Macrophage-Assisted Dissolution of Gold Nanoparticles - ACS

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Macrophage-assisted dissolution of gold nanoparticles Ulrika Carlander, Klara Midander, Yolanda Susanne Hedberg, Gunnar Johanson, Matteo Bottai, and Hanna L Karlsson ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00537 • Publication Date (Web): 24 Jan 2019 Downloaded from http://pubs.acs.org on January 26, 2019

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ACS Applied Bio Materials

Macrophage-assisted dissolution of gold nanoparticles Ulrika Carlander†, Klara Midander†, Yolanda S. Hedberg†, ‡, Gunnar Johanson†, Matteo Bottai§ and Hanna L. Karlsson∥* †Unit of Work Environment Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-17177 Stockholm, Sweden ‡KTH Royal Institute of Technology, School of Engineering Sciences in Chemistry, Biotechnology and Health, Dept. Chemistry, Div. Surface and Corrosion Science, Drottning Kristinas väg 51, SE-10044 Stockholm, Sweden §Unit of Biostatistics, Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-17177 Stockholm, Sweden ∥Unit of Biochemical Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-17177 Stockholm, Sweden. *Corresponding author: Hanna L. Karlsson: [email protected]

KEYWORDS: Dissolution, metal release, bio-solubility, macrophages, inflammation

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ABSTRACT Gold nanoparticles (AuNPs) are readily functionalized and considered biocompatible making them useful in a wide range of applications. Upon human exposure, AuNPs will to a high extent reside in macrophages, cells that are designed to digest foreign materials. To better understand the fate of AuNPs in the human body, their possible dissolution needs to be explored. In this study, we tested the hypothesis that macrophages, and especially stimulated macrophages, can impact the dissolution of AuNPs in a size dependent manner. We developed an in vitro method to compare the dissolution of citrate coated 5 and 50 nm-sized AuNPs, in terms of released gold ions as measured by inductive coupled mass spectrometry (ICP-MS), in i) cell medium (alone) ii) in medium with macrophages present, and iii) in medium with lipopolysaccharide (LPS) triggered macrophages (simulating inflammatory conditions). We found an evident, time-dependent dissolution of AuNPs in cell medium, corresponding to 3% and 0.6% of the added amounts of 5 and 50 nm AuNPs, respectively, after 1 week (168 h) of incubation. The dissolution of 5 nm AuNPs was further increased to 4% in the presence of macrophages and, most strikingly, 14% was dissolved in case of LPStriggering. In contrast, only a minor increase was observed for 50 nm AuNPs after 1 week in the presence of LPS-triggered macrophages compared to medium alone. Dissolution experiments in the absence of cells highlighted the importance of biomolecules. Our findings thus show dissolution of citrate coated AuNPs that is dependent on size, presence of macrophages and their inflammatory state. These findings have implications for understanding the transformation/dissolution and fate of AuNPs.


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INTRODUCTION Gold nanoparticles (AuNPs) are extensively investigated due to their potential applications in diagnosis and therapy. Biomedical applications include targeting tumours,1-3 imaging and photothermal therapies,4-6 use in anti-angionetic7 and anti-inflammatory treatments8-9 and for molecular sensing.6, 10-12 In general, AuNPs are regarded as non-toxic, although some questions regarding deleterious effects remain, particularly for the ultra-small AuNPs.10, 13 By using radioactively labeled AuNPs and various exposure routes, the biodistribution up to 24 h has been shown to be dependent on size; ultra-small AuNPs were retained in the blood circulation for a longer time and showed a more wide spread organ distribution compared with larger AuNPs.13 Recently, the accumulation and secretion of intravenously injected AuNPs were also reported to be greatly influenced by the shape of the AuNPs, when investigated up to 120 h after injection by using inductive coupled mass spectrometry (ICPMS).14 To understand NP fate, we and others have developed physiologically based pharmacokinetic (PBPK) models that can help predict the absorption, distribution, metabolism and excretion (ADME) of NPs.15-19 During the development of such PBPK models we observed distribution patterns that led us to hypothesize that even NPs considered to be inert might dissolve or transform to some extent in vivo.15 For example, following intravenous injection of titanium dioxide (TiO2) NPs in rats, an initial enrichment in certain organs was observed, where after the titanium concentration decreased slowly without any noticeable excretion in urine and feces.20 Moreover, the dissolution of NPs is regarded as a key element needed to understand NP fate in the body and to enable risk assessment.21-22 Despite this, the information on dissolution kinetics in relevant biological systems remains limited for most NPs.23 Dissolution of NPs has to some extent been tested in various model solutions, such as artificial sweat, lung lining and gastric fluids, artificial lysosomal fluid and also cell culture



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medium, at static or dynamic conditions.24-27 28-29 In such test systems, dissolution has been shown to depend on chemical and physical properties of the particles but also on the model solution including its ionic strength, pH, and temperature.23, 30-31 Except for material properties defined by elemental composition, smaller NPs are thermodynamically more soluble. The small size leads to greater surface to bulk atom ratios and changes in lattice parameters and surface energies that affect dissolution equilibrium constants.32 Furthermore, the importance of ligands was recently demonstrated in a study on gold biodissolution in a complicated system (freshwater wetland mesocosm), in which the nanoparticles were shown to be oxidized and complexed to cyanide, hydroxyls and thiol ligands.33 Another complicated system is the human body. Upon entering the human body, macrophages efficiently recognize and capture NPs in the lung and in circulation.34-35 The macrophages annihilate foreign substances by producing hydroxyl radicals at enhanced levels when cells are triggered causing a condition of oxidative stress.36-37 The NP-macrophage interaction is likely to affect the dissolution of NPs, yet, dissolution in contact with macrophages is rarely quantified. However, previous studies have shown that macrophages can impact the dissolution of micrometer-sized manganese dioxide particles.38 Furthermore, macrophages seem able to "digest" carbon nanotubes using a superoxide/peroxynitrite oxidative pathway39 and the presence of proteolytic enzymes in Kupffer cells can degrade a polymer shell of NPs.40 Regarding gold, macrophages have been demonstrated to facilitate gold release in the interface between a solid gold plate and the cells, leading to macrophage uptake of gold.41 Still, dissolution of AuNPs in different sizes in contact with macrophages with different activities has not yet been explored. Taken together, it appears important to further explore the role of macrophages in the dissolution process of NPs. This study aims to explore the hypothesis that macrophages as well as their inflammatory state affect the dissolution of NPs generally considered as “inert”. The specific aims were


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threefold; (1) to develop an in vitro method for monitoring the kinetics of AuNP dissolution in extra- and intracellular compartments, (2) to compare the dissolution of AuNPs in i) cell medium (alone) ii) in medium with macrophages present, and iii) in medium with lipopolysaccharide (LPS) triggered macrophages and (3) to investigate the size-dependence of dissolution of AuNPs.

RESULTS AND DISCUSSION

5 nm-sized Au NPs agglomerate in cell medium A specific aim of this study was to test size-dependent dissolution processes, hence we decided to use AuNPs with a primary size of 5 nm and 50 nm (citrate-coated). The sizes were consistent with the information from the manufacturer (NanoComposix) as determined using Transmission Electron Microscopy (TEM) (Figure 1a). AuNPs 5 nm agglomerated heavily in the cell medium (Figure 1a lower) which was also confirmed when agglomeration in the cell medium was tested with Nanoparticle Tracking Analysis (NTA). A main peak of around 65 nm was found for the 50 nm AuNPs, probably due to adsorption of proteins, whereas the 5 nm AuNPs were highly agglomerated, exhibiting sizes between 80 and 200 nm (Figure 1b). It is unclear whether all 5 nm particles were agglomerated or whether the non-agglomerated ones escaped detection by NTA. These results were reproducible with no noticeable difference between triplicates of the same sample, between replicate measurements, or between measurements after 0 and after 24 h incubation at 37° C (supplement Figure S1). Agglomeration of 5 nm AuNPs was also indicated by photon-cross-correlation-spectroscopy (PCCS) analyses (data not shown), although in this case the data was less reproducible, due to a greater influence of a few larger agglomerates.42



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Figure 1. Initial characterization of the gold nanoparticles in terms of size. a) The primary size of the gold nanoparticles (AuNPs) was confirmed using TEM imaging (upper). The 5 nm AuNPs agglomerated in cell medium (lower). b) The distribution of the mean hydrodynamic size of 5 and 50 nm AuNPs in diluted cell medium as determined by nanoparticle tracking analysis.

Development of an in vitro assay for dissolution One important aim of this study was to develop an assay that allowed for a direct comparison of NP dissolution in i) cell medium only ii) in medium with macrophages present, and iii) in medium with LPS triggered macrophages (simulating inflammatory conditions). We decided to base our method on RAW 264.7 murine macrophages and to include a time-point beyond 24 h since we expected only minor dissolution from the AuNPs. The separation of small AuNPs (5 nm) and dissolved gold ions/complexes constituted a crucial experimental challenge because released gold ions bound to proteins and cell constituents may be difficult to separate from small AuNPs. Furthermore, the separation method employed should not deteriorate the level of detection at chemical analysis of dissolved (released) gold.


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To investigate these aspects, we performed control experiments using cell medium with cell lysate prepared by mechanical shearing to avoid detergents that may affect the analysis. First, we tested the possibility to separate AuNPs added to the medium with cell lysate by using centrifugation with and without subsequent ultrafiltration. Hence, 5 nm AuNPs was added to the medium with cell lysate followed by centrifugation at 13.000 rpm (30 min), whereafter gold in the supernatant was analyzed. The results showed that the separation efficiency (assuming that no ionic gold was present) was high, although reaching 100% only after subsequent ultrafiltration using a 10 kDa filter (Figure 2a). After centrifugation in cell medium (alone), the separation efficiency was lower (Figure 2a). Furthermore, 5 nm AuNPs suspended in water was not possible to spin down (data not shown). We suggest that the proteins and cell debris appear to act as flocculants that enable separation of (small) NPs by centrifugation. Second, we explored whether dissolved gold would be possible to detect after using ultrafiltration for separation, in control experiments in which we added gold ions (in the form of HAuCl4) to the medium with cell lysate and analyzed gold in the supernatant after centrifugation (13.000 rpm). We found that approximately 50% of the added gold ions were detected in the supernatant. Following subsequent ultrafiltration, however, no gold ions could be detected in the filtrate (Figure 2b). This suggests that centrifugation may be employed, although released gold will be underestimated, whereas the 10 kDa filter is unusable.



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Figure 2. Development and verification of in vitro assay for analyzing dissolution of gold nanopartices. a) Separation efficiency when using centrifugation with or without subsequent ultrafiltration to separate 5 nm AuNPs added to cell medium with cell lysate, or to cell medium alone. Gold was analyzed in the supernatant following centrifugation at 13.000 rpm, alternatively in the filtrate of the supernatant after subsequent filtration using a 10 kDa filter. b) Gold ions detected after centrifugation at 13.000 rpm of medium with cell lysate spiked with gold ions (in the form of HAuCl4), with or without subsequent ultrafiltration. c) Gold ions (% of added) detected in the supernatant (centrifugation at 13.000 rpm) after exposing macrophages to gold ions (in the form of HAuCl4), followed by mechanical cell lysis and centrifugation.

Furthermore, since gold ions released from the NPs theoretically may be reduced back to the elemental form, or bind to cell debris with time, we made control experiments using ionic gold following incubation with macrophages at different time points. Thus, 0.25 µg/mL gold in the form of HAuCl4 (i.e. a concentration that corresponds to dissolution of 5% of the 8 ACS Paragon Plus Environment

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AuNPs) was added to macrophages with or without triggering with LPS, and (after different time points) approximately 70 % of the gold added could be detected by our centrifugation procedure (Figure 2c). This percentage was somewhat lower when higher concentrations of HAuCl4 (1 and 5 µg in 1 ml), were tested (supplement Table S2). Thus, we conclude that most of the ions released from the AuNPs will be detected using the centrifugation procedure. Adding cell lysate (from unexposed cells) before centrifugation of AuNPs incubated with cell medium (alone) will enable good separation and equal treatment of the samples with and without macrophages. This simple, yet novel, procedure for separation of small AuNPs from dissolved gold avoids flaws due to varying efficiency of centrifugation and ensures equal treatment of samples. In short, our experimental procedure is based on incubation of NPs with cell medium alone or with macrophages (RAW 264.7) in the presence or absence of LPS (0.1 µg/ml), for the timeperiods 0, 24 or 168 h (one week), see Figure 3.

Figure 3. Schematic overview of the experimental procedure. The scheme describes the different steps in the method used for detecting gold dissolution in the macrophage cell system. Au NPs (5 and 50 nm) are inclubated in cell medium (alone) or with unstimulated or LPS-triggered macrophages (simulating inflammation) for 0, 24 or 168 h (exposure). Cells are then lysed and followed by centrifugation (13.000 rpm, 30 min) of the the medium/lysate/AuNPs mix. The upper part of the supernatant is then collected, treated with aqua regia and gold content analyzed using ICP-MS.



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AuNPs are taken up by macrophages and cause limited effects on viability To explore AuNPs uptake, intracellular localization and characterization of the AuNPs intracellulary, TEM imaging was performed on macrophages exposed for 24 h to a AuNP concentration of 5 µg/ml. The images show that the AuNPs were taken up and were localized mainly within membrane-bound structures. The 5 nm-sized AuNPs were highly agglomerated and the endosomes typically contained hundreds of agglomerated AuNPs (Figure 4). In contrast, the 50 nm AuNPs were not agglomerated, and the endosomes contained only a few or tens of these NPs. The agglomeration states were thus similar to those found in cell medium (Figure 1). No signs of toxicity were observed for the AuNPs after 24 h, as also confirmed using viability test (supplement figure S2), but the LPS exposure caused decreased proliferation and a change in the morphology of the RAW 264.7 macrophages in that more vacuoles were observed (supplement figure S3). A small decrease in viability was, however, noted after 1 week exposure to the AuNPs, possibly due to decreased proliferation. No clear effect on viability due to the AuNPs was, however, observed in the presence of LPS (supplement figure S2). Taken together, TEM imaging and viability analysis showed, for both 5 and 50 nm-sized AuNPs, cellular uptake into membrane-bound structures and decreased viability after one week exposure.


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Figure 4. TEM imaging of gold nanoparticles in macrophages. TEM images were taken following 24 h exposure of RAW 264.7 macrophages (with or without LPS triggering) to 5 µg/ml gold nanoparticles (AuNPs). Both NPs were mainly observed in endosome-like structures and the 5 nmsized AuNPs were highly agglomerated. The bars indicate 200 nm.

LPS-triggered macrophages produce reactive oxygen species Due to the fact that LPS was used to trigger macrophages, we tested whether such stimulation leads to enhanced production of reactive oxygen species (ROS) in the cells since we reasoned that ROS indeed may have an impact of dissolution. Thus, we exposed macrophages to LPS for 24 h and measured the ROS formation by using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). There was no increased ROS production following exposure to AuNPs in the absence of LPS (data not shown). However, when each exposure (and the control) was 11 ACS Paragon Plus Environment


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compared to the same conditions with LPS a clear increase in intracellular ROS was observed (Figure 5). These results suggest that the LPS exposure causes increase ROS production and this may affect dissolution. Figure 5. Reactive oxygen species (ROS) induction by LPS. ROS increase was calculated as times increase of fluorescence in wells with LPS vs the same exposure without LPS. Results are presented as the mean +/- SD of three independent experiments.

Triggered macrophages can release gold ions from 5 nm AuNPs Next, we investigated the influence of particle size, time of incubation and exposure condition on dissolution of 5 and 50 nm citrate-coated AuNPs using median linear regression modeling with correlations between all parameters (for details, see supplement Table S4). By using quantile regression, all the available data could be used, including those below the limit of detection, without requiring imputing their values. First and foremost, gold ions were released from both AuNPs over time in cell medium alone, although significantly more (three- to sixfold) in the case of 5 nm AuNPs compared to 50 nm particles (Figure 6a). Thus, after one week of exposure, 3% and 0.6% of the 5 nm and 50 nm AuNPs, respectively, had been dissolved. The dissolution of the 5 nm AuNPs increased (from 3% to 4%) in the presence of macrophages and, most strikingly, to 14% after triggering these cells with LPS (Figure 6b). In contrast, non-triggered macrophages exerted no marked effect on the dissolution of 50 nm AuNPs. A small but significant (p = 0.023) increase from 0.6% to 0.8% dissolution was, however, observed in the in the presence of triggered macrophages when compared to cell medium (alone) after 168 h (Figure 6c and supplement Table S4). Control experiments 12 ACS Paragon Plus Environment

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showed that LPS in cell medium alone had no effect on dissolution (see supplement Table S2). Furthermore, a successful separation of AuNPs and dissolved gold was confirmed by low and similar amounts of dissolved gold between the test conditions at 0 h (Figure 6, supplement Table S2), lower than at all other time-points for the AuNPs of both sizes. Taken together, these results demonstrate an evident and size-dependent dissolution of AuNPs particularly under simulated inflammatory conditions.

Figure 6. Dissolution of 5 and 50 nm gold nanoparticles. The gold nanoparticles (AuNPs) were incubated for 0, 24 or 168 h in cell medium alone or with unstimulated or lipopolysaccharide (LPS)triggered macrophages. a) Tukey boxplot of the amount of gold released following incubation of 5 μg of AuNPs. Model predictions for b) 5 nm and c) 50 nm AuNPs, with error bars depicting the 95% confidence intervals (for additional information, see the supplementary material).



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The release of gold ions from AuNPs demonstrated in these experiments cannot be explained by classical models of thermodynamic equilibrium based on chemical composition of bulk materials in simple solutions.43 Several alternative explanations are suggested by the fact that gold ions bind to ligands such as thiol groups and phosphorus compounds, as well as to halides, oxygen and nitrogen donors.33, 43-44 These and many other groups/compounds are present in both the cell medium and cells and could probably promote gold dissolution. LPS acts via toll-like receptors to transform macrophages into the M1 phenotype characterized by extensive antigen presentation, and elevated production of various cytokines (IFN-γ, IL-12, IL-23, TNF, IL-6, IL-1), nitric oxide and reactive oxygen species.45 Hydroxyl radicals with a high redox potential possibly produced by LPS triggered macrophages can theoretically enable electrochemical dissolution of gold.43 Danscher and colleagues (2007) suggested that ions liberated from a gold surface incubated with macrophages are likely to be present in the form of aurocyanide (Au(CN)2–), generated from thiocyanate and hypochlorite during the oxidative burst by macrophages41.

Biomolecules are important for gold dissolution Next we used a simplified system to test the hypothesis that a combination of ROS and biomolecules is important for the gold dissolution. The biomolecules acting as complexing agents could, with the help of a high oxidative potential, weaken the stability of gold at the outermost AuNP surface, bind gold and keep it in solution thereby preventing its reduction or precipitation on the gold surface. To test this hypothesis for the dissolution of 5-nm sized AuNPs, we used a simple test system to produce hydroxyl radicals using iron ions (Fe2+) and hydrogen peroxide (H2O2) with and without biomolecules. Glutathione (0.2 mM) was used as a single biomolecule and was selected since it contains a thiol group, and cell medium, containing a mix of 15 amino acids (0.2-4 mM) was used as a biomolecule mix of relevance


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for this study. Thus, AuNPs were incubated together with Fe2+ and H2O2 in (1) water, (2) in water with glutathione, or (3) in cell medium (without serum) for 24 h or 1 week (168 h). The results showed no evident release of gold in the test system with only hydroxyl radicals in water or in the presence of glutathione. In contrast, a clear dissolution was observed in the cell medium (Figure 7). These results support the need for a combination of ROS and biomolecules, although the role of the different amino acids or other components present in the cell medium needs to be further studied.

Figure 7. Dissolution of 5 nm AuNPs in a simplified test system. 5 nm AuNPs were incubated together with iron ions and hydrogen peroxide (to produce hydroxyl radicals) in water, in water with glutathione (GSH), or in cell medium (without serum) for 24 h or 1 week (168 h).

The exact mechanisms underlying enhanced AuNP dissolution by LPS-triggered macrophages, as well as the speciation and fate of the gold ions dissolved remain to be elucidated.

Differences in cellular dose do not explain the size-dependent dissolution Since dissolution occurs at the surface, more pronounced dissolution in terms of wt% of the 5 nm AuNPs in cell medium is presumably related to their larger surface area to volume ratio.46 Our observation of four- to six-fold higher dissolution of 5 nm compared to 50 nm AuNPs particles in cell medium (alone) is less than the ten-fold difference in surface area, assuming non-agglomerated and naked NPs (5 µg AuNPs corresponds to a total surface area of 3 cm2 15 ACS Paragon Plus Environment


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for 5 nm and 0.3 cm2 for 50 nm AuNPs). The less than ten-fold difference in dissolution probably reflects the high degree of agglomeration of the 5 nm AuNPs. Furthermore, their coating/interaction with organic compounds can affect the dissolution in various ways Our finding that triggering macrophages with LPS mainly enhanced dissolution of the smaller 5 nm AuNP may be due to more pronounced cellular dose (NPs taken up by or attached to the cells) of these NPs after such triggering. This dose can be calculated using dosimetric models,47-49 but can also be measured experimentally. We analyzed the cellular dose using ICP-MS and found this to be approximately 60% and 80% of the amount added for the 5 nm and the 50 nm particles, respectively, and not evidently affected by the LPS treatment (Figure 8). Thus, despite the fact that the 5 nm AuNPs were highly agglomerated, the cell dose was higher for the 50 nm particles. This can probably be explained by the lower effective density of the small AuNP-protein agglomerates (hence lower sedimentation and cellular dose).49 It should be noted that we measured cell dose, which mainly is a measure of sedimentation onto the cells, and the uptake may still differ. A recent study showed, for example, a clear decrease in AuNPs uptake at 4 h in monocyte-derived macrophages at an inflammatory state (M1) when compared to regulatory (M2) phenotype50. Even though agglomerated, the small AuNPs appeared to retain the unique properties related to small size, such as high surface area-tovolume ratio and enhanced surface activity. This is most likely related to the fact that the NPs are loosely agglomerated and thus, there are more available surface atoms per mass, and a higher curvature (more corner atoms), as compared to the 50 nm AuNPs. The largely increasing surface to bulk atom ratio for the 5 nm as compared to the 50 nm AuNPs changes for instance lattice parameters, surface energies, thermodynamic parameters, potentials, and dissolution equilibrium constants explaining the higher dissolution. 32, 51-52


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Figure 8. Cellular dose of gold nanoparticles depending on size and LPS triggering. The bars (n=3) show the cellular dose of gold nanoparticles (AuNPs), i.e. AuNPs in the cells or attached to the cell surface, after 24 h exposure to 5 μg of 5 and 50 nm AuNPs. The cell dose was significantly higher for the 50 nm compared to 5 nm AuNPs (n=6, Mann–Whitney test).

Speciation modeling does not explain gold dissolution In aqueous solution, gold exists in an equilibrium between the metal (Au(0)) and the oxidized states (Au(I) and Au(III)). The Joint Expert Speciation System (JESS) predicts that the proportion of gold ions in the cell medium rises with increasing pH (pH 4.5-7 was tested) and redox potential (300-430 mV) (Figure 9). With a gold ion concentration of 0.25 µg/ml, pH of 7.4 and Eh of 380 mV, (i.e. our experimental parameters), approximately 8% of the gold added is predicted to be in solution. This value is in sharp contrast to the approximately 70% that we actually measured one week after addition of gold ions (Figures 2 and 9). Thus, the factors taken into account in the JESS modeling (pH, temperature, chloride concentration and redox potential) cannot explain our finding that a relatively large part of the added gold ions could be detected in cell medium. Moreover, the JESS prediction appears to contradict the conclusion by Sabella and colleagues that the acidic lysosomal pH is responsible for dissolving gold.30 Both our and other studies have demonstrated more pronounced dissolution of gold at neutral or towards basic pH, and the presence of biomolecules is likely an even more important aspect.53-54



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Figure 9. Theoretical gold in solution. The theoretical amount of gold in solution over a range of redox potentials and pHs calculated with the Joint Expert Speciation Software (JESS). The solid horizontal line represents the total amount of gold ions added to the test system (either JESS or experiment using ions, i.e., 0.25 μg in 1 ml cell medium). The amount of gold measured in solution in the experiments was considerably higher compared to that predicted by the model that does not consider gold interactions with biomolecules due to lack of input data.

Implications, strengths and weaknesses The implications of the released gold ions require further attention. Gold ions can contribute to toxicity, and/or exert an anti-inflammatory/immunosuppressive effect.30, 55 Indeed, gold salts have been successfully used to treat rheumatoid arthritis since the early twentieth century.56 However, this treatment is associated with side effects, such as nephrotoxicity,57 and a gradual release of gold ions from NPs to inflamed tissues, as suggested possible by our study, may prove clinically useful in the future. The fate of gold ions in humans remains unclear and is likely to depend on exposure route58. It is also evident that the biodistribution of gold ions is different from that of the AuNP, as observed e.g. following administration of gold ions and NPs to rats via the lungs.59 Furthermore, studies on patients treated with gold 18 ACS Paragon Plus Environment

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salts suggest high affinity for organs of the reticuloendothelial system, especially the lymph nodes, liver and bone marrow.60-61 The exact form of gold in these organs remains unknown, but elevated levels can be detected in tissue samples from patients treated with gold years earlier.61 A main challenge in the study was the separation of the dissolved gold from the remaining AuNPs in the complicated system used. Whereas a 100% efficient separation only was achieved when using 10kDa filters, these filters were not possible to use due to the fact that the dissolved gold binds to proteins and, apparently, results in complexes that cannot pass the filter. This was, however, thoroughly evaluated and the use of a relevant, novel, and robust system for exploring gold dissolution from NPs was elaborated. The results indicated that dissolution was mediated via ROS and biomolecules, although the exact mechanisms need to be further explored. In a broader perspective, our study highlights the importance of developing more physiologically relevant dissolution models to bridge the gap between in vitro and in vivo conditions and for improving modelling of biological systems. CONCLUSIONS This study aimed to explore the hypothesis that macrophages as well as their inflammatory state affect the dissolution of AuNPs generally considered as “inert”. A novel in vitro method was developed to monitor the kinetics of AuNPs dissolution, with which dissolution from AuNPs was rather underestimated than overestimated. We have demonstrated that AuNPs in sizes 5 and 50 nm can dissolve in cell medium and in the presence of macrophages. The inflammatory state of the macrophages strongly impacted the dissolution of the 5 nm AuNPs. The dissolution of the 5 nm AuNPs was as high as 14% after one week in the presence of LPS-triggered macrophages, compared to 4% without LPS. The effect of LPS-triggered macrophages was smaller for 50 nm AuNPs. These findings highlight the increased dissolution rate of ultra small nano-sized AuNPs, the role of biomolecules in their dissolution,



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and the impact of LPS-triggered macrophages. These insights could be important for applications and risk assessments of AuNPs as well as for the understanding of the biological fate of NPs in general.

EXPERIMENTAL SECTION Chemicals and cells. Dulbecco's Modified Eagle Medium (DMEM, Gibco Art. No. 41965036), phenol free DMEM (Art. No 21063-029), fetal bovine serum (FBS, Gibco Art. No 10270-106), penicillin/streptomycin (PEST, Gibco Art. No 15140-122), L-glutamine (Art. No 25030081), Phosphate-buffered saline (PBS, Gibco Art. No 100 10-015) and Hank’s buffered salt solution (HBSS, Gibco 14175-05) were purchased from Life Technologies (Thermo Fisher Scientific, USA). LPS (from Escherichia coli 0111:B4), glutathione (reduced form, 98-100%), dichlorodihydrofluorescein diacetate (DCFH-DA), potassium hydroxide (KOH) and H2O2 were from Sigma-Aldrich, Sweden. Iron sulfate (FeSO4×7H2O) was obtained from Merck, USA. Stock solution of gold ions ([HAuCl4], 1000±6 µg Au/ml in 2 % HNO3 (v/v), and Bismuth 1004±6 µg/ml in 5% HNO3 were obtained Spectrascan, Teknolab, Sweden. Nitric acid (67% HNO3 for trace metal analysis) were from Normatom; VWR, Leuven, Belgium) and hydrochloric acid (HCl 37% standard grade) from EMPARTA ACS for analysis, Merck, Darmstadt, Germany). Ultrapure water used in the study was produced by using PURELAB flex 3 from ELGA labwater (UK) (18.2 MΩ/cm). The murine macrophage RAW 264.7 cell line were obtained from ATCC (American Type Culture Collection). The cells were cultured in DMEM (Gibco 41965-039) supplemented with 10% FBS, 1% Lglutamine and 1% PEST at 37C under a humidified atmosphere containing 5% CO2. Nanoparticles. Citrate-coated 5 and 50 nm AuNPs were obtained as stock suspensions of approximate 1mg/ml in 2 mM sodium citrate from NanoComposix in USA (BioPure, (99,99% pure,) batch no JEA0027 and MGM2244, respectively). These suspensions are 20 ACS Paragon Plus Environment

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sterile and free from endotoxin (< 2.5 EU/ml). Our own ICP-MS analysis demonstrated concentrations of 1.16 and 1.37 mg/ml for the 5 and 50 nm AuNPs, respectively. In order to confirm the primary sizes and agglomeration in DMEM, TEM imaging was performed using a Hitachi HT 7700 (Hitachi, Tokyo, Japan) electron microscope at 80 kV and digital images was taken by a Veleta camera (Olympus, Münster, Germany). TEM analysis of cellular uptake. The murine macrophage RAW 264.7 cell line was cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine and 1% penicillin/streptomycin at 37 C under a humidified atmosphere containing 5% CO2. For TEM analysis, 1.25×106 cells were seeded per well in 6-well plates and were after 24 h exposed to the AuNPs (5 µg/mL) for 24 h. Cells were then washed, scraped, centrifuged and fixed in freshly prepared 0.1M glutaraldehyde solution. The pellets were then post fixed in 2% osmium tetroxide. Ultrathin sections (approximately 60–80 nm) were cut and contrasted with uranyl acetate followed by lead citrate. Samples were then examined using a FEI Tecnai 12 Spirit Bio TWIN transmission electron microscope at 100 kV. Detection of ROS production. Production of intracellular reactive oxygen spieces was measured by using the dichlorodihydrofluorescein diacetate (DCFH-DA) assay. DCFH-DA is a lipophilic cell permeable compound that is deacetylated in the cytoplasm to DCF by cellular esterases. DCF is then oxidized by radicals such as hydroxyl, peroxyl, alkoxyl, nitrate and carbonate to a fluorescent molecule. RAW 264.7 macrophages were seeded in black well plates (Costar, clear bottom, 55.000 cells/well) and were after 24 h incubated with LPS (0.1 μg/mL), AuNPs (5 μg/mL), or a both LPS and AuNPs, for 24 h. After exposure, cells were washed with HBSS and were then loaded with 20 μM DCFH-DA for 45 min at 37°C. Thereafter, cells were washed with HBSS, 100 μL HBSS was added to each well and fluorescence was recorded every 10 min over 50 min (excitation 485 nm, emission 535 nm) using a plate reader (Tecan Infinite F200) at 37°C. For each experiment, the mean of four



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wells with the same exposure was calculated. ROS increase was then calculated as times increase in fluorescence of wells exposed to AuNPs vs controls, and times increase of fluorescence of wells with LPS vs the same exposure without LPS. Results are presented as the mean +/- SD of three independent experiments. In vitro dissolution assay. RAW 264.7 cells were seeded into each well of 24-well plates (3527 cell culture plate, Corning Incorporated); 250 000 cells (for the 0- and 24-h exposure) or 10 000-15 000 cells (for the one-week exposure). After 3 h (in the case of the 24-h exposure) or 24 h (for the 0-h- and one-week exposures), 5 µg AuNPs were added to each well, containing 1 ml (the 0-h and 24-h exposures) and 0.75 ml (one-week exposure) cell medium. For the latter, 0.25 ml additional cell medium was added to each well after 3 days to provide fresh nutrients to the cells. The doses used should result in approx. 10 pg/macrophage, which usually is considered a relevant macrophage loading. To trigger the macrophages, LPS was added in at a final concentration of 0.1 µg/ml per well, immediately or on day 3, respectively. As a control, the same procedure was performed in cell medium without cells. After exposure, the cell medium was collected in 1.5 ml microtubes (Safe Lock Tubes, Eppendorf AG); ultrapure water (200 µl) added to the wells; and the cells lysed by mechanically shearing using 5 passages through a 27G needle (Microlance3, 27 G3/4, 0.4x19 Nr.20, reference number 302200, Becton Dickinson). The wells were rinsed with 200 µl HNO3 (20 vol%, diluted with ultrapure water from 67% HNO3) and this rinse and the cell lysate added to the tubes containing the corresponding cell medium. To separate dissolved gold from the remaining NPs and cell debris, these mixtures were centrifuged at 13 000 rpm (16 060 g) for 30 minutes at 4 C, 0.25 ml of the supernatant was withdrawn and prepared for metal analysis by inductively coupled plasma mass spectrometry (ICP-MS).


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Test of separation efficiency and ability to detect dissolved gold. To initially explore the separation efficiency at different conditions, gold was analyzed in the supernatant/filtrate immediately after addition of AuNPs to a solution of lysed cells (prepared from un-exposed cells according to above), or immediately after addition to medium (alone), followed by centrifugation at 13 000, with or without subsequent ultrafiltration through a 10 kDa filter (Amicon, Merk Millipore). The separation efficiency (%) was calculated as 100 minus percent of gold detected in the supernatant/filtrate for each sample (i.e. 100% in case no gold is detected). To initially confirm that the method allows detection of released ions, 0.25 µg gold ions (produced from stock solution of [HAuCl4]) was added to a solution of lysed cells (prepared from un-exposed cells according to above) followed by centrifugation (13 000 rpm) with or without subsequent ultrafiltration through a 10 kDa filter (Amicon, Merk Millipore). To further investigate detection of gold ions after different time-points in contact with macrophages, unstimulated or triggered macrophages were exposed to 0.25, 1 or 5 µg gold ions [HAuCl4] for 0, 24 and 168 h following the protocol described above for the AuNPs. Simple dissolution assay to test the role of biomolecules. To test if dissolution of AuNPs by ROS is promoted by biomolecules, a simplified dissolution assay was performed. 5 nm AuNPs were thus incubated together with 50 M Fe2+ (from FeSO4×7H2O) and 200 M H2O2 (to produce hydroxyl radicals) in three different solutions; 1) water (ultrapure), 2) water (ultrapure) and 200 M glutathione (diluted from a stock solution of 0.2 M and adjusted to pH 4-5 using KOH), and 3) cell medium (serum and phenol free). The samples were prepared by adding 10 L of 5 mM Fe2+, 2 l of 100 mM H2O2 (diluted from stock of 30% w/v) and 5 L of 1 mg/mL AuNPs (5 nm) to the the solutions in a final volume of 1 ml. The tubes were covered with parafilm, mixed by vortexing and incubated in room temperature for 24 h or 1 week (168 h). After 3-4 days, another 2 μL of the diluted H2O2 stock was added to samples aimed to be incubated for 168 h. After incubation, 500 L was added to 10 kDa centrifugation 23 ACS Paragon Plus Environment


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filters (VWR, cat no 82031-348) followed by centrifugation (13 000 rpm) for 30 minutes at 4 C. 200 L of the filtrate was transferred to an Eppendorf tube and treated with Aqua regia for at least 24 h before ICP-MS analysis. Gold analysis by ICP-MS. 0.25 ml of the samples containing gold ions released extra- and intracellularly was digested with 1 ml aqua regia for 24 h prior to gold analysis by ICP-MS. For the simplified dissolution assay, 0.2 ml was digested with 0.8 ml aqua regia. To correct for evaporation during this treatment, the volume remaining in each tube was measured afterwards. 0.5 ml of each treated sample was mixed with 4.5 ml ultrapure water and 20 µl of an internal bismuth standard (1.25 mg Bi/l diluted from a stock solution) in a Falcon tube prior to analysis by ICP-MS (iCAP Q; Thermo Scientific, Waltham, MA, USA). Standard solutions of 0, 0.1, 0.5, 1, 5, 10, 50 and 100 µg Au/l were prepared from a gold stock solution diluted with 2% aqua regia and bismuth (5 µg Bi/l) again added as the internal standard. The levels of 197Au and 209Bi isotopes were detected in triplicate for each sample using the flatapole collision/reaction cell and helium as the collision gas (at a flow of 5.1 ml/min). Argon gas was used for cooling (14 l/min) and as the auxiliary (0.79 l/min) and nebulizer gas (1.0 l/min). In the experiments related to separation efficiency and ability to detect dissolved gold, the limits of detection (LOD) and quantification (LOQ) were set at 0.092 and 0.276 µg Au/l, i.e., 3- and 10-fold, respectively, derived from the standard deviation (SD) of the blank samples (cells not exposed to AuNPs). Recovery of the internal standard, varied from 64 to101% (average 84 ± 7.2 %), and the gold concentrations were corrected accordingly. In the simplified dissolution assay, the LOD and LOQ were set to 0.009 and 0.029 µg Au/l / and the recovery of internal standard, varied from 70 to 114% (average 97 ± 11.8 %). In all other analysis, the LOD and LOQ were set at 0.0035 and 0.012 µg Au/l and the recovery of the internal standard, varied from 63-93% (average 82 ± 5.1 %).


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Nanoparticle tracking analysis (NTA) . To obtain the hydrodynamic size distribution of the AuNPs in the cell medium, a Nanosight NS300 instrument (Malvern, Uppsala, Sweden) with a 405 nm laser was used for nanoparticle tracking analysis (NTA). First, control measurement of 23-nm latex NPs gave reliable results and measurement of ultrapure water revealed little particle contamination. Since this procedure tracks individual particles and thus requires diluted solutions, the medium/NP mixture was diluted with ultrapure water and analysis performed immediately, as well as after standing for 24 h at 37° C (supplement Figure S1). The hydrodynamic size distribution of the 5 nm AuNPs in ultrapure water was also determined. Each measurement was performed three times at 25° C and with 60-second captures. A NTA 3.2 software was employed and the viscosity of water utilized as the input value. Cellular dose of AuNPs. To determine cellular dose, 45 000 cells in 1 ml cell medium were seeded in each well of 24-well plates. After 24 h, 5 µl 5 or 50 nm AuNPs (1mg/ml suspension) was added. For triggering macrophages, 10 µl LPS (0.01 mg/ml) was added immediately after the addition of NPs. After 24 h of exposure, the cell medium was removed and the cells were treated with 1 ml aqua regia for at least 24 h. Prior to ICP-MS analysis, 0.5 ml of each sample was diluted with 4.5 ml ultrapure water in 1 5 ml Falcon tubes. Equilibrium speciation modeling To theoretically examine the solubility of gold ions in the cell medium, the Joint Expert Speciation Software, JESS, (version 8.3)62 was applied. This software calculates the theoretical fraction of gold in solution at equilibrium based on thermodynamic principles, input and factors such as pH, potential, temperature, the concentration of gold, and composition of the medium (i.e. counter ions/ionic strength), as well as the gold reactions recorded in its database (based exclusively on pH, redox potential and chlorides). Most, but not all of the constituents of the cell medium we used were included in this database.



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Supplement Table S1 shows the input values utilized in the program. In addition, the concentration of gold ions, pH, and redox potential (Eh) were varied. Precipitation was allowed, so that the relative amounts of dissolved and precipitated gold could be assessed. The redox potential of the cell medium was measured with a Mettler Toledo InLab Redox ORP electrode, calibrated with ORP standard solution in accordance with the manufacturer’s instructions. After 3, 10, 15 min, and 6 days at 37° C with 5 % CO2 buffering, these potentials varied between 350 and 380 mV Eh (versus a standard hydrogen electrode, SHE). Accordingly, redox potentials of 300-430 mV Eh were chosen for the calculations. Since in the presence of cells a slight decrease in pH was observed (as indicated by the pH indicator in the cell medium) after one week, the pH input values were varied from 4.5 - 7.4. The input values for the concentrations of gold ion were set at 1.27, 5.08, and 25.4 µM, e.g., the same as those utilized experimentally. Statistical analyses Quantile regression with the amount of gold ion dissolved as dependent variable and a categorical variable as the only independent variable was employed.63 The categorical variable was included in the regression model by means of indicator variables. It comprised fourteen different categories defined by all possible combinations of size ( 5 and 50 nm), duration of incubation (0, 24, and 168 h), and sample composition (cell medium with and without unstimulated or LPS triggered macrophages), more information about sample size, see supplement Table S1, S2 and S4). The standard error for the median estimated was computed with a robust estimator assuming that the residual density is continuous and bounded away from zero and infinity at the median. P-values lower than 0.05 were considered statistically significant. All of these analyses were performed with Stata version 14 software (StataCorp, College Station, TX).


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Quantile regression was chosen here because some of the data were skewed, rather than normally distributed, in five cases the results were below LOD (50 nm AuNPs and incubated for 0 or 24 h) and assigned a value of LOD/2. Results below LOQ (15% of the values, all for samples containing 50 nm AuNPs and incubated for 0 or 24 h) were used without modification. ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Table S1. Gold ions detected after incubation with macrophages Table S2. Amount of dissolved gold in cell medium after incubation at different time points. Table S3. Set non-variable input parameters for JESS (37ºC) Table S4. Results from quartile regression in Stata version 14 (StataCorp, College Station, TX) Figure S1. Hydrodynamic number size distributions of the 5 nm and 50 nm AuNPs in diluted cell medium. Figure S2. Cell viability after exposure to AuNPs. Figure S3. TEM images of macrophages with or without LPS triggering AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Present Address †Unit of Work Environment Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-17177 Stockholm, Sweden 27 ACS Paragon Plus Environment


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Author Contributions UC was involved in the experimental design and data interpretation, performed parts of the cell culture work, conducted the ICP-MS analysis, made the figures and wrote the first version of the manuscript; KM was involved in the experimental design, supervised the ICPMS analysis /data interpretation and was involved in manuscript writing; YSH performed the NTA, PCCS, redox potential measurements and JESS predictions and was involved in data interpretation and manuscript writing; GJ was involved in data interpretation and manuscript writing; MB performed the statistics; HLK supervised the study, was involved in the experimental design and data interpretation, performed the main part of the cell culture work, performed the simple dissolution assay and finalized the manuscript. All authors contributed to and approved the final version of the manuscript. Funding Sources This project was supported financially by the Swedish Research Council for Health Working Life and Welfare (Forte, grant 2010-0702) and the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (Formas, grant 2017-00883). Funding from the Swedish Research Council (VR, grants 2013-5621, 2014-4598 and 201504177) and from the Institute of Environmental Medicine, Karolinska Insitutet, Sweden, is also acknowledged. ACKNOWLEDGMENT The authors gratefully acknowledge Dr. K. Hultenby, Karolinska Institutet, for help with TEM imaging. ABBREVIATIONS AuNPs – gold nanoparticles; FBS – fetal bovine serum; ICP-MS – inductively coupled plasma mass spectrometry; LPS – lipopolysaccharide; NTA – Nanoparticle Tracking


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Macrophage-Assisted Dissolution of Gold Nanoparticles - ACS

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