Pulmonary surfactant mitigates silver nanoparticle toxicity in human alveolar type-I-like epithelial cells

Colloids and Surfaces B: Biointerfaces 145 (2016) 167–175

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Pulmonary surfactant mitigates silver nanoparticle toxicity in human alveolar type-I-like epithelial cells Sinbad Sweeney a , Bey Fen Leo b,c , Shu Chen d , Nisha Abraham-Thomas a , Andrew J. Thorley a , Andrew Gow e , Stephan Schwander f , Junfeng Jim Zhang g , Milo S.P. Shaffer d , Kian Fan Chung h , Mary P. Ryan b , Alexandra E. Porter b , Teresa D. Tetley a,∗ a

Lung Cell Biology, Airways Disease, National Heart & Lung Institute, Imperial College London, London, UK Department of Materials and London Centre for Nanotechnology, Imperial College London, London, UK c Department of Mechanical Engineering, University of Malaya, Kuala Lumpur, Malaysia d Department of Chemistry and London Centre for Nanotechnology, Imperial College London, London, UK e Department of Toxicology, Ernst Mario School of Pharmacy, Rutgers University, Piscataway, NJ, USA f Department of Environmental and Occupational Health, School of Public Health, Rutgers University, Piscataway, NJ, USA g Division of Environmental Sciences & Policy, Nicholas School of the Environment and Duke Global Health Institute„ Duke University, Durham, USA h Respiratory Medicine and Experimental Studies Unit, Airways Disease, National Heart & Lung Institute, Imperial College London, London, UK b

a r t i c l e

i n f o

Article history: Received 13 November 2015 Received in revised form 4 April 2016 Accepted 19 April 2016 Available online 27 April 2016 Keywords: Silver nanoparticles Lung toxicity Surfactant Silver ions

a b s t r a c t Accompanying increased commercial applications and production of silver nanomaterials is an increased probability of human exposure, with inhalation a key route. Nanomaterials that deposit in the pulmonary alveolar region following inhalation will interact firstly with pulmonary surfactant before they interact with the alveolar epithelium. It is therefore critical to understand the effects of human pulmonary surfactant when evaluating the inhalation toxicity of silver nanoparticles. In this study, we evaluated the toxicity of AgNPs on human alveolar type-I-like epithelial (TT1) cells in the absence and presence of Curosurf® (a natural pulmonary surfactant substitute), hypothesising that the pulmonary surfactant would act to modify toxicity. We demonstrated that 20 nm citrate-capped AgNPs induce toxicity in human alveolar type I-like epithelial cells and, in agreement with our hypothesis, that pulmonary surfactant acts to mitigate this toxicity, possibly through reducing AgNP dissolution into cytotoxic Ag+ ions. For example, IL-6 and IL-8 release by TT1 cells significantly increased 10.7- and 35-fold, respectively (P < 0.01), 24 h after treatment with 25 ␮g/ml AgNPs. In contrast, following pre-incubation of AgNPs with Curosurf® , this effect was almost completely abolished. We further determined that the mechanism of this toxicity is likely associated with Ag+ ion release and lysosomal disruption, but not with increased reactive oxygen species generation. This study provides a critical understanding of the toxicity of AgNPs in target human alveolar type-I-like epithelial cells and the role of pulmonary surfactant in mitigating this toxicity. The observations reported have important implications for the manufacture and application of AgNPs, in particular for applications involving use of aerosolised AgNPs. © 2016 Elsevier B.V. All rights reserved.

1. Introduction According to the Project on Emerging Nanotechnologies (http:// www.nanotechproject.org), silver nanomaterials currently represent the greatest proportion of commercialised nanomaterials globally [1]. Silver nanomaterials have a wide range of biomedical applications, including therapeutics and diagnostics. Their antibac-

∗ Corresponding author. E-mail address: [email protected] (T.D. Tetley). http://dx.doi.org/10.1016/j.colsurfb.2016.04.040 0927-7765/© 2016 Elsevier B.V. All rights reserved.

terial, antiviral and antifungal activity has led to their use in wound dressings, catheter coatings and medical devices [2–5]. In diagnosis, the enhanced plasmon resonant scattering from silver nanomaterials promotes their use as significantly improved microscopic imaging labels [6]. Inhalation is potentially a key route of human exposure to silver nanomaterials. Focusing on the lung, the toxicity of silver nanomaterials has been investigated in a range of human in vitro cell models including bronchial epithelial cells, macrophages and human lung fibroblasts [7–10]. In the aforementioned studies and across the available literature in general, silver ions (released during

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dissolution of the silver nanomaterial) are suggested as the principal contributors to toxicity [11–14]. Understanding silver nanomaterial toxicity with cells of the alveolar region is crucial, where inhaled nanoparticle deposition rate is relatively high [15]. Over 95% of the alveolar surface is composed of alveolar type-I epithelial cells (ATI). ATI cells are highly attenuated, squamous cells (∼200 nm thick and 40–80 ␮m in diameter; facilitating efficient gas exchange across the alveolar wall) [16] and are coated by a pulmonary surfactant-containing compound that lowers surface tension at the alveolar air-liquid interface, preventing the lungs from collapsing on exhalation. Pulmonary surfactant (secreted by alveolar type-II epithelial cells) is largely composed of phospholipids (∼90% by mass) and proteins (∼10% by mass) [17]. Phosphatidylcholine accounts for ∼70% of total phospholipid weight, ∼50% of which is saturated dipalmitoylphosphatidylcholine (DPPC), primarily responsible for surfactant’s surface tension lowering capabilities [18]. DPPC has also been reported to promote particle aggregation [19–21] and, as we have recently shown, has the ability to delay the dissolution of AgNPs into Ag+ ions [21]. Curosurf® is a natural pulmonary surfactant, isolated from porcine lungs with properties similar to human pulmonary surfactant. It is available commercially and is used as a therapeutic intervention in neonatal respiratory distress syndrome [22]. According to the manufacturer, Curosurf® almost exclusively contains polar lipids, in particular DPPC (∼50% of the total phospholipid content). Inhaled silver nanomaterials that deposit in the alveolar unit will interact with pulmonary surfactant prior to interaction with ATI cells. It is therefore critical to consider the effects of pulmonary surfactant when determining the toxicity of silver nanomaterials in these cells. In the present study, we investigated the toxicity of citrate-capped silver nanoparticles (AgNP; spherical geometry, 20 nm diameter and thus capable of reaching the alveolar unit) in human alveolar type I-like epithelial cells (TT1), in the absence or presence of Curosurf® . We hypothesised that Curosurf® would act to modify AgNP toxicity and that this could be attributed to its ability to slow the kinetics of Ag+ ion release from inhaled AgNPs in the lung. 2. Methods 2.1. AgNP characterisation 20 nm citrate-capped BioPureTM silver nanoparticles, sterile and endotoxin-free (<5 EU/ml) were obtained from nanoComposix (San Diego, USA). The morphology and primary size of particles were characterised using a JEOL 2100 Field Emission Transmission Electron Microscope (FEG TEM), operated at an accelerating voltage of 200 kV. AgNP suspensions were sonicated in a bath sonicator for 10 s and a single drop of the suspension was deposited on to a 300Cu Mesh grid with a holey carbon support film and were left to dry under vacuum for TEM imaging. High-resolution transmission electron microscopy (HRTEM) is a highly sensitive technique that enables the atomic structure of the material to be analysed. The chemistry composition of the as-received AgNPs was examined using energy dispersive X-ray (Oxford Instruments EDX detector) and to ensure that sulfidation of AgNPs had not occurred in the environment. Moreover, their surface charge and size distribution in RPMI 1640 culture medium and Curosurf (2 and 24 h) were further investigated using Zeta potential and dynamic light scattering (DLS) measurements, respectively. 2.2. TT1 cell model Our laboratory has previously immortalised human ATI-like cells (derived from freshly isolated and cultured primary human

ATII cells) using transduction with the catalytic subunit of telomerase (human telomerase reverse transcriptase; hTERT) and a temperature sensitive mutant of simian virus 40 large-tumour antigen [23]. These ATI-like (transformed type-I-like; TT1) cells are negative for the ATII cell markers SP-C, alkaline phosphatase and thyroid transcription factor-1. Moreover, TT1s do not contain lamellar bodies. They display a thin, attenuated morphology containing vesicles, being caveolae positive. TT1 cells were cultured as previously described by us [24].

2.3. Preparation of Curosurf® pre-incubated AgNPs Curosurf® , a natural pulmonary surfactant prepared from porcine lungs with properties similar to human pulmonary surfactant (but lacking SP-A and SP-D), was kindly donated by Chiesi Pharmaceuticals (Cheadle UK). AgNPs were incubated (at room temperature and with agitation for 1 h) with a 1:1000 dilution of Curosurf® (in RPMI 1640 culture medium). The incubated AgNPs were then centrifuged at 10,000g for 10 min, the pellet was washed twice by suspension in PBS followed by centrifuging again at 10,000g for 10 min. Following washing, the Curosurf® preincubated AgNPs were suspended in RPMI 1640 culture medium, ready for cell exposure. The AgNPs alone sample was prepared exactly the same as the pre-incubated, except Curosurf® was omitted from the RPMI 1640 culture medium.

2.4. TT1 cell treatment with AgNPs TT1 cells were treated with AgNPs (alone or with Curosurf® ) at concentrations ranging from 1.56 to 50 ␮g/ml (prepared in RPMI 1640 culture medium) or AgNO3 at concentrations ranging from 0.078 to 1.25 ␮g/ml (prepared in RPMI 1640 culture medium). The AgNP concentration range equates to approximately 0.48–15.6 ␮g/cm2 of alveolar surface area and is considered relevant in terms of potential exposure when particle deposition “hotspots” [25] and the slow clearance rate of insoluble particles from the alveolar region [26] are considered. For reference, we have calculated the insoluble silver particle recommended occupational exposure to be 0.0036 ␮g/cm2 /h [27,28] (see Supplementary for calculation details).

2.5. Transmission electron microscopy analysis of AgNP uptake Following 24 h AgNP (+/− Curosurf® ) treatment, TT1 cells were rinsed with fresh RPMI 1640 culture medium and then fixed in 2.5%glutaraldehyde in 0.1 M PIPES buffer, pH 7.2 for 1 h at 4 ◦ C. The use of RPMI cell culture medium ensured that the AgNPs did not dissolve or transform to Ag2 S in the cell culture medium before any interactions with TT1 cells [29]. Cell sectioning and imaging was performed as described by us previously [24], using a bright field transmission electron microscope (JEOL 2000) operated at 80 kV.

2.6. Cell viability TT1 cells were seeded on 96-well plates as described above. Following 24 h treatment with AgNPs (+/− Curosurf® ) TT1 cells were rinsed with PBS and incubated with RPMI 1640 culture medium containing the MTS reagent according to the manufacture’s protocol for 45 min (CellTiter 96® AQueous One Solution Assay, Promega, USA). The absorbance of formazan product in the culture medium was then read using a spectrophotometer at a wavelength of 490 nm.

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TT1 cells were seeded on 96-well plates as described above. Following 24 h treatment with AgNPs (+/− Curosurf® ), TT1 cell mediator-conditioned medium was assayed for concentrations of the inflammatory mediators, human IL-6 and IL-8 using sandwich enzyme-linked immunosorbent assays (ELISA). The assays were performed using DuoSet® antibody kits, according to the manufacturer’s directions (R&D systems, USA).

the mean ± standard error (n = 3; where three independent experiments were performed using three separate TT1 cell passage generations). We elected a priori to compare treated groups to untreated groups; thus significant effects of AgNP treatment (+/− Curosurf® ) were determined using a one-way (cell viability and ROS generation data) or two-way (inflammatory meditator release and lysosomal disruption data) analysis of variance, with Bonferroni post tests. In all analyses, a P value <0.05 was considered significant.

2.8. Generation of reactive oxygen species

3. Results

TT1 cells were seeded on 96-well plates (black, with a clear bottom) as described above. Following 4 and 24 h treatment with AgNPs (+/− Curosurf® ), or tert-Butyl hydroperoxide (TBHP, a positive control), intracellular TT1 cell reactive oxygen species (ROS) were detected by measuring the oxidation of fluorescent dihydroethidium (DHE), as described by us previously [24].

3.1. AgNP material physicochemical characteristics

2.7. Measurement of inflammatory mediator release

2.9. AgNP dissolution into Ag+ Inductively coupled plasma optical emission spectroscopy (ICPOES) was used to determine the amount of dissolved Ag in the presence or absence of Curosurf® (100 ␮g/ml at pH 7). To minimise the impact of anions on the stability of the AgNPs, non-interacting buffers—either 0.1 M sodium perchlorate (NaClO4 ) or perchloric acid (HClO4 ) (Sigma-Aldrich), were used to adjust the pH. Each AgNP suspension was incubated in a temperature controller at 37 ◦ C and then centrifuged at high speed (13,000 rpm) with 2 kDa (<4 nm) filter tubes (Sartorius Stedim VIVACON 500) to separate the NPs from the solution. The concentration of released Ag+ ions was measured after the particles had been removed at different time intervals of 2, 6 and 24 h (n = 3). For the control experiment, pure water (no AgNPs) and supernatant from which the AgNPs had been removed were analysed to ensure that any residual AgNPs were removed by centrifugation and filtering. Our previous work showed that dissolved Ag+ ions will re-precipitate as insoluble Ag salts (e.g. Ag2 O, AgCl and Ag2 S) in the RPMI cell culture medium [20]. For this reason, we decided not to measure the amount of ionic Ag+ in the cell culture medium as it was expected that the amount of Ag+ ion release in this medium will be below the detection limit of ICP. 2.10. Contribution of lysosomal disruption to AgNP toxicity LysoSensor Green DND-189 was used to measure pH changes in lysosomes and hence lysosomal disruption following 2, 4, 6 and 24 h treatment with AgNP (+/− Curosurf® ) or with AgNO3 . The LysoSensor probe is a fluorescent pH indicator that partitions into acidic compartments (primarily lysosomes) as a result of protonation. Protonation of probe removes the fluorescent quenching ability of their weak base side chain, thus increasing fluorescence. LysoSensor Green DND-189 has a pKa of 5.2 and therefore becomes less florescent at higher pH values. Briefly, following treatment or nontreatment, TT1 cells were rinsed with PBS and incubated with RPMI 1640 culture medium containing 100 nM of the LysoSensor Green DND-189 probe, for 15 min (37 ◦ C with 5% CO2 ). At the end of the incubation period, TT1 cells were washed twice to remove residual probe and were then imaged using a fluorescent microscope and green fluorescence intensity was quantified using a fluorescence plate reader at Ex/Em: 485/530 nm (Tecan, Switzerland). 2.11. Statistical analyses Data from cell viability, inflammatory meditator release, ROS generation and lysosomal disruption experiments are presented as

Transmission electron microscopy (TEM) characterisation of the AgNPs revealed a spherical geometry with an average diameter of 19.5 ± 3.6 nm (mean +/− standard deviation; Fig. 1a). The hydrodynamic diameter of NPs in aqueous solution was also determined by DLS, which demonstrated that the average particles size was 22.2 ± 0.4 nm, in agreement with the TEM size measurement. TEM and DLS analysis confirmed that the AgNPs had an average diameter consistent with manufacturers’ specifications. The ␰ value of the as-received AgNPs/citrate was −39.4 ± 0.5 mV in deionised water. This negative surface charge is consistent with the electrostatic stabilisation against aggregation. The pure silver composition was confirmed by EDX analysis, showing sulfidation of AgNPs had not occurred and all impurities had been removed during washing (Fig. 1d). The AgNPs had a lattice spacing of 0.24 nm, consistent with the (111) interplanar spacing of bulk Ag as shown in standard reference structures (ref. # 01-087-0597). 3.2. Transmission electron microscopy Bright-field transmission microscopy (BFTEM) imaging showed that AgNPs were taken up by TT1 cells following 24 h exposure and were located both in the cytoplasm and in poorly-defined endosome/lysosome-like vesicles (Fig. 2). In contrast, AgNPs preincubated with Curosurf® were also internalised but were not seen in the cytoplasm and were located in large, well-defined lysosomal/endosomal structures following 24 h treatment of TT1 cells; these AgNPs were mainly found as agglomerates (Fig. 2). 3.3. Cell viability AgNPs only significantly reduced TT1 cell viability (measured using the MTS assay) at the highest exposure concentration, 50 ␮g/ml (14% reduction after 24 h exposure; P < 0.05; Fig. 3A). After pre-incubation of AgNPs with Curosurf® , TT1 cell viability following 24-h exposure was only significantly changed at the highest exposure concentration, 50 ␮g/ml. 3.4. Inflammatory mediator release Following 24 h treatment with 12.5, 25 and 50 ␮g/ml of AgNPs, TT1 cell IL-6 release significantly increased by 9.4-, 10.7 and 17.4fold respectively (P < 0.01; Fig. 3B); IL-6 release was unchanged following treatment with the lower concentrations of AgNPs. Preincubation of AgNPs with Curosurf® significantly reduced the magnitude of this induced IL-6 release. Specifically, following 24 h treatment with 12.5 and 25 ␮g/ml of AgNPs (+ Curosurf® ), the magnitude of IL-6 release was reduced by 73 and 74% (P < 0.01), respectively; at 50 ␮g/ml of AgNPs the reduction was 71% (P < 0.001). Following 24 h treatment with 12.5, 25 and 50 ␮g/ml of AgNPs, TT1 cell IL-8 release significantly increased by 18-, 35 and 52-fold respectively (P < 0.01; Fig. 3B); IL-8 release was unchanged following treatment with the lower concentrations

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Fig. 1. Physicochemical characteristics of AgNPs. (A) TEM images of AgNPs, (B) size distribution of AgNPs based on n = 450, (C) AgNPs showing a lattice spacing of 0.24 nm, consistent with the (111) interplanar spacing of bulk Ag and (D) background-corrected STEM-EDX analysis of AgNPs (area under HR-TEM, seen in image insert).

of AgNPs. Pre-incubation of AgNPs with Curosurf® significantly reduced the magnitude of this induced IL-8 release. Specifically, following 24-h treatment with 12.5 and 25 ␮g/ml of AgNPs (+ Curosurf® ), the magnitude of IL-8 release was reduced by 72 and 94% (P < 0.05), respectively; at 50 ␮g/ml of AgNPs the reduction was 93% (P < 0.001).

to approximately 0.3, 0.325 and 0.35 ␮g of Ag, respectively. Following pre-incubation of AgNPs with Curosurf® and after 2, 6 and 24 h incubation in ClO4 buffer solution at pH 7, the percentage of Ag that had dissolved from 25 ␮g of AgNPs was reduced to zero, zero and 0.4%, respectively (Fig. 4A).

3.5. AgNP dissolution into Ag±

3.6. Proportion of inflammatory mediator induction attributable to Ag± release

After 2, 6 and 24 h incubation in perchlorate (ClO4 ) buffer solution at pH 7, the percentage of Ag that had dissolved from 25 ␮g of AgNPs was 1.2, 1.4 and 1.3%, respectively; this equates

To determine what proportion of the inflammatory mediator release (induced by 25 ␮g AgNP) was potentially attributable to the release of Ag ions, we exposed TT1 cells to increasing

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Fig. 2. AgNP-treated TT1 cells under TEM. Intracellular localisation of 25 ␮g/ml AgNP (+/− Curosurf® ) following 24-h exposure in TT1 cells. N = nucleus, E/L = endosome/lysosome, ES = extracellular space, * = cytoplasmic AgNPs.

Fig. 3. (A) Cell viability. TT1 cell viability following 24-h treatment with AgNP (+/− Curosurf® ). Cell viability data are presented as a% of the non-treated (NT) control (n = 3) ± SEM; significant differences between non-treated and treated cells are indicated where * P < 0.05. (B) IL-6 and IL-8 release (pg/ml; normalised to cell number) from TT1 cells following 24-h treatment with AgNP (+/− Curosurf® ); n = 3; ± SEM; significant differences between non-treated and treated cells are indicated where # P < 0.05 and significant differences between responses in the absence and presence of Curosurf® are indicated where * P < 0.05, ** P < 0.01 and *** P < 0.001.

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Fig. 4. (A) Ag+ ion release from 25 ␮g AgNPs. AgNPs incubated in perchlorate acid/perchlorate buffer solutions (pH 7) over the course of 24 h (+/− Curosurf® ). Ag+ ion release from AgNPs (+ Curosurf® ) were below detection limit at 2 and 6 h, indicating negligible dissolution occurring at these times. (B) IL-6 and IL-8 release (pg/ml; normalised to cell number) from TT1 cells following 24-h treatment with AgNO3 . n = 3; ± SEM; significant differences between non-treated and treated cells are indicated where * P < 0.05, ** P < 0.01 and *** P < 0.001.

concentrations of AgNO3 (0, 0.078, 0.156, 0.312, 0.625, 1.25 ␮g/ml). An AgNO3 concentration of 0.312 ␮g/ml induced approximately the same IL-6 and IL-8 release as 25 ␮g/ml AgNPs (Fig. 4B); the proportion of inflammatory mediator release potentially attributable to these Ag ions was 88 and 79% for IL-6 and IL-8, respectively. TT1 cell viability following treatment with AgNO3 is shown in Supplementary Fig. S1, and 0.312 ␮g/ml did not cause noteworthy cell death, however 0.625 and 1.25 ␮g/ml induced a large amount of cell death, accounting for the large decrease in IL-6 and IL-8 release seen at these concentrations. 3.7. Generation of reactive oxygen species The fluorescent probe, DHE, was used to measure the generation of ROS (primarily, the superoxide anion) in TT1 cells, 4 and 24 h after treatment with 25 ␮g/ml AgNP or AgNP preincubated with Curosurf® . TT1 cell superoxide levels did not significantly change following either treatment at either time point (Supplementary Fig. S3). 3.8. Contribution of lysosomal disruption to AgNP toxicity Disruption of TT1 cell lysosomes following treatment with 25 ␮g/ml AgNPs, as determined by their alkalinisation, was

measured using the fluorescent pH indicator probe LysoSensor Green DND-189. Following 2-h treatment with AgNPs, Lysosensor fluorescence decreased significantly by 79% (P < 0.05; Fig. 5), indicating lysosomal pH had increased. Following 4-h treatment with AgNPs, Lysosensor fluorescence had increased however the pH was still not as acidic as the non-treatment at this time point. A similar trend occurred following 6 and 24 h treatment with AgNPs, with lysosomal pH significantly increasing and recovering to nontreatment levels, respectively. Neither the treatment of TT1 cells with AgNPs pre-incubated with Curosurf® nor AgNO3 resulted in any changes to lysosomal pH compared to the non-treatment at any of the time points.

4. Discussion Increased commercial applications and production of silver nanomaterials is accompanied by an increased probability of human exposure, with inhalation a key route. Nanomaterials that deposit in the pulmonary alveolar region following inhalation will interact firstly with pulmonary surfactant before they interact with the alveolar epithelium. It is therefore critical to determine how pulmonary surfactant might affect nanoparticle-cell interactions when evaluating the inhalation toxicity of nanomaterials. Thus, in

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Fig. 5. Contribution of lysosomal disruption to AgNP toxicity. Fluorescence of the LysoSensor Green DND-189 probe in TT1 cells following 2, 4, 6 or 24-h treatment with AgNP (+/− Curosurf® ) or AgNO3 is shown in (A). The corresponding quantification of green fluorescence is shown in (B); significant differences between treated and non-treated TT1 cells are indicated where * P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

this study, we evaluated the toxicity of AgNPs on human alveolar type-I epithelial cells in the absence and presence of Curosurf® . While exposure to AgNP, with or without Curosurf® preincubation, did not result in any substantial reduction in cell viability, these nanoparticles were clearly capable of inducing TT1 pro-inflammatory bioreactivity. Significant increases in IL-6 and IL-8 release from TT1 cells may have important consequences for pulmonary disease. Increased release of IL-6 is associated with the pathogenesis of lung diseases such as asthma [30], chronic obstructive pulmonary disease [31] and idiopathic pulmonary fibrosis [32]. IL-6 can also translocate from the lung into the systemic circulation upon exposure to environmental particles [33] and is used as a biomarker of cardiovascular disease in humans [34]. IL-8 is a proinflammatory chemokine, functioning in neutrophil attraction to sites of cellular inflammation. Thus, our finding that pre-incubating AgNPs with Curosurf® significantly reduces the release of IL-6 and IL-8 from exposed TT1 cells has important implications for our understanding of the putative bioreactivity and toxicological profile of nanomaterials. Silver ions (Ag+ ), released during dissolution of silver nanomaterials, have been suggested to be the causal factor in AgNP toxicity in mammalian cells in vitro and the relative contribution of release of Ag+ ions extracellularly remains unclear [7,35–37]. The quantity and rate of Ag+ ion dissolution can differ depending on the AgNP size, geometry and surface coating [38–41]. Indeed we have previously shown that DPPC, a major component of Curosurf® , has the ability to delay the dissolution of AgNPs into Ag+ ions [21]. Therefore, in the present study we were interested to discover what role release of extracellular Ag+ ions may play in the toxicity of AgNPs in TT1 cells and if Curosurf® could affect the quantity and rate of Ag+ ion dissolution, possibly modifying the toxicity. At physiologically relevant pH7 we determined that the quantity of Ag+ ion release from 25 ␮g AgNP was approximately 1.3% or 0.325 ␮g/ml. Strikingly, we found that a large proportion of the inflammatory mediator release from TT1 cells (induced by 25 ␮g AgNP) could potentially be attributed to this quantity of released Ag+ ions. Moreover, we determined that pre-incubating the AgNPs with Curosurf® almost completely abolished Ag+ ion release from AgNPs over a 24 h period. Therefore, these data indicate that extracellular Ag+ ions could be the causal factor in AgNP toxicity in TT1 cells and that Curosurf® mitigates this toxicity by reducing the quantity and rate of Ag+ ion release. However, the dynamic behaviour of the Ag+ ions released from AgNP may well be different to AgNO3 ; some studies have suggested that AgNPs are more toxic than the equivalent concentration of Ag+ ions [11,37,42]. Regardless of which, the finding that Curosurf® protects against Ag NP and Ag+ ions exposure indicates the potential importance of lung surfactant in modifying Ag

NP bioreactivity and poses the question, how might Ag NP impact on ATII cell function and would surfactant synthesis/function be compromised? These questions remain to be clarified. While the precise mechanism of this mitigation is unclear, we propose that the Curosurf® may form a protective membrane layer around the AgNPs as we have reported previously for DPPC [21]. This could act to impart an initial retarding effect on the AgNPs dissolution and increasing their chemical stability. The present study of AgNP bioreactivity with TT1 cells, when considered together with our previous work with silver nanowires [24], also provides an interesting observation to the Ag nanomaterial toxicity paradigm. Our previous study revealed that Curosurf® was unable to reduce silver nanowire uptake or toxicity in TT1 cells, suggesting the geometry type of the silver nanomaterial (NP vs nanowire) may be an important determinant for toxicity; this may be associated with differential particle uptake (as described for other metallic nanoparticles) [43] or indeed differential Ag+ ion release kinetics between NP and nanowire geometries. Interestingly, in contrast to observations by Foldbjerg et al. in pulmonary adenocarcinoma A549 cells (∼70 nm; up to 15 ␮g/ml) [36] and Kim et al. in bronchial BEAS-2B cells (∼100 nm; up to 10 ␮g/ml) [42], we determined that the toxicity of AgNPs in TT1 cells was not precipitated nor accompanied by an increase in the generation of intracellular ROS. However, this is not unique to the present study as Carlson et al. have also previously reported AgNP toxicity in rat alveolar macrophages without increased ROS generation (∼30 nm; up to 75 ␮g/ml) [44]. Because we observed (under TEM) AgNPs both inside endosomal/lysosomal-like bodies, as well as within the cytoplasm, following 24-h TT1 cell exposure, we were interested to establish if AgNPs could induce lysosomal disruption that could contribute to their toxicity. Using the LysoSensor Green DND-189 probe, we observed acidic-alkaline fluctuations in the pH of TT1 cell lysosomes over a 2–24 h period, indicating that the lysosomes were undergoing some form of disruption, which can lead to a leaky membrane whereby cytosolic fluid at pH7 can enter and increase the lysosomal pH or loss of lysosomal structures altogether and thus reduced flourescence. Repair of leaky lysosomal membrane integrity, and/or generation of new lysosomes to deal with continued exposure to Ag NP, resulted in increased fluorescent signal. In an acellular system, we determined that AgNPs alone cannot modify the pH of a solution over time (Supplementary Fig. S2), thus this effect was biologically driven. Consistent with the reduction in AgNP-induced TT1 cell toxicity and the measured decrease in Ag+ ion release from AgNPs, pre-incubation of AgNPs with Curosurf® also mitigated the observed lysosomal disruption. Importantly, AgNPs preincubated with Curosurf® were also observed (under TEM) in

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endosomal/lysosomal-like bodies following 24-h TT1 cell exposure, but the endosomal structures were well-defined and structurally intact, unlike those in the AgNP-treated TT1 cells. It is likely that the differential effect is related to the ability of Curosurf® to reduce the quantity and rate of Ag+ ion release from AgNPs and thus prevent the lysosomal disruption detected in the AgNP-exposed cells. Interestingly when we treated TT1 cells with the equivalent quantity of Ag+ ions released from 25 ␮g/ml of AgNP (i.e. 0.325 ␮g/ml AgNO3 ), we did not observe lysosomal disruption over the 2–24 h period. This supports the suggestion that endosomal uptake of AgNP, followed by release of intra-lysosomal Ag+ ions accounts for lysosomal disruption. In addition, we have shown here that, should even a very low percentage of Ag+ ions be released extracellularly from AgNPs, in the absence of sulphidation or a biological membrane such as lung surfactant, there is potential to trigger a pro-inflammatory response, possibly even in the absence of AgNP uptake. 5. Conclusion Because inhaled AgNPs that deposit in the alveolar unit will interact with pulmonary surfactant prior to interaction with the alveolar epithelium it is critical to consider the effects of pulmonary surfactant when determining the toxicity of AgNPs in these cells. We have demonstrated that 20 nm citrate-capped AgNPs induce toxicity in human alveolar type I-like epithelial cells and furthermore that pulmonary surfactant acts to mitigate this toxicity, likely through reducing AgNP dissolution into toxic Ag+ ions. The observations reported here have important implications for the manufacture and application of AgNPs, in particular where application and use require aerosolised AgNPs.

[6]

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[11]

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Acknowledgements

[18]

This work was funded by the NIEHS (grant number U19ES019536). AP acknowledges an individual ERC starting grant (Project number 257182) for additional support for AP, BFL and SC. The silver nanomaterials investigated in this study were procured, characterized and provided by NIEHS as part of NCNHIR consortium program. We would like to thank Richard Blackwell and Daniella Lindsay of Chiesi Pharmaceuticals, UK for arranging the kind donation of Curosurf® to this study. This research was also supported by the Medical Research Council and Public Health England, Centre for Environment and Health.

[19]

[20]

[21]

[22]

[23]

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.04. 040.

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