to the negative control (0 %). Silver nanoparticles tend to release Ag+ ions when in solution, which are (to some extent) responsible for their cytotoxic effect. So, a AgNO3 solution was also tested as a control. As shown for the Caco-2 and HT-29-MTX results a concentration of 1.5 µg/ml AgNO3 appeared to be already very toxic. Therefore, the effect of a concentration series of AgNO3 (digested an undigested) on the co-culture was tested in order to generate a standard reference curve for AgNO3 exposure. MTT, WST-1, and ATPlite assays were applied to investigate the effects of these ions. A B C
  19. 19. 19 The results were obtained for different concentrations of silver ions (AgNO3) that represented 6% of each AgNP concentration used in this study. The 6% was determined on basis of published data that indicated AgNPs were able to release about 6% Ag+ ions in water after 24 hours (van der Zande et al., 2012). Undigested silver nitrate was sub-toxic (≥80% viability) up to 0.6 μg/ml. However, from 1.5 μg/ml onwards there was a dose-dependent decrease in cell viability that reached 50 to 55% compared with the negative control at the highest concentration (6 µg/ml) using the MTT and WST-1 assays (Figure 7A ,B, and C). The ATPlite assay showed again higher cell mortality rates that reached 10% at the highest concentration for digested and undigested AgNO3 (Figure 7C). Digested AgNO3 showed higher cytotoxicity compared to the undigested AgNO3 but with the same dose-dependency. Figure 7. Cytotoxicity of different concentrations of digested and undigested AgNO3 on co- culture (caco-2cells, HT29-MTX) after 24hours of exposure measured using A) MTT, B) WST-1, and C) ATPlite assays. Data are expressed as a percentage of the (-) control and shown as a mean ± SEM (n=6). The * sign signifies p<0.05 compared to the negative control (0 %). A C B
  20. 20. 20 3.2 ROS results Figure 8 show that the AgNPs induced the production of ROS in the co-cultured cells after 24h of exposure. Silver nanoparticles (digested and undigested) were able to increase intracellular ROS from 5 μg/ml onwards. Undigested particles induced a progressive decline in intracellular ROS with increasing AgNP concentrations, reaching the lowest value of 110% compared with the positive control (PC; i.e. H2O2) at 100 μg/ml (Figure 8). The digested particles showed higher ROS production values reaching ~7 times the effect of the PC at 25 and 100 μg/ml (Figure 8). The chyme was able to induce 4 times ROS production higher than the PC. Figure 8. ROS production by Caco-2 and HT29-MTX cells (co-culture) caused by exposure to undigested and digested AgNPs for 24h. Data are expressed as a percentage of the positive control (0.125 nM H2O2) and shown as a mean ± SEM (n=3). The * sign signifies p<0.05 compared to the unexposed cells (0 μg/ml). Microscopic images of cells exposed to 5, 10, 25, 50, and 100 μg/ml of digested AgNPs in comparison with healthy cells (0 μg/ml) are provided in Figure 9. Image (A) shows the negative control in which the cells remain untreated. In the next images (Image B, C, D, E), the AgNPs seemed to induce cell mortality, which is visible by cell morphological changes and these images clearly emphasize a reduction of cell density. It should also be made clear that the healthy (unexposed cells) in Image A showed a confluence less than 80%.
  21. 21. 21 Figure 9. Microscopic images (×10 magnification) of co-culture (Caco-2cells, HT-29MTX) exposed to different concentrations of digested AgNPs. Image A: Negative control (0 µg/ml), Image B, C, D, E, F: 5, 10, 25, 50, 100 µg/ml digested AgNPs respectively. 4. Discussion The main aim of this study was to evaluate the potential toxicity of NM-300K silver nanomaterial on an in vitro intestinal epithelium composed of a co-culture of Caco-2 & HT29-MTX cells before and after human in vitro digestion. Since NP toxicity strongly depends on the size, shape and surface area of the NPs (Darolles et al., 2013b), the size of the AgNPs used in this study was investigated using DLS. Dispersion of the AgNPs was highly standardised using a sonication procedure based on the publicly available NanoGenotox dispersion protocol. DLS measurements were performed before each individual experiment to ensure the quality and comparability of the NP dispersions. The DLS results revealed a slightly bigger hydrodynamic diameter in BSA water (~34nm) than in DMEM+ (~28nm). A B C D E F
  22. 22. 22 The core diameter provided by the manufacturer (<20 nm) as measured with electron microscopy is smaller, but this was expected as the hydrodynamic diameter is usually higher. It should be kept in mind that although DLS provides an estimation of the diameter and several studies have used it and reached the expected results, there is no agreement regarding its credibility for measurement in media (Powers et al., 2007), as it is necessary to consider several factors when interpreting DLS results. Firstly, DLS is well-known for its tendency to overestimate the size as it is largely influenced by the presence of a small number of larger particles or aggregates (Dhawan and Sharma, 2010). Secondly, unlike TEM which measures the core size, DLS measures the hydrodynamic size (core and the corona), which is directly affected by the solvent molecules and biomolecules present in the medium (Srnova- Sloufova et al., 2000). The corona formation influencing the NP size using DLS can be explained as follows, when “naked” NPs are introduced into a physiological environment containing proteins i.e. cell culture medium supplemented with FCS, these proteins rapidly absorb onto the NP surface (Fadeel, 2012). This corona, also known as the biocorona, increases the hydrodynamic size, changes the surface charge, and makes the NPs visible to the immune cell receptors depending on the proteins that were absorbed (Hubbs et al., 2011). In this way the biocorona plays a crucial role in the identification and clearance by phagocytes (small particles can evade phagocytosis), translocation in the body, and as a result toxicity of the nanoparticles (Cheng et al., 2013; Dobrovolskaia et al., 2008; Dobrovolskaia and McNeil, 2013; Singh and Ramarao, 2012; Walczak et al., 2015). Once a NP is taken up, the biomolecules (corona) on its surface can be degraded in the acidic lysosomes (Fadeel, 2012; Lynch et al., 2009). Then either charged surface will disrupt the cell membrane and lead to inevitable death or toxic ions will be released into the acidic environment and move freely to the cytoplasm to interact with organelles like the mitochondria (Krug and Wick, 2011; Wang et al., 2013). In the present study there were minor differences in size in which the size of the AgNPs was always slightly smaller in DMEM+. In a previous study by Murdock, Braydich-Stolle et al. (2008) DLS was used to characterize the nanoparticle size of 80 nm AgNPs in solution. The size significantly increased to 1810 and 1640 nm after dispersion in deionized water and growth medium (RPMI-1640) supplemented with 20% FBS respectively (Murdock et al., 2008). This indicates strong effects of the composition of the media, so likely the differences in the present study were due to the different compositions of the media that were used (e.g. differences in ionic strength). However, Darolles et al., (2013) demonstrated a strong tendency of NPs to agglomerate as size increased to 1120 and 1110 nm in water and growth medium without FBS whereas in presence of serum, the size decreased to 340 nm (Darolles et al., 2013b). Furthermore, serum (FBS) was used in several studies to prevent agglomeration (aggregation) of AgNPs (Singh and Ramarao, 2012; Wells et al., 2012). Therefore, the smaller size (diameter) of AgNPs in media that contains 10% FBS could be explained using the aforementioned reported findings in which the FBS at ≥10% was clearly inhibiting the agglomeration and the ionic strength of the media could resulted in smaller diameter (Darolles et al., 2013b).
  23. 23. 23 As well, the serum (FBS) was used in several studies to prevent agglomeration (aggregation) of AgNPs (Singh and Ramarao, 2012; Wells et al., 2012). Therefore, the smaller size (diameter) of AgNPs in media that contains 10% FBS could be explained using the aforementioned reported findings in which the FBS at ≥10% was clearly inhibiting the agglomeration of the particles and the media constituents of DMEM+ (i.e. salt composition) are likely contributed to stabilization of the dispersion by means of steric forces (Darolles et al., 2013b). The potential cytotoxicity of digested and undigested AgNPs on a co-culture of Caco-2 and HT29- MTX cells was investigated in this study using three assays, namely WST-1, MTT, and ATPlite. All three assays measure cell viability, the WST-1 and MTT tests are measuring the mitochondrial activity as an end point, while the ATPlite assay is measuring presence of the ATP marker as an endpoint. The WST-1 and MTT assays revealed a significant impairment of mitochondrial function (<80% viability) from 50 μg/ml upwards for the undigested particles, reaching a decrease of 65-70% of cell viability at 100 μg/ml. The 5, 10, and 25 μg/ml concentrations of undigested AgNPs showed viability levels that were sub-toxic (i.e. between 80-100% viability). Applying the ATPlite assay the 50 and 100 μg/ml showed higher mortality values in comparison with the WST-1 and MTT assay, reaching 90% at the highest concentration, but below 50 μg/ml the viability levels were sub-toxic, which is in good correspondence with the other two assays. For the digested particles, toxicity started at very low concentrations in all three assays. At 5 μg/ml (digested particles), around 50% mortality was observed. This increased in a dose dependent manner reaching 20-25% viability at the highest concentration in MTT and WST-1 results and 10% using ATPlite assay. However, taking a closer look at Figure 4, the chyme effect cannot be neglected since the 10% chyme control was inducing 35-40% cell mortality already. The 10% chyme control was chosen based on experiments with Caco-2 cells (Figure 2A and B), but performing the chyme exposure experiment on the co-culture at a later stage, clearly showed that the co-culture is more sensitive. Higher sensitivity of the co-culture -Caco-2, HT29-MTX- compared with the Caco-2 cells has been frequently described in literature, especially for compounds undergoing passive intestinal absorption and compounds transported via the paracellular pathway (Hilgendorf et al., 2000a, b; van Breemen and Li, 2005). As the chyme already had a significant effect on cell viability it is difficult to determine whether the cytotoxicity of the digested AgNPs was higher than that of the undigested particles. Nevertheless, this is a very plausible scenario as AgNPs have the ability to release ions when in solution, which has been described to be even higher at low pH, like in the stomach phase of the digestion process (Behra et al., 2013; Bohmert et al., 2014; Walczak et al., 2013). Soluble silver has frequently been suggested to be the main cause of cytotoxicity and has been demonstrated to cause mitochondrial damage (Park et al., 2010b; Park et al., 2011b; Singh and Ramarao, 2012). As it is very likely that Ag ions are the main cause of AgNP cytotoxicity, the cytotoxic effect of AgNO3 was examined. The concentrations used to test the effect of the silver ions (AgNO3) were determined on basis of published data.
  24. 24. 24 Data demonstrated that NM-300K in water was capable of releasing about 6% Ag+ ions after 24 hours (Bouwmeester et al., 2011; van der Zande et al., 2012). Hence, the concentrations of AgNO3 used in this study were prepared to represent 6% of each AgNP concentration used. Undigested AgNO3 induced a significant dose-dependent cytotoxicity (≥20% mortality) of the co-culture starting at 3 µg/ml. The lowest viability was observed at 6 μg/ml of which 50-60% of the cells retained mitochondrial activity in MTT and WST-1. Digested ions showed cytotoxicity at the lowest concentration of 0.3 μg/ml and the reduction in cell viability was in a dose dependent pattern. As indicated earlier in the discussion this effect is most likely overestimated due to an additive effect of the chyme (30-35% mortality). Even though the dissolution of NM-300K has been reported to be 6% in water it is worth mentioning that the percentage of free ions in the digestive mixture is possibly lower. Unpublished data from RIKILT indicated that about 1% of free Ag+ ions were present after in vitro digestion. The 1% of free ions scenario will probably induce less toxicity. Nevertheless, the AgNO3 concentration series (6%) used in this study can be used as a reference for Ag+ toxicity. The cytotoxic effect of NM-300K on the intestinal model co-culture model of Caco-2 and HT29-MTX cells was starting from the lowest dose used. However, taking the cytotoxicity data of AgNO3 into account, these effects are most likely caused by the silver ions that were released from the nanoparticles and for the digested particles the additional toxicity is most likely mainly exerted by the chyme. In this study, one of the most discussed mechanisms of AgNPs induced injury to the cells (production of ROS) was investigated. Reactive oxygen species (superoxide anion, hydroxyl radicals and hydrogen peroxide) are produced from the mitochondrial electron transport chain and participate in immune defence responses against pathogens (Bulua et al., 2011; Park et al., 2011c; Zhang et al., 2012). However, elevated ROS levels can overwhelm the cells’ innate antioxidant capacity consequently damaging proteins, lipids and DNA and are associated with inflammatory diseases, cell apoptosis, and necrosis (Arora et al., 2012; Hsin et al., 2009; Karlsson et al., 2009). The results obtained show that digested AgNPs induce an increase in ROS production reaching about 5 to 8 times effect of the positive control for all the concentration tested (5 - 100 μg/ml). However, the chyme alone induced production of ROS ~4 times the effect of the positive control (H2O2). For that reason, it is hard to conclude that digested AgNPs were able to cause oxidant injury or if this was caused by the chyme without particles. Although digested AgNPs significantly induced ROS at all concentrations, this could again be due to Ag+ which also increased ROS production. On the other hand, undigested particles showed a dose dependent decrease in ROS levels (figure 6). This could be explained by AgNPs masking the fluorescence, as was demonstrated before using cobalt oxide nanoparticles, due to their black color (Darolles et al., 2013a). Thus, this might also apply for AgNPs as they have a dark color. Or this effect could be due to another possible reason related to the mode of action of AgNPs.
  25. 25. 25 Since AgNPs display cytotoxicity already at low concentrations (5 μg/ml) by affecting the mitochondria, ATP production stops and the cells undergo apoptosis (Hsin et al., 2008). Hence one important source of ROS (respiration chain) is damaged, and if the cytotoxicity is very high (as seen for the higher concentrations) most of the cells will likely be dying and not be producing any ROS anymore. As mentioned earlier, this experiment was performed only once and the discussed results are not conclusive. Comparing the cytotoxicity of NM-300K on Caco-2 cells and HT29-MTX (Figure 3) to the co-culture, a clear added toxicity of the digested particles on the co-culture was observed, where the 10% chyme control induced ~40% cells mortality. But on Caco-2 cells no added effect was observed (the chyme was not toxic), indicating that AgNPs are more toxic after digestion, which is something that could not be observed from the results of the co-culture. Therefore, one can suggest repeating this experiment using differentiated cells (less sensitive) to investigate whether the digested AgNPs are more toxic in a co-culture as well. For the HT29-MTX cells the lowest mortality of cells was observed for all concentrations of AgNPs used. These cells produce a mucus layer, which forms a significant barrier to absorption of compounds and possibly also AgNPs, which could be resulting in the observed lower cytotoxicity (Behrens et al., 2001; Gagnon et al., 2013). 5. Conclusion To conclude, the data from DLS showed a slightly bigger size of AgNPs in BSA water (0.05%) whereas in physiological medium (DMEM+) the size was always smaller. FBS and the media constituents of DMEM+ (i.e. salt composition) likely contributed to stabilization of the dispersion by means of steric forces. Future experiments should focus on quantifying the amount and type of protein in the biocorona and its effect on particle size. Further investigations to understand these phenomena should focus on answering how strongly attached the corona can be, as the conformation of the absorbed proteins and the corona-surface charge relate strongly to toxicity mechanism. From the cytotoxicity results it can be concluded that silver nanoparticles (NM-300K) had a cytotoxic effect on the intestinal model co-culture model of Caco-2 and HT29-MTX cells starting from the lowest dose used. These effects are most likely caused by the silver ions that were released from the nanoparticles. In vitro digestion increased the toxicity of these particles, but the observed increase in effects was likely caused mainly by the presence of chyme from the in vitro digestion. Caco-2 cells culture showed slightly higher viability values compared to the co-culture and showed clear added toxicity of the digested AgNPs without apparent toxicity of the 10% chyme. HT29-MTX demonstrated the highest viability values for all concentrations tested, which is likely due to the formation of a significant barrier to absorption through mucus secretion. For the 10% chyme control both cell types exhibit a higher survival rate of cells compared to the co-culture, indicating that the co-culture appears more sensitive. The underlying mechanism of action of AgNPs cytotoxicity is likely caused by induced oxidative stress, although ROS production findings in this study were not conclusive.
  26. 26. 26 6. Recommendations On basis of the results obtained and the discussion chapter of this study the following recommendations are formed: 1. Performing the DLS measurements, it is suggested to select a different size characterization technique that is not affected by the presence of big particles and proteins will also help in better characterizing the nanoparticles. 2. Investigate silver nanoparticle color interference with the cell viability assays. For example, it was obvious that AgNPs exerted a strong, concentration dependent, colour related effect on the WST-1 resulting in an increase in cell viability. Although this problem was partially overcome by protocol modification, further investigations of these assays suitability are still needed. 3. Since the biocorona drives the nanoparticle adverse effects and kinetics in the human organism, further investigations using techniques like the bicinchoninic acid assay and polyacrylamide gel electrophoresis (PAGE) will help to estimate the amount of proteins constituting the corona and PAGE can give information about the composition (Lundqvist et al., 2008). 4. Further studies on the relevance of the concentrations used and how nanoparticles behave in the human body in relation to their physicochemical attributes are crucial to understand the real life scenarios. 5. The 10% chyme control effect in the co-culture can be reduced by further dilution to 5% where the chyme toxicity is less than 20% (sub-toxic) (Figure 5). Or through use of differentiated cells (21 days old) that demonstrate absorptive and defensive properties of the intestinal mucosa. These mature cell structures function as the microvilli, goblet cells, mucous secretory granules, tight junctions, etc. in the small intestine (Hilgendorf et al., 2000a; Sambuy et al., 2005). 6. Other models can also be used such as the dynamic in vitro large intestinal model that contains fecal microflora of human origin (for digestion of the particles) (Krul et al., 2002). For cell exposure, a 3 dimensional intestinal model that contains more types of relevant cells like Caco-2 cells, HT29- MTX, M cells, enterocytes, dendritic cells, etc. may be used (Leonard et al., 2010). 7. Dissolution of AgNPs should be further investigated to determine whether the observed effects were due to the particles itself or to the ions made available by dissolution. Dissolution has to be checked in water, biological fluids, different cellular compartments, and in the different conditions available in the environment.
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  31. 31. 31 8. Appendices 8.1 Appendix 1. Dilution scheme for the Ag NPs stock solution 111.76mg/ml. 19000 ppm (1000 ppm in juice) 9500 ppm (500 ppm in juice) 4750 PPM (250 ppm in juice) 1900 ppm (100 ppm in juice) 950 ppm (50 ppm in juice) The concentrations presented are the Ag NPs concentrations in the digestion juices (conc./19) and the concentration in digestion juices is calculated 10 times higher, to allow 10 times dilution (10 µl+90 µl DMEM) prior to cells exposure in order to avoid the chyme toxic effect upon cells exposure (chyme is toxic at > 15% conc.). 1 ml BSA 1.5 ml BSA 1 ml BSA 1 ml BSA 1 ml 1 ml 90 ul EtOH and 2400 ul MilliQ + BSA (3ml) The starter concentration 19 mg/ml 1 ml 1 ml NM-300K (Stock) 111.76 mg/ml
  32. 32. 32 8.2 Appendix 2. Sonication setup for placement of sonication vial in ice-water bath (adopted from NANOGENOTOX, 2011). 8.3 Appendix 3. The constituents and concentrations of the digestive juices as described by (Versantvoort et al., 2005) and (adopted from RIKILT SOP-A-1124).
  33. 33. 33 8.4 Appendix 4. Cytotoxicity of different concentrations of digested and undigested on Caco-2 cells after 24hours of exposure measured using MTT and WST-1 assays (this experiment was carried out once in triplicate). Data are expressed as percentage of the (-) control and shown as mean ± SEM. The * sign signifies p<0.05 compared to the negative control (0 %).
  34. 34. 34 8.5 Appendix 6.Dilution scheme for the Ag NO3 stock solution 10 mg/ml. 1140 ppm (60 ppm in juice) 570 ppm (30 ppm in juice) 285 PPM (15 ppm in juice) 114 ppm (6 ppm in juice) 950 ppm (3 ppm in juice) *The concentrations presented are the Ag NO3 concentrations in the digestion juices and it is calculated 10 times higher, to allow 10 times dilution (10 µl+90 µl DMEM) prior to cells exposure in order to reach the desired concentration (6% of the NPs concentration). AgNO3 (Stock) 10 mg/ml 1 ml BSA 1.5 ml BSA 1 ml BSA 1 ml BSA 1 ml 1 ml 2331ul MilliQ + BSA 1 ml 1 ml

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