Transformation and Speciation Analysis of Silver Nanoparticles of

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Ecotoxicology and Human Environmental Health

Transformation and Speciation Analysis of Silver Nanoparticles of Dietary Supplement in Simulated Human Gastrointestinal Tract Wenhao Wu, Ruojie Zhang, David Julian McClements, Benny Chefetz, Tamara Polubesova, and Baoshan Xing Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01393 • Publication Date (Web): 03 Jul 2018 Downloaded from http://pubs.acs.org on July 4, 2018

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Environmental Science & Technology

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Transformation and Speciation Analysis of Silver Nanoparticles of

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Dietary Supplement in Simulated Human Gastrointestinal Tract

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Wenhao Wu1, Ruojie Zhang2, David Julian McClements2, Benny Chefetz3, Tamara Polubesova3,

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Baoshan Xing1,*

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Stockbridge School of Agriculture, University of Massachusetts, Amherst, MA 01003, USA

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Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA

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Department of Soil and Water Sciences, Hebrew University of Jerusalem, Rehovot 76100, Israel

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*Corresponding Author: Dr. Baoshan Xing, Phone: 413-545-5212, Email: [email protected]

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Abstract: Knowledge of the physicochemical properties of ingestible silver nanoparticles (AgNPs)

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in human gastrointestinal tract (GIT) is essential for assessing their bioavailability, bioactivity, and

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potential health risks. The gastrointestinal fate of AgNPs and silver ions from a commercial dietary

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supplement was therefore investigated using a simulated human GIT. In the mouth, no dissolution or

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aggregation of AgNPs occurred, which was attributed to the neutral pH and the formation of

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biomolecular corona, while the silver ions formed complexes with biomolecules (Ag-biomolecule).

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In the stomach, aggregation of AgNPs did not occur, but extensive dissolution was observed due to

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the low pH and the presence of Cl-. In the fed state (after meal), 72% AgNPs (by mass) dissolved,

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with 74% silver ions forming Ag-biomolecule and 26% forming AgCl. In the fasted state (before

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meal), 76% AgNPs dissolved, with 82% silver ions forming Ag-biomolecule and 18% forming AgCl.

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A biomolecular corona around AgNPs, comprised of mucin with multiple sulfhydryl groups,

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inhibited aggregation and dissolution of AgNPs. In the small intestine, no further dissolution or

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aggregation of AgNPs occurred, while the silver ions existed only as Ag-biomolecule. These results

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provide useful information for assessing the bioavailability of ingestible AgNPs and their

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subsequently potential health risks, and for the safe design and utilization of AgNPs in biomedical

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applications.

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Abstract Art:

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Introduction

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Silver nanoparticles (AgNPs) have been utilized in a number of commercial products due to their

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strong antimicrobial properties (1-3). The AgNPs present in these products may either intentionally

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or unintentionally get into the human food chain. AgNPs have been incorporated into commercially

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available supplements and functional foods intended for oral consumption due to their proposed

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health benefits (4-5). The AgNPs incorporated into the food containers or packaging materials for

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protecting foods may leach out into the foods and be consumed with the foods (3). Moreover, the

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increasingly manufactured AgNPs will be inevitably released into the environment, particularly

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during their use in agricultural systems, where they may eventually become associated with plants or

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animals intended for human consumption. Indeed, Frohlich et al. (6) estimated that the daily intake

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of AgNPs by humans is about 20-80 µg based on food consumption. The daily intake dose could be

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even higher when the silver supplements were involved (7-10). The ingestion of AgNPs could have

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severe health risks (7-10). For example, Asharani et al. (8) linked increased concentrations of

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ingested AgNPs to increased mortality and developmental defects such as cardiac arrhythmia and

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spinal deformation. Other researchers have reported that ingestion of AgNPs can cause argyria in

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humans (4,9,10). For these reasons, there has been increasing concern about the potential

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environmental and health risks associated with AgNPs.

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After being ingested, AgNPs will first pass through the gastrointestinal tract (GIT), which

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consists of mouth, esophagus, stomach, small intestine and large intestine regions, and ultimately

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either be directly excreted from the body within feces or urine, or be absorbed through the small

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intestinal epithelium and enter the circulatory system where they can cause toxic effects (11-12). The

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bioavailability of AgNPs depends on their initial physicochemical properties (e.g., concentration, 4 ACS Paragon Plus Environment

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particle size distribution, surface properties and chemical species), as well as any changes in these

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properties as they pass through the GIT (11-12). Therefore, knowledge of the fate of AgNPs within

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the human GIT is of great importance, because the AgNPs properties before, during, and after

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passing through the GIT could be significantly different (11-15). To date, only a few studies have

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investigated the fate of AgNPs in a simulated human GIT (13-15). Walczak et al. (13) showed that

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AgNPs dissolved and formed AgCl in the simulated gastric fluid and subsequently AgNPs

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concentration recovered in the simulated small intestinal fluid, but the AgNPs size distribution was

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not altered in simulated GIT. Kastner et al. (14) and Bohmert et al. (15) both observed that AgNPs

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partially aggregated when exposed to simulated GIT, but the size of the formed aggregates was still

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in the nanoscale. A number of other studies focused on the aggregation, dissolution, and

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transformation of AgNPs only under simulated gastric conditions (16-19). These previous

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investigations provide important information; however, knowledge of the fate of AgNPs in human

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GIT is still limited due to the following reasons. First, the AgNPs investigated in these studies (13-19)

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are all laboratory-synthesized; no ingestible AgNPs or commercial products actually from the market

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were used. The structural and physicochemical properties of commercial AgNPs, such as

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concentration, particle size distribution, interfacial properties and chemical species, may be

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appreciably different from those of laboratory-synthesized AgNPs. Thus, the results from previous

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studies may not accurately predict the fate of ingestible AgNPs in the human GIT. Second, it was

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reported that AgNPs could be oxidized into Ag+ and then combine with Cl- in the gastrointestinal

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fluids to form AgCl (13,18). Alternatively, Ag+ ions can also combine with the various types of

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biomolecules (e.g., mucin and digestive enzymes) in the gastrointestinal fluids and form

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Ag-biomolecule complexes, because most biomolecules contain sulfhydryl, carboxyl and hydroxyl 5 ACS Paragon Plus Environment


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groups that can strongly interact with Ag+ ions through complexation or electrostatic attraction (20).

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Thus, the chemical species of silver in human GIT could be AgNPs, AgCl, Ag-biomolecule and free

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Ag+ ions. However, the formation of Ag-biomolecule complexes was not examined in previous

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studies (13-19), and no comprehensive and quantitative speciation analysis of the silver in human

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GIT has previously been conducted. Third, the applicability of the results from previous studies

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(16-19) is rather limited in predicting the gastrointestinal fate of AgNPs because the effects of the

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saliva and small intestinal fluid were neglected. Therefore, further investigations are needed to more

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accurately predict the gastrointestinal fate of ingestible AgNPs under simulated human GIT

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conditions and their bioavailability.

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In this study, a commercially available silver nanoparticle dietary supplement (AgDS) was

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selected to investigate the fate of ingestible AgNPs in a simulated human GIT. According to the

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product specification provided by the commercial supplier, the AgDS is designed to support and

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boost a healthy human immune system through the antimicrobial function of AgNPs. It is a pure

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silver solution with about 75% of silver in nanoparticle form (AgNPs) and 25% in ionic form (Ag+),

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and the total silver concentration is around 20 mg/L. A simulated human GIT model containing

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mouth, stomach and small intestine phases was used in this study to examine the potential

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gastrointestinal fate of the AgDS. The specific objectives of this study were to: (i) investigate the

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gastrointestinal fate including aggregation, dissolution and transformation of AgNPs from the AgDS

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in the simulated GIT; (ii) quantitatively analyze the distribution of different silver species from

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AgDS in the simulated GIT; (iii) understand the transformation pathways and mechanisms of AgNPs

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from AgDS in the simulated GIT. The obtained results in this study will be useful for understanding

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the human exposure to AgNPs through ingestion and assessing the bioavailability of ingestible 6 ACS Paragon Plus Environment

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AgNPs in better-understood forms and their potential health risks, because the physicochemical

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properties of AgNPs can be significantly changed as they passed through the GIT.

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Materials and Methods

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Materials. The AgDS (a dietary supplement) was purchased online (Amazon). Gastrointestinal

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components, including mucin from porcine stomach, α-amylase from porcine pancreas (≥10

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units/mg), pepsin from porcine gastric mucosa (≥250 units/mg), pancreatin (4×USP) and bile salts

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from porcine pancreas, were purchased from the Sigma-Aldrich Co. (St. Louis, MO). Sodium

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chloride (>99.0%), sodium nitrate (>99.0%), sodium hydroxide (>97.0%), hydrochloric acid

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(36.5%-38.0%), nitric acid (68.0%-70.0%) were purchased from Fisher Scientific Co. (Fair Lawn,

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NJ). Ultrapure deionized water was used in the experiment.

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Simulated Gastrointestinal Tract Model. The AgDS was passed through a simulated GIT to

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mimic the passage through human mouth, stomach and small intestine. This simulated GIT model

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(Table S1) was based on the one used in previous studies (21-23) with some slight modifications as

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described below. (i) Mouth phase: Simulated saliva, containing mucin (3.0 g/L), α-amylase (0.5 g/L)

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and NaCl (2.0 g/L) was preheated to 37 °C, and then the pH was adjusted to 6.8 using NaOH or HCl

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solutions. Fifteen mL of AgDS (suggested daily dosage) were mixed with 15 mL of simulated saliva

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in a 100 mL glass beaker, and then placed in an incubator shaker at 37 °C for 2 minutes to mimic the

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mouth phase. (ii) Stomach phase: both fed state (i.e., after meal) and fasted state (i.e., before meal)

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were considered in this study. Fifteen mL “bolus” sample resulting from the mouth phase was mixed

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with 15 mL fed gastric fluid (3.2 g/L pepsin, 3.0 g/L mucin and 2.0 g/L NaCl) or fasted gastric fluid

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(0.4 g/L pepsin, 3.0 g/L mucin and 2.0 g/L NaCl) in a 100 mL glass beaker, and the pH of the 7 ACS Paragon Plus Environment


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mixture was adjusted to 2.5. Then, the mixture was placed in an incubator shaker at 37 °C for 2 hours

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to mimic the stomach phase. (iii) Small intestine phase: Twenty-five mL “chyme” sample from the

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fed or fasted stomach phase was adjusted to pH 7.0, and then mixed with 5 mL fed small intestinal

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fluid (30.0 g/L bile salts, 14.4 g/L pancreatin, and 52.6 g/L NaCl) or fasted small intestinal fluid (3.0

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g/L bile salts, 2.4 g/L pancreatin, and 52.6 g/L NaCl) in a 100 mL glass beaker. The pH of the

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mixture was adjusted to 7.0. Then, the mixture was placed in the incubator shaker at 37 °C for 2

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hours to mimic the small intestine phase. All the incubations in the simulated GIT model were

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conducted in the dark.

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Determination of AgNPs and Ag+ in the AgDS. To obtain the total silver concentration of

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AgDS, 1 mL of AgDS was totally dissolved in 4 mL concentrated HNO3 and then analyzed by

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Inductively Coupled Plasma Mass Spectrometry (ICPMS-2030, Shimadzu). The AgNPs and Ag+ ion

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concentrations in AgDS were quantified by ultrafiltration/ICPMS according to a previous study (24).

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The AgNPs in the AgDS was first removed by centrifugation (4000 g, 20 min) using Amicon Ultra-4

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3K centrifugal filter devices and then the Ag+ ion concentration was detected using ICPMS. The

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mass loss of Ag+ on the filter was determined to be negligible (300 nm, thus, the presence of the AgNPs can be

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detected and quantified. Second, the formation of AgCl in the simulated GIT could not influence the

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UV-Visible signals of AgNPs, because the AgNPs-AgCl core-shell structure was not formed

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(Supporting Information, Page S6). Third, the UV-Visible spectroscopy has already been successfully

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used to quantify the AgNPs in biological media in many previous studies (16,17,28). According to

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previous studies (16,17,28), the aggregation and dissolution of AgNPs can both be detected using

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UV-Visible spectroscopy. The dissolution of AgNPs reduces the intensity of the absorption peak at

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λmax around 400 nm, because there are less AgNPs adsorbing the incident light after dissolution.

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While the aggregation of AgNPs not only decreases the intensity of the absorption peak at λmax

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around 400 nm, but also increases the intensity of another much broader absorption peak at

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λmax >500 nm (16,17,28). This phenomenon occurs because the aggregation of AgNPs reduces its

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surface energy, resulting in significant reduction in the surface plasmon resonant frequency (ωmax)

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and significant increase in the λmax of AgNPs (29). Therefore, the absorption peak at λmax around 400

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nm is characteristic of non-aggregated monodisperse AgNPs, while the absorption peak at λmax >500

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nm is characteristic of aggregated AgNPs. Furthermore, the absorption peak at λmax >500 nm is much

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broader due to the random aggregation of AgNPs. Thus, the UV-Visible spectroscopy method 9 ACS Paragon Plus Environment


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(Agilent 8453) applied in this study was able to characterize both the aggregation and dissolution of

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the AgNPs from the AgDS collected from the simulated GIT.

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Silver Speciation Analysis. AgDS contains both AgNPs and silver ions. During passage through

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the GIT, the AgNPs from AgDS could be oxidized and dissolve into silver ions. The silver ions

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present in the gastrointestinal fluids may therefore come from the original AgDS or from AgNPs

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dissolution within the GIT, and may be present in three different forms: free Ag+, Ag-biomolecule

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and AgCl. The AgNPs concentration in the gastrointestinal fluids can be detected by UV-Visible

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spectroscopy as mentioned above, while the total silver ion concentration can be calculated by mass

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difference. In order to quantify the concentrations/distributions of different silver ion species in the

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gastrointestinal fluids, Ag+ (AgNO3) with a series of known concentrations (0, 5, 10, 15, 20 and 25

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mg/L) were passed through the simulated gastrointestinal tract. 4 mL of the resultant mouth, stomach,

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and small intestine digesta fluids were respectively added into Amicon Ultra-4 3K centrifugal filter

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devices and then centrifuged (4000 g, 30 min). The free Ag+ from the digesta was passed through the

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filter and analyzed by ICPMS, while the Ag-biomolecule and AgCl from the digesta that were

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blocked by the filter, were collected and rinsed three times using deionized water until no free Cl-

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can be detected by ion chromatography (Dionex, Thermo Scientific). Then, 4 mL of 20 mmol/L

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Na2S2O3 was added into the mixture of the Ag-biomolecule and AgCl in the centrifugal filter device

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and shaken (150 rpm, 1 h) to dissolve the AgCl, because the stability of the thiosulfate complexes of

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Ag+ are much higher than that of AgCl (20,30). The concentration of dissolved Cl- from AgCl can be

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detected by ion chromatography, and then the silver concentration from AgCl can be calculated

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correspondingly. According to a control experiment, >87% AgCl can be dissolved by Na2S2O3.

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Finally, the silver concentration from Ag-biomolecule was calculated by subtracting the silver 10 ACS Paragon Plus Environment

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concentration of free Ag+ and AgCl from the total silver ion concentration. Therefore, the

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quantitative relationship between the total silver ion concentration in the gastrointestinal fluids and

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the concentrations/distributions of different silver ion species was established. The method flowchart

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of the silver speciation analysis is presented in the Page S7 of Supporting Information. As the total

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silver ion concentration of AgDS in the GIT can be calculated by subtracting the total silver

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concentration measured as mentioned above from the AgNPs concentration quantified by the UV-vis

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spectroscopy, the concentrations/distributions of different silver ion species from AgDS in the GIT

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could be calculated using the established quantitative relationship above.

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Results and Discussion

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Analysis of Silver Nanoparticle Dietary Supplement. Analysis of the AgDS using

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ultrafiltration/ICPMS indicated that it contained 17.96±0.09 mg/L of AgNPs and 8.00±0.03 mg/L

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of silver ions. The particle size distribution of the AgNPs in the supplement were measured by both

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TEM (Figure 1A) and DLS (Figure 1B). The particle size distribution obtained from the TEM

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images (Figure S3) reflects the core-size distribution of AgNPs. The mean core diameter and

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standard deviation was 14.0±2.5 nm. The volume average particle size distribution obtained from

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DLS analysis reflects the hydrodynamic size distribution of AgNPs, which was 32.6±9.4 nm. The

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surface charge of the AgNPs was negative at neutral pH and the negative charge decreased in

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magnitude as the pH decreased, with a point of zero charge around pH 2.5 (Figure 1C). Meanwhile,

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trace amounts of Na+, Cl-, and NO3- and total organic carbon (TOC) were detected in the AgDS

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(Table S2). However, the effects of these trace ingredients on the fate of AgNPs in the GIT would be

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negligible, considering the much higher concentrations of NaCl and organic biomolecules in the 11 ACS Paragon Plus Environment


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simulated GIT (Table S1), and the much stronger interactions between AgNPs and organic

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biomolecules with sulfhydryl groups (20).

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UV-Visible Spectra of AgDS in the Simulated Gastrointestinal Tract. The UV-Visible spectra

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of the AgDS in the simulated GIT with both background and dilution corrections are displayed in

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Figure 2. The absorbance of AgDS only decreased slightly after exposure to the mouth phase (around

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2.0%), but there was a redshift of the λmax from 397 nm to 410 nm. In contrast, there was a steep

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decrease in the absorbance of the AgDS after exposure to the stomach phase (around 72% for the fed

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state and 76% for the fasted state), and there was a further redshift of λmax (from 410 nm to 420 nm

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for the fed state and from 410 nm to 416 nm for the fasted state). Finally, the absorbance of the

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AgDS decreased slightly after exposure to the small intestine phase (around 10% for the fed state and

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11% for the fasted state), but there was a blueshift of λmax (from 420 nm to 408 nm for the fed state

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and from 416 nm to 403 nm for the fasted state). However, this was still a net redshift when

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compared to the λmax value of the original AgDS (397 nm). Since the λmax of AgNPs depends on the

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surface plasmon resonant frequency (ωmax) (29), the shift of λmax is likely to reflect changes in the

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surface properties of AgNPs. The redshifts of the λmax of AgDS after mouth and stomach phases were

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relatively small (< 23 nm) and the λmax value was still around 400 nm, which suggests that most of

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the AgNPs were not aggregated (16,17,28). Additionally, the width of the shifted peak did not

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significantly increase. Thus, the redshifts observed in Figure 2 are probably not due to aggregation of

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the AgNPs. Alternatively, it has been reported that the binding of biomolecules to monodisperse

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AgNPs leads to the formation of biomolecular corona that induces a small redshift around the λmax of

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monodisperse AgNPs, due to the change of ωmax (29,31,32). The ωmax of AgNPs is highly sensitive

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to the refractive index of the surrounding medium. Proteins and other biomolecules have a higher 12 ACS Paragon Plus Environment

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refractive index than water, and therefore their adsorption to AgNPs surfaces can decrease ωmax and

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increase λmax, thereby leading to a redshift (29,31,32). Studies have shown that an increase in ionic

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strength can reduce the refractive index of biomolecules and thus reduce the magnitude of the

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redshift (33). Therefore, we assume that the binding of biomolecules to AgNPs in the saliva and

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gastric fluids could be the reason for the redshift of λmax, while the relatively high NaCl

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concentration in the small intestinal fluids could be responsible for the reduced redshift. The

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biomolecular corona around the AgNPs can be seen from the TEM images (Figures S3), and more

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evidence supporting this explanation can be found in the Supporting Information (Pages S9-S10).

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Aggregation and Dissolution of AgNPs from AgDS in the Simulated Gastrointestinal Tract.

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Mouth phase: The fact that there was little change in the maximum absorbance measured by

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UV-Visible spectrophotometry of the AgDS before and after exposure to the mouth phase (Figure 2)

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suggests that there was no dissolution of the AgNPs. Moreover, no absorption peak was observed at

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λmax >500 nm, which suggests that there was no aggregation of the AgNPs in the mouth phase. The

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lack of dissolution and aggregation of the AgNPs under simulated oral conditions is also supported

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by the SAXS data, i.e., the SAXS intensities and calculated particle size distributions of the original

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AgDS suspension and the AgDS placed in saliva were almost the same (Figure S1). The main change

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of the AgNPs observed in the mouth phase was the coating of the nanoparticle surfaces with

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biomolecules (mucin and α-amlyase), which can be seen from the TEM image (Figure S3). The

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presence of this biomolecular corona could enhance the stability of the AgNPs to aggregation and

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dissolution (34,35). Moreover, the neutral pH condition within the simulated saliva was not

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conducive to AgNPs dissolution (24).

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Stomach phase: The pronounced decrease in the maximum absorbance for the AgDS after being 13 ACS Paragon Plus Environment


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exposed to both the fed (71.8%) and the fasted (75.9%) stomach phases (Figure 2) indicates that

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significant dissolution of the AgNPs occurred. Interestingly, the degree of dissolution was not

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significantly different for the fed and fasted stomach phase conditions. Again, no absorption peak

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was observed at λmax >500 nm, implying that there was no aggregation of the AgNPs in both the fed

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and fasted stomach phases. Further insights into the fate of the AgNPs in the stomach phase were

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obtained by measuring changes in the UV-Visible spectra of the AgDS over time (Figures 3A, 3B).

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The λmax and peak width of the AgDS remained constant throughout 2 hours of incubation in both the

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fed and fasted stomach phases, again indicating that particle aggregation did not occur. Experiments

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were also carried out to determine the role of the biomolecules on the gastric fate of the nanoparticles.

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The maximum absorbance of the AgDS in the simulated gastric fluid without biomolecules (Figure

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3C) decreased much faster than that in the gastric fluid with biomolecules (Figure 3A). Moreover, in

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the absence of biomolecules, there was evidence of nanoparticle aggregation, with a broad

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absorption peak appearing at λmax >500 nm. Thus, the surface-bound biomolecules (Figure S3)

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played important roles in slowing down the dissolution rate and preventing aggregation of the AgNPs

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in the gastric fluid. To elucidate the roles of the different biomolecules (mucin, α-amylase, or pepsin)

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in protecting the AgNPs in the gastric fluids, AgDS was digested in the gastric fluids containing

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separate biomolecules (fed state) and the spectra are displayed in Figures 3D-3F. In the gastric fluid

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containing mucin, the λmax and the peak width of AgDS both kept constant during the 2 hours

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digestion, similar to that in the gastric fluid containing all biomolecules. Moreover, the decreasing

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rates of absorbance were almost the same (Figures 3A,3D). In the gastric fluid containing α-amylase,

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an absorption peak associated with aggregated AgNPs (about 500 nm) emerged and the absorbance

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decreased faster than in the gastric fluid containing all biomolecules (Figures 3A,3E). In the gastric 14 ACS Paragon Plus Environment

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fluid containing pepsin, a redshift of the λmax was observed and the peak width became broader

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(Figures 3F). Overall, the fairly similar UV-Visible spectra of the AgDS in the gastric fluid

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containing all biomolecules (Figure 3A) and in gastric fluid containing only mucin (Figure 3D)

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suggests that mucin played the main role in slowing down the dissolution rate and preventing the

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aggregation of AgNPs in the stomach phase. Mucin is rich in cysteines and contains plenty of

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sulfhydryl groups that could strongly interact with AgNPs and silver ions (20,36,37). Pepsin and

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α-amylase contain much less sulfhydryl groups as compared with mucin (38,39). Hence, the

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interaction between mucin and AgNPs in the dietary supplements was much stronger than the

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interaction with pepsin/α-amylase, and mucin primarily protected AgNPs in the stomach phase. The

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dissolution of AgNPs into silver ions has been reported in acidic solutions through their oxidation by

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oxygen (20,40). This dissolution could be enhanced by chloride ions in the surrounding solution

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leading to the formation of insoluble AgCl (20,41). An experiment was then carried out to confirm

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the role of the chloride ions and pH in the dissolution process. The decrease in the maximum

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absorbance of AgDS in the gastric fluid occurred much more slowly when nitric acid (NO3-, Figure

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3G) was used to control the pH (2.5) rather than hydrochloric acid (Cl-, Figure 3A). In addition, the

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decrease occurred much more slowly when the pH of the gastric fluid was adjusted to 5.0 (Figure 3H)

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rather than 2.5 (Figure 3A) using HCl. Overall, these results indicate that the dissolution rate of

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AgNPs was accelerated by H+ and Cl-, but slowed down by biomolecules in the stomach phase.

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Small intestine phase: The maximum absorbance of AgDS decreased slightly after exposure to

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both fed and fasted small intestine phases (Figure 2). This effect may have occurred because the

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AgNPs continued to dissolve during the short operation time (around 3 min) between the stomach

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and small intestine phases. These results therefore suggest that there was little further dissolution of 15 ACS Paragon Plus Environment


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the AgNPs in both fed and fasted small intestine phases. No absorption peak was observed at

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λmax >500 nm, which suggested that no aggregation of the AgNPs occurred in both the fed and fasted

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states. The lack of further dissolution of AgNPs can mainly be attributed to the fact that the pH was

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changed from 2.5 to 7.0, so that the concentration of H+ that could accelerate the dissolution reaction

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significantly decreased (20,40). Furthermore, the biomolecular corona around the AgNPs that can

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prevent AgNPs from aggregation and dissolution could still be the reason for the good stability of the

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AgNPs in the small intestine phase. The TEM images clearly showed the presence of a biomolecular

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corona on the AgNPs in the small intestine phase (Figure S3).

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Previous studies (13-15) have explored the aggregation and dissolution of chemically

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synthesized and surface-coated AgNPs in a simulated human GIT. However, the main differences of

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those previous studies from this study were observed. First, partial aggregation of AgNPs was

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reported in the mouth phase by Kastner et al. (14). Second, partial aggregation of AgNPs in the

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stomach phase was reported by Kastner et al. (14) and Bohmert et al. (15). Third, the particle number

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of AgNPs was reported to increase to its original value after exposure to the small intestine phase by

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Walczak et al. (13). The partial aggregation of AgNPs observed in the mouth and stomach phases by

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other workers may be due to the much higher and unrealistic dose of AgNPs used, i.e., 3 g/L (14) and

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10% (w/w) (15). As a consequence, more aggregation occurred because of the higher nanoparticle

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concentration in solution. In our study, the AgNPs concentration in the original dietary supplement

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was 17.96 mg/L, and became lower when passed through the simulated GIT due to dilution effects

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(Table S1). Consequently, AgNP aggregation may have been less likely because of the reduction in

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the particle-particle collision frequency and the increase in entropy of mixing effects. The particle

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size distribution of the AgNPs also remained constant throughout the whole GIT in the study by 16 ACS Paragon Plus Environment

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Walczak et al. (13), who also used a relatively low initial AgNPs concentration (10 mg/L). Thus, the

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aggregation state of AgNPs in the GIT appears to be sensitive to its initial concentration, and the lack

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of aggregation reported in this study would be more practically relevant since the used AgDS was a

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commercially available product containing AgNPs. Walczak et al. (13) attributed the recovery of

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AgNPs particle number in the small intestine to the reversible clustering and disassembling of

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AgNPs in the GIT. However, since there was substantial mass loss of AgNPs in the stomach phase

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due to the dissolution, only the reversible clustering and disassembling cannot result in the recovery

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of AgNPs particle number. In this study, recovery of AgNPs was not observed in the small intestine

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phase. Moreover, no other study has confirmed the AgNPs recovery in the small intestine phase.

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Besides the aforementioned studies (13-15), several other investigations (16-19) showed the

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aggregation and rapid dissolution of AgNPs in simulated gastric fluids. However, the lack of

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biomolecules (such as mucin and digestive enzymes) in their simulated gastric fluids limits the

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applicability and implication of their results because these biomolecules can inhibit the aggregation

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and slow down the dissolution rate of AgNPs as observed in this work. Furthermore, the impacts of

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the mouth and small intestine phases on the fate of the AgNPs in the human GIT were not considered

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in these earlier studies (16-19). Therefore, in order to explore the realistic fate of ingestible AgNPs in

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the human digestive system, a comprehensive GIT model needs to be applied.

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Transformation and Speciation Analysis of AgNPs and Silvers Ions from AgDS in the

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Simulated Gastrointestinal Tract. The quantitative relationship between the total silver ion

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concentration in the gastrointestinal fluids and the distributions of different silver ion species (free

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Ag+, Ag-biomolecule and AgCl) were established as follows. First, no free Ag+ ions were observed in

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any of the gastrointestinal fluids according to the ultrafiltration/ICPMS results. Second, the 17 ACS Paragon Plus Environment


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quantitative relationship between the percentage of silver ions as AgCl species (PAgCl) and the silver

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ion concentration (C) in the gastrointestinal fluids are listed below and displayed in Figure 4A.

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Mouth:

367 368

(0

Transformation and Speciation Analysis of Silver Nanoparticles of

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