In Vivo Bioimaging of Silver Nanoparticle Dissolution in the Gut

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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 ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b06003 • Publication Date (Web): 20 Nov 2018 Downloaded from http://pubs.acs.org on November 20, 2018

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In Vivo Bioimaging of Silver Nanoparticle Dissolution in the Gut Environment of Zooplankton Neng Yan1, Ben Zhong Tang2, Wen-Xiong Wang1,* 1Department

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 2Department

of Chemistry, Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, HKUST, Clear Water Bay, Kowloon, Hong Kong, China

*Correspondence author, [email protected]

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ABSTRACT Release of silver ions (Ag+) is often regarded as the major cause for silver nanoparticles (AgNPs) toxicity towards aquatic organisms. Nevertheless, differentiating AgNPs and Ag+ in complicated biological matrix and its dissolution remains a bottleneck in our understanding of AgNPs behavior in living organisms. Here, we directly visualized and quantified the timedependent 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 nm and 60 nm AgNPs. By applying a pH sensor, we further showed that the dissolution of AgNPs was partially related to the heterogenous distribution of pH in different gut sections of daphnids. Further, Ag+ was found to across the gills and enter the daphnids, which may be a potential pathway leading to AgNPs toxicity. Our findings provided fundamental knowledge about the transformation of AgNPs and distribution of Ag+ in daphnids.

KEYWORDS: AgNPs, Daphnia, guts, dissolution, AIE, bioimaging

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Silver nanoparticles (AgNPs) have been widely applied in our daily life because of their antibacterial properties,1, 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 was the releases 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, AgNPs 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 towards aquatic organisms. However, little is known regarding the ingested and internalized AgNPs and Ag+, as well as the tranformation, especially regarding the dissolution process. Despite the numerous studies on AgNPs toxicity in different species of aqutic 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 unique filter-feeding strategy, leading to an effective uptake of nanoparticles.13, 14 Earlier study found that the concentration of accumulated AgNPs could reach as high as 22.9 mg/g dry weight at the highest AgNPs 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 towards the 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 the 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,

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electron microscopy,20,

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synchrotron-radiation-based micro/nano X-ray fluorescence spectroscopy (μXRF/nano-XRF),22, 23 4


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isotope labeling techniques24-26 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 non-invasive detection appears to be the advanced methodology for in vivo probing the metallic ions.28-30 Owning to its excellent selectivity and sensitivity, low cost, and easy operation, fluorescence imaging has been widely applied for the in vivo detection and visulization 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 the dynamic events of interest.35 Contrary to the conventional fluorophore, a group of unique luminogens such as the aggregation-induced emission (AIE) which displays photostability and highly fluorescent when aggregated have been discovered for several years.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 sensing based on Ag-selective organic fluorophores, AIE luminogens (AIEgens) exhibit high quantum efficiency, good photostability and excellent biocompatibility, and could selectively detect the Ag+ at very low concentration (with a limit of detection at 2.3 nM).44 In a recent study,45 we demonstrated that the fluorogenic Ag ions sensor, tetrazole-functionalized tetraphenylethylene derivative 1 (TEZ-TPE-1) showed good selectivity and sensitivity towards Ag+, and could be applied for the real-time 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 represented the direct visualization of Ag ion and quantitative monitoring of the dissolution kinetics of AgNPs in any aquatic organisms.

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 firstly 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 5


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concentration of TEZ-TPE-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 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 the toxic effect.14 While most studies focused on the environmental behavior of AgNPs, knowledge regarding the behaviors in organisms is rather limited. To get an insight into the fate of AgNPs in daphnids, the bioaccumulation kinetics of manufactured AgNPs in daphnids was firstly 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 48h exposure to different sized AgNPs (20 nm and 60 nm) at a concentration of 500 μg/L, daphnids continuously accumulated AgNPs along with the time. Uptake rate of AgNPs decreased significantly with the increase of particles size. At the end of 48-h exposure, AgNPs in daphnids reached more than 15.4 mg/g and 9.41 mg/g for 20 nm and 60 nm, respectively. The AgNPs distribution showed that more than 60% of 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 guts and gradually eliminated. To gain a better insight into the toxic mechanisms of AgNPs towards Daphnia, typical distributions of AgNPs in the gut membrane and their retention in the guts were investigated. As shown in Figure 1b, AgNPs was detected in the gut cells, which was confirmed by the EDX analysis, implying that these accumulated AgNPs could penetrate the gut membrane and potentially induced a stronger cytotoxicity towards 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 futher study the behavior of AgNPs in the gut regions, these accumulated AgNPs (not entering the cells of Daphnia) were further characterzied 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 6


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complexes, which was gray matter surrounding AgNPs. EDX mapping technique also confirmed the coexistence of Ag with chloride and sulfide since these three different elements showed similar distribution patterns.

Figure 1. (a) Newly accumulated Ag (μg/g) in the daphnids during 48-h exposure to AgNPs (500 μg/L) in the SM7 medium. (b) TEM micrographs of 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 gut separated by sharp scalpel and the EDX mapping of selected elements (Ag, S and Cl). The scale bar is 0.5 μm. 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 containg 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

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previous study, the fluorogenic Ag+ sensor exhibited highly selectivity towards Ag+ rather than the AgNPs nor Ag0, and could selectively detect Ag+ even in the presence of other interfering substances (metallic ions and humic acid).45 Besides, binding affinity of Ag+ towards 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 interaction between Ag+ and SH-, Ag+ and Cl-. To explore the capability of the proposed AIE method for selectively sensing Ag+ in the 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 turn-on 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 of 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 was in the formation of Ag+ bound with SH- and Cl- complexes, and the detection was based on the strong binding affinity of TZE-TPE-1 towards Ag+. Micro-injection technique was used to explore the feasibility of the proposed AIE method for sensing the 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 daphnids. To test whether these ligands in guts could compete and snatch off Ag+ bound with TZE-TPE-1 complexes as well as the variation of fluorescence 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 digestive systems of daphnids after 2 h. Slight difference in the fluorescence intensity in 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 TZETPE-1 was highly stable in the gut. Therefore, TZE-TPE-1 could be utilized to selectively sense Ag+ even with the presence of SH- and Cl-. The 48-h 50% lethal concentrations (LC50) of AgNO3 and AgNPs towards 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 towards AgNO3 with a LC50 of 0.94 μg/L (Figure S4), which was much lower than the micro-injected concentration of AgNO3 (20 μg/L), implying that a direct exposure of Daphnia to AgNO3 could induce more severe toxic effect 8


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compared with micro-injection pathway. The addition of TZE-TPE-1 efficiently reduced the toxicity of both AgNO3 and AgNPs towards Daphnia. Therefore, the released of Ag+ could be one of major reasons contributing to the toxicity of AgNPs towards Daphnia.

Figure 2. (a) Chemical reaction of Ag+ with -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 with the presence of 10 µM and 50 µM of cysteine, NaCl and under different pH conditions. (c) Confocal images of daphnids firstly 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. Selective detection of Ag+ in daphnids. The capability of the proposed AIE method for the 9


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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 non-fluorescent 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 AIE phenomenon.36 Therefore, control experiments were also conducted by exposing daphnids to TEZ-TPE-1 and TEZ-TPE-1 mixed with some typical nanoparicles (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 3B, 3C and 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 TEZ-TPE-1 mixed with different nanoparicles (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 nor the ingested TEZ-TPE-1. The AIE method could also differentiate Ag+ in daphnids exposed to 20 nm and 60 nm AgNPs based on the difference in fluorescence intensity (Figure 3C and 3D). Smaller AgNPs owned much higher surface/volume ratio than the larger sized AgNPs, resulting in more atoms on the surface in contact with the oxidant.46 Correspondingly, much stronger fluorescence signal was detected for daphnids exposed to 20 nm AgNPs than to 60 nm AgNPs.

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Figure 3. (1) Transmitted light, (2) green, and (3) merged fluorescence microscopy images of daphnids 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). Verification of Ag+ distribution in daphnids by the 3D technique. Three-dimensional fluorescence imaging technique with high spatial resolution was used to determine the localization

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of Ag+ in daphnids (Figure 4). The fluorescent signal was transformed into a pseudo colors heat map for the comparison of the fluorescence intensity in different parts of 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 gills had rather weak fluorescent 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 localised 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 AgNPs bioaccumulation in daphnids.14, 23

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 pseudo colors heat map image (C).

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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+ in daphnids could be transported through epithelial cells and interfered with the ion regulation of whole body. Therefore, the time-dependent transportation of Ag+ derived from the AgNPs accumulation was investigated. Clearly, the accumulated AgNO3 was solely detected within the guts and homogeneously distributed after exposure for 8 h (Figure S7-C), whereas Ag+ derived from 20 nm AgNPs was heterogeneously distributed in the guts after 8 h exposure (Figure S7-A). After 48 h, Ag+ was found at both sides of guts as highlighted by the arrows in Figure S7-D, which might be derived from Ag+ accumulated in the guts 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 gut and gills after 48-h exposure (Figure S7-B). The concentration of dissolved Ag+ in the guts 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+ was highly concentrated in certain sites of guts after 8-h exposure and distributed much more uniformly after 48-h than that at 8-h, suggesting the transportation of Ag+ from anterior gut to hindgut. Some Ag+ was found at sites between 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 AgNPs uptake, and these absorbed AgNPs undergone dissolution to Ag+. Therefore, we concluded that accumulated Ag+ was homogeneously distributed in gut regions and could be transported from gills, surface body and gut regions to daphnids, while the dissolved Ag+ derived from AgNPs was heterogeneously distributed in gut regions and could be transported from gills and guts to daphnids. Correlation of fluorescence intensity and Ag+ concentration in guts 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 were applied to quantify the concentration of Ag+ in the guts 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 13


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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 the drawbacks of most bioimaging 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 the dissoultion kinetics of AgNPs in the guts and in the medium. We further quantified the time-dependent dissolution of AgNPs and the detailed data processes are described in 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 has strong affinity towards Ag+, and has been applied to control the Ag+ concentration in AgNPs suspension.14 Therefore, daphnids were firstly exposed to the solution containing the 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 guts 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 pseudo colors heat map and only 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 nm 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 hind-gut. 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 μg/g and 75-95 μg/g for 20 nm and 60 nm AgNPs, respectively. Besides, smaller sized AgNPs 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.

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Figure 5. Dissolution of AgNPs (20 nm and 60 nm) in daphnids as detected by the proposed method in 120 h. White arrows in figures denote the anterior gut of daphnids. 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 (Figures 6A and 6B). However, 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 Daphnia). As shown in Figure 6C, a relatively rapid clearance of the 20 nm and 60 nm AgNPs from daphnids was observed, and smaller sized AgNPs were depurated much faster than the larger 15


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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 (a 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 gut regions than that in the aqueous solution. However, these aggregated AgNPs could be excreted outside the guts and stimulated the re-dissolution of AgNPs in 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).

Figure 6. Dissolution kinetics of AgNPs (20 nm and 60 nm) in gut parts of daphnids (A) and in

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SM7 medium (B). Retention of AgNPs in Daphnia magna during 120 h depuration after being exposed to 500 µg/L 20 nm and 60 nm AgNPs (C). Proportional dissolution of Ag+ released from 20 nm and 60 nm AgNPs in SM7 medium and in guts of daphnids (D). 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 micro-environment and unique physiological structure of gut regions. Gut micro-environment contained proteins and sodium chloride (NaCl), which could potentially influence the dissolution processes of AgNPs in guts. Besides, the guts consisted of three parts: 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 a better insight into the dissolution mechanisms of AgNPs in gut regions, especially with the presence of proteins and chloride, an in vitro study was conducted by investigating the dissolution kinetics of AgNPs with the presence of proteins or NaCl. 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 (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 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 AgNPs dissolution were determined by the AIE method. Specifically, a pH sensitive dye (tetraphenyl-ethene−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 aquatic environment, TPE-Cy showed strong to medium red emissions at pH 5−7, weak to nil red emissions at pH 7−10, and nil to strong blue emissions at pH 10−14 (Figure 7a). To quantitatively estimate the pH in different regions of gut, a model 17


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phosphatidylcholine, 1,2-dioleoyl-glycero-3-phosphocholine (DOPC), was used as a standard compound since major lipid classes of most aquatic organisms were phosphoglycerides (one type of phospholipid). The preparation of lipid and measurements of pH were based on a previous method.

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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 presence of DOPC in the SM7 medium was established, enabling the pH sense within the range of 4.5 to 8.0 (Figure 7a), which covered the pH range of Daphnia guts. Based on the ratio of the I489/I615, it was possible to estimate the pH distribution in different regions of gut. Data acquired by LSM were processed by the imaging analysis MATLAB program, and data for each pixel was calculated by I489/I615 and then reconstructed based on the correlation between I489/I615 and 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

In Vivo Bioimaging of Silver Nanoparticle Dissolution in the Gut

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