In Vivo Bioimaging of Silver Nanoparticle Dissolution in the Gut

Nov 20, 2018 - Release of silver ions (Ag+) is often regarded as the major cause for silver nanoparticle (AgNP) toxicity toward aquatic organisms. Nev...
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In Vivo Bioimaging of Silver Nanoparticle Dissolution in the Gut Environment of Zooplankton Neng Yan,† Ben Zhong Tang,‡ and Wen-Xiong Wang*,†

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Department of Ocean Science, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong, and Marine Environmental Laboratory, HKUST Shenzhen Research Institute, Shenzhen 518057, China ‡ Department of Chemistry, Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, HKUST, Clear Water Bay, Kowloon, Hong Kong, China S Supporting Information *

ABSTRACT: Release of silver ions (Ag+) is often regarded as the major cause for silver nanoparticle (AgNP) toxicity toward aquatic organisms. Nevertheless, differentiating AgNPs and Ag+ in a complicated biological matrix and their dissolution remains a bottleneck in our understanding of AgNP behavior in living organisms. Here, we directly visualized and quantified the time-dependent release of Ag+ from different sized AgNPs in an in vivo model zooplankton (Daphnia magna). A fluorogenic Ag+ sensor was used to selectively detect and localize the released Ag+ in daphnids. We demonstrated that the ingested AgNPs were dissoluted to Ag+, which was heterogeneously distributed in daphnids with much higher concentration in the anterior gut. At dissolution equilibrium, a total of 8.3−9.7% of ingested AgNPs was released as Ag+ for 20 and 60 nm AgNPs. By applying a pH sensor, we further showed that the dissolution of AgNPs was partially related to the heterogeneous distribution of pH in different gut sections of daphnids. Further, Ag+ was found to cross the gills and enter the daphnids, which may be a potential pathway leading to AgNP toxicity. Our findings provided fundamental knowledge about the transformation of AgNPs and distribution of Ag+ in daphnids. KEYWORDS: AgNPs, Daphnia, gut, dissolution, AIE, bioimaging

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Despite the numerous studies on AgNPs toxicity in different species of aquatic organisms, our understanding of AgNPs is thus severely limited due to the lack of information differentiating AgNPs and Ag+ in living organisms. Bioaccumulation and toxicity of AgNPs in many aquatic organisms have been well documented.5,12,13 The freshwater zooplankton Daphnia magna has been extensively used as a model organism to assess the hazardous potentials of AgNPs because it responds most sensitively and possesses a unique filter-feeding strategy, leading to an effective uptake of nanoparticles.13,14 An earlier study found that the concentration of accumulated AgNPs could reach as high as 22.9 mg/ g dry weight at the highest AgNP concentration tested (500 μg/L), while daphnids were extremely sensitive to free Ag+, with a measured 48 h 50% lethal concentration of 2.51 μg/L.7 Other studies also showed that the toxic effect of AgNPs

ilver nanoparticles (AgNPs) have been widely applied in our daily life because of their antibacterial properties1,2 and may be eventually released into the surrounding environment,3,4 presenting potential hazards to aquatic organisms.5−7 Most previous studies focused on the environmental behavior of AgNPs, particularly the transformation between AgNPs and Ag+, and the sequential effects of such transformation (i.e., dissolution) on the toxicity to aquatic organisms.8,9 One major toxicity mechanism for AgNPs is the release of soluble Ag ions (Ag+) associated with the oxidation of AgNPs,10 typically by reacting with dissolved O2 in the presence of protons and other components from their surroundings.11 Therefore, AgNP toxicity is likely complicated by the coexistence of both NPs and ionic species, which exhibit different fates and independent or synergistic toxicity.10 Species analysis of AgNPs and Ag+ is thus of great importance in understanding the toxic mechanisms of AgNPs toward aquatic organisms. However, little is known regarding the ingested and internalized AgNPs and Ag+, as well as the transformation, especially regarding the dissolution process. © 2018 American Chemical Society

Received: August 7, 2018 Accepted: November 20, 2018 Published: November 20, 2018 12212

DOI: 10.1021/acsnano.8b06003 ACS Nano 2018, 12, 12212−12223

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Figure 1. (a) Newly accumulated Ag (μg/g) in the daphnids during a 48 h exposure to AgNPs (500 μg/L) in the SM7 medium. (b) TEM micrographs of a gut section of daphnids exposed to 60 nm citrate-coated AgNPs for 8 h, selected area in the left figure for EDX analysis of elemental abundance. (c) TEM images of AgNPs accumulated in the gut separated by a sharp scalpel and the EDX mapping of selected elements (Ag, S, and Cl). The scale bar is 0.5 μm.

the dynamic events of interest.35 Contrary to conventional fluorophores, a group of unique luminogens, such as those with aggregation-induced emission (AIE), which display photostability and are highly fluorescent when aggregated, were discovered several years ago.36,37 The AIE methods have been applied in the areas of electroluminescence,36,38 mechanochromism,39 chiral recognition,40 and ionic sensing.41−43 Compared with other fluorescent sensors based on Agselective organic fluorophores, AIE luminogens (AIEgens) exhibit high quantum efficiency, good photostability, and excellent biocompatibility and could selectively detect Ag+ ions at very low concentrations (with a limit of detection at 2.3 nM).44 In a recent study,45 we demonstrated that the fluorogenic Ag ion sensor tetrazole-functionalized tetraphenylethylene derivative 1 (TEZ-TPE-1) showed good selectivity and sensitivity toward Ag+ and could be applied for the realtime monitoring of Ag+ released from AgNPs and Ag nanowires. In the present study, fluorogenic TEZ-TPE-1 was used for selectively sensing the Ag+ released from AgNPs and monitoring the time-dependent dissolution of different sized Ag nanoparticles in daphnids. Besides, the distribution of Ag nanoparticles and the corresponding Ag+ in the daphnids were also investigated. This study represents the direct visualization of Ag ions and quantitative monitoring of the dissolution kinetics of AgNPs in any aquatic organism.

toward cells increased with increasing release of Ag+ in vitro, supporting the hypothesis that Ag+ was the major cause for AgNPs’ toxicity.15,16 The size distribution and exposure concentration of AgNPs have been regarded as two major factors influencing the bioaccumulation and biotransformation.14,17 The transformation of AgNPs, especially the dissolution of AgNPs in biological systems, has been seldom investigated. A major difficulty is the effective differentiation between AgNPs and Ag+ in complicated biological systems. Various techniques including laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS),18,19 electron microscopy,20,21 synchrotron-radiation-based micro/nano X-ray fluorescence spectroscopy (μXRF/nano-XRF),22,23 isotope labeling techniques,24−26 and fluorescence techniques27 have been applied to species analysis of nanoparticles and metallic ions in cells and aquatic organisms. Among these methods, imaging technology with real-time monitoring and noninvasive detection appears to be the advanced methodology for in vivo probing the metallic ions.28−30 Owing to its excellent selectivity and sensitivity, low cost, and easy operation, fluorescence imaging has been widely applied for the in vivo detection and visualization of nanoparticles and ions.28,31−34 However, identification and tracking of nanoparticles and metallic ions in biological systems remain a great challenge by fluorescent probes since most of the existing fluorescence probes suffer photodecomposition, limiting the time available for probing 12213

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Figure 2. (a) Chemical reaction of Ag+ with an −SH groups in the side groups of cysteine residues in the protein chain and Cl−. (b) Interference study of sensing Ag+ by the proposed AIE method in the presence of 10 and 50 μM cysteine and NaCl and under different pH conditions. (c) Confocal images of daphnids first exposed to 30 μM TZE-TPE-1 and then injected with 1 μL of 20 μg/L AgNO3 after 0 and 2 h and the corresponding fluorescence intensity at different time points.

tured AgNPs in daphnids was first investigated. Daphnids were exposed to AgNPs (see SI for characterization, Figure S2 and Figure S3), and at different time points the concentrations of AgNPs in daphnids were quantified. Figure 1a shows that during 48 h of exposure to different sized AgNPs (20 and 60 nm) at a concentration of 500 μg/L, daphnids continuously accumulated AgNPs with time. The uptake rate of AgNPs decreased significantly with the increase in particle size. At the end of the 48 h exposure, AgNPs in daphnids reached more than 15.4 and 9.41 mg/g for 20 and 60 nm, respectively. The AgNPs’ distribution showed that more than 60% of the AgNPs (64.2 ± 2.5% for 20 nm, 69.7 ± 3.8% for 60 nm) were in the gut, suggesting that ingestion was the predominant pathway for bioaccumulation. It should be noted that some of these accumulated AgNPs could probably penetrate the gut membrane through phagocytosis, endocytosis, or other pathways, which could induce varying degrees of cell deformity and membrane damage, while the majority of AgNPs would be detained in the

RESULTS AND DISCUSSION Biocompatibility Assay of TEZ-TPE-1. To investigate the potentials of the proposed AIE method for the in vivo differentiation of the AgNPs and Ag+, the biocompatibility of TEZ-TPE-1 was first assessed by exposing daphnids to different concentrations of TEZ-TPE-1 (5−100 μM). The results indicated that the survival of daphnids was not affected (above 85%) even when the exposed concentration of TEZTPE-1 was up to 100 μM in the simplified M7 (SM7) medium (Figure S1), suggesting that TEZ-TPE-1 was not toxic to daphnids and was highly biocompatible for application in the sensing of Ag+ in daphnids. Bioaccumulation and in Vivo Distribution of AgNPs in Daphnids. Bioaccumulation of AgNPs is a major pathway for these nanomaterials entering the food web and exerting toxic effects.14 While most studies focused on the environmental behavior of AgNPs, knowledge regarding the behaviors in organisms is rather limited. To get insight into the fate of AgNPs in daphnids, the bioaccumulation kinetics of manufac12214

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intensity, the fluorescence intensity in the gut regions of daphnids was analyzed at 0 and 2 h (Figure 2c). At 0 h, the fluorescence signals were mainly concentrated at the injection sites and then transferred to the posterior region of the gut through the digestive systems of the daphnids after 2 h. A slight difference in the fluorescence intensity in the gut was observed for these daphnids exposed for 0 and 2 h, implying that the amount of Ag+ detected by the AIE method did not change and the Ag+ bound with TZE-TPE-1 was highly stable in the gut. Therefore, TZE-TPE-1 could be utilized to selectively sense Ag+ even in the presence of SH− and Cl−. The 48 h 50% lethal concentrations (LC50) of AgNO3 and AgNPs toward daphnids were quantified. Compared with AgNPs (20 nm: LC50 of 6.60 μg/L; 60 nm: LC50 of 9.03 μg/ L), daphnids were highly sensitive toward AgNO3, with an LC50 of 0.94 μg/L (Figure S4), which was much lower than the microinjected concentration of AgNO3 (20 μg/L), implying that a direct exposure of Daphnia to AgNO3 could induce a more severe toxic effect compared with the microinjection pathway. The addition of TZE-TPE-1 efficiently reduced the toxicity of both AgNO3 and AgNPs toward Daphnia. Therefore, the released of Ag+ could be one of major reasons contributing to the toxicity of AgNPs toward Daphnia. Selective Detection of Ag+ in Daphnids. The capability of the proposed AIE method for the selective sensing of Ag+ in daphnids was tested. Daphnids were exposed to AgNPs and AgNO3 independently, and no fluorescence was detected under the microscope, suggesting that these accumulated AgNPs and AgNO3 localized mainly within the guts were nonfluorescent and had no interference with the proposed fluorescence technique (Figure S5). Compared with the detection of Ag+ in the medium, the in vivo analysis of AgNPs and AgNO3 by the proposed AIE method was much more complicated since the employed TEZ-TPE-1 could be absorbed or accumulated in daphnids and restricted the rotation of TEZ-TPE-1 by forming complex chemicals, potentially triggering the aggregation of TEZ-TPE-1 and subsequently leading to fluorescence enhancement based on the mechanisms of the AIE phenomenon.36 Therefore, control experiments were also conducted by exposing daphnids to TEZ-TPE-1 and TEZ-TPE-1 mixed with some typical nanoparticles (titanium dioxide NPs, TiO2 NPs; gold NPs, AuNPs; zinc oxide NPs, ZnO NPs, Figure S6), respectively. Strong fluorescence was detected for these daphnids exposed to AgNPs and Ag+ (Figure 3D) only, especially in the gills and guts as the major sites of AgNPs and Ag+ accumulation.14 No fluorescence was detected except for the body surface of daphnids exposed to TEZ-TPE-1 only (Figure 3A), nor for TEZ-TPE-1 mixed with different nanoparticles (TiO2 NPs, AuNPs, ZnO NPs) (Figure S6). The strong fluorescence in these daphnids exposed to Ag+ and AgNPs thus originated from Ag+, rather than the particles themselves or the ingested TEZ-TPE-1. The AIE method could also differentiate Ag+ in daphnids exposed to 20 and 60 nm AgNPs based on the difference in fluorescence intensity (Figure 3C and D). Smaller AgNPs had much higher surface/volume ratios than the larger sized AgNPs, resulting in more atoms on the surface in contact with the oxidant.46 Correspondingly, a much stronger fluorescence signal was detected for daphnids exposed to 20 nm AgNPs than to 60 nm AgNPs. Verification of Ag+ Distribution in Daphnids by the 3D Technique. The three-dimensional fluorescence imaging technique with high spatial resolution was used to determine

gut and gradually eliminated. To gain better insight into the toxic mechanisms of AgNPs toward Daphnia, typical distributions of AgNPs in the gut membrane and their retention in the gut were investigated. As shown in Figure 1b, AgNPs were detected in the gut cells, which was confirmed by EDX analysis, implying that these accumulated AgNPs could penetrate the gut membrane and potentially induce a stronger cytotoxicity toward Daphnia. Compared with the AgNPs in the medium, these AgNPs were aggregated with a rough surface, suggesting a higher degree of dissolution probably occurring in Daphnia. To further study the behavior of AgNPs in the gut regions, these accumulated AgNPs (not entering the cells of Daphnia) were further characterized by TEM and EDX mapping (Figure 1c). Compared with AgNPs in the medium, the size of accumulated AgNPs in the gut region was much larger (nearly 1.23 μm). In biological matrices, Ag+ released from AgNPs likely formed precipitates of Ag-chloride and Ag-sulfide complexes, which was the gray matter surrounding the AgNPs. EDX mapping technique also confirmed the coexistence of Ag with chloride and sulfide since these three different elements showed similar distribution patterns. AIE Method to Sense Ag+ in Complicated Biological Matrices. Speciation of Ag in the biological systems was complicated by the presence of chloride and biological thiols, which may interact with Ag+ released from AgNPs. In general, proteins and enzymes containing sulfide (S2−), organosulfur compounds (thiols-SH), and chloride (Cl−) could bind with Ag+, driving Ag+ to very low concentrations (Figure 2a). Therefore, these organic ligands could potentially compete with TEZ-TPE-1 and affected the detection of in vivo Ag+ by the proposed AIE method. Based on our previous study, the fluorogenic Ag+ sensor exhibited high selectivity toward Ag+ rather than the AgNPs or Ag0 and could selectively detect Ag+ even in the presence of other interfering substances (metallic ions and humic acid).45 Besides, the binding affinity of Ag+ toward TEZ-TPE-1 and some ligands (chloride, Cl−; thiol, −SH) widely found in biological matrices was investigated, and the interaction between TEZ-TPE-1 and Ag+ was the strongest among the interactions between Ag+ and SH−, Ag+, and Cl−. To explore the capability of the proposed AIE method for selectively sensing Ag+ in biological matrices, interference tests including SH− (cysteine) and Cl− (NaCl) and under different pH conditions (3−11) were also conducted (Figure 2b). All these interfering reagents did not trigger the fluorescence turnon of TZE-TPE-1, and the TZE-TPE-1 was able to snatch off Ag+ from Ag+ bound with SH− and Cl− complexes, which could initiate the aggregation of TZE-TPE-1 and lead to fluorescence enhancement. Besides, the proposed AIE method could detect Ag+ under pH conditions in the physiological range (4−9) with a recovery of more than 75%, as shown in Figure 2b. It should be noted that all these Ag+ ions detected in the biological system were in the form of Ag+ bound with SH− and Cl− complexes, and the detection was based on the strong binding affinity of TZE-TPE-1 toward Ag+. The microinjection technique was used to explore the feasibility of the proposed AIE method for sensing Ag+ in daphnids. Daphnids were exposed to medium containing 30 μM TZE-TPE-1 for 4 h, allowing it to be fully distributed throughout the gut, and 1 μL of 20 μg/L AgNO3 was injected at certain parts of the daphnids. To test whether these ligands in the gut could compete and snatch off Ag+ bound with TZETPE-1 complexes as well as the variation of fluorescence 12215

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signal, which agreed well with the two-dimensional images. It could be confirmed that the Ag+ was mainly distributed in the gut, while a small amount of Ag+ was localized in the gills, implying that absorption and ingestion were the two major routes for AgNPs and Ag+ to enter into daphnids. Ingestion was previously documented to be the predominant pathway for AgNP bioaccumulation in daphnids.14,23 Distribution and Transportation of Ag+ in Daphnids. The accumulated AgNPs could undergo dissolution spontaneously, and the resulting Ag+ contributed to the toxicity of AgNPs.7,47 These Ag+ ions in daphnids could be transported through epithelial cells and interfered with the ion regulation of the whole body. Therefore, the time-dependent transportation of Ag+ derived from the AgNP accumulation was investigated. Clearly, the accumulated AgNO3 was solely detected within the gut and homogeneously distributed after exposure for 8 h (Figure S7C), whereas Ag+ derived from 20 nm AgNPs was heterogeneously distributed in the gut after 8 h exposure (Figure S7-A). After 48 h, Ag+ was found at both sides of the gut, as highlighted by the arrows in Figure S7D, which might be derived from Ag+ accumulated in the gut or absorbed Ag+ in the gills and surface of daphnids. In contrast, Ag+ derived from 20 nm AgNPs was found in the gut and the areas between the gut and gills after 48 h of exposure (Figure S7B). The concentration of dissolved Ag+ in the gut was significantly higher after 48 h exposure than that after 8 h exposure, attributable to the dissolution of AgNPs during the exposure. These dissolved Ag+ ions were highly concentrated in certain sites of the gut after 8 h of exposure and distributed much more uniformly after 48 h than at 8 h, suggesting the transportation of Ag+ from the anterior gut to the hindgut. Some Ag+ was found at sites between the gills and gut, possibly originating from the absorbed AgNPs in the gills since these fluorescent dots just existed at one side of gut. Gills may therefore serve as another major site for AgNP uptake, and these absorbed AgNPs underwent dissolution to Ag+. Therefore, we concluded that accumulated Ag+ was homogeneously distributed in the gut regions and could be transported from the gills, surface body, and gut regions of the daphnids, while the dissolved Ag+ derived from AgNPs was heterogeneously distributed in the gut region and could be transported from the gills and gut of the daphnids. Correlation of Fluorescence Intensity and Ag + Concentration in the Gut of Daphnids. The proposed AIE method only qualitatively analyzed the Ag+ in daphnids, which has been regarded as one of the major drawbacks of fluorescence techniques. To provide detailed insights into the observed toxicity of AgNPs and distinguish the toxicity of AgNPs and Ag+, it was necessary to quantitatively determine the Ag+ in daphnids. Dissection followed by acid digestion was applied to quantify the concentration of Ag+ in the gut of daphnids.14 Specifically, after exposure to different concentrations of AgNO3 mixed with 30 μM TEZ-TPE-1, daphnids were collected and dissected using fine dissecting needles, and the guts were removed from the bodies. The concentration of Ag+ in the gut was measured by conventional ICP-MS after acid digestion. Fluorescence intensity of Ag+ measured by confocal laser scanning microscopy (LSM 710) was correlated with the concentration of Ag+ detected by ICP-MS, and the results are depicted in Figure S8. The fluorescence intensity− concentration was linearly correlated (R2 > 0.90, y = 1.4587x − 3.9384); thus it was possible to quantify Ag+ in daphnids based on the fluorescence intensity. The present study thus overcame

Figure 3. (1) Transmitted light, (2) green, and (3) merged fluorescence microscopy images of daphnid exposure in the SM7 medium containing TEZ-TPE-1 for 12 h. Daphnids exposed to TEZ-TPE-1 alone (A), AgNO3 (0.5 μg/L) (B), 20 nm AgNPs (500 μg/L) (C), and 60 nm AgNPs (500 μg/L) (D).

the localization of Ag+ in daphnids (Figure 4). The fluorescent signal was transformed into a pseudocolor heat map for the comparison of the fluorescence intensity in different parts of the daphnids, and the distribution pattern of Ag+ could be easily obtained. As shown in Figure 4, the fluorescence intensity varied at different layers. The most fluorescent part was in the gut, while the gills had a rather weak fluorescent

Figure 4. 3D data set collected with a laser scanning confocal microscope of Daphnia magna at different layers (A), 2D image of corresponding 3D data (B), transformed pseudocolor heat map image (C). 12216

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Figure 5. Dissolution of AgNPs (20 and 60 nm) in daphnids as detected by the proposed method in 120 h. White arrows denote the anterior gut of the daphnids.

dissolved much faster than the larger ones, probably as a result of decreased effective specific surface area of 60 nm AgNPs compared to 20 nm AgNPs. The in vitro and in vivo dissolution kinetics of different sized AgNPs were further compared. The dissolution rate of smaller sized AgNPs was much faster than the larger size of AgNPs both in the medium and in daphnids (Figure 6A and B). However, a greater difference in dissolution rate was observed between in vivo and in vitro dissolution, where the time for in vivo dissolution to reach equilibrium was much longer than the in vitro process. The percentage of Ag+ in daphnids was calculated based on the depuration of different sized AgNPs since the amount of AgNPs in daphnids was related to the elimination (some accumulated AgNPs could be excreted to the outside of the Daphnia). As shown in Figure 6C, a relatively rapid clearance of the 20 and 60 nm AgNPs from daphnids was observed, and smaller sized AgNPs were depurated much faster than the larger particles. The content of Ag dropped from 17246 μg/g to 1354 μg/g for 20 nm AgNPs (>90% decrease) over 120 h, whereas the concentration of 60 nm AgNPs decreased from 8528 μg/g to 1087 μg/g (an 85% decrease). As expected, the percentage of AgNPs dissolved in the SM7 medium was much higher than that in daphnids, especially within the first 72 h. Therefore, at dissolution equilibrium state, the concentration of released Ag+ reached 9.7% and 8.3% (wt % of NPs) for 20 nm AgNPs and 60 nm AgNPs, respectively. Compared with the AgNPs accumulated in the gills or aqueous solution, the AgNPs were highly aggregated and the active space was limited in the guts (SEM and TEM images in Figure S9); thus the dissolution was much slower in the gut regions than that in the aqueous solution. However, these aggregated AgNPs could be excreted outside the guts and stimulated the redissolution of AgNPs in

the drawbacks of most bioimaging techniques by allowing a quantitative detection of Ag+ in the organisms with great potential in quantitatively monitoring the dissolution kinetics of AgNPs in daphnids. Quantitative Monitoring of the Dissolution Kinetics of AgNPs in the Gut and in the Medium. We further quantified the time-dependent dissolution of AgNPs, and the detailed data processes are described in the Methods. Smaller sized AgNPs exert more toxic effects than the larger sizes of AgNPs, which could be attributed to the release of Ag+.48 Chelating ligands such as cysteine have strong affinity toward Ag+ and have been applied to control the Ag+ concentration in a AgNP suspension.14 Therefore, daphnids were first exposed to the solution containing AgNPs and cysteine, with all the Ag+ being complexed by cysteine, ensuring that almost all the Ag in daphnids was in the form of nanoparticles. The released Ag+ was mainly distributed in the gut, and the concentration of Ag+ was detected at different time points based on the correlation between the fluorescence intensity and the concentration detected by ICP-MS. All the fluorescent images were transformed into a pseudocolor heat map, and only the gut was retained for the comparison since it was the major accumulation site of AgNPs in daphnids. Figure 5 shows that the concentrations of Ag+ derived from 20 and 60 nm AgNPs increased with exposure time. After 8 h of exposure, Ag+ was mainly concentrated at the anterior gut and gradually diffused from the anterior gut to the hindgut. After 48 h, Ag+ was nearly homogeneously distributed throughout the gut. The dissolution process of different sized AgNPs reached equilibrium after 72 h, which was much slower than the dissolution in bulk solution (about 48 h). At dissolution equilibrium state, the concentration of Ag+ reached 120−140 and 75−95 μg/g for 20 and 60 nm AgNPs, respectively. Besides, smaller sized AgNPs 12217

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Figure 6. Dissolution kinetics of AgNPs (20 and 60 nm) in the gut parts of daphnids (A) and in SM7 medium (B). Retention of AgNPs in Daphnia magna during 120 h depuration after being exposed to 500 μg/L 20 and 60 nm AgNPs (C). Proportional dissolution of Ag+ released from 20 and 60 nm AgNPs in SM7 medium and in the gut of daphnids (D).

(Figure S10) showed that the presence of 20 μM BSA accelerated the dissolution of AgNPs, while the presence of 20 mM NaCl inhibited the dissolution of AgNPs. Besides, the total amount of Ag+ released from 20 nm AgNPs in the presence of BSA (85.4 μg/L, ∼17.1% of total amount of AgNPs) at the equilibrium state was greater than the 20 nm AgNPs in the presence of NaCl (28.6 μg/L, ∼5.72% of total amount of AgNPs) or the 20 nm AgNPs alone (52.0 μg/L, ∼10.4% of total amount of AgNPs). Such a difference may be explained by the sequestration of surface-sorbed Ag+ by thiol groups in BSA. In contrast, the presence of NaCl induced the formation of silver chloride (AgCl) on the NP surface, and the precipitate could form a layer on AgNPs surfaces and prevent further dissolution. The influences of pH conditions of different gut regions on AgNP dissolution were determined by the AIE method. Specifically, a pH-sensitive dye (tetraphenylethene−cyanine adduct, TPE−Cy) was utilized to determine the pH distribution in daphnids. TPE−Cy was capable of sensing pH over a broad range (1−13) and was previously applied for intracellular pH sensing of living HeLa cells.51,52 In an aquatic environment, TPE−Cy showed strong to medium red emissions at pH 5−7, weak to nil red emissions at pH 7−10,

the gut. Therefore, dissolution of AgNPs in the medium gradually reached equilibrium, while the percentage of Ag+ continuously increased, especially for the 20 nm AgNPs (Figure 6D). Mapping the Gut pH and Correlation with Dissolved Ag+. The distribution of Ag+ in the gut was highly heterogeneous, which could be attributed to the complex biological matrices in the gut microenvironment and unique physiological structure of gut regions. Gut microenvironment contained proteins and sodium chloride (NaCl), which could potentially influence the dissolution processes of AgNPs in the gut. Besides, the gut consists of three parts, an anterior part, middle part, and posterior part, and the pH condition in these three parts was different,49 which could potentially influence the dissolution process. To gain better insight into the dissolution mechanisms of AgNPs in the gut regions, especially in the presence of proteins and chloride, an in vitro study was conducted by investigating the dissolution kinetics of AgNPs in the presence of proteins or NaCl. Bovine serum albumin (BSA), a model protein, was employed to study the influence of proteins on the dissolution behaviors of AgNPs. NaCl was also chosen in our study since Cl− is one of the major parameters affecting the dissolution of AgNPs.8,50 Our results 12218

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Figure 7. (a) Left: PL spectra of TPE−Cy in aqueous solutions with different pH values. [TPE−Cy] = 10−5 M; excitation wavelength, 380 nm. Right: Emission spectra of TPE−Cy in the buffer solutions with different pH values in the presence of 1,2-dioleoyl-glycero-3phosphocholine (DOPC). [TPE−Cy] = 10−5 M; [DOPC] = 0.1 mg/mL. Inset: Plot of I489/I615 versus pH. I489 and I615 denote the emission intensities of the solution at 489 and 615 nm, respectively. Excitation wavelength, 380 nm. (b) Ratiometric fluorescent images of gut pH of daphnids exposed to TPE−Cy (10 μM) in SM7 medium under different pH conditions. Left: pH = 6.0; middle: pH = 6.8; right: pH = 7.8. Excitation wavelength, 405 nm. Emission ranges: 450−520 and 580−650 nm. (c) Left: Light microscope of gut dissected from Daphnia magna cultured in creek water; right: comparison of the concentration of Ag+ and pH distribution in different gut regions. 1: intestinal ceca, 2: anterior gut, 3: midgut, 4: posterior gut. Normalized Ag+ concentration by analysis of Ag+ concentration distribution from intestinal ceca to posterior gut. Normalized pH by analysis of pH distribution from intestinal ceca to posterior gut.

cerides (one type of phospholipid). The preparation of lipid and measurements of pH were based on a previous method.52 Then, a linear relationship between the emission intensity of TPE−Cy (fluorescence intensity ratio of two different channels, I489/I615) under different pH conditions in the

and nil to strong blue emissions at pH 10−14 (Figure 7a). To quantitatively estimate the pH in different regions of the gut, a model phosphatidylcholine, 1,2-dioleoyl-glycero-3-phosphocholine (DOPC), was used as a standard compound since major lipid classes of most aquatic organisms are phosphogly12219

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daphnids. The visualized Ag+ was heterogeneously distributed in daphnids during the dissolution processes (anterior part > middle part > posterior part), which was closely related to the pH distribution patterns in the gut. Furthermore, timedependent migration of Ag+ from the anterior to the posterior gut part was evident. The concentration of released Ag+ at the dissolution equilibrium was 9.7% and 8.3% (wt % of NPs) for 20 nm AgNPs and 60 nm AgNPs, respectively. Besides, the transportation of Ag+ across the gills was observed and may be a potential pathway leading to the toxicity of AgNPs. The present study provides strong evidence of the dissolution kinetics of AgNPs in daphnids.

presence of DOPC in the SM7 medium was established, enabling the pH sensing within the range of 4.5 to 8.0 (Figure 7a), which covers the pH range of the Daphnia gut. Based on the ratio of I489/I615, it was possible to estimate the pH distribution in different regions of the gut. Data acquired by LSM were processed by the imaging analysis MATLAB program, and data for each pixel were calculated by I489/I615 and then reconstructed based on the correlation between I489/ I615 and the pH value in the presence of DOPC (Figure 7a). Figure 7b shows a representative pH image of daphnids after the exposure of TPE−Cy under different pH conditions (left, pH = 6.0; middle, pH = 6.8; right, pH = 7.8). The pH followed the sequence of anterior part < midgut < posterior part (Figure 7b), similar to an earlier study.49 Specifically, the pH of the anterior gut, midgut, and posterior gut of Daphnia maintained at a pH of 6.8 followed the sequence of anterior part < midgut < posterior part and ranged from 4.75 to 5.75, 5.75 to 6.25, and 6.25 to 6.75, respectively (middle image of Figure 7b; corresponding confocal images are shown in Figure S11), which was consistent with the distribution of dissolved Ag+ (20 nm at 8 h, Figure 5). The dissolution behavior of AgNPs under different pH conditions was also evaluated (Figure S12). pH significantly influenced the dissolution rate and the total amount of released Ag+, while AgNPs at a lower pH (pH = 4.55) possessed a significantly higher dissolution rate than AgNPs at a higher pH (5.73−6.88). The total amount of Ag+ released at lower pH was also much higher. pH was therefore considered as an important parameter determining the dissolution of AgNPs.53 A comparison study between the distributions of Ag+ and pH in different regions of the gut was further conducted (Figure 7c). The concentration of Ag+ decreased from the anterior part to the posterior part, which corresponded inversely with the gut pH distribution (2, 3, and 4 in Figure 7c). The dissolution of AgNPs in the anterior gut was much faster than the midgut and posterior gut, resulting in a heterogeneous distribution of Ag+. It should be noted that the intestinal ceca (1 in Figure 7c) was close to the mouth and easily influenced by the environmental conditions with greater variation of pH in this region. The concentration of Ag+ in this region was also relatively higher, which might be attributed to the uptake of Ag+ from aqueous solution and the dissolved Ag+ at the site. Therefore, in vivo dissolution of AgNPs in the Daphnia gut microenvironment was influenced by complex biological matrices (such as proteins and NaCl) and unique physiological structure (pH distribution in different gut regions). Compared with AgNPs in the ambient solution, AgNPs in the gut of Daphnia always coexisted with a high concentration of proteins and low pH, both of which facilitated the dissolution. However, the total amount of AgNPs dissolved in the gut was less than that in the SM7 medium. These AgNPs in the gut were highly aggregated and dissolved much slower than those in the SM7 medium.

METHODS Daphnia Culture and Viability Test. Daphnia magna was cultured in our laboratory for about 20 years. Details of Daphnia culture were given in an earlier study.13 Based on the previous studies, the presence of certain chloride ions in the medium may cause the precipitation of Ag+ as AgCl, which may limit the imaging of Ag+ in live organisms. Therefore, a modified simplified M7 medium (SM7), containing 0.04 mM NaHCO3, 0.35 mM CaSO4, 0.50 mM MgSO4, and 0.05 mM KNO3 at pH 7.0−8.0, was used in the present study. Before the viability test, daphnids were acclimated in clean SM7 without food for 4 h. Then, 10 individuals were exposed to medium containing different concentrations of TZE-TPE-1 in a polyethylene beaker containing 100 mL of SM7. After 48 h, the dead daphnids in different treatments were recorded. Synthesis and Characterization of AgNPs. Different sizes of citrate-capped AgNPs were prepared according to a previous method.10 For the synthesis of 20 nm AgNPs, under constant stirring, 6.0 mL of freshly prepared 5 mM NaBH4 was added into a mixed solution containing AgNO3 (100 mM, 250 μL) and citrate (100 mM, 250 μL) under vigorous stirring. The resulting yellow solution was stirred for another 30 min. The AgNPs of 60 nm in diameter were prepared by injecting citrate (34 mM, 2 mL) to a boiling solution of AgNO3 (1 mM, 100 mL) under vigorous stirring. Following 3 h of additional stirring at room temperature, the soluble byproducts were removed by centrifugal ultrafiltration (Amicon Ultra15 3K, Millipore, MA) and DI water addition in two cycles, after which the AgNPs stock suspensions were stored at 4 °C for later use. AgNP synthesis was characterized by transmission electron microscopy (TEM, JEM 2010) equipped with an EDX attachment. Figure S2 shows the TEM of the self-prepared AgNPs with sizes of ∼17.3 and ∼54.9 nm. The total Ag concentrations of AgNP solutions and the contents of Ag+ after separation were measured by ICP-MS (NexION 300X, PerkinElmer, USA), which was conducted with Ag standards and after HNO3 digestion. The release kinetics of different sized AgNP concentrations in SM7 medium was determined by ultracentrifugation through a 3 kDa membrane (pore size around 1 nm, Millipore, USA). Specifically, the AgNP suspensions were centrifuged at 4000 rpm for 20 min. After that, the filtrate, which contained the soluble Ag (nanoparticles were trapped on the membrane), was sampled at different time points (0, 1, 2, 4, 8, 12, 16, 24, 30, 36, 48, 72, and 120 h). The Ag concentrations in the filtrate were measured by ICP-MS. The aggregation kinetics of different sized AgNPs in SM7 medium were measured using dynamic light scattering on a Malvern Zetasizer Nano-ZSat (UK). After about 48 h of exposure, the hydrodynamic diameter of 20 and 60 nm AgNPs could reach about 300 and 800 nm, respectively (Figure S3). Electron Microscopy Analysis of AgNPs in Daphnia magna. The gut was separated from Daphnia using a sharp scalpel. The AgNPs were collected and analyzed by TEM equipped with an EDX attachment. A small amount of AgNPs could also penetrate the daphnids, and the sample preparation was based on a previous study.54 To collect these AgNPs, the isolated guts were placed in 2% formaldehyde and 5% glutaraldehyde with 0.2 M potassium cacodylate buffer at pH 7.4 for 1 h. The specimens were washed

CONCLUSIONS In this study, we directly visualized the existence of Ag+ and monitored the dissolution kinetics of different sized AgNPs and the distribution pattern of Ag+ within the daphnids based on the AIE method. A good correlation between the concentration of Ag+ detected by ICP-MS and the fluorescence intensity measured by LSM was obtained, allowing quantitative monitoring of the time-dependent dissolution process of different sized AgNPs as well as the distribution of Ag+ in 12220

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ACS Nano twice in the cacodylate buffer and placed in 1% osmium tetroxide in 0.2 M phosphate buffer for 1 h. Tissue was dehydrated with an acetone series followed by infiltration and embedding with Eponate812 (Ted Pella, Inc., Redding, CA, USA). Gut tissue was segmented into regions and oriented with the anterior end at the top of the block prior to polymerization. A diamond knife was used to cut 60−80 nm thick ultrathin sections, which were placed on copper grids (Pelco International, Reddings, CA, USA) and imaged using TEM. Correlation of Ag Concentration and Fluorescence Intensity in Daphnids’ Gut. Ten individuals of daphnids were exposed in 100 mL of medium containing 30 μM TEZ-TPE-1 and 3.0 μg/L AgNO3 (the toxic effect of Ag+ was reduced due to its complexation with TEZ-TPE-1). At different time points the daphnids were collected. Their guts were removed from the bodies with forceps. Then, the guts were digested in 1 mL of 65% HNO3 at 80 °C for 12 h to analyze the Ag content in the guts. The fluorescence intensity of Ag+ in the gut was determined by a Zeiss laser scanning confocal microscope (channel: excitation: 405 nm, emission collected: 450− 550 nm). Finally, the correlation between the concentration and fluorescence intensity was obtained. Uptake of AgNPs and Distribution of AgNPs in Daphnia magna. Ten individuals of daphnids per treatment were exposed to 100 mL of medium containing 1 μM cysteine and 500 μg/L AgNPs. Cysteine was added into the AgNP suspensions to reduce the toxicity and uptake caused by soluble Ag released from the AgNPs since the thiol group in cysteine had high affinity for soluble Ag. At different time points, daphnids were collected, washed in uncontaminated SM7, filtered onto a polycarbonate membrane, and dried at 80 °C overnight. After weighing, the daphnids were digested in 1 mL of 65% HNO3 at 80 °C for 12 h. Based on the Ag concentrations in the digestion (determined by ICP-MS), the concentration of Ag accumulated in the daphnids during 48 h of exposure was calculated. To determine the distribution of AgNPs in the gut of D. magna, daphnids were collected at different time points, washed in uncontaminated SM7, and dissected under the microscope. Their guts were removed from the bodies with forceps and washed in the clean medium. Then, the guts were digested in 1 mL of 65% HNO3 at 80 °C for 12 h to analyze the Ag content in the guts. Quantitative Monitoring of Dissolution Kinetics of AgNPs in Daphnia. Ten individuals of daphnids were exposed in 100 mL of medium containing AgNPs and cysteine, and almost all the ingested Ag was in the form of AgNPs. When the AgNP uptake reached the steady state, daphnids were transferred to 10 mL of medium containing 30 μM TEZ-TPE-1. At different time points, the daphnids were collected, and the fluorescence intensity was acquired with a Zeiss laser scanning confocal microscope (model: LSM7 DUO, LSM 710). After the fluorescence images were captured by LSM (Figure S13A), fluorescence intensity of gut parts was directly measured by selecting the gut regions. Then a histogram that counted each pixel intensity in the selected area could be obtained. Afterward, these fluorescence images were transformed to 16-bit images by using ImageJ and then processed by MATLAB based on the correlation between fluorescence intensity and concentration of Ag+. Finally, images were transformed to a pseudocolor heat map and exported for comparison and quantitative analysis. To standardize the confocal imaging and imaging intensity between groups, a reference standard provided by Zeiss was employed to ensure that the fluorescence intensity of the whole optical field (measured by an image analyzer program) was almost the same (RSD within 10%) by adjusting the setting of the LSM. pH Mapping of the Gut. Ten individual daphnids per treatment were exposed in 100 mL of medium (pH = 6.0, 6.8, and 7.8) containing 10 μM TPE−Cy for 4 h, respectively. Before the bioimaging, daphnids were washed in uncontaminated SM7. For confocal images, channel 1: excitation: 405 nm, emission collected: 450−520 nm; channel 2: excitation: 405 nm, emission collected: 580−650 nm.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.8b06003. Figures S1−S13 (PDF)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Neng Yan: 0000-0003-4662-4348 Ben Zhong Tang: 0000-0002-0293-964X Wen-Xiong Wang: 0000-0001-9033-0158 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank the reviewers for their comments on this work. This study was supported by grants from the Basic Research Program of Shenzhen Science, Technology and Innovation Commission (JCYJ20170413173434280), the General Research Fund of Hong Kong Research Grants Council (16102918), the Natural Science Foundation of China (21577116), and the Collaborative Research Fund of Hong Kong Research Grants Council (C6009-17G). REFERENCES (1) Maynard, A. D.; Aitken, R. J.; Butz, T.; Colvin, V.; Donaldson, K.; Oberdörster, G.; Philbert, M. A.; Ryan, J.; Seaton, A.; Stone, V. Safe Handling of Nanotechnology. Nature 2006, 444, 267. (2) Morones, J. R.; Elechiguerra, J. L.; Camacho, A.; Holt, K.; Kouri, J. B.; Ramírez, J. T.; Yacaman, M. J. The Bactericidal Effect of Silver Nanoparticles. Nanotechnology 2005, 16, 2346. (3) Benn, T. M.; Westerhoff, P. Nanoparticle Silver Released into Water from Commercially Available Sock Fabrics. Environ. Sci. Technol. 2008, 42, 4133−4139. (4) Geranio, L.; Heuberger, M.; Nowack, B. The Behavior of Silver Nanotextiles during Washing. Environ. Sci. Technol. 2009, 43, 8113− 8118. (5) Navarro, E.; Piccapietra, F.; Wagner, B.; Marconi, F.; Kaegi, R.; Odzak, N.; Sigg, L.; Behra, R. Toxicity of Silver Nanoparticles to Chlamydomonas reinhardtii. Environ. Sci. Technol. 2008, 42, 8959− 8964. (6) Dos Santos, C. A.; Seckler, M. M.; Ingle, A. P.; Gupta, I.; Galdiero, S.; Galdiero, M.; Gade, A.; Rai, M. Silver Nanoparticles: Therapeutical Uses, Toxicity, and Safety Issues. J. Pharm. Sci. 2014, 103, 1931−1944. (7) Zhao, C. M.; Wang, W.-X. Comparison of Acute and Chronic Toxicity of Silver Nanoparticles and Silver Nitrate to Daphnia magna. Environ. Toxicol. Chem. 2011, 30, 885−892. (8) Levard, C.; Mitra, S.; Yang, T.; Jew, A. D.; Badireddy, A. R.; Lowry, G. V.; Brown, G. E. Effect of Chloride on the Dissolution Rate of Silver Nanoparticles and Toxicity to E. coli. Environ. Sci. Technol. 2013, 47, 5738−5745. (9) Adegboyega, N. F.; Sharma, V. K.; Siskova, K.; Zboril, R.; Sohn, M.; Schultz, B. J.; Banerjee, S. Interactions of Aqueous Ag+ with Fulvic Acids: Mechanisms of Silver Nanoparticle Formation and Investigation of Stability. Environ. Sci. Technol. 2013, 47, 757−764. (10) Liu, J. Y.; Hurt, R. H. Ion Release Kinetics and Particle Persistence in Aqueous Nano-Silver Colloids. Environ. Sci. Technol. 2010, 44, 2169−2175. (11) Ho, C. M.; Yau, S. K. W.; Lok, C. N.; So, M. H.; Che, C. M. Oxidative Dissolution of Silver Nanoparticles by Biologically Relevant 12221

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