Quantitatively Profiling the Dissolution and Redistribution of Silver

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Quantitatively Profiling the Dissolution and Redistribution of Silver Nanoparticles in Living Rats Using a Knotted Reactor–Based Differentiation Scheme Cheng-Kuan Su, Hsin-Tung Liu, Sheng-Chieh Hsia, and Yuh-Chang Sun Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac501691z • Publication Date (Web): 15 Jul 2014 Downloaded from http://pubs.acs.org on July 17, 2014

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Analytical Chemistry

Quantitatively Profiling the Dissolution and Redistribution of Silver Nanoparticles in Living Rats Using a Knotted Reactor–Based Differentiation Scheme Cheng-Kuan Su, Hsin-Tung Liu, Sheng-Chieh Hsia, and Yuh-Chang Sun* Department of Biomedical Engineering and Environmental Sciences, National Tsing-Hua University, Hsinchu, 30013, Taiwan. KEYWORDS: biodistribution, inductively coupled plasma mass spectrometry, knotted reactor, silver nanoparticle, SolvableTM Corresponding Author *To whom correspondence should be addressed. Fax: +886-3-5723883; Tel.: +886-3-5727309 E-mail: [email protected]

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ABSTRACT Whether silver nanoparticles (AgNPs) degrade and release silver ions (Ag+) in vivo has remained an unresolved issue. To evaluate the biodistribution and dissolution behavior of intravenously administered AgNPs in living rats, we employed a knotted reactor (KR) device to construct a differentiation scheme for quantitative assessment of residual AgNPs and their released Ag+ ions in complicated animal tissues; to do so, we adjusted the operating parameters of the KR, namely the presence/absence of a rinse solution and the sample acidity. After optimization, our proposed differentiation system was confirmed to be tolerant to rat tissue and organ matrix and provide superior reliability of differentiating AgNPs/Ag+ than the conventional centrifugal filtration method. We then applied this differentiation strategy to investigate the biodistribution and dissolution of AgNPs in rats one, three, and five days post-administration, and it was found that the administered AgNPs accumulated predominantly in the liver and spleen, then dissolved and released Ag+ ions that were gradually excreted, resulting in almost all of the Ag+ ions becoming deposited in the kidney, lung, and brain. Histopathological data also indicated that toxic responses were specifically located in the AgNP-rich liver, not in the Ag+-dominated tissues and organs. Thus, the full-scale chemical fate of AgNPs in vivo should be integrated into future assessments of the environmental health effects and utilization of AgNP-containing products.


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Introduction Exposure to silver nanoparticles (AgNPs) through inhalation, dermal contact, and ingestion is growing progressively in our daily lives because of their high degree of commercialization in customer-oriented products, primarily for their excellent antibacterial abilities.1–9 To date, it has been established that AgNPs elicit toxic effects at the cellular level as a result of increasing oxidative stress, altering mitochondrial normal functions, and inducing cell membrane leakage and apoptosis, because AgNPs and their released silver ions (Ag+) tend to disturb the biological activities of sulfur- and phosphorus-containing molecules (e.g., DNA, proteins).2, 4, 8–12 Although the debate about the source of AgNP toxicity toward cultured cells is ongoing, several critical reports have correlated recently regarding the toxic potential of AgNPs in the form of their released Ag+ ions, instead of their nanoparticulate forms, particularly under strictly anaerobic conditions.5, 13, 14 In contrast to the many toxicity studies that have been performed in vitro, our understanding of AgNP nanotoxicity in living animals is relatively poor.1,

5

Nevertheless, we know that

mammalian animals exposed to AgNPs can exhibit lung inflammation or dysfunction, the expression of specific genes associated with neurodegenerative disease and immunotoxicity, and oxidative stress related to reactive oxygen species (ROS).15–18 Most studies of AgNP toxicity toward living animals have, however, merely connected the diverse exposure routes and physicochemical properties of AgNPs to their time-dependent biodistribution profiles and associated site-specific physiological responses.18–25 An unanswered issue is whether the toxicities of AgNPs in living animals can be attributed solely to nanoparticulate forms of Ag or to their released Ag+ ions, or indeed whether both are required.5 Therefore, we wished to extend the capability of a conventional analytical strategy—one that has previously provided only the


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normalized elemental concentrations of total Ag—to quantitatively distinguish the fractions of AgNPs and Ag+ ions within the tissues or organs of tested animals. Unfortunately, the determination of Ag concentrations when using currently available elemental analysis instruments [e.g., atomic absorption spectrometry, inductively coupled plasma mass spectrometry (ICP-MS)] has required complete decomposition of the harvested animal samples through chemical digestion, revealing nothing about the integrity of the AgNPs or their dissolution behavior.26, 27 Thus, exactly differentiating coexisting AgNPs and their released Ag+ ions within intact organs or tissues would appear to be infeasible because of the unavailability of (i) appropriate sample digestion methods to release both the AgNPs and Ag+ ions from bulk animal tissues without altering their originalities; (ii) suitable sample pretreatment schemes to practically differentiate biomolecule-bound Ag+ ions from biomolecule-coated AgNPs; (iii) and high-throughput quantification procedures to efficiently handle large quantities of animal samples, particularly for labor-intensive biodistribution studies. Although treatments of animal tissues with tetramethylammonium hydroxide have been introduced to partially extract AgNPs, gold nanoparticles, carbon nanotubes, and quantum dots for subsequent identification,28–31 these methods still unsuccessfully homogenize animal samples with maintaining the equilibrium status between the residual nanosized materials (NMs) and their released ions, which have completely different physical and chemical forms. Moreover, the available strategies5, 32, 33 for separating AgNPs/Ag+ from liquid samples include centrifugation,34 ultrafiltration,35-37 chromatography,38 field-flow fractionation,28, extraction;41,

42

39,

40

and cloud point

in addition, the single particle ICP-MS analysis mode,43,

44

Ag+-specific

indicators,45, 46 and Ag+-selective electrodes47 have also been applied to identify Ag+ ions from AgNP-containing samples without the need for sophisticated pretreatment procedures. Because


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the properties of AgNPs/Ag+, especially in biological environments or in the presence of highaffinity thiol groups, can change as a result of strong interactions between Ag0/Ag+ species and various biomolecules, such analytical strategies might provide a distorted view when differentiating between the two distinct Ag species.2,

28, 32, 33, 40

Accordingly, we wished to

develop a more reliable analytical strategy that would enable the rapid identification of residual AgNPs and their released Ag+ ions in complicated animal tissues. Determining the fractions of Ag+ ions and AgNPs in animal tissues, as well as the chemical fate of AgNPs, should play an essential role in assessments of the health effects and safety of AgNPs for the future development of AgNP-related products.1, 4–9 To quantify the Ag+ ions released from administered AgNPs in living animals, in this study we employed a knotted reactor (KR) as a versatile solid phase extraction (SPE) device to develop a novel analytical scheme for the smart differentiation of AgNPs and their released Ag+ ions from intact organ and tissue samples. The application of KR, traditionally used to extract trace metal ions from samples containing high salt contents, necessitated selective conversion of the metal ions of interest into readily extractable species or precipitates through prior mixing of sample solutions with appropriate complexing or precipitating reagents. Because AgNPs can be considered as a precipitate form of Ag0, we suspected that they would be automatically retained on the KR’s walls by means of the net retention effect, driven by hydrophobic interactions and centrifugation of the fast-flowing sample solutions inside the knotted, coiled tubes. In addition, we wished to make the ionic Ag+ ions selectively extractable for the KR as well as provide critical experimental conditions for retention of Ag+ ions onto the walls of the KRs without any concomitant contributions of the AgNPs from the SolvableTM-solubilized tissue and organ samples. After optimizing the proposed online automatic KR-based differentiation scheme, we


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applied it practically to study the biodistribution and dissolution behavior of administered AgNPs (500 µg kg–1 body weight) through quantitative identification of the Ag+/Agtotal ratios in rat liver, spleen, kidney, lung, brain, and blood, one, three, and five days post-administration. In addition, we performed histopathological examinations to investigate the source of toxicity and health effects of AgNPs in living rats.


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Experimental Section Chemicals The AgNPs used in this study were purchased from Sigma–Aldrich (658804; St. Louis, USA). They were initially dispersed in pure ethylene glycol; the total Ag concentration analyzed after digestion in concentrated HNO3 was 44.3 g L–1. The 1000 mg L–1 Ag+ stock solution was obtained from Merck (Darmstadt, Germany). Dulbecco’s modified Eagle’s medium (DMEM; 11965-092), fetal bovine serum (FBS; 16000-044), and penicillin-streptomycin (15140-122; 10000 U mL–1) were purchased from Life Technologies (Carlsbad, CA, USA). Nitric acid (J. T. Baker, NJ, USA) and SolvableTM tissue solubilizer (6NE9100, PerkinElmer, IL, USA) were purchased ready-to-use. Apparatus and Methods Figure 1 and Table S1 (Supporting Information) provide a detailed schematic representation and step-by-step operating sequence for the proposed KR-based differentiation system. The hyphenated system consisted of two main parts: an online automatic sample pretreatment system and an ICP-MS analysis system. To double the sample throughput, the KR-based analytic system was run in a sequential dual-loading configuration, accomplished by using two identical KRs in the presence and absence of rinse solution to control the selective extraction of AgNPs and Ag+ ions, respectively. The samples, adjusted to appropriate acidities, were delivered to their respective KRs for extraction of the analytes (AgNPs and Ag+ ions) and removal of unwanted sample matrix. The extracted AgNPs and Ag+ ions were then detached, dissolved, and transported to an Agilent 7500a ICP mass spectrometer system (Agilent Technologies, CA, USA) through a Micromist nebulizer (AR35-1-EM04EX, Glass Expansion, Victoria, Australia)


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for time-resolved scanning at m/z 107. All the valves used to construct the hyphenated system were synchronized and programmed using a single laptop, connected through a serial valve interface (SIV-110, Valco, Lucerne, Switzerland), to eliminate any errors from manual operation. Animal Studies and Sample Preparation Adult male Sprague–Dawley rats (249 ± 17 g; n = 16) were obtained from BioLASCO (Taiwan). These animals, which were specifically pathogen-free, were acclimatized to their environmentally controlled quarters (25 °C; 12-h light/12-h dark cycle); water and food were available ad libitum. All animal treatments and experimental protocols were conducted in conformity with the guidelines and approval of the Institutional Animal Care and Use Committee at National Tsing-Hua University (approval number: 10212). Twelve rats were injected intravenously with AgNPs (125 µg; injection volume: 50 µL) dispersed in 10% FBS/DMEM; the others were injected with equal volume of 10% FBS/DMEM solution as the control group. The rats were sacrificed one, three, and five days post-administration; their blood, livers, spleens, kidneys, lungs, and brains were harvested. The collected rat samples (ca. 50 mg) were solubilized in SolvableTM (1:9 dilution, w/v) and placed in an oven for 2 h (60 °C). An additional 20-fold (v/v) dilution of the treated samples with 10% FBS/DMEM was then demanded to stabilize the two Ag species and equalize the matrix contents for our KR-based differentiation scheme. The rat blood that was free of AgNPs, collected from the control group, was subjected to the same procedure; it served as the sample matrix and was used to construct the calibration curves for the quantification of both the AgNPs and Ag+ ions. For the histopathological examinations, the tissues and organs were harvested, fixed in 10% neutral buffered formalin, processed, and embedded in paraffin. Sections 3–5 µm in


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thickness were cut and placed on slides for hematoxylin and eosin (H&E) staining and for further analysis by pathologists. Results and Discussion Properties of the Administered AgNPs To examine possible AgNP exposure from unanticipated commercial products or environmental resources,48 the AgNPs tested in this study had a relatively wide particle size distribution. These AgNPs were initially dispersed in pure ethylene glycol; before they were administered into living rats, 10% FBS/DMEM was selected as a dispersion medium to avoid salt-induced aggregation of the AgNPs and to dilute them to an appropriate concentration for injection. These AgNPs were approximately spherical (Figure S1, Supporting Information) with an average particle size of 35.3 ± 8.2 nm (n = 466). Their hydrodynamic diameters measured before and after dispersion in 10% FBS/DMEM were 43.5 ± 5.4 and 68.7 ± 5.2 nm, respectively; more detailed information about the AgNP preparation and characterization can be found in our previous report.49 Development of the KR-Based Differentiation Scheme We developed a novel sample pretreatment scheme—using a homemade polytetrafluoroethylene (PTFE) KR to selectively extract the two Ag species (Figure 1)—to differentiate the administered AgNPs and their released Ag+ ions rapidly from rat organs/tissues. The operation of the KR involved four steps: sample loading, KR rinsing, elution of the analyte, and KR reconditioning for the next loading. Table S1 provided the detailed system’s operation step by step for the dual-loading differentiation scheme, and Figure S2 and S3 (Supporting Information)


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showed the effects of sample loading flow rate and KR length on the extraction efficiencies of AgNPs and Ag+. We identified two factors that influenced the KR’s extraction behavior toward intact AgNPs and Ag+ ions: (i) the volume of the solution (pure water) used to rinse the KR following sample loading and (ii) the sample acidity for extraction. Figure 2A reveals that when the volume of the solution used to rinse the sample-loaded KRs exceeded 750 µL, the KR retained lesser amounts of the AgNPs than the Ag+ ions, offering an opportunity to differentiate the two Ag species by simply adjusting the KR’s operating parameters. When we applied operating conditions for the KR involving a 1000-µL rinse solution, Figure 2B illustrates that a sample pH of greater than 9 provided ideal conditions for extraction of only the Ag+ ions, without any discernible contributions from the coexisting AgNPs (both prepared in 10% FBS/DMEM; see Figure S4 in Supporting Information for the elution profiles). Thus, based on dissimilar extraction behavior of AgNPs and Ag+ species, we had found an experimental condition for the KRs to predominantly extract released Ag+ ions, but not residual AgNPs, merely through controlling the volume of the rinse solution and the acidity of the sample. In addition, Figure 2B also indicates that when we operated the KR in the absence of rinse solution (i.e., rinsed with air), the AgNPs and Ag+ ions were both retained efficiently under slightly acidic sample conditions (pH 5–6), providing favorable operating conditions to determine the total Ag concentrations from rat tissues/organs. As a result, through respective determinations of Ag+ ions (pH 10) and Agtotal (pH 6), through manipulation of the operating conditions of the KR, we had developed a pH-dependent sample pretreatment scheme for rapid quantification of Ag+/Agtotal ratios from biological samples. The on-wall adsorption or retention of neutral metal complexes or precipitates onto filterless KR-based SPE device is facilitated by mixing sample solutions with appropriate complexing or


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precipitating reagents in advance to ensure that the analyte is readily extractable.50, 51 In this study, we considered the AgNPs to be a form of Ag0 precipitate, such that they would be retained on the KR’s wall even when their surface was covered by a thick layer of protein corona. For the Ag+ ions in biological system, instead of forming the insoluble AgCl precipitate,52,

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the

biomolecules in FBS not only converted them into extractable species but also provided extraction efficiencies that were higher than those for the AgNPs in the KRs. Furthermore, the rinsing step was used to selectively remove those non-adsorbed or weakly bound concomitant species in KR’s operation. After careful optimization of sample acidity and volume of rinsing solution to adjust the stability of absorbed species (AgNPs and Ag+ ions) on the inner wall of KRs, a critical operation condition to allow the retention of the released Ag+ ions without the contributions of AgNPs was availably achieved. Our results suggested that, under well-optimized operating conditions, this KR-based differentiation scheme could be a powerful tool for identifying other kinds of NMs and their released constituent ions from a variety of biological and environmental samples. Table 1 summarizes the used system’s operating conditions. We constructed calibration curves for the quantification of the AgNPs and Ag+ ions individually (0.1–10 µg Ag L–1); the method’s detection limits (three times the standard deviation of the background noise; n = 7) were 0.006 µg L–1 for determination of the total AgNPs and Ag+ (sample run at pH 6 without rinse solution) and 0.234 µg L–1 for determination of the Ag+ ions alone (sample run at pH 10 with 1000-µL rinse solution). For the SolvableTM-treated whole rat blood diluted 20-fold (1:19 dilution) with 10% FBS/DMEM and spiked with a series of concentrations of AgNPs and Ag+ ions (different concentration ratios of Ag+/Agtotal), Figure 3A indicates that the slope between the expected and experimentally measured Ag+/Agtotal ratios was 0.9970 (ideally, the ratio would be 1). In other


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words, neither the presence of complicated biomolecules nor the high salt content of the matrix had a significant influence on the method’s accuracy when differentiating intact AgNPs and their released Ag+ ions. When we measured the fractions of released Ag+ ion using a centrifugal filtration method (molecular weight cut off: 3 kDa), Figure 3B reveals that the slope between the expected and measured ratios of Ag+/Agtotal for the same treated blood samples was only 0.0008. Thus, the separation of released Ag+ species through physical size-discrimination of samples in the presence of large amounts of biomolecules was suppressed significantly, presumably because of strong interactions between the Ag+ ions and sulfur- and phosphorus-containing compounds.2, 4, 8–12

Accordingly, the contributions of the released Ag+ ions might be seriously overestimated in

toxicity studies of AgNPs if these released Ag+ species were determined using physical sizeseparation strategies. In contrast, our developed KR-based differentiation scheme appears to be a much more accurate, versatile, and efficient means of evaluating AgNP dissolution profiles and their resulting toxic responses in living animals. To study AgNP dissolution in vivo, the challenge remained to maintain the equilibrium status or original physicochemical properties of the administered AgNPs and their released Ag+ ions in animal organs and tissues after sample homogenization. Here, we introduced a convenient solubilization protocol (samples were completely dissolved by tenfold dilution with SolvableTM and then heated at 60 °C for 2 h)30, 54 to ensure that the rat samples became solubilized while maintaining the original properties of the two distinct Ag species. To evaluate the applicability of this solubilization method for the rat samples, we performed spike analyses of harvested rat samples that had been treated with Ag+ ions or AgNPs individually prior to applying this advanced solubilization treatment; all data agreed reasonably with acceptable values (83–113%,


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Table 2), suggesting that the Ag+/AgNPs species in the rat tissues and organs maintained their properties for subsequent quantification using our developed KR-based differentiation scheme. Biodistribution and Dissolution Kinetics of AgNPs in Living Rats After giving rats an intravenous dosage of 500 µg AgNPs kg–1 body weight, we acquired the biodistributions of the total Ag concentrations (Figure 4A) one, three, and five days postadministration through use of our KR-based differentiation scheme in the total Ag extraction mode (sample pH 6, without rinse solution). The administered AgNPs accumulated primarily in the liver and spleen. By further applying our KR-based differentiation scheme, Figure 4B indicates that the Ag+/Agtotal ratios were approximately 100% in the kidney, lung, brain, and blood, less than 20% in the liver, but gradually decreased in the spleen within five days postadministration. These results reveal unambiguously that AgNP degradation had occurred with a systemic redistribution of the released Ag+ ions. From whole histopathological evaluations of these tested rats under this administration dose, we found, interestingly, no significant lesions in the spleen, kidney, lung, or brain. In the liver, however, the infiltration of inflammatory cells was evident from three days post-administration (Figure 5A), with focal necrosis further observed at five days post-administration (Figure 5B). Because our advanced AgNP biodistribution data revealed that, after systemic circulation and redistribution, the Ag species that accumulated predominantly in the rat liver were AgNPs (the Ag+/Agtotal ratios were constant and less than 20% within the observation time course), we propose that the hepatic inflammatory response should be attributable specifically to metabolism of the AgNPs by liver cells. In contrast, we observed no significant toxic responses from the Ag+-dominated kidney, lung, and brain. Therefore, these results imply explicitly that the toxicity


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of AgNPs in living animals arises from their nanoparticulate identity, possibly with a minor contribution from their released Ag+ ions. AgNPs are being used popularly in medical applications and commercial products because of their unique antimicrobial activity; thus, the large quantities of AgNPs and Ag+ ions released from these materials after mechanical stress or chemical treatment might subject us to a highexposure-risk environment.1–9 It was believed that the AgNPs underwent the following processes after various exposure routes: (i) immediate deposition in the liver and spleen; (ii) internalization and metabolization by local phagocytic activities; (iii) progressive release of constituent Ag+ ions; (iv) redistribution of the Ag+ ions. Because our developed KR-based differentiation scheme can be used to identify residual AgNPs and released Ag+ ions from intact animal tissues, it could open an avenue for methodical exploration of the more general dissolution behavior of AgNPs. In addition, our quantitative assessments of AgNP dissolution and Ag+ redistribution in living rats emphasize the need to pursue the chemical fate of administered AgNPs when performing future nanotoxicological studies in vivo.


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Conclusions By tuning the KR’s operating conditions, we have developed a novel analytical strategy for the rapid and exact differentiation of residual AgNPs and their released Ag+ ions in intact animal tissues, thereby allowing investigations of the time-dependent biodistribution and dissolution behaviors of intravenously administered AgNPs. We found that the administered AgNPs accumulated predominantly in the liver and spleen, with a gradual redistribution of Ag+ ions to the kidney, lung, brain, and bloodstream. A histopathological evaluation further indicated that the inflammation response occurred specifically in the AgNP-rich liver and not in the Ag+dominated organs and tissues. Accordingly, our results imply that the source of the toxicity of AgNPs is their intact nanostructure, with only minor contributions from their released Ag+ ions.


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Figure 1. Scheme representation and configuration of the online automatic dual-KR–based differentiation system. V1, V5: Six-port rotary valves; V2, V3, V4: eight-port rotary valves. V1, V3, V5, and KR1 were integrated for total Ag determination (sample pH: 6; without rinse solution); V2, V4, V5, and KR2 were integrated for Ag+ ion determination (sample pH: 10; with rinse solution). When the sample pH was adjusted to 6 and the system run without a rinsing step, the total Ag content, including AgNPs and Ag+ ions, was retained; when the sample pH was adjusted to 10 and the system run with a 1000-µL rinse solution, the Ag+ ions were extracted selectively. The two pretreated components were run through the operating sequence


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independently, but were synchronized with a well-defined time delay. The unmarked arrow indicates the outflow of liquid waste.


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A Releative intensity of Ag, %

100

AgNP Ag ion

80

60

40

20

0 0

200

400

600

800

1000

1200

Rinse solution volume, µL

B

AgNP (rinse)

100

Ag ion (rinse)

Relative intensity of Ag, %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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AgNP (w/o rinse) 80

Ag ion (w/o rinse)

60

40

20

0 3

4

5

6

7

8

9

10

11

12

pH

Figure 2. Relative intensities of AgNPs and Ag+ ions, plotted with respect to the (A) rinse solution volume and (B) sample pH, with and without a 1000-µL rinse solution for the established KR-based differentiation scheme. The signals of the two Ag species were both normalized to their respective operating conditions for Ag+ without rinsing solution. The two Ag


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species were prepared in 10% FBS/DMEM at concentrations equal to 10 µg Ag L–1. Error bars represent standard deviations (n = 5).


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A Measured ratio, %

100

y = 0.997x - 2.4009 R = 0.9944

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80

100

80

100

Expected ratio, %

0.5

B

0.4

Measured ratio, %

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0.3

0.2

y = 0.0008x + 0.0297 R = 0.6968

0.1

0 0

20

40

60

Expected ratio, %

Figure 3. Correlation between the expected and practically measured Ag+/Agtotal ratios obtained using (A) our proposed KR-based differentiation scheme and (B) a conventional centrifugal filtration method. The total Ag concentration was fixed at 10 µg Ag L–1. Error bars represent standard deviations (n = 5).


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10000

A

Day 1

Concentration of Ag, µg kg-1

Day 3 Day 5

1000

100

10

1

Liver

B

120

Spleen

Kidney

Lung

Brain

Blood

Lung

Brain

Blood

Day 1 Day 3

100

Ag+/Agtotal ratio, %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Day 5

80

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40

20

0

Liver

Spleen

Kidney

Figure 4. Quantitative biodistribution and dissolution profiles of AgNPs one, three, and five days post-administration in living rats (500 µg kg–1 body weight; n = 4). (A) Total Ag concentrations in rat organs/tissues determined using our developed KR-based sample pretreatment scheme with the sample solubilized in SolvableTM. (B) Dissolution kinetics of the administered AgNPs in vivo, represented with respect to Ag+/Agtotal ratios.


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A

B

Figure 5. Haematoxylin- and eosin-stained liver sections after exposure to AgNPs. (A) Day 3 (400×); inflammatory cells infiltrated. (B) Day 5 (200×); focal necrosis with inflammatory cells infiltrated.


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Table 1. Optimized operating conditions for the developed KR-based differentiation scheme KR-Based Differentiation Scheme for AgNPs and Ag+ KR

PTFE tubing (i.d.: 0.03 inch; 2.5 m; 25 knots m–1)

Sample volume

1 mL

Sample loading flow rate

1 mL min–1

Rinse flow rate

1 mL min–1

Rinse solution

1000-µL H2O/air

Dilution medium

DMEM+10 % FBS (pH 7.4)

Sample pH for Ag+ ions only

10

Sample pH for total Ag

6

Elution

1% HNO3, 1 mL min–1

Sampling frequency

10 h–1 ICP-MS

ICP mass spectrometer

Agilent 7500a

Ar gas flow rates Plasma

15 L min–1

Auxiliary

0.9 L min–1

Nebulizer

1.03 L min–1

Make up

0.13 L min–1

Plasma forward power

1500 W

Sampling cone

Ni, 1-mm orifice

Skimmer cone

Ni, 0.4-mm orifice

Analysis mode

Time-resolved analysis

Integration time

50 ms

Isotope monitored

107

Ag


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Page 24 of 32

Table 2. Spike recoveries of AgNPs and Ag+ ions in SolvableTM-treated rat organ/tissue samples AgNPs (pH 6)

Ag+ (pH 6)

Ag+ (pH 10)

Liver

89.5 ± 0.9

100.1 ± 3.4

88.5 ± 2.9

Spleen

99.5 ± 6.8

89.2 ± 3.9

97.3 ± 3.1

Kidney

83.1 ± 7.2

111.4 ± 2.1

98.7 ± 2.0

Lung

106.2 ± 5.3

113.0 ± 2.1

106.5 ± 2.8

Brain

111.1 ± 5.1

100.1 ± 1.4

103.2 ± 2.9

Blood

99.3 ± 5.0

100.0 ± 5.2

100.0 ± 3.1

*The AgNPs and Ag+ ions were spiked individually prior to the solubilization procedure, and the spike concentration for each of the Ag species was 10 µg Ag kg–1.


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Supporting Information Available: TEM imaging of the used AgNPs, the effect of sample loading flow rate and KR length on the extraction efficiencies of AgNPs and Ag+, the KR elution profiles of AgNPs and Ag+, and the operation sequence illustrated step by step of the proposed KR-based differentiation scheme are provided. This material is available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION *To whom correspondence should be addressed. Fax: +886-3-5723883; Tel.: +886-3-5727309 E-mail: [email protected]

ACKNOWLEDGMENT We thank Professor Mo-Hsiung Yang for providing helpful advice and the National Science Council of the Republic of China (Taiwan) for financial support (grant 102-2627-M-007-005MY3).


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For TOC only:


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Quantitatively Profiling the Dissolution and Redistribution of Silver

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